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Ectopic expression of Neuronatin potentiates adipogenesis through enhanced phosphorylation of cAMP-response element-binding protein in 3T3-L1 cells
Abstract
Neuronatin (Nnat) is selectively expressed in the neonatal brain and is involved in neuronal differentiation during brain development. However, Nnat also appears to be abundantly expressed in adipose tissue, and is conspicuously elevated in the adipose tissue of obese Zucker diabetic fatty rats compared with control lean Zucker lean control rats shown in our previous report. Here, we examined the expression of Nnat in adipose tissue and demonstrated that the ectopic expression of Nnat mediated by retroviral infection or stable transfection of 3T3-L1 pre-adipocytes stimulated differentiation into mature adipocytes with early induction of adipogenic transcription factors. Moreover, in 3T3-L1 cells overexpressing Nnat, increased intracellular free calcium levels and enhanced phosphorylation of cAMP-response element-binding protein (CREB) were observed, which appears to potentiate CCAAT/enhancer-binding protein (C/EBP)beta, C/EBPdelta, and C/EBPalpha transcriptional activities. Collectively, the data indicate that Nnat enhances CREB phosphorylation through increasing intracellular free calcium levels, which potentiates expression of adipogenic transcription factors resulting in heightened adipocyte differentiation. These findings contribute to a greater fundamental understanding of obesity, a clinically important risk factor in numerous diseases.
... In addition, NNAT β has been reported to play a critical role in the pathogenesis of type 2 diabetes by mediating the high glucose-induced apoptosis of pancreatic β-cells [16]. NNAT also plays a role in adipocyte differentiation by increasing the intracellular Ca ATPase in the ER [17]. ...
... To confirm the in silico results, Nnat β expression levels were examined using RT-PCR analysis in the adipose tissue of wild-type and db/db obese mice because it is known that Nnat α expression is increased in 3T3-L1 cells [17]. Nnat β expression was also significantly increased in the adipose tissue of db/db mice, similar to Nnat α expression. ...
... Next, to examine whether Nnat β expression is associated with adipogenesis, similar to Nnat α [17], 3T3-L1 preadipocytes were differentiated by treatment with a differentiation cocktail. The cells were harvested every 2 days after the beginning of differentiation, and the Nnat β expression level was examined by RT-PCR. ...
Obesity and related metabolic diseases are major problems worldwide. Some probiotics are currently considered potential therapeutic strategies for obesity. We aimed to investigate the anti-obesity efficacy of Latilactobacillus sakei WIKIM31 in obese mice induced by a high fat diet. The administration of a high-fat diet with L. sakei WIKIM31 reduced body weight gain, epididymal fat mass, triglyceride and total cholesterol levels in the blood, and remarkably decreased the expression of lipogenesis-related genes in the epididymal adipose tissue and liver. Interestingly, intake of L. sakei WIKIM31 improved gut barrier function by increasing the gene expression of tight junction proteins and suppressing the inflammatory responses. Additionally, L. sakei WIKIM31 enhanced the production of short-chain fatty acids, such as butyrate and propionate, in the intestinal tract. These results showed that L. sakei WIKIM31 can be used as a potential therapeutic probiotic for obesity.
... In addition, NNAT β has been reported to play a critical role in the pathogenesis of type 2 diabetes by mediating the high glucose-induced apoptosis of pancreatic β-cells [16]. NNAT also plays a role in adipocyte differentiation by increasing the intracellular Ca ATPase in the ER [17]. ...
... To confirm the in silico results, Nnat β expression levels were examined using RT-PCR analysis in the adipose tissue of wild-type and db/db obese mice because it is known that Nnat α expression is increased in 3T3-L1 cells [17]. Nnat β expression was also significantly increased in the adipose tissue of db/db mice, similar to Nnat α expression. ...
... Next, to examine whether Nnat β expression is associated with adipogenesis, similar to Nnat α [17], 3T3-L1 preadipocytes were differentiated by treatment with a differentiation cocktail. The cells were harvested every 2 days after the beginning of differentiation, and the Nnat β expression level was examined by RT-PCR. ...
Oligomeric proanthocyanidins (OPCs), classified as condensed tannins, have significant effects in anti-oxidation, anti-inflammation and anti-cancer. This study was performed to investigate anti-inflammatory effects of OPCs and the mechanism underlying these effects in lipopolysaccharide (LPS)-stimulated bovine mammary epithelial cells (MAC-T). Real-time PCR and ELISA assays indicated that OPCs treatment at 1, 3 and 5 μg/mL significantly reduced mRNA and protein respectively of oxidant indicators cyclooxygenase-2 (COX-2) (P<0.05) and inducible nitric oxide synthase (iNOS) (P<0.01) as well as inflammation cytokines interleukin (IL)-6 (P<0.01), IL-1β (P<0.01) and tumor necrosis factor-α (TNF-α) (P<0.05) in LPS-induced MAC-T cells. Moreover, OPCs down-regulated LPS-induced phosphorylation of p65 and inhibitor of nuclear factor kappa B (NF-κB) (IκB) in the NF-κB signaling pathway (P < 0.01), and they inhibited p65 translocation from the cytoplasm to the nucleus as revealed by immunofluorescence test and western blot. Additionally, OPCs decreased phosphorylation of p38, extracellular signal regulated kinase and c-jun NH2-terminal kinase in the MAPK signaling pathway (P < 0.01). In conclusion, the anti-inflammatory and antioxidant activities of OPCs involve NF-κB and MAPK signaling pathways, thus inhibiting expression of pro-inflammatory factors and oxidation indicators. These findings provide novel experimental evidence for the further practical application of OPCs in prevention and treatment of bovine mastitis.
... 26 Finally, high NNAT mRNA was found in adult adipocytes of obese rats. 27 Collectively, this has led to the notion that NNAT is a WAT marker. Indeed, cold exposure which causes browning in subcutaneous WAT, has been shown to reduce NNAT mRNA expression. ...
... 28 Conversely, warming mice leads to brown and beige adipose tissue "whitening" with a concomitant increase in NNAT mRNA. 29 Moreover, NNAT may not only be associated with the WAT phenotype but may also play an active role as studies in 3T3-L1 adipocytes have shown that NNAT stimulates adipogenesis through increasing [Ca 2+ ] I , 27 which is consistent with NNAT's putative role as a SERCA regulator. Finally, silencing NNAT in cultured adipocytes has been shown to induce a brown-like phenotype with increased expression of UCP-1. ...
... [43][44][45][46] NNAT shares 50% sequence homology with the allosteric SERCA regulator, PLN (Figure 2A). 27 changes that decrease SERCA's affinity for Ca 2+ . 11 In muscle, PLN inhibition of SERCA prolongs relaxation and reduces contractility via a reduction in SR Ca 2+ stores. ...
Neuronatin (NNAT) was originally discovered in 1995 and labelled as a brain developmental gene due to its abundant expression in developing brains. Over the past 25 years, researchers have uncovered NNAT in other tissues; notably, the hypothalamus, pancreatic β‐cells, and adipocytes. Recent evidence in these tissues indicates that NNAT plays a significant role in metabolism whereby it regulates food intake, insulin secretion, and adipocyte differentiation. Furthermore, genetic deletion of Nnat in mice lowers whole‐body energy expenditure and increases susceptibility to diet‐induced obesity and glucose intolerance; however, the underlying cellular mechanisms remain unknown. Based on its sequence homology with phospholamban, NNAT has a purported role in regulating the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump. However, NNAT also shares sequence homology with sarcolipin, which has the unique property of uncoupling the SERCA pump, increasing whole‐body energy expenditure and thus promoting adaptive thermogenesis in states of caloric excess or cold exposure. Thus, in this article, we discuss the accumulating evidence suggestive of NNAT’s role in whole‐body metabolic regulation, while highlighting its potential to mediate adaptive thermogenesis via SERCA uncoupling.
... The NNAT gene is expressed as two alternatively spliced mRNA isoforms, both of which encode an endoplasmic reticulum-associated proteolipid [18,19]. ...
... Sequence homology [18] and functional analyses [19][20][21] have suggested that NNAT acts as a regulator of the sarco/ endoplasmic reticulum Ca 2+ ATPase (ATP2A2, SERCA2), thereby participating in the regulation of intracellular Ca 2+ levels ([Ca 2+ ] i ) in some cells. Originally described as a gene selectively expressed in the developing brain, NNAT has indeed been shown to play a role in the induction of neural differentiation in embryonic stem cells via inhibition of SERCA2 [21]. ...
... Additional observations, however, suggest a more pleotropic role. NNAT expression has been shown to induce adipocytic differentiation in mesenchymal cells [19] and to induce apoptosis in pancreatic cells [20]. The roles ascribed to NNAT of induction of differentiation in cells of mesenchymal origin and silencing/suppression of neoplastic phenotypes in various cancers prompted us to explore a potential role in solid tumors in children, especially those of mesenchymal origin. ...
The paternally imprinted neuronatin (NNAT) gene has been identified as a target of aberrant epigenetic silencing in diverse cancers, but no association with pediatric bone cancers has been reported to date. In screening childhood cancers, we identified aberrant CpG island hypermethylation in a majority of osteosarcoma (OS) samples and in 5 of 6 human OS cell lines studied but not in normal bone-derived tissue samples. CpG island hypermethylation was associated with transcriptional silencing in human OS cells, and silencing was reversible upon treatment with 5-aza-2'-deoxycytidine. Expression of NNAT was detectable in osteoblasts and chondrocytes of human bone, supporting a potential role in bone homeostasis. Enforced expression of NNAT in human OS cells lacking endogenous expression resulted in significant reduction in colony formation and in vitro migration compared to nonexpressor control cells. We next analyzed the effect of NNAT expression on intracellular calcium homeostasis and found that was associated with an attenuated decay of calcium levels to baseline following ATP-induced release of calcium from endoplasmic reticulum (ER) stores. Furthermore, NNAT expression was associated with increased cytotoxicity in OS cells from thapsigargin, an inhibitor of calcium reuptake into ER and an inducer of the ER stress response. These results suggest a possible tumor suppressor role for NNAT in human osteosarcoma. Additional study is needed ascertain sensitization to ER stress-associated apoptosis as a mechanism of NNAT-dependent cytotoxicity. In that case, epigenetic modification therapy to effect NNAT transcriptional derepression may represent a therapeutic strategy potentially of benefit to a majority of osteosarcoma patients.
... Neuronatin (Nnat) is a paternally expressed imprinted proteolipid-encoding gene originally identified in the developing rat brain but also found in all placental mammals, including humans. It is expressed predominantly in the neuroendocrine systems of the developing fetus and adult but is also found in adipose tissue (3)(4)(5). The Nnat locus resides in a "microimprinted" region within the intronic sequence of the neighboring gene Blcap, with differential expression controlled by localized methylation of the silenced maternal allele (6)(7)(8)(9)(10). ...
... For example, fasting and leptin administration down-and upregulate Nnat mRNA levels in the hypothalamus, respectively (11)(12)(13). Nutrientdependent changes in expression are also seen in white adipose tissue and pancreatic β cells, and altered expression is found in the adipose tissue and islets of rodent models of obesity and diabetes (5,(14)(15)(16). Furthermore, recent evidence suggests that Nnat expression in mice, together with a cluster of other imprinted genes under the regulation of the chromatin-interacting protein TRIM28, underpins the "stochastic" development of obesity seen in inbred mouse strains (17). ...
... While to date no direct in vivo evidence has been provided for a role for Nnat in the regulation of glucose homeostasis, in vitro studies manipulating its expression in cultured pancreatic β cells suggest that Nnat may regulate glucose-stimulated insulin secretion (GSIS) (14,18). In terms of the potential mechanisms underlying this observation, in vitro studies have shown that NNAT is present in the endoplasmic reticulum (ER) (14,19) and, in part owing to a suggested structural resemblance to the calciumhandling protein phospholamban, have implicated this protein in the control of intracellular calcium dynamics (3,5,14,20). Involvement in the control of ion channels, Ca 2+ ATPases, and glucokinase has also been proposed (21). ...
Neuronatin (Nnat) is an imprinted gene implicated in human obesity and widely expressed in neuroendocrine and metabolic tissues in a hormone- and nutrient-sensitive manner. However, its molecular and cellular functions and precise role in organismal physiology remain only partly defined. Here we demonstrate that mice lacking Nnat globally or specifically in β cells display impaired glucose-stimulated insulin secretion leading to defective glucose handling under conditions of nutrient excess. In contrast, we report no evidence for any feeding or body weight phenotypes in global Nnat-null mice. At the molecular level neuronatin augments insulin signal peptide cleavage by binding to the signal peptidase complex and facilitates translocation of the nascent preprohormone. Loss of neuronatin expression in β cells therefore reduces insulin content and blunts glucose-stimulated insulin secretion. Nnat expression, in turn, is glucose-regulated. This mechanism therefore represents a novel site of nutrient-sensitive control of β cell function and whole-animal glucose homeostasis. These data also suggest a potential wider role for Nnat in the regulation of metabolism through the modulation of peptide processing events.
... Neuronatin (Nnat) is a paternally expressed imprinted proteolipid-encoding gene originally identified in the developing rat brain but also found in all placental mammals including humans. It is expressed predominantly in the neuroendocrine systems of the developing fetus and adult but is also found in adipose tissue (3)(4)(5). The Nnat locus resides in a 'microimprinted' region within the intronic sequence of the neighboring gene, Blcap, with differential expression controlled by localised methylation of the silenced maternal allele (6)(7)(8)(9)(10). ...
... Nutrient-dependent changes in expression are also seen in white adipose tissue and pancreatic beta cells, and altered expression is found in the adipose tissue and islets of 5 rodent models of obesity and diabetes (5,(14)(15)(16). Furthermore, recent evidence suggests that Nnat expression in mice, together with a cluster of other imprinted genes under the regulation of the chromatin interacting protein Trim28, underpins the "stochastic" development of obesity seen in inbred mouse strains (17). ...
... While to date no direct in vivo evidence has been provided for a role for Nnat in the regulation of glucose homeostasis, in vitro studies manipulating its expression in cultured pancreatic beta cells suggest that Nnat may regulate glucose-stimulated insulin secretion (GSIS) (14,18). In terms of the potential mechanisms underlying this observation, in vitro studies have shown that NNAT is present in the endoplasmic reticulum (ER) (14,19) and, in part due to a suggested structural resemblance to the calcium-handling protein phospholamban, have implicated this protein in the control of intracellular calcium dynamics (3,5,14,20). Involvement in the control of ion channels, Ca 2+ ATPases and glucokinase has also been proposed (21). ...
Neuronatin (Nnat) is an imprinted gene implicated in human obesity and widely expressed in neuroendocrine and metabolic tissues in a hormone and nutrient-sensitive manner. However, its molecular and cellular functions and precise role in organismal physiology remain only partly defined. Here we demonstrate that mice lacking Nnat globally or specifically in β cells display impaired glucose-stimulated insulin secretion leading to defective glucose handling under conditions of nutrient-excess. In contrast, we report no evidence for any feeding or body weight phenotypes in global Nnat null mice. At the molecular level neuronatin augments insulin signal peptide cleavage by binding to the signal peptidase complex and facilitates translocation of the nascent preprohormone. Loss of neuronatin expression in β cells therefore reduces insulin content and blunts glucose-stimulated insulin secretion. Nnat expression, in turn, is glucose-regulated. This mechanism therefore represents a novel site of nutrient-sensitive control of β cell function and whole animal glucose homeostasis. These data also suggest a potential wider role for Nnat in the regulation of metabolism through the modulation of peptide processing events.
... In addition to the neuronal expression of Nnat, it is also expressed in non-neuronal tissues such as the pituitary glands, lung, adrenal glands, uterus, skeletal muscles, ovaries, and pancreas [4,5]. Nnat is expressed in pancreatic b-cells and is possibly involved in ion-channel transport or channel modulation [6,7]. ...
... Nnat and insulin are co-localized in the cytoplasm of pancreatic b-cells, and knockdown of Nnat dramatically decreases insulin secretion after glucose challenge [6,7]. Additional data from pancreatic cells and 3T3-L1 cells showed that Nnat resides in the endoplasmic reticulum and modulates intracellular Ca 2+ stores [5][6][7]. ...
... Originally, it was believed to be a brain-specific developmental gene associated with neuronal differentiation [1,3,18,19]. However, it is also expressed in non-neuronal tissues such as the pituitary glands, lung, adrenal glands, uterus, skeletal muscles, ovaries, and pancreas [4,5]. The expression of neuronatin is higher in the aortic endothelium of diabetic and obese mice, while lower in several appetite regulatory hypothalamic nuclei during fasting [20,21]. ...
Neuronatin (Nnat), which is a neuronal developmental and differentiation molecule, is expressed in the endoplasmic reticulum of non-neuronal cells and is involved in insulin secretion from pancreatic β-cells by plausibly modulating their intracellular calcium concentration. However, the role of Nnat in keratinocyte differentiation remains unclear.
To unveil a possible integration of Nnat in controlling the keratinocyte differentiation markers such as involucrin, cytokeratin1, filaggrin, loricrin and S100A7.
Immunohistological staining was done using psoriasis, chronic eczema, lichen planus and normal skin. Immunofluorescence staining, Western blotting and semi-quantitative real-time PCR were performed for detecting Nnat, involucrin, cytokeratin1, filaggrin, loricrin and S100A7 using human keratinocytes with or without Nnat gene transfection. Small interference RNA was applied to knockdown the Nnat gene expression.
Nnat existed in normal human epidermis and cultured keratinocytes. In the hyperplastic epidermis of psoriasis, chronic eczema and lichen planus, over-expression of Nnat was evident along with involucrin and cytokeratin1 expression. Coordinate up-regulation of Nnat and involucrin, but not cytokeratin1, was demonstrated in cultured keratinocytes under differentiation stimuli such as extracellular calcium elevation, exposure to phorbol myristate acetate, and increased cell density. Transfection of small intereference RNA for Nnat decreased the mRNA levels of Nnat and involucrin, but not of cytokeratin1. Furthermore, a gene transfection assay showed increased involucrin expression in the Nnat-transfected keratinocytes than in mock-transfected counterparts, without any appreciable influence on cytokeratin1, filaggrin, loricrin and S100A7 expression.
These data indicate that Nnat is related to keratinocyte differentiation by up-regulating involucrin expression.
... NNAT, also known as paternally expressed gene 5 (PEG5), was first discovered in the rat neonatal brain and has significant roles in the differentiation of neurons [7]. Other studies in adipose tissue and pancreatic b cells showed that NNAT is involved in adipocyte differentiation and in regulating glucose-mediated insulin secretion [8,9]. ...
... NNAT was an important gene in regulating mouse brain and adipocyte differentiation and insulin secretion in response to glucose in mouse pancreatic cells [7,8,9]. NNAT was paternally expressed in human and mouse brains. ...
... The two transcripts of NNAT had different functions. NNAT was significantly increased in the adipose tissue of ZDF (fat) rats and ectopic expression of NNATa augmented adipocyte by increase adipogenic transcription factors in 3T3-L1 cell line [8]. Study in pancreas showed that NNATa and NNATb increased insulin secretion in the low glucose charge; under chronic high glucose conditions, the ratio of NNATb to NNATa increased in murine pancreatic b-cells, which increased hyperglycemia-induced apoptosis by inhibition of proteasome function. ...
Imprinted genes play important roles in placental and embryonic development. Neuronatin (NNAT), first identified as an imprinted gene in human and mouse brains, played important roles in neuronal differentiation in the brain and in glucose-mediated insulin secretion in pancreatic β cells. In the pig, NNAT was reported to be imprinted in eleven tissues. Our previous microarray hybridization study showed that NNAT was differentially expressed in Yorkshire and Meishan pig placentas, but the imprinting status and function of NNAT in the placenta have not been investigated. We demonstrated for the first time that NNAT was monoallelically expressed in the placenta. Immunochemistry analysis showed that NNAT was located in the uterine luminal and glandular epithelium in placentas. We also confirmed the differential expression of NNAT in Meishan and Yorkshire pig placentas by qPCR. Using IPA software and the published literature, we created a model network of the possible relationships between NNAT and glucose transporter genes. A dual luciferase reporter assay demonstrated that the crucial promoter region of NNAT contained a CANNTG sequence in the +210 to +215 positions, which corresponded to the E-box. Our findings demonstrated important roles of NNAT in placenta function.
... Local translation of dendritically localized transcripts such as calmodulins, CaMKIIá, visinin-like protein-1, NMDA (N-methyl-D-aspartic acid), and AMPA (2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid) receptors may be a means for regulating dendritic Ca2+ signaling [5],[33],[34],[35],[36],[37]. Although uncharacterized in neurons, NNAT may also belong to this group due to the dendritic localization of its mRNA and its ability to regulate intracellular Ca2+ [18],[19]. ...
... NNAT overexpression in non-neuronal cell types modulates intracellular Ca2+ storage, resulting in elevated cytoplasmic Ca2+ levels [18],[19].To see if NNAT could regulate dendritic Ca2+ levels, we overexpressed NNATβ, the predominant isoform, in mature hippocampal cultures followed by loading with Calcium Crimson-AM, a BAPTA-based Ca2+ indicator dye [55],[56]. NNATβ was overexpressed using a dual expression construct containing GFP and the full length rat Nnatβ mRNA sequence (including 5′ and 3′ UTRs to preserve translational control and localization) driven by separate promoters (GFP/NNATβ) (Fig. 6A). ...
... The Nnat gene is also maternally imprinted and contains a neuron-restrictive silencer element [16], [17]. Non-neuronal data from pancreatic beta and 3T3-L1 cells shows that NNAT resides in the endoplasmic reticulum (ER) and modulates intracellular Ca2+stores [18], [19]. NNAT is strikingly similar to phospholamban (PLN), an ER-resident Ca2+ regulator found in cardiac muscle. ...
Local translation of dendritic mRNAs plays an important role in neuronal development and synaptic plasticity. Although several hundred putative dendritic transcripts have been identified in the hippocampus, relatively few have been verified by in situ hybridization and thus remain uncharacterized. One such transcript encodes the protein neuronatin. Neuronatin has been shown to regulate calcium levels in non-neuronal cells such as pancreatic or embryonic stem cells, but its function in mature neurons remains unclear. Here we report that neuronatin is translated in hippocampal dendrites in response to blockade of action potentials and NMDA-receptor dependent synaptic transmission by TTX and APV. Our study also reveals that neuronatin can adjust dendritic calcium levels by regulating intracellular calcium storage. We propose that neuronatin may impact synaptic plasticity by modulating dendritic calcium levels during homeostatic plasticity, thereby potentially regulating neuronal excitability, receptor trafficking, and calcium dependent signaling.
... In adult rodents and humans Nnat is highly expressed in neurons in many brain regions, including hypothalamic nuclei concerned with appetite regulation, and is concerned in nutrient sensing, and responds to feeding/ fasting and leptin [10,11]. Nnat is also highly expressed in adult white adipose tissue and aortic endothelial cells, where expression is increased in obese and diabetic rodents [12,13]. Nnat is moreover expressed in pancreatic b-cell lines, where expression is glucose-dependent, and reduced in diabetic rodents [14][15][16]. ...
... Nnat is an amphipathic proteolipid. It has two isoforms, α of 81 and b of 54 amino acids, generated by alternative splicing of the middle exon, encoding a membrane-binding domain [13][14][15]19]. Both isoforms reside in the endoplasmic ER membranes, the b-isoform is implicated in ER stress and b-cell apoptosis [15], it is considered to regulate ER calcium ATPase through homology to other proteolipid. ...
... Both isoforms reside in the endoplasmic ER membranes, the b-isoform is implicated in ER stress and b-cell apoptosis [15], it is considered to regulate ER calcium ATPase through homology to other proteolipid. Nnat expression in vitro is associated with calcium-induced 3T3-L1 cell adipogenesis; glucose-stimulated, calcium-induced, insulin secretion from b-cells; activation of Pi3k, Erk, mTor and calcium signalling in medulloblastoma; Nfkb-regulated inflammation in aortic endothelial cells and protection against mitochondrial toxins and ionophores in PC12 cells and resolution of injury after ischemic/reperfusion of the kidney [12][13][14]19,20]. ...
Obesity causes insulin resistance in target tissues - skeletal muscle, adipose tissue, liver and the brain. Insulin resistance predisposes to type-2 diabetes (T2D) and cardiovascular disease (CVD). Adipose tissue inflammation is an essential characteristic of obesity and insulin resistance. Neuronatin (Nnat) expression has been found to be altered in a number of conditions related to inflammatory or metabolic disturbance, but its physiological roles and regulatory mechanisms in adipose tissue, brain, pancreatic islets and other tissues are not understood.
We identified transcription factor binding sites (TFBS) conserved in the Nnat promoter, and transcription factors (TF) abundantly expressed in adipose tissue. These include transcription factors concerned with the control of: adipogenesis (Pparγ, Klf15, Irf1, Creb1, Egr2, Gata3); lipogenesis (Mlxipl, Srebp1c); inflammation (Jun, Stat3); insulin signalling and diabetes susceptibility (Foxo1, Tcf7l2). We also identified NeuroD1 the only documented TF that controls Nnat expression. We identified KEGG pathways significantly associated with Nnat expression, including positive correlations with inflammation and negative correlations with metabolic pathways (most prominently oxidative phosphorylation, glycolysis and gluconeogenesis, pyruvate metabolism) and protein turnover. 27 genes, including; Gstt1 and Sod3, concerned with oxidative stress; Sncg and Cxcl9 concerned with inflammation; Ebf1, Lgals12 and Fzd4 involved in adipogenesis; whose expression co-varies with Nnat were identified, and conserved transcription factor binding sites identified on their promoters. Functional networks relating to each of these genes were identified.
Our analysis shows that Nnat is an acute diet-responsive gene in white adipose tissue and hypothalamus; it may play an important role in metabolism, adipogenesis, and resolution of oxidative stress and inflammation in response to dietary excess.
... NNAT is a widely expressed proteolipid with physiological and pathophysiological roles in neural development, metabolism, inflammation, and multiple malignancies [5]. NNAT is localized to the cytoplasm and endoplasmic reticulum in multiple cell types, including neurons [27,28], adipocytes [29], and pancreatic beta cells [30], and regulates intracellular Ca 2+ levels [28][29][30][31] through direct interaction with SERCA2 in the endoplasmic reticulum membrane [28]. ...
... NNAT is a widely expressed proteolipid with physiological and pathophysiological roles in neural development, metabolism, inflammation, and multiple malignancies [5]. NNAT is localized to the cytoplasm and endoplasmic reticulum in multiple cell types, including neurons [27,28], adipocytes [29], and pancreatic beta cells [30], and regulates intracellular Ca 2+ levels [28][29][30][31] through direct interaction with SERCA2 in the endoplasmic reticulum membrane [28]. ...
Purpose
Understanding the molecular mediators of breast cancer survival is critical for accurate disease prognosis and improving therapies. Here, we identified Neuronatin (NNAT) as a novel antiproliferative modifier of estrogen receptor-alpha (ER+) breast cancer.
Experimental design
Genomic regions harboring breast cancer modifiers were identified by congenic mapping in a rat model of carcinogen-induced mammary cancer. Tumors from susceptible and resistant congenics were analyzed by RNAseq to identify candidate genes. Candidates were prioritized by correlation with outcome, using a consensus of three breast cancer patient cohorts. NNAT was transgenically expressed in ER+ breast cancer lines (T47D and ZR75), followed by transcriptomic and phenotypic characterization.
Results
We identified a region on rat chromosome 3 (142–178 Mb) that modified mammary tumor incidence. RNAseq of the mammary tumors narrowed the candidate list to three differentially expressed genes: NNAT, SLC35C2, and FAM210B. NNAT mRNA and protein also correlated with survival in human breast cancer patients. Quantitative immunohistochemistry of NNAT protein revealed an inverse correlation with survival in a univariate analysis of patients with invasive ER+ breast cancer (training cohort: n = 444, HR = 0.62, p = 0.031; validation cohort: n = 430, HR = 0.48, p = 0.004). NNAT also held up as an independent predictor of survival after multivariable adjustment (HR = 0.64, p = 0.038). NNAT significantly reduced proliferation and migration of ER+ breast cancer cells, which coincided with altered expression of multiple related pathways.
Conclusions
Collectively, these data implicate NNAT as a novel mediator of cell proliferation and migration, which correlates with decreased tumorigenic potential and prolonged patient survival.
... ER Ca 2+ -ATPase isoform 2 (SERCA2). Involvement of NNAT in the differentiation of keratinocytes 11 and adipose tissue 12 has also been suggested. Moreover, several studies have demonstrated diverse roles of NNAT, such as insulin secretion, 8 synaptic plasticity, 13 calcium-induced cell migration, 14 stress responses, 15,16 and others. ...
... [17][18][19] Thus, NNAT performs diverse functions according to its expression in particular tissues, although the details have not yet been reported. Recently, the involvement of NNAT in disease development and regression, including tissue degeneration and cancer, has been reported, 20 particularly in relation to downstream signaling of NNAT, such as via increase of intracellular Ca 2+ level through the regulation of Ca 2+ ATPase in the ER, 12 mediation of the high glucoseinduced apoptosis of pancreatic β-cells, 8 reduction in glucose-mediated insulin secretion, 21 increase of the inflammatory response in the endothelial cells, 22 and decrease of the inflammatory signaling pathway. 23 Accordingly, in this study, we analyzed the localization of NNAT-positive cells using immunohistochemistry in embryonic and postnatal tissues, with a particular focus on the testes, whisker hair follicles, and tongue papillae, which are regulated by Ca 2+ signaling, and the pancreases, regulated by glucose signaling. ...
Neuronatin ( Nnat) is expressed in the pituitary, pancreas, and other tissues; however, the function of NNAT is still unclear. Recent studies have demonstrated that NNAT is localized in the sex-determining region Y-box 2-positive stem/progenitor cells in the developing rat pituitary primordium and is downregulated during differentiation into mature hormone-producing cells. Moreover, NNAT is widely localized in subcellular organelles, excluding the Golgi. Here, we further evaluated NNAT-positive cells and intracellular localization in embryonic and postnatal rat tissues such as the pancreas, tongue, whisker hair follicle, and testis. Immunohistochemistry revealed that NNAT was localized in undifferentiated cells (i.e., epithelial basal cells and basement cells in the papillae of the tongue and round and elongated spermatids of the testis) as well as in differentiated cells (insulin-positive cells and exocrine cells of the pancreas, taste receptor cells of the fungiform papilla, the inner root sheath of whisker hair follicles, and spermatozoa). In addition, NNAT exhibited novel intracellular localization in acrosomes in the spermatozoa. Because the endoplasmic reticulum (ER) is excluded from spermatozoa and sarco/ER Ca2+-ATPase isoform 2 (SERCA2) is absent from the inner root sheath, these findings suggested that NNAT localization in the ER and its interaction with SERCA2 are cell- or tissue-specific properties.
... Between P10 and P21, during which point mice begin to leave the nest, juveniles start to accumulate adipose depots, and therefore require a switch to a lipogenic state in which carbohydrate becomes the major source of fuel [11,54]. Nnat is highly expressed in adipose tissue and has been previously reported to potentiate adipogenesis [29]. At P20, even when normalized to body weight, Nnat þ/Àp mice had reduced fat pad mass in two distinct white adipose tissue (WAT) depots (epididymal and femoral subcutaneous) and reduced interscapular brown adipose tissue (BAT) mass, indicating disproportionate reductions in adipose tissue (Figure 2A). ...
... Although core body temperature was unaltered in Nnat null mice, growth restricted mutant mice had disproportionate reductions in adipose tissues when measured as percentage of body weight, in contrast to proportionate reductions in weights for other major organs. We show that NNAT is expressed in mature white adipocytes, and indeed previous in vitro studies have demonstrated that Nnat is able to potentiate adipogenesis [29]. After weaning, this body weight difference diminishes between Nnat deficient juveniles and their wild type littermates. ...
Objective:
Imprinted genes are crucial for the growth and development of fetal and juvenile mammals. Altered imprinted gene dosage causes a variety of human disorders, with growth and development during these crucial early stages strongly linked with future metabolic health in adulthood. Neuronatin (Nnat) is a paternally expressed imprinted gene found in neuroendocrine systems and white adipose tissue and is regulated by the diet and leptin. Neuronatin expression is downregulated in obese children and has been associated with stochastic obesity in C57BL/6 mice. However, our recent studies of Nnat null mice on this genetic background failed to display any body weight or feeding phenotypes but revealed a defect in glucose-stimulated insulin secretion due to the ability of neuronatin to potentiate signal peptidase cleavage of preproinsulin. Nnat deficiency in beta cells therefore caused a lack of appropriate storage and secretion of mature insulin.
Methods:
To further explore the potential role of Nnat in the regulation of body weight and adiposity, we studied classical imprinting-related phenotypes such as placental, fetal, and postnatal growth trajectory patterns that may impact upon subsequent adult metabolic phenotypes.
Results:
Here we find that, in contrast to the lack of any body weight or feeding phenotypes on the C57BL/6J background, deletion of Nnat in mice on 129S2/Sv background causes a postnatal growth restriction with reduced adipose tissue accumulation, followed by catch up growth after weaning. This was in the absence of any effect on fetal growth or placental development. In adult 129S2/Sv mice, Nnat deletion was associated with hyperphagia, reduced energy expenditure, and partial leptin resistance. Lack of neuronatin also potentiated obesity caused by either aging or high fat diet feeding.
Conclusions:
The imprinted gene Nnat plays a key role in postnatal growth, adult energy homeostasis, and the pathogenesis of obesity via catch up growth effects, but this role is dependent upon genetic background.
... Although the physiological function of NNAT is poorly understood, existing literature indicates its involvement in glucose-mediated insulin secretion from the pancreas, adipogenesis, and in metabolic regulation (Chu and Tsai, 2005;Suh et al., 2005;Joe et al., 2008;Mzhavia et al., 2008;Vrang et al., 2010). Few diseases have been identified to associate with abnormal NNAT expression. ...
... Over the years, the role of NNAT in Ca 2þ signaling has become more comprehensible (Fig. 3). Thus, direct evidences of NNAT contribution to intracellular Ca 2þ signaling have come from the studies of Joe et al. (2008) and Suh et al. (2005), who have demonstrated that NNAT overexpression in non-neuronal cell types modulates intracellular Ca 2þ storage, resulting in elevated cytoplasmic Ca 2þ levels. In another study performed by Lin et al. (2010) on embryonic stem cells (ESC), NNAT has been reported to be a neural initiator and a novel intrinsic factor for the neural differentiation of the ESC. ...
Neuronatin (NNAT) was first identified as a brain-specific gene crucial for brain development. Over the years, NNAT has been studied in different developing and post-developed tissues and organs. While NNAT manifests functional and structural similarities to the phospholamban gene, its physiological and pathological roles in healthy and diseased tissues have not been precisely identified. Ca(2+) signaling, glucose transport, insulin secretion, and inflammation modulated at different pathological conditions have been proposed to be governed by NNAT. This review describes the current findings of cellular molecular pathways known to be modified concomitantly with an alteration in NNAT expression, and it highlights the need to conduct extensive investigation regarding the role of NNAT in health and disease. This article is protected by copyright. All rights reserved.
... To clarify the potential role of NNAT during the transition from stem/ progenitor cell to each hormone-producing cell, investigations involving the physiologically changed pituitary, such as during the estrous cycle, pregnancy, lactation/weaning, stress and/or estrogen-induced prolactinoma, in the future may provide novel clues. In support of our hypothesis, several investigators have reported that NNAT participates in tissue differentiation, such as potentiation for adipogenesis (Suh et al. 2005), promotion of neural lineage induction from embryonic stem cells (Lin et al. 2010) and differentiation of keratinocytes (Dugu et al. 2014). ...
... NNAT has thus far been supposed to reside in ER (Joe et al. 2008;Suh et al. 2005). The presence of NNAT in another unidentified cellular structure has also been postulated (Chu and Tsai 2005). ...
The pituitary gland, an indispensable endocrine organ that synthesizes and secretes pituitary hormones, develops with the support of many factors. Among them, neuronatin (NNAT), which was discovered in the neonatal mouse brain as a factor involved in neural development, has subsequently been revealed to be coded by an abundantly expressing gene in the pituitary gland but its role remains elusive. We analyze the expression profile of Nnat and the localization of its product during rat pituitary development. The level of Nnat expression was high during the embryonic period but remarkably decreased after birth. Immunohistochemistry demonstrated that NNAT appeared in the SOX2-positive stem/progenitor cells in the developing pituitary primordium on rat embryonic day 11.5 (E11.5) and later in the majority of SOX2/PROP1 double-positive cells on E13.5. Thereafter, during pituitary embryonic development, Nnat expression was observed in some stem/progenitor cells, proliferating cells and terminally differentiating cells. In postnatal pituitaries, NNAT-positive cells decreased in number, with most coexpressing Sox2 or Pit1, suggesting a similar role for NNAT to that during the embryonic period. NNAT was widely localized in mitochondria, peroxisomes and lysosomes, in addition to the endoplasmic reticulum but not in the Golgi. The present study thus demonstrated the variability in expression of NNAT-positive cells in rat embryonic and postnatal pituitaries and the intracellular localization of NNAT. Further investigations to obtain functional evidence for NNAT are a prerequisite.
... Thus hypothalamic Nnat expression is responsive to acute nutrient and leptin signalling in a neurone-specific fashion, and may mediate intracellular signalling to determine appetite. In white adipose tissue (WAT), Nnat is over-expressed in obese and high-fat fed murine models, and is under-expressed in lipodystrophic and lean S6kko mice, as well as in mice fed conjugated linoleic acid to induce weight loss [27,28,29,30]. In the pancreas, Nnat is expressed at high levels in islets cells [31,32], where it is a target of the NeuroD1 a transcription factor that is important for neuronal and endocrine cell differentiation and survival [30,33] . ...
... In the pancreas, Nnat is expressed at high levels in islets cells [31,32], where it is a target of the NeuroD1 a transcription factor that is important for neuronal and endocrine cell differentiation and survival [30,33] . In vitro, Nnat expression is associated with calciuminduced 3T3-L1 cell adipogenesis [28]; activation of PI3k, Erk, mTor and calcium signalling in medulloblastoma cells [34] ; Nfkbregulated inflammation in aortic endothelial cells [35]; and protection against mitochondrial toxins and ionophors in PC12 cells [36]. Similarly, in b-cell lines Nnat expression is associated with glucose-stimulated, calcium-induced insulin secretion [24,33], whilst overexpression of the b-isoform may be concerned with ER stress and b-cell apoptosis [24]. ...
Neuronatin (NNAT) is an endoplasmic reticulum proteolipid implicated in intracellular signalling. Nnat is highly-expressed in the hypothalamus, where it is acutely regulated by nutrients and leptin. Nnat pre-mRNA is differentially spliced to create Nnat-α and -β isoforms. Genetic variation of NNAT is associated with severe obesity. Currently, little is known about the long-term regulation of Nnat.
Expression of Nnat isoforms were examined in the hypothalamus of mice in response to acute fast/feed, chronic caloric restriction, diet-induced obesity and modified gastric bypass surgery. Nnat expression was assessed in the central nervous system and gastrointestinal tissues. RTqPCR was used to determine isoform-specific expression of Nnat mRNA.
Hypothalamic expression of both Nnat isoforms was comparably decreased by overnight and 24-h fasting. Nnat expression was unaltered in diet-induced obesity, or subsequent switch to a calorie restricted diet. Nnat isoforms showed differential expression in the hypothalamus but not brainstem after bypass surgery. Hypothalamic Nnat-β expression was significantly reduced after bypass compared with sham surgery (P = 0.003), and was positively correlated with post-operative weight-loss (R(2) = 0.38, P = 0.01). In contrast, Nnat-α expression was not suppressed after bypass surgery (P = 0.19), and expression did not correlate with reduction in weight after surgery (R(2) = 0.06, P = 0.34). Hypothalamic expression of Nnat-β correlated weakly with circulating leptin, but neither isoform correlated with fasting gut hormone levels post- surgery. Nnat expression was detected in brainstem, brown-adipose tissue, stomach and small intestine.
Nnat expression in hypothalamus is regulated by short-term nutrient availability, but unaltered by diet-induced obesity or calorie restriction. While Nnat isoforms in the hypothalamus are co-ordinately regulated by acute nutrient supply, after modified gastric bypass surgery Nnat isoforms show differential expression. These results raise the possibility that in the radically altered nutrient and hormonal milieu created by bypass surgery, resultant differential splicing of Nnat pre-mRNA may contribute to weight-loss.
... Several distinguishing genes are related to adipocytic differentiation. One example is NNAT, which induces the activation of adipocytic transcription factors CREB and CEBP family 20 and was significantly methylated (hypermethylation) and upregulated in MLPS than in normal fat and other sarcomas 19,21 . Decreased methylation (hypomethylation) and downregulation of NNAT was observed in DDLPS and PLPS, which likely results in a more dedifferentiated state. ...
Soft tissue sarcomas STS are malignancies that show mesenchymal and neuroectodermal differentiation and thus most often resemble supportive and connective tissue including fat blood vessels muscle bone tendons and nerves Over 70 subtypes of sarcomas exist and pathologists have classified these broadly according to the degree to which they resemble differentiated cell types Figure 1 1 This review will focus on the most common subset of STS in adults liposarcoma which are tumors with histological features of specialized fat cells Liposarcoma are broken down into several subtypes The four with the highest incidence are welldifferentiated liposarcoma WDLPS dedifferentiated liposarcoma DDLPS myxoid liposarcoma MLPS and pleomorphic liposarcoma PLPS 1 Overall survival is highest for MLPS followed by WDLPS and DDLPS and then PLPS2-4 Figure 2 While WDLPS occurs predominantly in the deep soft tissues of the limbs and retroperitoneum DDLPS is located mostly in the retroperitoneum MLPS and PLPS are preferentially located within the limbs5 Despite these broad categories liposarcoma can also have mixed phenotypes and is often further subdivided into even more rare entities with other ultra-rare features For instance pleomorphic MLPS has attributes of both PLPS and MLPS6,7.
... Nnat is a paternally expressed canonically imprinted gene which encodes a proteolipid protein that localizes to the ER (Li et al., 2010). Nnat is diet-responsive and its overexpression in 3T3L1 pre-adipocytes promotes adipogenesis through increased free cytosolic calcium (Young et al., 2005). In pre-neural stem cells, Nnat binds sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) to block Ca 2+ uptake into the ER thereby increasing cytosolic Ca 2+ levels (Lin et al., 2010). ...
Parent-of-origin effects are unexpectedly common in complex traits, including metabolic and neurological traits. Parent-of-origin effects can be modified by the environment, but the architecture of these gene-by-environmental effects on phenotypes remains to be unraveled. Previously, quantitative trait loci (QTL) showing context-specific parent-of-origin effects on metabolic traits were mapped in the F 16 generation of an advanced intercross between LG/J and SM/J inbred mice. However, these QTL were not enriched for known imprinted genes, suggesting another mechanism is needed to explain these parent-of-origin effects phenomena. We propose that non-imprinted genes can generate complex parent-of-origin effects on metabolic traits through interactions with imprinted genes. Here, we employ data from mouse populations at different levels of intercrossing (F 0 , F 1 , F 2 , F 16 ) of the LG/J and SM/J inbred mouse lines to test this hypothesis. Using multiple populations and incorporating genetic, genomic, and physiological data, we leverage orthogonal evidence to identify networks of genes through which parent-of-origin effects propagate. We identify a network comprised of 3 imprinted and 6 non-imprinted genes that show parent-of-origin effects. This epistatic network forms a nutritional responsive pathway and the genes comprising it jointly serve cellular functions associated with growth. We focus on 2 genes, Nnat and F2r , whose interaction associates with serum glucose levels across generations in high fat-fed females. Single-cell RNAseq reveals that Nnat expression increases and F2r expression decreases in pre-adipocytes along an adipogenic trajectory, a result that is consistent with our observations in bulk white adipose tissue.
... In adult hypothalamic nuclei expression is influenced by metabolic status 8 and recognised to be downregulated in obese ob/ob and db/db mice 9,10 . Subsequent reports have shown expression in several peripheral sites including pituitary, pancreas and adipocytes [11][12][13] , although the physiological and cellular role of Nnat at these sites remains to be fully determined. Nnat has been characterised and reported to be an amphiphilic membrane proteolipid highly homologous to mammalian sarcoplasmic protein Phospholamban (Pln) and yeast protein PM1 [14][15][16] . ...
Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat+/−p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat+/−p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat+/−p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat+/−p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat+/−p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat+/−p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.
... Early in vitro work reported a potential role in glucose-stimulated insulin secretion (GSIS) in two different stable mouse pancreatic beta cell lines and expression of both known isoforms of neuronatin, Nnat-a and Nnat-b, found predominantly in the endoplasmic reticulum (ER), were increased after acute stimulation with high glucose (41,42). It has been postulated that NNAT plays a role in the regulation of the intracellular calcium dynamics in several cell types (42)(43)(44); however, primary islets from Nnat-null mutant mice displayed unaltered Ca 2+ signalling (45). Both global and beta cell-specific Nnat deficient mice demonstrate impaired GSIS due to reduced beta cell insulin content (45). ...
Beta cell failure lies at the centre of the aetiology and pathogenesis of type 2 diabetes and the epigenetic control of the expression of critical beta cell genes appears to play a major role in this decline. One such group of epigenetically-controlled genes, termed ‘imprinted’ genes, are characterised by transgenerational monoallelic expression due to differential allelic DNA methylation and play key functional roles within beta cells. Here, we review the evidence for this functional importance of imprinted genes in beta cells as well as their nutritional regulation by the diet and their altered methylation and/or expression in rodent models of diabetes and in type 2 diabetic islets. We also discuss imprinted genes in the context of the next generation, where dietary overnutrition in the parents can lead to their deregulation in the offspring, alongside beta cell dysfunction and defective glucose handling. Both the modulation of imprinted gene expression and the likelihood of developing type 2 diabetes in adulthood are susceptible to the impact of nutritional status in early life. Imprinted loci, therefore, represent an excellent opportunity with which to assess epigenomic changes in beta cells due to the diet in both the current and next generation.
... In adult white adipose tissue and aortic endothelial cells, Nnat is highly expressed and may be involved in the pathogenesis of various human diseases [23,24]. Suh et al. [25] have shown that Nnat could regulate preadipocyte differ-entiation via potentiation of cAMP-response element binding protein-(CREB-) mediated transcription of CCAA T/enhancer binding protein (C/EBP)b, C/EBPd, and C/EBPa by enhancing CREB phosphorylation through increasing the intracellular Ca 2+ level. Therefore, we believe that APN promotes the differentiation of preadipocytes and accelerates adipocyte metabolism which may involve the Nnat pathway. ...
A previous study has demonstrated that adiponectin (APN) could promote preadipocyte differentiation, and the present study further explored its mechanism. 3T3-L1 cells were infected with adenovirus holding human adiponectin gene apM1 and mouse neuronatin (Nnat) shRNA and initiated differentiation while coculturing with mature adipocytes stimulated with LPS. After 8 days, preadipocyte differentiation was observed by Oil Red O staining. Real-time quantitative PCR was used to evaluate mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1), interleukin- (IL-) 6, IL-8, and tumor necrosis factor α (TNF- α ). The levels of reactive oxygen species (ROS), total antioxidant capacity (T-AOC), malondialdehyde (MDA), and superoxide dismutase (SOD) in 3T3-L1 cells were detected. Western blotting was done to quantify the protein expression levels of Nnat, peroxisome proliferator-activated receptor (PPAR) γ , p65, and inhibitor of nuclear factor κ B (I κ B) α . Results demonstrated that APN overexpression markedly increased preadipocyte differentiation; inhibited gene expression of MCP-1, IL-6, IL-8, and TNF- α ; reduced ROS and MDA release; increased T-AOC and SOD levels; upregulated Nnat, PPAR γ , and I κ B α protein expressions; and downregulated p65 protein expression under LPS stimulation. However, the effects of APN were markedly attenuated when Nnat expression was knocked down. Taken together, the present study provided evidences that the effects of APN on promoting preadipocyte differentiation under inflammatory conditions via anti-inflammation and antioxidative stress may be regulated by the PPAR γ /Nnat/NF- κ B signaling pathway.
... In mice, NNAT is an inflammation and obesity-related gene and activates several signaling pathways such as p38 MAPK, Jun NH(2)-terminal kinase, extracellular signalrelated kinase (ERK), and AKT but not NF-kB [12]. This link to obesity was further underlined by the finding that NNAT potentiates adipogenesis in rats by increased phosphorylation of CREB [13]. ...
Neuronatin (NNAT) is a proteolipid involved in cation homeostasis especially in the developing brain. Its expression has been associated with the progression of lung cancer, glioblastoma, and neuroblastoma as well as glucose induced apoptosis in pancreatic cells. We performed a retrospective study of 148 breast cancer specimens for NNAT expression by immunohistochemistry to evaluate this protein as a prognostic marker for breast cancer. We found a high NNAT immunoreactivity score (by multivariate cox regression) to be an independent prognostic marker for relapse-free (hazard ratio HR = 3.55, p = 0.002) and overall survival (HR = 6.29, p < 0.001). However, NNAT expression was not associated with classical parameters such as hormone receptor expression (p = 0.86) or lymph node metastasis (p = 0.83). Additional independent risk factors in this study population were tumor size (≤2 cm; overall survival: HR = 0.36, p = 0.023; relapse-free survival: HR = 0.26, p < 0.01) and blood vessel
... As of today the precise role of NNAT in healthy and diseased adult human tissues has not been addressed. Thus, Zheng et al. have proposed the protective role for NNAT during neuronal development from the external toxic insults (Zheng et al., 2002), while other studies conducted with non-neuronal cells suggested that NNAT could act as an intracellular Ca 2+ modulator (Joe et al., 2008;Poon et al., 2006;Suh et al., 2005). In addition to maintaining cellular homeostasis, NNAT can play a role in pathological processes. ...
Neuronatin (NNAT) is a small transmembrane proteolipid that is highly expressed in the embryonic developing brain and several other peripheral tissues. This study is the first to provide evidence that NNAT is detected in the adult retina of various adult rod-dominant mammals, including wild-type (WT) rodents, transgenic rodents expressing mutant S334ter, P23H, or T17M rhodopsin, non-human primates, humans, and cone-dominant tree shrews. Immunohistochemical and qRT-PCR analyses were applied to detect NNAT. Confocal microscopy analysis revealed that NNAT immunofluorescence is restricted to the outer segments (OS) of photoreceptors without evidence of staining in other retinal cell types across all mammalian species. Moreover, in tree shrew retinas, we found NNAT to be co-localized with rhodopsin, indicating its predominant expression in rods. The rod-derived expression of NNAT was further confirmed by qRT-PCR in isolated rod photoreceptor cells. We also used these cells to mimic cellular stress in transgenic retinas by treating them with the endoplasmic reticulum stress inducer, tunicamycin. Thus, our data revealed accumulation of NNAT around the nucleus as compared to dispersed localization of NNAT within control cells. This distribution coincided with the partial intracellular mislocalization of NNAT to the outer nuclear layer observed in transgenic retinas. In addition, stressed retinas demonstrated an increase of NNAT mRNA and protein levels. Therefore, our study demonstrated that NNAT is a novel stress responsive protein with a potential structural and/or functional role in adult mammalian retinas.
... Hypothalamic neuronatin-beta levels are decreased following gastric bypass surgery (40). Neuronatin enhances CREB phosphorylation through increasing free intracellular calcium (41). Single nucleotide polymorphisms in the neuronatin gene are also associated with severe obesity in humans (42). ...
Neuronatin is a unique, ubiquitous and highly conserved mammalian gene involved in brain development. Its mRNA isoforms, chromosomal location, genomic DNA structure and regulation have been characterized. More recently there has been rapid progress in the understanding of its function in physiology and human disease. In particular there is fairly direct evidence implicating neuronatin in the causation of Lafora disease and diabetes. Neuronatin protein has a strong predisposition to misfold and form cellular aggregates that causes cell death by apoptosis. Aggregation of Neuronatin within cortical neurons and resulting cell death is the hallmark of Lafora disease, a progressive and fatal neurodegenerative disease. Under high glucose conditions simulating diabetes, neuronatin protein also accumulates and destroys pancreatic beta cells. The neuronatin gene is imprinted and only the paternal allele is normally expressed in the adult. However, changes in DNA methylation may cause the maternal allele to lose imprinting and trigger cell proliferation and metastasis. Neuronatin has also been shown to be translated peripherally within the dendrites of neurons, a finding of relevance in synaptic plasticity. The current understanding of the function of neuronatin raises the possibility that this gene may participate in the common downstream mechanisms associated with aberrant neuronal growth and death. A better understanding of these mechanisms may open new therapeutic targets to help modify the progression of devastating neurodegenerative conditions such as Alzheimer's and anterior horn cell disease.
... In the context of our findings, it is of interest that NNAT was previously reported to be upregulated in MLS compared to normal fat [93] and that ectopic expression of NNAT in pre-adipocytes stimulated differentiation into mature adipocytes by induction of adipogenic transcription factors [94]. Compared to normal fat samples we found DNA hypomethylation and high expression of NNAT in MLSs. ...
High-grade soft tissue sarcomas are a heterogeneous, complex group of aggressive malignant tumors showing mesenchymal differentiation. Recently, soft tissue sarcomas have increasingly been classified on the basis of underlying genetic alterations; however, the role of aberrant DNA methylation in these tumors is not well understood and, consequently, the usefulness of methylation-based classification is unclear.
We used the Infinium HumanMethylation27 platform to profile DNA methylation in 80 primary, untreated high-grade soft tissue sarcomas, representing eight relevant subtypes, two non-neoplastic fat samples and 14 representative sarcoma cell lines. The primary samples were partitioned into seven stable clusters. A classification algorithm identified 216 CpG sites, mapping to 246 genes, showing different degrees of DNA methylation between these seven groups. The differences between the clusters were best represented by a set of eight CpG sites located in the genes SPEG, NNAT, FBLN2, PYROXD2, ZNF217, COL14A1, DMRT2 and CDKN2A. By integrating DNA methylation and mRNA expression data, we identified 27 genes showing negative and three genes showing positive correlation. Compared with non-neoplastic fat, NNAT showed DNA hypomethylation and inverse gene expression in myxoid liposarcomas, and DNA hypermethylation and inverse gene expression in dedifferentiated and pleomorphic liposarcomas. Recovery of NNAT in a hypermethylated myxoid liposarcoma cell line decreased cell migration and viability.
Our analysis represents the first comprehensive integration of DNA methylation and transcriptional data in primary high-grade soft tissue sarcomas. We propose novel biomarkers and genes relevant for pathogenesis, including NNAT as a potential tumor suppressor in myxoid liposarcomas.
... The decrease in Nnat expression may explain the reduction in glucose stimulated insulin secretion that we observed. Nnat is also abundantly expressed in adipose tissue and has a role in the potentiation of adipocyte differentiation [45]. Nnat potentiates adipogenesis through enhanced phosphorylation of cAMP-response element-binding protein in 3T3-L1 cells [46], and is upregulated in Zucker diabetic rats compared to control lean Zucker rats [47]. ...
We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.
... In addition, an association between single nucleotide polymorphisms in the human neuronatin gene locus and severe obesity has been demonstrated in both children and adults (39). Interestingly, neuronatin expression in WAT and aortic endothelial cells is increased in obese and diabetic rodents (22, 32). In contrast, neuronatin expression in pancreatic β-cell lines is reduced in diabetic rodents (1). ...
Failure of white adipose tissue to appropriately store excess metabolic substrate seems to underpin obesity-associated type 2 diabetes. Encouraging 'browning' of white adipose has been suggested as a therapeutic strategy to dispose of excess stored lipid and ameliorate the resulting insulin resistance. Genetic variation at the DNA locus encoding the novel proteolipid neuronatin has been associated with obesity, and we recently observed that neuronatin expression is reduced in subcutaneous adipose tissue from obese humans. Thus, to explore the function of neuronatin further, we used RNAi to silence its expression in murine primary adipocyte cultures and examined the effects on adipocyte phenotype. We found that primary adipocytes only express the longer isoform of neuronatin. Loss of neuronatin led to increased mitochondrial biogenesis, indicated by greater intensity of MitoTracker Green staining. This was accompanied by increased expression of UCP1 and the key genes in mitochondrial oxidative phosphorylation, PGC-1α, Cox8b, and Cox4 in primary subcutaneous white adipocytes, indicative of a 'browning' effect. In addition, phosphorylation of AMPK and ACC was increased, suggestive of increased fatty acid utilisation. Similar, but less pronounced, effects of neuronatin silencing were also noted in primary brown adipocytes. In contrast, loss of neuronatin caused a reduction in both basal and insulin stimulated glucose uptake and glycogen synthesis, but had no effect on insulin signalling, likely mediated by a reduction in Glut1 protein upon silencing of neuronatin. Thus, neuronatin appears to be a novel regulator of 'browning' and metabolic substrate utilisation in white adipocytes.
... Neuronatin is also expressed in several other peripheral tissues including pancreas, adipose tissues, and skin (32)(33)(34). Although the physiological function of neuronatin is poorly understood, existing literature indicates its involvement in glucose-mediated insulin secretion from pancreas, in adipogenesis, and in metabolic regulation (31,32,(35)(36)(37). Interestingly, neuronatin has significant homology with phospholamban and PMP1 that function as regulatory subunit of ion channel and shown to promote neural lineage in embryonic stem cells through increasing the intracellular Ca 2ϩ by antagonizing serco/endoplasmic reticulum Ca 2ϩ -ATPase-2 (SERCA2) (27,38). ...
Lafora disease (LD) is a teenage onset inherited progressive myoclonus epilepsy characterized by the accumulations of intracellular inclusions called Lafora bodies and caused by mutations in protein phosphatase laforin or ubiquitin ligase malin. But how the loss-of-function of either laforin or malin causes disease pathogenesis is poorly understood. Recently, neuronatin was identified as a novel substrate of malin that regulates glycogen synthesis. Here we demonstrate that the level of neuronatin is significantly up-regulated in the skin biopsy sample of LD patients having mutations in both malin and laforin. Neuronatin is highly expressed in human fetal brain with gradual decrease in expression in developing and adult brain. However, in adult brain, neuronatin is predominantly expressed in parvalbumin-positive GABAergic interneurons and localized in their processes. The level of neuronatin is increased and accumulated as insoluble aggregates in the cortical area of LD brain biopsy samples and there is also a dramatic loss of parvalbumin-positive GABAergic interneurons. Ectopic expression of neuronatin in cultured neuronal cells result in increased intracellular Ca2+, endoplasmic reticulum stress, proteasomal dysfunction and cell death that can be partially rescued by malin. These findings suggest that the neuronatin-induced aberrant Ca2+ signaling and endoplasmic reticulum stress might underlie LD pathogenesis.
... Neuronatin is a membrane protein in the ER (Joseph et al., 1994) that resembles phospholamban, an inhibitor of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA). Neuronatin-mediated regulation of intracellular Ca 2+ has been implicated in neural induction in embryonic stem cells (Lin et al., 2010) and in adipogenesis (Suh et al., 2005). Notably, Ca 2+ is a critical regulator of cell migration (Pettit and Fay, 1998; Yang et al., 2009). ...
The progression of cancer to metastatic disease is a major cause of death. We identified miR-708 being transcriptionally repressed by polycomb repressor complex 2-induced H3K27 trimethylation in metastatic breast cancer. miR-708 targets the endoplasmic reticulum protein neuronatin to decrease intracellular calcium level, resulting in reduction of activation of ERK and FAK, decreased cell migration, and impaired metastases. Ectopic expression of neuronatin refractory to suppression by miR-708 rescued cell migration and metastasis defects. In patients with breast cancer, miR-708 expression was decreased in lymph node and distal metastases, suggesting a metastasis-suppressive role. Our findings uncover a mechanistic role for miR-708 in metastasis and provide a rationale for developing miR-708 as a therapeutic agent against metastatic breast cancer.
... GATA2 is expressed in the osteoblasts and is a regulator involved in osteogenesis (21). The up-regulation of GATA2 expression is required for diverting preadipocytes as well as multipotent mesenchymal precursors away from adipogenesis and toward osteogenesis (22). The present in vivo/in vitro observations together with our genetic association data with spine BMD strongly suggest that the MPP7 gene is important in the regulation of bone metabolism. ...
Our previous genome-wide association study (GWAS) in a Hong Kong Southern Chinese population with extreme bone mineral density (BMD) scores revealed suggestive association with MPP7, which ranked second after JAG1 as a candidate gene for BMD. To follow-up this suggestive signal, we replicated the top single-nucleotide polymorphism rs4317882 of MPP7 in three additional independent Asian-descent samples (n= 2684). The association of rs4317882 reached the genome-wide significance in the meta-analysis of all available subjects (P(meta)= 4.58 × 10(-8), n= 4204). Site heterogeneity was observed, with a larger effect on spine than hip BMD. Further functional studies in a zebrafish model revealed that vertebral bone mass was lower in an mpp7 knock-down model compared with the wide-type (P= 9.64 × 10(-4), n= 21). In addition, MPP7 was found to have constitutive expression in human bone-derived cells during osteogenesis. Immunostaining of murine MC3T3-E1 cells revealed that the Mpp7 protein is localized in the plasma membrane and intracytoplasmic compartment of osteoblasts. In an assessment of the function of identified variants, an electrophoretic mobility shift assay demonstrated the binding of transcriptional factor GATA2 to the risk allele 'A' but not the 'G' allele of rs4317882. An mRNA expression study in human peripheral blood mononuclear cells confirmed that the low BMD-related allele 'A' of rs4317882 was associated with lower MPP7 expression (P= 9.07 × 10(-3), n= 135). Our data suggest a genetic and functional association of MPP7 with BMD variation.
... The Nnat gene was originally discovered from a differential display on the developing rat brain. However, Nnat also appears to be abundantly expressed in adipose tissue, and is significantly elevated in adipose tissue of obese Zuker diabetic fatty rats, compared with control lean Zuker rats [45]. Tpm1 is known to associate with microfilaments, also a component of the cytoskeleton in preadipocytes. ...
Different responses to a high fat diet (HFD) can occur even within a group of animals with the same genetic background, such as obesity-prone (OP) or obesity-resistant (OR) phenotypes, on the same feeding. To explain these phenotypes, we performed an analysis of gene expression differences in brown (BAT) and white adipose tissue (WAT) of OP and OR rats. Microarray analysis of transcripts revealed that 91 and 53 genes showed significant differences in expression between the BAT and WAT gene, respectively. Surprisingly, a majority of these genes were significantly down-regulated in adipose tissues in response to HFD feeding. K-means clustering of the expression levels of these genes identified 4 distinct groupings of genes with significant expression levels. Only a limited number of genes were significantly regulated in adipose tissues in response to HFD feeding, whereas expression levels of a large number of genes differed significantly between OP and OR rat. Our observations support that distinct discrepancies exist in gene-expression regulations in adipose tissues, and that alteration likely resulted from significant differences in genes encoding metabolic enzymes. To the best of our knowledge, this study provided the first direct comparison of gene-expression changes between OP and OR rats.
Human genetic studies have associated Neuronatin gene variants with anorexia nervosa (AN) and obesity. Studies on the expression of the Neuronatin gene product, a proteolipid, are lacking. We investigated the relationship between circulating Neuronatin, body mass index (BMI), body composition (BC), physical activity (PA), and psychometric outcomes in patients with AN, normal weight, and obesity. Plasma Neuronatin was measured by ELISA in (1) 79 subjects of five BMI categories (AN/BMI < 17.5 kg/m2; normal weight/BMI 18.5–25 kg/m2; obesity/BMI 30–40 kg/m2; obesity/BMI 40–50 kg/m2; obesity/BMI > 50 kg/m2) with assessment of BC (bioimpedance analysis; BIA); (2) 49 women with AN (BMI 14.5 ± 1.8 kg/m2) with measurements of BC (BIA) and PA (accelerometry); (3) 79 women with obesity (BMI 48.8 ± 7.8 kg/m2) with measurements of anxiety (GAD-7), stress (PSQ-20), depression (PHQ-9) and eating behavior (EDI-2). Overall, a positive correlation was found between Neuronatin and BMI (p = 0.006) as well as total fat mass (FM; p = 0.036). In AN, Neuronatin did not correlate with BMI, FM, or PA (p > 0.05); no correlations were found between Neuronatin and psychometric outcomes in obesity (p > 0.05). The findings suggest an FM-dependent peripheral Neuronatin expression. The decreased Neuronatin expression in AN provides evidence that Neuronatin is implicated in the pathogenesis of eating disorders.
Termination codon readthrough (TCR) is a process in which ribosomes continue to translate an mRNA beyond a stop codon generating a C-terminally extended protein isoform. Here, we demonstrate TCR in mammalian NNAT mRNA, which encodes NNAT, a proteolipid important for neuronal differentiation. This is a programmed event driven by cis-acting RNA sequences present immediately upstream and downstream of the canonical stop codon, and is negatively regulated by NONO, an RNA-binding protein known to promote neuronal differentiation. Unlike the canonical isoform NNAT, we determined that the TCR product (NNATx) does not show detectable interaction with the SERCA2 Ca2+ pump, cannot increase cytoplasmic Ca2+ levels, and therefore does not enhance neuronal differentiation in Neuro-2a cells. Additionally, an antisense oligonucleotide that targets a region downstream of the canonical stop codon reduced TCR of NNAT and enhanced the differentiation of Neuro-2a cells to cholinergic neurons. Furthermore, NNATx-deficient Neuro-2a cells, generated using CRISPR-Cas9, showed increased cytoplasmic Ca2+ levels and enhanced neuronal differentiation. Overall, these results demonstrate regulation of neuronal differentiation by TCR of NNAT. Importantly, this process can be modulated using a synthetic antisense oligonucleotide.
Studies in genetically ‘identical’ individuals indicate that as much as 50% of complex trait variation cannot be traced to genetics or to the environment. The mechanisms that generate this ‘unexplained’ phenotypic variation (UPV) remain largely unknown. Here, we identify neuronatin (NNAT) as a conserved factor that buffers against UPV. We find that Nnat deficiency in isogenic mice triggers the emergence of a bi-stable polyphenism, where littermates emerge into adulthood either ‘normal’ or ‘overgrown’. Mechanistically, this is mediated by an insulin-dependent overgrowth that arises from histone deacetylase (HDAC)-dependent β-cell hyperproliferation. A multi-dimensional analysis of monozygotic twin discordance reveals the existence of two patterns of human UPV, one of which (Type B) phenocopies the NNAT-buffered polyphenism identified in mice. Specifically, Type-B monozygotic co-twins exhibit coordinated increases in fat and lean mass across the body; decreased NNAT expression; increased HDAC-responsive gene signatures; and clinical outcomes linked to insulinemia. Critically, the Type-B UPV signature stratifies both childhood and adult cohorts into four metabolic states, including two phenotypically and molecularly distinct types of obesity. Yang et al. show that neuronatin (NNAT) can explain part of the phenotypic variation of complex traits, independently of genetics or the environment. Such NNAT-dependent variations can stratify human cohorts into four metabolic sub-types, including two distinct types of obesity.
Objective:
The authors hypothesize that an untargeted metabolomics study will identify novel mechanisms underlying smoking-associated weight loss.
Methods:
This study performed cross-sectional analyses among 1,252 participants in the Bogalusa Heart Study and assessed 1,202 plasma metabolites for mediation effects on smoking-BMI associations. Significant metabolites were tested for associations with smoking genetic risk scores among a subset of participants (n = 654) with available genomic data, followed by direction dependence analysis to investigate causal relationships between the metabolites and smoking and BMI. All analyses controlled for age, sex, race, education, alcohol drinking, and physical activity.
Results:
Compared with never smokers, current and former smokers had a 3.31-kg/m2 and 1.77-kg/m2 lower BMI after adjusting for all covariables, respectively. A total of 22 xenobiotics and 94 endogenous metabolites were significantly associated with current smoking. Eight xenobiotics were also associated with former smoking. Forty metabolites mediated the smoking-BMI associations, and five showed causal relationships with both smoking and BMI. These metabolites, including 1-oleoyl-GPE (18:1), 1-linoleoyl-GPE (18:2), 1-stearoyl-2-arachidonoyl-GPE (18:0/20:4), α-ketobutyrate, and 1-palmitoyl-GPE (16:0), mediated 26.0% of the association between current smoking and BMI.
Conclusions:
This study cataloged plasma metabolites altered by cigarette smoking and identified five metabolites that partially mediated the association between current smoking and BMI.
Imprinted genes play important regulatory roles in the growth and development of placentas and foetuses during pregnancy. In a previous study, we found that the imprinted gene Neuronatin (NNAT) is involved in foetal development; NNAT expression was significantly lower in the placentas of piglets that died neonatally compared to the placentas of surviving piglets. However, the function and mechanism of NNAT in regulating porcine placental development is still unknown. In this study, we collected the placentas of high- and low-weight foetuses at gestational day (GD 65, 90), (n = 4–5 litters/GD) to investigate the role of NNAT in regulating foetal growth and development. We found that the mRNA and protein levels of NNAT were significantly higher in the placentas of high-weight than low-weight foetuses. We then overexpressed NNAT in porcine placental trophoblast cell lines (pTr2) and demonstrated that NNAT activated the PI3K-AKT pathway, and further promoted the expression of glucose transporter 1 (GLUT1) and increased cellular calcium ion levels, which improved glucose transport in placental trophoblast cells in vitro. To conclude, our study suggests that NNAT expression impacts porcine foetal development by regulating placental glucose transport.
Neuronatin (NNAT) is a transmembrane protein in the endoplasmic reticulum involved in metabolic regulation. It shares sequence homology with sarcolipin (SLN), which negatively regulates the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) that maintains energy homeostasis in muscles. Here, we examined whether NNAT could uncouple the Ca2+ transport activity of SERCA from ATP hydrolysis, similarly to SLN. NNAT significantly reduced Ca2+ uptake without altering SERCA activity, ultimately lowering the apparent coupling ratio of SERCA. This effect of NNAT was reversed by the adenylyl cyclase activator forskolin. Furthermore, soleus muscles from high fat diet (HFD)‐fed mice showed a significant downregulation in NNAT content compared with chow‐fed mice, whereas an upregulation in NNAT content was observed in fast‐twitch muscles from HFD‐ versus chow‐ fed mice. Therefore, NNAT is a SERCA uncoupler in cells and may function in adaptive thermogenesis. Neuronatin reduces sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA)‐mediated calcium uptake without altering SERCA activity, thereby uncoupling calcium transport from ATP hydrolysis. In C57BL/6 mice, 12 weeks of high fat feeding leads to a reduction in neuronatin content in the soleus but an upregulation in neuronatin content in fast muscle types including the extensor digitorum longus and red and white gastrocnemius.
Bisphenol A (BPA) is an endocrine disrupting chemical that promotes obesity. It acts on the hypothalamus by increasing expression of the orexigenic neuropeptides, Npy and Agrp. Exactly how BPA dysregulates energy homeostasis is not completely clear. Since microRNAs (miRNA) have emerged as crucial weight regulators, the question of whether BPA could alter hypothalamic miRNA profiles was examined. Treatment of the mHypoA-59 cell line with 100 μM BPA altered a specific subset of miRNAs, and the most upregulated was miR-708-5p. BPA was found to increase the levels of miR-708-5p, and its parent gene Odz4, through the ER stress-related protein Chop. Overexpression of an miR-708-5p mimic resulted in a reduction of neuronatin, a proteolipid whose loss of expression is associated with obesity, and an increase in orexigenic Npy expression, thus potentially increasing feeding through converging regulatory pathways. Therefore, hypothalamic exposure to BPA can increase miR-708-5p that controls neuropeptides directly linked to obesity.
Parent-of-origin effects are unexpectedly common in complex traits, including metabolic and neurological diseases. Parent-of-origin effects can be modified by the environment, but the architecture of these gene-by-environmental effects on phenotypes remains to be unraveled. Previously, quantitative trait loci (QTL) showing context-specific parent-of-origin effects on metabolic traits were mapped in the F 16 generation of an advanced intercross between LG/J and SM/J inbred mice. However, these QTL were not enriched for known imprinted genes, suggesting another mechanism is needed to explain these parent-of-origin effects phenomena. We propose that non-imprinted genes can generate complex parent-of-origin effects on metabolic traits through interactions with imprinted genes. Here, we employ data from mouse populations at different levels of intercrossing (F 0 , F 1 , F 2 , F 16 ) of the LG/J and SM/J inbred mouse lines to test this hypothesis. Using multiple populations and incorporating genetic, genomic, and physiological data, we leverage orthogonal evidence to identify networks of genes through which parent-of-origin effects propagate. We identify a network comprised of 3 imprinted and 6 non-imprinted genes that show parent-of-origin effects. This epistatic network forms a nutritional responsive pathway and the genes comprising it jointly serve cellular functions associated with growth. We focus on 2 genes, Nnat and F2r , whose interaction associates with serum glucose levels across generations in high fat-fed females. Single-cell RNAseq reveals that Nnat and F2r are negatively correlated in pre-adipocytes along an adipogenic trajectory, a result that is consistent with our observations in bulk white adipose tissue.
Neuronatin (Nnat) is involved in the regulation of cellular molecular signaling and appears to be also linked to metabolic processes. The gastrointestinal peptides cholecystokinin (CCK) and bombesin (BN) have an effect on the short-term inhibition of food intake and induce neuronal activation in different brain nuclei, prominently in the nucleus of the solitary tract (NTS) involved in the modulation of food intake. The aim of the study was to examine if whether Nnat immunoreactivity is detectable in the NTS, and whether peripheral CCK-8S or BN cause c-Fos activation of Nnat neurons. Non-fasted male Sprague-Dawley rats received an intraperitoneal (i.p.) injection of 5.2 or 8.7 nmol CCK-8S/kg or 26 or 32 nmol BN/kg (n=4 all groups) or vehicle solution (0.15M NaCl; n=7). The number of c-Fos neurons was determined 90 minutes post injection in the NTS and dorsal motor nucleus of the vagus (DMV). We observed Nnat immunoreactive neurons in the NTS and DMV. CCK-8S (25-fold and 51-fold, p = 0.025 and p = 0.001) and BN (31-fold and 59-fold, p = 0.007 and p = 0.001) at both doses increased the number of c-Fos positive neurons in the NTS. CCK and BN did not show a significant effect in the DMV. Both doses of CCK-8S (24-fold and 48-fold p=0.011 and p=0.001) and bombesin (31-fold and 56-fold, p=0.002 and p=0.001) increased the number of activated Nnat neurons in the NTS (p=0.001) compared to the vehicle group, while in the DMV no significant increase of c-Fos activation was detected. In conclusion, i.p. injected CCK-8S or BN induce an increased neuronal activity in NTS Nnat neurons, giving rise that Nnat may play a role in the regulation of food intake mediated by peripheral CCK-8S or BN.
Neuronatin (NNAT) was first identified as a gene selectively and abundantly expressed in the cytoplasm of the newborn mouse brain, and involved in neonatal neurogenesis. However, the particular roles of NNAT in the developing prenatal brain have not been identified, especially in mid to late stages. In this study, we performed immunohistochemical analyses of NNAT and SOX2 proteins, a nuclear transcription factor and neural stem/progenitor marker, in the rat brain on embryonic days 13.5, E16.5, and E20.5. NNAT signals were broadly observed across the developing brain on E13.5 and gradually more localized in later stages, eventually concentrated in the alar and basal parts of the terminal hypothalamus, the alar plate of prosomere 2 of the thalamus, and the choroid plexus in the lateral and fourth ventricles on E20.5. In particular, the mammillary body in the basal part of the terminal hypothalamus, a region with a high number of SOX2-positive cells, evidenced intense NNAT signals on E20.5. The intracellular localization of NNAT showed diverse profiles, suggesting that NNAT was involved in various cellular functions, such as cell differentiation and functional maintenance, during prenatal neurogenesis in the rat brain. Thus, the present observations suggested diverse and active roles of the NNAT protein in neurogenesis. Determining the function of this molecule may assist in the elucidation of the mechanisms involved in brain development.
Parent-of-origin effects (POE) are unexpectedly common in complex traits, including metabolic and neurological diseases. POE can also be modified by the environment, but the architecture of these gene-by-environmental effects on phenotypes remains to be unraveled. Previously, quantitative trait loci (QTL) showing context-specific POE on metabolic traits were mapped in the F16 generation of an advanced intercross between LG/J and SM/J inbred mice. However, these QTL were not enriched for known imprinted genes, suggesting another mechanism is needed to explain these POE phenomena. Here, we use a simple yet powerful F1 reciprocal cross model to test the hypothesis that non-imprinted genes can generate complex POE on metabolic traits through genetic interactions with imprinted genes. Male and female mice from a F1 reciprocal cross of LG/J and SM/J strains were fed either high or low fat diets. We generated expression profiles from three metabolically-relevant tissues: hypothalamus, white adipose, and liver. We identified two classes of parent-of-origin expression biases: genes showing parent-of-origin-dependent allele-specific expression and biallelic genes that are differentially expressed by reciprocal cross. POE patterns of both gene classes are highly tissue- and context-specific, sometimes occurring only in one sex and/or diet cohort in a particular tissue. We then constructed tissue-specific interaction networks among genes from these two classes of POE. Some gene pairs show significant epistasis in the F16 LG/J x SM/J advanced intercross data in cases where the biallelic gene fell within a previously-identified metabolic POE QTL interval. We highlight one such interaction in adipose, between Nnat and Mogat1, which associates with variation in multiple adiposity traits. The genes and networks we present here represent a set of actionable interacting candidates that can be probed to further identify the machinery driving POE on complex traits.
The endoplasmic reticulum (ER) is a cellular compartment that has a key function in protein translation and folding. Maintaining its integrity is of fundamental importance for organism's physiology and viability. The dynamic regulation of intraluminal ER Ca(2+) concentration directly influences the activity of ER-resident chaperones and stress response pathways that balance protein load and folding capacity. We review the emerging evidence that microRNAs play important roles in adjusting these processes to frequently changing intracellular and environmental conditions to modify ER Ca(2+) handling and storage and maintain ER homeostasis.
Adipose tissue plays an important role in energy metabolism and related diseases. The content of intramuscular fat significantly influences the pork quality. In this study, the whole gene expression of dorsal subcutaneous (s.c.) adipose tissue and intramuscular (i.m.) adipose tissue isolated from longissimus dorsi muscle tissue were compared using Affymetrix Gene-Chip microarray technology. The result revealed that 1228 genes were more highly expressed in s.c. adipose tissue, whereas 965 genes were higher expressed in i.m. adipose tissue. We found that the s.c. adipose tissue had a stronger capacity of lipid metabolism and fatty acid metabolism compared with i.m. adipose tissue, and angiopoietin-like-4, neuronatin, neuron-derived orphan receptor-1 alfa, and chloride intracellular channel 5 may play important roles in the regulation of fat deposition between i.m. and s.c. adipose tissues.
We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5–14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level. Citation: Goldsworthy M, Absalom NL, Schrö ter D, Matthews HC, Bogani D, et al. (2013) Mutations in Mll2, an H3K4 Methyltransferase, Result in Insulin Resistance and Impaired Glucose Tolerance in Mice. PLoS ONE 8(6): e61870.
Background:
Ewing sarcoma (ES), a highly aggressive tumor of children and young adults, is characterized most commonly by an 11;22 chromosomal translocation that fuses EWSR1 located at 22q12 with FLI1, coding for a member of the ETS family of transcription factors. Although genetic changes in ES have been extensively researched, our understanding of the role of epigenetic modifications in this neoplasm is limited.
Procedure:
In an effort to improve our knowledge in the role of epigenetic changes in ES we evaluated the in vitro antineoplastic effect of the DNA methyltransferase inhibitor 5-Aza-deoxycytidine (5-Aza-dC) and identified epigenetically silenced genes by pharmacologic unmasking of DNA methylation coupled with genome-wide expression profiling.
Results:
Comparisons between untreated and 5-Aza-dC treated ES cell lines (n = 5) identified 208 probe sets with at least twofold difference in expression (P ≤ 0.05). The 208 probe sets represented 145 upregulated and 31 down-regulated genes. Of the 145 genes upregulated after 5-Aza-dC treatment, four: were further characterized. ACRC, CLU, MEST, and NNAT were found to be hypermethylated and transcriptionally down-regulated in ES cell lines. Further studies revealed that ACRC, CLU, MEST, and NNAT were often hypermethylated in primary ES tumors. Transfection-mediated reexpression of ACRC, CLU, MEST, and NNAT in ES cell lines resulted in decreased growth in culture.
Conclusions:
This study demonstrated epigenetically modified genes in ES cell lines and primary tumors and suggested that epigenetic dysregulation may contribute to disease pathogenesis in ES.
Bridge structures crossing navigable waterways must not only be designed to resist gravity, wind, and earthquake loads, but must also be capable of resisting ship and barge collision loads. Design specifications used both in the US and internationally employ empirical models of vessel crush behavior to produce codified procedures for computing equivalent static design loads due to vessel impact. In this paper, the ADINA finite element code is used to compute force-deformation relationships for several hopper barge crushing scenarios. Results obtained from the nonlinear finite element crush analyses are then compared to empirical crush models found in bridge design specifications.
Lafora disease (LD) is the inherited progressive myoclonus epilepsy caused by mutations in either EPM2A gene, encoding the protein phosphatase laforin or the NHLRC1 gene, encoding the ubiquitin ligase malin. Since malin is an ubiquitin ligase and its mutations cause LD, it is hypothesized that improper clearance of its substrates might lead to LD pathogenesis. Here, we demonstrate for the first time that neuronatin is a novel substrate of malin. Malin interacts with neuronatin and enhances its degradation through proteasome. Interestingly, neuronatin is an aggregate prone protein, forms aggresome upon inhibition of cellular proteasome function and malin recruited to those aggresomes. Neuronatin is found to stimulate the glycogen synthesis through the activation of glycogen synthase and malin prevents neuronatin-induced glycogen synthesis. Several LD-associated mutants of malin are ineffective in the degradation of neuronatin and suppression of neuronatin-induced glycogen synthesis. Finally, we demonstrate the increased levels of neuronatin in the skin biopsy sample of LD patients. Overall, our results indicate that malin negatively regulates neuronatin and its loss of function in LD results in increased accumulation of neuronatin, which might be implicated in the formation of Lafora body or other aspect of disease pathogenesis.
A knowledge of beta cell-specific gene expression provides a basis for identifying proteins potentially involved in beta cell function and pathology. To identify candidate beta cell-specific genes, we applied the PCR-based subtractive hybridization technique of representational difference analysis (RDA) to the mouse SV40-transformed endocrine cell lines, betaTC3 and alphaTC1. Following three successive subtractions of alphaTC1 complementary DNA from betaTC3 complementary DNA, difference products were cloned into pUC19 and nucleotide sequences determined. Comparison of 91 sequences against the databases identified 11 known and 8 novel genes. Known genes included previously reported beta cell-specific genes, insulin I/II and islet amyloid polypeptide, as well as other non-beta cell-specific genes such as those for insulin-like growth factor II, selenoprotein P, neuronatin, prohormone convertase, and type 1 protein kinase A regulatory subunit. By Northern blot hybridization, expression of the majority of known and novel genes was restricted to betaTC3 cells. Novel genes BA-12, -13, -14, and -18 were expressed not only in betaTC3 cells, but also in normal pancreatic islets and a limited number of other tissues. The deduced amino acid sequence of BA-14 showed significant homology with members of the cadherin superfamily indicating that BA-14 may encode a cadherin-like molecule potentially involved in beta cell adhesion events during islet ontogeny. In betaTC3 cells, none of the novel genes were regulated at the RNA level by high glucose. However, in parallel studies, transcription of BA-12 was significantly increased by both sodium butyrate and nicotinamide, suggesting that this gene may play a role in pancreatic beta cell growth and/or differentiation. In this study, we have demonstrated that cRDA is an effective strategy for systematically mapping differences in gene expression between two related but functionally-distinct endocrine cells. Its application to experimental animal models of islet-cell regeneration may facilitate the discovery of potential factors that mediate beta cell growth and differentiation.
LAP/C/EBP beta is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Recently we showed that, besides its posttranscriptional regulation, LAP/C/EBP beta mRNA is modulated during liver regeneration. Therefore, in this study we investigated mechanisms which control LAP/C/EBP beta gene transcription. Deletion analysis of the 5'-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonstrated that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. In gel shift experiments two sites were identified which are important for specific complex formation within this region. Further analysis by cross-linking, super shift, and competition experiments was performed with liver cell nuclear extracts, hepatoma cell nuclear extracts, or recombinant CREB protein. These experiments conclusively demonstrated that CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that with the CREB consensus sequence. Transfection experiments with promoter constructs where the CREB sites were mutated showed that these sites are important to maintain both basal promoter activity and LAP/C/EBP beta inducibility through CREB. Northern blot analysis and runoff transcription assays demonstrated that the protein kinase A pathway not only stimulated the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types. Western blot analysis of rat liver cell nuclear extracts and runoff transcription assays of rat liver cell nuclei after two-thirds hepatectomy showed a functional link between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP beta transcription in vivo. As several pathways control CREB phosphorylation, our results provide evidence for the transcriptional regulation of LAP/C/EBP beta via CREB under different physiological conditions.
We have established a systematic screen for imprinted genes using a subtraction-hybridization method with day 8.5 fertilized and parthenogenetic embryos. Two novel imprinted genes, Peg1/Mest and Peg3, were identified previously by this method, along with the two known imprinted genes, Igf2 and Snrpn. Recently three additional candidate imprinted genes, Peg5-7 , were detected and Peg5 is analyzed further in this study. The cDNA sequence of Peg5 is identical to Neuronatin, a gene recently reported to be expressed mainly in the brain. Two novel spliced forms were detected with some additional sequence in the middle of the known Neuronatin sequences. All alternatively spliced forms of Peg5 were expressed only from the paternal allele, confirmed using DNA polymorphism in a subinterspecific cross. Peg5/Neuronatin maps to sub-distal Chr 2, proximal to the previously established imprinted region where imprinted genes cause abnormal shape and behavior in neonates.
The adipocyte plays a critical role in energy balance. Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from precursor cells. For the last 20 years, the cellular and molecular mechanisms of adipocyte differentiation have been extensively studied using preadipocyte culture systems. Committed preadipocytes undergo growth arrest and subsequent terminal differentiation into adipocytes. This is accompanied by a dramatic increase in expression of adipocyte genes including adipocyte fatty acid binding protein and lipid-metabolizing enzymes. Characterization of regulatory regions of adipose-specific genes has led to the identification of the transcription factors peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer binding protein (C/EBP), which play a key role in the complex transcriptional cascade during adipocyte differentiation. Growth and differentiation of preadipocytes is controlled by communication between individual cells or between cells and the extracellular environment. Various hormones and growth factors that affect adipocyte differentiation in a positive or negative manner have been identified. In addition, components involved in cell-cell or cell-matrix interactions such as preadipocyte factor-1 and extracellular matrix proteins are also pivotal in regulating the differentiation process. Identification of these molecules has yielded clues to the biochemical pathways that ultimately result in transcriptional activation via PPAR-gamma and C/EBP. Studies on the regulation of the these transcription factors and the mode of action of various agents that influence adipocyte differentiation will reveal the physiological and pathophysiological mechanisms underlying adipose tissue development.
Identification and characterization of genes expressed preferentially in pancreatic beta-cells will clarify the mechanisms involved in the specialized properties of these cells, as well as providing new markers of the development of type 1 diabetes. Despite major efforts, relatively few beta-cell-specific genes have been characterized. We applied representational difference analysis to identify genes expressed selectively in the pancreatic beta-cell line betaTC1 compared with the pancreatic alpha-cell line alphaTC1 and isolated 26 clones expressed at higher levels in the beta-cells than in the alpha-cells. DNA sequencing revealed that 14 corresponded to known genes (that is, present in GenBank). Only four of those genes had been shown previously to be expressed at higher levels in beta-cells (insulin, islet amyloid polypeptide, neuronatin, and protein kinase A regulatory subunit [RIalpha]). The known genes include transcription factors (STAT6) and mediators of signal transduction (guanylate cyclase). The remaining 12 genes are absent from the GenBank database or are present as expressed sequence tag (EST) sequences (4 clones). Some of the genes are expressed in a highly specific pattern-expression in betaTC1 and islet cells and in relatively few of the non-beta-cell types examined; others are expressed in most cell types tested. The identification of these differentially expressed genes may aid in attaining a clearer understanding of the mechanisms involved in beta-cell function and of the possible immunogens involved in development of type 1 diabetes.
NIDDM is characterized by islet amyloid deposits and decreased beta-cell mass. Islet amyloid is derived from the locally expressed protein islet amyloid polypeptide (IAPP). While it is now widely accepted that abnormal aggregation of IAPP has a role in beta-cell death in NIDDM, the mechanism remains unknown. We hypothesized that small IAPP aggregates, rather than mature large amyloid deposits, are cytotoxic. Consistent with this hypothesis, freshly dissolved human (h)-IAPP was cytotoxic when added to dispersed mouse and human islet cells, provoking the formation of abnormal vesicle-like membrane structures in association with vacuolization and cell death. Human islet cell death occurred by both apoptosis and necrosis, predominantly between 24 and 48 h after exposure to h-IAPP. In contrast, the addition to dispersed islet cells of matured h-IAPP containing large amyloid deposits of organized fibrils was seldom associated with vesicle-like structures or features of cell death, even though the cells were often encased in the larger amyloid deposits. Based on these observations, we hypothesized that h-IAPP cytotoxicity is mediated by membrane damage induced by early h-IAPP aggregates. Consistent with this hypothesis, application of freshly dissolved h-IAPP to voltage-clamped planar bilayer membranes (a cell-free in vitro system) also caused membrane instability manifested as a marked increase in conductance, increased membrane electrical noise, and accelerated membrane breakage, effects that were absent using matured h-IAPP or rat IAPP solutions. Light-scattering techniques showed that membrane toxicity corresponded to h-IAPP aggregates containing approximately 25-6,000 IAPP molecules, an intermediate-sized amyloid particle that we term intermediate-sized toxic amyloid particles (ISTAPs). We conclude that freshly dissolved h-IAPP is cytotoxic and that this cytotoxicity is mediated through an interaction of ISTAPs with cellular membranes. Once ISTAPs mature into amyloid deposits comprising >10(6) molecules, the capacity of h-IAPP to cause membrane instability and islet cell death is significantly reduced or abolished. These data may have implications for the mechanism of cell death in other diseases characterized by local amyloid formation (such as Alzheimer's disease).
Obesity is the result of numerous, interacting behavioral, physiological, and biochemical factors. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding and/or large increases in body fat composition. The generation of new adipocytes is controlled by several "adipocyte-specific" transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed only following the induction of adipogenesis. The transcription factor(s) that are involved in initiating adipocyte differentiation have not been identified. Here we demonstrate that the transcription factor, CREB, is constitutively expressed in preadipocytes and throughout the differentiation process and that CREB is stimulated by conventional differentiation-inducing agents such as insulin, dexamethasone, and dibutyryl cAMP. Stably transfected 3T3-L1 preadipocytes were generated in which we could induce the expression of either a constitutively active CREB (VP16-CREB) or a dominant-negative CREB (KCREB). Inducible expression of VP16-CREB alone was sufficient to initiate adipogenesis as determined by triacylglycerol storage, cell morphology, and the expression of two adipocyte marker genes, peroxisome proliferator activated receptor gamma 2, and fatty acid binding protein. Alternatively, KCREB alone blocked adipogenesis in cells treated with conventional differentiation-inducing agents. These data indicate that activation of CREB was necessary and sufficient to induce adipogenesis. Finally, CREB was shown to bind to putative CRE sequences in the promoters of several adipocyte-specific genes. These data firmly establish CREB as a primary regulator of adipogenesis and suggest that CREB may play similar roles in other cells and tissues.
Intracellular calcium ([Ca(2+)](i)) modulates adipocyte lipid metabolism and inhibits the early stages of murine adipogenesis. Consequently, we evaluated effects of increasing [Ca(2+)](i) in early and late stages of human adipocyte differentiation. Increasing [Ca(2+)](i) with either thapsigargin or A23187 at 0-1 h of differentiation markedly suppressed differentiation, with a 40-70% decrease in triglyceride accumulation and glycerol-3 phosphate dehydrogenase (GPDH) activity (P < 0.005). However, a 1-h pulse of either agent at 47-48 h only modestly inhibited differentiation. Sustained, mild stimulation of Ca(2+) influx with either agouti protein or 10 mM KCl-induced depolarization during 0-48 h of differentiation inhibited triglyceride accumulation and GPDH activity by 20-70% (P < 0.05) and markedly suppressed peroxisome proliferator-activated receptor gamma (PPARgamma) expression. These effects were reversed by Ca(2+) channel antagonism. In contrast, Ca(2+) pulses late in differentiation (71-72 h or 48-72 h) markedly increased these markers of differentiation. Thus increasing [Ca(2+)](i) appears to exert a biphasic regulatory role in human adipocyte differentiation, inhibiting the early stages while promoting the late stage of differentiation and lipid filling.
Insulin is a potent adipogenic hormone that triggers an induction of a series of transcription factors governing differentiation of pre-adipocytes into mature adipocytes. However, the exact link between the insulin signaling cascade and the intrinsic cascade of adipogenesis remains incompletely understood. Herein we demonstrate that inhibition of prenylation of p21ras and Rho-A arrests insulin-stimulated adipogenesis. Inhibition of farnesylation of p21ras also blocked the ability of insulin to activate mitogen-activated protein (MAP) kinase and cyclic AMP response element-binding (CREB) protein. Expression of two structurally different inducible constitutively active CREB constructs rescued insulin-stimulated adipocyte differentiation from the inhibitory influence of prenylation inhibitors. Constitutively active CREB constructs induced expression of PPARgamma2, fatty acid synthase, GLUT-4, and leptin both in control and prenylation inhibitors-treated cells. It appears that insulin-stimulated prenylation of the Ras family GTPases assures normal phosphorylation and activation of CREB that, in turn, triggers the intrinsic cascade of adipogenesis.
White adipose tissue mass is governed by competing processes that control lipid synthesis and storage, the development of new adipocytes, and their survival. We have shown that the transcription factor cAMP-response element-binding protein (CREB) participates in adipogenesis, with constitutively active forms of CREB inducing adipocyte differentiation and dominant negative forms of CREB blocking this process. In other cell types, CREB and related factors have been shown to play important roles in survival and apoptosis. Here we demonstrate that reduction of CREB activity by ectopic expression of the dominant negative CREB, KCREB, induces apoptosis of mature 3T3-L1 adipocytes in culture. Death by apoptosis was confirmed by increased nuclear condensation, changes in membrane morphology, and increased DNA fragmentation. Gene microarray analysis indicated that KCREB expression increased expression of several pro-apoptotic genes like Interleukin Converting Enzyme and decreased the expression of the anti-apoptotic signaling molecule, Akt/protein kinase B. Finally, introduction of constitutively active CREB, CREB-DIEDML, blocked death of mature adipocytes treated with TNF-alpha. The data indicate that CREB plays a central role in adipocyte survival, perhaps by regulating the expression of certain pro- and anti-apoptotic genes. These results not only extend the role of CREB in adipocyte biology but also highlight the general developmental and survival role of this factor in numerous cell and tissue types.
Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling, a process that takes place during obesity-mediated adipose tissue formation. Here, we examine expression profiles and the potential role of MMPs and their tissue inhibitors (TIMPs) in adipose tissue remodeling during obesity. Expression patterns are studied by Northern blot and real-time PCR in two genetic models of obesity (ob/ob and db/db mice) and in a diet-induced model of obesity (AKR mice). Of the MMPs and TIMPs studied, mRNA levels for MMP-2, MMP-3, MMP-12, MMP-14, MMP-19, and TIMP-1 are strongly induced in obese adipose tissues compared with lean tissues. In contrast, MMP-7 and TIMP-3 mRNAs are markedly decreased in obesity. Interestingly, enzymatic activities of MMP-12 and of a new identified adipocyte-derived 30-kDa metalloproteinase are enhanced in obese adipose tissue fractions, demonstrating that MMP/TIMP balance is shifted toward increased matrix degradation in obesity. Finally, we analyze the modulation of MMP-2, MMP-19, and TIMP-1 during 3T3-L1 preadipocyte differentiation, and we explore the effect of inhibition of MMP activity on in vitro adipogenesis. We find that the synthetic MMP inhibitor BB-94 (Batimastat) decreases adipose conversion of 3T3-L1 and primary rat preadipocytes. BB-94 represses differentiation without affecting mitotic clonal expansion but prevents the early expression of CCAAT/enhancer-binding protein beta, a transcription factor that is thought to play a major role in the adipogenic program. Such findings support a role for the MMP/TIMP system in the control of proteolytic events and adipogenesis during obesity-mediated fat mass development.
Insulin resistance occurs early in the disease process, preceding the development of type 2 diabetes. Therefore, the identification of molecules that contribute to insulin resistance and leading up to type 2 diabetes is important to elucidate the molecular pathogenesis of the disease. To this end, we characterized gene expression profiles from insulin-sensitive tissues, including adipose tissue, skeletal muscle, and liver tissue of Zucker diabetic fatty (ZDF) rats, a well characterized type 2 diabetes animal model. Gene expression profiles from ZDF rats at 6 weeks (pre-diabetes), 12 weeks (diabetes), and 20 weeks (late-stage diabetes) were compared with age- and sex-matched Zucker lean control (ZLC) rats using 5000 cDNA chips. Differentially regulated genes demonstrating > 1.3-fold change at age were identified and categorized through hierarchical clustering analysis. Our results showed that while expression of lipolytic genes was elevated in adipose tissue of diabetic ZDF rats at 12 weeks of age, expression of lipogenic genes was decreased in liver but increased in skeletal muscle of 12 week old diabetic ZDF rats. These results suggest that impairment of hepatic lipogenesis accompanied with the reduced lipogenesis of adipose tissue may contribute to development of diabetes in ZDF rats by increasing lipogenesis in skeletal muscle. Moreover, expression of antioxidant defense genes was decreased in the liver of 12-week old diabetic ZDF rats as well as in the adipose tissue of ZDF rats both at 6 and 12 weeks of age. Cytochrome P450 (CYP) genes were also significantly reduced in 12 week old diabetic liver of ZDF rats. Genes involved in glucose utilization were downregulated in skeletal muscle of diabetic ZDF rats, and the hepatic gluconeogenic gene was upregulated in diabetic ZDF rats. Genes commonly expressed in all three tissue types were also observed. These profilings might provide better fundamental understanding of insulin resistance and development of type 2 diabetes.
Gregoire, Francine M., Cynthia M. Smas, and Hei Sook Sul. Understanding Adipocyte Differentiation. Physiol. Rev. 78: 783–809, 1998. — The adipocyte plays a critical role in energy balance. Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from precursor cells. For the last 20 years, the cellular and molecular mechanisms of adipocyte differentiation have been extensively studied using preadipocyte culture systems. Committed preadipocytes undergo growth arrest and subsequent terminal differentiation into adipocytes. This is accompanied by a dramatic increase in expression of adipocyte genes including adipocyte fatty acid binding protein and lipid-metabolizing enzymes. Characterization of regulatory regions of adipose-specific genes has led to the identification of the transcription factors peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein (C/EBP), which play a key role in the complex transcriptional cascade during adipocyte differentiation. Growth and differentiation of preadipocytes is controlled by communication between individual cells or between cells and the extracellular environment. Various hormones and growth factors that affect adipocyte differentiation in a positive or negative manner have been identified. In addition, components involved in cell-cell or cell-matrix interactions such as preadipocyte factor-1 and extracellular matrix proteins are also pivotal in regulating the differentiation process. Identification of these molecules has yielded clues to the biochemical pathways that ultimately result in transcriptional activation via PPAR-γ and C/EBP. Studies on the regulation of the these transcription factors and the mode of action of various agents that influence adipocyte differentiation will reveal the physiological and pathophysiological mechanisms underlying adipose tissue development.
We have established a systematic screen for imprinted genes using a subtraction-hybridization method with day 8.5 fertilized
and parthenogenetic embryos. Two novel imprinted genes, Peg1/Mest and Peg3, were identified previously by this method, along with the two known imprinted genes, Igf2 and Snrpn. Recently three additional candidate imprinted genes, Peg5–7, were detected and Peg5 is analyzed further in this study. The cDNA sequence of Peg5 is identical to Neuronatin, a gene recently reported to be expressed mainly in the brain. Two novel spliced forms were detected with some additional
sequence in the middle of the known Neuronatin sequences. All alternatively spliced forms of Peg5 were expressed only from the paternal allele, confirmed using DNA polymorphism in a subinterspecific cross. Peg5/Neuronatin maps to sub-distal Chr 2, proximal to the previously established imprinted region where imprinted genes cause abnormal shape
and behavior in neonates.
A knowledge of β cell-specific gene expression provides a basis for identifying proteins potentially involved in β cell function and pathology. To identify candidate β cell-specific genes, we applied the PCR-based subtractive hybridization technique of representational difference analysis (RDA) to the mouse SV40-transformed endocrine cell lines, βTC3 and αTC1. Following three successive subtractions ofα TC1 complementary DNA from βTC3 complementary DNA, difference products were cloned into pUC19 and nucleotide sequences determined. Comparison of 91 sequences against the databases identified 11 known and 8 novel genes. Known genes included previously reported β cell-specific genes, insulin I/II and islet amyloid polypeptide, as well as other non-β cell-specific genes such as those for insulin-like growth factor II, selenoprotein P, neuronatin, prohormone convertase, and type 1 protein kinase A regulatory subunit. By Northern blot hybridization, expression of the majority of known and novel genes was restricted to βTC3 cells. Novel genes BA-12, -13, -14, and -18 were expressed not only in βTC3 cells, but also in normal pancreatic islets and a limited number of other tissues. The deduced amino acid sequence of BA-14 showed significant homology with members of the cadherin superfamily indicating that BA-14 may encode a cadherin-like molecule potentially involved in β cell adhesion events during islet ontogeny. In βTC3 cells, none of the novel genes were regulated at the RNA level by high glucose. However, in parallel studies, transcription of BA-12 was significantly increased by both sodium butyrate and nicotinamide, suggesting that this gene may play a role in pancreatic β cell growth and/or differentiation.
In this study, we have demonstrated that cRDA is an effective strategy for systematically mapping differences in gene expression between two related but functionally-distinct endocrine cells. Its application to experimental animal models of islet-cell regeneration may facilitate the discovery of potential factors that mediate β cell growth and differentiation.
When cells of the established preadipose line 3T3-L1 enter a resting state, they accumulate triglyceride and convert to adipose cells. The adipose conversion is brought about by a large increase in the rate of triglyceride synthesis, as measured by the incorporation rate of labeled palmitate, acetate, and glucose. In a resting 3T3 subline which dose not undergo the adipose conversion, the rate of triglyceride synthesis from these precursors is very low, and similar to that of growing 3T3-L1 cells, before their adipose conversion begins. If 3T3-L1 cells incorporate bromodeoxyuridine during growth, triglyceride synthesis does not increase when the cells reach a stationary state, and triglycerides do not accumulate. As would be expected from their known actions on tissue adipose cells, lipogenic and lipolytic hormones and drugs affect the rate of synthesis and accumulation of triglyceride by 3T3-L1 cells, but in contrast to bromodeoxyuridine, these modulating agents do not seem to affect the proportion of cells which undergoes the adipose conversion. Insulin markedly increases the rate of synthesis and accumulation of triglyceride by fatty 3T3-L1 cells, and produces a related increase in cell protein content. Of 20 randomly selected clones isolated from the original 3T3 stock, 19 are able to convert to adipose cells. The probability of such a conversion varies greatly among the different clones, in most cases being much lower than for 3T3-L1; but once the conversion takes place, the adipose cells produced from all of the 19 clones appear similar. The adipose conversion would seem to depend on an on-off switch, which is on with a different probability in different clones. This probability is quasistably inherited by the clonal progeny.
Neurogenesis begins with the closure of the neural tube around mid gestation and continues in the rat for about two weeks postnatally. Therefore, we investigated the role of neuronatin, a novel cDNA that we cloned from neonatal rat brain (Joseph et al., Biochem. Biophys. Res. Commun., 201 (1994) 1227-1234), in brain development. Further studies described in the present manuscript, lead to the identification of two alternatively spliced forms of neuronatin mRNA, alpha and beta, with the same open reading frame. Neuronatin-alpha encoded a novel protein of 81 aa, and the beta-form encoded 54 aa. Both forms were identical, except that the alpha-form had an additional 81 bp sequence inserted into the middle of the coding region. On Northern analyses, neuronatin mRNA was relatively selective for the brain. It first appeared at E11-14, a time when the neural tube has closed and neuroepithelial proliferation initiated, became pronounced at E16-19 with a surge in neurogenesis, and declined postnatally to adult levels with the completion of neurogenesis. In order to determine whether there were other forms of neuronatin mRNA, and to study the expression of the alpha and beta forms separately during development, reverse transcriptase-polymerase chain reaction was carried out using primers flanking the coding region of the alpha and beta forms. The RT-PCR results clearly indicated that there were only two forms of neuronatin. The beta-form first appeared at E11-14, whereas the alpha-form was present even earlier at E7-10. Together, these findings indicate that the two forms of neuronatin mRNA are regulated differently during brain development.(ABSTRACT TRUNCATED AT 250 WORDS)
The developing hindbrain is segmented in a series of repetitive bulges called neuromeres or rhombomeres. In the mouse, first molecular evidence for segmentation of the hindbrain came from rhombomeres 3- and 5-specific expression of the Krox-20 gene. The hindbrain segments are linked with the expression of different Hox genes which have a role in patterning the hindbrain and branchial region of the vertebrate head. Here we identified by subtractive hybridization a gene, mouse neuronatin, that is downregulated in P19 embryo carcinoma cells that have undergone a partial differentiation process. Neuronatin encodes putative transmembrane proteins of 54, 55, and 81 amino acids that might serve as protein ligands, cofactors, or small cell adhesion molecules. The neuronatin gene is transiently expressed in rhombomeres 3 and 5 during early hindbrain development and in the floor of the foregut pocket. In addition, expression is observed in the early Rathke's pouch, in the derived adenohypophysis, and in the developing inner ear. During later embryogenesis the neuronatin gene is strongly expressed in the major part of the central and peripheral nervous system. These results suggest that neuronatin participates in the maintenance of segment identity in the hindbrain and pituitary development and maturation or maintenance of the overall structure of the nervous system.
Neuronatin is a brain-specific human gene that we recently isolated and observed to be selectively expressed during brain development. In this report, the genomic structure and organization of human neuronatin is described. The human gene spans 3973 bases and contains three exons and two introns. Based on primer extension analysis, a single cap site is located 124 bases upstream from the methionine (ATG) initiation codon, in good context, GAACCATGG. The promoter contains a modified TATA box, CATAAA (-27), and a modified CAAT box, GGCGAAT (-59). The 5'-flanking region contains putative transcription factor binding sites for SP-1, AP-2 (two sites), delta-subunit, SRE-2, NF-A1, and ETS. In addition, a 21-base sequence highly homologous to the neural restrictive silence element that governs neuron-specific gene expression is observed at -421. Furthermore, SP-1 and AP-3 binding sites are present in intron 1. All splice donor and acceptor sites conformed to the GT/AG rule. Exon 1 encodes 24 amino acids, exon 2 encodes 27 amino acids, and exon 3 encodes 30 amino acids. At the 3'-end of the gene, the poly(A) signal, AATAAA, poly(A) site, and GT cluster are observed. The neuronatin gene is expressed as two mRNA species, alpha and beta, generated by alternative splicing. The alpha-form contains all three exons, whereas in the beta-form, the middle exon has been spliced out. The third nucleotide of all frequently used codons, except threonine, of neuronatin is either G or C, consistent with codon usage expected for Homo sapiens. This information about the structure of the human neuronatin gene will help in understanding the significance of this gene in brain development and human disease.
Human brain development is a continuum governed by differential gene expression. Therefore, we proceeded to identify genes selectively expressed in the developing brain. Using differential display and library screening, a novel rat cDNA, neuronatin, was identified and used to screen a human fetal brain cDNA library. Human neuronatin cDNA was isolated and sequenced. The cDNA was 1159 bp long and corresponded in size to the 1.25 kb message detected on Northern analysis. Neuronatin mRNA was selectively expressed in human brain during fetal development, but became repressed in adulthood. When studied in the rat, neuronatin mRNA first appeared at mid-gestation in association with the onset of neurogenesis, becoming most pronounced later in development when neuroepithelial proliferation and neuroblast commitment are manifest, and declined postnatally coinciding with the completion of neurogenesis. The deduced protein has two distinct domains, a hydrophobic N-terminal and basic C-terminal rich in arginine residues. Both the amino acid sequence and secondary structure of this amphipathic polypeptide exhibited homology to PMP1 and phospholamban, members of the "proteolipid' class of proteins which function as regulatory subunits of membrane channels. The neuronatin gene, 3973 bases long, contains in its 5'-flanking region a neural restrictive silencer element which may govern neuron-specific expression. Based on screening a somatic cell hybrid panel, neuronatin gene was assigned to chromosome-20. And, using deletion constructs of chromosome-20 and fluorescence in situ hybridization, neuronatin was localized to chromosome-20q11.2-12. In conclusion, neuronatin is a novel human gene that is developmentally regulated and expressed in the brain. The deduced protein is a proteolipid that may function as a unique regulator of ion channels during brain development. The definitive localization of neuronatin to human chromosome 20q11.2-12 provides the basis to investigate this gene as a candidate in neuro-developmental diseases that may also map to this region.
CCAAT-enhancer binding protein-delta (C/EBP delta) is a transcriptional nuclear factor, and belongs to basic region-leucine zipper class DNA binding proteins. One genomic clone containing a 12-kb sequence of the C/EBP delta gene was isolated from a rat genomic library, and a 2,056-bp fragment containing the 5'-flanking region was characterized. Sequence analysis of this fragment revealed that there were a TATA-like sequence (TAGAAAA) and many transcriptional regulatory elements. The transcription start site of the gene was determined by both primer extension analysis and riboprobe mapping. Both analyses indicated that the transcription start site was located at 31-bp downstream of the TATA-like sequence. Transient transfection experiments showed that the fragment cloned in this study was able to act as a functional promoter in rat vascular smooth muscle cells. The 5'-deletion analysis of this fragment revealed that the sequence spanning -235 through -82, which was designated as an upstream control element (UCE), remarkably increased a basal promoter activity of the C/EBP delta gene, and was also able to act as a promoter by itself. In addition, we also studied effects of the UCE on the heterologous gene promoter including rat alpha-actin gene promoter or SV40 virus promoter. Interestingly, the UCE specifically increased the promoter activity of the rat alpha-actin gene suggesting that the C/EBP delta gene may be positively controlled by the UCE via a cell-type or promoter-type specific manner.
Functional differences between parental genomes are due to differential expression of parental alleles of imprinted genes. Neuronatin (Nnat) is a recently identified paternally expressed imprinted gene that is initially expressed in the rhombomeres and pituitary gland and later more widely in the central and peripheral nervous system mainly in postmitotic and differentiating neuroepithelial cells. Nnat maps to distal chromosome (Chr) 2, which contains an imprinting region that causes morphological abnormalities and early neonatal lethality. More detailed mapping analysis of Nnat showed that it is located between the T26H and T2Wa translocation breakpoints which is, surprisingly, proximal to the reported imprinting region between the T2Wa and T28H translocation breakpoints, suggesting that there may be two distinct imprinting regions on distal chromosome 2. To investigate the potential role of Nnat, we compared normal embryos with those which were PatDp.dist2.T26H (paternal duplication/maternal deficiency of chromosome 2 distal to the translocation breakpoint T26H) and MatDp.dist2.T26H. Expression of Nnat was detected in the PatDp.dist2.T26H embryos, where both copies of Nnat are paternally inherited, and normal embryos but no expression was detected in the MatDp.dist2.T26H embryos with the two maternally inherited copies. The differential expression of Nnat was supported by DNA methylation analysis with the paternally inherited alleles being unmethylated and the maternal alleles fully methylated. Although experimental embryos appeared grossly similar phenotypically in the structures where expression of Nnat was detected, differences in folding of the cerebellum were observed in neonates, and other more subtle developmental or behavioral effects due to gain or loss of Nnat cannot be ruled out.
Nine regions on six mouse autosomes are subject to imprinting and uniparental inheritance of any one of these regions results in mice with phenotypic anomalies. So far on distal Chromosome (Chr) 2 there is a unique imprinting region between 2H3 and 2H4 associated with two behavioural disorders and neonatal lethality. A maternally imprinted gene, Nnat, has been identified which is expressed in the nervous system and maps to distal Chr 2. Nnat has been excluded as a candidate for either or both the behavioural phenotypes as it lies proximal to the 2H3-2H4 imprinting region. Here we have mapped Nnat to band 2H1 which is at least 18 Mb proximal to the previously described imprinting region. It maps close to agouti, some alleles of which show differential expression according to parental origin. The localisation of Nnat to band H1 confirms and refines the map location of a second imprinting region on mouse Chr 2.
Studies from the past several years have revealed that adipogenesis is controlled by an interplay of transcription factors, including members of the CCAAT/enhancer binding protein family and peroxisome proliferator activated receptor gamma. In addition to providing a new understanding of this aspect of the energy balance systems, these factors provide potential new targets for therapeutic intervention in metabolic diseases, such as obesity and type 2 diabetes mellitus.
Obesity and its antithesis, starvation, have always been part of the human condition, and for most of human history have been seen as resulting simply from availability of food, or acts of will related to attainment of desired body shape. Although this view persists in some quarters to this day, the last 5 years of the millennium have witnessed a dramatic increase in our understanding of the biology of regulated energy balance and body weight. Physiologic pathways whose existence was debated 10 years ago are now being characterized in molecular detail, with immediate implications for understanding of pathogenesis of human obesity and other disorders of energy balance. The roadmap provided by these advances establishes a clear direction for future research, but critical details remain to be discovered, and therapeutic applications remain to be realized. In particular, the mechanisms by which environmental factors, including diet and exercise, interact with molecular pathways in the common polygenic forms of obesity is largely unknown at present. Insights from the sequencing of the human genome and the coming advances in proteomics are likely to fuel the next wave of progress. It is likely that both new genes and new regulatory pathways will be identified. It may seem unlikely that the recent wave of progress can be matched in the early years of the current millennium, but we would not choose to make that bet.Smith 2000xThe controls of eating (a shift from nutritional homeostasis to behavioral neuroscience) . Smith, G.P. Nutrition. 2000; 16: 814–820Abstract | Full Text | Full Text PDF | PubMed | Scopus (111)See all ReferencesSmith 2000
Neuronatin (Nnat) is an imprinted gene that is expressed exclusively from the paternal allele while the maternal allele is silent and methylated. The Nnat locus exhibits some unique features compared with other imprinted domains. Unlike the majority of imprinted genes, which are organised in clusters and coordinately regulated, Nnat does not appear to be closely linked to other imprinted genes. Also unusually, Nnat is located within an 8-kb intron of the Bc10 gene, which generates a biallelically expressed, antisense transcript. A similar organisation is conserved at the human NNAT locus on chromosome 20. Nnat expression is first detected at E8.5 in rhombomeres 3 and 5, and subsequently, expression is widespread within postmitotic neuronal tissues. Using modified BAC transgenes, we show that imprinted expression of Nnat at ectopic sites requires, at most, an 80-kb region around the gene. Furthermore, reporter transgenes reveal distinct and dispersed cis-regulatory elements that direct tissue-specific expression and these are predominantly upstream of the region that confers allele-specific expression.
A small fraction of the genome contains genes that are imprinted and thus expressed exclusively from one parental allele. We report here that the human neuronatin gene (NNAT) on chromosome 20q11.2 is imprinted and transcribed specifically from the paternal allele. The region containing NNAT has multiple CpG islands, and methylation analysis showed that a 1.8-kb CpG island in its promoter region exhibits differential methylation in all tissues examined. This finding is consistent with the island acting as a component of the NNAT imprint control domain. NNAT lies within the singular 8.5-kb intron of the gene encoding bladder cancer-associated protein (BLCAP), which, as we demonstrate, is not imprinted. This study provides the first example, to our knowledge, in humans of an imprinted gene contained within the genomic structure of a nonimprinted gene. Thus, NNAT is in an imprinted "microdomain," making this locus uniquely suited for the investigation of mechanisms of localized imprint regulation.
The gene transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding (CREB) protein is a nuclear protein that regulates synaptic plasticity via modulating the expression of several (cAMP)-inducible genes. Alcohol addiction is a complex psychiatric disorder and is characterized by a compulsive and uncontrolled pattern of alcohol drinking by an individual in spite of the adverse consequences of its abuse. Ethanol produces both euphoric (reward and reinforcing) and dysphoric (negative withdrawal reactions) effects and these are most likely involved in the initiation and maintenance of alcohol use and abuse. Several neurotransmitter systems in the brain might be involved in the effects of alcohol but the exact molecular mechanisms of both the positive and negative affective states of alcohol abuse are still unclear. Recent research in molecular neurosciences using animal models have identified the role of extended amygdaloid (shell structures of nucleus accumbens [NAc] and central and medial amygdaloid nuclei) CREB signaling in positive and negative affective states of alcohol drinking behaviors. This review article highlights the current findings on the role of nucleus accumbal and amygdaloid CREB signaling in behavioral consequences of alcohol use and abuse.
BETA2 (NeuroD1) is a member of the basic helix-loop-helix transcription factor family. BETA2 plays an important role in the development of the pancreas and the nervous system. Using microarray technology, we identified neuronatin (Nnat) as differentially expressed between wild-type (WT) and knockout (KO) pancreatic RNA from embryonic day 14 (e14.5). NNAT is a member of the proteolipid family of amphipathic polypeptides and is believed to be involved in ion channel transport or channel modulation. Northern blot and in situ hybridization analysis of WT and KO samples confirmed the downregulation of Nnat in pancreas of mutant BETA2 embryos. Chromatin immunoprecipitation and gel shift assays were performed and demonstrated the presence of BETA2 on the Nnat promoter, thus confirming the direct transcriptional regulation of Nnat by BETA2. To assess NNAT potential function, we performed knockdown studies by siRNA in NIT cells and observed a reduction in the ability of the NIT cells to respond to glucose. These results suggest for the first time an important role for NNAT in insulin secretion and for proper beta-cell function.