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![Adipose-specific production of glucokinase and in vivo basal glucose uptake in adipose tissue of Gck-expressing transgenic mice (Tg) and control mice (Con). a Schematic representation of the Ap2– Gck chimeric gene. b Expression of the transgene in WAT and BAT. Representative northern blots from epididymal WAT, interscapular BAT and liver from control and heterozygous transgenic mice from line 1 (Tg1) and line 2 (Tg2), hybridised with a Gck probe are shown. c Protein levels of GK were detected by Western blots of epididymal WAT and liver from control and heterozygous transgenic mice from line 2 (Tg2). A representative western blot is shown. d The glucose utilisation index was measured in epididymal white adipose tissue of fed control and transgenic mice, as indicated in the text and the ESM. Results are presented as percentage of basal glucose utilisation in WAT of control mouse (302.13 pmol[mg protein] −1 min −1 ). The results are the means ± SEMs from at least four mice for each group. *p<](profile/Sergio-Munoz-23/publication/45152179/figure/fig1/AS:394379094183970@1471038711929/Adipose-specific-production-of-glucokinase-and-in-vivo-basal-glucose-uptake-in-adipose.png)
Adipose-specific production of glucokinase and in vivo basal glucose uptake in adipose tissue of Gck-expressing transgenic mice (Tg) and control mice (Con). a Schematic representation of the Ap2– Gck chimeric gene. b Expression of the transgene in WAT and BAT. Representative northern blots from epididymal WAT, interscapular BAT and liver from control and heterozygous transgenic mice from line 1 (Tg1) and line 2 (Tg2), hybridised with a Gck probe are shown. c Protein levels of GK were detected by Western blots of epididymal WAT and liver from control and heterozygous transgenic mice from line 2 (Tg2). A representative western blot is shown. d The glucose utilisation index was measured in epididymal white adipose tissue of fed control and transgenic mice, as indicated in the text and the ESM. Results are presented as percentage of basal glucose utilisation in WAT of control mouse (302.13 pmol[mg protein] −1 min −1 ). The results are the means ± SEMs from at least four mice for each group. *p<
Source publication
In adipocytes, triacylglycerol synthesis depends on the formation of glycerol 3-phosphate, which originates either from glucose, through glycolysis, or from lactate, through glyceroneogenesis. However, glucose is traditionally viewed as the main precursor of the glycerol backbone and thus, enhanced glucose uptake would be expected to result in incr...
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... Alexa 488 and mitochondria with orange rosamine MitoTracker. Nuclei of adipocytes were marked with blue Hoechst stain solution. Note that hexokinases and mitochondria surround an empty space corresponding to the unilocular lipid droplet. Scale bar, 11 μm Tg2) expressing hepatic Gck under control of the adipose- specific aP2 promoter were obtained (Fig. 1a). Transgenic mice showed high levels of Gck mRNA in white adipose tissue and brown adipose tissue (BAT; Fig. 1b). Further- more, GK protein was detected in the WAT of Tg2 mice, whereas no GK was noted in the controls (Fig. 1c). In addition, the level of GK protein in the WAT of Tg2 mice was 20% that of the endogenous hepatic GK level. ...
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... solution. Note that hexokinases and mitochondria surround an empty space corresponding to the unilocular lipid droplet. Scale bar, 11 μm Tg2) expressing hepatic Gck under control of the adipose- specific aP2 promoter were obtained (Fig. 1a). Transgenic mice showed high levels of Gck mRNA in white adipose tissue and brown adipose tissue (BAT; Fig. 1b). Further- more, GK protein was detected in the WAT of Tg2 mice, whereas no GK was noted in the controls (Fig. 1c). In addition, the level of GK protein in the WAT of Tg2 mice was 20% that of the endogenous hepatic GK level. While skeletal muscle from both control and transgenic mice did not produce GK (results not shown), liver from ...
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... droplet. Scale bar, 11 μm Tg2) expressing hepatic Gck under control of the adipose- specific aP2 promoter were obtained (Fig. 1a). Transgenic mice showed high levels of Gck mRNA in white adipose tissue and brown adipose tissue (BAT; Fig. 1b). Further- more, GK protein was detected in the WAT of Tg2 mice, whereas no GK was noted in the controls (Fig. 1c). In addition, the level of GK protein in the WAT of Tg2 mice was 20% that of the endogenous hepatic GK level. While skeletal muscle from both control and transgenic mice did not produce GK (results not shown), liver from both groups showed a 2.3 kb mRNA transcript (Fig. 1b) resulting from the expression of the endogenous hepatic Gck ...
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... detected in the WAT of Tg2 mice, whereas no GK was noted in the controls (Fig. 1c). In addition, the level of GK protein in the WAT of Tg2 mice was 20% that of the endogenous hepatic GK level. While skeletal muscle from both control and transgenic mice did not produce GK (results not shown), liver from both groups showed a 2.3 kb mRNA transcript (Fig. 1b) resulting from the expression of the endogenous hepatic Gck gene. Moreover, similar levels of GK protein were detected in the livers of both control and transgenic mice, indicating the adipose specificity of the transgene (Fig. 1c). The transgenic line with highest Gck expression, Tg2, was used in this ...
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... and transgenic mice did not produce GK (results not shown), liver from both groups showed a 2.3 kb mRNA transcript (Fig. 1b) resulting from the expression of the endogenous hepatic Gck gene. Moreover, similar levels of GK protein were detected in the livers of both control and transgenic mice, indicating the adipose specificity of the transgene (Fig. 1c). The transgenic line with highest Gck expression, Tg2, was used in this ...
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... of adipocyte cell surface area from epididymal white adipose tissue of control mice (f) (mean=1,024±812 μm 2 ) and heterozygous transgenic mice (g) (mean=940±743 μm 2 ). Results are means ± SEMs from 3,748 adipocytes from control mice and 5,015 adipocytes from transgenic mice [1-3 H]glucose uptake in vivo, was observed in transgenic mice (Fig. 1d). In addition, production of glucose trans- porters 1 and 4 in the adipose tissue of transgenic mice was similar to that in controls (Fig. 1e). This suggests that the specific increase in adipose tissue glucose uptake in transgenic mice was due to the presence of GK in ...
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... mice (g) (mean=940±743 μm 2 ). Results are means ± SEMs from 3,748 adipocytes from control mice and 5,015 adipocytes from transgenic mice [1-3 H]glucose uptake in vivo, was observed in transgenic mice (Fig. 1d). In addition, production of glucose trans- porters 1 and 4 in the adipose tissue of transgenic mice was similar to that in controls (Fig. 1e). This suggests that the specific increase in adipose tissue glucose uptake in transgenic mice was due to the presence of GK in ...
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... order to examine the subcellular localisation of GK, the percentage of co-localisation of GK with mitochondria was determined and compared with that of endogenous hexoki- nase, HKII. To this end, both enzymes were immunodetected in isolated adipocytes from transgenic mice and visualised by laser-scanning confocal microscopy ( Fig. 1f-k). In transgenic mice, similar percentages of HKII and GK co-localised with mitochondria (HKII, 41.05±9.58% vs GK, 39.37±15.88%; n=12), indicating a similar subcellular ...
Citations
... Nowadays, adipose tissue has become a widely analyzed subject of MS-related disorders, mainly diabetes and obesity, often because of its non-physiological excess rather than for adipopenia, which is usually linked to nutrient deprivation and the pathologies of scarcity and cachexia. This interest is slowly changing our perception of WAT from an inert energy dumping site to a tissue (organ) sporting many more active functions in addition to the handling of TAG depots, such as the conversion of 6C to 3C, an active metabolism of 3C units (i.e., producing lactate [810][811][812], glycerol [813,814], or alanine [815]), and a less-known ability to metabolize amino acids [816][817][818]. This high activity in relation to a relatively limited cytosol mass is compounded in WAT by a normally low pO2 [819] and relative mitochondrial scarcity, both in agreement with a marked glycolytic shift of its 6C ↔ 3C metabolism, which allows for its operation with a markedly low consumption of oxygen [820]. ...
This review focuses on the question of metabolic syndrome (MS) being a complex, but essentially monophyletic, galaxy of associated diseases/disorders, or just a syndrome of related but rather independent pathologies. The human nature of MS (its exceptionality in Nature and its close interdependence with human action and evolution) is presented and discussed. The text also describes the close interdependence of its components, with special emphasis on the description of their interrelations (including their syndromic development and recruitment), as well as their consequences upon energy handling and partition. The main theories on MS’s origin and development are presented in relation to hepatic steatosis, type 2 diabetes, and obesity, but encompass most of the MS components described so far. The differential effects of sex and its biological consequences are considered under the light of human social needs and evolution, which are also directly related to MS epidemiology, severity, and relations with senescence. The triggering and maintenance factors of MS are discussed, with especial emphasis on inflammation, a complex process affecting different levels of organization and which is a critical element for MS development. Inflammation is also related to the operation of connective tissue (including the adipose organ) and the widely studied and acknowledged influence of diet. The role of diet composition, including the transcendence of the anaplerotic maintenance of the Krebs cycle from dietary amino acid supply (and its timing), is developed in the context of testosterone and β-estradiol control of the insulin-glycaemia hepatic core system of carbohydrate-triacylglycerol energy handling. The high probability of MS acting as a unique complex biological control system (essentially monophyletic) is presented, together with additional perspectives/considerations on the treatment of this ‘very’ human disease.
... Glycogen content quantification Glycogen content in skeletal muscles, diaphragm, heart, liver, brain and spinal cord samples was measured in perchloric extracts as previously described. Perchloric extracts were adjusted to pH 5 with 5 M K 2 CO 3 and 10% HClO 4 , and glycogen content was determined using the a-amyloglucosidase method [43]. Glucose was measured enzymatically (Glucose HK CP; Horiba Medical ABX Diagnostics) using a Pentra 400 Analyzer (Horiba Medical-ABX) and glycogen content was expressed as mg glucose per gram of tissue biopsies. ...
Objective
Pompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy.
Methods
PD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured.
Results
PD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate.
Conclusions
This gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.
... In line with a decrease of medium-and long-chain AACs, an increase of glucose uptake by AT may be viewed as an index of insulin sensitivity. 61 In other words, an upregulation of the glycolysis pathway in AT appears to be a downstream effect of the insulin-dependent glucose uptake in AT upon glucose transporter 4 (GLUT4) translocation. Both increase of glycolysis and expression of PPAR-g after nerve injury in male mice should facilitate fat storage in adipocytes and energy sparing as suggested by the decrease of energy expenditure found at D7, which was reduced as compared to both baseline heat (Kcal/Kg) and heat emitted at the same time point by female animals. ...
Epidemiological data and research highlight increased neuropathy and chronic pain prevalence among females, spanning metabolic and normometabolic contexts, including murine models. Prior findings demonstrated diverse immune and neuroimmune responses between genders in neuropathic pain (NeP), alongside distinct protein expression in sciatic nerves. This study unveils adipose tissue’s (AT) role in sex-specific NeP responses after peripheral nerve injury. Metabolic assessments, metabolomics, energy expenditure evaluations, AT proteomic analyses, and adipokine mobilization depict distinct AT reactions to nerve damage. Females exhibit altered lipolysis, fatty acid oxidation, heightened energy expenditure, and augmented steroids secretion affecting glucose and insulin metabolism. Conversely, male neuropathy prompts glycolysis, reduced energy expenditure, and lowered unsaturated fatty acid levels. Males’ AT promotes regenerative molecules, oxidative stress defense, and stimulates peroxisome proliferator-activated receptors (PPAR-γ) and adiponectin. This study underscores AT’s pivotal role in regulating gender-specific inflammatory and metabolic responses to nerve injuries, shedding light on female NeP susceptibility determinants.
... To this point, our attempts to express HK2 in WAT of HFD-fed mice were unsuccessful (unpublished data), most likely because HK2 expression is tightly controlled at the post-transcriptional level (Figure 7). Importantly, expression of glucokinase (also known as HK4, hepatic hexokinase) in adipose tissue has been shown to prevent insulin insensitivity in HFD-fed mice (Muñoz et al., 2010). Elucidating the molecular mechanism of HFD-induced translational repression of HK2 may lead to a novel strategy in the treatment of insulin insensitivity and type 2 diabetes. ...
Chronically high blood glucose (hyperglycemia) leads to diabetes and fatty liver disease. Obesity is a major risk factor for hyperglycemia, but the underlying mechanism is unknown. Here, we show that a high-fat diet (HFD) in mice causes early loss of expression of the glycolytic enzyme Hexokinase 2 (HK2) specifically in adipose tissue. Adipose-specific knockout of Hk2 reduced glucose disposal and lipogenesis and enhanced fatty acid release in adipose tissue. In a non-cell-autonomous manner, Hk2 knockout also promoted glucose production in liver. Furthermore, we observed reduced hexokinase activity in adipose tissue of obese and diabetic patients, and identified a loss-of-function mutation in the hk2 gene of naturally hyperglycemic Mexican cavefish. Mechanistically, HFD in mice led to loss of HK2 by inhibiting translation of Hk2 mRNA. Our findings identify adipose HK2 as a critical mediator of local and systemic glucose homeostasis, and suggest that obesity-induced loss of adipose HK2 is an evolutionarily conserved mechanism for the development of selective insulin resistance and thereby hyperglycemia.
... Adipocyte area was determined as previously described. 82 A minimum of seven animals per group were used and at least 250 adipocytes per animal were analyzed. ...
Type 2 diabetes, insulin resistance and obesity are strongly associated and are a major health problem worldwide. Obesity largely results from a sustained imbalance between energy intake and expenditure. Therapeutic approaches targeting metabolic rate may counteract body weight gain and insulin resistance. Bone morphogenic protein 7 (BMP7) has proven to enhance energy expenditure by inducing non-shivering thermogenesis in short-term studies in mice treated with the recombinant protein or adenoviral vectors encoding BMP7. To achieve long-term BMP7 effects, the use of adeno-associated viral (AAV) vectors would provide sustained production of the protein after a single administration. Here, we demonstrated that treatment of high fat diet-fed mice and ob/ob mice with liver-directed AAV-BMP7 vectors enabled a long-lasting increase in circulating levels of this factor. This rise in BMP7 concentration induced browning of white adipose tissue (WAT) and brown adipose tissue activation, that enhanced energy expenditure, and reversed WAT hypertrophy, hepatic steatosis and WAT and liver inflammation, ultimately resulting in normalization of body weight and insulin resistance. This study underscores the potential of AAV-BMP7-mediated gene therapy for the treatment of insulin resistance, type 2 diabetes and obesity.
... This would in turn shift FFA to the liver, where they serve as substrates for lipogenesis and allosterically stimulate gluconeogenesis, ultimately explaining the lower hepatic IS of GCK +/--carriers [10]. Transgenic mice with GCK-overexpression in adipose tissue present with improved adipocyte glucose-uptake and higher whole-body IS [11]. Thus, GCK-deficiency could directly affect adipose tissue IS. ...
Carriers heterozygous for the D124N (c.370, GAC>AAC in exon 4) variant of GCK not only exhibit reduced insulin-secretion, but also impaired adipose insulin sensitivity, which may shift fatty acids towards the liver. This could contribute to increased hepatic lipid-accumulation and alterations of liver energy metabolism resulting in dysglycemia.
ClinicalTrial.gov registration no: NCT01055093
... Tables 3 and 4 compared the glucose and lactate concentrations obtained from our DWE biosensor with previously reported glucose and lactate concentrations in blood. These values of glucose and lactate concentrations from normal mice and obese mice in our results were comparable with those of previously reported concentrations [33][34][35][36][37][38][39][40][41][42][43][44][45]. Increasing ratios were calculated with the following equation: Increasing ratio = Obese−Normal Normal × 100. ...
... Glucose increase is a well-known phenomenon in HFD-induced obesity [35]. However, changes in lactate were not significant during obesity monitoring because a relative short term of diet such as 8~12 weeks usually provided a negligible increase (0.5 mM, equivalent to 10% of increasing ratio) in lactate in the case of HFD-induced obesity [33,36,41]. Thus, our high increase (2.5 mM, equivalent to 61% of increasing ratio) of lactate in HFD-induced obesity would be a turning point to consider lactate as an important factor for obesity monitoring [42]. ...
... The values of glucose and lactate concentrations obtained with the DWE biosensor could differentiate the obese mice group from the normal mice group. These values were comparable with those of previously reported concentrations [33][34][35][36][37][38][39][40]. Using glucose and lactate concentrations in normal and obese cases measured with the biosensor, L/G was calculated and used to obtain a new obesity diagnostic plot. ...
Understanding the levels of glucose (G) and lactate (L) in blood can help us regulate various chronic health conditions such as obesity. In this paper, we introduced an enzyme-based electrochemical biosensor adopting glucose oxidase and lactate oxidase on two working screen-printed carbon electrodes (SPCEs) to sequentially determine glucose and lactate concentrations in a single drop (~30 µL) of whole blood. We developed a diet-induced obesity (DIO) mouse model for 28 weeks and monitored the changes in blood glucose and lactate levels. A linear calibration curve for glucose and lactate concentrations in ranges from 0.5 to 35 mM and 0.5 to 25 mM was obtained with R-values of 0.99 and 0.97, respectively. A drastic increase in blood glucose and a small but significant increase in blood lactate were seen only in prolonged obese cases. The ratio of lactate concentration to glucose concentration (L/G) was calculated as the mouse’s gained weight. The results demonstrated that an L/G value of 0.59 could be used as a criterion to differentiate between normal and obesity conditions. With L/G and weight gain, we constructed a diagnostic plot that could categorize normal and obese health conditions into four different zones. The proposed dual electrode biosensor for glucose and lactate in mouse whole blood showed good stability, selectivity, sensitivity, and efficiency. Thus, we believe that this dual electrode biosensor and the diagnostic plot could be used as a sensitive analytical tool for diagnosing glucose and lactate biomarkers in clinics and for monitoring obesity.
... Adipose tissue provides lactate for hepatic gluconeogenesis during fasting, as well as for hepatic glycogen synthesis after food ingestion [112]. The rate of glucose conversion to lactate increases with adipocyte size, and obese adipocytes may metabolize 50-70% of their glucose to lactate [111,113]. Lactate production is an anaerobic process, and adipose tissue is hypoxic under physiological conditions. Obesity increases adipose tissue hypoxia, favoring lactate production, and so obesity and diabetes are associated with markedly increased lactate production in adipocytes [112]. ...
The safe removal of apoptotic debris by macrophages-often referred to as efferocytosis-is crucial for maintaining tissue integrity and preventing self-immunity or tissue damaging inflammation. Macrophages clear tissues of hazardous materials from dying cells and ultimately adopt a pro-resolving activation state. However, adipocyte apoptosis is an inflammation-generating process, and the removal of apoptotic adipocytes by so-called adipose tissue macrophages triggers a sequence of events that lead to meta-inflammation and obesity-associated metabolic diseases. Signals that allow apoptotic cells to control macrophage immune functions are complex and involve metabolites released by the apoptotic cells and also metabolites produced by the macrophages during the digestion of apoptotic cell contents. This review provides a concise summary of the adipocyte-derived metabolites that potentially control adipose tissue macrophage immune functions and, hence, may induce or alleviate adipose tissue inflammation.
... The ABC peroxidase kit (Pierce) was used for immunodetection, and sections were counterstained in Mayer's hematoxylin. Morphometric analysis of adipocyte size was performed in WAT sections stained with hematoxylin-eosin as previously described [33]. A minimum of four animals per group was used and at least 250 adipocytes per animal were analyzed. ...
Background/objectives
During obesity, hypertrophic enlargement of white adipose tissue (WAT) promotes ectopic lipid deposition and development of insulin resistance. In contrast, WAT hyperplasia is associated with preservation of insulin sensitivity. The complex network of factors that regulates white adipogenesis is not fully understood. Bone morphogenic protein 7 (BMP7) can induce brown adipogenesis, but its role on white adipogenesis remains to be elucidated. Here, we assessed BMP7-mediated effects on white adipogenesis in ob/ob mice.
Methods
BMP7 was overexpressed in either WAT or liver of ob/ob mice using adeno-associated viral (AAV) vectors. Analysis of gene expression, histological and morphometric alterations, and metabolites and hormones concentrations were carried out.
Results
Overexpression of BMP7 in adipocytes of subcutaneous and visceral WAT increased fat mass, the proportion of small-size adipocytes and the expression of adipogenic and mature adipocyte genes, suggesting induction of adipogenesis irrespective of fat depot. These changes were associated with reduced hepatic steatosis and improved insulin sensitivity. In contrast, liver-specific overproduction of BMP7 did not promote WAT hyperplasia despite BMP7 circulating levels were similar to those achieved after genetic engineering of WAT.
Conclusions
This study unravels a new autocrine/paracrine role of BMP7 on white adipogenesis and highlights that BMP7 may modulate WAT plasticity and increase insulin sensitivity.
... Decrease in glucose uptake accounts for most of the insulin insensitivity observed in obesity and diabetic nephropathy. [48,49] As shown in Figure 7A and B, we examined the glucose uptake capacity of HK2 cells after exposure to BP-QDs using a 2-deoxy-2-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) uptake assay. 2-NBDG is a fluorescence-labeled 2-deoxy-glucose analog used to track glucose uptake in cells. ...
Quantum dots (QDs) have numerous potential applications in lighting, engineering, and biomedicine. QDs are mainly excreted through the kidney due to their ultrasmall sizes; thus, the kidneys are target organs of QD toxicity. Here, an organoid screening platform is established and used to study the nephrotoxicity of QDs. Organoids are templated from monodisperse microfluidic Matrigel droplets and found to be homogeneous in both tissue structure and functional recapitulation within a population and suitable for the quantitative screening of toxic doses. Kidney organoids are proved displaying higher sensitivity than 2D‐cultured cell lines. Similar to metal‐containing QDs, black phosphorus (BP)‐QDs are found to have moderate toxicity in the kidney organoids. The nephrotoxicity of BP‐QDs are validated in both mice and human renal tubular epithelial cells. BP‐QDs are also found to cause insulin insensitivity and endoplasmic reticulum (ER) stress in the kidney. Furthermore, ER stress‐related IRE1α signaling is shown to mediate renal toxicity and insulin insensitivity caused by BP‐QDs. In summary, this work demonstrates the use of constructed kidney organoids as 3D high‐throughput screening tools to assess nanosafety and further illuminates the effects and molecular mechanisms of BP‐QD nephrotoxicity. The findings will hopefully enable improvement of the safety of BP‐QD applications.
