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Adipogenesis: lipid accumulation. (A) Oil red O staining (day 14). (B) Lipid accumulation under different medium conditions without TZD treatment. (C) TZD treatment in AM with PPARg antagonist (GW9662). (D) TZD treatment in AM without indomethacin. (E) TZD treatment in AM without indomethacin and with added GW9662. Statistical analysis: * P!0.01, ***P!0.001 indicate significant difference from AM (B) $ P!0.05, $$ P!0.01, $$$ P!0.001 indicate significant difference between cell types within the same media and TZD treatment conditions (B and D).
Source publication
Chronic administration of the insulin-sensitizing drugs, thiazolidinediones (TZDs) results in low bone mineral density and "fatty bones". This is thought to be due, at least in part, to aberrant differentiation of progenitor mesenchymal stem cells (MSCs) away from osteogenesis towards adipogenesis. Objective: This study directly compared the effect...
Citations
... The sub-cultured hADSCs were allowed to reach 2 days' post confluence (passage 4-6) before differentiation was initiated (Supplementary Figure 1). Adipogenic differentiation medium (AM) consisted of DMEM (Life Technologies, California, USA), supplemented with 10 µM insulin (Sigma-Aldrich®, Missouri, USA), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich®, Missouri, USA), 1 µM dexamethasone (Sigma-Aldrich®, Missouri, USA), 56 µM indomethacin (Sigma-Aldrich®, Missouri, USA), and 1 ml FBS (Scientific Group, South Africa) [45]. AM was replaced every second day for a period of 14 days. ...
... The differentiation protocol was repeated six times to create enough adipocyte-conditioned media for subsequent experiments. Following the collection of adipocyteconditioned media (prior to its use), successful differentiation of mature adipocytes was confirmed by quantitation of triglycerides by the Oil Red O staining method as previously described [45]. Mature adipocytes were stained with Oil Red O stain (0.7% Oil Red diluted in 70% (v/v) isopropanol), 30-minute incubation at room temperature (22°C). ...
Adipocytes in the breast tumour microenvironment promotes acquired treatment resistance. We used an in vitro adipocyte-conditioned media approach to investigate the direct paracrine effects of adipocyte secretory factors on MDA-MB-231 breast cancer cells treated with doxorubicin to clarify the underlying treatment resistance mechanisms. Cell-viability assays, and Western blots were performed to determine alterations in apoptotic, proliferation and lipid metabolism protein markers. Free fatty acids (FFA) and inflammatory markers in the collected treatment-conditioned media were also quantified. Adipocyte secretory factors increased the cell-viability of doxorubicin-treated cells (p < 0.0001), which did not correspond to apoptosis or proliferation pathways. Adipocyte secretory factors increased the protein expression of hormone-sensitive lipase (p < 0.05) in doxorubicin-treated cells. Adipocyte secretory factors increased the utilization of leptin (p < 0.05) and MCP-1 (p < 0.01) proteins and possibly inhibited release of linoleic acid by doxorubicin-treated cells (treatment-conditioned media FFA profiles). Adipocyte secretory factors induced doxorubicin treatment resistance, by increasing the utilization of inflammatory mediators and inhibiting the release of FFA by doxorubicin-treated cells. This further promotes inflammation and lipid metabolic reprogramming (lipid storage) in the tumour microenvironment, which breast cancer cells use to evade the toxic effects induced by doxorubicin and confers to acquired treatment resistance.
... Systemically administered ASCs have the potential to enhance wound repair distally from their site of entrapment in the lungs, whereas locally administered ASCs migrate into the wound bed. GlutaMAX, 4.5 g/L glucose) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin (10 000 units/mL)-streptomycin (10 000 μg/mL; pen/strep; Gibco, Life Technologies, NY). [46][47][48] After 24 h, non-adherent cells were removed and the medium changed to complete growth medium (CGM, high glucose DMEM supplemented with 10% FBS and 1% pen/strep). Isolated cells were maintained in CGM (37 C, 5% CO 2 ) until 80% confluent before being trypsinized. ...
There is increasing interest in the use of adipose-derived mesenchymal stromal cells (ASCs) for wound repair. As the fate of administered cells is still poorly defined, we aimed to establish the location, survival, and effect of ASCs when administered either systemically or locally during wound repair under physiological conditions. To determine the behavior of ASCs, a rat model with wounds on the dorsal aspect of the hind paws was used and two treatment modes were assessed: ASCs administered systemically into the tail vein or locally around the wound. ASCs were transduced to express both firefly luciferase (Fluc) and green fluorescent protein to enable tracking by bioluminescence imaging and immunohistological analysis. Systemically administered ASCs were detected in the lungs 3 hours after injection with a decrease in luminescent signal at 48 hours and signal disappearance from 72 hours. No ASCs were detected in the wound. Locally administered ASCs remained strongly detectable for 7 days at the injection site and became distributed within the wound bed as early as 24 hours post injection with a significant increase observed at 72 hours. Systemically administered ASCs were filtered out in the lungs, whereas ASCs administered locally remained and survived not only at the injection site but were also detected within the wound bed. Both treatments led to enhanced wound closure. It appears that systemically administered ASCs have the potential to enhance wound repair distally from their site of entrapment in the lungs whereas locally administered ASCs enhanced wound repair as they became redistributed within the wound bed.
Significance statement The exogenous administration of adipose-derived mesenchymal stromal cells (ASCs) holds promise as a treatment strategy for wound healing by promoting tissue repair and regeneration. However, the best route of administration is still not well defined. Herein, this article describes the biodistribution and survival of systemically vs locally administered ASCs. Interestingly, both routes of administration led to enhanced wound repair as seen by earlier wound closure. Systemically administered ASCs have the potential to enhance wound repair distally from their site of entrapment in the lungs, whereas locally administered ASCs migrate into the wound bed.
... For adipogenic differentiation, ADSCs were exposed to AM in the presence of participant-derived serum for a period of 14 days with media being changed twice weekly. The extent of lipid accumulation was quantified using Oil Red O staining as previously described (van de Vyver et al., 2014;Jacobs et al., 2016). AM consisted of: high-glucose (4.5 g/L) DMEM with ultra-glutamine (BioWhittaker, Lonza, Basel, Switzerland), 1% penicillin/streptomycin (BioWhittaker, Lonza, Basel, Switzerland), 10 mM insulin (SLBD5980, Sigma Life Sciences), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, STBC7632V, Sigma Life Sciences), 1 mM dexamethasone (BCBK1265V, Sigma Life Sciences), 56 mM indomethacin (064K1207, Sigma Life Sciences), and 20% participant-derived serum. ...
It is known that advanced metabolic disorders such as type 2 diabetes compromise the functional and regenerative capacity of endogenous adipose-tissue resident stem cells (ADSCs). It is, however, still unclear at which stage of disease progression ADSCs become compromised and whether systemic factors contribute to their functional decline. It was therefore hypothesized that inflammatory changes in the systemic microenvironment during distinct stages of disease progression negatively affect the functional capacity of ADSCs. A total of forty-seven (n = 47) black African reproductive aged females (32 ± 8 years; mean ± SD) were included in this study and subdivided into: (a) healthy lean (C; body mass index, BMI ≤ 25 kg/m2), (b) healthy overweight/obese (OB; BMI ≥ 25 kg/m2), (c) obese metabolic syndrome (MetS; BMI ≥ 30 kg/m2), and (d) type 2 diabetes mellitus (T2DM; previously diagnosed and on treatment) groups. Participants underwent anthropometric assessments and a DXA scan to determine their body composition and adipose indices. Each persons’ systemic metabolic- (cholesterol, HDL, LDL, triglycerides, and blood glucose) and inflammatory profiles (CRP, SDF1α, TNFα, IL6, IL8, IL10, and IFNy) were also evaluated. Participant-derived serum was then used to treat an ADSC cell line in vitro and its effect on viability (MTT-based assay), proliferation (BrdU), migration (wound healing assay), and osteogenic differentiation assessed. When exposed to serum derived from overweight/obese individuals (with or without metabolic syndrome), both the proliferative and migratory responses of ADSCs were less pronounced than when exposed to healthy control serum. Serum IL6 concentrations were identified as a factor influencing the proliferation of ADSCs, suggesting that long-term disruption to the systemic cytokine balance can potentially disrupt the proliferative responses of ADSCs. Obese participant-derived serum (with and without metabolic syndrome) furthermore resulted in lipid accumulation during osteogenic differentiation. This study, therefore demonstrated that systemic factors in obese individuals, regardless of the presence of metabolic syndrome, can be detrimental to the multifunctional properties of ADSCs.
... Although the effect of the other two ARVs (LVP/r and TDF) assessed in this study was less pronounced, both NRTI-based treatments (Stavudine and TDF) resulted in significant marrow adiposity. This suggests that osteoblast numbers may be depleted by skewing progenitor cell differentiation away from osteoblastogenesis towards adipogenesis: it is well established that osteoblasts and adipocytes are derived from a common mesenchymal progenitor [38] and there is evidence that a number of other pharmaceutical agents (glucocorticoids and thiazolidinediones) cause bone loss by stimulating adipogenesis at the expense of osteoblastogenesis [39,40], thereby depleting the pool of both osteoblast progenitors as well as functional osteoblasts. It is therefore attractive to hypothesize that in addition to the imbalance at tissue level (higher resorption and lower formation parameters), this cellular bone-fat relationship underlies NRTI-induced bone disease. ...
Antiretroviral (ARV) treatment may induce metabolic complications in HIV patients on long-term therapy that can affect bone health. In this study, the effects of the ARVs Stavudine (d4T), Tenofovir (TDF) and Lopinavir/ritonavir (LPV/r) on bone metabolism and lipodystrophy were directly compared in rats to negate the consequences of HIV-associated confounding factors. Healthy 12-14-week-old male Wistar rats (n = 40) were divided into four treatment groups and received an oral animal equivalent dose of either Stavudine (6.2 mg/kg/day), TDF (26.6 mg/kg/day), LPV/r (70.8 mg/kg/day) or water (Control 1.5 mL water/day) for a period of 9 weeks. Whole-body DXA measurements, a biomechanical three-point breaking test and histomorphometric analysis were performed on the femurs and tibias at the end of the treatment period. Stavudine monotherapy was found to be associated with decreased femoral bone mineral density that translated into reduced bone strength, whereas histomorphometric analysis demonstrated that Stavudine induces an imbalance in bone metabolism at tissue level, evident in higher resorption (eroded surfaces, osteoclast surfaces and osteoclast number) and lower formation parameters (osteoblast surfaces and osteoid surfaces). This was less clear in the rats treated with either TDF or LPV/r. Furthermore, both Stavudine and TDF treatment resulted in significant bone marrow adiposity, although no significant redistribution of body fat was noted in the treated rats compared to controls. The data from this study suggest that in the absence of HIV-associated factors, LPV/r is less detrimental to bone metabolism compared to Stavudine and TDF.
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... The causes of the emergence of this phenomenon still remain unclear. The possible reason is that different conditions of microenvironment and nature of progenitor cells affects the differentiation outcome [31][32][33]. These studies also further explain that the role of proinflammatory cytokines, such as IL-17A, in osteogenic differentiation of BMSCs is very complicated and understanding the mechanism of this in osteogenic differentiation of BMSCs is necessary and meaningful. ...
Background
Interleukin-17A (IL-17A) is not only an important modulator of inflammatory reactions, but also affects bone metabolism, which is involved in osteogenic differentiation of stem cells. However, the role and mechanism of IL-17A in osteogenic differentiation of bone mesenchymal stem cells (BMSCs) are not fully understood. In this study, we investigated the role and mechanism of IL-17A in osteogenic differentiation of BMSCs.
Material/Methods
The osteogenic differentiation of BMSCs was induced by osteoblast-induction medium with IL-17A or without IL-17A. The osteogenic differentiation of BMSCs was confirmed by the alkaline phosphatase and alizarin red staining. The lentiviral plasmid was used to construct the sFRP1-shRNA expression vector. The associated osteogenic differentiation marks (RUNX2, ALP, OPN), Wnt signaling pathway inhibitor (sFRP1), and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR and Western blot method.
Results
The results showed that the addition of IL-17A inhibited osteogenic differentiation of BMSCs. IL-17A induced up-regulated expression of sFRP1 and down-regulated expression of Wnt3 and Wnt6 in BMSCs. In addition, sFRP1-shRNA abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs and induced up-regulated expression of Wnt3 and Wnt6 in the Wnt signaling pathway in BMSCs.
Conclusions
Our findings show that IL-17A inhibits osteogenic differentiation of bone mesenchymal stem cells via the Wnt signaling pathway.
... To determine whether any of these cells correspond to adipocyte progenitors, we exposed cultures to adipogenic conditions. Because the activation of peroxisome proliferator-activated receptor gamma (PPARγ) by ligands such as thiazolidenediones can induce lipid accumulation in cells independently of adipogenic conversion 16 , we used a minimal adipogenic cocktail of 3-isobutyl-1-methylxanthine, dexamethasone and insulin (MDI). After ~6 d, we observed a loss of continuity between the cells that form the capillary structure and the lipid droplets that are in cells within the capillaries (Fig. 1c). ...
Uncoupling protein 1 (UCP1) is highly expressed in brown adipose tissue, where it generates heat by uncoupling electron transport from ATP production. UCP1 is also found outside classical brown adipose tissue depots, in adipocytes that are termed 'brite' (brown-in-white) or 'beige'. In humans, the presence of brite or beige (brite/beige) adipocytes is correlated with a lean, metabolically healthy phenotype, but whether a causal relationship exists is not clear. Here we report that human brite/beige adipocyte progenitors proliferate in response to pro-angiogenic factors, in association with expanding capillary networks. Adipocytes formed from these progenitors transform in response to adenylate cyclase activation from being UCP1 negative to being UCP1 positive, which is a defining feature of the beige/brite phenotype, while displaying uncoupled respiration. When implanted into normal chow-fed, or into high-fat diet (HFD)-fed, glucose-intolerant NOD-scid IL2rg(null) (NSG) mice, brite/beige adipocytes activated in vitro enhance systemic glucose tolerance. These adipocytes express neuroendocrine and secreted factors, including the pro-protein convertase PCSK1, which is strongly associated with human obesity. Pro-angiogenic conditions therefore drive the proliferation of human beige/brite adipocyte progenitors, and activated beige/brite adipocytes can affect systemic glucose homeostasis, potentially through a neuroendocrine mechanism.
... For example, clinical and experimental agonists of the peroxisome proliferator activated receptor (PPAR ), a fatty acid-activated member of the nuclear *Address correspondence to this author at the Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Luzhou Medical College, No.2, Jiang Yang Nan Road, Luzhou 646000, P.R. China; Tel: 86-0830-3165521; Fax: 86-0830-3165520; E-mail: drxiaojg@sohu.com hormone receptor superfamily, have been shown to reduce osteogenesis [6] or increase adipogenesis [7]. This phenomenon indicates that repression of adipogenic factors like PPAR may favor the differentiation of mesenchymal stem cells (MSCs) into the osteoblast lineage rather than the adipocyte lineage [8]. ...
Mesenchymal stem cells (MSCs) arise from a variety of tissues, including bone marrow and adipose tissue and, accordingly, have the potential to differentiate into multiple cell types, including osteoblasts and adipocytes. Research on MSCs to date has demonstrated that a large number of transcription factors and ectocytic or intrastitial signaling pathways regulate adipogenic and osteogenic differentiation. A theoretical inverse relationship exists in adipogenic and osteogenic lineage commitment and differentiation, such that signaling pathways induce adipogenesis at the expense of osteogenesis and vice versa. For example, peroxisome proliferator-activated receptor γ (PPARγ), which belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors, is known to function as a master transcriptional regulator of adipocyte differentiation, and inhibit osteoblast differentiation. Moreover, recent studies have demonstrated that inducers of osteogenic differentiation, such as bone morphogenetic protein (BMP) and Wnt, inhibit the function of PPARγ transactivation during MSC differentiation towards adipocytes through a variety of mechanisms. To illustrate this, the canonical Wnt/β-catenin pathway represses expression of PPARγ mRNA, whereas the noncanonical wnt pathway activates histone methyltransferases that inhibit PPARγ transactivation via histone H3 lysine 9 (H3K9) methylation of its target genes. The role of microRNAs (miRNAs) in adipogenesis and osteoblastogenesis is garnering increased attention, and studies in this area have shed light on the integration of miRNAs with Wnt signaling and transcription factors such as Runx2 and PPARγ. This review summarizes our current understanding of the mechanistic basis of these signaling pathways, and indicates future clinical applications for stem cell-based cell transplantation and regenerative therapy.
... The resulting metabolic improvement is also contributed to by increased adiponectin secretion; the net result being improved insulin sensitivity and prevention/improvement of type 2 diabetes (106). Of course the weight gain has negative musculoskeletal and cosmetic implications and the enhanced adipocyte differentiation may be at the expense of reduced osteoblast differentiation in bone with consequent reduction of bone density (107). ...
Adipocyte differentiation and its impact on restriction or expansion of particular adipose tissue depots have physiological and pathophysiological significance in view of the different functions of these depots. Brown or “beige” fat [brown adipose tissue (BAT)] expansion can enhance thermogenesis, lipid oxidation, insulin sensitivity, and glucose tolerance; conversely expanded visceral fat [visceral white adipose tissue (VAT)] is associated with insulin resistance, low grade inflammation, dyslipidemia, and cardiometabolic risk. The largest depot, subcutaneous white fat [subcutaneous white adipose tissue (SAT)], has important beneficial characteristics including storage of lipid “out of harms way” and secretion of adipokines, especially leptin and adiponectin, with positive metabolic effects including lipid oxidation, energy utilization, enhanced insulin action, and an anti-inflammatory role. The absence of these functions in lipodystrophies leads to major metabolic disturbances. An ability to expand white adipose tissue adipocyte differentiation would seem an important defense mechanism against the detrimental effects of energy excess and limit harmful accumulation of lipid in “ectopic” sites, such as liver and muscle. Adipocyte differentiation involves a transcriptional cascade with PPARγ being most important in SAT but less so in VAT, with increased angiogenesis also critical. The transcription factor, Islet1, is fairly specific to VAT and in vitro inhibits adipocyte differentiation. The physiological importance of Islet1 requires further study. Basic control of differentiation is similar in BAT but important differences include the effect of PGC-1α on mitochondrial biosynthesis and upregulation of UCP1; also PRDM16 plays a pivotal role in expression of the BAT phenotype. Modulation of the capacity or function of these different adipose tissue depots, by altering adipocyte differentiation or other means, holds promise for interventions that can be helpful in human disease, particularly cardiometabolic disorders associated with the world wide explosion of obesity.
The advancement of autologous mesenchymal stem cell (MSC) therapy for the treatment of non‐healing diabetic wounds is hampered by endogenous MSC dysfunction and limited viability of cells post transplantation into the pathological wound environment. The development of effective strategies to restore the functional capabilities of these impaired MSCs prior to transplantation may be a key to their ultimate success as wound repair mediators. The current study therefore investigated whether antioxidant preconditioning [7.5 mM N‐acetylcysteine (NAC) + 0.6 mM ascorbic 2‐phosphate (AAP)] could restore the growth rate, migration ability and viability of impaired MSCs and whether this restored state is maintained in the presence of diabetic wound fluid (DWF). Healthy control (source: wild type, C57BL/6J mice) (n = 12) and impaired/ diabetic MSCs (source: obese pre‐diabetic, B6.Cg‐Lepob/J mice) (n = 12) were isolated from the bone marrow of mice. Treatment groups post isolation were as follow: 1) No treatment (baseline phenotype): MSCs expanded in standard growth media (SGM) (±8 days) and only exposed to growth media. 2) DWF (baseline response): MSCs expanded in SGM (±8 days) followed by exposure to DWF (24 h, 48 h, 96 h). 3) Antioxidant preconditioning (preconditioned phenotype): MSCs expanded in the presence of NAC/AAP (±8 days). 4) Antioxidant preconditioning + DWF (preconditioned response): MSCs expanded in the presence of NAC/AAP (±8 days) followed by exposure to DWF (24 h, 48 h, 96 h). The results demonstrated that expansion of MSCs (both healthy control and impaired diabetic) in the presence of combined NAC/AAP treatment improved ex vivo MSC viability and protected MSCs in the presence of DWF. Despite improved viability, AAP/NAC could however not rescue the reduced proliferation and migration capacity of impaired diabetic MSCs. The protective effect of NAC/AAP preconditioning against the toxicity of DWF could however be a potential strategy to improve cell number post transplantation. This article is protected by copyright. All rights reserved.
In this study, the pro-osteogenic effects of EPA-containing phosphatidylcholine (EPA-PC), a natural agonist of peroxisome proliferator-activated receptor (PPARγ), were first investigated both in vitro and in vivo. In vitro experiments showed that EPA-PC promoted mesenchymal stem cells (MSCs) differentiation into osteoblasts over adipocytes, which was contrary to the role of rosiglitazone, a well-known full PPARγ agonist. qRT-PCR and western blotting indicated that EPA-PC significantly increased the mRNA and protein levels of osteogenesis-related genes, such as Runx2, ALP and COL-I. Although EPA-PC promoted mRNA expression of PPARγ, it didn’t affect the protein level of PPARγ. Furthermore, EPA-PC promoted ERK-mediated phosphorylation of PPARγ at serine 112, which was positively associated with osteogenesis. In vivo experiments indicated that EPA-PC increased bone formation rate and enhanced bone biomechanical properties of adolescent mice. In summary, our study showed that EPA-PC promoted osteogenesis by up-regulating Runx2 expression and ERK-regulated serine 112 phosphorylation of PPARγ, which enhanced our understanding about the action mode of PPARγ agonists.
