IL-4 activates the futile triacylglyceride cycle for glucose utilization in white adipocytes.

The development of cardiometabolic complications during obesity is strongly associated with chronic latent inflammation in hypertrophied adipose tissue (AT). IL-4 is an anti-inflammatory cytokine, playing a protective role against insulin resistance, glucose intolerance and weight gain. The positive effects of IL-4 are associated not only with the activation of anti-inflammatory immune cells in AT, but also with the modulation of adipocyte metabolism. IL-4 is known to activate lipolysis and glucose uptake in adipocytes, but the precise regulatory mechanisms and physiological significance of these processes remain unclear. In this study, we detail IL-4 effects on glucose and triacylglycerides (TAGs) metabolism and propose mechanisms of IL-4 metabolic action in adipocytes. We have shown that IL-4 activates glucose oxidation, lipid droplet (LD) fragmentation, lipolysis and thermogenesis in mature 3T3-L1 adipocytes. We found that lipolysis was not accompanied by fatty acids (FAs) release from adipocytes, suggesting FA re-esterification. Moreover, glucose oxidation and thermogenesis stimulation depended on adipocyte triglyceride lipase (ATGL) activity, but not the uncoupling protein (UCP1) expression. Based on these data, IL-4 may activate the futile TAG-FA cycle in adipocytes, which enhances the oxidative activity of cells and heat production. Thus, the positive effect of IL-4 on systemic metabolism can be the result of the activation of non-canonical thermogenic mechanism in AT, increasing TAG turnover and utilization of excessive glucose.

Visceral mesenchymal stem cells from type 2 diabetes donors activate triglycerides synthesis in healthy adipocytes via metabolites exchange and cytokines secretion.

BACKGROUND: In recent years, there has been an increase in the prevalence of obesity and type 2 diabetes mellitus (T2DM). Development of visceral instead of subcutaneous adipose tissue is pathogenic and increases the risk of metabolic abnormalities. We hypothesize that visceral adipocytes and stromal cells are able to deteriorate other fat depots metabolism via secretory mechanisms. METHODS: We study the regulatory role of visceral adipose-derived stem cells (vADSC) from donors with obesity and T2DM or normal glucose tolerance (NGT) on healthy subcutaneous ADSC (sADSC) in the Transwell system. Lipid droplets formation during adipogenesis was assessed by confocal microscopy. Cell metabolism was evaluated by 14C-glucose incorporation analysis and western blotting. vADSC secretome was assessed by Milliplex assay. RESULTS: We showed that both NGT and T2DM vADSC had mesenchymal phenotype, but expression of CD29 was enhanced, whereas expressions of CD90, CD140b and IGF1R were suppressed in both NGT and T2DM vADSC. Co-differentiation with T2DM vADSC increased lipid droplet size and stimulated accumulation of fatty acids in adipocytes from healthy sADSC. In mature adipocytes T2DM vADSC stimulated triglyceride formation, whereas NGT vADSC activated oxidative metabolism. Secretome of NGT vADSC was pro-inflammatory and pro-angiogenic in comparison with T2DM vADSC. CONCLUSIONS: The present study has demonstrated the critical role of secretory interactions between visceral and subcutaneous fat depots both in the level of progenitor and mature cells. Mechanisms of these interactions are related to direct exchange of metabolites and cytokines secretion.

Transplantation of Adipose-Tissue-Engineered Constructs with CRISPR-Mediated UCP1 Activation.

Thermogenic adipocytes have potential utility for the development of approaches to treat type 2 diabetes and obesity-associated diseases. Although several reports have proved the positive effect of beige and brown adipocyte transplantation in obese mice, translation to human cell therapy needs improvement. Here, we describe the application of CRISPR activation (CRISPRa) technology for generating safe and efficient adipose-tissue-engineered constructs with enhanced mitochondrial uncoupling protein 1 (UCP1) expression. We designed the CRISPRa system for the activation of UCP1 gene expression. CRISPRa-UCP1 was delivered into mature adipocytes by a baculovirus vector. Modified adipocytes were transplanted in C57BL/6 mice, followed by analysis of grafts, inflammation and systemic glucose metabolism. Staining of grafts on day 8 after transplantation shows them to contain UCP1-positive adipocytes. Following transplantation, adipocytes remain in grafts and exhibit expression of PGC1α transcription factor and hormone sensitive lipase (HSL). Transplantation of CRISPRa-UCP1-modified adipocytes does not influence glucose metabolism or inflammation in recipient mice. We show the utility and safety of baculovirus vectors for CRISPRa-based thermogenic gene activation. Our findings suggest a means of improving existing cell therapy approaches using baculovirus vectors and CRISPRa for modification and transplantation of non-immunogenic adipocytes.

Type 2 Diabetes Mellitus Facilitates Shift of Adipose-Derived Stem Cells Ex Vivo Differentiation toward Osteogenesis among Patients with Obesity.

OBJECTIVE: Sedentary behavior with overnutrition provokes the development of obesity, insulin resistance, and type 2 diabetes mellitus (T2DM). The main progenitor cells of adipose tissue are adipose-derived stem cells (ADSCs) which can change differentiation, metabolic, and secretory phenotypes under obesity conditions. The purpose of this study was to evaluate ADSC osteogenesis activity among patients with obesity in normal glucose tolerance (NGT) and T2DM conditions. METHODS: In the study, ADSCs from donors with obesity were used. After clinical characterization, all patients underwent bariatric surgery and ADSCs were isolated from subcutaneous fat biopsies. ADSCs were subjected to osteogenic differentiation, stained with Alizarin Red S, and harvested for real-time PCR and Western blotting. Cell senescence was evaluated with a ß-galactosidase-activity-based assay. RESULTS: Our results demonstrated the significantly increased calcification of ADSC on day 28 of osteogenesis in the T2DM group. These data were confirmed by the statistically significant enhancement of RUNX2 gene expression, which is a master regulator of osteogenesis. Protein expression analysis showed the increased expression of syndecan 1 and collagen I before and during osteogenesis, respectively. Moreover, T2DM ADSCs demonstrated an increased level of cellular senescence. CONCLUSION: We suggest that T2DM-associated cellular senescence can cause ADSC differentiation to shift toward osteogenesis, the impaired formation of new fat depots in adipose tissue, and the development of insulin resistance. The balance between ADSC adipo- and osteogenesis commitment is crucial for the determination of the metabolic fate of patients and their adipose tissue.

Regulation of Glucose Transport in Adipocytes by Interleukin-4.

Metabolic abnormalities such as obesity, insulin resistance, and type 2 diabetes mellitus are known to be associated with adipose tissue inflammation and impaired secretion of cytokines. Anti-inflammatory cytokine interleukin-4 (IL-4) was found to promote insulin sensitivity, glucose tolerance, and reduce lipid accumulation in vivo through multiple mechanisms, including direct regulation of lipolysis in adipocytes. However, little is known about its role in adipocyte glucose metabolism. This study reveals that IL-4 upregulates glucose uptake in adipocytes without additional activation of the insulin-dependent IRS1 (insulin receptor substrate 1)-Akt (protein kinase B) pathway. Moreover, the main transcription factor STAT6 (signal transducer and activator of transcription 6), regulated by IL-4, was not involved in adipocyte glucose uptake. The proteomic results showed that IL-4 upregulates expression of proteins involved in mitochondrial biogenesis, renewal, and glucose oxidation. Our study provides a new hypothesis, explaining protective effects of IL-4 against metabolic abnormalities through activation of adipocytes glucose utilization and maintenance of mitochondrial function under metabolic overload conditions.

NDRG1 Activity in Fat Depots Is Associated With Type 2 Diabetes and Impaired Incretin Profile in Patients With Morbid Obesity.

Objective: We aimed to investigate insulin-, mTOR- and SGK1-dependent signaling basal states in morbidly obese patients' fat. We analyzed the correlation between the signaling activity, carbohydrate metabolism, and incretin profiles of patients. Methods: The omental and subcutaneous fat was obtained in patients with obesity. The omental study included 16 patients with normal glucose tolerance (NGT) and 17 patients with type 2 diabetes mellitus (T2DM); the subcutaneous study included 9 NGT patients and 12 T2DM patients. Insulin resistance was evaluated using the hyperinsulinemic euglycemic clamp test and HOMA-IR index. The oral glucose tolerance test (OGTT) for NGT patients and mixed meal tolerance test (MMTT) for T2DM patients were performed. The levels of incretins (GLP-1, GIP, oxyntomodulin) and glucagon were measured during the tests. Signaling was analyzed by Western blotting in adipose tissue biopsies. Results: We have shown equal levels of basal phosphorylation of insulin- and mTOR-dependent signaling in omental fat depot in NGT and T2DM obese patients. Nevertheless, pNDRG1-T346 was decreased in omental fat of T2DM patients. Correlation analysis has shown an inverse correlation of pNDRG1-T346 in omental fat and diabetic phenotype (HbA1c, impaired incretin profile (AUC GLP-1, glucagon)). Moreover, pNDRG1-T346 in subcutaneous fat correlated with impaired incretin levels among obese patients (inverse correlation with AUC glucagon and AUC GIP). Conclusions: According to results of the present study, we hypothesize that phosphorylation of pNDRG1-T346 can be related to impairment in incretin hormone processing. pNDRG1-T346 in adipose tissue may serve as a marker of diabetes-associated impairments of the systemic incretin profile and insulin sensitivity.

Direct Effect of the Synthetic Analogue of Glucagon-Like Peptide Type 1, Liraglutide, on Mature Adipocytes Is Realized through Adenylate-Cyclase-Dependent Enhancing of Insulin Sensitivity.

Incretin hormones analogues, including glucagon-like peptide type 1 (GLP-1), exhibit complex glucose-lowering, anorexigenic, and cardioprotective properties. Mechanisms of action of GLP-1 and its analogues are well known for pancreatic ß-cells, hepatocytes, and other tissues. Nevertheless, local effects of GLP-1 and its analogues in adipose tissue remain unclear. In the present work effects of the GLP-1 synthetic analogue, liraglutide, on adipogenesis and insulin sensitivity of the 3T3-L1 adipocytes were examined. Enhancement of insulin sensitivity of mature adipocytes by the GLP-1 synthetic analogue liraglutide mediated by adenylate cyclase was demonstrated. The obtained results imply existence of the positive direct insulin-sensitizing effect of liraglutide on mature adipocytes.

AICAR Protects Vascular Endothelial Cells from Oxidative Injury Induced by the Long-Term Palmitate Excess.

Hyperlipidemia manifested by high blood levels of free fatty acids (FFA) and lipoprotein triglycerides is critical for the progression of type 2 diabetes (T2D) and its cardiovascular complications via vascular endothelial dysfunction. However, attempts to assess high FFA effects in endothelial culture often result in early cell apoptosis that poorly recapitulates a much slower pace of vascular deterioration in vivo and does not provide for the longer-term studies of endothelial lipotoxicity in vitro. Here, we report that palmitate (PA), a typical FFA, does not impair, by itself, endothelial barrier and insulin signaling in human umbilical vein endothelial cells (HUVEC), but increases NO release, reactive oxygen species (ROS) generation, and protein labeling by malondialdehyde (MDA) hallmarking oxidative stress and increased lipid peroxidation. This PA-induced stress eventually resulted in the loss of cell viability coincident with loss of insulin signaling. Supplementation with 5-aminoimidazole-4-carboxamide-riboside (AICAR) increased endothelial AMP-activated protein kinase (AMPK) activity, supported insulin signaling, and prevented the PA-induced increases in NO, ROS, and MDA, thus allowing to maintain HUVEC viability and barrier, and providing the means to study the long-term effects of high FFA levels in endothelial cultures. An upgraded cell-based model reproduces FFA-induced insulin resistance by demonstrating decreased NO production by vascular endothelium.

Grain-Based Dietary Background Impairs Restoration of Blood Flow and Skeletal Muscle During Hindlimb Ischemia in Comparison With Low-Fat and High-Fat Diets.

Background: Among vascular pathologies associated with obesity, peripheral artery disease (PAD) occupies the important position. In clinical practice, nutritional interventions are recommended for patients with PAD. In this work, we investigated how the different dietary backgrounds affect the regeneration rate of ischemic hindlimb in mice. Methods: Male C57BL/6J mice were housed on three types of diet: low-fat (LFD), high-fat (HFD), and grain-based diet (GBD) for 13 weeks. Metabolic parameters including FBG level, ITT, and GTT were evaluated. The blood flow was assessed by laser Doppler scanning on 7, 14, and 21 days after hindlimb ischemia. Necrotic area of m.tibialis, macrophage infiltration, and angiogenesis/arteriogenesis were evaluated by histology. Glucose uptake in recovered skeletal muscle was analyzed using [3H]-2-deoxyglucose, and GLUT1 and GLUT4 expression were assessed by Western blotting. Results: In our work, we developed three experimental groups with different metabolic parameters: LFD with normal glucose metabolism, GBD with mild hyperglycemia, and HFD with impaired glucose tolerance. GBD-fed mice had a tendency to increase necrosis of m. tibialis and significantly higher macrophage infiltration than LFD and HFD groups. Moreover, GBD-fed mice had a trend to decreased blood flow recovery and significantly impaired arteriogenesis. Recovered skeletal muscle of GBD-fed mice had lower glucose uptake and decreased level of GLUT4 expression. Conclusion: Thus, we conclude that dietary background and metabolic status determine the rate of post-ischemic regeneration including angiogenesis, skeletal muscle recovery and metabolic activity. The most effective regeneration is supported by LFD, while the lowest rate of regeneration occurs on GBD.

The Effects of Glucagon-Like Peptide Type 1 (GLP-1) and its Analogues in Adipose Tissue: Is there a way to Thermogenesis?

Obesity is a global problem and the most common metabolic disorder leading to many associated diseases, such as arterial hypertension, ischemic heart disease, type 2 diabetes, certain types of cancer, impaired lipid and uric acid metabolism. The prevalence of obesity has risen globally in the past four decades in both children and adults, and it accounts for the rapid increase in the prevalence of diabetes. Currently, the study of thermogenic tissues, brown and beige adipose tissues, is of extreme value from the point of view of therapeutic potential for obesity and its associated diseases. An analogue of the glucagon-like peptide-1 (GLP-1) liraglutide, used in the treatment of type 2 diabetes, has been proven to have a positive effect on weight loss through appetite suppression. However, this mechanism of weight loss is not the only one involved. This article discusses the main molecular and cellular mechanisms of adipogenesis, as well as the effect of GLP-1 and its analogues, in particular liraglutide on this process through various transcription factors, signaling pathways, and hormones, including brown and beige adipose tissue. Also, the twincretins have had a positive effect on insulin resistance and fat beiging activation. The results of numerous studies have helped us to better understand the peripheral mechanisms of lipid metabolism regulation, and have demonstrated the effectiveness of GLP-1 analogues for the treatment of diabetes and obesity.

Decreased UCP-1 expression in beige adipocytes from adipose-derived stem cells of type 2 diabetes patients associates with mitochondrial ROS accumulation during obesity.

OBJECTIVE: Adipose derived stem cells (ADSC) are defective in metabolic disorders in various functionalities and properties including differentiation, multipotent state, metabolism and immunomodulation. However, the role of ADSC beiging potential in promoting of type 2 diabetes mellitus (T2DM) development remains unclear. Here we uncover association between potential of subcutaneous ADSC to beige differentiation and T2DM in patients with obesity. METHODS: ADSC were isolated from subcutaneous adipose tissue of patients with long morbid obesity (BMI > 35 kg/m2) and normal glucose tolerance (NGT) or T2DM. ADSC were differentiated into white or beige adipocytes and levels of thermogenic markers, lipid metabolism and electron transport chain (ETC) genes was analyzed by Western blotting and RT-PCR. ROS production was estimated by fluorescent microscopy. RESULTS: We have shown decreased UCP-1 expression in beige adipocytes from T2DM patients. Nevertheless, signal and expression activities of lipolysis were equal in NGT and T2DM beige adipocytes. Expression analysis of ETC genes also has not shown any statistically significant differences. Interestingly, we revealed increased mitochondrial ROS production in T2DM beige adipocytes during beige differentiation. CONCLUSIONS: In summary, compromised UCP1 expression in beige adipocytes of T2DM patients may cause increase of mitochondrial ROS. Elevated oxidative level is liable to act as damaging mechanism leading to insulin resistance or, alternatively, serve as compensatory mechanism for thermogenesis activation.

Role of MicroRNAs in the Regulation of Subcutaneous White Adipose Tissue in Individuals With Obesity and Without Type 2 Diabetes.

Obesity is a high-risk factor for such comorbidities as cardiovascular disease, several types of cancer, and type 2 diabetes; however not all individuals with obesity have such complications. Approximately 20% of individuals with obesity are metabolically healthy. This study focused on differences between obese individuals with and without type 2 diabetes (T2D+ and T2D-, respectively) on the transcriptome level. Subjects included were 35 T2D- patients with obesity and 35 T2D+ patients with obesity with the same body mass index (BMI). The study was based on the transcription analysis of mRNA and microRNAs (miRs) by RNAseq. In the first step, we performed RNAseq of miRs, in the second step, we analyzed only those mRNA, which appeared targets for significant miRs from the first step. All RNAseq results were validated by qPCR. There were seven miRs differently expressed with adjusted p-value <0.1, which were confirmed by qPCR. Five among them: miR-204-5p, miR125b-5p, miR-125a-5p, miR320a, miR-99b-were upregulated in T2D+ patients with obesity, while only two miRs, miR-23b-3p, and miR197-3p, were increased in T2D- patients with obesity. These seven miRs target two groups of genes: matrix metalloproteinases and TGFß signal pathway genes. According to the results of transcriptome analysis, the main difference between T2D+ and T2D- patients with obesity was in adipogenesis and fibrosis regulation by matrix metalloproteinases and SMAD4-RUNX2 signal cascade. Based on the data about transcription profiles of both groups, we suggested that the process of fibrosis in T2D+ patients with obesity is more pronounced than in T2D- patients with obesity.

Low AS160 and high SGK basal phosphorylation associates with impaired incretin profile and type 2 diabetes in adipose tissue of obese patients.

OBJECTIVE: To compare basal insulin and mTOR signaling in subcutaneous fat of obese T2DM vs. obese subjects with normal glucose tolerance (NGT), and correlate it with clinical parameters of carbohydrate metabolism and incretin secretion profiles. METHODS: Recruited were 22 patients with long (>10 years) and morbid (BMI > 35 kg/m2) obesity, 12 of which had NGT and 10 had T2DM. Hyperinsulinemic-euglycemic clamp test and HOMA-IR were used to measure insulin resistance. Blood samples taken at 0, 30 and 120 min of food load test were used to assess incretin profile, insulin and glucose levels. Amount of total and visceral fat was determined by bioelectrical impedance analysis. Subcutaneous fat biopsies were obtained during bariatric surgery for all patients and analyzed by western blots. RESULTS: As assessed by western blots of insulin receptor substrate (IRS), Akt, Raptor, Rictor, mTOR and S6K1, the basal insulin signaling and mTORC activities were comparable in NGT and T2DM groups, whereas phosphorylation of AS160 was significantly lower and that of serum and glucocorticoid-induced kinase (SGK) was significantly higher in T2DM group. Various correlations were found between the degree of insulin resistance and amount of visceral fat, changes in incretin profile, glucose metabolic parameters and phosphorylation level of AS160, incretin secretion profile and phosphorylated levels of AS160 or SGK1. CONCLUSION: Altered phosphorylation of AS160 and SGK1 is associated with obese T2DM phenotype.