Augmentation of RBP4/STRA6 signaling leads to insulin resistance and inflammation and the plausible therapeutic role of vildagliptin and metformin.

A role of Retinol Binding Protein-4 (RBP4) in insulin resistance is widely studied. However, there is paucity of information on its receptor viz., Stimulated by Retinoic Acid-6 (STRA6) with insulin resistance. To address this, we investigated the regulation of RBP4/STRA6 expression in 3T3-L1 adipocytes exposed to glucolipotoxicity (GLT) and in visceral adipose tissue (VAT) from high fat diet (HFD) fed insulin-resistant rats. 3T3-L1 adipocytes were subjected to GLT and other experimental maneuvers with and without vildagliptin or metformin. Real-time PCR and western-blot experiments were performed to analyze RBP4, STRA6, PPARγ gene and protein expression. Adipored staining and glucose uptake assay were performed to evaluate lipid and glucose metabolism. Oral glucose tolerance test (OGTT) and Insulin Tolerance Test (ITT) were performed to determine the extent of insulin resistance in HFD fed male Wistar rats. Total serum RBP4 was measured by quantitative sandwich enzyme-linked immunosorbent assay kit. Adipocytes under GLT exhibited significantly increased RBP4/STRA6 expressions and decreased insulin sensitivity/glucose uptake. Vildagliptin and metformin not only restored the above but also decreased the expression of IL-6, NFκB, SOCS-3 along with lipid accumulation. Furthermore, HFD fed rats exhibited significantly increased serum levels of RBP4 along with VAT expression of RBP4, STRA6, PPARγ, IL-6. These molecules were significantly altered by the vildagliptin/ metformin treatment. We conclude that RBP4/STRA6 pathway is primarily involved in mediating inflammation and insulin resistance in adipocytes and visceral adipose tissues under glucolipotoxicity and in insulin resistant rats.

Linking a role of lncRNAs (long non-coding RNAs) with insulin resistance, accelerated senescence, and inflammation in patients with type 2 diabetes.

BACKGROUND: Studying epigenetics is expected to provide precious information on how environmental factors contribute to type 2 diabetes mellitus (T2DM) at the genomic level. With the progress of the whole-genome resequencing efforts, it is now known that 75-90% of the human genome was transcribed to generate a series of long non-coding RNAs (lncRNAs). While lncRNAs are gaining widespread attention as potential and robust biomarkers in the genesis as well as progression of several disease states, their clinical relevance and regulatory mechanisms are yet to be explored in the field of metabolic disorders including diabetes. Despite the fact that Asian Indians are highly insulin resistant and more prone to develop T2DM and associated vascular complications, there is virtually lack of data on the role of lncRNAs in the clinical diabetes setting. Therefore, we sought to evaluate a panel of lncRNAs and senescence-inflammation signatures in peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes (T2DM; n = 30) compared to individuals with normal glucose tolerance (NGT; n = 32). RESULTS: Compared to control subjects, expression levels of lncRNAs in PBMCs from type 2 diabetes patients showed significantly (p < 0.05) increased levels of HOTAIR, MEG3, LET, MALAT1, MIAT, CDKN2BAS1/ANRIL, XIST, PANDA, GAS5, Linc-p21, ENST00000550337.1, PLUTO, and NBR2. In contrast, lncRNA expression patterns of THRIL and SALRNA1 were significantly (p < 0.05) decreased in patients with T2DM compared to control subjects. At the transcriptional level, senescence markers (p53, p21, p16, and ß-galactosidase), proinflammatory markers (TNF-α, IL6, MCP1, and IL1-ß), and epigenetic signature of histone deacetylase-3 (HDAC3) were significantly (p < 0.05) elevated in patients with type 2 diabetes compared to control subjects. Interestingly, mRNA expression of Sirt1 and telomere length were significantly (p < 0.05) decreased in patients with type 2 diabetes compared to control subjects. Majority of the altered lncRNAs were positively correlated with poor glycemic control, insulin resistance, transcriptional markers of senescence, inflammation, and HDAC3 and negatively correlated with telomere length. Logistic regression analysis revealed a significant association of altered lncRNA signatures with T2DM, but this association was lost after adjusting for insulin resistance (HOMA-IR) and senescence markers. CONCLUSION: Our study provides a clinically relevant evidence for the association of altered lncRNAs with poor glycemic control, insulin resistance, accelerated cellular senescence, and inflammation.

Improvement in glucose tolerance and insulin sensitivity by probiotic strains of Indian gut origin in high-fat diet-fed C57BL/6J mice.

PURPOSE: Diabetes and obesity are characterized by glucose intolerance, fat deposition, inflammation, and dyslipidemia. Recent reports postulated that distinct gut microbiota alterations were observed in obese/diabetic subjects and modulating gut microbiota beneficially through specific probiotics could be a potential therapeutic option for type 2 diabetes/obesity. Therefore, we attempted to study the efficacy of probiotics of Indian gut origin (Lactobacillus plantarum MTCC5690 and Lactobacillus fermentum MTCC5689) along with a positive control, Lactobacillus rhamnosus (LGG) on glucose/lipid homeostasis in high-fat-diet-induced diabetic animal model. METHODS: C57BL/6J male mice were divided into seven groups (n = 6 per group) comprising feeding on: (1) Normal Pellet Diet (NPD), (2) High-Fat Diet (HFD), (3) HFD with LGG, (4) HFD with MTCC5690, (5) HFD with MTCC5689, (6) HFD with metformin, and 7) HFD with vildagliptin for a period of 6 months. Biochemical markers, glucose tolerance, insulin resistance, and GLP-1 and LPS levels were assessed by standard protocols. Gut integrity was measured by intestinal permeability test. Transcriptional levels of tight junction proteins (TJPs) were probed in small intestinal tissues while inflammatory signals and other pathway specific genes were profiled in liver, visceral adipose tissue, and skeletal muscle. RESULTS: Mice fed with HFD became insulin resistant, glucose intolerant, hyperglycemic, and dyslipidemic. Diabetic mice were characterized to exhibit decreased levels of GLP-1, increased gut permeability, increased circulatory levels of LPS, decrease in the gene expression patterns of intestinal tight junction markers (occludin and ZO-1), and increased proinflammatory gene markers (TNFα and IL6) in visceral fat along with decreased mRNA expression of FIAF and adiponectin. Diabetic mice also exhibited increased mRNA expression of ER stress markers in skeletal muscle. In addition, liver from HFD-fed diabetic mice showed increased gene expressions of proinflammation, lipogenesis, and gluconeogenesis. Probiotic interventions (most prominently the MTCC5689) resisted insulin resistance and development of diabetes in mice under HFD feeding and beneficially modulated all the biochemical and molecular alterations in a mechanistic way in several tissues. The metabolic benefits offered by the probiotics were also more or less similar to that of standard drugs such as metformin and vildagliptin. CONCLUSION: Native probiotic strains MTCC 5690 and MTCC 5689 appear to have potential against insulin resistance and type 2 diabetes with mechanistic, multiple tissue-specific mode of actions.

Augmentation of histone deacetylase 3 (HDAC3) epigenetic signature at the interface of proinflammation and insulin resistance in patients with type 2 diabetes.

BACKGROUND: A role of proinflammation has been implicated in the pathogenesis of diabetes, but the up-stream regulatory signals and molecular signatures are poorly understood. While histone modifications such as changes in histone deacetylase (HDAC) are emerging as novel epigenetic biomarkers, there is lack of studies to demonstrate their clinical relevance in diabetes. Therefore, we investigated the extent of HDAC machinery and inflammatory signals in peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes mellitus (T2DM) compared to control subjects. RESULTS: HDAC3 activity was significantly (p < 0.05) increased in patients with T2DM compared to control subjects. While subtypes of HDACs were differentially expressed at their transcriptional levels in patients with type 2 diabetes, the most prominent observation is the significantly (p < 0.05) elevated messenger RNA (mRNA) levels of HDAC3. Expression levels of Sirt1 which represents the class III HDAC were decreased significantly in T2DM (p < 0.05). Plasma levels of both TNF-α and IL-6 were significantly higher (p < 0.05) in patients with type 2 diabetes compared to control subjects. Among the proinflammatory mediators, the mRNA expression of MCP-1, IL1-ß, NFκB, TLR2, and TLR4 were also significantly (p < 0.05) increased in T2DM. Transcriptional levels of DBC1 (deleted in breast cancer 1, which is a negative regulator of HDAC3) were seen significantly reduced in PBMCs from T2DM. Interestingly, HDAC3 activity/HDAC3 mRNA levels positively correlated to proinflammation, poor glycemic control, and insulin resistance. CONCLUSIONS: Striking message from this study is that while looking for anti-inflammatory strategies and drugs with novel mode of action for T2DM, discovering and designing specific inhibitors targeted to HDAC3 appears promising.

Circulating MiRNAs of 'Asian Indian Phenotype' Identified in Subjects with Impaired Glucose Tolerance and Patients with Type 2 Diabetes.

Several omics technologies are underway worldwide with an aim to unravel the pathophysiology of a complex phenotype such as type 2 diabetes mellitus (T2DM). While recent studies imply a clinically relevant and potential biomarker role of circulatory miRNAs in the etiology of T2DM, there is lack of data on this aspect in Indians--an ethnic population characterized to represent 'Asian Indian phenotype' known to be more prone to develop T2DM and cardiovascular disease than Europeans. We performed global serum miRNA profiling and the validation of candidate miRNAs by qRT-PCR in a cohort of subjects comprised of normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and patients with T2DM. Our study revealed 4 differentially expressed miRNAs (miR-128, miR-130b-3p, miR-374a-5p, miR-423-5p) in subjects with IGT and T2DM patients compared to control subjects. They were positively or negatively correlated to cholesterol levels, HbA1C, HOMA-IR and fasting insulin. Interestingly, circulating level of miR-128 and miR-130b-3p were also altered in serum of diet-induced diabetic mice compared to control animals. Among the altered circulating miRNAs, miR-128 had never been described in previous studies/populations and appeared to be a 'New Lead' in Indians. It was positively correlated with cholesterol both in prediabetic subjects and in diet-induced diabetic mice, suggesting that its increased level might be associated with the development of dyslipedemia associated with T2DM. Our findings imply directionality towards biomarker potential of miRNAs in the prevention/diagnosis/treatment outcomes of diabetes.

Accelerated fat cell aging links oxidative stress and insulin resistance in adipocytes.

Telomere shortening is emerging as a biological indicator of accelerated aging and aging-related diseases including type 2 diabetes. While telomere length measurements were largely done in white blood cells, there is lack of studies on telomere length in relation to oxidative stress in target tissues affected in diabetes. Therefore, the aim of this study is to induct oxidative stress in adipocytes and to test whether these adipocytes exhibit shortened telomeres, senescence and functional impairment. 3T3-L1 adipocytes were subjected to oxidative stress and senescence induction by a variety of means for 2 weeks (exogenous application of H2O2, glucose oxidase, asymmetric dimethylarginine (ADMA) and glucose oscillations). Cells were probed for reactive oxygen species generation (ROS), DNA damage, mRNA and protein expression of senescent and pro-inflammatory markers, telomere length and glucose uptake. Compared to untreated cells, both ROS generation and DNA damage were significantly higher in cells subjected to oxidative stress and senescence. Adipocytes subjected to oxidative stress also showed shortened telomeres and increased mRNA and protein expression of p53, p21, TNF alpha and IL-6. Senescent cells were also characterized by decreased levels of adiponectin and impaired glucose uptake. Briefly, adipocytes under oxidative stress exhibited increased ROS generation, DNA damage, shortened telomeres and switched to senescent/pro-inflammatory phenotype with impaired glucose uptake.

Convergence of adipocyte hypertrophy, telomere shortening and hypoadiponectinemia in obese subjects and in patients with type 2 diabetes.

OBJECTIVE: Although telomere shortening has been linked with type 2 diabetes and most variables of adiposity, a shortcoming of such studies is the measurement of telomere length in leukocytes. Therefore, we tested the association among adipocyte cell size, telomere length (both subcutaneous and visceral adipose tissue) and systemic levels of adiponectin in obese subjects and patients with type 2 diabetes compared to control subjects. METHODS: Human subcutaneous and visceral adipose tissues were obtained from the subjects who have undergone bariatric surgery or other abdominal surgeries. The study groups comprised: i) control subjects, ii) type 2 diabetes patients, iii) obese subjects without diabetes and iv) obese subjects with diabetes. Adipocyte cell size was measured by histological staining. Adiponectin levels were measured by ELISA. Telomere length was determined by Real-time PCR and lipid peroxidation was assessed by fluorimetry. RESULTS: Compared to control subjects, adipocyte size (both subcutaneous and visceral) from obese, diabetic and obese-diabetic subjects was significantly larger [p<0.001]. Individuals with adipose hypertrophy also exhibited shortened telomeres and hypoadiponectinemia. Pearson correlation analysis revealed that both visceral and subcutaneous fat cell size showed a positive correlation with FBS, HbA1c, HOMA-IR, LDL, total cholesterol, triglycerides and negatively correlated with HDL and adiponectin. Regression analysis revealed that the association between shortened telomeres and hypoadiponectinemia was lost when adjusted for adipocyte cell size. CONCLUSION: Adipocyte hypertrophy appears to be strongly associated with shortened telomeres, hypoadiponectinemia and poor glycemic and lipid control. Interestingly, these molecular alterations seen in lean diabetics reflect a state of 'metabolic obesity'.

Accelerated aging as evidenced by increased telomere shortening and mitochondrial DNA depletion in patients with type 2 diabetes.

Although shortened telomeres were shown associated with several risk factors of diabetes, there is lack of data on their relationship with mitochondrial dysfunction. Therefore, we compared the relationship between telomere length and mitochondrial DNA (mtDNA) content in patients with type 2 diabetes mellitus (T2DM; n = 145) and in subjects with normal glucose tolerance (NGT; n = 145). Subjects were randomly recruited from the Chennai Urban Rural Epidemiology Study. mtDNA content and telomere length were assessed by Real-Time PCR. Malonodialdehyde, a marker of lipid peroxidation was measured by thiobarbituric acid reactive substances (TBARS) using fluorescence methodology. Adiponectin levels were measured by radioimmunoassay. Oxidative stress as determined by lipid peroxidation (TBARS) was significantly (p < 0.001) higher in patients with T2DM compared to NGT subjects. In contrast, the mean telomere length, adiponectin and mtDNA content were significantly (p < 0.001) lower in patients with T2DM compared to NGT subjects. Telomere length was positively correlated with adiponectin, HDL, mtDNA content and good glycemic/lipid control and negatively correlated with adiposity and insulin resistance. On regression analysis, shortened telomeres showed significant association with T2DM even after adjusting for waist circumference, insulin resistance, triglyceride, HDL, adiponectin, mtDNA & TBARS. mtDNA depletion showed significant association with T2DM after adjusting for waist circumference and adiponectin but lost its significance when further adjusted for telomere length, TBARS and insulin resistance. Our study emphasizes the clustering of accelerated aging features viz., shortened telomeres, decreased mtDNA content, hypoadiponectinemia, low HDL, and increased oxidative stress in Asian Indian type 2 diabetes patients.