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ObjectivesMetabolic syndrome is a cluster of conditions—increased blood pressure, a high blood sugar level, abdominal fat accumulation and abnormal cholesterol levels—that occur together, increasing the risk of heart disease, stroke and type 2 diabetes mellitus (T2DM). Mitochondria play a central role in the energy metabolism. Mitochondrial dysfunction contributes to the pathogenesis of metabolic disorders. Abnormality in mitochondrial function is a leading cause for development of insulin resistance and is promoted, at least partly, through lipid accumulation in ectopic tissues, including liver and skeletal muscle. Many growth and transcription factors involved in the regulation of mitochondrial gene expression also contribute to the pathophysiology of obesity, insulin resistance and type-2 diabetes. The role of mitochondria as functional organelles, and the signaling molecules produced by them are critical in the regulation of cellular energy metabolism and homeostasis. MOTS-c (mitochondrial open reading frame of the 12S rRNA type-c) encoding a 16-amino-acid peptide is a recently identified peptide. The skeletal muscle appears to be the main target organ for MOTS-c and its cellular actions inhibit the folate cycle and its tethered de novo purine biosynthesis, leading to AMPK activation. MOTS-c increases glucose utilization, promotes insulin sensitivity and metabolic homeostasis through activation of AMPK dependent mechanisms in skeletal muscle. In rodent studies MOTS-c has been demonstrated to protect against insulin resistance induced by diet and ageing. However the role of MOTS-c in adipocytes is largely unknown. In this current study we aimed to study the effect of MOTS-c on adipocyte development and metabolism in cultured mouse adipocytes.Methods3T3L1 cells were grown in DMEM media containing 10% FBS. Cells were stimulated with various concentrations of MOTS-c (100 nM to 50 μM) to study the effect of MOTS-c on cell viability. For differentiation pre-adipocytes were grown in differentiation induction medium containing dexamethasone, IBMX and insulin for two days. The media was replaced with medium containing insulin only for the next six days. About 60–70% differentiation was achieved by day 8. For treatment group pre-adipocytes were pre-treated with MOTS-c for 24 hours, and then differentiated into adipocytes for eight days. Quantitative real-time PCR was used to investigate the effect of MOTS-c on adipogenic genes expression:, Peroxisome Proliferator-Activated Receptor Gamma (PPARg), AP2, adiponectin and leptin in mouse 3T3-L1 pre-adipocytes.ResultMOTS-c treatment did not induce any significant changes in cell viability at 24h following treatment. Pretreatment with MOTS-c prior to induction of differentiation down regulated expression of adipogenic markers including leptin, adiponectin, PPARg and PPARg-coactivator 1-alpha (PGC1a) mRNA levels, compared to differentiated adipocyte controls.ConclusionOur primary result indicates that MOTS-c inhibits adipogenesis through down-regulation of multiple genes involved in development of adipocytes, and this inhibition could contribute to the reduced abdominal adiposity. Future studies are currently in progress to further assess the effects of MOTS-c on adipocytes lipolysis, glucose uptake, glucose transporter (GLUT) family and other insulin-signaling pathway protein including GSK3B, IGF1R, IGF2R, and PIK3K are needed to understand the role of MOTS-c in mouse 3T3-L1 adipocytes.

SummaryInsulin resistance manifest in skeletal muscle, liver and adipocytes. Insulin stimulates muscle glucose uptake, suppresses the rate of hepatic glucose production (HGP) and restrains lipolysis. In insulin resistant individuals, insulin-stimulated glucose uptake is markedly reduced in skeletal muscle, impaired insulin-mediated suppression of hepatic glucose production (HGP) and inhibition of lipolysis. It is evident from human and animal studies that diabetes alters miRNAs expression in metabolically important organs (adipose, liver and skeletal muscle).micRNA are small non coding RNA molecules. However, they play critical role in the regulation of gene expression via post-transcriptional mechanisms. They are small in size (21?23 nucleotides) and recent studies have demonstrated that they play important role in many human complex diseases including diabetes. The ability of miRNA that simultaneously regulate many target genes makes them attractive candidates for regulating insulin production/secretion and action. mRNA are secreted to the circulation and are taken up from the circulation by several tissues, making them an attractive mechanism for inter-tissue signaling molecules. Thus, if micro RNA play role in the pathogenesis of insulin resistance or in communicating between insulin resistant tissues and the beta cell to regulate the level of beta cell function in concert with the prevailing level of insulin resistance, we anticipate a change in the circulatory profile of micro RNA in insulin resistant individuals compared to insulin sensitive individuals. Such a distinct circulatory profile of micro RNA in insulin sensitive versus insulin resistant individuals could provide a signature for the identification of insulin resistant individuals. Moreover, differentially expressed micro RNA molecules in insulin resistant individuals could serve signaling molecules which convey messages between insulin resistant tissues, i.e. liver, skeletal muscle and adipocytes and the beta cell. Insulin sensitivity is commonly assessed to predict the risk and evaluate the management of type 2 diabetes in clinical and research settings. Insulin sensitivity can be measured using a variety of techniques that are commonly employed in diabetes research and care. Among them, euglycemic hyperinsulemic clamp is the gold-standard method to quantify the sensitivity to insulin.The aim of the present study is to determine the profile of miRNA in the plasma in insulin sensitive Arab individuals and compare the result to that in insulin resistant Arab individuals.specific aims are;1. To discover the circulatory profile of microRNA in insulin sensitive and insulin resistant Arab individuals by using next generation sequencing technology.2. To identify novel microRNA/targets which are differentially expressed in insulin resistant individuals by real time Taq-Man PCR.3. To explore the effects changes of these miRNAs in insulin resistance diabetes β-Cell Biology. The experiments range from relevant tissue and cell cultures systems to human physiology. Design and methods: The circulating miRNA profile was assessed in a pilot study of 20 healthy volunteers whom glucose tolerance status was determined by the OGTT, and their insulin sensitivity was quantitated by the gold standard method the euglycemic hyperinsulemic clamp. Based on Glucose Infusion rate (GIR: mg/kg/min), before and after a 6-h hyperinsulinemeic euglycemic clamp, we selected two groups: 10 with high levels of GIR (insulin-sensitive group) and 10 with Very low levels of GIR (insulin resistant group).The serum samples was collected from the two groups for miRNA extraction and subsequent sequencing using the True-seq Illumina protocol. A small RNA library was generated from samples using the Illumina Truseq™ Small. The purified cDNA library was used for cluster generation on Illumina's Cluster Station and then sequenced on Illumina GAIIx following vendor's instruction for running the instrument. Raw sequencing reads (40 nts) were obtained using Illumina's Sequencing Control Studio software version 2.8 (SCS v2.8) following real-time sequencing image analysis and base-calling by Illumina's Real-Time Analysis version 1.8.70 (RTA v1.8.70). Result: Sequencing of the small circulating RNA isolated from insulin-resistant and insulin-sensitive healthy samples produced 69,559,764 raw reads. A total of 41,697,300 mappable reads was obtained after sequence filtering. Using a 2-fold expression difference as a cutoff, 33 miRNAs showed significally differential expression between insulin sensitive and insulin-resistant group. Among which, 10 were up-regulated and 22 were down-regulated. This RNA-seq discovery study shows that the most changes in miRNAs expression is in accordance with previous studies performed to explore circulating miRNAs in human insulin resistance or T2DM. Indeed, among the 54 selected miRNAs, we have identified the same changes in mir-126 expression that has described by Zampetaki and colleagues. This group reported that mir-126 is a unique plasma miRNA signature in 800 patients with T2D and observed that the reduced levels of mir-126 showed the strongest association with T2D. From the 33 miRNAs, a relative number of them are much documented to be associated with insulin resistance and type 2 diabetes. Nevertheless, some identified miRNAs are potential novel candidates and need to be deeply exploring during the event of the pathogenesis of T2D. Conclusion: This study provides a comparative profiling of circulating miRNAs in insulin-resistant versus insulin–sensitive subjects and the identification of specific miRNAs in circulation that changes are associated with insulin action. To our knowledge, this is the first study to investigate insulin resistance in Arab population using euglycemic hyperinsulemic clamp the gold-standard method to assess insulin sensitivity and aim to identify changes in circulating miRNAs expression associated to insulin action and signaling. The study also provides evidence that Insulin Resistance in human is related to changes in multiples microRNAs. Functional Validation studies of the differentially expressed miRNAs and relevant target and signaling pathways are ongoing.

Diabetes is highly prevalent in Qatar and about 1/3 of patients are not aware of their diseases. Screening for undiagnosed diabetes is essential for effective management and prevention of diabetes and its complications. The effectiveness and cost analysis of several diabetes screening programs have been the subject of intensive investigation. Point of care (POC) measurement of capillary blood glucose (CBG) is very simple and can be applied widely, however, random CBG values are hard to interpret unless a high cut-off point is utilized, such as >200 mg/dL. A high cut off point would miss a large number of diabetics and pre-diabetics. POC CBG measurement of fasting and post-prandial values is difficult to organize in a community sitting. In Qatar, and all Muslim countries, most people observe fasting. If POC CBG is applied between 12:00 and sunset, then all values are at least 9 h after the morning meal and are considered fasting values. Similarly, POC CBG measured after evening prayer (Taraweeh) is equivalent to a late post-prandial value (~3 h after evening meal). Here we piloted a study to evaluate the usefulness of POC CBG to screen for diabetes in Ramadan after Juma'a prayer (~9 h of fast) and after Taraweeh prayer (3-4 h after evening meal - Iftar) in the large Mosq in Doha. We also administered a questionnaire to learn about known diabetes, co-morbidities and family history. A total of 2177 individuals were screened in 2 days, 75% were men and 25% were women representative of 40 different nationalities with most from Egypt (743, 38%), India (348, 23%) and Qatar (146, 7%). The distribution of CBG values were not statistically different between afternoon and evening values and were pooled for this analysis. 57% of all values were normal (

Insulin resistance and beta cell dysfunction are core defects in type 2 diabetes (T2DM). Impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) are intermediate states in the transition in glucose tolerance from normal to T2DM, both of which are associated with increased conversion rate to T2DM. Understanding the metabolic abnormalities that lead to the development of IFG and IGT will help develop strategies to prevent the development of T2DM. in the present study, we have examined the metabolic abnormalities responsible for the development of IFG and IGT in Arab individuals. 43 subjects with NGT (n=17), isolated IFG (N=17) and isolated IGT (n=9) received 75-gram OGTT and plasma glucose, insulin and C-peptide concentrations were measured at baseline and every 15 minutes for 3 hours after the glucose drink. Insulin secretion was measured with the incremental area under the plasma insulin and C-peptide concentration curves and insulin sensitivity was measured with the Matsuda Index. Beta cell function was measured with the Disposition Index as the product of insulin secretion and insulin sensitivity indices. IGT subjects manifested severe insulin resistance compared to IFG and NGT subjects. The Matsuda Index was 3.7±0.4, 7.2±1.0 and 8.4±1.1, respectively (p

Effect of non-nutritive sweetener stevioside on regulation of adipogenesis and oxidative stress in adipocytes Jayakumar Jerobin, Manjunath Ramanjaneya, Ilham Bettahi, Siveen Kodappully Sivaraman, Ramzi M Mohammad, Monica Skarulis Young, Abdul Badi Abou Samra Interim Translational Research Institute, Qatar Metabolic Research Institute & Department of Medicine, Hamad Medical Corporation, Doha, Qatar. Introduction The metabolic syndrome which includes obesity, diabetes, polycystic ovarian syndrome and non-alcoholic fatty liver disease is a major health risk in Middle Eastern countries including Qatar. The metabolic syndrome is worsened by unhealthy diet and sedentary lifestyle. Both nutritive and nonnutritive sweeteners (NNS) are consumed by people worldwide, and the consumption of sweeteners above the recommended level contributes to metabolic syndrome. In adipocytes, artificial NNS such as saccharin and acesulfame potassium promotes adipogenesis and suppresses adipose tissue lipolysis. The consumption of artificial NNS saccharin modulates the gut microbiota and promotes glucose intolerance. Currently the artificial NNS are replaced with natural NNS stevioside in foods and beverages since it has low caloric value. Studies have shown that stevioside possess glucose uptake properties which may have a potential role in type 2 diabetes mellitus treatment. This proposed study mainly focuses on the effect of NNS stevioside on regulation of adipogenesis and oxidative stress in matured adipocytes. Methods The 3T3L1 preadipocytes cells were cultured in DMEM medium, and the preadipocyte cells were differentiated in to mature adipocytes by the addition of IBMX, Insulin and Dexamethasone on 0th day. It was replaced with maintenance medium containing insulin on 2,4 and 6th days of differentiation. The stevioside at different dosage (10, 100 and 200 μM) were treated on 0, 2, 4 and 6th days of differentiation in matured adipocytes. Cell viability was analyzed using MTS solution. Oil red¬ O staining was carried out to study the accumulation of lipids in the undifferentiated and matured adipocyte cells. The different oxidative stress markers such as Reactive oxygen species (ROS) and reduced Glutathione were measured using flow cytometry. Western blot and gene expression studies were carried out to study the adipogenesis and insulin sensitivity markers. Results The stevioside was found to be non-toxic to both preadipocyte and adipocytes. On eighth day of differentiation, a significant increase in the accumulation of lipids was observed in the differentiated adipocytes compared to the undifferentiated preadipocytes as analyzed by Oil Red O. The generation of oxidative stress was higher in adipocytes compared to the undifferentiated adipocytes. This was confirmed by increase in the level of reactive oxygen species radicals and reduced glutathione in untreated adipocytes. Treatment with stevioside resulted in inhibition of oxidative stress markers in a dose dependent manner. The protein expression level of early adipogenesis marker such as PPAR-γ and C/EBP-alpha was significantly decreased in stevioside treated adipocytes. Adiponectin, an adipokine found to have association with insulin sensitivity was significantly increased in stevioside treated adipocytes. The protein and gene expression levels of GLUT4 was also found to be significantly increased in stevioside treated matured adipocytes. Conclusion Stevioside reduces oxidative stress by modulating the level of free radicals in maturing adipocytes. Adiponectin an insulin sensitizer which was found to be lower in obese individuals was significantly upregulated in stevioside treated adipocytes. Similarly, the increase in the expression of insulin regulated glucose transporter GLUT-4 in adipocytes indicates that stevioside may possess insulin sensitivity properties in matured adipocytes.