Session 04 : Diabetes and Heart

Genetics of Type 2 Diabetes in India: Association to causality

posted Jan 26, 2017, 3:11 AM by sourav ghosh   [ updated Jan 26, 2017, 3:12 AM ]

Dwaipayan Bharadwaj
1 Systems Genomics Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, 2 CSIR-Institute of Genomics & Integrative Biology, New Delhi, India

The rapid increase in the prevalence of type 2 diabetes in the 21st century has earned it’s name as a global epidemic, affecting over 347 million people worldwide. Several risk factors for T2D including age, sex, obesity, low physical activity, smoking and diet have already been identified. Exploring the hereditary components of T2D has been highlighted by several research groups till date, majorly involving genome wide association studies to find the causal route to this disease. Our lab at CSIR-IGIB has carried out a type 2 diabetes GWAS in Indian population, taking about 12,535 human samples under consideration. We found five novel signals at the 2q21 position of human genome within TMEM163 gene that encodes for a synaptic vesicular membrane protein.Further experimentations are being done in order to find out the biological role of TMEM163 with respect to T2D etiology.

Tackling the Diabetes epidemic in India

posted Jan 26, 2017, 1:13 AM by sourav ghosh   [ updated Jan 26, 2017, 3:10 AM ]

Nihal Thomas
Department of Endocrinology, Diabetes and Metabolism,Christian Medical College, Vellore, India

The questions which concern the pathogenesis of diabetes mellitus in the Indian Subcontinent are many, owing to the high prevalence of the disease, the comparatively lower age of onset and a relatively low body mass index amongst Asian Indians.
The foetal origins of diseases- propounded by Barker and colleagues may in part be responsible. This maybe a critical factor, considering that the prevalence of low birth weight (LBW) in India is amongst the highest in the world and approaches a figure of 15 to 20% of all live births in some parts of the country. The fetal insulin hypothesis (FIH) proposed by Hattersley states that genetic variants associated with insulin resistance may lead to impaired insulin – mediated growth prenatally, leading to low birth weight and adverse metabolic outcomes in adulthood. Thus, low birth weight is part of a syndrome that may be associated with in utero insulin resistance which in turn may manifest with diabetes and hypertension at the time of adulthood.
We successfully enrolled 60 LBW and 60 NBW males, born in and still living in rural environments around the city of Vellore. We confirmed that low birth weight adult males who had fully completed puberty were shorter in height and lighter in body weight when compared to their NBW counterparts. Moreover, the LBW individuals had a significantly lower lean body mass when compared to their NBW counterparts. This difference was present in the total body lean mass and extended to include the upper and lower limbs of these subjects and was associated with a lower bone mineral content in the LBW group.
Interestingly, 8% of the LBW individuals had impaired glucose tolerance, which was not present in the NBW individuals. However, this was not reflected in the 'm' values (measure of insulin sensitivity) - that were obtained from the hyperinsulinemic euglycaemic clamp studies done on these individuals, who were all incidentally associated with a low median BMI (19.5kg/m2) in both the LBW and NBW groups. The LBW subjects had a marginally significant higher supine resting diastolic blood pressure level when compared to NBW subjects, when adjusted for height and weight. Measurement of High Density Lipoprotein Levels (HDL levels) showed values which were low, which bore similarity to what is seen in the Southern Indian population, moreover there was no difference in the measurements in HDL between the LBW and NBW subjects.
There was no difference in resting energy expenditure when measured by indirect calorimetry between the LBW and the NBW group of subjects, nor was there any difference in Glucose or Fat oxidation between the groups. When the data of all 120 subjects were taken as a whole, and the m-value correlated with the insulin resistance indices including HOMA-IR, QUICKI, Fasting Insulin levels, Glucose insulin ratio, McCauley’s index and Matsuda Index: the strongest correlation was obtained in the association between the m-value and the McCauley’s index and the Matsuda index. In fact more recent studies by our group have shown that complex calculations apart, that the fasting glucose-insulin ratio correlates well with the hyperinsulinemic euglycaemic clamp studies and is superior to HOMA-IR, QUICKI and McCauley's index.
The Dietary intake of protein was significantly lower in quantum in the LBW subjects when compared to the normal birth weight subjects at the time of recruitment into the study. This was associated with a lower proportion of energy being extracted from the protein intact per se. The mothers and the fathers of the LBW subjects were shorter than the NBW subjects, suggesting the possibility of an intergenerational influence on birth weight (though the difference did not achieve statistical significance.
The LBW group had agreater Fat mass/ fat free mass reduction when compared to their pre-exercise baseline status and a significant decline in FM/body weight followinga 45 minute exercise intervention for a 6 week period on a bicycle. The NBW subjects had a small but significant increase in fat percentage. Moreover, there was a statistically significant reduction in fasting plasma insulin levels in the LBW group, while the reduction was not statistically significant in the NBW group. Regarding reductions in insulin secretion, HOMA-IS a change were significant in the LBW and NBW groups, while reductions in HOMA-IR was only significant in the NBW group.
The subjects had spectroscopic assessment of micro-quantitities of fat in the liver and the muscle, through NMR spectroscopy. It was found that there was a negligible quantum of ectopic fat storage in the liver in particular, and to some extent in the muscle, which is unlike what is seen in Caucasian subjects at a similar age. There was no difference in ectopic fat storage between the NBW and LBW subjects. Measurements of insulin resistance (HOMA-IR) , did not have any relationship with hepatic, intramyocellular or extramyocellular fat content In factthe only independent predictor of intra-myocellular and extramyocellular fat content was with the total body fat percentage. Our study has shown a significant elevation in diastolic blood pressure, alterations in systolic pressure have been shown in LBW subjects in a number of cohorts, and occur in both gender groups.
MODY genetic testing to identify mutations in a comprehensive panel of ten MODY genes was carried out in 80 subjects of Asian-Indian origin with young onset diabetes A novel multiplex polymerase chain reaction (PCR) based target enrichment was established, followed by Next Generation Sequencing (NGS) on the Ion Torrent Personal Genome Machine (PGM). All the mutations and rare variants were confirmed by Sanger sequencing.We identified mutations in 11 (19%) of the 56 clinically diagnosed MODY subjects and seven of these mutations were novel.The identified mutations include p.H241Q, p.E59Q, c.-162G>A 5’ UTR in NEUROD1, p.V169I co-segregating with c.493-4G>A and c.493-20C>T, p.E271Kin HNF4A, p.A501S in HNF1A, p.E440Xin GCK, p.V177M in PDX1, p.L92F in HNF1B and p.R31L in PAX4 genes.. Interestingly two patients with NEUROD1 mutation were also positive for the p.E224K mutation in PDX1 gene. These patients with co-existing NEUROD1-PDX1 mutations showed a marked reduction in glucose induced insulin secretion. None of the 24 subjects who had not met the clinical criteria of MODY were positive for mutations. To the best of our knowledge, this is the first report of PDX1, HNF1B, NEUROD1 and PAX4 mutations from India. Multiplex PCR coupled with NGS provides a rapid, cost-effective and accurate method for comprehensive parallelized genetic testing of MODY. When compared to earlier reports, we have identified a higher frequency and a novel digenic mutation pattern involving NEUROD1 and PDX1 genes.
The work performed above clearly established next generation sequencing as he modality of choice for looking to the genetic profile of young onset diabetes, MODY, syndromic disorders and neonatal diabetes and at present CMC, Vellore as a single library preparation capable of handling 30 genes simultaneously in a cost effective manner.
In summary, there are a number of factors which are responsible for that shift in the phenotype of towards the left in India with regards to the leanness of body habitus as well as the age of these patients. More work is required to be done to identify these pathogenic factors at a cellular level to establish the reasons for this propensity.

Roles of altered hepatic miRNA signatures during diabetes

posted Jan 26, 2017, 1:10 AM by sourav ghosh   [ updated Jan 26, 2017, 3:08 AM ]

Malabika Datta
Functional Genomics Unit, CSIR-Institute of Genomics & Integrative Biology, Delhi, India.

microRNAs (miRNAs) are a class of small (~ 22 nucleotides) non-coding RNA species that act by regulating the expression of their target genes by either mRNA degradation or translational repression. miRNAs are now widely believed to participate in several physiological cellular pathways and aberrant signatures are associated with the onset and progression of diverse diseases. We have been studying altered miRNA patterns in the livers of diabetic mice and the mechanisms by which these alterations precipitate into the observed metabolic deregulations during diabetes. Specifically, levels of miR-107 and miR-22-3p are abnormally increased in the livers of diabetic db/db mice. We show that miR-22-3p binds to the 3' UTR of TCF7 and downregulates it and this miR-mediated regulation of TCF7 increases the expression of enzymes of the gluconeogenic pathway in HepG2 cells. siRNA-mediated knockdown of TCF7 in HepG2 cells also cause similar upregulation of gluconeogenic genes. In vivo silencing of miR-22-3p by antagomiR administration lowered random as well as fasting glucose levels in diabetic mice. miR-22-3p antagonism improved glucose tolerance and insulin sensitivity. Importantly, the hepatic Tcf7 levels were restored along with reduced hepatic glucose output which was also reflected by decreased expression of gluconeogenic genes. Our results support a critical role for miR-22-3p and its target, Tcf7 in the pathogenesis of diabetes by up-regulating gluconeogenesis. miR-107, on the other hand, regulates fatty acid synthase (FASN) and reduces its protein levels. Overexpression of miR-107 led to significant accumulation of malonyl CoA, accompanied by ER stress induction. This was followed by increased triglyceride formation and lipid accumulation in the presence of miR-107. These indicate that miR-107 inhibits FASN levels and this interaction promotes ER stress induction and malonyl CoA and lipid accumulation in HepG2 cells and primary hepatocytes.miR-107 also significantly inhibited the mitochondrial β-oxidation enzyme, HADHA, in HepG2 cells and this was rescued by ER stress inhibition. Over-expression of miR-107 in mice increased random blood glucose levels, impaired oral glucose tolerance and promoted hepatic lipid accumulation. These suggest that altered miRNA levels regulate metabolic processes that are reflected in terms of hyperglycemia and increase hepatic lipid accumulation during diabetes.

Metabolic syndrome, systemic autoimmunity and type I interferons:a ménage à trois

posted Jan 26, 2017, 1:08 AM by sourav ghosh   [ updated Jan 26, 2017, 3:06 AM ]

Dipyaman Ganguly
Senior Scientist & Ramanujan Fellow, IICB-Translational, Research Unit of Excellence, CSIR-Indian Institute of Chemical Biology, Kolkata India

Plasmacytoid dendritic cells (pDC) are major producers of type I interferons (IFN-I) in response to recognition of pathogen-derived nucleic acid molecules by endosomal toll-like receptors (TLRs). Involvement of pDC-derived IFN-I in a number of autoimmune diseases as well is established. Recognition of self-nucleic acids leads to induction of IFN-I from pDCs and drives innate initiation of inflammation in Systemic Lupus Erythematosus (SLE), Psoriasis and a number of other autoimmune contexts. Thus induction of pDC-derived IFN-I is a shared initiator event in discreet clinical contexts, leading to a model where these clinical contexts are grouped together as a syndrome of inadvertent pDC activation. We propose the name ‘Plasmacytoidopathy’.
Interestingly, we also identified role of pDC-derived IFN-I in obesity associated metaflammation and insulin resistance. We found chemerin, an adipose-derived chemokine, recruits pDCs in obese adipose tissue and initiates the metaflammation cascade. TLR9 activation in recruited PDC induces IFN-I, which drive in situ macrophage polarization and adipose tissue insulin resistance. Of note, systemic autoimmune contexts are often associated with insulin resistance. We actually could link systemic IFN-I response with susceptibility to insulin resistance in an autoimmune context. In rodent models of the disease TLR9 deficiency has also recently been shown to ameliorate insulin resistance, thus validating our model. Moreover, a distinct autoantibody response can be detected in obese individuals, like IFN-I-driven autoimmunities.
We think that a syndromic description (‘Plasmacytoidopathy’) of these different clinical contexts, from systemic autoimmune diseases to metabolic derangements, will enable identification of important biomarkers as well as novel therapeutic targets.

The impact of lower vitamin D metabolites levels on coronary artery disease in type 2 diabetes patients in India

posted Jan 26, 2017, 1:05 AM by sourav ghosh

Sanjay K Banerjee
Drug Discovery Research Center, Translational Health Science and Technology Institute, Faridabad, India

The association of vitamin D insufficiency and increased risk of metabolic diseases has been reported in several scientific journals. However, it is not clear which vitamin D metabolite should be measured and the association of each metabolite with increased risk of metabolic diseases. Among all vitamin D metabolites, only 25(OH)D and 1,25(OH)2D have received great attention. Hence in the present study six vitamin D (D2/D3) metabolites have been quantified by LC-MS method. The purpose of the present study is to measure six vitamin D metabolites, total 25(OH)D and total 1,25(OH)2D levels and to find the association between vitamin D deficiency and coronary artery diseases in diabetes. Four groups [control, type 2 diabetes (T2DM), coronary artery diseases (CAD), T2DM with CAD (T2DM_CAD)] were included for the study. Glycated hemoglobin (HbA1c) and fasting blood sugar (FBS) levels were increased significantly (p < 0.05) in patients having 20–30 ng/ml and <20 ng/ml at 25(OH)D levels as compared with subjects having >30 ng/ml of 25(OH)D. Our data revealed that all the vitamin D metabolites were significantly (p < 0.05) decreased in T2DM_CAD as compared to both control and T2DM subjects. However, only two metabolites i.e., 25(OH)D3 and total 25(OH)D were significantly (p < 0.05) decreased in the T2DM subjects as compared with the control subjects. Multiple logistic regression analysis revealed that total 25(OH)D and total 1,25(OH)2D can be used to predict T2DM and T2DM with CAD respectively. This study concludes that lower vitamin D metabolites levels is associated with type 2 diabetes coexisting with coronary artery diseases in Indian subjects.

Understanding Endothelial Dysfunction in Diabetic Cardiovascular Complications Using Mass Spectrometry-based Proteomics

posted Jan 26, 2017, 12:58 AM by sourav ghosh

Mahesh Kulkarni
Proteomics Facility, CSIR-National Chemical Laboratory, Pune, India

One of the initial steps in the development of cardiovascular diseases involves endothelial dysfunction (ED), wherein the regulatory functions of the vascular endothelium are disrupted. ED can be caused by chemically-modified plasma proteins that are present in vivo whose levels are elevated by metabolites and drugs found in the bloodstream. Under conditions such as prolonged hyperglycemia in diabetes, there are elevated plasma levels of glucose-modified proteins, also known as advanced glycation end-products (AGEs). The interaction of these AGEs with their receptor RAGE has been implicated in different complications and could also be major contributory factors towards the development of ED in diabetes leading to cardiovascular disease and clinical complications. Since cardiovascular disease is a major pathological outcome in patients that exhibit diabetes, there is importance in understanding the mechanism underlying of how AGEs cause ED. 
In our study, we have synthesized AGE-HSA, characterized mass spectrometrically, developed ion library for targeted quantification in the clinical plasma. Furthermore, AGE-HSA was used to study AGE-RAGE signaling in human umbilical vein endothelial cells (HUVECs) cells. Differential total cell proteomics of control and stimulated endothelial cells was performed using mass spectrometry. Total cell proteomic analysis shows differential expression of numerous proteins regulating endothelial function including those affecting barrier function, inflammation and angiogenesis. This shows that AGEs can elicit ED, thus predisposing diabetic patients to increased risk of cardiovascular complications.

1-6 of 6