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1.  Stimulation of glucagon-like peptide-1 secretion downstream of the ligand-gated ion channel TRPA1 
Diabetes  2014;64(4):1202-1210.
Stimulus-coupled incretin secretion from enteroendocrine cells plays a fundamental role in glucose homeostasis, and could be targeted for the treatment of type-2 diabetes. Here, we investigated the expression and function of transient receptor potential (TRP) ion channels in enteroendocrine L-cells producing glucagon-like peptide-1 (GLP-1). By microarray and qPCR analysis we identified trpa1 as an L-cell enriched transcript in the small intestine. Calcium imaging of primary L-cells and the model cell line GLUTag revealed responses triggered by the TRPA1 agonists allyl-isothiocyanate (AITC, mustard oil), carvacrol and polyunsaturated fatty acids, that were blocked by TRPA1 antagonists. Electrophysiology in GLUTag cells showed that carvacrol induced a current with characteristics typical of TRPA1 and triggered the firing of action potentials. TRPA1 activation caused an increase in GLP-1 secretion from primary murine intestinal cultures and GLUTag cells; an effect that was abolished in cultures from trpa1−/− mice or by pharmacological TRPA1 inhibition. These findings present TRPA1 as a novel sensory mechanism in enteroendocrine L-cells, coupled to the facilitation of GLP-1 release, which may be exploitable as a target for treating diabetes.
PMCID: PMC4375100  PMID: 25325736
2.  β-cell-specific CD8 T cell phenotype in type 1 diabetes reflects chronic autoantigen exposure 
Diabetes  2014;64(3):916-925.
Autoreactive CD8 T cells play a central role in the destruction of pancreatic islet β-cells that leads to type 1 diabetes, yet the key features of this immune-mediated process remain poorly defined. In this study, we combined high definition polychromatic flow cytometry with ultrasensitive peptide-human leukocyte antigen class I (pHLAI) tetramer staining to quantify and characterize β-cell-specific CD8 T cell populations in patients with recent onset type 1 diabetes and healthy controls. Remarkably, we found that β-cell-specific CD8 T cell frequencies in peripheral blood were similar between subject groups. In contrast to healthy controls, however, patients with newly diagnosed type 1 diabetes displayed hallmarks of antigen-driven expansion uniquely within the β-cell-specific CD8 T cell compartment. Molecular analysis of selected β-cell-specific CD8 T cell populations further revealed highly skewed oligoclonal T cell receptor (TCR) repertoires comprising exclusively private clonotypes. Collectively, these data identify novel and distinctive features of disease-relevant CD8 T cells that inform the immunopathogenesis of type 1 diabetes.
PMCID: PMC4557541  PMID: 25249579
3.  Inorganic Nitrate Promotes the Browning of White Adipose Tissue through the Nitrate-Nitrite-Nitric Oxide Pathway 
Diabetes  2014;64(2):471-484.
Inorganic nitrate was once considered an oxidation end-product of nitric oxide metabolism with little biological activity. However, recent studies have demonstrated that dietary nitrate can modulate mitochondrial function in man and is effective in reversing features of the metabolic syndrome in mice. Using a combined histological, metabolomics, and transcriptional and protein analysis approach we mechanistically define that nitrate not only increases the expression of thermogenic genes in brown-adipose tissue but also induces the expression of brown adipocyte-specific genes and proteins in white adipose tissue, substantially increasing oxygen consumption and fatty acid β-oxidation in adipocytes. Nitrate induces these phenotypic changes through a mechanism distinct from known physiological small molecule activators of browning, the recently identified nitrate-nitrite-nitric oxide pathway. The nitrate-induced browning effect was enhanced in hypoxia, a serious co-morbidity affecting white adipose tissue in obese individuals, and corrected impaired brown adipocyte-specific gene expression in white adipose tissue in a murine model of obesity. Since resulting beige/brite cells exhibit anti-obesity and anti-diabetic effects, nitrate may be an effective means of inducing the browning response in adipose tissue to treat the metabolic syndrome.
PMCID: PMC4351918  PMID: 25249574
Nitrate; White Adipose Tissue; Thermogenesis; Hypoxia; Nitrite; Metabolomics
4.  Increasing Pyruvate Dehydrogenase Flux as a Treatment for Diabetic Cardiomyopathy: A Combined 13C Hyperpolarized Magnetic Resonance and Echocardiography Study 
Diabetes  2015;64(8):2735-2743.
Although diabetic cardiomyopathy is widely recognised, there are no specific treatments available. Altered myocardial substrate selection has emerged as a candidate mechanism behind the development of cardiac dysfunction in diabetes. As pyruvate dehydrogenase (PDH) activity appears central to the balance of substrate utilisation, we aimed to investigate the relationship between PDH flux and myocardial function in a rodent model of type-II diabetes and to explore whether or not increasing PDH flux, with dichloroacetate, would restore the balance of substrate utilisation and improve cardiac function.
All animals underwent in vivo hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopy and echocardiography to assess cardiac PDH flux and function respectively. Diabetic animals showed significantly higher blood glucose (10.8±0.7mM vs 8.4±0.5mM), lower PDH flux (0.005±0.001s−1 vs 0.017±0.002s−1) and significantly impaired diastolic function (E/E’ 12.2±0.8 vs 20±2) in keeping with early diabetic cardiomyopathy. Twenty-eight days treatment with dichloroacetate restored PDH flux to normal levels (0.018±0.002s−1), reversed diastolic dysfunction (E/E’ 14±1) and normalized blood glucose (7.5±0.7mM).
Treatment of diabetes with dichloroacetate therefore restored the balance of myocardial substrate selection, reversed diastolic dysfunction and normalised blood glucose levels. This suggests that PDH modulation could be a novel therapy for the treatment and/or prevention of diabetic cardiomyopathy.
PMCID: PMC4516266  PMID: 25795215
Diastolic Dysfunction; Echocardiography; Fuel Selection; Magnetic Resonance Spectroscopy; Metabolism; MRI; Pyruvate Dehydrogenase; Pyruvate Dehydrogenase Kinase; Type 2 Diabetes
5.  A Type I Interferon Transcriptional Signature Precedes Autoimmunity in Children Genetically at Risk for Type 1 Diabetes 
Diabetes  2014;63(7):2538-2550.
Diagnosis of the autoimmune disease type 1 diabetes (T1D) is preceded by the appearance of circulating autoantibodies to pancreatic islets. However, almost nothing is known about events leading to this islet autoimmunity. Previous epidemiological and genetic data have associated viral infections and antiviral type I interferon (IFN) immune response genes with T1D. Here, we first used DNA microarray analysis to identify IFN-β–inducible genes in vitro and then used this set of genes to define an IFN-inducible transcriptional signature in peripheral blood mononuclear cells from a group of active systemic lupus erythematosus patients (n = 25). Using this predefined set of 225 IFN signature genes, we investigated the expression of the signature in cohorts of healthy controls (n = 87), patients with T1D (n = 64), and a large longitudinal birth cohort of children genetically predisposed to T1D (n = 109; 454 microarrayed samples). Expression of the IFN signature was increased in genetically predisposed children before the development of autoantibodies (P = 0.0012) but not in patients with established T1D. Upregulation of IFN-inducible genes was transient, temporally associated with a recent history of upper respiratory tract infections (P = 0.0064), and marked by increased expression of SIGLEC-1 (CD169), a lectin-like receptor expressed on CD14+ monocytes. DNA variation in IFN-inducible genes altered T1D risk (P = 0.007), as exemplified by IFIH1, one of the genes in our IFN signature for which increased expression is a known risk factor for disease. These findings identify transient increased expression of type I IFN genes in preclinical diabetes as a risk factor for autoimmunity in children with a genetic predisposition to T1D.
PMCID: PMC4066333  PMID: 24561305
6.  Association of Organic Cation Transporter 1 with Intolerance to Metformin in Type 2 Diabetes: A GoDARTS Study 
Diabetes  2014;64(5):1786-1793.
Metformin is the most widely prescribed medication for the treatment of type 2 diabetes (T2D). However ~25% of patients treated with metformin develop gastrointestinal (GI) side-effects leading to discontinuation of therapy in approximately 5% cases. We hypothesised that reduced transport of metformin via Organic Cation Transporter 1 (OCT1) could increase metformin concentration in the intestine, leading to increased risk of severe GI side-effects and drug discontinuation. We compared the phenotype, carriage of reduced-function OCT1 variants, and concomitant prescribing of drugs known to inhibit OCT1 transport in 251 intolerant and 1915 fully metformin tolerant T2D patients. We showed that women and older people were more likely to be intolerant to metformin. Concomitant use of medications, known to inhibit OCT1 activity, was associated with intolerance (odds ratio OR=1.63, 95% CI 1.22-2.17, p=0.001) as was carriage of two reduced-function OCT1 alleles compared to carriage of one or no deficient allele (OR=2.41, 95% CI 1.48-3.93, p < 0.001). Individuals with two reduced function OCT1 alleles who were treated with OCT1 inhibitors were over four times more likely to develop intolerance (OR=4.13, 95% CI 2.09-8.16, p < 0.001). Our results suggest that reduced OCT1 transport is an important determinant of metformin intolerance.
PMCID: PMC4452716  PMID: 25510240
7.  Evidence of Interaction Between PPARG2 and HNF4A Contributing to Variation in Insulin Sensitivity in Mexican Americans 
Diabetes  2007;57(4):1048-1056.
We hypothesized that interaction between PPARG2 Pro12Ala and variants in the promoter region of HNF4A are associated with type 2 diabetes–related quantitative traits in Mexican-American families of a proband with previous gestational diabetes.
The BetaGene project genotyped PPARG2 Pro12Ala and nine HNF4A single nucleotide polymorphisms (SNPs) in 473 individuals in 89 families. Members of the proband generation had fasting glucose <126 mg/dl and were phenotyped by oral and intravenous glucose tolerance tests.
Neither PPARG2 Pro12Ala nor any of the nine HNF4A SNPs were independently associated with type 2 diabetes–related quantitative traits. However, the interaction between PPARG2 Pro12Ala and HNF4A rs2144908 was significantly associated with both insulin sensitivity (SI) (Bonferroni P = 0.0006) and 2-h insulin (Bonferroni P = 0.039). Subjects with at least one PPARG2 Ala allele and homozygous for the HNF4A rs2144908 A allele had 40% higher SI compared with individuals with at least one G allele. SI did not vary by rs2144908 genotype among PPARG2 Pro/Pro. The interaction result for SI was replicated by the Insulin Resistance Atherosclerosis Family Study (P = 0.018) in their San Antonio sample (n = 484) where subjects with at least one PPARG2 Ala allele and homozygous for the HNF4A rs2144908 A allele had a 29% higher SI compared with individuals with at least one G allele. However, the interaction was not replicated in their San Luis Valley sample (n = 496; P = 0.401).
Together, these results suggest that variation in PPARG2 and HNF4A may interact to regulate insulin sensitivity in Mexican Americans at risk for type 2 diabetes.
PMCID: PMC4447520  PMID: 18162503
8.  5-Lipoxygenase, but Not 12/15-Lipoxygenase, Contributes to Degeneration of Retinal Capillaries in a Mouse Model of Diabetic Retinopathy 
Diabetes  2008;57(5):1387-1393.
Lipoxygenases are regulators of chronic inflamation and oxidative stress generation. We evaluated the role of 5-and 12-lipoxygenases in the development of diabetic retinopathy.
Wild-type mice, 5-lipoxygenase– deficient mice, and 12/15-lipoxygenase– deficient mice were assessed 1) after 9 months of diabetes for retinal histopathology and leukotriene receptor expression and 2) after 3 months of diabetes for leukostasis and retinal superoxide generation.
Diabetic wild-type mice developed the expected degeneration of retinal capillaries and pericytes and increases in both leukostasis and superoxide production (P < 0.006). We found no evidence of diabetes-induced degeneration of retinal ganglion cells in these animals. The vascular histopathology was significantly inhibited in 5-lipoxygenase– deficient mice, but not in 12/15-lipoxygenase– deficient mice. Retinas from diabetic 5-lipoxygenase– deficient mice also had significantly less leukostasis, superoxide production, and nuclear factor-κB (NF-κB) expression (all P < 0.006), whereas retinas from diabetic 12/15-lipoxygenase– deficient mice had significantly less leukostasis (P < 0.005) but not superoxide production or NF-κB expression. Retinas from diabetic wild-type mice were enriched with receptors for the 5-lipoxygenase metabolite leukotriene B4. Diabetes-induced histological and biochemical alterations were significantly reduced in 5-lipoxygenase– deficient mice, but not 12/15-lipoxygenase– deficient mice.
5-Lipoxygenase represents a novel pathway for therapeutic intervention of diabetic retinopathy.
PMCID: PMC4444435  PMID: 18346986
9.  Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes 
Diabetes  2014;63(11):3835-3845.
Studies in type 1 diabetes indicate potential disease heterogeneity, notably in the rate of β-cell loss, responsiveness to immunotherapies and, in limited studies, islet pathology. We sought evidence for different immunological phenotypes using two approaches. First, we defined blood autoimmune response phenotypes by combinatorial, multi-parameter analysis of autoantibodies and autoreactive T-cell responses in 33 children/adolescents with newly-diagnosed disease. Multi-dimensional cluster analysis showed two equal-sized patient agglomerations, characterized by pro-inflammatory (IFN-γ+, multi-autoantibody-positive) and partially-regulated (IL-10+, pauci-autoantibody-positive) responses. Multi-autoantibody-positive non-diabetic siblings at high-risk of disease progression showed similar clustering. Second, pancreas samples obtained post mortem from a separate cohort of 21 children/adolescents with recently-diagnosed type 1 diabetes were examined immunohistologically. This revealed two distinct types of insulitic lesion, distinguishable by degree of cellular infiltrate and presence of B-lymphocytes, that we term “hyper-immune CD20Hi” and “pauci-immune CD20Lo”. Notably, subjects had only one infiltration phenotype and were partitioned by this into two equal-size groups that differed significantly by age of diabetes diagnosis, hyper-immune CD20Hi subjects being 5 years younger. These data indicate potentially related islet and blood autoimmune response phenotypes that coincide with, and precede disease. We conclude that different immunopathological processes (endotypes) may underlie type 1 diabetes, carrying important implications for treatment/prevention strategies.
PMCID: PMC4207393  PMID: 24939426
10.  Adiponectin Induces A20 Expression in Adipose Tissue To Confer Metabolic Benefit 
Diabetes  2014;64(1):128-136.
Obesity is a major risk factor for metabolic disease, with white adipose tissue (WAT) inflammation emerging as a key underlying pathology. We detail that mice lacking Reverbα exhibit enhanced fat storage without the predicted increased WAT inflammation or loss of insulin sensitivity. In contrast to most animal models of obesity and obese human patients, Reverbα−/− mice exhibit elevated serum adiponectin levels and increased adiponectin secretion from WAT explants in vitro, highlighting a potential anti-inflammatory role of this adipokine in hypertrophic WAT. Indeed, adiponectin was found to suppress primary macrophage responses to lipopolysaccharide and proinflammatory fatty acids, and this suppression depended on glycogen synthase kinase 3β activation and induction of A20. Attenuated inflammatory responses in Reverbα−/− WAT depots were associated with tonic elevation of A20 protein and ex vivo shown to depend on A20. We also demonstrate that adipose A20 expression in obese human subjects exhibits a negative correlation with measures of insulin sensitivity. Furthermore, bariatric surgery–induced weight loss was accompanied by enhanced WAT A20 expression, which is positively correlated with increased serum adiponectin and improved metabolic and inflammatory markers, including C-reactive protein. The findings identify A20 as a mediator of adiponectin anti-inflammatory action in WAT and a potential target for mitigating obesity-related pathology.
PMCID: PMC4396702  PMID: 25190567
11.  Bilirubin as a potential causal factor in type 2 diabetes risk: a Mendelian randomization study 
Diabetes  2014;64(4):1459-1469.
Circulating bilirubin, a natural antioxidant, is associated with decreased risk of type 2 diabetes (T2D), but the nature of the relationship remains unknown. We performed Mendelian randomization in a prospective cohort of 3,381 participants free of diabetes at baseline (aged 28-75 years; women, 52.6%). We used rs6742078 located in UDP-glucuronosyltransferase (UGT1A1) locus as instrumental variable (IV) to study a potential causal effect of serum total bilirubin on T2D risk. T2D developed in a total of 210 (6.2%) participants during a median follow-up of 7.8 years. In adjusted analyses, rs6742078, which explained 19.5% of bilirubin variation, was strongly associated with total bilirubin (a 0.68-SD increase in bilirubin levels per T allele; P<1×10−122) and was also associated with T2D risk (OR 0.69 [95%CI, 0.54-0.90]; P=0.006). Per 1-SD increase in log-transformed bilirubin levels, we observed a 25% (OR 0.75 [95%CI, 0.62-0.92]; P=0.004) lower risk of T2D. In Mendelian randomization analysis, the causal risk reduction for T2D was estimated to be 42% (causal ORIVestimation per 1-SD increase in log-transformed bilirubin 0.58 [95%CI, 0.39-0.84]; P=0.005), which was comparable to the observational estimate (Durbin-Wu-Hausman chi-square test Pfor difference =0.19). These novel results provide evidence that elevated bilirubin is causally associated with risk of T2D and support its role as a protective determinant.
PMCID: PMC4346199  PMID: 25368098
Bilirubin; liver; epidemiology; type 2 diabetes; Mendelian randomization
12.  Clinical and molecular characterization of a novel PLIN1 frameshift mutation identified in patients with familial partial lipodystrophy 
Diabetes  2014;64(1):299-310.
Perilipin-1 is a lipid droplet coat protein predominantly expressed in adipocytes, where it inhibits basal and facilitates stimulated lipolysis. Loss-of-function mutations in PLIN1 were recently reported in patients with a novel subtype of familial partial lipodystrophy, designated as FPLD4. We now report the identification and characterization of a novel heterozygous frameshift mutation affecting the carboxy-terminus (439fs) of perilipin-1 in two unrelated families. The mutation co-segregated with a similar phenotype including partial lipodystrophy, severe insulin resistance and type 2 diabetes, extreme hypertriglyceridaemia and non-alcoholic fatty liver disease in both families. Poor metabolic control despite maximal medical therapy prompted two patients to undergo bariatric surgery, with remarkably beneficial consequences. Functional studies indicated that expression levels of the mutant protein were lower than wild type protein and in stably tranfected pre-adipocytes the mutant protein was associated with smaller lipid droplets. Interestingly, unlike the previously reported 398 and 404 frameshift mutants, this variant binds and stabilizes ABHD5 expression, but still fails to inhibit basal lipolysis as effectively as wild type perilipin-1. Collectively, these findings highlight the physiological need for exquisite regulation of neutral lipid storage within adipocyte lipid droplets, as well as the possible metabolic benefits of bariatric surgery in this serious disease.
PMCID: PMC4361744  PMID: 25114292
13.  Generation of L-cells in mouse and human small intestine organoids 
Diabetes  2013;63(2):410-420.
Upon a nutrient challenge, L-cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate functional L-cells from 3D cultures of mouse and human intestinal crypts. We show that short-chain fatty acids (SCFAs) selectively increase the number of L-cells resulting in an elevation of GLP-1 release. This is accompanied by up-regulation of transcription factors, associated with the endocrine lineage of intestinal stem cell development. Thus, our platform allows us to study and modulate the development of L-cells in mouse and human crypts as a potential basis for novel therapeutic strategies in type 2 diabetes.
PMCID: PMC4306716  PMID: 24130334
14.  The Link Between Nutritional Status and Insulin Sensitivity Is Dependent on the Adipocyte-Specific Peroxisome Proliferator–Activated Receptor-γ2 Isoform 
Diabetes  2005;54(6):1706-1716.
The nuclear receptor peroxisome proliferator–activated receptor-γ (PPARγ) is critically required for adipogenesis. PPARγ exists as two isoforms, γ1 and γ2. PPARγ2 is the more potent adipogenic isoform in vitro and is normally restricted to adipose tissues, where it is regulated more by nutritional state than PPARγ1. To elucidate the relevance of the PPARγ2 in vivo, we generated a mouse model in which the PPARγ2 isoform was specifically disrupted. Despite similar weight, body composition, food intake, energy expenditure, and adipose tissue morphology, male mice lacking the γ2 isoform were more insulin resistant than wild-type animals when fed a regular diet. These results indicate that insulin resistance associated with ablation of PPARγ2 is not the result of lipodystrophy and suggests a specific role for PPARγ2 in maintaining insulin sensitivity independently of its effects on adipogenesis. Furthermore, PPARγ2 knockout mice fed a high-fat diet did not become more insulin resistant than those on a normal diet, despite a marked increase in their mean adipocyte cell size. These findings suggest that PPARγ2 is required for the maintenance of normal insulin sensitivity in mice but also raises the intriguing notion that PPARγ2 may be necessary for the adverse effects of a high-fat diet on carbohydrate metabolism.
PMCID: PMC4304004  PMID: 15919792
15.  Pharmacological Inhibition of Glucosylceramide Synthase Enhances Insulin Sensitivity 
Diabetes  2007;56(5):1341-1349.
A growing body of evidence implicates ceramide and/or its glycosphingolipid metabolites in the pathogenesis of insulin resistance. We have developed a highly specific small molecule inhibitor of glucosylceramide synthase, an enzyme that catalyzes a necessary step in the conversion of ceramide to glycosphingolipids. In cultured 3T3-L1 adipocytes, the iminosugar derivative N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM) counteracted tumor necrosis factor-α-induced abnormalities in glycosphingo-lipid concentrations and concomitantly reversed abnormalities in insulin signal transduction. When administered to mice and rats, AMP-DNM significantly reduced glycosphin-golipid but not ceramide concentrations in various tissues. Treatment of ob/ob mice with AMP-DNM normalized their elevated tissue glucosylceramide levels, markedly lowered circulating glucose levels, improved oral glucose tolerance, reduced A1C, and improved insulin sensitivity in muscle and liver. Similarly beneficial metabolic effects were seen in high fat-fed mice and ZDF rats. These findings provide further evidence that glycosphingolipid metabolites of ceramide may be involved in mediating the link between obesity and insulin resistance and that interference with glycosphingolipid biosynthesis might present a novel approach to the therapy of states of impaired insulin action such as type 2 diabetes.
PMCID: PMC4298701  PMID: 17287460
16.  [No title available] 
PMCID: PMC4295171  PMID: 17259371
17.  A type I interferon transcriptional signature precedes autoimmunity in children genetically at-risk of type 1 diabetes 
Diabetes  2014;63(7):2538-2550.
Diagnosis of the autoimmune disease type 1 diabetes (T1D) is preceded by the appearance of circulating autoantibodies to pancreatic islets. However, almost nothing is known about events leading to this islet autoimmunity. Previous epidemiological and genetic data have associated viral infections and anti-viral type I interferon (IFN) immune response genes with T1D. Here, we first used DNA microarray analysis to identify IFN-β inducible genes in vitro and then used this set of genes to define an IFN-inducible transcriptional signature in peripheral blood mononuclear cells from a group of active systemic lupus erythematosus patients (N=25). Using this predefined set of 225 IFN signature genes, we investigated expression of the signature in cohorts of healthy controls (N=87), T1D patients (N=64) and a large longitudinal birth cohort of children genetically predisposed to T1D (N=109; 454 microarrayed samples). Expression of the IFN signature was increased in genetically-predisposed children prior to the development of autoantibodies (P=0.0012), but not in established T1D patients. Upregulation of IFN-inducible genes was transient, temporally associated with a recent history of upper respiratory tract infections (P=0.0064) and marked by increased expression of SIGLEC-1 (CD169), a lectin-like receptor expressed on CD14+ monocytes. DNA variation in IFN-inducible genes altered T1D risk (P=0.007), as exemplified by IFIH1, one of the genes in our IFN signature and for which increased expression is a known disease risk factor. These findings identify transient increased expression of type I IFN genes in pre-clinical diabetes as a risk factor for autoimmunity in children with a genetic predisposition to T1D.
PMCID: PMC4066333  PMID: 24561305
18.  Common genetic variants highlight the role of insulin resistance and body fat distribution in type 2 diabetes, independently of obesity 
Diabetes  2014;63(12):4378-4387.
We aimed to validate genetic variants as instruments for insulin resistance and secretion, to characterise their association with intermediate phenotypes, and to investigate their role in T2D risk among normal-weight, overweight and obese individuals.We investigated the association of genetic scores with euglycaemic-hyperinsulinaemic clamp- and OGTT-based measures of insulin resistance and secretion, and a range of metabolic measures in up to 18,565 individuals. We also studied their association with T2D risk among normal-weight, overweight and obese individuals in up to 8,124 incident T2D cases. The insulin resistance score was associated with lower insulin sensitivity measured by M/I value (β in SDs-per-allele [95%CI]:−0.03[−0.04,−0.01];p=0.004). This score was associated with lower BMI (−0.01[−0.01,−0.0;p=0.02) and gluteofemoral fat-mass (−0.03[−0.05,−0.02;p=1.4×10−6), and with higher ALT (0.02[0.01,0.03];p=0.002) and gamma-GT (0.02[0.01,0.03];p=0.001). While the secretion score had a stronger association with T2D in leaner individuals (pinteraction=0.001), we saw no difference in the association of the insulin resistance score with T2D among BMI- or waist-strata(pinteraction>0.31). While insulin resistance is often considered secondary to obesity, the association of the insulin resistance score with lower BMI and adiposity and with incident T2D even among individuals of normal weight highlights the role of insulin resistance and ectopic fat distribution in T2D, independently of body size.
PMCID: PMC4241116  PMID: 24947364
Genetics; type 2 diabetes; insulin resistance; insulin secretion; adipose expandability
19.  The association between circulating lipoprotein(a) and type 2 diabetes: is it causal? 
Diabetes  2013;63(1):332-342.
Epidemiological evidence supports a direct and causal association between lipoprotein(a) [Lp(a)] levels and coronary risk, but the nature of the association between Lp(a) levels and risk of type 2 diabetes (T2D) is unclear. In this study, we assessed the association of Lp(a) levels with risk of incident T2D, and tested whether Lp(a) levels are causally linked to T2D. We analysed data on 18,490 participants from the EPIC-Norfolk cohort that included adults aged 40-79 years at baseline 1993-1997. During average 10 years of follow-up, 593 participants developed incident T2D. Cox regression models were used to estimate the association between Lp(a) levels and T2D. In Mendelian randomisation analyses, based on EPIC-Norfolk combined with DIAGRAM data involving a total of 10,088 diabetes cases and 68,346 controls, we used a genetic variant (rs10455872) as an instrument to test whether the association between Lp(a) levels and T2D is causal. In adjusted analyses there was an inverse association between Lp(a) levels and T2D: hazard ratio (HR) was 0.63 (95% confidence interval 0.49-0.81; p-trend=0.003) comparing the top versus bottom quintile of Lp(a). In EPIC-Norfolk, a 1-SD increase in logLp(a) was associated with a lower risk of T2D (OR=0.88, 95%CI: 0.80-0.95). However, in Mendelian randomisation analyses, a 1-SD increase in logLp(a) due to rs10455872, which explained 26.8% of the variability in Lp(a) levels, was not associated with risk of T2D (OR=1.03, 95%CI: 0.96-1.10, p = 0.41). These prospective findings demonstrate a strong inverse association of Lp(a) levels with risk of T2D. However, a genetic variant that elevated Lp(a) levels was not associated with risk of T2D, suggesting that elevated Lp(a) levels are not causally associated with a lower risk of T2D.
PMCID: PMC4246060  PMID: 24089516
lipoprotein(a); type 2 diabetes; causal association; coronary heart disease; hazard ratio; Mendelian randomisation; prospective study
20.  Mechanisms of Glomerulosclerosis in Diabetic Nephropathy 
Diabetes  2008;57(6):1439-1445.
PMCID: PMC4239998  PMID: 18511444
21.  HLA DR-DQ Haplotypes and Genotypes and Type 1 Diabetes Risk 
Diabetes  2008;57(4):1084-1092.
The Type 1 Diabetes Genetics Consortium has collected type 1 diabetic families worldwide for genetic analysis. The major genetic determinants of type 1 diabetes are alleles at the HLA-DRB1 and DQB1 loci, with both susceptible and protective DR-DQ haplotypes present in all human populations. The aim of this study is to estimate the risk conferred by specific DR-DQ haplotypes and genotypes.
Six hundred and seven Caucasian families and 38 Asian families were typed at high resolution for the DRB1, DQA1, and DQB1 loci. The association analysis was performed by comparing the frequency of DR-DQ haplotypes among the chromosomes transmitted to an affected child with the frequency of chromosomes not transmitted to any affected child.
A number of susceptible, neutral, and protective DR-DQ haplotypes have been identified, and a statistically significant hierarchy of type 1 diabetes risk has been established. The most susceptible haplotypes are the DRB1*0301-DQA1*0501-DQB1*0201 (odds ratio [OR] 3.64) and the DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0401-DQA1*0301-DQB*0302, and DRB1*0402-DQA1*0301-DQB1*0302 haplotypes (ORs 11.37, 8.39, and 3.63), followed by the DRB1*0404-DQA1*0301-DQB1*0302 (OR 1.59) and the DRB1*0801-DQB1*0401-DQB1*0402 (OR 1.25) haplotypes. The most protective haplotypes are DRB1*1501-DQA1*0102-DQB1*0602 (OR 0.03), DRB1*1401-DQA1*0101-DQB1*0503 (OR 0.02), and DRB1*0701-DQA1*0201-DQB1*0303 (OR 0.02).
Specific combinations of alleles at the DRB1, DQA1, and DQB1 loci determine the extent of haplotypic risk. The comparison of closely related DR-DQ haplotype pairs with different type 1 diabetes risks allowed identification of specific amino acid positions critical in determining disease susceptibility. These data also indicate that the risk associated with specific HLA haplotypes can be influenced by the genotype context and that the trans-complementing heterodimer encoded by DQA1*0501 and DQB1*0302 confers very high risk.
PMCID: PMC4103420  PMID: 18252895
22.  Identification and characterisation of glucagon-like peptide-1 receptor expressing cells using a new transgenic mouse model 
Diabetes  2013;63(4):1224-1233.
Glucagon-like peptide-1 (GLP-1) is an intestinal hormone with widespread actions on metabolism. Therapies based on GLP-1 are highly effective because they increase glucose-dependent insulin secretion in people with type 2 diabetes, but many reports suggest that GLP-1 has additional beneficial, or in some cases potentially dangerous, actions on other tissues, including the heart, vasculature, exocrine pancreas, liver and central nervous system. Identifying which tissues express the GLP-1 receptor (GLP1R) is critical for the development of GLP-1 based therapies. Our objective was to identify and characterise the targets of GLP-1 in mice, using a method independent of GLP1R antibodies. Using newly-generated glp1r-cre mice crossed with fluorescent reporter strains, we show that major sites of glp1r expression include pancreatic β and δ-cells, vascular smooth muscle, cardiac atrium, gastric antrum/pylorus, enteric neurones and vagal and dorsal root ganglia. In the central nervous sytem, glp1r-fluorescent cells were abundant in the area postrema, arcuate nucleus, paraventricular nucleus and ventromedial hypothalamus. Sporadic glp1r-fluorescent cells were found in pancreatic ducts. No glp1r-fluorescence was observed in ventricular cardiomyocytes. Glp1r-positive enteric and vagal neurons were activated by GLP-1, and may contribute to intestinal and central responses to locally-released GLP-1, such as regulation of intestinal secretomotor activity and appetite.
PMCID: PMC4092212  PMID: 24296712
23.  The iddm4 Locus Segregates With Diabetes Susceptibility in Congenic WF.iddm4 Rats 
Diabetes  2002;51(11):3254-3262.
Viral antibody–free BBDR and WF rats never develop spontaneous diabetes. BBDR rats, however, develop autoimmune diabetes after perturbation of the immune system, e.g., by viral infection. We previously identified a disease-susceptibility locus in the BBDR rat, iddm4, which is associated with the development of autoimmune diabetes after treatment with polyinosinic:polycytidylic acid and an antibody that depletes ART2+ regulatory cells. We have now developed lines of congenic WF.iddm4 rats and report that in an intercross of N5 generation WF.iddm4 rats, ∼70% of animals either homozygous or heterozygous for the BBDR origin allele of iddm4 became hyperglycemic after treatment to induce diabetes. Fewer than 20% of rats expressing the WF origin allele of iddm4 became diabetic. Testing the progeny of various recombinant N5 WF.iddm4 congenic rats for susceptibility to diabetes suggests that iddm4 is centered on a small segment of chromosome 4 bounded by the proximal marker D4Rat135 and the distal marker D4Got51, an interval of <2.8 cM. The allele at iddm4 has 79% sensitivity and 80% specificity in prediction of diabetes in rats that are segregating for this locus. These characteristics suggest that iddm4 is one of the most powerful non–major histocompatibility complex determinants of susceptibility to autoimmune diabetes described to date.
PMCID: PMC4034451  PMID: 12401717
24.  Role of the Fatty Acid Binding Protein mal1 in Obesity and Insulin Resistance 
Diabetes  2003;52(2):300-307.
The metabolic syndrome is a cluster of metabolic and inflammatory abnormalities including obesity, insulin resistance, type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis. The fatty acid binding proteins aP2 (fatty acid binding protein [FABP]-4) and mal1 (FABP5) are closely related and both are expressed in adipocytes. Previous studies in aP2-deficient mice have indicated a significant role for aP2 in obesity-related insulin resistance, type 2 diabetes, and atherosclerosis. However, the biological functions of mal1 are not known. Here, we report the generation of mice with targeted null mutations in the mal1 gene as well as transgenic mice overexpressing mal1 from the aP2 promoter/enhancer to address the role of this FABP in metabolic regulation in the presence or absence of obesity. To address the role of the second adipocyte FABP in metabolic regulation in the presence and deficiency of obesity, absence of mal1 resulted in increased systemic insulin sensitivity in two models of obesity and insulin resistance. Adipocytes isolated from mal1-deficient mice also exhibited enhanced insulin-stimulated glucose transport capacity. In contrast, mice expressing high levels of mal1 in adipose tissue display reduced systemic insulin sensitivity. Hence, our results demonstrate that mal1 modulates adipose tissue function and contributes to systemic glucose metabolism and constitutes a potential therapeutic target in insulin resistance.
PMCID: PMC4027060  PMID: 12540600
25.  Reduced Incorporation of Fatty Acids Into Triacylglycerol in Myotubes From Obese Individuals With Type 2 Diabetes 
Diabetes  2014;63(5):1583-1593.
Altered skeletal muscle lipid metabolism is a hallmark feature of type 2 diabetes (T2D). We investigated muscle lipid turnover in T2D versus BMI-matched control subjects (controls) and examined whether putative in vivo differences would be preserved in the myotubes. Male obese T2D individuals (n = 6) and BMI-matched controls (n = 6) underwent a hyperinsulinemic-euglycemic clamp, VO2max test, dual-energy X-ray absorptiometry scan, underwater weighing, and muscle biopsy of the vastus lateralis. 14C-palmitate and 14C-oleate oxidation rates and incorporation into lipids were measured in muscle tissue as well as in primary myotubes. Palmitate oxidation (controls: 0.99 ± 0.17 nmol/mg protein; T2D: 0.53 ± 0.07 nmol/mg protein; P = 0.03) and incorporation of fatty acids (FAs) into triacylglycerol (TAG) (controls: 0.45 ± 0.13 nmol/mg protein; T2D: 0.11 ± 0.02 nmol/mg protein; P = 0.047) were significantly reduced in muscle homogenates of T2D. These reductions were not retained for palmitate oxidation in primary myotubes (P = 0.38); however, incorporation of FAs into TAG was lower in T2D (P = 0.03 for oleate and P = 0.11 for palmitate), with a strong correlation of TAG incorporation between muscle tissue and primary myotubes (r = 0.848, P = 0.008). The data indicate that the ability to incorporate FAs into TAG is an intrinsic feature of human muscle cells that is reduced in individuals with T2D.
PMCID: PMC4023412  PMID: 24487026

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