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ObjectiveProinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology.Research design and methodsWe have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates.ResultsNine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets.ConclusionsWe have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis.

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet beta cells and is a major autoantigen in both mouse and human type I diabetes. It is possible that suppression oflGRP gene transcription may prevent or delay the progression of this disease. We have previously shown using chromatin immunoprecipitation (CHIP) assays that the IGRP promoter binds, directly or indirectly, the islet-enriched transcription factors Pdx- 1, Pax-6 and BETA2 in beta TC-3 cells in situ. We show here, again using ChIP assays, that the IGRP promoter also binds the islet-enriched transcription factors MafA and Foxa2 in beta TC-3 cells in situ. Single binding sites for these factors were identified in the proximal IGRP promoter mutation of which resulted in decreased IGRP fusion gene expression. Gel retardation studies demonstrate that MafA binds to the IGRP promoter with a five fold higher affinity than it binds the well-characterized C 1 element in the insulin promoter. Co-transfection experiments in HeLa ceils, in which neither these islet-enriched transcription factors nor the IGRP gene are endogenously expressed, were used to investigate the mechanism by which these factors activate IGRP gene expression. Individually Pdx-1, Pax-6, BETA2, MafA and Foxa2 have little effect on IGRP fusion gene expression in HeLa cells whereas combined expression of all five factors results in a synergistic induction of expression. An identical result is seen in HepG2 hepatoma cells in which Foxa2 is endogenously expressed. Interestingly in both HeLa and HepG2 cells the action of Foxa2 is biphasic in that it both stimulates and inhibits IGRP fusion gene expression through mechanisms that are DNA binding dependent and independent, respectively. The observation that Pdx-1 stimulates IGRP fusion gene expression in HeLa cells was surprising because mutation of the four Pdx-1 binding sites in the IGRP promoter has little effect on IGRP fusion gene expression in either HeLa or beta-TC-3 cells. This suggests that in both cell types Pdx-1 stimulates IGRP gene expression through a mechanism that is independent of DNA binding. [ABSTRACT FROM AUTHOR]

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet beta cells and is a major autoantigen in both mouse and human type I diabetes. It is possible that suppression of IGRP gene transcription may prevent or delay the progression of this disease. With this in mind we have used a combination of in vivo transgenic and in situ transfection strategies to characterize the DNA elements that confer expression of the IGRP gene. Transgenic mice were generated containing the IGRP promoter sequence from -306, -911 or -3911 to +3 ligated to a beta-galactosidase reporter gene. Multiple independent founder lines were analyzed for each transgene construct. In addition, IGRP knockout mice were generated in which a LacZ/Neo cassette replaced exons 1-3 such that the endogenous IGRP promoter drives beta-galactosidase expression. Transgene expression was monitored by XGal staining of pancreatic tissue isolated from newborn, 3 week old, 6 week old and 6 month old mice. In both the Iransgenic and knockout mice robust beta-galactosidase expression was detected in newborn mouse islets. However, in the -306 and -911 mice beta-galactosidase expression became mosaic by 3 weeks of age, remained mosaic at 6 weeks of age but was largely absent in the pancreas of 6 month old mice. Similar results were obtained with the -3911 and IGRP knockout mice except that mosaic islet beta-galactosidase expression was still detected in the pancreas at 6 months of age. In contrast the endogenous IGRP gene remains expressed in all beta cells at 6 months of age. These results suggest that that non-promoter sequences are required for the maintenance of IGRP gene expression in adult mice. To identify candidate sequences a VISTA analysis was performed in which 20 kbp of DNA spanning the entire mouse and human IGRP loci were compared. This analysis revealed a ∼780 bp region in the third intron of the IGRP gene whose sequence is >75% identical between the mouse and human IGRP genes. Transient transfection experiments in beta TC-3 cells showed that when this region was ligated to an IGRP-luciferase fusion gene containing human promoter sequence from -324 to +3 it conferred enhanced reporter gene expression. These data suggest that the ∼780 bp conserved region represents an intronic enhancer that is required for the maintenance of IGRP gene expression in adult mice. [ABSTRACT FROM AUTHOR]

Islet autoimmunity may contribute to β-cell dysfunction in type 2 diabetes (T2D). Its prevalence and clinical significance have not been rigorously determined. In this ancillary study to the Glycemia Reduction Approaches in Diabetes-A Comparative Effectiveness (GRADE) Study, we investigated the prevalence of cellular and humoral islet autoimmunity in patients with T2D duration 4·0±3·0 y, HbA1c 7·5±0·5% on metformin alone. We measured T cell autoreactivity against islet proteins, islet autoantibodies against GAD65, IA2, ZnT8, and β-cell function. Cellular islet autoimmunity was present in 41·3%, humoral islet autoimmunity in 13·5%, and both in 5·3%. β-cell function calculated as iAUC-CG and ΔC-peptide(0- 30)/Δglucose(0-30) from an oral glucose tolerance test was lower among T cell-positives (T+) than T cell-negatives (T-) using two different adjustments for insulin sensitivity (iAUC-CG: 13·2% [95% CI 0·3, 24·4%] or 11·4% [95% CI 0·4, 21·2%] lower; ΔC-peptide(0-30)/Δglucose(0-30)) 19% [95% CI 3·1, 32·3%] or 17·7% [95% CI 2·6, 30·5%] lower). T+ patients had 17% higher HbA1c (95% CI 0·07, 0·28) and 7·7 mg/dL higher fasting plasma glucose levels (95% CI 0·2,15·3) than T- patients. We conclude that islet autoimmunity is much more prevalent in T2D patients than previously reported. T cell-mediated autoimmunity is associated with diminished β-cell function and worse glycemic control., (© 2022 by the American Diabetes Association.)

Gene-lifestyle interactions have been suggested to contribute to the development of type 2 diabetes. Glucose levels 2 h after a standard 75-g glucose challenge are used to diagnose diabetes and are associated with both genetic and lifestyle factors. However, whether these factors interact to determine 2-h glucose levels is unknown. We meta-analyzed single nucleotide polymorphism (SNP) × BMI and SNP × physical activity (PA) interaction regression models for five SNPs previously associated with 2-h glucose levels from up to 22 studies comprising 54,884 individuals without diabetes. PA levels were dichotomized, with individuals below the first quintile classified as inactive (20%) and the remainder as active (80%). BMI was considered a continuous trait. Inactive individuals had higher 2-h glucose levels than active individuals (β = 0.22 mmol/L [95% CI 0.13-0.31], P = 1.63 × 10(-6)). All SNPs were associated with 2-h glucose (β = 0.06-0.12 mmol/allele, P ≤ 1.53 × 10(-7)), but no significant interactions were found with PA (P > 0.18) or BMI (P ≥ 0.04). In this large study of gene-lifestyle interaction, we observed no interactions between genetic and lifestyle factors, both of which were associated with 2-h glucose. It is perhaps unlikely that top loci from genome-wide association studies will exhibit strong subgroup-specific effects, and may not, therefore, make the best candidates for the study of interactions.

Objective: Fixed genomic variation explains only a small proportion of the risk of adiposity. In animal models, maternal diet alters offspring body composition, accompanied by epigenetic changes in metabolic control genes. Little is known about whether such processes operate in humans., Research Design and Methods: Using Sequenom MassARRAY we measured the methylation status of 68 CpGs 5' from five candidate genes in umbilical cord tissue DNA from healthy neonates. Methylation varied greatly at particular CpGs: for 31 CpGs with median methylation ≥5% and a 5-95% range ≥10%, we related methylation status to maternal pregnancy diet and to child's adiposity at age 9 years. Replication was sought in a second independent cohort., Results: In cohort 1, retinoid X receptor-α (RXRA) chr9:136355885+ and endothelial nitric oxide synthase (eNOS) chr7:150315553+ methylation had independent associations with sex-adjusted childhood fat mass (exponentiated regression coefficient [β] 17% per SD change in methylation [95% CI 4-31], P = 0.009, n = 64, and β = 20% [9-32], P < 0.001, n = 66, respectively) and %fat mass (β = 10% [1-19], P = 0.023, n = 64 and β =12% [4-20], P = 0.002, n = 66, respectively). Regression analyses including sex and neonatal epigenetic marks explained >25% of the variance in childhood adiposity. Higher methylation of RXRA chr9:136355885+, but not of eNOS chr7:150315553+, was associated with lower maternal carbohydrate intake in early pregnancy, previously linked with higher neonatal adiposity in this population. In cohort 2, cord eNOS chr7:150315553+ methylation showed no association with adiposity, but RXRA chr9:136355885+ methylation showed similar associations with fat mass and %fat mass (β = 6% [2-10] and β = 4% [1-7], respectively, both P = 0.002, n = 239)., Conclusions: Our findings suggest a substantial component of metabolic disease risk has a prenatal developmental basis. Perinatal epigenetic analysis may have utility in identifying individual vulnerability to later obesity and metabolic disease.

Objective: Approximately 25% of children and adolescents with type 1 diabetes will develop diastolic dysfunction. This defect, which is characterized by an increase in time to cardiac relaxation, results in part from a reduction in the activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), the ATP-driven pump that translocates Ca(2+) from the cytoplasm to the lumen of the sarcoplasmic reticulum. To date, mechanisms responsible for SERCA2a activity loss remain incompletely characterized., Research Design and Methods: The streptozotocin (STZ)-induced murine model of type 1 diabetes, in combination with echocardiography, high-speed video detection, confocal microscopy, ATPase and Ca(2+) uptake assays, Western blots, mass spectrometry, and site-directed mutagenesis, were used to assess whether modification by reactive carbonyl species (RCS) contributes to SERCA2a activity loss., Results: After 6-7 weeks of diabetes, cardiac and myocyte relaxation times were prolonged. Total ventricular SERCA2a protein remained unchanged, but its ability to hydrolyze ATP and transport Ca(2+) was significantly reduced. Western blots and mass spectroscopic analyses revealed carbonyl adducts on select basic residues of SERCA2a. Mutating affected residues to mimic physio-chemical changes induced on them by RCS reduced SERCA2a activity. Preincubating with the RCS, methylglyoxal (MGO) likewise reduced SERCA2a activity. Mutating an impacted residue to chemically inert glutamine did not alter SERCA2a activity, but it blunted MGO's effect. Treating STZ-induced diabetic animals with the RCS scavenger, pyridoxamine, blunted SERCA2a activity loss and minimized diastolic dysfunction., Conclusions: These data identify carbonylation as a novel mechanism that contributes to SERCA2a activity loss and diastolic dysfunction during type 1 diabetes.

Objective: Loss of lipin 1 activity causes lipodystrophy and insulin resistance in the fld mouse, and LPIN1 expression and common genetic variation were recently suggested to influence adiposity and insulin sensitivity in humans. We aimed to conduct a comprehensive association study to clarify the influence of common LPIN1 variation on adiposity and insulin sensitivity in U.K. populations and to examine the role of LPIN1 mutations in insulin resistance syndromes., Research Design and Method: Twenty-two single nucleotide polymorphisms tagging common LPIN1 variation were genotyped in Medical Research Council (MRC) Ely (n = 1,709) and Hertfordshire (n = 2,901) population-based cohorts. LPIN1 exons, exon/intron boundaries, and 3' untranslated region were sequenced in 158 patients with idiopathic severe insulin resistance (including 23 lipodystrophic patients) and 48 control subjects., Results: We found no association between LPIN1 single nucleotide polymorphisms and fasting insulin but report a nominal association between rs13412852 and BMI (P = 0.042) in a meta-analysis of 8,504 samples from in-house and publicly available studies. Three rare nonsynonymous variants (A353T, R552K, and G582R) were detected in severely insulin-resistant patients. However, these did not cosegregate with disease in affected families, and Lipin1 protein expression and phosphorylation in patients with variants were indistinguishable from those in control subjects., Conclusions: Our data do not support a major effect of common LPIN1 variation on metabolic traits and suggest that mutations in LPIN1 are not a common cause of lipodystrophy in humans. The nominal associations with BMI and other metabolic traits in U.K. cohorts require replication in larger cohorts.

The Hertfordshire Cohort Study based in the U.K. was the first to report associations between fetal or infant growth and the prevalence of adult glucose intolerance and diabetes. Many studies have replicated the findings with respect to birth weight, but there have been fewer observations in relationship to infant growth, because this is infrequently recorded in routine datasets. Recently, we carried out glucose tolerance tests in a more recently born group of men and women from the Hertfordshire Cohort Study. The objective was to determine whether the associations with weights at birth and 1 year of age reported in the original study of people born between 1920 and 1930 were observed in people born between 1931 and 1939. Birth weight was inversely related to the overall prevalence of diabetes (comprising newly diagnosed as well as existing cases) in men and women. However, weight at 1 year of age was not associated with diabetes in either sex. Analysis of data from the glucose tolerance tests showed that both sexes had evidence of higher insulin and glucose concentrations in people who were small at birth or during infancy. Finally, direct comparison of 2-h plasma glucose concentrations in the previous and current Hertfordshire study suggested that both surveys showed broad similarity of the trends in glucose tolerance with birth or infant weights; most differences arose at the extremes of the birth weight, possibly because of the small numbers of subjects studied in these groups.