Kristi Silver

Polymorphisms in the Transcription Factor 7-Like 2 (TCF7L2) Gene Are Associated With Type 2 Diabetes in the Amish: Replication and Evidence for a Role in Both Insulin Secretion and Insulin Resistance

Transcription factor 7-like 2 (TCF7L2) regulates genes involved in cell proliferation and differentiation. The TCF7L2 gene is located on chromosome 10q25 in a region of replicated linkage to type 2 diabetes. Recently, a microsatellite marker in intron 3 (DG10S478) and five correlated single nucleotide polymorphisms (SNPs) were identified in Icelandic individuals that showed strong association with type 2 diabetes, which was replicated in Danish and European-American cohorts. We genotyped four of the SNPs (rs7901695, rs7903146, rs11196205, and rs12255372) in Amish subjects with type 2 diabetes (n = 137), impaired glucose tolerance (IGT; n = 139), and normal glucose tolerance (NGT; n = 342). We compared genotype frequencies in subjects with type 2 diabetes with those with NGT and found marginal association for rs7901695 (P = 0.05; odds ratio [OR] 1.51); comparison between NGT control subjects and the combined type 2 diabetes/IGT case group showed strong association with rs7901695 and rs7903146 (P = 0.008–0.01; OR 1.53–1.57) and marginal association with rs11196205 and rs12255372 (P = 0.07 and P = 0.04, respectively). In an expanded set of 698 Amish subjects without diabetes, we found no association with insulin and glucose levels during a 3-h oral glucose tolerance test. We also genotyped these SNPs in nondiabetic, non-Amish subjects (n = 48), in whom intravenous glucose tolerance tests were performed, and found an association between rs7901695 and rs7903146 and insulin sensitivity (P = 0.003 and P = 0.005, respectively) and disposition index (P = 0.04 and P = 0.007, respectively). These data provide replicating evidence that variants in TCF7L2 increase the risk for type 2 diabetes and novel evidence that the variants likely influence both insulin secretion and insulin sensitivity.

Variations in the G6PC2/ABCB11 genomic region are associated with fasting glucose levels

Identifying the genetic variants that regulate fasting glucose concentrations may further our understanding of the pathogenesis of diabetes. We therefore investigated the association of fasting glucose levels with SNPs in 2 genome-wide scans including a total of 5,088 nondiabetic individuals from Finland and Sardinia. We found a significant association between the SNP rs563694 and fasting glucose concentrations (P = 3.5 x 10(-7)). This association was further investigated in an additional 18,436 nondiabetic individuals of mixed European descent from 7 different studies. The combined P value for association in these follow-up samples was 6.9 x 10(-26), and combining results from all studies resulted in an overall P value for association of 6.4 x 10(-33). Across these studies, fasting glucose concentrations increased 0.01-0.16 mM with each copy of the major allele, accounting for approximately 1% of the total variation in fasting glucose. The rs563694 SNP is located between the genes glucose-6-phosphatase catalytic subunit 2 (G6PC2) and ATP-binding cassette, subfamily B (MDR/TAP), member 11 (ABCB11). Our results in combination with data reported in the literature suggest that G6PC2, a glucose-6-phosphatase almost exclusively expressed in pancreatic islet cells, may underlie variation in fasting glucose, though it is possible that ABCB11, which is expressed primarily in liver, may also contribute to such variation.

A Central Role for GRB10 in Regulation of Islet Function in Man

Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

A Genome-Wide Linkage Scan of Insulin Level–Derived Traits The Amish Family Diabetes Study

OBJECTIVE—Serum insulin levels are altered in insulin resistance and insulin deficiency, states that are associated with the development of type 2 diabetes. The goal of our study was to identify chromosomal regions that are likely to harbor genetic determinants of these traits. RESEARCH DESIGN AND METHODS—We conducted a series of genetic analyses, including genome-wide and fine-mapping linkage studies, based on insulin levels measured during an oral glucose tolerance test (OGTT) in 552 nondiabetic participants in the Amish Family Diabetes Study. Indices of insulin secretion included the insulinogenic index and insulin at 30 min postglucose load (insulin 30), while indices of insulin resistance included homeostasis model assessment of insulin resistance (HOMA-IR) and fasting insulin. Insulin area under the curve, a measure of both insulin secretion and insulin resistance, was also examined. RESULTS—All traits were modestly heritable, with heritability estimates ranging from 0.1 to 0.4 (all P < 0.05). There was significant genetic correlation between fasting insulin and HOMA-IR (ρG > 0.86, P < 0.05), as well as insulin 30 and insulinogenic index (ρG = 0.81, P < 0.0001), suggesting that common genes influence variation in these pairs of traits. Suggestive linkage signals in the genome scan were to insulin 30 on chromosome 15q23 (logarithm of odds [LOD] 2.53, P = 0.00032) and to insulinogenic index on chromosome 2p13 (LOD 2.51, P = 0.00034). Fine-mapping study further refined our signal for insulin 30 on chromosome 15 (LOD 2.38 at 68 cM). CONCLUSIONS—These results suggest that there may be different genes influencing variation in OGTT measures of insulin secretion and insulin resistance.

Arsenic exposure is associated with diminished insulin sensitivity in non-diabetic Amish adults.

Substantial evidence supports an association between diabetes and arsenic at high exposure levels, but results are mixed at low exposure levels. The aetiology of diabetes involves insulin resistance and β‐cell dysfunction. However, only a few epidemiologic studies have examined measures of insulin resistance and β‐cell function in relation to arsenic exposure, and no studies have tested for associations with the oral glucose tolerance test (OGTT). We examined the association between urinary total arsenic and OGTT‐based markers of insulin sensitivity and β‐cell function.

Computerisation of a paper-based intravenous insulin protocol reduces errors in a prospective crossover simulated tight glycaemic control study

BACKGROUND Paper-based continuous intravenous insulin protocols for tight glycaemic control (TGC) are typically complex, error-prone, time-consuming and burdensome. Little is known about the errors that occur as a result of misinterpretation and whether computerised protocols reduce errors. OBJECTIVE To compare the errors resulting from protocol misinterpretation, time required to manage insulin infusions and nursing satisfaction between a computerised insulin protocol and a paper-based protocol. METHODS In a crossover study, 62 ICU nurses completed 10 TGC simulated scenarios for the computerised and paper protocols. Scenarios evaluated three phases of insulin management: initiation, titration and transition. Scenarios response errors, time to completion and user satisfaction were examined. RESULTS A total of 620 responses were recorded using both protocols. The computerised protocols were associated with higher user satisfaction, as well as: fewer errors in the titration (13 vs. 113 errors, p=.0001) and transition phases (9 vs. 23 errors, p=.001), fewer dosing errors, although not statistically significant (p=.096), in the initiation phase, and less time to complete in the titration phase (6 vs. 9.5 min, p=.0001). CONCLUSIONS In a simulated environment, a computerised protocol for TGC resulted in significant insulin dosing error reduction, saved time and improved nurse satisfaction.

Analysis of coding variants in the betacellulin gene in type 2 diabetes and insulin secretion in African American subjects

BackgroundBetacellulin is a member of the epidermal growth factor family, expressed at the highest levels predominantly in the pancreas and thought to be involved in islet neogenesis and regeneration. Nonsynonymous coding variants were reported to be associated with type 2 diabetes in African American subjects. We tested the hypotheses that these previously identified variants were associated with type 2 diabetes in African Americans ascertained in Arkansas and that they altered insulin secretion in glucose tolerant African American subjects.MethodsWe typed three variants, exon1 Cys7Gly (C7G), exon 2 Leu44Phe (L44F), and exon 4 Leu124Met (L124M), in 188 control subjects and 364 subjects with type 2 diabetes. We tested for altered insulin secretion in 107 subjects who had undergone intravenous glucose tolerance tests to assess insulin sensitivity and insulin secretion.ResultsNo variant was associated with type 2 diabetes, and no variant altered insulin secretion or insulin sensitivity. However, an effect on lipids was observed for all 3 variants, and variant L124M was associated with obesity measures.ConclusionWe were unable to confirm a role for nonsynonymous variants of betacellulin in the propensity to type 2 diabetes or to impaired insulin secretion.

A case of congenital generalized lipodystrophy: metabolic effects of four dietary regimens. Lack of association of CGL with polymorphism in the lamin A/C Gene

Sirs, Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder, characterized by severe metabolic derangement associated with the absence of subcutaneous adipose tissue. The exact genetic defect is unknown, and systematic studies of optimal dietary and pharmacological therapy for CGL have not been performed. We report a case of CGL, in which the patients dietary/pharmacological management was optimized with the help of metabolic testing. In addition, DNA from this patient was screened for mutations in several candidate genes. A 36-year-old female presented for treatment of diabetes mellitus uncontrolled with 0·8 units per kg of insulin per day. The diabetes was diagnosed 10 years earlier and was complicated by macroalbuminuria (533 µg/minutes), retinopathy, neuropathy, and peripheral vascular disease. Her parents were first cousins. She had generalized fat loss, prominent musculature, hepatomegaly, clitoromegaly, mild hirsutism and no acanthosis nigricans. Her height was 151 cm and weight was 49·2 kg (body mass index 21 kg/m2). Tetrapolar bioelectric impedance revealed a fat mass of 3% (1·5 kg). CT demonstrated no subcutaneous fat in the abdominal area, and visceral abdominal fat was approximately one third of that in age, height and body weight matched females. Her blood chemistry profile, pituitary, gonadal, adrenal and thyroid tests were normal. Serum leptin was 0·8 µg/l (10·8–16·6 µg/l is normal for females with BMI 21 ± 5 kg/m2). All research procedures were approved by the Institutional Review Board of the Hospital das Clinicas at the University, Sao Paulo, Brazil. After the patient was hospitalized, baseline studies were performed, and four dietary regimens (Reg. 1–4, see Table 1) varying in energy and fat content, and with the addition of dL-fenfluramine with intensive insulin therapy were Instituted under carefully controlled conditions with a goal to maintain body weight and optimize glycaemic control. After each intervention, energy substrate metabolism was tested with a euglycaemic hyperinsulinaemic clamp (insulin infusion rate 10 mU × m−2 × minutes−1) and indirect calorimetry using a flow-through canopy gas analyser system in the resting state and after a glucose load (Felber et al., 1987). The respiratory exchange ratios (RER) indicate (Table 1) that in all but the hypercaloric moderate-fat diet (Reg. 2), glucose was the primary fuel source after an overnight fast. This finding was supported by the low fasting free fatty acid availability in all regimens except for the hypercaloric moderate-fat regimen. The thermic effect of glucose did not change with any of the interventions, including dL-fenfluramine (Table 1). As measured by euglycaemic hyperinsulinaemic clamp exogenous glucose disposal did not correlate with energy content in the diet. According to the clamp data (Table 1), treatment with a hypercaloric very low-fat diet (Reg. 3) and hypercaloric low-fat diet (Reg. 4) resulted in markedly greater exogenous glucose disposal than treatment with a hypercaloric moderate-fat diet (Reg. 2). However, the total glucose disposal determined by indirect calorimetry was not considerably different in the four regimens, suggesting that the disposal of glucose produced endogenously accounts for the difference between glucose disposal measured by the clamp vs. indirect calorimetry. Indeed, basal plasma glucose was higher after treatment with a moderate-fat diet (Reg. 2 vs. 3). Interestingly, with slightly higher basal plasma glucose levels, basal plasma insulin and C-peptide levels after an overnight fast (Table 1) were almost twice as low in Reg. 2 compared to Reg. 3. The lower endogenous insulin levels in Reg. 2 may be due to suppression by free fatty acids, which were at higher levels in Reg. 2. Table 1 Effects of dietary modifications, insulin therapy and addition of dL-fenfluramine in a patient with CGL Energy restriction in the diet has limitations in patients with CGL because of undesirable weight loss and persistent hunger (Reg. 1). However, as in our patients case, modestly hypercaloric diets (Reg. 2 and 3, see Table 1) add a risk of hyperglycaemia that responds poorly to insulin. The exogenous glucose disposal rate was the highest and basal plasma glucose after an overnight fast normalized after the addition of dL-fenfluramine to a hypercaloric low-fat diet (Reg. 4). In addition, this drug in sharp contrast to dietary treatments alone, seems to be responsible for increased lipid oxidation after an oral glucose load, apparently at the expense of suprabasal glucose oxidation. However, even this decrease in suprabasal glucose oxidation may be a positive phenomenon in a patient with CGL if it reflects an overall decrease in glucose recycling and hepatic glucose production. Our findings are supported by data from Andersen et al. (1993), who have shown that lipid oxidation is increased and glucose oxidation is decreased during dL-fenfluramine treatment in obese nondiabetic patients. We suggest that the improvement in insulin sensitivity, despite no change in total energy intake and virtually the same nutrient composition after transition from dietary Reg. 3–4, is related to a dL-fenfluramine induced augmentation in fat oxidation and diminished cycling of the nutritional substrates. Clinically this positive effect was reflected by achievement of euglycaemia with just 1·1 U/kg of insulin per day. We performed single-stranded conformational polymorphism (SSCP) and DNA analysis to search for mutations in the coding regions, exon-intron junctions and proximal promoter regions of the β3-adrenergic receptor (β3AR) and the peroxisome proliferator-activated receptor-gamma (PPARγ) genes. We did not detected mutations in the β3-AR gene in this patient, similar to studies by Silver et al. (1997) and Vigouroux et al. (1997) in other patients with CGL. The only polymorphism in the PPARγ gene in this patient was a silent nucleotide substitution at the position 1431 (CACHis→CATHis). This variant was also present in several nonlipodystrophic controls. Similarly, we (unpublished) and colleagues (Vigouroux et al., 1998) did not find unique mutations in the PPARγ gene in other patients with CGL. Heterozygosity for several missense mutations in the lamin A/C gene on chromosome 1 has been associated with familial partial lipodystrophy (Cao & Hegele 2000). DNA sequence analysis of the lamin A/C gene revealed that our patient was heterozygous for the previously reported polymorphism at codon 583 [GCCAla→GACAla] (Speckman et al. 2000). The patient was also heterozygous for a C2737T polymorphism in intron 8, which introduces a potential new splice donor site (GC→GT). However, this novel intron variant was also present in three of nine normal controls. In conclusion, we show that a low-fat diet with supplemental dL-fenfluramine and insulin therapy may be an effective method to manage hyperglycaemia in a patient with CGL. Studies of the genes for lamin A/C, PPARγ and β3-AR in our patient failed to detect pathogenic alterations. Uncovering the genetic defect of CGL will allow a better understanding of the pathogenesis of this disease and provide insights into adipocyte development and function, as well as potential treatments for CGL and perhaps other metabolic disorders such as obesity and type 2 diabetes.

Association of a polymorphism in the betacellulin gene with type 1 diabetes mellitus in two populations

Betacellulin, a member of the epidermal growth factor family, is expressed in fetal and adult pancreas. In vitro and in vivo studies suggest a role for betacellulin in islet neogenesis and regeneration. Therefore, a mutation in the betacellulin gene might lead to fewer beta cells. With reduced beta cell reserve, beta cells may not be able to compensate for an autoimmune attack, and in turn, susceptibility to type 1 diabetes mellitus (T1DM) would increase. Previous mutational analysis identified seven polymorphisms in the betacellulin gene [5′ UT (−233G>C, −226A>G), exon 1 (TGC19GGC, Cys7Gly), exon 2 (CTC130TTC, Leu44Phe), exon 4 (TTG370ATG, Leu124Met), intron 2 (-31T>C), and intron 4 (-4C>T)]. An association study of these variants with T1DM was first carried out in 100 Caucasian subjects with T1DM and 282 Caucasian subjects without diabetes recruited at the University of Maryland. The frequency of the intron 4 T-4 allele was significantly higher among nondiabetic controls than that among diabetic cases (0.29 vs 0.21, p=0.04). Allele frequencies for the other polymorphisms did not differ significantly between cases and controls. The intron 4 T-4 association was then replicated by transmission disequilibrium testing in a separate population of Caucasian parent/offspring with T1DM trios (n=168 trios, 113 informative) recruited at the Medical College of Wisconsin (p=0.024). An interaction of the intron 4 T-4 allele and human leukocyte antigen (HLA) was also detected with undertransmission of the T allele in those T1DM subjects with susceptible HLA types as compared to those T1DM subjects without susceptible HLA types (p=0.018). RNA studies of the intron T-4 variant showed similar RNA levels for intron 4 T-4 and intron 4 C-4 alleles. Additionally, there was no evidence for an effect of this variant on exon–intron splicing. We conclude that the intron 4 T-4 allele in the betacellulin gene is associated with lower risk of T1DM and may interact with HLA. Further studies will be necessary to establish the significance of this association.

Sequence variation in IGF1R is associated with differences in insulin levels in nondiabetic Old Order Amish

Insulin growth factor‐1 receptor (IGF1R) encodes the insulin‐like growth factor 1 receptor, a transmembrane tyrosine kinase receptor located on chromosome 15q26.3, in a region of linkage (LOD = 2.53, P = 0.00032) to Insulin30 on an OGTT in the Old Order Amish. Mouse models with beta‐cell‐specific deficiency of IGF1R demonstrate defects in glucose‐stimulated insulin secretion.