M. A. Permutt

Missense mutations in the pancreatic islet beta cell inwardly rectifying K+ channel gene (KIR6.2/BIR): a meta-analysis suggests a role in the polygenic basis of Type II diabetes mellitus in Caucasians.

Summary The K+ inwardly rectifier channel (KIR) is one of the two sub-units of the pancreatic islet ATP-sensitive potassium channel complex (IKATP), which has a key role in glucose-stimulated insulin secretion and thus is a potential candidate for a genetic defect in Type II (non-insulin-dependent) diabetes mellitus. We did a molecular screening of the KIR6.2 gene by single strand conformational polymorphism (SSCP) and direct sequencing in 72 French Caucasian Type II diabetic families. We identified three nucleotide substitutions resulting in three amino acid changes (E23K, L270V and I337V), that have also been identified in other Caucasian Type II diabetic subjects. These variants were genotyped in French cohorts of 191 unrelated Type II diabetic probands and 119 normoglycaemic control subjects and association studies were done. The genotype frequencies of the L270V and I337V variants were not very different between Type II diabetic subjects and control groups. In contrast, analysis of the E23K variant showed that the KK homozygocity was more frequent in Type II diabetic than in control subjects (27 vs 14 %, p = 0.015). Analyses in a recessive model (KK vs EK/EE) tended to show a stronger association of the K allele with diabetes (p = 0.0097, corrected p-value for multiple testing < 0.02). The data for the E23K variant obtained here and those obtained from three other Caucasian groups studied so far were combined and investigated by meta-analysis. Overall, the E23K variant was found to be significantly associated with Type II diabetes (0.001 ≤p≤ 0.0016, corrected p-values for multiple testing p≤ 0.01). This study shows that KIR6.2 polymorphisms are frequently associated with Type II diabetes in French Caucasians. Furthermore, a meta-analysis combining different Caucasian groups suggests an significant role of KIR6.2 in the polygenic context of Type II diabetes. [Diabetologia (1998) 41: 1511–1515]

A Nonsense Mutation in the Inward Rectifier Potassium Channel Gene, Kir6.2, Is Associated With Familial Hyperinsulinism

ATP-sensitive potassium (KATP) channels are an essential component of glucose-dependent insulin secretion in pancreatic islet β-cells. These channels comprise the sulfonylurea receptor (SUR1) and Kir6.2, a member of the inward rectifier K+ channel family. Mutations in the SUR1 subunit are associated with familial hyperinsulinism (HI) (MIM:256450), an inherited disorder characterized by hyperinsulinism in the neonate. Since the Kir6.2 gene maps to human chromosome 11p15.1 (1,2), which also encompasses a locus for HI, we screened the Kir6.2 gene for the presence of mutations in 78 HI probands by single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses. A nonsense mutation, Tyr→Stop at codon 12 (designated Y12X) was observed in the homozygous state in a single proband. 86Rb+ efflux measurements and single-channel recordings of COS-1 cells co-expressing SUR1 and either wild-type or Y12X mutant Kir6.2 proteins confirmed that KATP channel activity was abolished by this nonsense mutation. The identification of an HI patient homozygous for the Kir6.2/Y12X allele affords an opportunity to observe clinical features associated with mutations resulting in an absence of Kir6.2. These data provide evidence that mutations in the Kir6.2 sub-unit of the islet β-cell KATP channel are associated with the HI phenotype and also suggest that the majority of HI cases are not attributable to mutations in the coding region of the Kir6.2 gene.

Post Genome-Wide Association Studies of Novel Genes Associated with Type 2 Diabetes Show Gene-Gene Interaction and High Predictive Value

Background Recently, several Genome Wide Association (GWA) studies in populations of European descent have identified and validated novel single nucleotide polymorphisms (SNPs), highly associated with type 2 diabetes (T2D). Our aims were to validate these markers in other European and non-European populations, then to assess their combined effect in a large French study comparing T2D and normal glucose tolerant (NGT) individuals. Methodology/Principal Findings In the same French population analyzed in our previous GWA study (3,295 T2D and 3,595 NGT), strong associations with T2D were found for CDKAL1 (ORrs7756992 = 1.30[1.19–1.42], P = 2.3×10−9), CDKN2A/2B (ORrs10811661 = 0.74[0.66–0.82], P = 3.5×10−8) and more modestly for IGFBP2 (ORrs1470579 = 1.17[1.07–1.27], P = 0.0003) SNPs. These results were replicated in both Israeli Ashkenazi (577 T2D and 552 NGT) and Austrian (504 T2D and 753 NGT) populations (except for CDKAL1) but not in the Moroccan population (521 T2D and 423 NGT). In the overall group of French subjects (4,232 T2D and 4,595 NGT), IGFBP2 and CXCR4 synergistically interacted with (LOC38776, SLC30A8, HHEX) and (NGN3, CDKN2A/2B), respectively, encoding for proteins presumably regulating pancreatic endocrine cell development and function. The T2D risk increased strongly when risk alleles, including the previously discovered T2D-associated TCF7L2 rs7903146 SNP, were combined (8.68-fold for the 14% of French individuals carrying 18 to 30 risk alleles with an allelic OR of 1.24). With an area under the ROC curve of 0.86, only 15 novel loci were necessary to discriminate French individuals susceptible to develop T2D. Conclusions/Significance In addition to TCF7L2, SLC30A8 and HHEX, initially identified by the French GWA scan, CDKAL1, IGFBP2 and CDKN2A/2B strongly associate with T2D in French individuals, and mostly in populations of Central European descent but not in Moroccan subjects. Genes expressed in the pancreas interact together and their combined effect dramatically increases the risk for T2D, opening avenues for the development of genetic prediction tests.

Sequence Variants in the Pancreatic Islet β-Cell Inwardly Rectifying K+ Channel Kir6.2 (Bir) Gene: Identification and Lack of Role in Caucasian Patients with NIDDM

Signals derived from the metabolism of glucose in pancreatic β-cells lead to insulin secretion via the closure of ATP-sensitive K+ channels (KATP). The cloning of the gene encoding the (β-cell inward rectifier Kir6.2 (Bir), a subunit of the β-cell KATP channel, provided the opportunity to look for mutations in this gene that might contribute to the impaired insulin secretion of NIDDM. By single-strand conformational polymorphism (SSCP) analysis on 35 Northern-European Caucasian patients with NIDDM, six sequence variants were detected: Glu10gag→Lys10aag (E10K), Glu23gag→Lys23aag (E23K), Leu270ctg→Val270gtg (L270V), Ile337atc→Val337gtc (I337V), and two silent mutations. Allelic frequencies for the missense variants were compared between the NIDDM group (n = 306) and nondiabetic control subjects (n = 175) and did not differ between the two groups. Pairwise allelic associations indicated significant linkage disequilibrium between the variants in Kir6.2 and between them and a nearby pancreatic β-cell sulfonylurea receptor (SUR1) missense variant (S1370A), but these linkage disequilibria did not differ between the NIDDM and control groups. The results of these studies thus revealed that mutations in the coding region of Kir6.2 1) were not responsible for the previously noted association of the SUR1 variants with NIDDM (Inoue H et al., Diabetes 45:825–831, 1996) and 2) did not contribute to the impaired insulin secretion characteristic of NIDDM in Caucasian patients.

Glucose transporter levels in spontaneously obese (db/db) insulin-resistant mice.

In the present study we examined mRNA and protein levels for the muscle/adipose tissue glucose transporter (GLUT-4) in various tissues of spontaneously obese mice (C57BL/KsJ, db/db) and their lean littermates (db/+). Obese (db/db) mice were studied at 5 wk of age, when they were rapidly gaining weight and were severely insulin resistant, evidenced by hyperglycemia (plasma glucose 683 +/- 60 vs. 169 +/- 4 mg/dl in db/+, P less than 0.05) and hyperinsulinemia (plasma insulin 14.9 +/- 0.53 vs. 1.52 +/- 0.08 ng/ml in db/+, P less than 0.05). The GLUT-4 mRNA was reduced in quadriceps muscle (67.5 +/- 8.5%, P = 0.02), but unaltered in adipose tissue (120 +/- 19%, NS), heart (95.7 +/- 6.1%, NS), or diaphragm (75.2 +/- 12.1%, NS) in obese (db/db) mice relative to levels in lean littermates. The GLUT-4 protein, measured by quantitative immunoblot analysis using two different GLUT-4 specific antibodies, was not different in five insulin-sensitive tissues including diaphragm, heart, red and white quadriceps muscle, and adipose tissue of obese (db/db) mice compared with tissue levels in lean littermates; these findings were consistent when measured relative to tissue DNA levels as an index of cell number. These data suggest that the marked defect in glucose utilization previously described in skeletal muscle of these young obese mice is not due to a decrease in the level of the major muscle glucose transporter. An alternate step in insulin-dependent activation of the glucose transport process is probably involved.

Effects of altered glucose homeostasis on glucose transporter expression in skeletal muscle of the rat.

Previous studies have suggested that alteration in the expression of the insulin-regulatable glucose transporter of muscle (GLUT-4 protein) may be an important determinant of insulin action. In the present studies, we have examined GLUT-4 mRNA and protein concentrations in muscle after variations in the metabolic status of the intact animal (i.e., 7 d streptozotocin-induced diabetes, 7 d insulin-induced hypoglycemia, and 3 d fasting). These changes in glucose homeostasis were associated with the following changes in GLUT-4 gene products: a decrease of approximately 30% in both mRNA and protein with diabetes; a 50% increase in mRNA and a 2.4-fold increase in protein with insulin injection; and normal mRNA in spite of a 2.7-fold increase in protein with fasting. Fasted diabetics exhibited an increase of 50% in GLUT-4 mRNA and a 2.4-fold increase in protein relative to fed diabetics. In diabetic and insulin-injected groups, the changes in GLUT-4 protein were similar to changes in mRNA, but in fasting, GLUT-4 protein increased without a concomitant change in mRNA. Overall there was no correlation between muscle concentrations of GLUT-4 protein and mRNA. Muscle GLUT-4 protein concentration tended to correlate with plasma glucose (r = -0.57, P less than 0.001), but not with plasma insulin. These results indicate that (a) chronic changes in glucose homeostasis are associated with changes in expression of GLUT-4 protein in muscle; (b) GLUT-4 protein increased in fasted soleus muscle without change in mRNA, thereby differing from fasted adipocytes in which both GLUT-4 products diminish; and (c) no simple relationship exists between total muscle GLUT-4 protein content and whole-body insulin sensitivity.

Genetic analysis of glucose tolerance in inbred mouse strains. Evidence for polygenic control.

To determine genetic factors involved in diabetes susceptibility in inbred strains of mice, we initially evaluated differences in fed plasma glucose and insulin concentrations among six strains (AKR/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J). There was considerable variation in fed plasma glucose concentration, with C3H/HeJ mice the most glucose tolerant (174 ± 7 mg/dl) and C57BL/6J mice the least glucose tolerant (252 ± 7 mg/dl, P < .0001 vs. C3H/HeJ mice). Glycosylated hemoglobin of C57BL/6J mice (4.0 ± 0.06%) was also higher than that of C3H/HeJ mice (3.52 ± 0.06%, P < .0001). The fed plasma insulin concentration did not differ between these two strains. Glucose tolerance was further evaluated in overnight-fasted C3H/HeJ and C57BL/6J mice by an intraperitoneal glucose tolerance test (IPGTT). Although fasting plasma glucose did not differ, the most remarkable difference in plasma glucose during IPGTT between C57BL/6J and C3H/HeJ mice was noted at 30 min (489 ± 29 vs. 227 ± 20 mg/dl, P < .001). To determine the number of genes involved in the phenotypic difference in glucose tolerance, C57BL/6J males were crossed with C3H/HeJ females (F1, C3H/HeJ × C57BL/6J), and the F1 hybrid females were backcrossed with C57BL/6J males (backcrossed, F1 × C57BL/6J). Plasma glucose after 30 min on IPGTT was 219 ± 8 (n = 21), 456 ± 18 (n = 23), and 292 ± 13 (n = 23) mg/dl for C3H/HeJ, C57BL/6J, and F1 mice, respectively (P < .001 for all comparisons). The range of glucose levels on IPGTT of the backcrossed mice was 250–500 mg/dl. If a single gene were determining the difference in glucose tolerance in C57BL/6J mice, analysis of the plasma glucose level of individual backcrossed mice would show segregation into two classes, one like the F1 hybrid and the other like the inbred parent. This segregation was not observed; therefore, we conclude that modification of glucose tolerance in these two inbred strains is under polygenic control. Plasma insulin levels after 15 min on IPGTT for C57BL/6J mice were lower than those for C3H/HeJ mice (1.0 ± 0.09 vs. 1.3 ± 0.09 ng/ml, n = 6, P < .001). On the other hand, insulin levels after 30 min on IPGTT for C3H/HeJ (0.9 ± 0.08 ng/ml, n = 21), C57BL/6J (1.05 ± 0.1 ng/ml, n = 23), F1 (1.05 ± 0.1 ng/ml, n = 23), and backcrossed mice (1.0 ± 0.1 ng/ml, n = 23) were not different from each other. A positive correlation between plasma glucose and insulin concentrations was observed in C3H/HeJ (r = .42, P < .05) and F1 (r = .46, P < .05) mice, whereas a negative correlation was seen in C57BL/6J mice (r = −.45, P < .05). This suggests that glucose-stimulated insulin secretion may be significantly impaired in C57BL/6J mice, indicating one of the possible mechanisms for the phenotypic expression of glucose intolerance.

An E23K single nucleotide polymorphism in the islet ATP-sensitive potassium channel gene (Kir6.2) contributes as much to the risk of Type II diabetes in Caucasians as the PPARγ Pro12Ala variant

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Effects of Glucose on Proinsulin Messenger RNA in Rats In Vivo

The purpose of these studies was to determine whether glucose, the principal regulator of insulin biosynthesis in mammals, controls synthesis through alterations in levels of proinsulin mRNA in whole animals. Rats were starved for 3 days and then either refed or injected with glucose or saline for 24 h. Glucose injection raised plasma glucose levels equivalent to levels seen with refeeding but provided less than 20% of caloric replacement. Pancreatic RNA was extracted and the relative concentration of proinsulin mRNA was determined by blot hybridization with a cloned rat proinsulin cDNA probe. In starved animals proinsulin mRNA levels were 15–20% that of fed controls. Glucose injection produced a specific three- to fourfold increase in proinsulin mRNA levels relative to total pancreatic RNA, within 24 h. The effect was measurable 2 h after glucose injection and appeared largely complete by 12 h. Actinomycin D blocked the glucose-induced increase in proinsulin mRNA. These studies demonstrate effects of changes of plasma glucose on levels of proinsulin mRNA. Their rapidity of onset and large magnitude are comparable to effects of glucose on rates of insulin biosynthesis in isolated islets and suggest that insulin biosynthesis is regulated at least in part by levels of proinsulin mRNA.

Glucokinase and NIDDM: A Candidate Gene That Paid Off

Glucokinase, the major enzyme that phosphorylates glucose upon entry into liver and islet β-cells, has been considered a prime candidate for inherited defects predisposing to NIDDM. Now that the human gene has been isolated, this question has been addressed directly. Polymorphic markers flanking the gene were identified. These markers (microsatellites) are composed of variable numbers of dinucleotide repeats that vary in size, resulting in different alleles. Variably sized alleles can be typed rapidly from genomic DNA of individuals by the PCR. Studies of inheritance of glucokinase genes have revealed significant linkage in families with early-onset NIDDM, or MODY, and mutations have been identified within the coding region of the gene in some families. These studies are extremely encouraging, as they indicate that genes can be identified even in this heterogeneous genetic disorder. This study considers the phenotypes that result from glucokinase defects and the relationship of MODY to NIDDM, and it estimates the role of glucokinase defects in NIDDM in general.