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The clinical characteristics of subjects with a missense glucokinase mutation, gly299-->arg, were studied in a large pedigree, BX, initially characterized by some members having Maturity Onset Diabetes of the Young (MODY). Glucose tolerance, beta cell function and insulin sensitivity were measured with Homeostasis Model Assessment (HOMA) and with a 'Continuous Infusion of Glucose with Model Assessment' (CIGMA) test. Diabetic complications were clinically assessed. Subjects with glucokinase gly299-->arg were the same age, height, and obesity as the subjects without the mutation. Diabetes was usually asymptomatic at diagnosis and was treated with diet alone in 15 of the 18 subjects. Five of the 11 adult females had been diagnosed when they developed gestational diabetes. The fasting plasma glucose concentrations at the time of study were 4.3-12.6 mmol l-1, with the higher levels being in the more obese (p < 0.05) and in the older subjects (p < 0.05). In subjects with the mutation, beta cell function was impaired, being geometric mean 63% (normal-100%) compared with 126% in the subjects without the mutation (p < 0.001) measured by HOMA and in a subset assessed by CIGMA 59% and 127% (p < 0.01), respectively. There was no difference in fasting insulin concentrations, insulin sensitivity, lipid concentrations or blood pressure between the groups. The haemoglobin A1c was raised (mean 6.5% compared with 5.5% in the subjects without the mutation), but microvascular and macrovascular complications were uncommon. The subjects with the mutation did not have microalbuminuria but had an impaired vibration perception threshold compared with subjects without the mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Glut2, the facilitative glucose transporter isoform expressed in pancreatic beta cells, is believed to play a role in glucose-stimulated insulin secretion. Two polymorphisms that result in amino acid substitutions have been reported in the human Glut2 gene (Tanizawa, Y., Riggs, A. C., Chiu, K. C., Janssen, R. C., Bell, D. S. H., Go, R. P. C., Roseman, J. M., Acton, R. T., and Permutt, M. A. (1994) Diabetologia 37, 420-427). A threonine 110-->isoleucine substitution was present at equal frequency in diabetic and control populations, and a valine 197-->isoleucine substitution was discovered in a single allele of a patient with non-insulin-dependent diabetes. The effect of these amino acid changes on glucose transport activity was tested by expression of the mutant proteins in Xenopus oocytes. The polymorphism at threonine 110 had no effect on the expression of Glut2 protein or the uptake of 2-deoxyglucose. Remarkably, however, the highly conservative valine 197-->isoleucine amino acid change abolished transport activity of the Glut2 transporter expressed in Xenopus oocytes. This represents the first known dysfunctional mutation in a human facilitative glucose transporter protein. The presence of this mutation in a diabetic patient suggests that defects in Glut2 expression may be causally involved in the pathogenesis of non-insulin-dependent diabetes.

The islet-1 (Isl-1) gene encodes a protein that binds to the enhancer region of the insulin gene. Isl-1 is a member of the LIM/homeodomain family of transcription factors. Because insulin deficiency, either relative or absolute, is a cardinal feature of non-insulin-dependent diabetes mellitus (NIDDM), this study addressed the question of whether mutations in genes that regulate insulin production could be involved. Rat Isl-1 was the first insulin enhancer binding protein to be isolated, and, in this study, the rat gene was used to isolate a partial human islet Isl-1 cDNA and subsequently to isolate genomic clones. A simple sequence repeat was found in the Isl-1 gene, and polymerase chain reaction amplification of this region of genomic DNA revealed 12 alleles in St. Louis African-Americans (het = 0.87), 14 alleles in black Nigerians (het = 0.89), 8 alleles in Japanese (het = 0.69), and 8 alleles in Caucasians (het = 0.81). Genetic linkage analysis uniquely placed Isl-1 on chromosome 5q (D5S395[12.8 cM]Isl-1 [11.6 cM]D5S407). The simple sequence repeat polymorphism at the Isl-1 locus was used to evaluate mutations in this gene as a possible contributor to the pathogenesis of NIDDM. Allelic frequencies did not differ between patients with NIDDM (n = 165) and nondiabetic control subjects (n = 163) in two black populations (St. Louis African-Americans and Nigerians). Linkage analyses in 15 nonglucokinase maturity-onset diabetes of the young pedigrees indicated that linkage could be rejected (LOD score < -3.0) over a distance of 15 cM.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucagon-like polypeptides, GLP-1-(7-36)-amide and GLP-1-(7-37), are important regulators of insulin synthesis and secretion by islet beta-cells. The hypothesis to be tested in this study was that defects in the islet beta-cell GLP-1 receptor gene contribute to the impaired glucose-regulated insulin secretion of non-insulin-dependent diabetes mellitus (NIDDM). Human islet GLP-1 receptor genomic clones were isolated, and two highly polymorphic simple sequence repeat regions (GLP-1R-CA1 and GLP-1R-CA3) were identified. Polymerase chain reaction assays were developed to define alleles. For GLP-1R-CA1, 14 alleles were observed in African-Americans (heterozygosity [het] = 0.78) and 6 alleles in Caucasians (het = 0.67). For GLP-1R-CA3, 16 alleles were observed in African Americans (het = 0.89) and 8 alleles in Caucasians (het = 0.83). By genotyping all members of the 40 reference Centre d'Etude du Polymorphisme Humain pedigrees at GLP-1R-CA3, the human GLP-1 receptor gene was uniquely placed on chromosome 6p between GLO1 and D6S19, 20.4 cM from human leukocyte antigen. To assess the possible role of the GLP-1 receptor gene in determining the genetic susceptibility to NIDDM, allelic frequencies of GLP-1R-CA1 and GLP-1R-CA3 were compared between African-American NIDDM patients (n = 95) and control subjects (n = 93). The frequencies did not differ between the two groups at either GLP-1R-CA1 or GLP-1R-CA3. The GLP-1 receptor gene simple-sequence repeat polymorphisms were used for linkage analysis in Utah Mormon pedigrees (n = 16) with NIDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of these experiments was to test the hypothesis that impaired glucose-stimulated insulin secretion in NIDDM is due to mutations in the islet beta cell/liver glucose transporter (GLUT 2) gene. Using oligonucleotide primers flanking each of the 11 exons, the structural portion of the gene was studied by PCR-SSCP analysis. DNA from African-American females (n = 48), who had gestational diabetes but developed overt NIDDM after delivery, was studied. Each SSCP variant was sequenced directly from genomic DNA. Two amino acid substitutions from the previously reported sequence were found, one in exon 3 and the other in exon 4B. Four additional silent mutations in the coding region, and six intron mutations outside the splice junction consensus sequences, were also identified. The mutation GTC x ATC in exon 4B substituted Val197 to Ile197. This amino acid substitution was found in only one NIDDM patient in a single allele, and was not found in 52 control subjects. This residue exists in the fifth membrane spanning domain, and Val at this position is conserved in mouse and rat GLUT 2, and human GLUT 1 to GLUT 4. The other codon change in exon 3, ACT x ATT, substituted Thr110 to Ile110 in the second membrane spanning domain. To determine the frequency of this non-conservative amino acid substitution, a PCR-LCR assay was developed. This assay was simple and highly specific for detection of this single nucleotide substitution. The allelic frequency of the ATT (Ile110) in NIDDM patients (39.6%, n = 48) and that in controls (47.1%, n = 52) did not differ (p = 0.32, Fisher's exact test).(ABSTRACT TRUNCATED AT 250 WORDS)

The glucokinase locus has been implicated by linkage studies in several Caucasian pedigrees with early onset, autosomal dominant diabetes, and mutations have been identified in a large number of these pedigrees. Although mutations have been reported in some pedigrees with late onset Type 2 (non-insulin-dependent) diabetes mellitus, linkage studies of typical familial Type 2 diabetes did not suggest a major role for this locus. Nonetheless, linkage studies were consistent with the hypothesis that mutations of the glucokinase gene were responsible for the pathogenesis of Type 2 diabetes in a minority of pedigrees or one gene in a polygenic disorder. To systematically address this hypothesis, we examined 60 diabetic members of 18 pedigrees ascertained for two or more Type 2 diabetic siblings and eight unrelated diabetic spouses. Initially, the coding regions from each of the 11 glucokinase exons were examined by the sensitive technique of single strand conformation polymorphism analysis to screen for single nucleotide substitutions. Subsequently, we also sequenced each exon from an affected member of the single pedigree in which a glucokinase allele was most likely to segregate with diabetes. Single strand conformation polymorphism analysis detected only three variants, none of which altered the amino acid sequence. No coding or splice site mutations were detected. Likewise, no additional mutations were detected upon direct sequence analysis. However, additional screening of promoter and 3' untranslated regions detected a variant pattern in the untranslated region of exon 10 which appeared to segregate with diabetes and impaired glucose tolerance in one pedigree.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations of the glucokinase gene result in early-onset familial Type 2 (non-insulin-dependent) diabetes mellitus, and several members of the mutant glucokinase kindreds were originally diagnosed as having gestational diabetes. This study examined the glucokinase gene in 270 American Black women, including 94 with gestational diabetes whose diabetes resolved after pregnancy (gestational diabetes only), 77 with gestational diabetes who developed Type 2 diabetes after pregnancy (overt diabetes), and 99 normal control subjects who were recruited during the peripartum period. Two simple sequence repeat polymorphisms flanking either end of the glucokinase gene were evaluated. No association was found between glucokinase alleles and gestational diabetes only or overt diabetes, after adjustment for multiple comparisons. To detect single base changes, all 11 exons and proximal islet and liver promoter regions were examined by polymerase chain reaction plus single-stranded conformational polymorphism analysis in 45 gestational diabetes only patients who had not yet developed Type 2 diabetes. Nine coding region variants were identified: Ala11 (GCC) to Thr11 (ACC) in islet exon 1, and 8 variants either in untranslated regions or in the third base of a codon. Four variant sites were found in introns, but none in splicing consensus sequences. Analysis of the promoter regions revealed two common variants, G-->A at islet -30 (24%), and G-->A at liver -258 (42%). The frequencies of the promoter variants, determined by allele specific polymerase chain reaction analysis, but did not differ among the three groups. Thus, no significant coding sequence glucokinase mutations were found in 90 alleles from 45 patients with gestational diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucokinase has a central role in glucose metabolism in pancreatic beta cells and hepatocytes and is an important candidate gene for Type 2 diabetes. Mutations of the glucokinase gene have been reported in Caucasian pedigrees with maturity-onset diabetes of the young and late-onset Type 2 diabetes. In population studies of American Blacks and Mauritian Creoles an association between alleles of a glucokinase polymorphism and Type 2 diabetes has been described. Two microsatellite polymorphisms (GCK 1 and GCK 2) flanking the glucokinase gene were investigated in Caucasian subjects. There was no significant linkage disequilibrium between the alleles of the two polymorphisms. The overall allelic frequencies for GCK 1 and the combined haplotyes did not significantly differ between 95 Type 2 diabetic and 76 normoglycaemic subjects. In an expanded cohort of 151 diabetic subjects the allelic frequencies at GCK 2 were also similar to controls. These results suggest that a single mutation of the glucokinase gene is not a common cause of Type 2 diabetes in English Caucasians.

Truncated forms of glucagon-like peptide-1 are the most potent endogenous stimuli of insulin secretion and have powerful antidiabetogenic effects. To determine the structure and coupling mechanisms of the human GLP-1 receptor we have isolated two pancreatic islet cDNAs, encoding the 463 amino acid receptor and differing mainly in their 3' untranslated regions. The deduced amino acid sequence is 90% homologous with the rat GLP-1 receptor. Northern blot analysis shows expression of a single 2.7 kb transcript in pancreatic tissue. When expressed in COS-7 cells the recombinant receptor conferred specific, high affinity GLP-1(7-37) binding. GLP-1(7-37) increased intracellular cAMP in a concentration dependent manner and caused an increase in the free cytosolic calcium ([Ca2+]i) from an intracellular pool, characteristic of phospholipase C (PLC) activation. Thus, like the structurally related glucagon and parathyroid hormone receptors, the human GLP-1 receptor can activate multiple intracellular signaling pathways including adenylyl cyclase and PLC. Knowledge of the GLP-1 receptor structure will facilitate the development of receptor agonists and elucidation of the important role of GLP-1 in normal physiology and disease states.

To assess the possible role of glucokinase defects contributing to a genetic susceptibility to NIDDM in Japanese, allelic frequencies of two microsatellite repeat polymorphisms, one in the 3'-flanking region (GCK1) and the other in the 5'-flanking region (GCK2) of the human glucokinase gene, were analyzed in subjects with NIDDM and in nondiabetic control subjects. After typing 107 diabetic and 74 nondiabetic subjects, we found four GCK1 alleles (Z, Z2, Z4, Z6) and six GCK2 alleles (0, -4, -2, 2, 4, 8). The frequency distribution of GCK1 alleles was different between the two groups (P = 0.005), although not significant after correction for multiple comparisons. The Z4 allele was found more frequently in diabetic than in nondiabetic subjects (23 vs. 10%, P = 0.002). This was still significant after correction for multiple comparisons (P < 0.05). The frequency distribution of GCK2 alleles was not different between the two groups. However, the -2 allele was more common in diabetic than in nondiabetic subjects (P = 0.044), although not significant after adjusting for multiple comparisons. Clinical characteristics were compared between the diabetic subjects with Z4 and/or -2 allele and those without either of these two alleles. No differences were found in the age of diagnosis, positive family history, mode of therapy, current HbA1c, or daily urinary C-peptide immunoreactivity excretion between the two groups. We demonstrated a significant association between GCK1 and GCK2 alleles and NIDDM. The results indicate that the polymorphic alleles GCK1 and GCK2 could be genetic markers in NIDDM in Japanese, suggesting a relationship between glucokinase defects and the susceptibility to NIDDM in this population.

The purpose of this study was to evaluate the role of potential glucokinase defects contributing to susceptibility to Type 2 (non-insulin-dependent) diabetes mellitus in Welsh Caucasians. For this analysis, two microsatellite repeat polymorphisms flanking opposite ends of the gene were employed. For a recently described microsatellite (GCK2), located 6 kilobases upstream of islet exon 1, six different sized alleles were observed, with heterozygosity of 0.50 and polymorphism information content 0.44. Combined heterozygosity with another microsatellite repeat (GCK1) was 0.72. Significant linkage disequilibrium was noted between GCK2 and GCK1, suggesting that haplotypes may be a better predictor of Type 2 diabetes than analysis with either microsatellite alone. Using these two markers, the association with Type 2 diabetes was examined. The frequencies of alleles and genotypes at GCK1 did not differ between the patients with Type 2 diabetes (n = 157) and control subjects (n = 73). Similarly no differences were observed in GCK2 alleles or genotypes. The frequencies of haplotypes, derived from the two markers, also did not differ between the two groups. To investigate the possibility of minor metabolic effects of glucokinase defects, we also studied the association between the GCK alleles or haplotypes and the response profiles to meal tolerance tests. No association was observed between plasma glucose or insulin responses to meal tolerance tests with GCK haplotypes or alleles. These results suggest that glucokinase mutations in Welsh Caucasians are not major determinants of susceptibility to the common type of Type 2 diabetes.

To determine whether a structural defect in glucokinase could be a primary cause of glucose intolerance in the common form of NIDDM, the prevalence of mutations in the gene in 60 American black NIDDM patients was investigated. First, by Southern blot analysis of DNA from a subset of randomly selected subjects (n = 20), no gross deletions, insertions, or rearrangements of the gene were detected. Next, the 5'-untranslated and coding regions of the gene were amplified directly from genomic DNA by the polymerase chain reaction. PCR products were screened for mutations by using single-strand conformational polymorphism analysis. A total of nine variants were identified, with two in the 5'-UT regions of islet exon 1, two in the 5'-UT region of liver exon 1, and five in the coding regions. For islet exon 1, 5 of 60 NIDDM patients had both variants in the 5'-UT region; and for liver exon 1, two variants each occurred in 1 of 60 NIDDM patients. The coding region variants included a missense mutation in islet exon 1, substitution of Ala11 (GCC) with Thr11 (ACC), found in 2 patients. The biological consequences of this mutation and the mutations in the 5'-UT portion of the gene have yet to be determined. The rest of the variants were third base pair changes of codons, i.e., silent. A common polymorphism, which was in linkage equilibrium with microsatellite repeats GCK1 and GCK2, was found in intron 9, and a variant in intron 2 in both alleles of 1 patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucokinase is among the few genes which may play a key role in both insulin secretion and insulin action. Glucokinase is present in pancreatic beta cells where it may have a key role in the glucose sensing mechanism, and it is present in hepatocytes, where it may participate in glucose flux. Glucokinase defects have recently been implicated in maturity-onset diabetes of the young. To examine the hypothesis that glucokinase plays a key role in the predisposition to common familial Type 2 (non-insulin-dependent) diabetes mellitus, we typed 399 members of 18 Utah pedigrees with multiple Type 2 diabetic individuals for two markers in the 5' and 3' flanking regions of the glucokinase gene. Linkage analysis was performed under both dominant and recessive models. We also repeated these analyses with individuals with impaired glucose tolerance who were considered affected if their stimulated (2-h) glucose exceeded age-specific normal levels for 95% of the population. Under several dominant models, linkage was significantly excluded, and under recessive models log of the odds (LOD) score was less than -1. We were also unable to demonstrate statistical support for the hypothesis that a small subgroup of pedigrees had glucokinase defects, but the most suggestive pedigree (individual pedigree LOD 1.8-1.9) ranked among the youngest and leanest in our cohort. We can exclude a major role for glucokinase in familial Type 2 diabetes, but our data cannot exclude a role for this locus in a minority of pedigrees.(ABSTRACT TRUNCATED AT 250 WORDS)

Familial NIDDM probably results from combined inherited defects of insulin secretion and action. Members of the facilitative glucose transporter family are strong candidates for both defects, and RFLPs for both GLUT1 (erythrocyte) and GLUT2 (liver/islet) genes have been associated with NIDDM in some populations. To test the hypothesis that GLUT1 and GLUT2 mutations contribute to the inherited predisposition to NIDDM, we examined linkage of these loci with NIDDM in 18 large Utah white pedigrees (two and three generation) ascertained for > or = 2 NIDDM siblings. We used two RFLPs detected with Xba1 and Stu1 for the GLUT1 transporter. For the GLUT2 (liver/beta-cell) transporter gene, we used an RFLP detected with EcoR1 and a highly polymorphic (6-allele) dinucleotide (microsatellite) repeat. Analysis was performed with the MLINK program of the LINKAGE package. We tested four models for each locus: dominant and recessive, with IGT alternately considered as unknown affection status, or affected if IGT was diagnosed < or = 45 yr of age and unknown if > 45 yr. Disease gene frequencies were chosen to give approximate disease prevalence in American whites (q = 0.03, dominant; q = 0.25, recessive). Linkage of GLUT1 and NIDDM was strongly and significantly rejected under all models, with total (pooled) LOD scores of -5.7 to -8.9, indicating > 500,000:1 odds against linkage. Pooled LOD scores were significantly negative (< -2.0, or 100:1 odds against linkage) to a recombination fraction of > 5%. No heterogeneity was apparent. Analysis of GLUT2 gave similar results, with LOD scores of < -4.0 under each model, indicating at least 10,000:1 odds against linkage.(ABSTRACT TRUNCATED AT 250 WORDS)

The question posed by these studies was whether chronic adaptive changes in glucose-stimulated insulin secretion are accompanied by comparable changes in islet Beta-cell glucose transporter (GLUT 2) gene expression. Control, fasted (3-day), insulin-injected hypoglycaemic (5-day), and dexamethasone-treated (4-day) rats (n = 5 for each condition), were studied. After fasting significant decrements in proinsulin mRNA/microgram RNA (-32%, p < 0.05) and islet amyloid polypeptide mRNA/microgram RNA (-44%, p < 0.05) were observed, while there was no change in GLUT 2 mRNA/microgram RNA (-13%, p > 0.05). After insulin-induced hypoglycaemia, decrements in proinsulin mRNA/microgram RNA (-49%, p < 0.01) and islet amyloid polypeptide mRNA/microgram RNA (-44%, p < 0.01) were also observed, with no change in islet GLUT 2 mRNA/microgram RNA (-18%, p > 0.05). Dexamethasone treatment resulted in a marked stimulatory effect on proinsulin mRNA/microgram RNA (+236%, p < 0.001) and islet amyloid polypeptide mRNA/microgram RNA (+221%, p < 0.01), while again there was no change in islet GLUT 2 mRNA/microgram RNA (+0.3%, p > 0.05). Quantitative immunoblot analysis with a GLUT 2 specific antibody revealed no change in islet GLUT 2 protein with fasting, but a small decrease (-39 +/- 11%) in islet GLUT 2/microgram protein after insulin-induced hypoglycaemia. These results do not support the hypothesis that chronic changes in glucose-stimulated insulin secretion are accompanied by changes in GLUT 2 expression. In contrast to the lack of correlation with GLUT 2, there was a striking correlation between proinsulin and islet amyloid polypeptide mRNAs for all experimental conditions (r = 0.974, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

NIDDM has a strong genetic component, as evidenced by the high level of concordance between identical twins. The nature of the genetic predisposition has remained largely unknown. Recently, the glucokinase gene locus on chromosome 7p has been shown to be linked to a subtype of NIDDM known as MODY in French and British pedigrees, and glucokinase mutations have been identified. To study the relationship between the glucokinase gene and NIDDM, we performed a linkage analysis in 12 Caucasian pedigrees ascertained through a proband with classical NIDDM. The LINKAGE program was used under four models, including autosomal dominant and recessive, with individuals with glucose intolerance counted as either affected or of unknown status. Linkage was significantly rejected with the dominant models (LOD scores -4.65, -4.25), and was unlikely with the recessive model when glucose intolerance was considered as affected (LOD score -1.38). These findings suggest that mutations in or near the glucokinase gene are unlikely to be the major cause of the inherited predisposition to NIDDM in Caucasian pedigrees, but do not exclude a role for this locus with a polygenic model, or a major role in some pedigrees.

Glucokinase, the major enzyme that phosphorylates glucose upon entry into liver and islet beta-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.

The gene encoding human glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), a major component of glucose sensing in pancreatic islet beta-cells, was isolated and characterized. The gene was shown by Southern blotting to exist as a single copy in the genome which mapped to chromosome 7p. It contained 12 exons including two tissue-specific first exons, one active in islet beta-cells (1B), and the other active in liver (1H), and one optional cassette exon which was expressed as a minor form in the liver. Thus the three previously reported isoforms of glucokinase mRNA were the result of tissue-specific activation of separate liver and islet promoters and subsequent alternative splicing events. Eleven exons, including 1H and the optional cassette exon 2A, were scattered over 16 kilobase (kb) in the genome, while exon 1B was separated from the rest by at least 20 kb. Although the islet promoter was found to lack a TATA box, a major transcript from the islet promoter was mapped 486 nucleotides upstream of the translation initiation site. The presence in the islet glucokinase promoter of the potential control element GCCACCAG, a homology of the regulatory element present in both human insulin (GCCACCGG) and rat insulin (GCCATCTG) genes, implied a possible tissue-specific regulatory role of this element. The liver promoter was found to contain a TATA box-like sequence, and transcription was initiated predominantly at 168 nucleotides upstream of the translation initiation site of the major isoform. A new highly polymorphic microsatellite, composed of a compound imperfect dinucleotide repeat [GT]15[GA]8CA[GA]7CA[GA]3AA[GA]2, was mapped 6 kb upstream of islet exon 1. A polymerase chain reaction-based assay was developed, and seven different sized alleles were identified in American Blacks. The sequence information reported here, along with the new polymorphic marker, will make it possible to clarify the molecular basis of potential glucokinase defects in noninsulin-dependent diabetes mellitus patients and may further elucidate the nature of genetic susceptibility to the development of this common metabolic disease.

Glucokinase (ATP:D-glucose-6-phosphotransferase), expressed exclusively in liver and pancreatic islet beta-cells, catalyzes the first step of glycolysis and acts as glucose sensor and metabolic signal generator in these tissues. The enzyme plays a key role in glucose homeostasis and as such is an excellent candidate for inherited defects predisposing to non-insulin-dependent diabetes mellitus (NIDDM). A compound-imperfect dinucleotide (CA)n repeat element was found approximately 10-kb 3' of the human glucokinase gene on chromosome 7p, which revealed polymorphism with alleles differing in size by 2-15 nucleotides in unrelated individuals. A polymerase chain reaction assay was developed, and genomic DNA from 275 biologically unrelated American black individuals was typed for glucokinase alleles. The differences in allelic frequencies between individuals with NIDDM and nondiabetic individuals were compared. After typing 112 diabetic and 163 nondiabetic subjects, we found five different-sized alleles, with Z defined as the most common allele, Z + 2, Z + 4, Z + 10, and Z - 15. The Z allele was more common in nondiabetic subjects than in diabetic patients (60.4 vs. 49.6%, P = 0.012). The Z + 4 allele was more common in diabetic patients than in nondiabetic subjects (20.1 vs. 12.0%, P = 0.009). After adjusting for age, sex, and body mass index, the Z + 4 allele continued to have a positive association with NIDDM (P = 0.0018), and the Z allele had a negative association with NIDDM (P = 0.0334). The Z + 4 allele, transmitted as an autosomal dominant trait, appeared to be the most significant one at this locus.(ABSTRACT TRUNCATED AT 250 WORDS)

The human liver/islet glucose transporter (GLUT2), a candidate gene for diabetes, has been incorporated into a genetic linkage map for chromosome 3q using a (CA)n dinucleotide repeat polymorphism adjacent to the 3'-end of exon 4a. We have found a total of nine alleles ranging in length from 153 to 169 nucleotides in three racial groups and have determined the precise structure of the variable region for four of the alleles by DNA sequencing. Five alleles were found to be common to the American Black, Caucasian, and Pima Indian racial groups studied. One allele (169 bp) was unique to American Blacks, and another rare allele (153 bp) was found only in the Caucasian population studied. Observed heterozygosity of the polymorphism in the Caucasian (CEPH) reference pedigree collection is 60%, for American Blacks 71%, and for Pima Indians 53%. An independent study recently identified the same dinucleotide repeat and found six alleles in a Caucasian population (Froguel et al., 1991), a result that we confirm; however, our sequencing data indicate a different molecular structure for the polymorphism for some of the alleles. We have constructed a new genetic linkage map of chromosome 3q uniquely placing the GLUT2 gene between flanking markers D3S26 and D3S43. The genetic map consists of 23 loci (25 RFLPs and 2 (CA)n dinucleotide repeat markers) with 14 markers uniquely localized with odds of at least 1000:1. Three genes (FTHL4, TF, GLUT2) are integrated into the map, which spans a sex-average distance of 147.3 cM, 103.8 cM in males and 227.0 cM in females.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic islet glucokinase (ATP:D-hexose-6-phosphotransferase) cDNAs were isolated from a human islet cDNA library in lambda-gt11. One clone (hlGLK2), 2723 bp plus additional poly(A) residues, appeared to be full length because its size was consistent with a single 2.9-kb glucokinase mRNA on Northern-blot analysis of islet RNA. This cDNA contained an open reading frame of 1395 bp from an ATG codon at position 459, encoding a predicted protein of 465 amino acids (52,000 M(r)). Comparison of the nucleotide sequences of the human islet glucokinase cDNA with that of the recently isolated human liver glucokinase cDNA revealed that the two cDNAs differed completely on their 5'-ends, followed by an identical 2204-bp overlap extending to the 3'-ends. The 5'-ends of islet and liver glucokinase cDNAs predicted proteins that differ by 15 NH2-terminal residues. The overall sequence identity (70%) between the first exons of the human islet and rat islet cDNAs suggested that the islet promoter regions, like the liver promoter regions, have been conserved through evolution. Thus, NH2-terminal differences for human liver and islet enzymes might be explained by use of alternate promoters between the two tissues, analogous to the NH2-terminal differences of the rat liver and rat islet enzymes. If so, this relationship predicts important tissue-specific regulatory functions of these regions. Variations in the glucokinase gene are likely to occur in humans. Isolation of a human islet glucokinase cDNA has provided the sequence necessary to determine whether these variants are important determinants in the genetic predisposition for diabetes mellitus.

The prevalence of Type 2 (non-insulin-dependent) diabetes mellitus is high in Mauritius, a multiethnic island nation in the southwestern Indian Ocean. Evaluation of candidate genes in the different ethnic groups represents a means of assessing the genetic component. As glucokinase is known to be a key regulator of glucose homeostasis in liver and pancreatic Beta-cells, the human gene was isolated and a dinucleotide repeat (CA)n marker was identified at this locus. A polymerase chain reaction assay was developed, and alleles differing in size were observed in individuals, according to the number of repeats in the amplified fragment. Eighty-five Creoles and 63 Indians of known glucose tolerance status were typed by amplification of genomic DNA for this dinucleotide (CA)n repeat marker. Four different alleles were observed including Z, the most common allele, and Z + 2, Z + 4, and Z + 10, which differed from Z by 2, 4, and 10 nucleotides respectively. In Mauritian Creoles, the frequency of the Z + 2 allele was greater in Type 2 diabetic subjects than in control subjects (23.8% vs 8.9%, p = 0.008), and the frequency of the Z allele was lower in Type 2 diabetic subjects (60% vs 75.6%, p = 0.03). Analysis with univariate logistic regression models indicated that the Z + 2 allele had the highest odds ratio, 3.08 (95% confidence interval 1.14-8.35, p = 0.0416), among the other risk factors (age, sex, body mass index, and waist/hip ratio). The multivariate odds ratio for Type 2 diabetes was 2.88 (95% confidence interval 0.98-8.50, p = 0.0551).(ABSTRACT TRUNCATED AT 250 WORDS)

Obese individuals are hyperinsulinemic and insulin resistant. Because amylin is cosecreted with insulin and may contribute to the insulin resistance of obesity, this study tested the hypothesis that insulin and amylin genes are coordinately regulated by obesity and carbohydrate feeding. Insulin and amylin gene expression were measured during the suckling/weaning transition in lean (Fa/Fa) and obese (fa/fa) Zucker rats, a period associated with marked changes in tissue insulin sensitivity. There was a decline in insulin mRNA (-90 +/- 15%, P less than 0.01) and amylin mRNA (-72 +/- 21%, P less than 0.01) content in pancreases of lean rats maintained on a high-fat diet from days 15 to 30, probably reflecting the relative increase in exocrine/endocrine development during this neonatal period and the effects of fat feeding. Weaning on high-carbohydrate versus high-fat diets resulted in enhanced expression of both insulin (P less than 0.05) and amylin (P less than 0.05) mRNAs. In contrast to the decline in pancreatic insulin and amylin mRNA content observed in lean rats, there was an increase in insulin mRNA (421.3 +/- 57.5%, P less than 0.05) and no change in amylin mRNA in obese rats maintained on a high-fat diet from days 15 to 30. There was no enhancement of insulin or amylin gene expression in obese rats with high carbohydrate relative to high-fat feeding, perhaps reflecting maximum rates of transcription in these obese insulin-resistant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies of the insulin gene--utilized restriction-fragment--length polymorphisms as markers for potential mutations at this locus. This indirect type of analysis could not define the number of variants that might exist within the structural portions and regulatory regions of the gene in non-insulin-dependent diabetes mellitus (NIDDM) patients. New technology has allowed us to examine insulin genes at the single nucleotide level from 100 American black NIDDM patients. Genomic DNA from patients was amplified by the polymerase chain reaction with primers flanking four regions of the gene: 1) the proximal promoter from positions -182 to 42 (including most of exon 1); 2) exon 1 from 14 to 259, which included the rest of exon 1 and all of the 1st intron; 3) exon 2 from 216 to 452; and 4) exon 3 from 1188 to 1433. One of the primers in each reaction was 32P-end labeled and the resulting products denatured into single strands and electrophoresed on nondenaturing sequencing gels such that mobility was a function of composition and size (single-strand conformational polymorphism or SSCP). Under these conditions, single-base changes in fragments up to 245 nucleotides were detected. Analysis of the proximal promoter region revealed several SSCP patterns in individuals. Direct genomic sequencing of DNA representative of these patterns showed the presence of a common C to G change at position -56 and a C deletion at position -90 in three patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Maturity-onset diabetes of the young (MODY) is a subtype of type 2 diabetes that presents from the second decade and has an autosomal dominant mode of inheritance. We have investigated the glucokinase gene, a candidate gene for diabetes, in two MODY pedigrees. In a large 5-generation pedigree (BX) with 15 diabetic members, use of a microsatellite polymorphism revealed linkage of diabetes to the glucokinase locus on chromosome 7p. A peak lod score of 4.60 was obtained at a recombination fraction (theta) of zero. This finding suggests that a defective glucokinase gene contributes to the diabetes phenotype in this pedigree. This is not universal in MODY since linkage to the glucokinase locus was excluded in a second pedigree M (lod score = -7.36 at theta = 0). The affected members in pedigree BX were diagnosed either when young (in pregnancy or on screening) or when they presented symptomatically in middle and old age; most of them were treated by diet alone. Defects in the glucokinase gene may play an important part in the pathogenesis of type 2 diabetes.

To test the hypothesis that alterations in regulatory regions of the insulin gene occur in a subset of patients with non-insulin-dependent diabetes mellitus (NIDDM), the promoter region was studied by polymerase chain reaction (PCR) amplification directly from genomic DNA, followed by high-resolution polyacrylamide gel electrophoresis under nondenaturing conditions. By using this method a previously identified HincII polymorphism (GTTGAC to GTTGAG at position-56) in American Blacks was readily detected, indicating that single base changes could be observed. In the course of screening the insulin promoter from 40 American Black subjects with NIDDM, an apparent larger allele was found in two individuals. Both patients were shown to have in addition to a normal allele, a larger allele containing an 8-bp repeat, TGGTCTAA from positions -322 to -315 of the insulin promoter. To facilitate rapid screening for the 8-bp repeat, a high-resolution agarose gel electrophoretic analysis was adopted. DNA from American Black NIDDM subjects (n = 100) and nondiabetic subjects (n = 100) was PCR amplified and analyzed. The 8-bp repeat was present in five NIDDM subjects, and one nondiabetic subject. DNA from Mauritius Creoles, also of African ancestry, was analyzed, and the 8-bp repeat was present in 3 of 41 NIDDM subjects, and 0 of 41 nondiabetic subjects. Analysis of glucose metabolism in three presumed normal sibs of an NIDDM patient with an 8-bp repeat revealed that one sib had overt diabetes, and two sibs were glucose intolerant, but there was no consistent segregation of the insulin promoter variant with the diabetes phenotype. The variant promoter was not present in 35 Caucasian NIDDM patients or in 40 Pima Indians. To test the biological consequences of the 8-bp repeat sequence in the insulin promoter, a normal and variant promoter were subcloned into a luciferase plasmid, and reporter gene activity assessed by transient transfection into mouse insulinoma (beta TC1) and hamster insulinoma (HIT) cells. The promoter activity of the variant allele was found to be reduced to 37.9 +/- 10.3% of the activity of the normal promoter in HIT cells (P less than 0.01, n = 4), and 49.1 +/- 6.4% in beta TC1 cells (P less than 0.01, n = 6). These data thus suggest that a naturally occurring variant of the insulin promoter may contribute to the diabetes phenotype in 5-6% of Black NIDDM patients.

To examine the possible involvement of insulin and glucose in regulation of pancreatic islet gene expression, hyperinsulinemic (insulin infusion 4.1 mU/kg per min) clamps were performed for 12 h in rats at two different levels of glycemia (either 3 or 8 mM). A control group received a saline infusion for 12 h. At the end of the 12-h study period, pancreatic RNA was extracted, proinsulin and amylin mRNAs were measured on total RNA, and glucokinase and glucose transporter (GLUT-2) mRNAs were measured on poly(A)+ RNA by dot blot analysis. In insulin-infused hypoglycemic rats, there was a 58% decrement in proinsulin mRNA (P less than 0.01) relative to levels in controls, with no change in amylin, glucokinase, or islet GLUT-2 mRNAs. In insulin-infused hyperglycemic rats, there was a comparable decrement (44%, P less than 0.01) in proinsulin mRNA and a smaller decrement in GLUT-2 mRNA (32%, P less than 0.05), with no change in amylin or glucokinase mRNAs relative to levels in control animals. These studies suggest that insulin has a negative feedback inhibitory effect on its own synthesis. The mechanism of inhibition is unknown. It could be a direct effect of insulin on its own transcription, or alternatively an indirect effect mediated by humoral or neural factors. Sustained hyperinsulinemia may lead to suppression of normal islet beta cells and may contribute to the ultimate hypoinsulinemia of noninsulin-dependent diabetes mellitus.

A new high-resolution genetic linkage map for human chromosome 7p has been constructed. The map is composed of 47 loci (54 polymorphic systems), 19 of which are uniquely placed with odds of at least 1000:1. Four genes are represented, including glucokinase (GCK, ATP:D-hexose-6-phosphotransferase, EC 2.7.1.2) which was mapped via a (CA)n dinucleotide repeat polymorphism. The sex-average map measures 94.4 cM and the male and female maps measure 73.2 and 116.1 cM, respectively. We believe that the genetic map extends nearly the full length of the short arm of chromosome 7 since a centromere marker has been incorporated, and the most distal marker, D7S21, has been cytogenetically localized by in situ hybridization to 7p22-pter. The average marker spacing is 2 cM, and the largest interval between uniquely placed markers is 13 cM (sex-average map). Overall, female recombination was observed to be about 1.5 times that of males, and a statistically significant sex-specific recombination frequency was found for a single interval. The map is based on genotypic data gathered from 40 CEPH reference pedigrees and was constructed using the CRI-MAP program package. The map presented here represents a combined and substantially expanded dataset compared to previously published chromosome 7 maps, and it will serve as a "baseline" genetic map that should prove useful for future efforts to develop a 1-cM map and for construction of a contiguous clone-based physical map for this chromosome.

A compound imperfect dinucleotide repeat element, [CA]4TTTGT[CT]7[CA]9AA[CA]4CCACATA[CA]3, was found approximately 10 kb 3' to the human glucokinase gene (GCK) from analysis of contiguous genomic DNA obtained from a bacteriophage lambda chromosome walk. Direct human genomic sequencing revealed the source of polymorphism to be variable numbers of CT and CA repeats. Altogether six alleles that range in length from +10 to -15 nucleotides compared to the most common (Z) allele have been identified. Alleles Z, Z + 2, and Z + 4 were present in American Blacks, Pima Indians, and Caucasians, with somewhat varied frequencies among the groups. Two alleles, Z + 10 and Z - 15, appear to be unique to American Blacks, while a Z + 6 allele was observed only in the Caucasian population studied. Observed heterozygosity of the polymorphism in the CEPH reference pedigree collection is 44% and the PIC 0.44. The polymorphism is assayed by PCR amplification and resolution of 32P-end-labeled products (ranging in length from 180 to 205 bp) on denaturing polyacrylamide sequencing gels. Using the PCR assay, the human glucokinase gene was physically localized to chromosome 7 in a panel of rodent/human somatic cell lines. Genetic analysis in CEPH pedigrees placed the dinucleotide repeat element, and thereby the human glucokinase gene, on chromosome 7p between TCRG and a RFLP locus D7S57. The glucokinase dinucleotide repeat genetic marker can now be used to assess the role of the glucokinase gene in diabetes by population association studies. In addition, this repeat marker and others flanking it on chromosome 7 can be used in linkage studies with families segregating the disorder.

The content of GLUT4 glucose transporter mRNA and protein were measured in samples of the vastus lateralis muscle of normal volunteers subjected to a 4-h hyperinsulinaemic, euglycaemic clamp. Plasma glucose concentration was clamped at 5.3 +/- 0.1 mmol/l, and serum insulin concentration was maintained at 740 +/- 5 pmol/l. Whole body glucose uptake averaged 38.3 +/- 2.2 mumol.kg-1.min-1, 62% of this being due to disposal via non-oxidative pathways. A significant correlation existed between basal levels of GLUT4 protein and the rate of whole body glucose disposal (r = 0.77, p less than 0.02) and non-oxidative glucose disposal (r = 0.80, p less than 0.02). There was no correlation between GLUT4 protein content and oxidative glucose disposal (r = 0.08, NS). These observations are consistent with an important role for skeletal muscle GLUT4 protein in whole body glucose disposal.

Polymorphisms occur on the average of one out of every 500 base pairs of DNA, and these polymorphisms provide useful markers for genetic analysis. Hundreds of RFLP markers have been mapped at regular intervals throughout the human genome. Diabetes genes have not been mapped with these markers, however, only one MODY family has been partially evaluated. This type of analysis is further complicated if NIDDM is multigenic and/or polygenic. RFLPs have been used to evaluate specific candidate loci for NIDDM, e.g. the insulin, insulin receptor and glucose transporter genes. For these analyses, population and family studies (limited in number) have suggested that none of these loci are major contributors to the genetic susceptibility to NIDDM. In no case, however, could a contribution of 10% or less of these loci be confidently excluded, because of variable penetrance, different degrees of linkage disequilibrium between RFLPs and putative mutations, the frequencies of the RFLPs in non-diabetic populations, and inadequate sample size. The conclusions are clear: either (1) the correct candidate gene(s) has not been found, or (2) sample sizes need to be increased by at least an order of magnitude, or (3) newer methods of analysis must be adopted (e.g. use of extended haplotypes and associations with subphenotypes, or screening with allele specific oligonucleotide probes, denaturing gradient gel electrophoresis or direct genomic sequencing of polymerase chain reaction amplified DNA).

A human liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) cDNA was isolated from a liver cDNA library. This cDNA (hLGLK1) appeared to be full length [2548 base pairs (bp) plus additional poly(A) residues], as its size was consistent with a single 2.8-kilobase (kb) glucokinase mRNA on Northern blot analysis of liver poly(A)+ RNA. The cDNA contained an open reading frame of 1392 bp that predicted a protein of 464 amino acids and a molecular mass of 52 kDa; this protein has 97% identity to rat liver glucokinase. Fourteen residues on the amino terminus of the predicted human liver glucokinase, however, differed completely from those of the predicted rat liver enzyme and could be explained by alternative splicing of a 124-bp cassette exon in human cDNA. A second glucokinase cDNA (hLGLK2), missing the 124-bp cassette exon, was isolated by PCR amplification of human liver cDNA. The hLGLK2 cDNA contained an open reading frame of 1398 bp from an ATG codon at position 164, encoding a predicted protein of 466 residues, 98% identical to the rat enzyme, but different from the predicted protein of hLGLK1 cDNA by 16 amino-terminal residues. In contrast, hLGLK1 cDNA contains multiple initiator codons upstream of the predicted initiator codon at position 294 within the cassette exon. Translation of the two mRNAs in vitro by a reticulocyte lysate system resulted in proteins of the expected size (52 kDa) for both mRNAs; yet hLGLK2 mRNA was translated four to six times more efficiently. These results suggested that the alternative splicing of a cassette exon in hLGLK1 resulted in an mRNA with an upstream initiator codon and reduced function. The relative biological activity of the two isoforms of human glucokinase and their possible developmental and/or metabolic regulation remain to be determined.

Steady-state levels of the major glucose transporter gene (GLUT-1) of the brain were evaluated under three conditions that induced chronic changes in plasma glucose and insulin in adult rats: (i) repeated injection of insulin for 5 days, resulting in plasma glucose levels of 60-70 mg/dl for at least 3 days; (ii) fasting for 3 days; and (iii) moderate streptozotocin-induced diabetes of 1 week duration. Brain GLUT-1 mRNA was measured by dot blot hybridization with a HepG2/erythrocyte (GLUT1) [(32)P]cRNA probe, and GLUT-1 protein by immunoblot analysis with a polyclonal antibody (11493). Insulin injection resulted in hypoglycemia, increased GLUT-1 mRNA (143 +/- 15%, P < 0.05), and increased GLUT-1 protein (141 +/- 6%, P < 0.05). The increase in GLUT-1 mRNA was specific for brain, as no change was observed in liver or kidney. Fasting resulted in mild hypoglycemia, lower plasma insulin, increased GLUT-1 mRNA (131 +/- 17%, P < 0.05 vs control), and no change in GLUT-1 protein (125 +/- 9%, N.S.). Mild streptozotocin diabetes resulted in hyperglycemia, undetectable plasma insulin, decreased GLUT-1 mRNA (65 +/- 6%, P < 0.05 vs control), and no change in GLUT-1 protein (84 +/- 9%, N.S.). A negative correlation (r = -0.61, P < .0001) between GLUT-1 mRNA levels in brain and plasma glucose concentrations was observed among the three experimental groups and control animals, suggesting that the plasma glucose concentration may be at least one determinant of GLUT-1 levels in rat brain. The importance of these results is the finding that GLUT-1 gene expression in rat brain is regulated in vivo by the nutritional and endocrine status of the animal.

Exercise training results not only in enhanced insulin sensitivity but also in a reduction in insulin secretion. In this study, we examined the effects of exercise training on the expression of genes potentially related to insulin synthesis and glucose-stimulated insulin release by measuring pancreatic islet proinsulin, glucose-transporter (GLUT2), and glucokinase mRNAs. Female Wistar rats were subjected to 100 min of running at 25 m.min-1 up a 15% incline for 90 min/day for 6 days/wk for 3 wk. Pancreatic mRNA was evaluated by Northern- and dot-blot analysis with [32P]cRNA probes. We found no change in the pancreatic content of GLUT2 mRNA but found marked decreases in the content of proinsulin mRNA (78%, P less than 0.005) and glucokinase mRNA (65%, P less than 0.001). These results suggest that exercise modulates both islet glucose metabolism and insulin synthesis at the level of gene expression. Furthermore, there was a significant correlation between the decreases in glucokinase and proinsulin mRNA concentrations (r = 0.95, P less than 0.001), suggesting that expression of these genes is regulated in parallel.

These studies compared measurements of in vivo insulin secretion, insulin stores, and insulin synthesis. Rats were studied at 24 wk of age, either 1 or 20 wk after a sham operation (Sham) or 50% pancreatectomy (Px), reducing beta-cell number. By 20 wk after surgery, an adaptation to pancreatectomy was apparent from results of serial glucose tolerance tests, done in a preliminary protocol. Some of the rats also received dexamethasone (ShamDex and PxDex, respectively), imposing insulin resistance. Insulin secretion was assessed with the acute insulin response to arginine under basal (AIRbas) and maximum glucose-potentiated (AIRmax) states. Pancreatic insulin was measured, and insulin synthesis was estimated by measurement of proinsulin mRNA. At 1 wk after surgery, there was no difference among Sham and Px rats in AIRbas, but in the Px rats, expected reductions of AIRmax, pancreatic insulin, and proinsulin mRNA were found. ShamDex rats had a markedly augmented AIRbas and increased AIRmax and proinsulin mRNA. However, pancreatic insulin was reduced both in ShamDex and PxDex rats. At 20 wk after surgery, the predicted adaptation to Px was substantiated by AIRmax and proinsulin mRNA in Px rats not different from those in Sham rats, but pancreatic insulin in the Px rats remained low. AIRbas and proinsulin mRNA were augmented in ShamDex and PxDex rats, but pancreatic insulin was again reduced, and in PxDex rats, low AIRmax and fed hyperglycemia were seen. Linear correlations of AIRbas and AIRmax with proinsulin mRNA were observed over a roughly fourfold range of secretion and synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The liver/islet (GLUT2) and muscle/adipose tissue (GLUT4) glucose-transporter gene products, membrane proteins that facilitate glucose uptake into cells, are important molecules for normal carbohydrate metabolism. Recent isolation of the genes encoding these proteins provides a means to assess the role of possible defects that might contribute to impaired glucose-stimulated insulin secretion or impaired insulin-mediated glucose uptake, both prominent phenotypic features of non-insulin-dependent diabetes (NIDDM). A GLUT2 cDNA clone was isolated from a human liver cDNA library to search for polymorphisms at this locus in American Blacks. Three highly polymorphic sites were identified, one of which (EcoRI-Hae III) appears to be due to an insertion and/or deletion of 200 base pairs of DNA. Significant linkage disequilibrium between these sites over approximately 30 kilobases of genomic DNA suggested that these polymorphisms could be in linkage disequilibrium with mutations at this locus if they exist. A GLUT4 cDNA clone was also utilized to search for polymorphisms at this locus, but only one previously described polymorphism was observed. GLUT2 and GLUT4 cDNA probes were used to evaluate DNA polymorphisms in genomic DNA from American Blacks with NIDDM. The allelic, genotypic, and haplotypic frequencies of the DNA polymorphisms at these loci did not differ from the frequencies in nondiabetic subjects. Because no associations with NIDDM were found, it appears unlikely that mutations at these loci contribute in a major way to the genetic susceptibility to NIDDM observed in American Blacks.

This brief review discusses the current level of understanding of the role of genetic defects in the etiology of non-insulin-dependent diabetes mellitus (NIDDM) and the use of molecular-genetic methods for this study. Evidence for genetic susceptibility is strong, and defects in both insulin production and action are suspect. With restriction-fragment-length polymorphisms and genomic sequencing, various candidate loci are being evaluated. Evidence that multiple genes are involved is only circumstantial. If NIDDM is genetically heterogeneous and also influenced by environmental components, population associations and linkage analyses in families may not be as easily interpreted as for diseases involving single major gene defects.

The relationships among fiber type, glucose transporter (GLUT-4) protein content, and glucose transport activity stimulated maximally with insulin and/or contractile activity were studied by use of the rat epitrochlearis (15% type I-20% type II2a-65% type IIb), soleus (84-16-0%), extensor digitorum longus (EDL, 3-57-40%), and flexor digitorum brevis (FDB, 7-92-1%) muscles. Insulin-stimulated 2-deoxy-D-glucose (2-DG) uptake was greatest in the soleus, followed (in order) by the FDB, EDL, and epitrochlearis. On the other hand, contractile activity induced the greatest increase in 2-DG uptake in the FDB, followed by the EDL, soleus, and epitrochlearis. The effects of insulin and contractile activity on 2-DG uptake were additive in all the muscle preparations, with the relative rates being FDB greater than soleus greater than EDL greater than epitrochlearis. Quantitation of the GLUT-4 protein content with the antiserum R820 showed the following pattern: FDB greater than soleus greater than EDL greater than epitrochlearis. Linear regression analysis showed that whereas a relatively low and nonsignificant correlation existed between GLUT-4 protein content and 2-DG uptake stimulated by insulin alone, significant correlations existed between GLUT-4 protein content and 2-DG uptake stimulated either by contractions alone (r = 0.950) or by insulin and contractions in combination (r = 0.992). These results suggest that the differences in maximally stimulated glucose transport activity among the three fiber types may be related to differences in their content of GLUT-4 protein.

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.

Insulin deficiency is a prominent feature of non-insulin-dependent (NIDDM) and insulin-dependent (IDDM) diabetes mellitus that could result from defects in the insulin gene. Cloning of this gene has permitted molecular-genetic studies including the definition of multiple-DNA-sequence polymorphisms detected with restriction endonucleases, or restriction-fragment-length polymorphisms (RFLPs), and the mapping of the insulin gene to the short arm of chromosome 11 adjacent to the insulinlike growth factor II (IGF-II) and tyrosine hydroxylase genes. The combined RFLPs for the insulin, IGF-II, and tyrosine hydroxylase genes make this a highly informative locus for genetic studies of the insulin gene in diabetes. Early studies of an RFLP consisting of variable-number tandem repeats (VNTR) of DNA near the insulin gene suggested an association of certain alleles with approximately 170 copies of the repeat unit with NIDDM. Although subsequent studies in NIDDM did not confirm this association, an association of different alleles defined by approximately 40 copies of the repeat unit in this VNTR region with IDDM has been demonstrated in multiple studies. This VNTR region and the multiple other RFLPs for this region have been used in linkage analysis to study the segregation of insulin genes in families. These studies have failed to demonstrate a major significant role for insulin-gene defects in NIDDM, maturity-onset diabetes of the young, or IDDM in American Blacks and Whites and under various models of inheritance. Several pedigrees with diabetes and defects of the insulin gene have been described, however, and a minor role for this gene in NIDDM cannot be eliminated from available studies. Similarly, the association studies of the insulin gene and IDDM suggest a minor modifying role undetectable in pedigree studies. The role of defects in or near the insulin gene in a small subset of NIDDM or in IDDM must await direct investigation of the insulin gene in diabetic individuals with the most recent methods for gene amplification and sequence analysis.

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.

To assess the contribution of the HepG2/erythrocyte glucose-transporter (HepG2 GT) gene to the inherited susceptibility to non-insulin-dependent diabetes mellitus (NIDDM), cDNA and genomic probes were used to search for restriction-endonuclease polymorphisms at this locus. Analysis of DNA from 16 unrelated Black American individuals with 19 enzymes and as many as six different probes, defined four polymorphisms over a 45-kilobase region. Nucleotide diversity (pi = 0.006) was low relative to that at other loci, with an average of 1 in 1700 base pairs different between two chromosomes at this locus. The observed combined heterozygosity for these four sites was 0.69, which indicates that the markers at this locus could be useful for linkage analysis in families. Linkage-disequilibrium values between the four polymorphisms were evaluated by pairwise analysis and extended haplotypes. Calculating pairwise associations by the disequilibrium statistic delta or by another measure of disequilibrium, D' (the maximum likelihood of disequilibrium, which is less dependent on frequency), significant linkage disequilibrium could not be demonstrated. However, the frequencies of the observed extended haplotypes were shown to differ (chi 2 = 9.1, df = 2, P less than 0.025) from predicted frequencies if the sites were in linkage equilibrium in Blacks. The frequencies of these four polymorphisms were determined in Black nondiabetic (n = 44) and NIDDM (n = 63) subjects. Neither the allelic nor genotypic frequencies of the polymorphisms differed between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

We sought to determine whether differences in the human insulin gene or its immediate flanking sequences could be found in diabetes. Peripheral leukocyte DNA from 217 unrelated persons, including blacks, whites, and Pima Indians, was analyzed by restriction-enzyme digestion, blotting to nitrocellulose filters, and hybridization to cloned [32P]insulin-gene probes. A region of length variation including deletions (0.1 to 0.2 kilobase pairs) or insertions (0.6 to 5.5 kb) of DNA was found only in the immediate 5' flanking region in 33 per cent of the genes examined. A 1.6-kb insertion accounted for 80 per cent of the polymorphism. This variant was found more often in subjects with non-insulin-dependent diabetes than in nondiabetics, regardless of race (P = 0.011). Length polymorphism in the 5' flanking region of the insulin gene may provide a genetic marker for non-insulin-dependent diabetes.

The purpose of these studies was to determine whether insulin gene expression at the level of proinsulin mRNA could be studied in human pancreas. RNA was isolated from autopsy specimens and analyzed by RNA-blot hybridization with various 32P-human insulin gene probes spanning either the entire gene or the second intervening sequence. A major band 0.62 kilobases (kb) in length accounted for over 95% of the mRNA, consistent in size with presumed mature proinsulin mRNA. In addition, minor bands of 1.5 and 1.3 kb were seen, consistent with an initial gene transcript containing both intervening sequences and with a processed intermediate. The 1.5- and 1.3-kb RNA were confirmed to be proinsulin mRNA precursors by hybridization specifically with the IVS II probe. Total RNA and polyadenylated RNA from five normal pancreata and two insulinomas revealed the same pattern. This method provides a means of determining whether altered insulin gene expression is one cause of diabetes.

The purpose of these experiments was to determine whether insulin-related peptides, larger than proinsulin, could be detected in pancreatic islet cells. Catfish pancreatic islets were incubated with radiolabeled amino acids. After 15- to 60-min incubation, two acid-alcohol-extractable peptides, larger than proinsulin, were detected which were approximately of Mr = 12,000 and 11,000 (12 K and 11K, respectively). They migrated as single polypeptide chains by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis under reducing conditions, and were therefore not aggregates of insulin or proinsulin. The 12 K protein had identical mobility with catfish preoproinsulin synthesized in a wheat germ cell-free system. On standard electrophoresis at pH 8.9, the 12 K protein migrated separately from proinsulin and was at least 65% one protein with two to three minor contaminants. The 12 K and 11 K proteins were chemically related to insulin and proinsulin as shown by tryptic peptide analysis, using cation exchange resin chromatography, and by two-dimensional tryptic peptide maps. Analysis of the tryptic digest of the 12 K protein, compared to proinsulin after leucine aminopeptidase treatment, suggested that the NH2 terminus of the larger protein was different from that of proinsulin. These peptides were specifically bound to anti-insulin antibody. The binding was only 5 to 8% of the protein added, but was specific for the 12 K and 11 K proteins when the immunoprecipitates were examined by electrophoresis and not from contaminating proinsulin. During the continuous incubation of the islets with [3H]leucine, 12 K and 11 K proteins were synthesized in the cell before proinsulin. When islets were first incubated with [3H]leucine for 30 min followed by incubation with excess unlabeled leucine, the 12 K and 11 K proteins appeared to show a precursor-product relationship to proinsulin and insulin. Even when total islet protein synthesis was inhibited by cycloheximide (100 microgram/ml), proinsulin continued to be synthesized for up to 2 h. This suggested that the conversion of the proinsulin precursors to proinsulin in the fish is a post-translational event.

The purpose of these experiments was to obtain proinsulin mRNA from catfish pancreatic islets and synthesize its cDNA. Poly(A)-rich mRNA was electrophoresed on preparative agarose-urea gels. One RNA fraction was obtained which translated predominantly preproinsulin. This mRNA was estimated to be approx. 210 000 Mr (650 nucleotides) when electrophoresed under denaturing conditions. [3H]Proinsulin cDNA was hybridized to excess RNA to monitor purification of mRNA from total islet RNA. Greater than 94% of proinsulin messenger contained poly(A) sequences. [3H]Proinsulin cDNA hybridized to its template mRNA with a Rot 1/2 of 4.4 x 10(-3) mole x sec/l. The overall purification was 80-fold by this type of analysis. Thermal denaturation studies indicated a high degree of fidelity of hybrid formation between [3H]proinsulin cDNA and proinsulin mRNA. Proinsulin comprised 20% of total islet protein when synthesis was measured in vivo (Albert and Permutt, 1979) and 12-20% when total islet mRNA was translated in a cell-free system. Using the [3H]proinsulin cDNA probe it was estimated that proinsulin mRNA accounted for approx. 15% of total islet mRNA.