Robert Turner

UKPDS 25: autoantibodies to islet-cell cytoplasm and glutamic acid decarboxylase for prediction of insulin requirement in type 2 diabetes

BACKGROUNDnAutoantibodies to islet-cell cytoplasm (ICA) and glutamic acid decarboxylase (GADA) can occur in apparently typical, non-insulin dependent diabetes mellitus (type 2). We investigated whether the presence of either or both antibodies characterises a subtype of diabetes and provides better prediction of requirement for insulin therapy by 6 years follow-up than clinical variables.nnnMETHODSnWe measured ICA and GADA at diagnosis of diabetes in a representative population of 3672 white patients with type 2 diabetes, aged between 25 and 65 years. The phenotype was assessed by age of onset, body-mass index, percentage haemoglobin A1c (HbA1c), and islet beta-cell function. We investigated the need for insulin therapy among 1538 patients not assigned insulin and followed up for 6 years from diagnosis.nnnFINDINGSnThe proportion of patients with ICA and GADA decreased with increasing age at diagnosis (from 33 [21%] of 157 patients aged 25-34 [corrected] to 66 [4%] of 1769 aged 55-65 for ICA; from 53 [34%] to 122 [7%] for GADA). Among patients younger than 35 at diagnosis, those with ICA or GADA had lower body-mass index than those without (mean 24.9 [SD 6.0] vs 31.7 [7.3] kg/m2; p < 0.0001 and had higher percentage of HbA1c (9.7 vs 8.7%, p < 0.05). 94% of patients with ICA and 84% of those with GADA required insulin therapy by 6 years, compared with 14% of those without the antibodies (p < 0.0001). Among patients older than 55 at diagnosis, the difference between those with and without antibodies in body-mass index was smaller (27.2 [5.4] vs 28.6 [4.8] kg/m2, p < 0.001); 44% of those with ICA, 34% of those with GADA, and 5% with neither antibody required insulin therapy by 6 years (p < 0.0001). Among patients older than 45 years, body-mass index and HbA1c provided little predictive information for insulin requirement, whereas the positive predictive values of GADA (> or = 60 U/L) alone, or both GADA (> or = 20 U/L) and ICA (> 5 U/L), for insulin therapy were 52% and 68%.nnnINTERPRETATIONnAmong young adults with type 2 diabetes, the phenotype of those with ICA or GADA antibodies was similar to that of classic juvenile-onset insulin-dependent diabetes, and either phenotype or antibodies predicted insulin requirement. In older adults, the phenotype was closer to that of patients without antibodies and only the presence of antibodies predicted an increased likelihood of insulin requirement.

Impaired Pulsatile Secretion of Insulin in Relatives of Patients with Non-Insulin-Dependent Diabetes

In fasting nondiabetic subjects, insulin is secreted in regular pulses every 12 to 15 minutes, but patients with non-insulin-dependent diabetes lack regular oscillatory insulin secretion. To investigate whether abnormal insulin oscillations are an early feature of diabetes, we studied 10 minimally glucose-intolerant first-degree relatives of patients with non-insulin-dependent diabetes and 10 controls matched for age and obesity. We performed a time-series analysis of fasting plasma insulin levels in blood samples obtained at 1-minute intervals for 150 minutes. Fasting plasma glucose levels were higher in the relatives than in the controls (mean +/- SD, 5.4 +/- 0.7 vs. 4.4 +/- 0.3 mmol per liter). Autocorrelation of pooled data showed no regular oscillatory activity in the relatives but a 13-minute cycle in the controls (r = 0.23, P less than 0.001). Similarly, Fourier transform analysis showed no significant peak in the relatives but the expected significant peak at 13 to 14 minutes in the controls (P less than 0.05). First-phase (0 to 10 minutes) insulin secretory responses to glucose administered intravenously were not significantly impaired in the relatives (geometric mean, 188 pmol per liter [26.2 mU per liter]; range of SD, +103 to -67 pmol per liter [+14.4 to -9.3 mU per liter]), as compared with the controls (geometric mean, 231 pmol per liter [32.2 mU per liter]; range of SD, +131 to -83 pmol per liter [+18.2 to -11.6 mU per liter]). We conclude that abnormal oscillatory insulin secretion may be an early phenomenon in the development of non-insulin-dependent diabetes.

Comparison of insulin sensitivity tests across a range of glucose tolerance from normal to diabetes

Aims/hypothesis. Adequate comparison of the relative performance of insulin sensitivity tests is not yet available. We compared the discrimination of four insulin sensitivity tests, commonly used in vivo, across a range of glucose tolerance. Methods. Normal (n = 7), impaired glucose tolerant (n = 8) and Type II (non-insulin-dependent) diabetic subjects (n = 9) had in random order two tests from the following: frequently sampled insulin-modified intravenous glucose tolerance test (FSIVGTT-MinMod); homeostasis model assessment (HOMA) and 2-h continuous infusion of glucose with model assessment (CIGMA) with immunoreactive or specific insulin; short insulin tolerance tests (ITT). The discriminatory power of tests was assessed by the ratio of the within-subject standard deviation to the underlying between-subject standard deviation (discriminant ratio –DR). The degree to which tests measured the same variable was assessed by comparing rank correlation with the maximum expected correlation given the imprecision of the tests. The unbiased lines of equivalence taking into account the precision of tests were constructed. Results. Reciprocal fasting plasma insulin (FPI–1), HOMA %S and 2-h CIGMA %S, had similar DRs with ITT being less informative. The FSIVGTT-MinMod analysis was able to assess 13 out of 24 subjects and had a performance similar to ITT. Using specific rather than immunoreactive insulin for HOMA-CIGMA did not improve the DR. Reciprocal fasting plasma insulin FPI–1, HOMA %S, 2-h CIGMA %S and SI FSIVGTT intercorrelated more than 90 % of the expected rank correlation given the imprecision of the tests, but ITT gave only limited correlation. Conclusion/interpretation. The HOMA-CIGMA test with immunoreactive insulin provides similar information in distinguishing insulin sensitivity between subjects with normal glucose tolerance, those with impaired glucose tolerance and those with Type II diabetes as does FSIVGTT, whereas ITT is less informative. [Diabetologia (1999) 42: 678–687]

Sequence Variants in the Sulfonylurea Receptor (SUR) Gene Are Associated With NIDDM in Caucasians

NIDDM is a common heterogeneous disorder, the genetic basis of which has yet to be determined. The sulfonylurea receptor (SUR) gene, now known to encode an integral component of the pancreatic β-cell ATP-sensitive potassium channel, IKATP, was investigated as a logical candidate for this disorder. The two nucleotide-binding fold (NBF) regions of SUR are known to be critical for normal glucose regulation of insulin secretion. Thus, singlestrand conformational polymorphism analysis was used to find sequence changes in the two NBF regions of the SUR gene in 35 NIDDM patients. Eight variants were found; and three were evaluated in two Northern European white populations (Utah and the U.K.): 1) a missense mutation in exon 7 (S1370A) was found with equal frequency in patients (n = 223) and control subjects (n = 322); 2) an ACC→ACṮ silent variant in exon 22 (T761T) was more common in patients than in control subjects (allele frequencies 0.07 vs. 0.02, P = 0.0008, odds ratio (OR) 3.01, 95% CI 1.54–5.87); and 3) an intronic t→c change located at position –3 of the exon 24 splice acceptor site was also more common in patients than in control subjects (0.62 vs. 0.46, P < 0.0001, OR 1.91, 95% Cl 1.50–2.44). The combined genotypes of exon 22 C/T or T/T and intron 24 –3c/–3c occurred in 8.9% of patients and 0.5% of control subjects (P < 0.0001, OR 21.5,95% CI 2.91–159.6). These results suggest that defects at the SUR locus may be a major contributor to the inherited basis of NIDDM in Northern European Caucasians.

High Prevalence of Hepatitis C Infection in Afro-Caribbean Patients with Type 2 Diabetes and Abnormal Liver Function Tests

Moderate elevations of serum transaminases are frequently found in patients with diabetes mellitus and are often attributed to fatty infiltration of the liver without further investigation. Recent studies of patients with end‐stage liver disease have suggested a possible association between Hepatitis C virus (HCV) antibody positivity and the development of diabetes (mostly Type 2). As a first step in the examination of any potential association between HCV and Type 2 diabetes in subjects without overt liver disease, we examined 200 British patients with Type 2 diabetes (100 White Caucasians, 50 Asians, and 50 Afro‐Caribbeans), recruited from the United Kingdom Prospective Study of Diabetes, half of whom had a significant elevation of alanine aminotransferase (ALT) on at least two occasions and half of whom had consistently normal ALT levels. In Afro‐Caribbean Type 2 diabetic subjects 7/25 (28%) patients with abnormal ALT and 1/25 (4%) with normal ALT were HCV antibody positive. Among White Caucasian subjects 6/50 (12%) patients with abnormal LFTs and 0/50 with normal LFTs were HCV antibody positive and in Asians the prevalence was 2/25 (8%) and 0/25, respectively. This study suggests that persistent mild to moderate elevation of serum transaminases in a patient with Type 2 diabetes should not automatically be attributed to the metabolic disturbances of diabetes. Particularly in Afro‐Caribbean subjects, HCV infection is a major diagnostic consideration. The question of whether HCV infection itself may have a diabetogenic action is worthy of further investigation.

Sequence variations in the human Kir6.2 gene, a subunit of the beta-cell ATP-sensitive K-channel: no association with NIDDM in while Caucasian subjects or evidence of abnormal function when expressed in vitro.

SummaryThe ATP-sensitive K-channel plays a central role in insulin release from pancreatic beta cells. This channel consists of two subunits: a sulphonylurea receptor, SUR1, and an inwardly rectifying K-channel subunit, Kir6.2. We screened 135 white Caucasian patients with non-insulin-dependent diabetes mellitus (NIDDM) and 90 non-diabetic subjects for mutations in the Kir6.2 gene by single-stranded conformational polymorphism (SSCP) analysis. We identified one silent mutation (A190A) and four missense mutations (E23K, L270V, I337V and S385C) in normal and diabetic individuals. In a single diabetic subject, we identified a two-amino acid insertion (380KP). We also screened 39 Afro-Caribbean diabetic subjects and identified one additional missense (L355P) and one more silent (S363S) mutation. The E23K and I337V variants were completely linked. The common variants (E23K, I337V and L270V) were found with similar frequency in diabetic and normal subjects. Diabetic subjects with the variants responded normally to sulphonylurea therapy. When mutant Kir6.2 subunits were coexpressed with SUR1 inXenopus oocytes, there was no difference in the sensitivity of the whole-cell currents to metabolic inhibition or to the sulphonylurea tolbutamide. We therefore conclude that mutations in Kir6.2 are unlikely to be a major cause of NIDDM.

Mutations in the Hepatocyte Nuclear Factor-1α Gene in MODY and Early-Onset NIDDM: Evidence for a Mutational Hotspot in Exon 4

We have recently shown that mutations in the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1α are the cause of one form of maturity-onset diabetes of the young (MODY3). Here, we report the exon-intron organization and partial sequence of the human HNF-1α gene. In addition, we have screened the ten exons and flanking introns of this gene for mutations in a group of 25 unrelated white subjects from Germany who presented with NIDDM before 35 years of age and had a first-degree relative with NIDDM. Mutations were identified in nine of these individuals, suggesting that mutations in the HNF-1α gene are a common cause of diabetes in German subjects with early-onset NIDDM and a family history of diabetes. Thus, screening for mutations in this gene may be indicated in subjects with early-onset NIDDM. Interestingly, three of the nine mutations occurred at the same site in exon 4 with insertion of a C in a polyCtract, centered around codon 290 (designated Pro291fsinsC), thereby resulting in a frameshift during translation and premature termination. Analyses of linked DNA polymorphisms in the HNF-1α gene indicated that the Pro291fsinsC mutation was present on a different haplotype in each subject, implying that the polyC tract represents a mutational hot spot. We have also identified the mutation in the HNF-1α gene in the Jutland pedigree, one of the original MODY pedigrees reported in the literature, as being a T→G substitution in codon 241, resulting in the replacement of a conserved Cys by Gly (C241G). The information on the sequence of the HNF-1α gene and its promoter region will facilitate the search for mutations in other subjects and studies of the role of the gene in determining normal β-cell function.

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.

Detection of Mutations in Insulin-Receptor Gene in NIDDM Patients by Analysis of Single-Stranded Conformation Polymorphisms

We used the recently described technique of single-stranded conformation-polymorphism (SSCP) analysis to examine the insulin-receptor locus. First, the ability of the method to detect known mutations and polymorphisms in the insulin-receptor coding sequence was assessed. Regions of the insulin-receptor sequence containing 16 different nucleotide changes, 9 in patient genomic DNA and 7 as cloned cDNA in plasmids, were analyzed. All 9 patient genomic DNA mutants and 5 of 7 plasmid mutants exhibited variant SSCP patterns. To investigate the potential of the technique for screening many patients, the 5 exons that encode the tyrosine kinase domain of the insulin receptor were examined in 30 unrelated white subjects with non-insulin-dependent diabetes mellitus (NIDDM). Exons 17–21 were amplified from genomic DNA with polymerase chain reaction and subjected to SSCP analysis. Exons 19, 20, and 21 revealed no bands of aberrant migration, suggesting a high degree of conservation of these sequences. One diabetic subject had an SSCP variant in exon 18. Direct sequencing of this subjects genomic DNA revealed a heterozygous missense mutation (Lys1068 → Glu1068). Five different SSCP patterns were detected in exon 17. Based on direct sequencing, these patterns were explained by combinations of three different nucleotide substitutions, two of which were common silent polymorphisms. One subject had a heterozygous missense mutation Val985 → Met985. Allele-specific oligonucleotide hybridization confirmed the presence of these mutations in the appropriate diabetic subjects and also detected the Val985 mutation in heterozygous form in 1 of 13 nondiabetic white subjects. SSCP analysis is a sensitive rapid method for screening for mutations in the insulin-receptor gene. Using SSCP, we detected two previously unreported amino acid substitutions in the highly conserved tyrosine kinase domain of the insulin receptor. These represent the first potentially significant mutations found by screening candidate genes in NIDDM. The detection of one of these variants in a normoglycemic subject suggests that it is unlikely to cause the diabetic state, but given the complex genetic basis for NIDDM, a possible contributory role of each of these mutations mandates further study.

LOCALISATION OF INSULINOMAS

Small insulinomas may be undetectable by arteriography or palpation of the pancreas. They can be identified, however, by the point at which high insulin levels are detected in the venous effluent sampled at several sites by catheterisation of the splenic and portal veins at laparotomy or via the percutaneous transhepatic route. Diagnosis by catheterisation techniques made it possible to resect only that part of the pancreas containing the tumour (in one patient the head and in another the body of the pancreas). Improved localisation is essential in planning an operation, and often removes the need for lengthy trials of medical therapy before laparotomy.