Isolation and characterization of immunoreactive somatostatin from fish pancreatic islets.

Using a radioimmunoassay with labeled synthetic tetradecapeptide somatostatin, a large amount of immunoreactive somatostatin was found in the principal pancreatic islet of the channel catfish (Ictalurus punctata). The purpose of these experiments was to isolate and characterize the somatostatin-like material. Extracts of islets were chromatographed on a Bio-Gel P-30 column, and over 90% of the immunoreactive somatostatin migrated with proteins at least twice the size of synthetic tetradecapeptide somatostatin. This fraction was further purified by ion-exchange chromatography on carboxymethyl-cellulose and DEAE-cellulose columns. Two peptides were obtained with identical immunoreactivity, which was approximately 25% that of the synthetic somatostatin. Each peptide was judged to be >95% pure by thin-layer electrophoresis, polyacrylamide gel electrophoresis at pH 8.9, and highpressure liquid chromatography. Further criteria of purity included amino-terminal analysis of fraction IV yielding only aspartic acid. A total of 1.3 mg of fraction II, and 3.8 mg of fraction IV somatostatin-like peptides were obtained from 10 g of fresh frozen islets. Characterization of the two peptides revealed both peptides slightly more acidic than synthetic tetradecapeptide somatostatin. Fraction II had an isoelectric point of 8.0-8.3, and fraction IV 8.3-9.0. Molecular weight estimation by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis revealed similar mobility of both peptides, between pancreatic polypeptide (mol wt 4,500) and glucagon (mol wt 3,500). The mobility was not altered by reduction, and was approximately twice the size of synthetic tetradecapeptide somatostatin (mol wt 1,800). This confirmed that the peptides were single polypeptide chains and not aggregates, or somatostatin bound to larger proteins. Molecular weight determination by gel filtration chromatography on Bio-Gel P-6 in 8 M urea gave an estimated mol wt of 3,700. Amino acid analysis of the two immunoreactive somatostatins indicated that they were very similar in composition. Both pancreatic somatostatins (1 muM) had full biological activity relative to synthetic somatostatin measured as inhibition of growth hormone release from rat anterior pituitary cells.

Linkage analysis and molecular scanning of glucokinase gene in NIDDM families.

Mutations in the glucokinase gene are a major cause of maturity-onset diabetes of the young. To evaluate the contribution of this gene to the development of late-onset NIDDM, linkage analyses between DNA polymorphisms at the glucokinase locus and NIDDM were performed in 79 multigenerational French families. In addition, all exons and the islet promoter region of glucokinase gene from 1 affected member from each family as well as from 17 unrelated women with previous gestational diabetes were amplified by polymerase chain reaction and screened for mutations by single-strand conformational polymorphism and DNA sequencing. Linkage of glucokinase and NIDDM was significantly rejected under all models tested. However, in 1 family, the lod score was 2.30, and we found a nucleotide substitution at the position -30 in the islet promoter region that cosegregated with diabetes. The proband of this family was a gestational diabetic individual. No other mutation in glucokinase was found in the 79 NIDDM families. We identified a missense mutation (TGG257-->CGG257) in exon 7 of glucokinase gene from 1 of 17 women with gestational diabetes, which was present in all diabetic members of her family. This family is likely to be a cryptic maturity-onset diabetes of the young, as 4 younger members, carrying this mutation, were subsequently found to be hyperglycemic. In conclusion, no evidence was obtained to incriminate glucokinase as a major gene for late age of onset NIDDM. Diabetic families with mutations in glucokinase must be carefully investigated, to differentiate cryptic maturity-onset diabetes of the young from late-onset NIDDM. Furthermore, pregnancy reveals diabetes in women carrying a glucokinase defect.

Cell-mediated synthesis of somatostatin.

Messenger RNA from catfish pancreatic islets was translated in a wheat germ cell-free protein synthesizing system. A protein of 12,000 mol wt, preprosomatostatin, was identified by specific immunoprecipitaton with anti-catfish pancreatic somatostatin and sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis.

Diabetes mellitus and optic atrophy: a study of Wolfram syndrome in the Lebanese population.

Wolfram syndrome (WFS) is a rare hereditary neurodegenerative disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). WFS seems to be a heterogeneous disease that has not yet been fully characterized in terms of clinical features and pathophysiological mechanisms because the number of patients in most series was small. In this study we describe 31 Lebanese WFS patients belonging to 17 families; this, to our knowledge, is the largest number of patients reported in one series so far. Criteria for diagnosis of WFS were the presence of insulin-dependent diabetes mellitus and optic atrophy unexplained by any other disease. Central diabetes insipidus was found in 87% of the patients, and sensorineural deafness confirmed by audiograms was present in 64.5%. Other less frequent features included neurological and psychiatric abnormalities, urodynamic abnormalities, limited joint motility, cardiovascular and gastrointestinal autonomic neuropathy, hypergonadotropic hypogonadism in males, and diabetic microvascular disease. New features, not reported in previous descriptions, such as heart malformations and anterior pituitary dysfunction, were recognized in some of the patients and participated in the morbidity and mortality of the disease. Genetic analysis revealed WFS1 gene mutations in three families (23.5%), whereas no abnormalities were detected in mitochondrial DNA. In conclusion, WFS is a devastating disease for the patients and their families. More information about WFS will lead to a better understanding of this disease and hopefully to improvement in means of its prevention and treatment.

Defect in insulin action on expression of the muscle/adipose tissue glucose transporter gene in skeletal muscle of type 1 diabetic patients.

Recently several members of the glucose transporter family have been identified by molecular cloning techniques. We determined the effect of a 4-h insulin infusion on the expression of the muscle/adipose tissue (GLUT-4) glucose transporter mRNA and protein in 14 insulin-treated type 1 diabetic patients and 15 matched nondiabetic subjects. GLUT-4 mRNA and protein concentrations were determined in muscle biopsies taken before and at the end of the insulin infusion during maintenance of normoglycemia. In response to insulin, muscle GLUT-4 mRNA increased in the nondiabetic subjects from 24 +/- 3 to 36 +/- 4 pg/microgram RNA (P less than 0.001) but remained unchanged in the insulin-resistant diabetic patients (24 +/- 2 vs. 26 +/- 2 pg/microgram RNA, before vs. after insulin). The glucose transporter protein concentrations were similar in the basal state and decreased by 21 +/- 7% (P less than 0.02) in the normal subjects but remained unchanged in the diabetic patients. The increase of the GLUT-4 mRNA and the decrease in the GLUT-4 protein correlated with the rate of glucose uptake [correlation coefficient (r) = -0.55, P less than 0.01, and r = -0.44, P less than 0.05, respectively]. We conclude that the insulin response of both the GLUT-4 glucose transporter mRNA and protein are absent in skeletal muscle of insulin-resistant type 1 diabetic patients. Thus, impaired insulin regulation of glucose transporter gene expression can be one of the underlying mechanisms of insulin resistance in type 1 diabetes.

Altered glucose tolerance, insulin response, and insulin sensitivity after massive weight reduction subsequent to gastric bypass.

We have studied an otherwise normal group of morbidly obese subjects and compared them with patients who had experienced massive weight loss after loop gastric bypass. Compared to normal controls (NLCs), morbidly obese control patients (OBCs) had abnormal glucose tolerance curves (after glucose ingestion), elevated basal insulin levels, and increased plasma insulin concentrations, suggesting insulin insensitivity. The latter has been corroborated by the measurement of decreased insulin binding in these patients. Postoperative (PO) patients were hyperglycemic after taking oral glucose, but all PO patients had a rapid decrease in plasma glucose concentration, half reaching hypoglycemic levels. PO basal insulin levels and insulin receptor number were not statistically different from those in NLCs, indicating up-regulation of insulin receptors (and therefore, increased insulin sensitivity) postoperatively. Hyperinsulinemia seen in the PO group (greater than that in OBCs, P less than 0.001) after administration of oral glucose occurred simultaneously with a doubling of plasma concentration of gastric inhibitory polypeptide. Massive weight loss in patients after gastric bypass was accompanied by an improvement in insulin receptor number, basal hyperinsulinemia, and glucose tolerance. In addition, postoperative patients demonstrated symptomatic reactive hypoglycemia which may have resulted from the hyperinsulinemia seen subsequent to ingestion of glucose.

The biological activity of catfish pancreatic somatostatin.

Catfish pancreatic somatostatin, which contains eight additional amino acids on the amino terminus of a tetradecapeptide with considerable homology to tetradecapeptide somatostatin (SRIF), is a naturally occurring homolog of the hypothalamic peptide. The purpose of these studies was to determine the biological activity of this somatostatin homolog. Inhibition of 125I-labelled tyr1-SRIF binding to bovine pituitary plasma membranes by catfish pancreatic somatostatin was approximately 33% that of SRIF. Pancreatic somatostatin had full biological activity measured by inhibition of growth hormone release from isolated rat pituitary cells, but 0.01-0.1% the potency of SRIF. Pancreatic somatostatin at 100 ng/ml produced a 50-60% inhibition of insulin and glucagon secretion from perfused rat pancreas, while SRIF produced comparable inhibition at 10 ng/ml. This report demonstrates that a larger molecular form and natural homolog of SRIF, isolated from fish pancreas, has the same (but reduced) biological activities in rat assay systems as somatostatin originally isolated from sheep hypothalamus.

Insulin biosynthesis and diabetes mellitus.

This review reports the use of recombinant DNA techniques in the study of the structure and regulation of expression of insulin genes in man and experimental animals. Insulin biosynthesis by pancreatic islet cells is predominantly regulated by change in plasma glucose concentration. Using a cell-free protein synthesizing system as an assay of functional proinsulin messenger RNA (mRNA), and hybridization analysis with a cloned DNA complementary to proinsulin mRNA, it has been determined that through changes in proinsulin mRNA levels. Insulin genes of the rat, chicken and human have been isolated and sequenced. The 5' ends of the genes have similar sequences suggesting areas important for regulation of transcription. There are two non-allelic insulin genes in the rat, but only one in chickens and humans. Intervening sequences, areas of DNA transcribed into precursor mRNA but which do not appear in mature mRNA, have been described within insulin genes. The insulin gene resides on chromosome 11 of humans as determined by DNA hybridization analysis of mouse human hybrid cells. The structure of the insulin gene in genomic DNA of humans has been analyzed in diabetics and non-diabetics. Insertions of DNA between 1500 and 3400 base pairs have been detected near the transcription initiation site in 65% of type II diabetics, and 25-30% of non-diabetics (this difference is significant at the p less than 0.001 level). Limitation of these insertions to this potential promotor region of the insulin gene suggests that they may alter gene expression in type II diabetes. These insertions of DNA may prove to be useful genetic markers for diabetes.

Amino acid sequence of catfish pancreatic somatostatin I.

Two peptides, pancreatic somatostatins I and II, larger and more acidic than synthetic tetradecapeptide somatostatin, have been purified from pancreatic islets of channel catfish (Ictalurus punctata). These peptides have reduced immunoreactivity in a radioimmunoassay for synthetic somatostatin, but full biological activity was measured as inhibition of growth hormone released from isolated rat anterior pituitary cells. Pancreatic somatostatin I is composed of 22 amino acids. Eight additional amino acids are found as an NH2-terminal extension of the segment which is homologous to synthetic tetradecapeptide somatostatin. Seven of fourteen residues of tetradecapeptide somatostatin are present in the COOH-terminal portion of catfish pancreatic somatostatin I. The sequence is NH2-Asp-Asn-Thr-Val-Arg-Ser-Lys-Pro-Leu-Asn-Cys-Met-Asn-Tyr-Phe-Trp-Lys-Ser-Ser-Thr-Ala-Cys-COOH. There is considerable homology with the carboxyl end of synthetic tetradecapeptide somatostatin.

Insulin resistance in type 2 (non-insulin-dependent) diabetic patients and their relatives is not associated with a defect in the expression of the insulin-responsive glucose transporter (GLUT-4) gene in human skeletal muscle.

To study whether insulin resistance in Type 2 (non-insulin-dependent) diabetes mellitus is due to a defect in the expression of the insulin-responsive glucose transporter gene (GLUT-4) in human skeletal muscle, we measured the level of GLUT-4 mRNA and (in some of the subjects) its protein in muscle biopsies taken from 14 insulin-resistant patients with Type 2 diabetes, 10 first-degree relatives of the diabetic patients and 12 insulin-sensitive control subjects. Insulin sensitivity was measured with a + 45 mU.m2(-1).min-1 euglycaemic insulin clamp in combination with indirect calorimetry and infusion of [3-3H]glucose. GLUT-4 mRNA was measured using a human GLUT-4 cDNA probe and GLUT-4 protein with a polyclonal antibody specific for the 15 amino acid carboxy-terminal peptide. Both Type 2 diabetic patients and their relatives showed impaired stimulation of total-body glucose disposal by insulin compared with control subjects (29.5 +/- 2.1 and 34.0 +/- 4.8 vs 57.9 +/- 3.1 mumol.kg lean body mass-1.min-1; p less than 0.01). This impairment in glucose disposal was primarily accounted for by a reduction in insulin-stimulated storage of glucose as glycogen (13.0 +/- 2.4 and 15.6 +/- 3.9 vs 36.9 +/- 2.2 mumol.kg lean body mass-1.min-1; p less than 0.01). The levels of GLUT-4 mRNA expressed both per microgram of total RNA and per microgram DNA, were higher in the diabetic patients compared with the control subjects (116 +/- 25 vs 53 +/- 10 pg/microgram RNA and 177 +/- 35 vs 112 +/- 29 pg/microgram DNA; p less than 0.05, p less than 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic islet cells express a family of inwardly rectifying K+ channel subunits which interact to form G-protein-activated channels.

Insulin secretion is associated with changes in pancreatic beta-cell K+ permeability. A degenerate polymerase chain reaction strategy based on the conserved features of known inwardly rectifying K+ (KIR) channel genes was used to identify members of this family expressed in human pancreatic islets and insulinoma. Three related human KIR transcript sequences were found: CIR (also known as cardiac KATP-1), GIRK1, and GIRK2 (KATP-2). The pancreatic islet CIR and GIRK2 full-length cDNAs were cloned, and their genes were localized to human chromosomes 11q23-ter and 21, respectively. Northern blot analysis detected CIR mRNA at similar levels in human islets and exocrine pancreas, while the abundance of GIRK2 mRNA in the two tissues was insufficient for detection by this method. Using competitive reverse-transcription polymerase chain reaction, CIR was found to be present at higher levels than GIRK2 mRNA in native purified beta-cells. Xenopus oocytes injected with M2 muscarinic receptor (M2) plus either GIRK2 or CIR cRNA expressed only very small carbachol-induced currents, while co-injection of CIR plus GIRK2 along with M2 resulted in expression of carbachol-activated strong inwardly rectifying currents. Activators of KATP channels failed to elicit currents in the presence or absence of co-expressed sulfonylurea receptor. These results show that two components of islet cell KIR channels, CIR and GIRK2, may interact to form heteromeric G-protein-activated inwardly rectifying K+ channels that do not possess the typical properties of KATP channels.