Adrenergically mediated intrapancreatic control of the glucagon response to glucopenia in the isolated rat pancreas.

Alpha adrenergic blockade with phentolamine (10 microM) reduces the glucagon response to severe glucopenia (from 150 to 25 mg/dl) to 22% of the control values in the isolated perfused rat pancreas. Propranolol (10 microM) had no significant effect. Neither alpha nor beta adrenergic blockade reduced the magnitude of glucopenic suppression of insulin secretion, but phentolamine increased insulin levels before and during glucopenia. The pattern of somatostatin secretion in these experiments resembled that of insulin. Depletion of norepinephrine from sympathetic nerve endings by pretreatment with 6-hydroxydopamine lowered the pancreatic norepinephrine content to less than 20% of control values and reduced the glucagon response to glucopenia to 69% of the controls. Combined alpha and beta adrenergic blockade during less severe glucopenia (from 120 to 60 mg/dl) reduced the glucagon response to 21% of controls. However, slight glucopenia (from 100 to 80 mg/dl), which elicited only 11% increase in glucagon in the control experiments, was not altered significantly by combined alpha and beta adrenergic blockade. Morphologic studies of adrenergic nerve terminals labeled with [3H]norepinephrine revealed associations with alpha cells. It is concluded that in the isolated rat pancreas adrenergic mediation accounts for most of the glucagon but not insulin response to glucopenia. It is controlled within the pancreas itself, possibly through a direct enhancement by glucopenia of norepinephrine release from nerve endings.

Insulin within islets is a physiologic glucagon release inhibitor.

To determine if glucagon secretion is under physiological control of intra-islet insulin, pancreata from normal rats were perfused at a 100 mg/dl glucose concentration with either guinea pig antiinsulin serum or normal guinea pig serum in a nonrecirculating system. Perfusion of antiserum was followed within 3 min by a significant rise in glucagon that reached peak levels three times the base-line values and assumed a hectic pattern that returned rapidly to base-line levels upon termination of the antiserum perfusion. Nonimmune guinea pig serum had no effect. To gain insight into the probable site of insulin neutralization, 125I-labeled human gamma-globulin was added to antiserum or nonimmune serum and perfused for 3 min. More than 83% of the radioactivity was recovered in the effluent within 3 min after termination of the infusion, and only 0.05 +/- 0.015% of the radioactivity injected was present in the pancreas 10 min after the perfusion. The maximal amount of insulin that could be completely bound to insulin antibody at a dilution and under conditions simulating those of the perfusion experiments was 20 mU/min. It is concluded that insulin maintains an ongoing restraint upon alpha cell secretion and in its absence causes hectic hypersecretion of glucagon. This restraint probably occurs largely in the intravascular compartment. Loss of this release-inhibiting action of insulin may account for initiation of hyperglucagonemia in insulin-deficient states.

Defective processing of insulin-receptor precursor in cultured lymphocytes from a patient with extreme insulin resistance.

We have studied a patient with extreme insulin resistance, acanthosis nigricans, and decreased erythrocyte insulin binding. EBV-transformed lymphocytes from this patient exhibited markedly reduced binding of 125I-insulin. Radioiodination of cell surface receptors followed by immunoprecipitation with anti-receptor antibodies revealed the presence of increased amounts of a 210-kD protein but no detectable alpha or beta subunits. Continuous labeling with 2-[3H]mannose revealed the synthesis of a 190-kD precursor and a 210-kD protein. The 210-kD protein was phosphorylated in an insulin-dependent manner at high insulin concentrations. These results suggest that in this patient the biosynthesis of 190-kD receptor precursor, its terminal glycosylation, and intracellular transport to the cell surface proceed normally, while proteolytic maturation to alpha and beta subunits does not occur. We postulate that this defect either results from mutation(s) within the insulin-receptor gene, which render the precursor resistant to cleavage, or from a defect in the receptor processing enzyme.

Involvement of the Ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma.

Although matrix metalloproteinases (MMPs) are thought to be involved in the invasion and metastasis of a variety of malignant tumors, including human hepatocellular carcinoma (HCC), the mechanisms for the expression of MMPs in HCC are not known. To understand the mechanism(s) of MMP expression, the expression of matrilysin (MMP-7) and several genes of the Ets transcription factor family was investigated in human HCC and hepatoma-derived cell lines. The role of Ets-1 gene expression in HCC was also studied. Analysis by semiquantitative reverse transcription-PCR revealed that MMP-7 and Ets-1 are overexpressed and closely associated in HCC. To clarify the role of Ets-1, hepatoma cells were transduced with human Ets-1 or targeted with the Ets-1-specific antisense oligonucleotides. Cells stably transduced with the Ets-1 gene showed increased MMP-7 expression compared to parental and mock-transfected cells. Cells targeted with Ets-1-specific antisense oligonucleotides showed reduced expression of MMP-7. Cotransfection of cells with a MMP-7 promoter-reporter gene plasmid and an Ets-1 expression vector yielded an increase in MMP-7 promoter activity in an Ets-1-responsive element-dependent manner. Taken together, these data suggested that the Ets-1 oncogene is up-regulated and involved in the overexpression of MMP-7 in human HCC and may contribute to the progression of HCC.

Secretin inhibits glucagon in the isolated perfused dog pancreas.

To investigate the effect of secretin upon islet hormone secretion, highly purified porcine secretin was perfused at 0.0001, 0.001, 0.01, and 0.1 clinical unit (CU)/ml in the isolated normal dog pancreas. The lowest concentration of secretin, a level which is within the physiologic range, significantly suppressed glucagon release and stimulated somatostatin release but was without significant effect on insulin release. At each concentration of secretion tested glucagon was dramatically suppressed and somatostatin was significantly stimulated, both in a dose-dependent manner. B-cell activity was first augmented by the concentration of 0.001 CU/ml (94 pM) of secretin. These data demonstrate that physiologic levels of secretin suppress pancreatic A-cell activity in the isolated dog pancreas.

Release of amylin from perfused rat pancreas in response to glucose, arginine, beta-hydroxybutyrate, and gliclazide.

Amylin is a 37-amino acid peptide isolated from the islet amyloid of patients with non-insulin-dependent diabetes mellitus. The isolated perfused normal rat pancreas was used to evaluate the effects of glucose and insulin secretagogues, such as arginine, beta-hydroxybutyrate, and gliclazide, on amylin secretion. Glucose and the other stimulants tested elicited a significant release of amylin from the rat pancreas in a biphasic pattern, similar to that of insulin. Dose-response studies of the glucose-induced release of amylin and insulin revealed that they possessed a similar dependency on glucose. However, the release of amylin induced by high concentrations of glucose was partially dissociated from that of insulin; that is, the amylin-insulin molar ratios induced by 22.2 and 33.3 mM glucose (1.11 +/- 0.05 and 1.05 +/- 0.04%, respectively) were significantly higher than those induced by 16.7 mM glucose (0.90 +/- 0.04%, P less than 0.01 vs. 22.2 mM glucose, P less than 0.05 vs. 33.3 mM glucose). Additionally, when the basal concentration of glucose in the perfusate was increased from 5.6 to 11.1 mM, the response of amylin was unchanged. These data suggest that amylin may be an islet hormone whose abundant response to high concentrations of glucose might contribute to the oversecretion of amylin in the hyperglycemia that accompanies diabetes mellitus.

Relative hypersecretion of amylin to insulin from rat pancreas after neonatal STZ treatment.

With isolated perfused pancreases from normal and diabetic model rats, we studied alterations of the secretion of islet amyloid polypeptide, or amylin, which has been recently identified as a major component of amyloid deposits in the pancreatic islets of patients with non-insulin-dependent diabetes mellitus. Neonatal (n) Wistar-King albino rats given streptozocin (STZ) on the 2nd (n2STZ) or 5th (n5STZ) neonatal day exhibited moderate and marked elevations, respectively, of plasma glucose and HbA1 as adults compared with control rats given the vehicle. The release of amylin from the perfused pancreases in response to glucose and arginine paralleled that of insulin in all three groups. However, the molar ratio of secreted amylin to insulin in response to 16.7 mM glucose by n5STZ pancreases (6.55 +/- 0.71%) was significantly greater than that for either n2STZ (1.71 +/- 0.24%, P less than 0.05) or the control (0.60 +/- 0.03%, P less than 0.05) pancreases. The secreted amylin-insulin ratio of n2STZ pancreases also was significantly greater than that of the controls (P less than 0.05). The increased amylin-insulin molar ratios of both n2STZ and n5STZ pancreases also occurred during infusions of 33.3 mM glucose and 10 mM arginine. These findings suggest that amylin secretion may be preserved in diabetic rats with reduced beta-cell mass and that hyperglycemia may increase amylin production independently of that of insulin, which may be significant in the pathogenesis of non-insulin-dependent diabetes mellitus.

Insulin resistance by uncleaved insulin proreceptor. Emergence of binding site by trypsin.

Two sisters presented with severe insulin resistance and markedly decreased insulin binding to erythrocytes, cultured fibroblasts, and transformed lymphocytes. The dose-response curve of insulin-stimulated amino acid uptake in the fibroblasts was shifted to the right. The molecular weight of the insulin receptor on the transformed lymphocytes from the patients was 210,000 and could not be dissociated to alpha- and beta-subunits by dithiothreitol treatment. However, the proreceptor was cleaved by trypsin, and this led to production of a 135,000-Mr alpha-subunit. Insulin binding to the trypsin-treated cells increased to the normal level, and insulin action was normalized. These results suggest that the failure of proreceptor cleavage produces hormone-resistant states and that a proreceptor syndrome may be a unique disease entity for hormone resistance.

Clarification of signaling pathways mediated by insulin and insulin-like growth factor I receptors in fibroblasts from patients with specific defect in insulin receptor.

Receptor binding and biological action of insulin and insulin-like growth factor I (IGF-I) were studied in fibroblasts from a patient with leprechaunism and a patient with type A syndrome of insulin resistance. Insulin binding was reduced to 18.8 and 27.7% of control value, respectively. In contrast, IGF-I binding was normal in both patients. In competitive binding studies, IGF-I had 0.2% of the ability of insulin to compete with 125I-labeled insulin binding, and insulin had 0.1% of the ability of IGF-I to compete with 125I-labeled IGF-I binding in control subjects and patient fibroblasts. The dose-response curves of insulin stimulation assessed by glucose incorporation and alpha-aminoisobutyric acid uptake showed normal responsiveness, and ED50 was significantly shifted to the right in fibroblasts from both patients. However, normal responsiveness and sensitivity were observed in thymidine incorporation studies. For IGF-I, dose-response curves of glucose incorporation, alpha-aminoisobutyric acid uptake, and thymidine incorporation were all normal in both patients. These results indicate that 1) the defect is specific to the insulin-receptor binding in these patients, 2) insulin and IGF-I activate glucose incorporation and alpha-aminoisobutyric acid uptake mainly through their own specific receptors, but 3) the IGF-I receptor appears to have a more important role in stimulating thymidine incorporation than the insulin receptor in physiological condition or, alternatively, an unknown postreceptor process with cascade signal transmission may overcome the decreased insulin-receptor binding to produce a normal dose-response curve.

Decreased VIP content in peripheral nerve from streptozocin-induced diabetic rats.

After induction of diabetes with streptozocin (STZ-D) in rats, we measured vasoactive intestinal polypeptide (VIP) content in sciatic nerve and spinal cord obtained from nondiabetic, untreated STZ-D, and insulin-treated STZ-D rats. Eight weeks after the onset of diabetes, caudal nerve conduction velocity (NCV) in the untreated STZ-D rats (n = 13) was slower than in the controls (n = 11; mean +/- SE 30.9 +/- 0.6 vs. 41.4 +/- 1.8 m/s, P less than 0.001). The decrease in NCV was less marked in the insulin-treated STZ-D rats (n = 11; 36.3 +/- 0.9 m/s, P less than 0.05 vs. control). VIP content in sciatic nerve decreased in the untreated STZ-D rats (1.33 +/- 0.23 ng/g wet wt) compared with the other groups (control, 3.10 +/- 0.44, P less than 0.01; insulin-treated STZ-D, 2.44 +/- 0.55, P less than 0.05). However, in spinal cord, VIP content was not significantly different among the three groups. The VIP levels in sciatic nerve showed a positive correlation with NCV (r = 0.430, P less than 0.01). In addition, an inverse correlation between VIP levels and blood glucose levels was observed (r = -0.5624, P less than 0.001). NCV was also inversely correlated with blood glucose levels (r = -0.7662, P less than 0.001). Together with a previous morphological study, these findings suggest a possible causal relationship between reduced VIP content and diabetic neuropathy.

Point mutation in a family with hyperproinsulinemia detected by single stranded conformational polymorphism.

We previously described a case of familial hyperproinsulinemia, the fifth to be reported. In the present study we characterized the genetic defect carried by this family and demonstrated that it could be detected by polymerase chain reaction-single stranded conformational polymorphism. Since the serum proinsulin molecule from the propositus, a 63-yr-old Japanese man, was eluted on the same fraction of human proinsulin intermediate cleaved only at the B-C junction, we sequenced exon 3 of his insulin gene, including the C-A junction. A point mutation was discovered that changed codon 65 from arginine (CGT) to histidine (CAT) in one allele. This was the same point mutation as that described previously in three unrelated kindreds representing two races, consistent with the hypothesis that the dinucleotide sequence CpG may be a "hot spot" for mutations. Recently, developed polymerase chain reaction-single stranded conformational polymorphism proved useful in detecting this mutation in the family members. The daughter of the propositus and one of his two grandsons were also demonstrated to be heterozygous for this point mutation by this method.

The effects of FK506 and cyclosporine on the exocrine function of the rat pancreas.

The effects of FK506 on exocrine function of the pancreas in Sprague-Dawley rats were examined and compared with those of cyclosporine. FK506 did not affect the levels of amylase and lipase in the pancreas of rats given the drug orally in a daily dose of 1 or 5 mg/kg for 2 weeks. Furthermore, the pancreas of the rats showed good exocrine secretion of enzymes such as amylase and lipase on stimulation with carbachol in vitro. Cyclosporine 50 mg/kg given in the same way as FK506 also was free of effect on enzyme levels in the rat pancreas, but the exocrine secretion of the enzyme from the pancreatic lobules on stimulation with carbachol was impaired. It is concluded that FK506 does not affect pancreatic exocrine function in rats.

Hypersecretion of amylin from the perfused pancreas of genetically obese (fa/fa) rats and its alteration with aging.

To evaluate the relationship between the development of obesity and the hypersecretion of amylin by the pancreas, we examined the effects of 16.7 mmol/L glucose and 10 mmol/L arginine on the secretion of amylin and insulin by isolated perfused pancreata from genetically obese (fa/fa) and lean (Fa/?) Zucker rats at 9, 18, and 54 weeks of age. Concentrations of amylin and insulin in the effluent were measured by radioimmunoassay (RIA). Pancreata of obese rats secreted greater amounts of amylin in response to 16.7 mmol/L glucose and 10 mmol/L arginine than did those of lean rats at all ages. The hypersecretion of amylin by obese rats was particularly marked at 18 weeks of age, when they showed the most rapid increase in body fat mass. This hypersecretion became obscure at 54 weeks of age, when obese rats showed the maximum body weight. The pattern of amylin release resembled that of insulin in all groups. However, the relative amount of amylin to insulin secreted following stimulation with 16.7 mmol/L glucose and 10 mmol/L arginine in obese rats exceeded that in lean rats at all ages. Differences in the secreted amylin to insulin molar ratios between obese and lean rats were significant when pancreata were stimulated with glucose at 18 weeks (obese, 1.23% +/- 0.05%; lean, 0.99% +/- 0.04%; P < .01), glucose at 54 weeks (P < .01), and arginine at 54 weeks (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Unprocessed insulin proreceptor in cultured fibroblasts from a patient with extreme insulin resistance.

Insulin receptors and IGF-I receptors in cultured fibroblasts were investigated in a patient with extreme insulin resistance due to unprocessed insulin receptors. Insulin binding to cultured fibroblast monolayers and partially purified insulin receptors was extremely decreased to 27% and 18% of control value, respectively. Affinity cross-linking study revealed that molecular weight of the insulin receptor was 210 kDa and that it could not be dissociated to alpha- and beta-subunit with dithiothreitol treatment. Because IGF-I binding to the fibroblasts from the patient was normal and alpha-subunit of IGF-I receptor was 135 KDa, the defect was specific to the insulin receptor. Autophosphorylation of the 210 kDa unprocessed insulin proreceptor was stimulated by insulin in a dose-dependent manner. In the fibroblasts from the patient, insulin-stimulated alpha-aminoisobutyric acid uptake was fivefold shifted to the right in the dose-response curve (ED50 20 ng/mL for the patient v 3.5 ng/mL for the control subjects), but the maximally stimulated uptake was normal. With 0.025% trypsin treatment, insulin binding and alpha-aminoisobutyric acid uptake were normalized. These results suggested that (1) abnormal processing of insulin proreceptor also occurred in the cultured fibroblasts, (2) the postreceptor steps of insulin action were totally intact, and (3) IGF-I receptors were normally processed in this patient.

Suppression of apoptosis is responsible for increased thickness of intestinal mucosa in streptozotocin-induced diabetic rats.

Intestinal mucosal growth is a common, but uncharacterized, observation associated with diabetes mellitus. Epithelial homeostasis is balanced by regulation of cell proliferation and cell death. To determine the contribution of apoptosis to the overall maintenance of intestinal growth, we examined intestinal apoptosis in the well-characterized streptozotocin (STZ)-induced diabetes rat model. Rats were injected with STZ (75 mg/kg body weight), thereafter they were allowed free feeding or restricted feeding for 3 weeks. Food intake and intestinal mucosal height were evaluated. In a second experiment, additional groups of animals were injected with STZ and were fed ad libitum for 1 or 3 weeks. Ornithine decarboxylase (ODC) activity, ratio of fragmented DNA to total DNA, electrophoresis of fragmented DNA, and Western blot analysis of caspase-3 were examined. Food intake gradually increased in free-feeding rats after induction of diabetes. Intestinal mucosal height in free-feeding diabetic rats was approximately 25% longer than controls, but this increase in mucosal height was not observed in restricted-fed diabetic rats (25 g/d). ODC activity in intestinal mucosa in diabetic rats did not differ from that of control rats. Percent fragmented DNA of diabetic rats 1 week after STZ injection was significantly lower than that of control rats, and this decrease returned to the control level 3 weeks after STZ treatment. Active form of caspase-3 was attenuated 1 week after drug treatment. Attenuated effect of diabetic rats on intestinal apoptosis did not affect increased apoptosis after ischemia-reperfusion. Suppression of apoptosis in the early days of STZ-induced diabetes was responsible for the increased mucosal height in the small intestine in STZ-induced diabetic animals.

Gln27Glu beta2-adrenergic receptor variant is associated with hypertriglyceridemia and the development of fatty liver.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is associated with the metabolism of lipid, glucose and energy. Beta-adrenergic receptors play an important role in the regulation of energy expenditure, in part, by stimulating lipid mobilization through lipolysis. METHODS: To assess whether it is common for the beta2-adrenergic receptor (B2AR) gene polymorphisms in codons 16 and 27 to play a role in the development of fatty liver, we investigated 251 unrelated healthy Japanese males who were drug-free and showed no signs of heavy drinking. RESULTS: The allelic frequency of B2AR gene mutation in codons 16 and 27 did not differ between obese subjects (BMI>25.0 kg/m(2), n=151) and non-obese subjects (BMI

Association of the Pro90Ser CD36 mutation with elevated free fatty acid concentrations but not with insulin resistance syndrome in Japanese.

BACKGROUND: CD36 deficiency is reportedly an underlying factor about insulin resistance, defective fatty acid metabolism and hypertriglyceridemia in spontaneously hypertensive rat (SHR), and may be involved in the pathogenesis of insulin resistance and hyperlipidemia in humans. METHODS: We examined 831 adults undergoing health screening. The majority (780) was Pro90 homozygous for the CD36 gene product, but 51 displayed a CD36 mutation (2 homozygous and 49 heterozygous for Ser90). This is the major mutation site involved in CD36 deficiency in Japanese. RESULTS: Among parameters related to insulin resistance, there were no differences in body mass index (BMI), HDL cholesterol, total cholesterol, triglycerides, insulin and insulin resistance index (HOMA IR), or blood pressure between 91 normal subjects (45 male and 46 female) randomly selected from the 780 Pro90 homozygotes and the 51 (29 male and 22 females) CD36-deficient subjects (Ser90 homozygote and Pro90Ser heterozygote). Free fatty acid concentrations, however, were higher in Ser90 CD36 subjects than in Pro90 control subjects. CONCLUSIONS: The CD36Pro90Ser mutation is not necessarily related to the insulin resistance syndrome, but is associated with high free fatty acid concentrations in Japanese.

Release of amylin from perfused rat pancreas in response to glucose and glucagon.

The effects of glucose and glucagon on the release of amylin from the isolated perfused rat pancreas were studied. Amylin is a 37-amino acid peptide isolated from pancreatic islet amyloid of patients with non-insulin-dependent diabetes mellitus (NIDDM). Glucose dose-dependently stimulated a biphasic release of amylin from the pancreas in parallel with that of insulin. However, the release of amylin induced by high concentrations of glucose was partially dissociated from that of insulin. The amylin-insulin molar ratios induced by 22.2 mM and 33.3 mM glucose (1.11 +/- 0.05%, 1.05 +/- 0.04%, respectively) were significantly higher than that induced by 16.7 mM glucose (0.90 +/- 0.04%, P less than 0.01 vs 22.2 mM glucose, P less than 0.05 vs 33.3 mM glucose). In the presence of 5.6 mM glucose, glucagon also stimulated the release of amylin from the perfused pancreas in parallel with that of insulin. These findings suggest that amylin may be a secretory protein from the pancreas and that the concomitant secretion of amylin and insulin might contribute to glucose homeostasis.

Amylin release from perfused rat pancreas in response to glucose and arginine.

The effects of glucose and arginine on the release of amylin from the perfused rat pancreas were studied. Amylin, or islet amyloid polypeptide, is a 37-amino acid peptide isolated from pancreatic islet amyloid of patients with non-insulin-dependent diabetes mellitus (NIDDM). Glucose stimulated dose-dependently amylin release, showing a typical biphasic pattern. Additionally, 10 mM arginine in the presence of 5.5 mM glucose also stimulated amylin release. These findings suggest that amylin is a secretory protein and its release from the pancreas is regulated by glucose and other nutrients.

A case of insulin allergy: the crystalline human insulin may mask its antigenicity.

We report an unusual case of insulin allergy. A 48-year-old man with non-insulin-dependent diabetes mellitus receiving biosynthetic isophane human insulin (Humulin N) developed itchy wheal-and-flare reactions at the sites of injection. When Humulin N was changed to a semi-synthetic crystalline human insulin zinc (Novolin U), the allergic reactions completely disappeared. Evaluation of his serum showed a high level of insulin-specific IgE. Skin testing with all commercially available insulins showed immediate local reactions to all agents tested except for Novolin U. In addition, decrystallized Novolin U prepared by lowering the pH with acetic acid also induced a positive reaction. These observations suggest that the crystallized structure of human insulin may mask its antigenicity for allergic reactions.