Inhibition of glycogenolysis and glycogen phosphorylase by insulin and proinsulin in rat hepatocyte cultures

The inhibitory action of insulin and proinsulin on basal and glucagon-activated glycogenolysis was studied in cultured rat hepatocytes containing [14C]glycogen. Insulin or proinsulin given as sole hormones in the presence of 5 mM glucose decreased basal release of [14C]glucose from [14C]glycogen to 20%. Half-maximal effective concentration of insulin was approximately 0.15 nM and of proinsulin was approximately 5 nM. Inhibition of [14C]lactate release from [14C]glycogen required slightly higher hormone concentrations with a similar difference in potency for insulin and proinsulin. The glucagon-stimulated release of [14C]glucose was completely blocked by insulin or proinsulin with half-maximal effective concentrations of approximately 0.2 and approximately 8 nM, respectively. In contrast, release of [14C]lactate in the presence of glucagon was increased slightly by insulin and proinsulin. Basal and glucagon-activated phosphorylase activity was inhibited by approximately 50% in a dose-dependent manner by both hormones, with differences in potency similar to those for the inhibition of glycogenolysis. These data point to a direct regulatory role of insulin in the control of hepatic glycogen breakdown even when acting as sole hormone. The results do not support the notion of a preferential inhibitory potency of proinsulin on hepatic glycogenolysis.     

Control of glycogen synthesis by glucose, glycogen, and insulin in cultured human muscle cells

A key feature of type 2 diabetes is impairment in the stimulation of glycogen synthesis in skeletal muscle by insulin. Glycogen synthesis and the activity of the enzyme glycogen synthase (GS) have been studied in human myoblasts in culture under a variety of experimental conditions. Incubation in the absence of glucose for up to 6 h caused an approximately 50% decrease in glycogen content, which was associated with a small decrease in the fractional activity of GS. Subsequent reincubation with physiological concentrations of glucose led to a dramatic increase in the rate of glycogen synthesis and in the fractional activity of GS, an effect which was both time- and glucose concentration-dependent and essentially additive with the effects of insulin. This effect was seen only after glycogen depletion. Inhibitors of signaling pathways involved in the stimulation of glycogen synthesis by insulin were without significant effect on the stimulatory action of glucose. These results indicate that at least two distinct mechanisms exist to stimulate glycogen synthesis in human muscle: one acting in response to insulin and the other acting in response to glucose after glycogen depletion, such as that which results from exercise or starvation.     

Regulation of ketogenesis by epinephrine and norepinephrine in the overnight-fasted, conscious dog

The effects on ketogenesis and lipolysis of a norepinephrine (0.04 microgram/kg-min), epinephrine (0.04 microgram/kg-min), or saline infusion were examined in the overnight-fasted, conscious dog. Plasma insulin and glucagon levels were maintained constant by means of a somatostatin infusion (0.8 microgram/kg-min) and intraportal replacement infusions of insulin and glucagon. In saline-infused dogs, plasma epinephrine (62 +/- 8 pg/ml), norepinephrine (92 +/- 29 pg/ml), blood glycerol (87 +/- 10 microM), and plasma nonesterified fatty acid (NEFA) (0.82 +/- 0.17 mM) levels did not change. Total blood ketone body levels tended to rise (62 +/- 10 to 83 +/- 11 microM) by 3 h as did total ketone body production (1.5 +/- 0.4 to 2.2 +/- 0.4 mumol/kg-min) over the same time interval. Norepinephrine infusion to produce plasma levels of 447 +/- 86 pg/ml caused a sustained 50% rise in glycerol levels (66 +/- 17 to 99 +/- 15 mumol/L, P less than 0.05) and 53% rise in nonesterified fatty acids (0.53 +/- 0.07 to 0.81 +/- 0.15 mumol/L, P less than 0.05). Total ketone body levels rose by 43% (51 +/- 8 to 73 +/- 10 mumol/L) and ketone body production rose by a similar proportion (1.5 +/- 0.2 to 2.2 +/- 0.3 mumol/kg-min), changes that did not differ significantly from control animals. A similar increment in plasma epinephrine levels (75 +/- 15 to 475 +/- 60 pg/ml) caused glycerol levels to rise by 82% (105 +/- 23 to 191 +/- 26 mumol/L) in 30 min, but this rise was not sustained and the level fell to 146 +/- 14 mumol/L by 120 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of halothane on human and rat hepatocyte cultures

The aim of this study was to investigate direct cytotoxicity to human and rat hepatocytes in primary culture from halothane and compare it with that of isoflurane, which is known to be minimally metabolized and less toxic in vivo. Both human and rat parenchymal cells were isolated by the two-step collagenase perfusion method and after attachment to plastic were incubated with either volatile anesthetic for 24 h. All the cultures were maintained in 20% O2 condition and were not induced prior to anesthetic treatment. Temperature, atmosphere conditions, and anesthetic concentrations were kept constant during the study period. Evaluation of cytotoxicity was based on morphologic, biologic (determination of both extracellular and intracellular lactate dehydrogenase activity), and metabolic (protein synthesis and secretion) end points. Protein synthesis and secretion rates were found to be the most sensitive parameters in hepatocyte cultures from both species. Protein synthesis was inhibited by 18% and protein secretion by 50% in the presence of 1 and 1.25 mM halothane, respectively, in human cell cultures (P less than 0.05). With 1.25 mM halothane intracellular lactate dehydrogenase was also decreased; lactate dehydrogenase leakage and morphologic alterations were detected only beyond 5 mM halothane. By contrast, in rat hepatocyte cultures protein secretion was inhibited by 26% and protein synthesis by 20% in the presence of 0.1 and 0.75 mM halothane, respectively, whereas morphologic alterations and a 37% lactate dehydrogenase leakage increase were observed with the concentration of 1 mM (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of glucagonlike peptide I-(7-36) on release of insulin, glucagon, and somatostatin by rat pancreatic islet cell monolayer cultures

Glucagonlike peptide I (GLP-I-(7-36] is cleaved from proglucagon in ileal epithelial cells and increases in human plasma after nutrient ingestion. This peptide has been shown to stimulate insulin secretion in vitro and in vivo and thus potentially acts as an incretin. To characterize its action on islet cells, the release of insulin, glucagon, and somatostatin by rat pancreatic islet monolayer cultures at varying concentrations of GLP-I-(7-36) was measured. The interaction of GLP-I-(7-36) with nutrient substrates was assessed by adding amino acids and differing glucose concentrations to the cultures. Islet cell cultures (n = 5) were incubated for 1 h in medium containing 1.67 or 16.7 mM glucose or 1.67 mM glucose supplemented with amino acids and GLP-I-(7-36) at 10(-13)-10(-7) M. Hormone release was compared with control cultures containing no GLP-I-(7-36); 1.67-16.7 mM glucose with and without GLP-I-(7-36) at 10(-11) M; and 1.67, 3.3, 8.3, or 11.1 mM glucose alone or supplemented with amino acids, GLP-I-(7-36) 10(-11) M, or both amino acids and GLP-I-(7-36). In medium with 1.67 or 16.7 mM glucose or 1.67 mM glucose and amino acids, GLP-I-(7-36) increased insulin secretion two- to threefold over control at concentrations of 10(-9), 10(-11), and 10(-12) M, respectively. In medium with increasing concentrations of glucose, GLP-I-(7-36) at 10(-11) M significantly increased insulin secretion at glucose concentrations greater than or equal to 3.34 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition of human insulin and proinsulin by monoclonal antibodies

High-affinity monoclonal antibodies (MAB) were obtained from lymph node cell fusions. Affinities ranging from 0.8 X 10(9) L/M to 5.2 X 10(9) L/M were calculated from binding studies with monoiodinated human, bovine, and porcine insulins and human proinsulin. Two monoclonal antibodies were specific for human insulin, recognizing an epitope involving the amino acid B-30 (Thr). Another two monoclonal antibodies were bound to the C-terminal end of the B-chain near B-30. The B-chain-specific monoclonal antibodies did not bind human proinsulin. One monoclonal antibody recognized the A-chain loop in the positions A-8 to A-10. This antibody bound also to human proinsulin. It was concluded that the A-chain loop is exposed on the surface of proinsulin, while the C-terminal B-chain is not available for binding. The study shows that monoclonal antibodies can be used to characterize structures of insulin and proinsulin. In contrast to x-ray studies, the molecules can be used at low concentrations in soluble form. It is suggested to use monoclonal antibodies for the screening of atypical insulins in the serum of diabetic patients and for the further refinement of insulin and proinsulin measurements.     

In vitro activity of biosynthetic human proinsulin. Receptor binding and biologic potency of proinsulin and insulin in isolated rat adipocytes

The receptor binding characteristics and biologic protency of biosynthetic human proinsulin (rDNA) were determined in isolated rat adipocytes and compared with those of insulin. In competition with 125I(A14)-iodoinsulin for binding to adipocyte receptors at 15 degrees C, proinsulin showed a 100-fold lower affinity for binding than did insulin. A proinsulin concentration of 3.2 +/- 0.8 X 10(-7) M was required for 50% inhibition of tracer binding as compared with a concentration of 1.7 +/- 0.3 X 10(-9) M for insulin. These results were confirmed in direct competition studies using proinsulin and 125I-iodoproinsulin. A similar 100-fold difference was also observed in competitive binding experiments conducted at 37 degrees C. The biologic potency of human proinsulin was evaluated by its ability to stimulate glucose incorporation into total fat cell lipid and also by its antilipolytic activity. Glucose incorporation into lipid was half-maximal at a proinsulin concentration of 1.5 +/- 0.4 X 10(-8) M, whereas the same response was observed at an insulin concentration of 5.2 +/- 1 X 10(-11) M. Proinsulin also demonstrated an antilipolytic potency that was less than 1% that of insulin. The time course over which insulin and proinsulin stimulated glucose incorporation into lipid was the same, as was the time course over which the stimulation dissipated after removal of the hormones. No synergism of insulin and proinsulin stimulation of lipogenesis was observed when fat cells were incubated with submaximal concentrations of the two hormones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of rat pancreatic CCKB receptor and somatostatin expression by insulin

The cholecystokinin B receptor (CCK(B)R) is localized on pancreatic endocrine somatostatin delta-cells. Pancreatic somatostatin content was increased in diabetic rats. The mechanisms involved in this phenomenon are unknown, and we believe insulin is involved. In this study, four groups of rats were used: controls, streptozotocin-induced diabetic, streptozotocin-induced diabetic with insulin, and streptozotocin-induced diabetic with insulin and its cessation. Rats were killed after 7-28 days of treatment for diabetes, and somatostatin mRNA expression and pancreatic somatostatin content, CCK(B)R mRNA and protein expression evaluation in total pancreas and purified islets, and the cellular localization of somatostatin and CCK(B)R in islets was measured. Data indicate that diabetes is established after 7 days, is controlled by insulin, and reappears after treatment cessation. Pancreatic somatostatin mRNA expression and somatostatin content were increased during diabetes, normalized during insulin treatment, and reaugmented after treatment cessation. Gland and islet CCK(B)R mRNA and protein almost disappeared during diabetes; CCK(B) mRNA reappeared in response to insulin, but the protein did not. Confocal microscopy confirmed data obtained on somatostatin and CCK(B)R as established biochemically in the course of the treatments. In conclusion, these data strongly suggest that insulin can negatively control pancreatic somatostatin mRNA and hormone content and positively control CCK(B)R mRNA; the CCK(B)R protein appears to be delayed.     

Effects of monensin on conversion of proinsulin to insulin and secretion of newly synthesized insulin in isolated rat islets

When isolated rat islets were incubated with 10(-10) - 10(-6) M monensin, a sodium and proton ionophore, glucose-stimulated insulin release was inhibited in a concentration- and time-dependent manner. After removal of monensin, inhibition of insulin secretion persisted during stimulation with a variety of secretagogues, including 5 mM glucose plus 15 mM arginine, 20 mM glucose, and 20 mM glucose plus 1 mM 3-isobutyl-1-methylxanthine. Within the same low range of monensin concentrations, proteolytic conversion of newly synthesized proinsulin to insulin was also blocked. At each concentration, prohormone-to-hormone conversion was inhibited to almost the same extent as inhibition of insulin secretion. Therefore, both processes may have equal or common dependency on a subcellular ionic gradient. Although monensin decreased total insulin secretion, the glucose-regulated marking process was unaffected. Regardless of the monensin concentration or the overall rate of insulin secretion, the percentage of secreted newly synthesized versus older insulin remained the same, and the threefold differences in the fractional secretory rates of newly synthesized versus total insulin also remained the same. Thus, rather than specifically blocking protein traffic through the Golgi apparatus of the beta cell, monensin probably first inhibited insulin secretion by disrupting proton gradients in secretory vesicles and, thereby, also inhibited other processes occurring within this organelle.     

Tris(hydroxymethyl)aminomethane inhibits the synthesis and processing of proinsulin in isolated rat pancreatic islets without affecting release of insulin stores

Isolated rat islets of Langerhans were pulse-labeled (5 min, [3H]leucine) and then exposed to 10 or 50 mM tris(hydroxymethyl)aminomethane (Tris) at pH 7.4 during an 85-min chase period. There was a dose-related inhibition of the conversion of labeled proinsulin to insulin by Tris. At 50 mM, Tris also inhibited the release of newly synthesized (labeled) proinsulin and insulin. These inhibitory effects of Tris were almost absent if the islets were exposed to 50 mM Tris during only the last 60 min of the 85-min chase period. Both proinsulin and total islet protein synthesis (as indexed by incorporation of [3H]leucine) were inhibited acutely by 50 mM Tris (5-min exposure); after 85 min of exposure to 50 mM Tris, the inhibition of proinsulin biosynthesis was more marked than that of total islet protein. In contrast to its effects on newly synthesized products, 50 mM Tris failed to inhibit the release of immunoreactive insulin during an 85-min incubation. However, when islets were exposed to 50 mM Tris for a longer period, a partial inhibition of immunoreactive insulin release was observed as from 120 min. Insulin released from islets consists of a mixture of older stored material and of newly synthesized products, the latter being released preferentially. These results are consistent with a selective effect of 50 mM Tris on the production of newly synthesized insulin. During the first 120 min of exposure to Tris, islet reserves of newly synthesized products will be depleted thereby leading to a new, reduced, rate of release of immunoreactive material consisting only of older insulin stores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of insulin and glucose on cultured rat hepatocyte gene expression

We previously used isolated adult rat hepatocyte cultures to study the ability of glucose to induce several hepatic mRNAs. However, we found that the optimal insulin concentration required to obtain the glucose effect was greater than 10,000 microU/ml. To test the hypothesis that the requirement for high concentrations of insulin in the culture was due to rapid loss of insulin in hepatocyte cultures, serial measurements of insulin were made at different media insulin concentrations (0-500,000 microU/ml) and glucose concentrations (5.5 and 2.75 mM). In addition, a dose-response relationship was established between media insulin concentrations and the pattern of mRNAs present in the hepatocytes determined by two-dimensional gel electrophoresis of in vitro translation products. We found that at low insulin concentrations (less than 1000 microU/ml), greater than 80% of the insulin was lost to the glassware, whereas at high initial insulin concentrations, approximately 23% of the insulin was lost to the glassware. Placement of media into the hepatocyte culture led to further insulin disappearance with a half-life for insulin of 41.5 h at 10,000 microU/ml and 13.8 h at 100 microU/ml. We found 16 mRNAs were altered by insulin at 5.5 mM glucose and 9 mRNAs were changed by insulin at 27.5 mM glucose. After taking into consideration the distributional and metabolic losses of insulin, all but one mRNA responded to insulin within the physiologic range of portal insulin (less than 1-94 microU/ml). Our data indicate that the hepatocyte culture is an excellent model to study the physiologic effects of insulin on hepatic gene expression.     

Hepatic glycogen metabolism and insulin receptor status after long-term peripheral insulin delivery in the islet-transplanted diabetic rat

Severely diabetic (0.15 g/kg streptozocin) rats were transplanted with fetal pancreatic islets under the renal capsule to model peripheral insulin delivery, or into the splenic pulp to model portal delivery. In both groups of transplanted rats, weight gain and blood glucose concentrations were normal. Peripheral insulin delivery abolished the physiologic portal-peripheral insulin concentration gradient but was not associated with peripheral hyperinsulinemia. Incorporation of 3H2O into liver glycogen and the increase in hepatic glycogen concentration after a meal were normal in animals receiving insulin peripherally for 10 wk. Activation of liver glycogen synthase in response to the meal was also normal. Hepatic insulin receptor status in animals with peripheral insulin delivery was identical to that of normal control and splenic pulp islet-transplanted rats. The findings indicate that portal insulin delivery is not a prerequisite for normal hepatic glycogen metabolism in the rat, and that receptor upregulation and increased hepatic extraction of insulin are unlikely to explain the normal hepatic metabolism with peripheral insulin delivery.