R. P. Robertson

Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations.

Glucose toxicity of the pancreatic beta cell is considered to play a secondary role in the pathogenesis of type II diabetes mellitus. To gain insights into possible mechanisms of action of glucose toxicity, we designed studies to assess whether the loss of insulin secretion associated with serial passages of HIT-T15 cells might be caused by chronic exposure to high glucose levels since these cells are routinely cultured in media containing supramaximal stimulatory concentrations of glucose. We found that late passages of HIT cells serially cultured in media containing 11.1 mM glucose lost insulin responsivity and had greatly diminished levels of insulin content and insulin mRNA. In marked contrast, late passages of HIT cells cultured serially in media containing 0.8 mM glucose retained insulin mRNA, insulin content, and insulin responsivity to glucose in static incubations and during perifusion with glucose. No insulin gene mutation or alteration of levels of GLUT-2 were found in late passages of HIT cells cultured with media containing 11.1 mM glucose. These data uniquely indicate that loss of beta cell function in HIT cells passed serially under high glucose conditions is caused by loss of insulin mRNA, insulin content, and insulin secretion and is preventable by culturing HIT cells under low glucose conditions. This strongly suggests potential genetic mechanisms of action for glucose toxicity of beta cells.

Systemic Venous Drainage of Pancreas Allografts as Independent Cause of Hyperinsulinemia in Type I Diabetic Recipients

To evaluate the metabolic consequences of pancreas transplantation with systemic venous drainage on beta-cell function, we examined insulin and C-peptide responses to glucose and arginine in type I (insulin-dependent) diabetic pancreas recipients (n = 30), nondiabetic kidney recipients (n = 8), and nondiabetic control subjects (n = 28). Basal insulin levels were 66 +/- 5 pM in control subjects, 204 +/- 18 pM in pancreas recipients (P less than 0.0001 vs. control), and 77 +/- 17 pM in kidney recipients. Acute insulin responses to glucose were 416 +/- 44 pM in control subjects, 763 +/- 91 pM in pancreas recipients (P less than 0.01 vs. control), and 589 +/- 113 pM in kidney recipients (NS vs. control). Basal and stimulated insulin levels in two pancreas recipients with portal venous drainage were normal. Integrated acute C-peptide responses were not statistically different (25.3 +/- 4.3 nM/min in pancreas recipients, 34.2 +/- 5.5 nM/min in kidney recipients, and 23.7 +/- 2.1 nM/min in control subjects). Similar insulin and C-peptide results were obtained with arginine stimulation, and both basal and glucose-stimulated insulin-C-peptide ratios in pancreas recipients were significantly greater than in control subjects. We conclude that recipients of pancreas allografts with systemic venous drainage have elevated basal and stimulated insulin levels and that these alterations are primarily due to alterations of first-pass hepatic insulin clearance, although insulin resistance secondary to immunosuppressive therapy (including prednisone) probably plays a contributing role. To avoid hyperinsulinemia and its possible long-term adverse consequences, transplantation of pancreas allografts into sites with portal rather than systemic venous drainage should be considered.

Glucagon, catecholamine and pancreatic polypeptide secretion in type I diabetic recipients of pancreas allografts.

Successful pancreas transplantation in type I diabetic patients restores normal fasting glucose levels and biphasic insulin responses to glucose. However, virtually no data from pancreas recipients are available relative to other islet hormonal responses or hormonal counterregulation of hypoglycemia. Consequently, glucose, glucagon, catecholamine, and pancreatic polypeptide responses to insulin-induced hypoglycemia and to stimulation with arginine and secretin were examined in 38 diabetic pancreas recipients, 54 type I diabetic nonrecipients, and 26 nondiabetic normal control subjects. Glucose recovery after insulin-induced hypoglycemia in pancreas recipients was significantly improved. Basal glucagon levels were significantly higher in recipients compared with nonrecipients and normal subjects. Glucagon responses to insulin-induced hypoglycemia were significantly greater in the pancreas recipients compared with nonrecipients and similar to that observed in control subjects. Glucagon responses to intravenous arginine were significantly greater in pancreas recipients than that observed in both the nonrecipients and normal subjects. No differences were observed in epinephrine responses during insulin-induced hypoglycemia. No differences in pancreatic polypeptide responses to hypoglycemia were observed when comparing the recipient and nonrecipient groups, both of which were less than that observed in the control subjects. Our data demonstrate significant improvement in glucose recovery after hypoglycemia which was associated with improved glucagon secretion in type I diabetic recipients of pancreas transplantation.

G Proteins and Modulation of Insulin Secretion

Guanine nucleotide–binding proteins (G proteins) are critically important mediators of many signal-transduction systems. Several important sites regulating stimulus-secretion coupling and release of insulin from pancreatic β-cells are modulated by G proteins. Gs mediates increases in intracellular cAMP associated with hormone-induced stimulation of insulin secretion. GI, mediates decreases in intracellular cAMP caused by inhibitors of insulin secretion, e.g., epinephrine, somatostatin, prostaglandin E2, and galanin. G proteins also regulate ion channels, phospholipases, and distal sites in exocytosis. Cholera and pertussis toxins irreversibly ADP ribosylate G proteins and are important tools that can be used both to manipulate G-protein–dependent modulators of insulin secretion and detect and quantify G proteins by electrophoretic techniques. The stage is set to pursue these initial observations in greater depth and ascertain whether G-protein research will provide important new insights into normal and abnormal regulation of insulin secretion.

Diminished Insulin Secretory Reserve in Diabetic Pancreas Transplant and Nondiabetic Kidney Transplant Recipients

Although both kidney and pancreas transplantation can restore renal and pancreatic endocrine functions, the accompanying immunosuppression may cause diminished glucose tolerance in some individuals. Therefore, we determined to what extent pancreas transplantation itself and the triple immunosuppressive therapy used in pancreas transplant recipients have adverse effects on insulin secretory reserve. β-cell secretory reserve was assessed by the method of glucose potentiation of arginine-induced insulin secretion in 25 normoglycemic pancreas recipients, 12 nondiabetic kidney recipients using the same immunosuppressive therapy, 3 psoriasis patients treated long term with cyclosporine, 5 arthritis patients treated long term with prednisone, and their respective sex-, age-, and body mass index-matched control subjects. Levels of fasting glucose, HbA1c, and glucose disappearance rates were normal in all subjects. During the glucose potentiation study, pancreas recipients had significantly less insulin secretion than control subjects (maximal acute response [ARmax] = 1,083 ± 93% vs. 3,938 ± 355%, P < 0.001). Insulin responses were also decreased in kidney recipients (ARmax = 2,296 ± 290%) vs. control subjects (4,691 ± 554%, P = 0.001) and in psoriasis patients treated with cyclosporine (ARmax = 2,153 ± 390%) vs. control subjects (3,962 ± 88%, P = 0.011), but not as extreme as that seen in pancreas recipients. No abnormalities were observed in arthritis patients treated with steroids. We conclude that normoglycemic pancreas and kidney transplant recipients receiving triple immunosuppressive therapy have diminished β-cell secretory reserve. Because this defect was present in psoriasis patients treated long term with cyclosporine, but not in arthritis patients treated long term with prednisone, this adverse effect was probably caused in part by cyclosporine.

Pancreas Transplantation in Diabetic Humans Normalizes Hepatic Glucose Production During Hypoglycemia

Although successful pancreas transplantation in humans with type I diabetes mellitus restores glucose-induced insulin secretion, provides freedom from insulin treatment, and normalizes fasting glucose levels, much less is known about its effects on counterregulation of hypoglycemia. To determine whether pancreas transplantation normalizes glucagon secretion and hepatic glucose production (HGP) during hypoglycemia, we performed hyperinsulinemic hypoglycemic clamps in successful recipients of pancreas allografts. Recipients were found to have glucagon secretory responses during hypoglycemia that were similar to those of control subjects (incremental glucagon response: recipients, 147 ± 34 ng/L; control subjects, 161 ± 43 ng/L, NS) but were significantly higher than those of matched subjects with type I diabetes (23 ± 9 ng/L, P < 0.01). HGP rates at the end of 120 min of hypoglycemia were also significantly higher in recipients and control subjects than in subjects with diabetes (pancreas recipients, 1.92 ± 0.33 mg.kg−1 · min−1; control subjects, 2.05 ± 0.18 mg.kg−1 · min−1; subjects with type I diabetes, 0.58 ± 0.12 mg.kg−1 · min−1). A comparison with a third group of nondiabetic kidney transplant recipients demonstrated that the beneficial effects on glucose counterregulation were a result of pancreas transplantation and not the associated immunosuppressive therapy. We conclude that pancreas transplantation restores hypoglycemia-induced glucagon secretion and HGP, thereby allowing for normalization of glucose recovery from hypoglycemia.

G-Proteins and Hormonal Inhibition of Insulin Secretion From HIT-T15 Cells and Isolated Rat Islets

G-proteins are important mediators of hormonal inhibition of insulin secretion. To characterize the pertussis toxin–sensitive substrates present in HIT cell membranes, we performed immunoblots with specific antisera and found evidence for the presence of GIα1, GIα2, GIα3, and three forms of GOoα. We observed that pertussis toxin–sensitive substrates mediate all of the effects of SRIF, and a major portion of the effects of EPI, on insulin secretion from rat islets during static incubations. These results agree with our previously reported studies examining phasic glucose-induced insulin secretion from HIT cells. To ascertain whether inhibition of adenylate cyclase, presumably involving coupling of the catalytic subunit to GI, may be a common mechanism for both hormones, we studied the effects of 8-bromo-cyclic AMP and found that this agent partially prevented the inhibitory effects of both hormones. We also observed that the inhibitory effects of SRIF and EPI on insulin were nonadditive, that both hormones were additive to nickel chloride during inhibition of insulin release, and that they noncompetitively inhibited glipizide-induced insulin secretion through pertussis toxin-sensitive mechanisms. Together, these results suggest that both hormones exert their effects on insulin secretion at multiple G- protein -regulated sites including adenylate cyclase and sites distal to the glipizide-binding site on the KATP channel.

No effect of deferoxamine therapy on glucose homeostasis and insulin secretion in individuals with NIDDM and elevated serum ferritin

Deferoxamine has been proposed as a potentially important therapy for individuals with NIDDM and mild elevations in serum ferritin. Previously, iron chelation therapy with intravenous deferoxamine over a 5–13-wk period has been reported to normalize serum ferritin and markedly improve glycemic control. To confirm these results and to study potential beneficial effects of deferoxamine on insulin secretion, 9 individuals with NIDDM and elevated serum ferritin levels were treated twice weekly with deferoxamine infusion, following a previously described protocol. Although 8 of 9 subjects achieved normal or near-normal serum ferritin values after deferoxamine therapy, we found little evidence that it produced beneficial effects on glycemic control. Fasting glucose levels pre– and post-deferoxamine therapy were unchanged (11.6 ± 1.2 and 11.3 ± 1.5 mM, respectively, P = 0.80). GHb levels declined slightly after deferoxamine therapy (9.3 ± 0.7 vs. 8.8 ± 0.7%, P < 0.05); however, this effect was small and was not associated with elimination of or even substantial reduction in insulin or oral hypoglycemic therapy. Deferoxamine therapy did not significantly alter fasting insulin or C-peptide levels, nor stimulated insulin or C-peptide responses to intravenous arginine or glucose. During follow-up studies 1.5–8 mo after deferoxamine therapy, serum ferritin levels again were elevated in 5 of 8 subjects who showed an initial response. Thus, although deferoxamine therapy reduced serum ferritin levels in our subjects, we were unable to confirm a previous report that this effect was associated with any meaningful improvement in glycemic control or insulin secretion.

Pertussis Toxin–Sensitive G Protein Mediation of PGE2 Inhibition of cAMP Metabolism and Phasic Glucose-Induced Insulin Secretion in HIT Cells

Although prostaglandin E2 (PGE2) is known to inhibit glucose-induced insulin secretion, it is uncertain whether PGE2 actions on the β-cell are direct, whether they are equipotent for both phases of hormone secretion, and whether the same mechanism of action prevails throughout. Study of the HIT cell, a clonai line of pancreatic β-cells, provides answers to these questions because perifusion with glucose and 3-isobutyl-1-methylxanthine stimulates biphasic insulin secretion. Perifusion with PGE2 decreased both the first and second phases of glucose-induced insulin release to 47 ± 4% of controls. Pretreatment with pertussis toxin partly prevented PGE2 inhibition to 80 ± 4% of controls for first phase and 79 ± 4% of controls for second phase. To evaluate whether the partial prevention of PGE2 inhibition seen with pertussis toxin pretreatment was caused by G1 heterotrimer association between the preincubation period and the end of perifusion, PGE2 actions were also examined during continuous treatment with pertussis toxin. Under these conditions, PGE2 inhibition of both phases was totally prevented. However, no difference was observed in membrane protein ADP ribosylation when cells were examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis after pretreatment or continuous treatment with pertussis toxin. Cyclic AMP (cAMP) accumulation was inhibited by PGE2 (from 3263 ± 153 to 1549 ± 158 fmol/106 cells) but less so after pretreatment with pertussis toxin (correlation between insulin release and cAMP accumulation during perifusion; n = 18, r = .85, P < .001). Thus, PGE2 equally inhibits both phases of glucose-induced insulin secretion and cAMP generation through a pertussis toxin–sensitive G protein–mediated direct effect on the pancreatic β-cell.

Glucose homeostasis and insulin secretion in human recipients of pancreas transplantation.

To ascertain the consequences of pancreas transplantation with systemic venous drainage on glucose homeostasis and insulin secretion, glucose and insulin responses to intravenous glucose were compared in 10 recipients and 15 normal control subjects. There were no differences in fasting glucose levels or intravenous glucose disappearance rates. However, basal insulin levels and acute insulin responses to glucose were threefold greater in the recipients. It is not clear whether this consequence of hyperinsulinemia in the recipients is due to the abnormal circulatory drainage, the lack of autonomie input, or concurrent immunosuppressive drug therapy.