Inhibitory effects of immunosuppressive drugs on insulin secretion from HIT-T15 cells and Wistar rat islets.

Until recently, islet allotransplantation for type 1 diabetic patients has been largely unsuccessful. Previous pharmacologic studies of single drugs have suggested that one factor contributing to this poor success is toxicity of immunosuppressive drugs on transplanted islets. However, no comprehensive study of agents currently used for islet transplantation has been previously reported. Consequently, we exposed HIT-T15 cells and Wistar rat islets to various concentrations of five immunosuppressive agents for 48 and 24 hr, respectively, and measured glucose-stimulated insulin secretion during subsequent static incubations. Results are expressed as percent reduction of insulin secretion at the lower and upper limits, respectively, of plasma drug concentrations used in clinical transplantation compared with control (no drug exposure). Insulin secretion from HIT-T15 cells was significantly inhibited by 74% and 90% after exposure to methylprednisolone (P<0.05), 11% and 24% after exposure to cyclosporine (P<0.01), 60% and 83% after exposure to mycophenolate (P<0.05), 56% and 63% after exposure to sirolimus (P<0.001), and 10% and 20% after exposure to tacrolimus (P<0.001). Insulin secretion from Wistar rat islets was reduced by 0% and 48% after exposure to mycophenolate (P<0.001) and 20% and 31% after exposure to tacrolimus (P<0.05). No reduction in insulin secretion was observed from either HIT-T15 cells or rat islets after exposure to daclizumab. The results support the hypothesis that toxicity of certain immunosuppressive drugs on beta-cell function plays a role in the poor success of islet allotransplantation. This is especially true of intrahepatically transplanted islets, which are exposed to higher portal concentrations of immunosuppressive agents. These findings support the use of low-dose immunosuppressive drug protocols in clinical islet transplantation.

Antecedent hyperglycemia, not hyperlipidemia, is associated with increased islet triacylglycerol content and decreased insulin gene mRNA level in Zucker diabetic fatty rats.

Type 2 diabetes is caused by a combination of beta-cell dysfunction and insulin resistance. Over time, hyperglycemia worsens, a phenomenon that has been attributed to deleterious effects of chronic hyperglycemia (glucotoxicity) or chronic hyperlipidemia (lipotoxicity) on beta-cell function and is often accompanied by increased islet triacylglycerol (TAG) content and decreased insulin gene expression. To examine these two potentially pathogenic forces, we studied Zucker rats (leptin receptor wild type, +/+; heterozygous, +/-; and mutant, -/-). First, +/+ and +/- Zucker rats were compared metabolically. At 6 weeks of age, the +/- rats had a lower level of islet insulin mRNA compared with +/+. At 12 weeks of age, differences were found in body weight and islet TAG content; however, levels of insulin mRNA were equivalent. Second, we examined whether worsening of the diabetic state in the homozygous mutant (-/-) Zucker diabetic fatty (ZDF) rat is related more to chronic hyperglycemia or to hyperlipidemia. The ZDF rats were treated for 6 weeks with either bezafibrate, a lipid-lowering drug that does not affect plasma glucose levels, or phlorizin, a drug that reduces plasma glucose without lowering lipid levels. Bezafibrate treatment lessened the rise in plasma TAG observed in nontreated rats (239 +/- 16 vs. 388 +/- 36 mg/dl, treated versus nontreated; P < 0.0001) but did not prevent the rise in fasting plasma glucose. Despite lowering plasma TAG, bezafibrate was not effective in preventing an increased islet TAG content and did not prevent the associated decrease in insulin mRNA levels. Phlorizin treatment prevented hyperglycemia (61 +/- 2 vs. 145 +/- 7 mg/dl, treated versus nontreated; P < 0.0001) and lowered islet TAG content (32.7 +/- 0.7 vs. 47.8 +/- 2.7 ng/islet, treated versus nontreated; P < 0.0001) and preserved insulin mRNA levels without preventing hypertriglyceridemia. Plasma free fatty acid level did not correlate with changes in islet TAG or insulin mRNA levels. We conclude that antecedent elevated plasma glucose levels, not plasma lipid levels, are associated with elevated islet TAG content and decreased insulin mRNA levels in ZDF animals.

Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids.

Prolonged exposure of isolated islets to supraphysiologic concentrations of palmitate decreases insulin gene expression in the presence of elevated glucose levels. This study was designed to determine whether or not this phenomenon is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids. Gene expression of sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase (DGAT), and hormone-sensitive lipase (HSL), three key enzymes of lipid metabolism, was detected in isolated rat islets. Their levels of expression were not affected after a 72-h exposure to elevated glucose and palmitate. To determine the effects of glucose on palmitate-induced neutral lipid synthesis, isolated rat islets were cultured for 72 h with trace amounts of [14C]palmitate with or without 0.5 mmol/l unlabeled palmitate, at 2.8 or 16.7 mmol/l glucose. Glucose increased incorporation of [14C]palmitate into complex lipids. Addition of exogenous palmitate directed lipid metabolism toward neutral lipid synthesis. As a result, neutral lipid mass was increased upon prolonged incubation with elevated palmitate only in the presence of high glucose. The ability of palmitate to increase neutral lipid synthesis in the presence of high glucose was concentration-dependent in HIT cells and was inversely correlated to insulin mRNA levels. 2-Bromopalmitate, an inhibitor of fatty acid mitochondrial beta-oxidation, reproduced the inhibitory effect of palmitate on insulin mRNA levels. In contrast, palmitate methyl ester, which is not metabolized, and the medium-chain fatty acid octanoate, which is readily oxidized, did not affect insulin gene expression, suggesting that fatty-acid inhibition of insulin gene expression requires activation of the esterification pathway. These results demonstrate that inhibition of insulin gene expression upon prolonged exposure of islets to palmitate is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids.

Determinants of glucose toxicity and its reversibility in the pancreatic islet beta-cell line, HIT-T15.

HIT-T15 cells, a clonal beta-cell line, were cultured and passaged weekly for 6 mo in RPMI 1640 media containing various concentrations of glucose. Insulin content decreased in the intermediate- and late-passage cells as a continuous rather than a threshold glucose concentration effect. In a second series of experiments, cells were grown in media containing either 0.8 or 16.0 mM glucose from passages 76 through 105. Subcultures of passages 86, 92, and 99 that had been grown in media containing 16.0 mM glucose were switched to media containing 0.8 mM glucose and also carried forward to passage 105. Dramatic increases in insulin content and secretion and insulin gene expression were observed when the switches were made at passages 86 and 92 but not when the switch was made at passage 99. These findings suggest that glucose toxicity of insulin-secreting cells is a continuous rather than a threshold function of glucose concentration and that the shorter the period of antecedent glucose toxicity, the more likely that full recovery of cell function will occur.

In vivo prevention of hyperglycemia also prevents glucotoxic effects on PDX-1 and insulin gene expression.

Chronic exposure of pancreatic islet beta-cell lines to supraphysiologic glucose concentrations causes defects in insulin gene expression and insulin secretion. To determine whether these in vitro phenomena have pathophysiologic relevance in vivo, we studied the Zucker diabetic fatty (ZDF) rat, an animal model of type 2 diabetes. The ZDF animals had relatively higher levels of glycemia and islet insulin mRNA at 6 weeks of age than age-matched Zucker lean control (ZLC) rats. As glycemia increased in 12- and 16-week-old ZDF rats, we observed decrements in glucose-induced insulin secretion during static incubations of pancreatic islets and in insulin mRNA levels, PDX-1 mRNA levels, and PDX-1 protein binding to the insulin promoter compared with age-matched ZLC rats and 6-week-old ZDF rats. To determine whether normalization of blood glucose levels would prevent these defects, ZDF rats were treated with troglitazone beginning at 6 weeks of age. Troglitazone prevented ZDF rats from becoming hyperglycemic and preserved glucose-induced insulin responses. Furthermore, troglitazone-treated ZDF animals had greater levels of insulin and PDX-1 mRNAs compared with untreated ZDF animals of the same ages at 12 and 16 weeks. Our results demonstrate that chronic and progressive hyperglycemia resulting from type 2 diabetes in ZDF rats is associated with loss of insulin and PDX-1 mRNAs and loss of glucose-stimulated insulin secretion. Prevention of hyperglycemia prevented the associated defects in insulin and PDX-1 gene expression and improved insulin secretion. These findings provide the first in vivo evidence that prevention of progressive hyperglycemia in a model of type 2 diabetes preserves insulin and PDX-1 gene expression.

Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants.

Chronic exposure of pancreatic islets to supraphysiologic concentrations of glucose causes adverse alterations in beta cell function, a phenomenon termed glucose toxicity and one that may play a secondary pathogenic role in type 2 diabetes. However, no mechanism of action has been definitively identified for glucose toxicity in beta cells. To ascertain whether chronic oxidative stress might play a role, we chronically cultured the beta cell line, HIT-T15, in medium containing 11.1 mM glucose with and without the antioxidants, N-acetyl-L-cysteine (NAC) or aminoguanidine (AG). Addition of NAC or AG to the culture medium at least partially prevented decreases in insulin mRNA, insulin gene promoter activity, DNA binding of two important insulin promoter transcription factors (PDX-1/STF-1 and RIPE-3b1 activator), insulin content, and glucose-induced insulin secretion. These findings suggested that one mechanism of glucose toxicity in the beta cell may be chronic exposure to reactive oxygen species, i.e., chronic oxidative stress. To ascertain the effects of these drugs on diabetes, NAC or AG was given to Zucker diabetic fatty rats, a laboratory model of type 2 diabetes, from 6 through 12 weeks of age. Both drugs prevented a rise in blood oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde + 4-hydroxy-2-nonenal), and partially prevented hyperglycemia, glucose intolerance, defective insulin secretion as well as decrements in beta cell insulin content, insulin gene expression, and PDX-1 (STF-1) binding to the insulin gene promoter. We conclude that chronic oxidative stress may play a role in glucose toxicity, which in turn may worsen the severity of type 2 diabetes.

Reconstitution of glucotoxic HIT-T15 cells with somatostatin transcription factor-1 partially restores insulin promoter activity.

We have reported that chronic culture of HIT-T15 cells in medium containing supraphysiologic glucose concentrations (11.1 mmol/l) causes a decrease in insulin mRNA levels, insulin content, and insulin release. Furthermore, decreases in insulin gene transcription and binding activity of two essential beta-cell transcription factors, somatostatin transcription factor-1 (STF-1; also known as GSTF, IDX-1, IPF-1, PDX-1, and GSF) and RIPE-3b1 activator, are associated with this glucotoxic effect. In this study, we observed that the loss of RIPE-3b1 occurs much earlier (79% decrease at passage [p]81) than the loss of STF-1 (65% decrease at p104), with abolishment of both factors by p122. Since the STF-1, but not the RIPE-3b1 activator, gene has been cloned, we examined its restorative effects on insulin gene promoter activity after reconstitution with STF-1 cDNA. Basal insulin promoter activities normalized to early (p71-74) passage cells (1.000 +/- 0.069) were 0.4066 +/- 0.093 and 0.142 +/- 0.034 for intermediate (p102-106) and late (p118-122) passage cells, respectively. Early, intermediate, and late passage cells, all chronically cultured in medium containing 11.1 mmol/l glucose, were transfected with STF-1 alone or cotransfected with E2-5, an E-box factor known to be synergistically associated with STF-1. Compared with basal levels, we observed a trend toward an increase in insulin promoter activity in intermediate passage cells with STF-1 transfection (1.43-fold) that became a significant increase when E2-5 was cotransfected (1.78-fold). In late passage cells, transfection of STF-1 alone significantly stimulated a 2.2-fold increase in the insulin promoter activity. Cotransfection of STF-1 and E2-5 in late passage cells stimulated insulin promoter activity 2.8-fold, which was 40% of the activity observed in early passage cells. Control studies in glucotoxic betaTC-6 cells deficient in RIPE-3b1 activator but not STF-1 did not demonstrate an increase in insulin promoter activity after STF-1 transfection. We conclude that loss of RIPE-3b1 activity precedes loss of STF-1 activity in glucotoxic HIT-T15 cells and that defective promoter activity can be partially restored by STF-1 transfection and predict that eventual cloning of the RIPE-3b1 gene will allow cotransfection studies with both factors that will allow full reconstitution of insulin promoter activity.

Differentiation of glucose toxicity from beta cell exhaustion during the evolution of defective insulin gene expression in the pancreatic islet cell line, HIT-T15.

Chronic exposure of HIT-T15 cells to supraphysiologic glucose concentration diminishes insulin gene expression and decreased binding of two critical insulin gene transcription factors, STF-1 and RIPE-3b1 activator. To distinguish whether these changes are caused by glucose toxicity or beta cell exhaustion, HIT-T15 cells grown from passage 75 through 99 in media containing 11.1 mM glucose were switched to 0.8 mM glucose at passage 100. They regained binding of STF-1 and RIPE-3b1 activator and had a partial but minimal return of insulin mRNA expression. In a second study, inclusion of somatostatin in the media-containing 11.1 mM glucose inhibited insulin secretion; however, despite this protection against beta cell exhaustion, dramatic decreases in insulin gene expression, STF-1 and RIPE-3b1 binding, and insulin gene promoter activity still occurred. These data indicate that the glucotoxic effects caused by chronic exposure to supraphysiologic concentration of glucose are only minimally reversible and that they are not due simply to beta cell exhaustion. These observations carry with them the clinical implication that Type II diabetic patients who remain hyperglycemic for prolonged periods may have secondary glucose toxic effects on the beta cell that could lead to defective insulin gene expression and worsening of hyperglycemia.

Identification of a carbohydrate response element in rat S14 gene.

To characterize the mechanism by which carbohydrate feeding regulates S14 gene transcription, we created mutations within footprinted sequences of the carbohydrate response region of the S14 gene and either transiently transfected these mutations into rat primary hepatocytes or end-labeled them for gel mobility shift assays. The wild type DNA gave a 2.9 +/- 1.0-fold response to raising the media glucose concentration. Mutations within the 3' footprint eliminated the glucose response and also eliminated hepatic nuclear factor binding. However, mutations within the 5' footprint did not inhibit the glucose responsiveness and had no effect on hepatic nuclear binding. The gel mobility shift assays were confirmed by appropriate competition gel assays. The 3' footprint contains a sequence similar to the USF (upstream stimulating factor) binding site (CACGTG) that has been suggested to play a role in carbohydrate regulation. However, the gel shift pattern with purified USF1 is distinctly different from that of rat hepatic nuclear extract. These studies demonstrate that the sequence GGCACCCGTGT is required for the carbohydrate response in the S14 gene.

Glucagon and insulin regulate lipolysis in trout liver by altering phosphorylation of triacylglycerol lipase.

Rainbow trout were used to investigate the hormonal regulation by glucagon and insulin of hepatic triacylglycerol (TG) lipase activation. Two purified preparations of the trout hepatic TG lipase enzyme, the 110,000-g preparation and the resuspended ammonium sulfate fraction (ASF), were activated up to 58% with (in mM) 0.5 ATP, 0.01 cAMP, 5 MgCl2, and exogenous protein kinase over control levels. ATP or cAMP alone had no effect on activation. Activation of the trout hepatic lipase was reversible; complete inactivation of the ASF was obtained within 3 h in the presence of exogenous phosphorylase phosphatase. Adenosine 3',5'-cyclic monophosphate (cAMP)/ATP-dependent 32P-phosphorylation of trout hepatic lipase was observed within 5 min of incubation with the cAMP/ATP-Mg2+ activation system and 25 microCi [32P]ATP. Hormonal modulation of trout hepatic lipase phosphorylation was studied in isolated hepatocytes. Hepatocytes were incubated with [32P]-monopotassium phosphate for 3 h, then exposed to mammalian glucagon (GLU). Within 5 min, increased lipolysis was accompanied by a 95% increase in phosphorylation of the enzyme. Mammalian insulin (INS) depressed GLU-stimulated phosphorylation by 56% and inhibited GLU-stimulated lipolysis. These results indicate that GLU and INS modulate lipolysis in trout liver by altering phosphorylation of the TG lipase enzyme.

Nurses' care during labor: its effect on the cesarean birth rate of healthy, nulliparous women.

This retrospective study was designed to determine the influence of nurses' care during labor and delivery on the cesarean birth rate of healthy, nulliparous women. Labor and delivery nurses in a large, nonprofit hospital were grouped according to the cesarean birth rates of their healthy, nulliparous patients in spontaneous labor. Large differences in cesarean birth rates between nurses in the lowest quintile (near 4.9%) and the highest quintile (near 19%) were not explained by differences in maternal age and gravidity, attendance of mother at childbirth class, insurance status, reliance on public assistance, physician who attended labor, use of epidural anesthesia, augmentation of labor, dilation when the nurse assumed care, infant weight, or gestational age. In multivariate analysis, the elapsed time between when the nurse assumed care and birth was significantly shorter for patients of nurses in the lowest quintile of cesarean birth rate (4.4 hrs) compared with patients of nurses in the highest quintile (5.6 hrs). The former were also less likely to have forceps used to assist vaginal delivery than the latter (13% vs 26%). Nurses in the lowest quintile of cesarean birth rates were more likely to use a form to record psychosocial data than nurses in the highest quintile (35% vs 15%). The study suggests that nurses' care during labor is an important factor influencing cesarean birth rates.

Effects of nutritional state, insulin, and glucagon on lipid mobilization in rainbow trout, Oncorhynchus mykiss.

The effects of nutritional state, insulin, and glucagon on lipid mobilization were determined in rainbow trout, Oncorhynchus mykiss. In nutritional state experiments, fish were either fed continuously (except 24 to 36 hr prior to experimentation) with commercial trout chow or fasted for 4 weeks. Lipase activity in liver tissue isolated from fasted fish and cultured for 5 hr was greater than that in tissue isolated from fed fish and cultured. The presence of glucose (5.55 mM) in the incubation medium accentuates lipolytic activity in both liver and adipose tissue. Hormone response was assessed both in vivo and in vitro. Salmon insulin was injected into anesthetized fish (fed continuously except 24 hr prior to injections) in 10 microliters of saline/g body weight; final hormone dose was 100 ng/g body weight. Tissue and plasma were sampled 1 and 3 hr after injection. Insulin resulted in depressed plasma FA concentration and reduced hepatic triacylglycerol lipase activity. In vitro effects of hormones were evaluated by incubating liver and adipose tissue pieces in Hanks-MEM. Glucagon (bovine/porcine) directly stimulated lipid breakdown in both liver and adipose tissue. These actions were manifested by enhanced FA and glycerol released into the culture medium and by elevated triacylglycerol lipase activity. Insulin (bovine) generally appeared antilipolytic as this agent inhibited glucagon-stimulated lipase activity and glucagon-stimulated FA release. Furthermore, insulin (in the presence of glucose) reduced net lipolysis, as indicated by glycerol release, compared to control cultures. These results indicate that nutritional state and glucose are important modulators of lipid mobilization and that glucagon and insulin act directly on lipid storage sites to coordinate lipolysis in rainbow trout.

Glucose-stimulated lipolysis in rainbow trout, Oncorhynchus mykiss, liver.

Rainbow trout, Oncorhynchus mykiss, were used to characterize further the influence of glucose on hepatic lipolysis. Liver was removed from fed fish, cut into 1 mm(3) pieces and incubated for up to 5 h in Hanks medium containing either 2 mM, 5.5 mM, 10 mM, or 25 mM glucose. Glucose-stimulated lipolysis was indicated by tissue triacylglycerol (TG) lipase activity and by medium concentrations of glycerol and fatty acids (FA). Triacylglycerol lipase activity in liver pieces incubated in the presence of higher concentrations (25 mM) of glucose was significantly higher than that in liver pieces incubated in lower concentrations (2 mM) of glucose, rising from 0.075 ± 0.002 (mean ± SEM) nmol FA released/h/mg protein to 0.092 ± 0.004 units. Similarly, higher concentrations of glucose stimulated significantly more FA release and glycerol release from liver pieces than that stimulated by lower concentrations of glucose. Glycerol release from liver pieces incubated in the presence of 10 mM glucose and 25 mM glucose was ca. 2-fold to 2.8-fold, respectively, higher than that from liver pieces incubated in the presence of either 2 mM or 5.5 mM glucose. Fatty acid release from liver pieces incubated in the presence of 10 mM or 25 mM glucose was ca. 1.8-fold higher than that from liver pieces incubated in the presence of either 2 mM or 5.5 mM glucose. Notably, increased glycerol release was not accompanied by a parallel increase in FA. Fatty acid reesterification was more pronounced in liver pieces exposed to higher glucose (10 mM and 25 mM) than in liver pieces exposed to lower glucose (2 mM and 5.5 mM). (14)C-incorporation studies indicated that glucose serves as a carbon source for reesterified FA in trout liver. The route of reesterification appears to be from glucose to glycerophosphate to phosphatidic acid to diacylglycerol to TG. Increasing concentrations of glucose did not affect glycerol kinase activity, indicating that glucose-stimulated lipolysis was not accompanied by increased glycerol recycling within the liver. These results suggest that glucose stimulates fatty acid reesterification and directly enhances net lipolysis in trout liver incubated in vitro.

Insulin suppression is associated with hypersomatostatinemia and hyperglucagonemia in glucose-injected rainbow trout.

Rainbow trout, Oncorhynchus mykiss, were used to evaluate the effects of carbohydrate loading on plasma levels of pancreatic hormones and associated changes in metabolic indexes in a carnivorous fish. Glucose (3,000 mg/dl, 10 microliters/g body wt) was injected intraperitoneally into fish (mean wt 54 +/- 5 g) that were killed 0.5-24 h after administration. Glucose injection resulted in hyperglycemia with maximum glucose levels of 306 +/- 13 mg/dl observed 60 min after injection. Glucose administration also resulted in hyperlipidemia. Plasma fatty acids increased twofold in glucose-injected animals. Alterations in plasma metabolites reflected changes in energy stores. Although total lipid concentration was unaffected by glucose injection, lipolytic enzyme activity in the liver was enhanced. Biosynthetic capacity, as indicated by NADPH production from glucose-6-phosphate dehydrogenase, was decreased by glucose injection. Liver glycogen content was reduced in glucose-injected animals 1 h after injection. Glucose injection was attended by increases in the plasma levels of gene II somatostatin-25 (predominant form of pancreatic somatostatin in salmonids) and of glucagon. Insulin levels were initially suppressed after glucose injection. These results indicate that metabolic adjustments caused by glucose administration can be related to the regulatory action of pancreatic hormones. Furthermore, these results suggest that the somatostatin-secreting cells of the trout are sensitive to glucose and that somatostatin-suppressed insulin secretion contributes to the glucose intolerance of trout.

Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss.

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring(14)C-oleic acid release from(14)C-triolein.(14)C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200-400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40-43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that Vmax=0.016 nmol/h/mg protein and that Km=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg(2+). These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.

Antenatal testing, mobile outpatient monitoring service.

At Good Samaritan Medical Center in Phoenix, Arizona, a group of perinatal nurses examined the potential for improving care for antepartum, high-risk pregnant women on restricted activities in their homes. An antenatal testing, mobile outpatient monitoring service was developed and implemented within three months. The goal is to reduce frequent admissions to the hospital caused by the interruption of bed rest for weekly and biweekly outpatient evaluation and monitoring. The service has resulted in reduced hospital admissions and improved patient awareness of the need for and adherence to activity restrictions.