Long-chain fatty acids amplify insulin secretion from the pancreatic beta cell. The G protein-coupled receptor GPR40 is specifically expressed in beta cells and is activated by fatty acids. Loss of function of GPR40 was shown to markedly inhibit fatty-acid stimulation of insulin secretion in vitro. However, the role of GPR40 in acute regulation of insulin secretion in vivo remains unclear. To this aim, we generated GPR40 knock-out (KO) mice and examined glucose homeostasis, insulin secretion in response to glucose and Intralipid in vivo, and insulin secretion in vitro after short-and long-term exposure to fatty acids. Our results show that GPR40 KO mice have essentially normal glucose tolerance and insulin secretion in response to glucose. Insulin secretion in response to Intralipid was reduced by approximately 50%. In isolated islets, insulin secretion in response to glucose and other secretagogues was unaltered, but fatty-acid potentiation of insulin release was markedly reduced. Islets from GPR40 KO mice were as sensitive to fatty-acid inhibition of insulin secretion upon prolonged exposure as islets from wild-type animals. We conclude that GPR40 contributes approximately half of the full insulin secretory response to fatty acids in mice, but does not play a role in the mechanisms of lipotoxicity.Long-chain fatty acids are essential regulators of normal pancreatic beta-cell function, and are likely to play a role in the pathogenesis of beta-cell dysfunction in type 2 diabetes (reviewed in (1)). Under normal circumstances, fatty acids do not initiate insulin release, but amplify glucose-stimulated insulin secretion (GSIS) (2-5). Fatty-acid potentiation of insulin secretion has physiological implications, particularly after a period of fasting (6). Until recently, the prevailing model postulated that the effects of fatty acids on the beta cell were mediated by their intracellular metabolism and the generation of lipid derived signals which, in turn, potentiate GSIS (2;7). According to this hypothesis, fatty acids are transported across the plasma membrane and activated into their long-chain coenzyme A esters, which in turn modulate a number of intracellular targets that influence insulin secretion. Moreover, evidence suggests that intracellular fatty-acid metabolism is a key component of both nutrient-and nonnutrient-induced insulin secretion (7). In contrast to their acute, stimulatory effect on GSIS, prolonged exposure to elevated levels of fatty acids impairs beta-cell function, a phenomenon referred to as lipotoxicity (reviewed in (1)). The mechanisms of lipotoxicity remain poorly understood but have been proposed to also involve intracellular metabolism of fatty acids and the generation of lipid-derived metabolites (8).The models described above have been challenged by the observation that fatty acids activate the G-protein coupled receptor (GPCR) GPR40 (9-11), also referred to as the free fatty-acid 1 receptor (FFA 1 R) (12;13). GPR40 belongs to a class of GPCR with high structural conservation, o...

Chronic hyperglycemia causes oxidative stress, which contributes to damage in various tissues and cells, including pancreatic beta-cells. The expression levels of antioxidant enzymes in the islet are low compared with other tissues, rendering the beta-cell more susceptible to damage caused by hyperglycemia. The aim of this study was to investigate whether increasing levels of endogenous glutathione peroxidase-1 (GPx-1), specifically in beta-cells, can protect them against the adverse effects of chronic hyperglycemia and assess mechanisms that may be involved. C57BLKS/J mice overexpressing the antioxidant enzyme GPx-1 only in pancreatic beta-cells were generated. The biological effectiveness of the overexpressed GPx-1 transgene was documented when beta-cells of transgenic mice were protected from streptozotocin. The transgene was then introgressed into the beta-cells of db/db mice. Without use of hypoglycemic agents, hyperglycemia in db/db-GPx(+) mice was initially ameliorated compared with db/db-GPx(-) animals and then substantially reversed by 20 wk of age. beta-Cell volume and insulin granulation and immunostaining were greater in db/db-GPx(+) animals compared with db/db-GPx(-) animals. Importantly, the loss of intranuclear musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) that was observed in nontransgenic db/db mice was prevented by GPx-1 overexpression, making this a likely mechanism for the improved glycemic control. These studies demonstrate that enhancement of intrinsic antioxidant defenses of the beta-cell protects it against deterioration during hyperglycemia.

The glucagon response is the first line of defense against hypoglycemia and is lost in insulin-dependent diabetes. The ␤-cell "switch-off" hypothesis proposes that a sudden cessation of insulin secretion from ␤-cells into the portal circulation of the islet during hypoglycemia is a necessary signal for the glucagon response from downstream ␣-cells. Although indirect evidence exists to support this hypothesis, it has not been directly tested in vivo by provision and then discontinuation of regional reinsulinization of ␣-cells at the time of a hypoglycemic challenge. We studied streptozotocin (STZ)-induced diabetic Wistar rats that had no glucagon response to a hypoglycemic challenge. We reestablished insulin regulation of the ␣-cell by regionally infusing insulin (0.025 U/min) directly into the superior pancreaticoduodenal artery (SPDa) of STZ-administered rats at an infusion rate that did not alter systemic venous glucose levels. SPDa insulin infusion was switched off simultaneously when blood glucose fell to <60 mg/dl after a jugular venous insulin injection. This maneuver restored the glucagon response to hypoglycemia (peak change within 5-10 min ‫؍‬ 326 ؎ 98 pg/ml, P < 0.05; and peak change within 15-20 min ‫؍‬ 564 ؎ 148 pg/ml, P < 0.01). No response was observed when the SPDa insulin infusion was not turned off (peak change within 5-10 min ‫؍‬ 44 ؎ 85 pg/ml, P ‫؍‬ NS; and peak change within 15-20 min ‫؍‬ 67 ؎ 97 pg/ml, P ‫؍‬ NS) or when saline instead of insulin was infused and then switched off (peak change within 5-10 min ‫؍‬ ؊44 ؎ 108 pg/ml, P ‫؍‬ NS; and peak change within 15-20 min ‫؍‬ ؊13 ؎ 43 pg/ml, P ‫؍‬ NS). No responses were observed during euglycemia (peak change within 5-10 min ‫؍‬ 48 ؎ 35 pg/ml, P ‫؍‬ NS; and peak change within 15-20 min ‫؍‬ 259 ؎ 129 pg/ml, P ‫؍‬ NS) or hyperglycemia (peak change within 5-10 min ‫؍‬ 49 ؎ 62 pg/ml, P ‫؍‬ NS; and peak change within 15-20 min ‫؍‬ 138 ؎ 87 pg/ml, P ‫؍‬ NS). Thus, the glucagon response to hypoglycemia that was absent in rats made diabetic by STZ was restored by regional infusion and then discontinuation of insulin. These data provide direct in vivo support for the ␤-cell "switch-off" hypothesis and indicate that the ␣-cell is not intrinsically abnormal in insulin-dependent diabetes because of STZ-induced destruction of ␤-cells.

The "switch-off" hypothesis to explain ␤-cell regulation of ␣-cell function during hypoglycemia has not been assessed previously in isolated islets, largely because they characteristically do not respond to glucose deprivation by secreting glucagon. We examined this hypothesis using normal human and Wistar rat islets, as well as islets from streptozotocin (STZ)-administered ␤-celldeficient Wistar rats. As expected, islets perifused with glucose and 3-isobutryl-1-methylxanthine did not respond to glucose deprivation by increasing glucagon secretion. However, if normal rat islets were first perifused with 16.7 mmol/l glucose to increase endogenous insulin secretion, followed by discontinuation of the glucose perifusate, a glucagon response to glucose deprivation was observed (peak change within 10 min after switch off ‫؍‬ 61 ؎ 15 pg/ml [mean ؎ SE], n ‫؍‬ 6, P < 0.01). A glucagon response from normal human islets using the same experimental design was also observed. A glucagon response (peak change within 7 min after switch off ‫؍‬ 31 ؎ 1 pg/ml, n ‫؍‬ 3, P < 0.01) was observed from ␤-cell-depleted, STZ-induced diabetic rats whose islets still secreted small amounts of insulin. However, when these islets were first perifused with both exogenous insulin and 16.7 mmol/l glucose, followed by switching off both the insulin and glucose perifusate, a significantly larger (P < 0.05) glucagon response was observed (peak change within 7 min after switch off ‫؍‬ 71 ؎ 11 pg/ml, n ‫؍‬ 4, P < 0.01). This response was not observed if the insulin perifusion was not switched off when the islets were deprived of glucose or when insulin was switched off without glucose deprivation. These data uniquely demonstrate that both normal, isolated islets and islets from STZ-administered rats can respond to glucose deprivation by releasing glucagon if they are first provided with increased endogenous or exogenous insulin. These results fully support the ␤-cell switch-off hypothesis as a key mechanism for the ␣-cell response to hypoglycemia.

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.

We used intravenous arginine with measurements of insulin, C-peptide, and glucagon to examine β-cell and α-cell survival and function in a group of 10 chronic pancreatitis recipients 1–8 years after total pancreatectomy and autoislet transplantation. Insulin and C-peptide responses correlated robustly with the number of islets transplanted (correlation coefficients range 0.81–0.91; P < 0.01–0.001). Since a wide range of islets were transplanted, we normalized the insulin and C-peptide responses to the number of islets transplanted in each recipient for comparison with responses in normal subjects. No significant differences were observed in terms of magnitude and timing of hormone release in the two groups. Three recipients had a portion of the autoislets placed within their peritoneal cavities, which appeared to be functioning normally up to 7 years posttransplant. Glucagon responses to arginine were normally timed and normally suppressed by intravenous glucose infusion. These findings indicate that arginine stimulation testing may be a means of assessing the numbers of native islets available in autologous islet transplant candidates and is a means of following posttransplant α- and β-cell function and survival.

We reported earlier that β-cell–specific overexpression of glutathione peroxidase (GPx)-1 significantly ameliorated hyperglycemia in diabetic db/db mice and prevented glucotoxicity-induced deterioration of β-cell mass and function. We have now ascertained whether early treatment of Zucker diabetic fatty (ZDF) rats with ebselen, an oral GPx mimetic, will prevent β-cell deterioration. No other antihyperglycemic treatment was given. Ebselen ameliorated fasting hyperglycemia, sustained nonfasting insulin levels, lowered nonfasting glucose levels, and lowered HbA1c levels with no effects on body weight. Ebselen doubled β-cell mass, prevented apoptosis, prevented expression of oxidative stress markers, and enhanced intranuclear localization of pancreatic and duodenal homeobox (Pdx)-1 and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), two critical insulin transcription factors. Minimal β-cell replication was observed in both groups. These findings indicate that prevention of oxidative stress is the mechanism whereby ebselen prevents apoptosis and preserves intranuclear Pdx-1 and MafA, which, in turn, is a likely explanation for the beneficial effects of ebselen on β-cell mass and function. Since ebselen is an oral antioxidant currently used in clinical trials, it is a novel therapeutic candidate to ameliorate fasting hyperglycemia and further deterioration of β-cell mass and function in humans undergoing the onset of type 2 diabetes.

OBJECTIVEThe intraislet insulin hypothesis proposes that glucagon secretion during hypoglycemia is triggered by a decrease in intraislet insulin secretion. A more recent hypothesis based on in vivo data from hypoglycemic rats is that it is the decrease in zinc cosecreted with insulin from β-cells, rather than the decrease in insulin itself, that signals glucagon secretion from α-cells during hypoglycemia. These studies were designed to determine whether closure of the α-cell ATP-sensitive K+ channel (KATP channel) is the mechanism through which the zinc switch-off signal triggers glucagon secretion during glucose deprivation.RESEARCH DESIGN AND METHODSAll studies were performed using perifused isolated islets.RESULTSIn control experiments, the expected glucagon response to an endogenous insulin switch-off signal during glucose deprivation was observed in wild-type mouse islets. In experiments with streptozotocin-treated wild-type islets, a glucagon response to an exogenous zinc switch-off signal was observed during glucose deprivation. However, this glucagon response to the zinc switch-off signal during glucose deprivation was not seen in the presence of nifedipine, diazoxide, or tolbutamide or if KATP channel knockout mouse islets were used. All islets had intact glucagon responses to epinephrine.CONCLUSIONSThese data demonstrate that closure of KATP channels and consequent opening of calcium channels is the mechanism through which the zinc switch-off signal triggers glucagon secretion during glucose deprivation.