Jamie S. Harmon

Inactivation of specific β cell transcription factors in type 2 diabetes

Type 2 diabetes (T2DM) commonly arises from islet β cell failure and insulin resistance. Here, we examined the sensitivity of key islet-enriched transcription factors to oxidative stress, a condition associated with β cell dysfunction in both type 1 diabetes (T1DM) and T2DM. Hydrogen peroxide treatment of β cell lines induced cytoplasmic translocation of MAFA and NKX6.1. In parallel, the ability of nuclear PDX1 to bind endogenous target gene promoters was also dramatically reduced, whereas the activity of other key β cell transcriptional regulators was unaffected. MAFA levels were reduced, followed by a reduction in NKX6.1 upon development of hyperglycemia in db/db mice, a T2DM model. Transgenic expression of the glutathione peroxidase-1 antioxidant enzyme (GPX1) in db/db islet β cells restored nuclear MAFA, nuclear NKX6.1, and β cell function in vivo. Notably, the selective decrease in MAFA, NKX6.1, and PDX1 expression was found in human T2DM islets. MAFB, a MAFA-related transcription factor expressed in human β cells, was also severely compromised. We propose that MAFA, MAFB, NKX6.1, and PDX1 activity provides a gauge of islet β cell function, with loss of MAFA (and/or MAFB) representing an early indicator of β cell inactivity and the subsequent deficit of more impactful NKX6.1 (and/or PDX1) resulting in overt dysfunction associated with T2DM.

Chronic oxidative stress as a mechanism for glucose toxicity of the beta cell in Type 2 diabetes

Type 2 diabetes is characterized by a relentless decline in pancreatic islet beta cell function and worsening hyperglycemia despite optimal medical treatment. Our central hypothesis is that residual hyperglycemia, especially after meals, generates reactive oxygen species (ROS), which in turn causes chronic oxidative stress on the beta cell. This hypothesis is supported by several observations. Exposure of isolated islets to high glucose concentrations induces increases in intracellular peroxide levels. The beta cell has very low intrinsic levels of antioxidant proteins and activities and thus is very vulnerable to ROS. Treatment with antioxidants protects animal models of type 2 diabetes against complete development of phenotypic hyperglycemia. The molecular mechanisms responsible for the glucose toxic effect on beta cell function involves disappearance of two important regulators of insulin promoter activity, PDX-1 and MafA. Antioxidant treatment in vitro prevents disappearance of these two transcription factors and normalizes insulin gene expression. These observations suggest that the ancillary treatment with antioxidants may improve outcomes of standard therapy of type 2 diabetes in humans.

Pancreatic islet β-cell and oxidative stress: The importance of glutathione peroxidase

Pancreatic β‐cell function continuously deteriorates in type 2 diabetes despite optimal treatment regimens, which has been attributed to hyperglycemia itself via formation of excess levels of reactive oxygen species (ROS). Glutathione peroxidase GPx), by virtue of its ability to catabolize both H2O2 and lipid peroxides, is uniquely positioned to protect tissues from ROS. The level of this antioxidant in β cells is extremely low and overexpression of GPx in islets provides enhanced protection against oxidative stress. This suggests that GPx mimetics may represent a valuable ancillary treatment that could add a novel layer of protection for the β‐cell.

β-Cell-Specific Overexpression of Glutathione Peroxidase Preserves Intranuclear MafA and Reverses Diabetes in db/db Mice

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.

Zinc, Not Insulin, Regulates the Rat α-Cell Response to Hypoglycemia In Vivo

The intraislet insulin hypothesis proposes that the decrement in β-cell insulin secretion during hypoglycemia provides an activation signal for α-cells to release glucagon. A more recent hypothesis proposes that zinc atoms suppress glucagon secretion via their ability to open α-cell ATP-sensitive K+ channels. Since insulin binds zinc, and zinc is cosecreted with insulin, we tested whether decreased zinc delivery to the α-cell activates glucagon secretion. In streptozotocin-induced diabetic Wistar rats, we observed that switching off intrapancreatic artery insulin infusions in vivo during hypoglycemia greatly improved glucagon secretion (area under the curve [AUC]: control group 240 ± 261 and experimental group 4,346 ± 1,259 pg · ml−1 · 90 min−1; n = 5, P < 0.02). Switching off pancreatic artery infusions of zinc chloride during hypoglycemia also improved the glucagon response (AUC: control group 817 ± 107 and experimental group 3,445 ± 573 pg · ml−1 · 90 min−1; n = 6, P < 0.01). However, switching off zinc-free insulin infusions had no effect. Studies of glucose uptake in muscle and liver cell lines verified that the zinc-free insulin was biologically active. We conclude that zinc atoms, not the insulin molecule itself, provide the switch-off signal from the β-cell to the α-cell to initiate glucagon secretion during hypoglycemia.

ATP-Sensitive K+ Channel Mediates the Zinc Switch-Off Signal for Glucagon Response During Glucose Deprivation

OBJECTIVE The 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 METHODS All studies were performed using perifused isolated islets. RESULTS In 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. CONCLUSIONS These 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.

Intrahepatic Glucose Flux as a Mechanism for Defective Intrahepatic Islet α-Cell Response to Hypoglycemia

OBJECTIVE— Glucagon responses to hypoglycemia from islets transplanted in the liver are defective. To determine whether this defect is related to intrahepatic glycogen, islets from inbred Lewis rats were transplanted into the hepatic sinus (H group), peritoneal cavity (P group), omentum (O group), and kidney capsule (K group) of recipient Lewis rats previously rendered diabetic with streptozotocin (STZ). RESEARCH DESIGN AND METHODS— Glucagon responses to hypoglycemia were obtained before and after transplantation under fed conditions and after fasting for 16 h and 48 h to deplete liver glycogen. RESULTS— Glucagon (area under the curve) responses to hypoglycemia in the H group (8,839 ± 1,988 pg/ml per 90 min) were significantly less than in normal rats (40,777 ± 8,192; P < 0.01). Fasting significantly decreased hepatic glycogen levels. Glucagon responses in the H group were significantly larger after fasting (fed 8,839 ± 1,988 vs. 16-h fasting 24,715 ± 5,210 and 48-h fasting 29,639 ± 4,550; P < 0.01). Glucagon response in the H group decreased after refeeding (48-h fasting 29,639 ± 4,550 vs. refed 10,276 ± 2,750; P < 0.01). There was no difference in glucagon response to hypoglycemia between the H and the normal control group after fasting for 48 h (H 29,639 ± 4,550 vs. control 37,632 ± 5,335; P = NS). No intragroup differences were observed in the P, O, and K groups, or normal control and STZ groups, when comparing fed or fasting states. CONCLUSIONS— These data suggest that defective glucagon responses to hypoglycemia by intrahepatic islet α-cells is due to dominance of a suppressive signal caused by increased glucose flux and glucose levels within the liver secondary to increased glycogenolysis caused by systemic hypoglycemia.

Ebselen treatment prevents islet apoptosis, maintains intranuclear Pdx-1 and MafA levels, and preserves β-cell mass and function in ZDF rats

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.

d-Glyceraldehyde Causes Production of Intracellular Peroxide in Pancreatic Islets, Oxidative Stress, and Defective Beta Cell Function via Non-mitochondrial Pathways

d-Glyceraldehyde (d-GLYC) is usually considered to be a stimulator of insulin secretion but theoretically can also form reactive oxygen species (ROS), which can inhibit beta cell function. We examined the time- and concentration-dependent effects of d-GLYC on insulin secretion, insulin content, and formation of ROS. We observed that a 2-h exposure to 0.05–2 mm d-GLYC potentiated glucose-stimulated insulin secretion (GSIS) in isolated Wistar rat islets but that higher concentrations inhibited GSIS. A 24-h exposure to 2 mm d-GLYC inhibited GSIS, decreased insulin content, and increased intracellular peroxide levels (2.14 ± 0.31-fold increase, n = 4, p < 0.05). N-Acetylcysteine (10 mm) prevented the increase in intracellular peroxides and the adverse effects of d-GLYC on GSIS. In the presence of 11.1 but not 3.0 mm glucose, koningic acid (10 μm), a specific glyceraldehyde-3-phosphate dehydrogenase inhibitor, increased intracellular peroxide levels (1.88 ± 0.30-fold increase, n = 9, p < 0.01) and inhibited GSIS (control GSIS = p < 0.001; koningic acid GSIS, not significant). To determine whether oxidative phosphorylation was the source of ROS formation, we cultured rat islets with mitochondrial inhibitors. Neither rotenone or myxothiazol prevented d-GLYC-induced increases in islet ROS. Adenoviral overexpression of manganese superoxide dismutase also failed to prevent the effect of d-GLYC to increase ROS levels. These observations indicate that exposure to excess d-GLYC increases reactive oxygen species in the islet via non-mitochondrial pathways and suggest the hypothesis that the oxidative stress associated with elevated d-GLYC levels could be a mechanism for glucose toxicity in beta cells exposed chronically to high glucose concentrations.

Glutathione peroxidase protein expression and activity in human islets isolated for transplantation

Abstract:  Background:  Overexpression of antioxidant enzymes has been reported to protect rodent beta cells from oxidative stress. However, very little is known about protein expression and activity of antioxidant enzymes in human islets.