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.
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.
The intraislet insulin hypothesis proposes that the decrement in -cell insulin secretion during hypoglycemia provides an activation signal for ␣-cells to release glucagon. A more recent hypothesis proposes that zinc atoms suppress glucagon secretion via their ability to open ␣-cell ATPsensitive K ؉ channels. Since insulin binds zinc, and zinc is cosecreted with insulin, we tested whether decreased zinc delivery to the ␣-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 ⅐ ml ؊1 ⅐ 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 ⅐ ml ؊1 ⅐ 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 -cell to the ␣-cell to initiate glucagon secretion during hypoglycemia. Diabetes 56:1107-1112, 2007 G lucagon secretion into the hepatic portal circulation is critical for counterregulation of hypoglycemia by virtue of its stimulatory effect on hepatic glycogenolysis, which releases glucose into the systemic circulation to return blood glucose to normal levels. The decrement in insulin release from -cells has been proposed to signal the ␣-cell to release glucagon (1-3). Recently, we demonstrated this phenomenon directly in vivo in streptozotocin-induced diabetic rats and in vitro using isolated islets from these animals. In the in vivo studies, an insulin infusion into the pancreatic artery was switched off when the animals were made hypoglycemic by a jugular-vein infusion of insulin (4).Glucagon secretion, absent in control diabetic animals, was fully restored by this maneuver. Glucagon release was not initiated by this maneuver if saline rather than insulin was used, or if insulin was infused but not switched off. The glucagon response was not observed during normoglycemic or hyperglycemic conditions; the switch-off signal was effective only in the setting of hypoglycemia. We corroborated these findings with in vitro islet perifusion studies (5).Recent work from several laboratories has demonstrated that zinc atoms are capable of suppressing ␣-cell function via activation of rat ␣-cell ATP-sensitive K ϩ (K ATP ) channels (6 -9). Since zinc atoms are bound by and cosecreted with insulin, we hypothesized that zinc might provide the switch-off signal during hypoglycemia. To evaluate this hypothesis, we performed in vivo studies using intrapancreatic artery infusions of zinc-containing insulin, zinc chloride alone, or zinc-free insulin in streptozo...
HOX antisense intergenic RNA (HOTAIR), a long non-coding RNA, plays an important role in the development of many types of cancers. Its function in acute leukemia (AL), however, has not been examined. The present study investigated the role of HOTAIR and its downstream genes in AL, and determined whether it could act as a molecular marker for prediction of leukemia development and prognosis. Real-time quantitative PCR was used to examine the expression of each gene in the HOTAIR signaling pathway in AL patients. The relationship between expression of HOTAIR and downstream genes and AL prognosis was analyzed. Expression of HOTAIR in patients with acute monocytic leukemia (M5) was increased as compared to controls (P<0.05). Compared to patients with low HOTAIR expression, overall survival and event-free survival of patients with high HOTAIR expression was significantly reduced. In addition, the expression of downstream genes in the HOTAIR signaling pathway including EZH2, LSD1, DNMT3A and DNMT3B was significantly increased in AL patients, and showed a significant positive correlation with high expression of HOTAIR (P<0.05). In conclusion, HOTAIR was closely related with a poor prognosis in AL patients. It may be involved in the development of leukemia by mediating methylation of DNA and histones.
The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 (IL-1). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 up-regulated GCLC expression (10 ng/ml IL-1, 3.76 ؎ 0.86; 100 ng/ml IL-1, 4.22 ؎ 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF B and also increased reactive oxygen species levels (10 ng/ml IL-1, 5.41 ؎ 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 ؎ 0.1; 10 ng/ml IL-1, 8.0 ؎ 0.5; 100 ng/ml IL-1, 8.2 ؎ 0.5-fold control; p < 0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1 on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 significantly decreased glucose-stimulated insulin secretion (control, 123.8 ؎ 17.7; IL-1, 40.2 ؎ 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1.Glutamylcysteine ligase (GCL) 1 is the primary and ratelimiting enzyme responsible for de novo synthesis of intracellular glutathione (GSH). This enzyme catalyzes ATP-dependent ligation of L-glutamate and L-cysteine to form ␥-glutamyl-L-cysteine, which undergoes another ATP-dependent ligation with glycine catalyzed by glutathione synthetase to form the final GSH product (1, 2). GCL is a heterodimer, composed of a light regulatory subunit as well as a heavy catalytic subunit. Although both are required for optimal enzyme activity, overexpression of the catalytic subunit GCLC alone is sufficient to increase enzyme activity significantly over control levels (1). The regulation of GCL expression and activity has been studied in many other cell types, including mesangial and endothelial cells, but not the pancreatic islet. Long term exposure to high glucose levels decreases GCL expression in mesangial, retinal Muller, and endothelial cells, which results in a decrease in GSH levels (3-5). Long term exposure to high glucose conditions inhibits endothelial cells from responding to cytokine exposure with an increase in GCL expression and activity (3). That the pathogenesis of diabetes mellitus involves interactions with IL-1 and oxidative stress secondary to chronic hyperglycemia (6, 7) raises the questions of whether GCL is normally expressed in the islet and whe...
Background:The mechanism whereby IL-1 induces islet PGE 2 production and inhibition of insulin secretion is unclear. Results: Basal COX-2 protein levels are stimulated by IL-1; mPGES-1 levels are not. Conclusion: COX-2, not mPGES-1, is the final regulatory enzyme for PGE 2 production. Significance: COX-2, not mPGES-1, is the pharmacologic target to protect islet function from IL-1 in type 2 diabetes.
Converting lignin components into a single product is a promising way to upgrade lignin. Here, an efficient biocatalyst was developed to selectively produce gallate from lignin components by integrating three main reactions: hydroxylation, O-demethylation, and aryl side-chain oxidation. A rationally designed hydroxylase system was first introduced into a gallate biodegradation pathway-blocked Rhodococcus opacus mutant so that gallate accumulated from protocatechuate and compounds in its upper pathways. Native and heterologous O-demethylation systems were then used, leading to multiple lignin-derived methoxy aromatics being converted to gallate. Furthermore, an aryl side-chain oxidase was engaged to broaden the substrate spectrum. Consequently, the developed biocatalyst showed that gallate yields as high as 0.407 and 0.630 g of gallate per gram of lignin when alkaline-pretreated lignin and base-depolymerized ammonia fiber explosion lignin were applied as substrates, respectively. These results suggested that this rationally developed biocatalyst enabled the lignin valorization process to be simple and efficient.
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