Biomedical subjects

C B Newgard

Publications and source records attributed to C B Newgard.

At least 19 recordsLinked to original sources

PubMed2003

Inflammatory mechanisms in diabetes: lessons from the beta-cell.

Inflammation plays an important role in the destruction of pancreatic islet beta-cells that leads to type I diabetes. This involves infiltration of T-cells and macrophages into the islets and local production of inflammatory cytokines such as interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. Our laboratory has developed several strategies for protecting beta-cells against oxidative stress and cytokine-induced cytotoxicity. These include a cytokine selection strategy that results in cell lines that are resistant to the combined effects of IL-1 beta+IFN-gamma. More recently, we have combined the cytokine selection procedure with overexpression of the antiapoptotic gene bcl-2, resulting in cell lines with greater resistance to oxidative stress and cytokine-induced damage than achieved with either procedure alone. This article summarizes this work and the remarkably divergent mechanisms by which protection is achieved in the different model systems. We also discuss the potential relevance of insights gained from these approaches for enhancing islet cell survival and function in both major forms of diabetes.

Animals↗

PubMed2001

Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1.

Blood glucose levels are maintained by the balance between glucose uptake by peripheral tissues and glucose secretion by the liver. Gluconeogenesis is strongly stimulated during fasting and is aberrantly activated in diabetes mellitus. Here we show that the transcriptional coactivator PGC-1 is strongly induced in liver in fasting mice and in three mouse models of insulin action deficiency: streptozotocin-induced diabetes, ob/ob genotype and liver insulin-receptor knockout. PGC-1 is induced synergistically in primary liver cultures by cyclic AMP and glucocorticoids. Adenoviral-mediated expression of PGC-1 in hepatocytes in culture or in vivo strongly activates an entire programme of key gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase, leading to increased glucose output. Full transcriptional activation of the PEPCK promoter requires coactivation of the glucocorticoid receptor and the liver-enriched transcription factor HNF-4alpha (hepatic nuclear factor-4alpha) by PGC-1. These results implicate PGC-1 as a key modulator of hepatic gluconeogenesis and as a central target of the insulin-cAMP axis in liver.

3T3 Cells↗

PubMed2001

Protein synthesis. The perks of balancing glucose.

What do the regulation of translation initiation and glucose metabolism have to do with each other? Quite a lot, it seems, according to Sonenberg and Newgard in their Perspective. They discuss new findings that identify the kinase responsible for inactivating eIF2--a factor that is required for translation initiation (and hence protein synthesis)--when the endoplasmic reticulum is under stress. Loss of this kinase results in destruction of insulin-producing b cells in the pancreas and dysregulation of glucose homeostasis.

Animals↗

PubMed2001

Overexpression of the P46 (T1) translocase component of the glucose-6-phosphatase complex in hepatocytes impairs glycogen accumulation via hydrolysis of glucose 1-phosphate.

The final step of gluconeogenesis and glycogenolysis is catalyzed by the glucose-6-phosphatase (Glc-6-Pase) enzyme complex, located in the endoplasmic reticulum. The complex consists of a 36-kDa catalytic subunit (P36), a 46-kDa glucose 6-phosphate translocase (P46), and putative glucose and inorganic phosphate transporters. Mutations in the genes encoding P36 or P46 have been linked to glycogen storage diseases type Ia and type Ib, respectively. However, the relative roles of these two proteins in control of the rate of glucose 6-phosphate hydrolysis have not been defined. To gain insight into this area, we have constructed a recombinant adenovirus containing the cDNA encoding human P46 (AdCMV-P46) and treated rat hepatocytes with this virus, or a virus encoding P36 (AdCMV-P36), or the combination of both viruses, resulting in large and equivalent increases in expression of the transgenes within 8-24 h of viral treatment. The overexpressed P46 protein was appropriately targeted to hepatocyte microsomes and caused a 58% increase in glucose 6-phosphate hydrolysis in nondetergent-treated (intact) microsomal preparations relative to controls, whereas overexpression of P36 caused a 3.6-fold increase. Overexpression of P46 caused a 50% inhibition of glycogen accumulation in hepatocytes from fasted rats incubated at 25 mm glucose relative to cells treated with a control virus (AdCMV-betaGAL). Furthermore, in hepatocytes from fed rats cultured at 25 mm glucose and then exposed to 15 mm glucose, AdCMV-P46 treatment activated glycogenolysis, as indicated by a 50% reduction in glycogen content relative to AdCMV-betaGAL-treated controls. In contrast, overexpression of P46 had only small effects on glycolysis, whereas overexpression of P36 had large effects on both glycogen metabolism and glycolysis, even in the presence of co-overexpressed glucokinase. Finally, P46 overexpression enhanced glucose 1-phosphate but not fructose 6-phosphate hydrolysis in intact microsomes, providing a mechanism by which P46 overexpression may exert its preferential effects on glycogen metabolism.

Animals↗

PubMed2001

Expression of the transcription factor STAT-1 alpha in insulinoma cells protects against cytotoxic effects of multiple cytokines.

Destruction of pancreatic islet beta-cells in type 1 diabetes appears to result from direct contact with infiltrating T-cells and macrophages and exposure to inflammatory cytokines such as interferon (IFN)-gamma, interleukin (IL)-1 beta, and tumor necrosis factor TNF-alpha that such cells produce. We recently reported on a method for selection of insulinoma cells that are resistant to the cytotoxic effects of inflammatory cytokines (INS-1(res)), involving their growth in progressively increasing concentrations of IL-1 beta plus IFN-gamma, and selection of surviving cells. In the current study, we have investigated the molecular mechanism of cytokine resistance in INS-1(res) cells. By focusing on the known components of the IFN-gamma receptor signaling pathway, we have discovered that expression levels of signal transducer and activator of transcription (STAT)-1 alpha are closely correlated with the cytokine-resistant and -sensitive phenotypes. That STAT-1 alpha is directly involved in development of cytokine resistance is demonstrated by an increase of viability from 10 +/- 2% in control cells to 50 +/- 6% in cells with adenovirus-mediated overexpression of STAT-1 alpha (p < 0.001) after culture of both cell groups in the presence of 100 units/ml IFN-gamma plus 10 ng/ml IL-1 beta for 48 h. The resistance to IL-1 beta plus IFN-gamma in STAT-1 alpha-expressing cells is due in part to interference with IL-1 beta-mediated stimulation of inducible nitric-oxide synthase expression and nitric oxide production. Furthermore, overexpression of STAT-1 alpha does not impair robust glucose-stimulated insulin secretion in the INS-1-derived cell line 832/13. We conclude that expression of STAT-1 alpha may be a means of protecting insulin-producing cell lines from cytokine damage, which, in conjunction with appropriate cell-impermeant macroencapsulation devices, may allow such cells to be used for insulin replacement in type 1 diabetes.

Adenoviridae↗

PubMed2001

Overexpression of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase in mouse liver lowers blood glucose by suppressing hepatic glucose production.

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is an important regulatory enzyme of glucose metabolism. By controlling the level of fructose-2,6-bisphosphate, an allosteric activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase regulates hepatic glucose output. We studied the effects of adenovirus-mediated overexpression of this enzyme on hepatic glucose metabolism in normal or diabetic mice. These animals were treated with virus encoding either wild-type or bisphosphatase activity-deficient 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase. Seven days after virus injection, hepatic fructose-2,6-bisphosphate levels increased significantly in both normal and diabetic mice, with larger increases observed in animals with overexpression of the mutant enzyme. Blood glucose levels in normal mice overexpressing either enzyme were lowered, accompanied by increased plasma lactate, triglycerides, and FFAs. Blood glucose levels were markedly reduced in diabetic mice overexpressing the wild-type enzyme, and still more so in mice overexpressing the mutant form of the enzyme. The lower blood glucose levels in diabetic mice were accompanied by partially normalized plasma triglycerides and FFAs, increased plasma lactate, and increased liver glycogen levels, relative to diabetic mice treated with a control adenovirus. Our findings underscore the critical role played by hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in control of fuel homeostasis and suggest that this enzyme may be considered as a therapeutic target in diabetes.

Adenoviridae↗

PubMed2000

Glucose activates mitogen-activated protein kinase (extracellular signal-regulated kinase) through proline-rich tyrosine kinase-2 and the Glut1 glucose transporter.

Glucose serves as both a nutrient and regulator of physiological and pathological processes. Presently, we found that glucose and certain sugars rapidly activated extracellular signal-regulated kinase (ERK) by a mechanism that was: (a) independent of glucose uptake/metabolism and protein kinase C but nevertheless cytochalasin B-inhibitable; (b) dependent upon proline-rich tyrosine kinase-2 (PYK2), GRB2, SOS, RAS, RAF, and MEK1; and (c) amplified by overexpression of the Glut1, but not Glut2, Glut3, or Glut4, glucose transporter. This amplifying effect was independent of glucose uptake but dependent on residues 463-468, IASGFR, in the Glut1 C terminus. Accordingly, glucose effects on ERK were amplified by expression of Glut4/Glut1 or Glut2/Glut1 chimeras containing IASGFR but not by Glut1/Glut4 or Glut1/Glut2 chimeras lacking these residues. Also, deletion of Glut1 residues 469-492 was without effect, but mutations involving serine 465 or arginine 468 yielded dominant-negative forms that inhibited glucose-dependent ERK activation. Glucose stimulated the phosphorylation of tyrosine residues 402 and 881 in PYK2 and binding of PYK2 to Myc-Glut1. Our findings suggest that: (a) glucose activates the GRB2/SOS/RAS/RAF/MEK1/ERK pathway by a mechanism that requires PYK2 and residues 463-468, IASGFR, in the Glut1 C terminus and (b) Glut1 serves as a sensor, transducer, and amplifier for glucose signaling to PYK2 and ERK.

3T3 Cells↗

PubMed2000

Overexpression of protein targeting to glycogen in cultured human muscle cells stimulates glycogen synthesis independent of glycogen and glucose 6-phosphate levels.

There is growing evidence that glycogen targeting subunits of protein phosphatase-1 play a critical role in regulation of glycogen metabolism. In the current study, we have investigated the effects of adenovirus-mediated overexpression of a specific glycogen targeting subunit known as protein targeting to glycogen (PTG) in cultured human muscle cells. PTG was overexpressed both in muscle cells cultured at high glucose (glycogen replete) or in cells incubated for 18 h in the absence of glucose and then incubated in high glucose (glycogen re-synthesizing). In both glycogen replete and glycogen resynthesizing cells, PTG overexpression caused glycogen to be synthesized at a linear rate 1-5 days after viral treatment, while in cells treated with a virus lacking a cDNA insert (control virus), glycogen content reached a plateau at day 1 with no further increase. In the glycogen replete PTG overexpressing cells, glycogen content was 20 times that in controls at day 5. Furthermore, in cells undergoing glycogen resynthesis, PTG overexpression caused a doubling of the initial rate of glycogen synthesis over the first 24 h relative to cells treated with control virus. In both sets of experiments, the effects of PTG on glycogen synthesis were correlated with a 2-3-fold increase in glycogen synthase activity state, with no changes in glycogen phosphorylase activity. The alterations in glycogen synthase activity were not accompanied by changes in the intracellular concentration of glucose 6-phosphate. We conclude that PTG overexpression activates glycogen synthesis in a glucose 6-phosphate-independent manner in human muscle cells while overriding glycogen-mediated inhibition. Our findings suggest that modulation of PTG expression in muscle may be a mechanism for enhancing muscle glucose disposal and improving glucose tolerance in diabetes.

Cells, Cultured↗

PubMed2000

Overexpression of a modified human malonyl-CoA decarboxylase blocks the glucose-induced increase in malonyl-CoA level but has no impact on insulin secretion in INS-1-derived (832/13) beta-cells.

The long-chain acyl-CoA (LC-CoA) model of glucose-stimulated insulin secretion (GSIS) holds that secretion is linked to a glucose-induced increase in malonyl-CoA level and accumulation of LC-CoA in the cytosol. We have previously tested the validity of this proposal by overexpressing goose malonyl-CoA decarboxylase (MCD) in INS-1 cells, but these studies have been criticized due to: 1) the small insulin secretion response (2-4-fold) of the INS-1 cells used; 2) unknown contribution of the ATP-sensitive K(+) (K(ATP)) channel-independent pathway of GSIS in INS-1 cells, which has been implicated as the site at which lipids regulate insulin granule exocytosis; and 3) deletion of the N-terminal mitochondrial targeting sequence, but not the C-terminal peroxisomal targeting sequence in the goose MCD construct, raising the possibility that a significant fraction of the overexpressed enzyme was localized to peroxisomes. To address these outstanding concerns, INS-1-derived 832/13 cells, which exhibit robust K(ATP) channel-dependent and -independent pathways of GSIS, were treated with a new adenovirus encoding human MCD lacking both its mitochondrial and peroxisomal targeting sequences (AdCMV-MCD Delta 5), resulting in large increases in cytosolic MCD activity. Treatment of 832/13 cells with AdCMV-MCD Delta 5 completely blocked the glucose-induced rise in malonyl-CoA and attenuated the inhibitory effect of glucose on fatty acid oxidation. However, MCD overexpression had no effect on K(ATP) channel-dependent or -independent GSIS in 832/13 cells. Furthermore, combined treatment of 832/13 cells with AdCMV-MCD Delta 5 and triacsin C, an inhibitor of long chain acyl-CoA synthetase that reduces LC-CoA levels, did not impair GSIS. These findings extend our previous observations and are not consistent with the LC-CoA hypothesis as originally set forth.

Adenoviridae↗

PubMed2000

Glucose down-regulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta -cell.

To better understand the action of glucose on fatty acid metabolism in the beta-cell and the link between chronically elevated glucose or fatty acids and beta-cell decompensation in adipogenic diabetes, we investigated whether glucose regulates peroxisomal proliferator-activated receptor (PPAR) gene expression in the beta-cell. Islets or INS(832/13) beta-cells exposed to high glucose show a 60-80% reduction in PPARalpha mRNA expression. Oleate, either in the absence or presence of glucose, has no effect. The action of glucose is dose-dependent in the 6-20 mm range and maximal after 6 h. Glucose also causes quantitatively similar reductions in PPARalpha protein and DNA binding activity of this transcription factor. The effect of glucose is blocked by the glucokinase inhibitor mannoheptulose, is partially mimicked by 2-deoxyglucose, and is not blocked by the 3-O-methyl or the 6-deoxy analogues of the sugar that are not phosphorylated. Chronic elevated glucose reduces the expression levels of the PPAR target genes, uncoupling protein 2 and acyl-CoA oxidase, which are involved in fat oxidation and lipid detoxification. A 3-day exposure of INS-1 cells to elevated glucose results in a permanent rise in malonyl-CoA, the inhibition of fat oxidation, and the promotion of fatty acid esterification processes and causes elevated insulin secretion at low glucose. The results suggest that a reduction in PPARalpha gene expression together with a rise in malonyl-CoA plays a role in the coordinated adaptation of beta-cell glucose and lipid metabolism to hyperglycemia and may be implicated in the mechanism of beta-cell "glucolipotoxicity."

Animals↗

PubMed2000

Distinctive regulatory and metabolic properties of glycogen-targeting subunits of protein phosphatase-1 (PTG, GL, GM/RGl) expressed in hepatocytes.

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. We have shown that overexpression of one member of the family, protein targeting to glycogen (PTG), causes large increases in glycogen storage in isolated hepatocytes or intact rat liver. In the current study, we have compared the metabolic and regulatory properties of PTG (expressed in many tissues), with two other members of the gene family, G(L) (expressed primarily in liver) and G(M)/R(Gl) (expressed primarily in striated muscle). Adenovirus-mediated expression of these proteins in hepatocytes led to the following key observations. 1) G(L) has the highest glycogenic potency among the three forms studied. 2) Glycogen synthase activity ratio is much higher in G(L)-overexpressing cells than in PTG or G(M)/R(Gl)-overexpressing cells. Thus, at moderate levels of G(L) overexpression, glycogen synthase activity is increased by insulin treatment, but at higher levels of G(L) expression, insulin is no longer required to achieve maximal synthase activity. In contrast, cells with high levels of PTG overexpression retain dose-dependent regulation of glycogen synthesis and glycogen synthase enzyme activity by insulin. 3) G(L)- and G(M)/R(Gl)-overexpressing cells exhibit a strong glycogenolytic response to forskolin, whereas PTG-overexpressing cells are less responsive. This difference may be explained in part by a lesser forskolin-induced increase in glycogen phosphorylase activity in PTG-overexpressing cells. Based on these results, we suggest that expression of either G(L) or G(M)/R(Gl) in liver of diabetic animals may represent a strategy for lowering of blood glucose levels in diabetes.

Animals↗

PubMed2000

Correction of diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance by moderate hyperleptinemia.

Human obesity and high fat feeding in rats are associated with the development of insulin resistance and perturbed carbohydrate and lipid metabolism. It has been proposed that these metabolic abnormalities may be reversible by interventions that increase plasma leptin. Up to now, studies in nongenetic animal models of obesity and in human obesity have concentrated on multiple injection therapy with mixed results. Our study sought to determine whether a sustained, moderate increase in plasma leptin, achieved by administration of a recombinant adenovirus containing the leptin cDNA (AdCMV-leptin) would be effective in reversing the metabolic abnormalities of the obese phenotype. Wistar rats fed a high-fat diet (HF) were heavier (P < 0.05), had increased fat mass and intramuscular triglycerides (mTG), and had elevated plasma glucose, insulin, triglyceride, and free fatty acids compared with standard chow-fed (SC) control animals (all P < 0.01). HF rats also had impaired glucose tolerance and were markedly insulin resistant, as demonstrated by a 40% reduction in insulin-stimulated muscle glucose uptake (P < 0.001). Increasing plasma leptin levels to 29.0 +/- 1.5 ng/ml (from 7.0 +/- 1.4 ng/ml, P < 0.001) for a period of 6 days decreased adipose mass by 40% and normalized plasma glucose and insulin levels. In addition, insulin-stimulated skeletal muscle glucose uptake was normalized in hyperleptinemic rats, an effect that correlated closely with a 60% (P < 0.001) decrease in mTG. Importantly, HF rats that received a control adenovirus containing the beta-galactosidase cDNA and were calorically matched to AdCMV-leptin-treated animals remained hyperglycemic, hyperinsulinemic, insulin resistant, and maintained elevated mTG. We conclude that a gene-therapeutic intervention that elevates plasma leptin moderately for a sustained period reverses diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance, and that these improvements are tightly linked to leptin-induced reductions in mTG.

Animals↗

PubMed2000

Activation of direct and indirect pathways of glycogen synthesis by hepatic overexpression of protein targeting to glycogen.

Glycogen-targeting subunits of protein phosphatase-1, such as protein targeting to glycogen (PTG), direct the phosphatase to the glycogen particle, where it stimulates glycogenesis. We have investigated the metabolic impact of overexpressing PTG in liver of normal rats. After administration of PTG cDNA in a recombinant adenovirus, animals were fasted or allowed to continue feeding for 24 hours. Liver glycogen was nearly completely depleted in fasted control animals, whereas glycogen levels in fasted or fed PTG-overexpressing animals were 70% higher than in fed controls. Nevertheless, transgenic animals regulated plasma glucose, triglycerides, FFAs, ketones, and insulin normally in the fasted and fed states. Fasted PTG-overexpressing animals receiving an oral bolus of [U-(13)C]glucose exhibited a large increase in hepatic glycogen content and a 70% increase in incorporation of [(13)C]glucose into glycogen. However, incorporation of labeled glucose accounted for only a small portion of the glycogen synthesized in PTG-overexpressing animals, consistent with our earlier finding that PTG promotes glycogen synthesis from gluconeogenic precursors. We conclude that hepatic PTG overexpression activates both direct and indirect pathways of glycogen synthesis. Because of its ability to enhance glucose storage without affecting other metabolic indicators, the glycogen-targeting subunit may prove valuable in controlling blood glucose levels in diabetes.

Adenoviridae↗

PubMed2000

Organizing glucose disposal: emerging roles of the glycogen targeting subunits of protein phosphatase-1.

Glucose is stored in mammalian tissues in the form of glycogen. Glycogen levels are markedly reduced in liver or muscle cells of patients with insulin-resistant or insulin-deficient forms of diabetes, suggesting that impaired glycogen synthesis may contribute to development of hyperglycemia. Recently, interest in this area has been further stimulated by new insights into the spatial organization of metabolic enzymes within cells and the importance of such organization in regulation of glycogen metabolism. It is now clear that a four-member family of glycogen targeting subunits of protein phosphatase-1 (PP1) plays a major role in coordinating these events. These proteins target PP1 to the glycogen particle and also bind differentially to glycogen synthase, glycogen phosphorylase, and phosphorylase kinase, thereby serving as molecular scaffolds. Moreover, the various glycogen-targeting subunits have distinct tissue expression patterns and can influence regulation of glycogen metabolism in response to glycogenic and glycogenolytic signals. The purpose of this article is to summarize new insights into the structure, function, regulation, and metabolic effects of the glycogen-targeting subunits of PP1 and to evaluate the possibility that these molecules could serve as therapeutic targets for lowering of blood glucose in diabetes.

Animals↗

PubMed2000

Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.

The biochemical mechanisms involved in regulation of insulin secretion are not completely understood. The rat INS-1 cell line has been used to gain insight in this area because it secretes insulin in response to glucose concentrations in the physiological range. However, the magnitude of the response is far less than that seen in freshly isolated rat islets. In the current study, we have stably transfected INS-1 cells with a plasmid containing the human proinsulin gene. After antibiotic selection and clonal expansion, 67% of the resultant clones were found to be poorly responsive to glucose in terms of insulin secretion (< or =2-fold stimulation by 15 mmol/l compared with 3 mmol/l glucose), 17% of the clones were moderately responsive (2- to 5-fold stimulation), and 16% were strongly responsive (5- to 13-fold stimulation). The differences in responsiveness could not be ascribed to differences in insulin content. Detailed analysis of one of the strongly responsive lines (832/13) revealed that its potent response to glucose (average of 10-fold) was stable over 66 population doublings (approximately 7.5 months of tissue culture) with half-maximal stimulation at 6 mmol/l glucose. Furthermore, in the presence of 15 mmol/l glucose, insulin secretion was potentiated significantly by 100 pmol/l isobutylmethylxanthine (320%), 1 mmol/l oleate/palmitate (77%), and 50 nmol/l glucagon-like peptide 1 (60%), whereas carbachol had no effect. Glucose-stimulated insulin secretion was also potentiated by the sulfonylurea tolbutamide (threefold at 3 mmol/l glucose and 50% at 15 mmol/l glucose) and was abolished by diazoxide, which demonstrates the operation of the ATP-sensitive K+ channel (K(ATP)) in 832/13 cells. Moreover, when the K(ATP) channel was bypassed by incubation of cells in depolarizing K+ (35 mmol/l), insulin secretion was more effectively stimulated by glucose in 832/13 cells than in parental INS-1 cells, which demonstrates the presence of a K(ATP) channel-independent pathway of glucose sensing. We conclude that clonal selection of INS-1 cells allows isolation of cell lines that exhibit markedly enhanced and stable responsiveness to glucose and several of its known potentiators. These lines may be attractive new vehicles for studies of beta-cell function.

Adenosine Triphosphate↗

PubMed2000

Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity.

Engineered insulinoma cell lines may represent an alternative to isolated islets for transplantation therapy of type 1 diabetes. Success of this approach may require development of cell lines that can withstand cytokine-mediated damage. To this end, we have cultured INS-1 insulinoma cells in increasing concentrations of interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma), with approximate weekly iterations over an 8-week period. Based on the C,N diphenyl-N'-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium+ ++ bromide (MTT) viability assay, the selected cells, termed INS-1res, were 100% viable after 5 days of treatment with 10 ng/ml of IL-1beta. These cells were also 78 +/- 1.2% viable after 5 days of exposure to the combination of 10 ng/ml IL-1beta and 100 U/ml IFN-gamma, whereas parental INS-1 cells treated in the same manner were only 0.3 +/- 0.03% viable. INS-1res cells were also resistant to treatment with supernatants from activated rat peripheral blood mononuclear cells, whereas only 20% of parental INS-1 cells survived such treatment. The resistance to IL-1beta conferred by this procedure was stable, whereas the partial resistance to IFN-gamma was transient but reinducible by culture in the presence of cytokines. Stable transfection of INS-1res cells with a plasmid containing the human insulin cDNA and expansion of the transfected colonies in the absence of cytokines produced cell lines that were on average more resistant to IL-1beta + IFN-gamma (53 +/- 11%) than similarly transfected clones derived from parental INS-1 cells (15 +/- 7%). Importantly, several INS-1res-derived clones retained the capacity to secrete insulin in response to glucose concentrations over the normal physiological range. With regard to the mechanism by which selection was conferred, we found normal levels of IFN-gamma receptor mRNA, but a 60% reduction in expression of the IL-1 receptor type I (IL-1RI) in INS-1res cells compared with parental INS-1 cells. IL-1beta signaling through p38 MAP kinase was found to be normal in INS-1res cells, suggesting that their expression of IL-1RI is sufficient to maintain cytokine action. However, normal IL-1beta-mediated translocation of NF-kappaB and induction of inducible nitric oxide synthase expression and nitric oxide production was severely impaired in the INS-1res cell lines, suggesting a mechanism for the IL-1beta resistance. In sum, this study defines a strategy for isolation of cytokine-resistant beta-cell lines and provides a new system for studying the mechanisms by which such resistance can be achieved.

Animals↗

PubMed1999

Glucokinase overexpression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats.

Zucker diabetic fatty rats develop type 2 diabetes concomitantly with peripheral insulin resistance. Hepatocytes from these rats and their control lean counterparts have been cultured, and a number of key parameters of glucose metabolism have been determined. Glucokinase activity was 4.5-fold lower in hepatocytes from diabetic rats than in hepatocytes from healthy ones. In contrast, hexokinase activity was about 2-fold higher in hepatocytes from diabetic animals than in healthy ones. Glucose-6-phosphatase activity was not significantly different. Despite the altered ratios of glucokinase to hexokinase activity, intracellular glucose 6-phosphate concentrations were similar in the two types of cells when they where incubated with 1-25 mM glucose. However, glycogen levels and glycogen synthase activity ratio were lower in hepatocytes from diabetic animals. Total pyruvate kinase activity and its activity ratio as well as fructose 2,6-bisphosphate concentration and lactate production were also lower in cells from diabetic animals. All of these data indicate that glucose metabolism is clearly impaired in hepatocytes from Zucker diabetic fatty rats. Glucokinase overexpression using adenovirus restored glucose metabolism in diabetic hepatocytes. In glucokinase-overexpressing cells, glucose 6-phosphate levels increased. Moreover, glycogen deposition was greatly enhanced due to the activation of glycogen synthase. Pyruvate kinase was also activated, and fructose-2,6-bisphosphate concentration and lactate production were increased in glucokinase-overexpressing diabetic hepatocytes. Overexpression of hexokinase I did not increase glycogen deposition. In conclusion, hepatocytes from Zucker diabetic fatty rats showed depressed glycogen and glycolytic metabolism, but glucokinase overexpression improved their glucose utilization and storage.

Animals↗

PubMed1999

Glucose-6-phosphatase overexpression lowers glucose 6-phosphate and inhibits glycogen synthesis and glycolysis in hepatocytes without affecting glucokinase translocation. Evidence against feedback inhibition of glucokinase.

In hepatocytes glucokinase (GK) and glucose-6-phosphatase (Glc-6-Pase)(1) have converse effects on glucose 6-phosphate (and fructose 6-phosphate) levels. To establish whether hexose 6-phosphate regulates GK binding to its regulatory protein, we determined the effects of Glc-6-Pase overexpression on glucose metabolism and GK compartmentation. Glc-6-Pase overexpression (4-fold) decreased glucose 6-phosphate levels by 50% and inhibited glycogen synthesis and glycolysis with a greater negative control coefficient on glycogen synthesis than on glycolysis, but it did not affect the response coefficients of glycogen synthesis or glycolysis to glucose, and it did not increase the control coefficient of GK or cause dissociation of GK from its regulatory protein, indicating that in hepatocytes fructose 6-phosphate does not regulate GK translocation by feedback inhibition. GK overexpression increases glycolysis and glycogen synthesis with a greater control coefficient on glycogen synthesis than on glycolysis. On the basis of the similar relative control coefficients of GK and Glc-6-Pase on glycogen synthesis compared with glycolysis, and the lack of effect of Glc-6-Pase overexpression on GK translocation or the control coefficient of GK, it is concluded that the main regulatory function of Glc-6-Pase is to buffer the glucose 6-phosphate concentration. This is consistent with recent findings that hyperglycemia stimulates Glc-6-Pase gene transcription.

Adenoviridae↗