Guim Kwon

Cytokines and Nitric Oxide in Islet Inflammation and Diabetes

Abstract Cytokines released by both T lymphocytes and activated macrophages, in particular interleukin-1 (IL-1), have been implicated as immunological effector molecules that both inhibit insulin secretion from the pancreatic β cell and induce β-cell destruction. Recent findings have demonstrated that production of the free radical nitric oxide (NO), resulting from the expression of the cytokine-inducible isoform of NO synthase (iNOS), mediates these deleterious effects. The cellular mechanism responsible for inhibition of β-cell function and destruction by NO involves, in part, inactivation of enzymes specifically localized to the β-cell mitochondria that contain iron-sulfur centers or clusters. Intraislet release of IL-1 also inhibits β-cell function by this same cellular mechanism involving the overproduction of NO. In addition, the cytokine, IL-1, induces the co-expression of both iNOS and the cytokine-inducible isoform of cyclooxygenase, COX-2. The expression of COX-2 results in the overproduction of the proinflammatory prostaglandins and thromboxanes. Furthermore, NO produced by iNOS directly stimulates the activities of both constitutive and inducible isoforms of COX, further augmenting the overproduction of these proinflammatory mediators, NO and prostaglandins, which may be important in initiating or maintaining the inflammatory response and destruction of the β cell associated with autoimmune diabetes. [P.S.E.B.M. 1996, Vol 211]

Branched-chain Amino Acids Are Essential in the Regulation of PHAS-I and p70 S6 Kinase by Pancreatic β-Cells A POSSIBLE ROLE IN PROTEIN TRANSLATION AND MITOGENIC SIGNALING

Amino acids have been identified as important signaling molecules involved in pancreatic β-cell proliferation, although the cellular mechanism responsible for this effect is not well defined. We previously reported that amino acids are required for glucose or exogenous insulin to stimulate phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein regulated by insulin), a recently discovered regulator of translation initiation during cell mitogenesis. Here we demonstrate that essential amino acids, in particular branched-chain amino acids (leucine, valine, and isoleucine), are largely responsible for mediating this effect. The transamination product of leucine, α-ketoisocaproic acid, also stimulates PHAS-I phosphorylation although the transamination products of isoleucine and valine are ineffective. Since amino acids are secretagogues for insulin secretion by β-cells, we investigated whether endogenous insulin secreted by β-cells is involved. Interestingly, branched-chain amino acids stimulate phosphorylation of PHAS-I independent of endogenous insulin secretion since genistein (10 μm) and herbimycin A (1 μm), two tyrosine kinase inhibitors in the insulin signaling pathway, exert no effect on amino acid-induced phosphorylation of PHAS-I. Furthermore, branched-chain amino acids retain their ability to induce phosphorylation of PHAS-I under conditions that block insulin secretion from β-cells. In exploring the signaling pathway responsible for these effects, we find that rapamycin (25 nm) inhibits the ability of branched-chain amino acids to stimulate the phosphorylation of PHAS-I and p70s6 kinase, suggesting that the mammalian target of rapamycin signaling pathway is involved. The branched-chain amino acid, leucine, also exerts similar effects on PHAS-I phosphorylation in isolated pancreatic islets. In addition, we find that amino acids are necessary for insulin-like growth factor (IGF-I) to stimulate the phosphorylation of PHAS-I indicating that a requirement for amino acids may be essential for other β-cell growth factors in addition to insulin and IGF-I to activate this signaling pathway. We propose that amino acids, in particular branched-chain amino acids, may promote β-cell proliferation either by stimulating phosphorylation of PHAS-I and p70s6k via the mammalian target of rapamycin pathway and/or by facilitating the proliferative effect mediated by growth factors such as insulin and IGF-I.

cAMP Dose-dependently Prevents Palmitate-induced Apoptosis by Both Protein Kinase A- and cAMP-Guanine Nucleotide Exchange Factor-dependent Pathways in β-Cells

Lipid accumulation in pancreatic β-cells is thought to cause its dysfunction and/or destruction via apoptosis. Our studies show that incubation of the β-cell line RINm5F with the saturated free fatty acids (FFA) palmitate caused apoptosis based on increases in caspase 3 activity, Annexin V staining, and cell death. Furthermore, exposure of RINm5F cells to cAMP-increasing agents, 3-isobutyl-1-methylxanthine (IBMX), and forskolin completely abolished palmitate-mediated caspase 3 activity and significantly inhibited Annexin V staining and cell death. The cyclic AMP analogs cpt-cAMP and dibutyryl-cAMP mimicked the protective effects of IBMX and forskolin, suggesting that cAMP is the mediator of the anti-apoptotic effects. The protective action of IBMX and forskolin was rapid and did not appear to require gene transcription or new protein synthesis. However, these protective effects were clearly independent of protein kinase A (PKA) because of the lack of inhibition by the PKA inhibitors H-89 and KT5720. In attempts to identify this PKA-independent mechanism, we found that the newly developed cAMP analog 8CPT-2Me-cAMP, which selectively activates the cAMP-dependent guanine nucleotide exchange factor (cAMP-GEF) pathway, mimicked the protective effects of IBMX and forskolin, suggesting that the cAMP-GEF pathway is involved. In addition, both glucagon-like peptide (GLP-1) and its receptor agonist, Exenatide, inhibited palmitate-mediated caspase 3 activation in a dose-dependent manner. Unexpectedly, H-89 partially reversed the protective effects of GLP-1 and Exenatide, suggesting that PKA may play a role in the protective effects of these incretins. To explain these seemingly conflicting results, we demonstrated that low concentrations of cAMP produced by GLP-1 and Exenatide preferentially activate the PKA pathway, whereas higher cAMP concentrations produced by IBMX and forskolin activate the more dominant cAMP-GEF pathway. Taken together, these results indicate that intracellular concentrations of cAMP may play a key role in determining divergent signaling pathways that lead to antiapoptotic responses.

Tumor Necrosis Factor α-induced Pancreatic β-Cell Insulin Resistance Is Mediated by Nitric Oxide and Prevented by 15-Deoxy-Δ12,14-prostaglandin J2 and Aminoguanidine A ROLE FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ ACTIVATION AND iNOS EXPRESSION

Recent studies have identified a β-cell insulin receptor that functions in the regulation of protein translation and mitogenic signaling similar to that described for insulin-sensitive cells. These findings have raised the novel possibility that β-cells may exhibit insulin resistance similar to skeletal muscle, liver, and fat. To test this hypothesis, the effects of tumor necrosis factor-α (TNFα), a cytokine proposed to mediate insulin resistance by interfering with insulin signaling at the level of the insulin receptor and its substrates, was evaluated. TNFα inhibited p70s6k activation by glucose-stimulated β-cells of the islets of Langerhans in a dose- and time-dependent manner, with maximal inhibition observed at ∼20–50 ng/ml, detected after 24 and 48 h of exposure. Exogenous insulin failed to prevent TNFα-induced inhibition of p70s6k , suggesting a defect in the insulin signaling pathway. To further define mechanisms responsible for this inhibition and also to exclude cytokine-induced nitric oxide (NO) as a mediator, the ability of exogenous or endogenous insulin ± inhibitors of nitric-oxide synthase (NOS) activity, aminoguanidine or N-monomethyl-l-arginine, was evaluated. Unexpectedly, TNFα and also interleukin 1 (IL-1)-induced inhibition of p70s6k was completely prevented by inhibitors that block NO production. Western blot analysis verified inducible NOS (iNOS) expression after TNFα exposure. Furthermore, the ability of IL-1 receptor antagonist protein, IRAP, to block TNFα-induced inhibition of p70s6k indicated that activation of intra-islet macrophages and the release of IL-1 that induces iNOS expression in β-cells was responsible for the inhibitory effects of TNFα. This mechanism was confirmed by the ability of the peroxisome proliferator-activated receptor-γ agonist 15-deoxy-Δ12,14-prostaglandin J2 to attenuate TNFα-induced insulin resistance by down-regulating iNOS expression and/or blocking IL-1 release from activated macrophages. Overall, TNFα-mediated insulin resistance in β-cells is characterized by a global inhibition of metabolism mediated by NO differing from that proposed for this proinflammatory cytokine in insulin-sensitive cells.

Glycogen Synthase Kinase-3 and Mammalian Target of Rapamycin Pathways Contribute to DNA Synthesis, Cell Cycle Progression, and Proliferation in Human Islets

OBJECTIVE—Our previous studies demonstrated that nutrient regulation of mammalian target of rapamycin (mTOR) signaling promotes regenerative processes in rodent islets but rarely in human islets. Our objective was to extend these findings by using therapeutic agents to determine whether the regulation of glycogen synthase kinase-3 (GSK-3)/β-catenin and mTOR signaling represent key components necessary for effecting a positive impact on human β-cell mass relevant to type 1 and 2 diabetes. RESEARCH DESIGN AND METHODS—Primary adult human and rat islets were treated with the GSK-3 inhibitors, LiCl and the highly potent 1-azakenpaullone (1-Akp), and with nutrients. DNA synthesis, cell cycle progression, and proliferation of β-cells were assessed. Measurement of insulin secretion and content and Western blot analysis of GSK-3 and mTOR signaling components were performed. RESULTS—Human islets treated for 4 days with LiCl or 1-Akp exhibited significant increases in DNA synthesis, cell cycle progression, and proliferation of β-cells that displayed varying degrees of sensitivity to rapamycin. Intermediate glucose (8 mmol/l) produced a striking degree of synergism in combination with GSK-3 inhibition to enhance bromodeoxyuridine (BrdU) incorporation and Ki-67 expression in human β-cells. Nuclear translocation of β-catenin responsible for cell proliferation was found to be particularly sensitive to rapamycin. CONCLUSIONS—A combination of GSK-3 inhibition and nutrient activation of mTOR contributes to enhanced DNA synthesis, cell cycle progression, and proliferation of human β-cells. Identification of therapeutic agents that appropriately regulate GSK-3 and mTOR signaling may provide a feasible and available approach to enhance human islet growth and proliferation.

Glucose-stimulated DNA synthesis through mammalian target of rapamycin (mTOR) is regulated by KATP channels: effects on cell cycle progression in rodent islets.

The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) leads to [3H]thymidine incorporation and that mTOR activation is mediated, in part, through the KATP channel and changes in cytosolic Ca2+, we determined whether glyburide, an inhibitor of KATP channels that stimulates Ca2+ influx, modulates [3H]thymidine incorporation. Glyburide (10–100 nm) at basal glucose stimulated [3H]thymidine incorporation to the same magnitude as elevated glucose and further enhanced the ability of elevated glucose to increase [3H]thymidine incorporation. Diazoxide (250 μm), an activator of KATP channels, paradoxically potentiated glucose-stimulated [3H]thymidine incorporation 2–4-fold above elevated glucose alone. Cell cycle analysis demonstrated that chronic exposure of islets to basal glucose resulted in a typical cell cycle progression pattern that is consistent with a low level of proliferation. In contrast, chronic exposure to elevated glucose or glyburide resulted in progression from G0/G1 to an accumulation in S phase and a reduction in G2/M phase. Rapamycin (100 nm) resulted in an ∼62% reduction of S phase accumulation. The enhanced [3H]thymidine incorporation with chronic elevated glucose or glyburide therefore appears to be associated with S phase accumulation. Since diazoxide significantly enhanced [3H]thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation.

Relative hypoglycemia and hyperinsulinemia in mice with heterozygous lipoprotein lipase (LPL) deficiency. Islet LPL regulates insulin secretion.

Lipoprotein lipase (LPL) provides tissues with fatty acids, which have complex effects on glucose utilization and insulin secretion. To determine if LPL has direct effects on glucose metabolism, we studied mice with heterozygous LPL deficiency (LPL+/−). LPL+/− mice had mean fasting glucose values that were up to 39 mg/dl lower than LPL+/+ littermates. Despite having lower glucose levels, LPL+/− mice had fasting insulin levels that were twice those of +/+ mice. Hyperinsulinemic clamp experiments showed no effect of genotype on basal or insulin-stimulated glucose utilization. LPL message was detected in mouse islets, INS-1 cells (a rat insulinoma cell line), and human islets. LPL enzyme activity was detected in the media from both mouse and human islets incubated in vitro. In mice, +/− islets expressed half the enzyme activity of +/+ islets. Islets isolated from +/+ mice secreted less insulin in vitro than +/− and −/− islets, suggesting that LPL suppresses insulin secretion. To test this notion directly, LPL enzyme activity was manipulated in INS-1 cells. INS-1 cells treated with an adeno-associated virus expressing human LPL had more LPL enzyme activity and secreted less insulin than adeno-associated virus-β-galactosidase-treated cells. INS-1 cells transfected with an antisense LPL oligonucleotide had less LPL enzyme activity and secreted more insulin than cells transfected with a control oligonucleotide. These data suggest that islet LPL is a novel regulator of insulin secretion. They further suggest that genetically determined levels of LPL play a role in establishing glucose levels in mice.

Glucose and insulin stimulate heparin-releasable lipoprotein lipase activity in mouse islets and INS-1 cells: a potential link between insulin resistance and β-cell dysfunction

Lipoprotein lipase (LpL) provides tissues with triglyceride-derived fatty acids. Fatty acids affect β-cell function, and LpL overexpression decreases insulin secretion in cell lines, but whether LpL is regulated in β-cells is unknown. To test the hypothesis that glucose and insulin regulate LpL activity in β-cells, we studied pancreatic islets and INS-1 cells. Acute exposure of β-cells to physiological concentrations of glucose stimulated both total cellular LpL activity and heparin-releasable LpL activity. Glucose had no effect on total LpL protein mass but instead promoted the appearance of LpL protein in a heparin-releasable fraction, suggesting that glucose stimulates the translocation of LpL from intracellular to extracellular sites in β-cells. The induction of heparin-releasable LpL activity was unaffected by treatment with diazoxide, an inhibitor of insulin exocytosis that does not alter glucose metabolism but was blocked by conditions that inhibit glucose metabolism. In vitro hyperinsulinemia had no effect on LpL activity in the presence of low concentrations of glucose but increased LpL activity in the presence of 20 mm glucose. Using dual-laser confocal microscopy, we detected intracellular LpL in vesicles distinct from those containing insulin. LpL was also detected at the cell surface and was displaced from this site by heparin in dispersed islets and INS-1 cells. These results show that glucose metabolism controls the trafficking of LpL activity in β-cells independent of insulin secretion. They suggest that hyperglycemia and hyperinsulinemia associated with insulin resistance may contribute to progressive β-cell dysfunction by increasing LpL-mediated delivery of lipid to islets.

A Role for Nitric Oxide and Other Inflammatory Mediators in Cytokine-Induced Pancreatic β-Cell Dysfunction and Destruction

Insulin-dependent diabetes mellitus is an autoimmune disease characterized by the selective destruction of the pancreatic β-cell. The autoimmune response that is ultimately responsible for the destruction of the β-cell consists of a number of complex components. Cytokines released during this inflammatory reaction have been implicated as effector molecules which mediate β-cell destruction1–3. Our studies have focused primarily in attempting to define the role of cytokines released by both T-lymphocytes and activated macrophages to produce deleterious effects directly on the β-cell during the initial inflammatory response associated with autoimmune diabetes. Our studies have described the ability of the cytokine, interleukin-1 (IL-1), to both inhibit insulin secretion from the β-cell and also to produce cytotoxic effects via the formation of the free radical nitric oxide (NO). Our more recent studies have revealed that the ability of cytokines such as IL-1 to produce nitric oxide by islets is also directly linked to the production of the proinflammatory prostaglandins and thromboxanes that may play an important role in the initial inflammatory response that is the hallmark of autoimmune diabetes.

Potential Autoantigens In IDDM: Expression of Carboxypeptidase-H and Insulin But Not Glutamate Decarboxylase on the β-Cell Surface

Insulin, carboxypeptidase-H (CP-H), and glutamate decarboxylase (GAD) have been identified as potential autoantigens in insulin-dependent diabetes mellitus (IDDM). Previous studies have described immunoreactive insulin as a surface molecule on the plasma membrane of rat islet cells and suggested that cell-surface insulin was derived during exocytosis by the fusion of insulin secretory granules with the β-cell plasma membrane. These findings predict that insulin and other secretory granule-derived proteins such as the putative autoantigen CP-H may be colocalized with insulin at specific sites of exocytosis on the β-cell surface. In studies to test this hypothesis, cell-surface staining of dispersed rat islet cells occurred in a granule-like pattern with antibodies for CP-H and insulin. The specificity of the CP-H antiserum was confirmed by immunoblotting and indicated that the antiserum was essentially monospecific for CP-H. Confocal laser microscopy confirmed that immunoreactive staining for CP-H and insulin was confined to the β-cell surface. Colocalization of CP-H and insulin on the cell surface of β-cells was demonstrated by double staining with antibodies to CP-H and insulin, and the percentage of β-cells positive for both of these autoantigens increased twofold with increases in insulin secretion. In contrast, islet cells failed to reveal cell-surface staining for GAD65, another putative autoantigen in IDDM, under either basal or insulin stimulatory conditions or following exposure of islet cells to the cytokines interleukin-1β, tumor necrosis factor-α, and recombinant human interferon-γ. These results demonstrate that the insulin secretory granule–derived proteins, insulin and CP-H, colocalize on the cell surface of β-cells during exocytosis and in this manner could be recognized by components of the immune system under certain disease settings. These findings further delineate a cellular mechanism whereby the functional activity of the pancreatic (β-cell, i.e., resting versus actively secreting, may correlate with cell-surface localization of antigens and raises the possibility that other unidentified granule derived antigens also may colocalize at sites of exocytosis on the β-cell membrane.