Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by A^-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 p-induced 1) nitrite formation (an oxidation product of NO) and cGMP accumulation by the rat p-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is ~40 times more potent than aminoguanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO production and suggest a role for NO in the pathogenesis of diabetic vascular complications. Diabetes 41:552-56, 1992 N itric oxide synthase catalyzes the mixed functional oxidation of a guanidino nitrogen atom of L-arginine to yield L-citrulline and NO-(1,2). The constitutive isoform of NO-synthase is Ca 2+ dependent and produces small amounts of NO-that activate guanylate cyclase, resulting in the formation of cGMP, which mediates endothelium-dependent relaxation (2) and neural transmission (3). NO-is produced in much larger amounts by the cytokine-and endotoxininducible isoform of NO-synthase, which is Ca 2+ inde-

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Cytokines have been implicated s immunological effector molecules that mediate beta cell destruction asciated with insulin-dependent diabetes mellitus. In this report we demonstrate that the cytokine combination of human recombinant interieukin lp (IL-1*), tumor necrosis factor a (TNF-a), and interferon y (IFN-y) induces the formation of nitric oxide by human islets. This combination of cytokines stimulates both the formation of the nitric oxide derivative, nitrite, and the accumulation of cGMP by human iWets. The nitric oide synthase inhibitor NG.monomethyl-L-argIne prevents formation of both cGMP and nitrite. IL-1,B and IFN-y are sufcient to induce nitric oide formation by human islets, whereas TNF-a potentiates nitrite production. This combination of cytokines (IL-1f3, TNF-a, and IFN-y) also influences insulin secretion by human idets. Pretreatment of human iWets with low concentrations of this cytokne combination (IL-lp at 15 units/ml, 0.7 nM TNF-a, and IFN-y at 150 units/ml) appears to slightly stimulate insulin secretion. Higher concentrations (IL-l1 at 75 units/nl, 3.5 nM TNF-a, and IFN-yat 750 units/ml) inhibit inlin secretion from human islets, and the inhibitory effect is prevented by NG-monomethyl-L-argine. This higher concentration of cytokines also induces the formation of an electron paramagnetic resonance-detectable g = 2.04 axial feature by human islets that is characteristic of the formation of an iron-diho-dinitrosyl complex. The formation of this complex is prevented by NG-monomethyl-L-arginine, thus confirming that this cytokine combination induces the formation of nitric oxide by human islets. These results indicate that nitric oxide mediates the inhibitory effects of cytoldnes on glucosestimulated insulin secretion by human Wets and suggest that nitric oidde may participate in beta-cell dysfunction associated with insulin-dependent diabetes mellitus.Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by specific destruction of the pancreatic islet beta cell (1). The destruction of beta cells is believed to be mediated by infiltrating lymphocytes. The ability of T cells to adoptively transfer diabetes in diabetesprone BB rats (2) and in the NOD mouse indicates that T cells participate in beta-cell destruction (3). Cytokines, released by infiltrating lymphocytes, have also been implicated as possible mediators of beta-cell destruction. Pretreatment of isolated rat islets with the cytokine human recombinant interleukin 1,B (IL-1*8) results in a concentration-and timedependent inhibition of glucose-stimulated insulin secretion that is followed by islet destruction after prolonged exposures to this cytokine (4, 5).The free-radical nitric oxide has been implicated as the cellular effector molecule that mediates the inhibitory and cytotoxic effects of IL-1,3 on rat islets (6). Pretreatment of rat islets for 18-24 hr with IL-1lB results in nearly complete inhibition of glucose-stimulated insulin secretion that is prevented by the nitric oxide synthase inhibitor...

In B cells from dispersed rat islet of Langerhans we have identified an inward rectifying voltageindependent K+ channel whose behavior parallels the metabolically regulated potassium permeability (PK) found in tracer flux and microelectrode recording studies. In cell-attached patches of membrane, the channel is closed when any one of several substrates (glucose, mannose, leucine, or glyceraldehyde) is added to the cell's bathing solution but is reopened on addition of an appropriate metabolic inhibitor, which prevents utilization of that substrate. In inside-out excised patches, a K+ channel with nearly identical kinetic features is closed by addition of micromolar concentrations of ATP to the "cytoplasmic" solution. The ATP sensitivity of channel activity is modified by addition of ADP, suggesting competition at a nucleotide binding site. These results suggest the presence of a metabolically regulated K+ channel gated by intracellular concentrations of ATP or the ratio of ATP/ADP concentrations.A popular hypothesis for stimulus-secretion coupling in pancreatic islet B cells is: nutrient metabolism by B cells decreased K+ permeability (PK) -* membrane depolarization --voltage-dependent Ca2 entry --insulin granule exocytosis (1, 2). Using single-channel recording techniques, several groups have identified, in cell-attached patches of pancreatic islet cells, a potassium-selective channel whose activity is reduced by raising the glucose concentration of the medium bathing the rest of the cell (3-6). Others have reported the existence in inside-out excised patches of a potassium channel of similar conductance whose opening frequency is reduced by micromolar concentrations of ATP (4-8). Here we demonstrate that both channels represent the same "metabolite-regulated" K+ channel in two different recording configurations and describe some regulatory effects of cytoplasmic nucleotides on channel gating. METHODS AND MATERIALSRat pancreatic islets, isolated by collagenase digestion of chopped pancreases of adult male Sprague-Dawley rats were dispersed into small clumps of cells by incubation with the enzyme "dispase" (9). Cells prepared in this manner increase their insulin secretion nearly 3-fold above baseline in the presence of 10 mM glucose media (10). Clumps of cells were added to 35-mm tissue culture dishes "seeded" with appropriately sized glass coverslips and were cultured for 1-10 days at 37°C in modified CMRL medium (GIBCO) containing 10% heat-inactivated fetal bovine serum, 0.5% penicillin, and 0.5% streptomycin in 5% C02/95% air. Individual coverslip chips were then transferred to the recording chamber containing modified physiological saline solutions at 20-23°C. The basic zero-glucose extracellular saline solution (zeroglucose ES solution) consisted of 138 mM NaCl, 5.5 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, and 20 mM Hepes titrated with NaOH to pH 7.35. The solution used to fill patch pipettes as well as bathe inside-out excised patches (IS solution) consisted of 138 mM KCl, 2 mM Mg2+, 5-20 AM CaCl2, and 2...

IntroductionNitric oxide has recently been implicated as the effector molecule that mediates IL-1(3-induced inhibition of glucose-stimulated insulin secretion and (3-cell specific destruction. The pancreatic islet represents a heterogeneous cell population con-

A combination of insulin resistance and pancreatic beta-cell dysfunction underlies most cases of type 2 diabetes. While the interplay of these two impairments is believed to be important in the development and progression of type 2 diabetes, the mechanisms involved are unclear. A number of factors have been suggested as possibly linking insulin resistance and beta-cell dysfunction in the pathogenesis of type 2 diabetes mellitus. Pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) have deleterious effects on both glucose homeostasis and beta-cell function, and can disrupt insulin signalling pathways in both pancreatic beta cells and liver and adipose tissue. The anti-inflammatory activity of the thiazolidinedione anti-diabetic agents is potentially beneficial, given the possible role of pro-inflammatory cytokines in linking insulin resistance with beta-cell dysfunction.

Recent findings have demonstrated that the branchedchain amino acid leucine can activate the translational regulators, phosphorylated heat-and acid-stable protein regulated by insulin (PHAS-I) and p70 S6 kinase (p70 s6k ), in an insulin-independent and rapamycin-sensitive manner through mammalian target of rapamycin (mTOR), although the mechanism for this activation is undefined. It has been previously established that leucine-induced insulin secretion by ␤-cells involves increased mitochondrial metabolism by oxidative decarboxylation and allosteric activation of glutamate dehydrogenase (GDH). We now show that these same intramitochondrial events that generate signals for leucine-induced insulin exocytosis are required to activate the mTOR mitogenic signaling pathway by ␤-cells. Thus, a minimal model consisting of leucine and glutamine as substrates for oxidative decarboxylation and an activator of GDH, respectively, confirmed the requirement for these two metabolic components and mimicked closely the synergistic interactions achieved by a complete complement of amino acids to activate p70 s6k in a rapamycinsensitive manner. Studies using various leucine analogs also confirmed the close association of mitochondrial metabolism and the ability of leucine analogs to activate p70 s6k . Furthermore, selective inhibitors of mitochondrial function blocked this activation in a reversible manner, which was not associated with a global reduction in ATP levels. These findings indicate that leucine at physiological concentrations stimulates p70 s6k phosphorylation via the mTOR pathway, in part, by serving both as a mitochondrial fuel and an allosteric activator of GDH. Leucine-mediated activation of protein translation through mTOR may contribute to enhanced ␤-cell function by stimulating growth-related protein synthesis and proliferation associated with the maintenance of ␤-cell mass. Diabetes 50:353-360, 2001

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 beta cell and induce beta-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 beta-cell function and destruction by NO involves, in part, inactivation of enzymes specifically localized to the beta-cell mitochondria that contain iron- sulfur centers or clusters. Intraislet release of IL-1 also inhibits beta-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 beta cell associated with autoimmune diabetes.