Meal-derived glucagon responses are related to lower hepatic phosphate concentrations in obesity and type 2 diabetes.

AIM: Type 2 diabetes (T2D) alters glucagon, glucagon-like peptide (GLP)-1, glucose-dependent insulinotropic polypeptide (GIP) and hepatic energy metabolism, yet the possible relationships remain unclear. METHODS: In this observational study, lean insulin-sensitive control subjects (BMI: 23.2±1.5kg/m2), age-matched insulin-resistant obese subjects (BMI: 34.3±1.7kg/m2) and similarly obese elderly T2D patients (BMI: 32.0±2.4kg/m2) underwent mixed-meal tolerance tests (MMTTs), and assessment of hepatic γATP, inorganic phosphate (Pi) and lipids using 31P/1H magnetic resonance spectroscopy. Meal-induced secretion of glucagon and incretins was calculated from incremental areas under the concentration-time curves (iAUCs). Peripheral and adipose tissue insulin sensitivity were assessed from time courses of circulating glucose, insulin and free fatty acids. RESULTS: MMTT-derived peripheral insulin sensitivity was lowest in T2D patients (P<0.001), while glucagon concentrations were comparable across all three groups. At 260min, GLP-1 was lower in T2D patients than in controls, whereas GIP was lowest in obese individuals. Fasting glucagon concentrations correlated positively with fasting (r=0.60) and postprandial hepatocellular lipid levels (160min: r=0.51, 240min: r=0.59), and negatively with adipose tissue insulin sensitivity (r=-0.73). Higher meal-induced glucagon release (iAUC0-260min) correlated with lower fasting (r=-0.62) and postprandial Pi levels (160min: r=-0.43, 240min: r=-0.42; all P<0.05). Higher meal-induced release of GIP (iAUC0-260min) correlated positively with fasting (r=0.54) and postprandial serum triglyceride concentrations (iAUC0-260min, r=0.54; all P<0.01). CONCLUSION: Correlations between fasting glucagon and hepatic lipids and between meal-induced glucagon and hepatic Pi suggest a role for glucagon in hepatic energy metabolism.

Inverse association of insulin antibody levels with insulin sensitivity in adults with Type 1 diabetes.

AIMS: Insulin resistance may contribute to the pathogenesis of autoimmune-mediated diabetes. Antibodies against ß-cell-associated molecules, comprising islet cell antigen (ICA), glutamic acid decarboxylase (GAD) and insulin, characterize the autoimmune process. Because the link between insulin resistance and autoimmunity might be relevant for disease progression and treatment, we hypothesized that insulin resistance associates positively with ß-cell-directed antibodies in newly diagnosed Type 1 diabetes. METHODS: Within the German Diabetes Study, an observational study including adults with newly diagnosed diabetes, 142 adults [84 men, 58 women; age 33.1 (26.4, 41.9) years; diabetes duration 6.3 (4.2, 9.1) months] positive for at least one antibody against ICA, GAD or insulin underwent hyperinsulinaemic-euglycaemic clamp tests to assess insulin sensitivity (M-value) in a cross-sectional setting. RESULTS: Insulin-directed antibodies were inversely correlated with M-values (ß = -0.039). Albeit not strong, the association persisted after adjustment for age, sex and BMI, and even after further adjustment for confounders reflecting exposure to exogenous insulin and residual ß-cell secretory capacity. Correlation network-based analyses revealed a complex interaction between levels of fasting insulin and of insulin antibodies with respect to their relationship with the M-value. GAD- or ICA-directed antibodies did not correlate with insulin sensitivity. CONCLUSIONS: In adults with recent-onset Type 1 diabetes expressing at least one ß-cell-directed antibody, insulin sensitivity is inversely related to insulin antibody titres, but not to other autoantibodies. Our finding may allow for the identification of insulin resistance in adults with high levels of insulin antibodies.

Fatty acids modulate cytokine and chemokine secretion of stimulated human whole blood cultures in diabetes.

Fatty acids, uric acid and glucose are thought to contribute to subclinical inflammation associated with diabetes mellitus. We tested whether co-incubation of free fatty acids and uric acid or glucose influences the secretion of immune mediators from stimulated human whole blood in vitro. Fresh whole blood samples from 20 healthy subjects, 20 patients with type 1 diabetes and 23 patients with type 2 diabetes were incubated for 24 h with palmitic acid (PAL), linolenic acid (LIN) or eicosapentaenoic acid (EPA) alone or together with elevated concentrations of uric acid or glucose. Concentrations of proinflammatory cytokines interleukin (IL)-1ß, IL-2, IL-12(p70), IL-18, IFN-γ, of regulatory cytokines IL-4, IL-10, IL-17 and chemokine CCL2 (MCP-1) were measured by multiplex-bead technology from supernatants. Co-incubation of fatty acids with uric acid resulted in a significant reduction of IL-10, IL-12(p70), IFN-γ and CCL2 (MCP-1) concentrations in supernatants compared to incubation with uric acid alone (P < 0·0001). In contrast, IL-18 was up-regulated upon co-stimulation with fatty acids and uric acid. Similarly, co-incubation of fatty acids with glucose diminished secretion of IL-10, IFN-γ and CCL2 (monocyte chemotactic protein-1), while IL-8 was up-regulated (P < 0·001). Samples from healthy and diabetic subjects did not differ after adjustment for age, sex, body mass index and diabetes type. All three fatty acids similarly influenced whole blood cytokine release in vitro and modulated uric acid or glucose-stimulated cytokine secretion. Although the ω-3-fatty acid EPA showed slightly stronger effects, further studies are required to elaborate the differential effects of PAL, LIN and EPA on disease risk observed previously in epidemiological studies.

Heat shock protein 60: regulatory role on innate immune cells.

Human heat shock protein 60 (Hsp60) exhibits immunoregulatory properties, primarily by inducing pro-inflammatory responses in innate immune cells. Extensive analyses identified specific receptor structures for the interaction of Hsp60 with these cells. The existence of distinct receptor structures responsible for Hsp60 binding and for Hsp60-induced release of pro-inflammatory mediators has been demonstrated, implying that the interaction of Hsp60 with innate immune cells is a multifaceted process. Distinct Hsp60 epitopes responsible for binding to innate immune cells and for the activation of these cells have been identified. Depending on the cell-type, the amino acid (aa) region 481-500 or the regions aa241-260, aa391-410 and aa461-480 are involved in Hsp60-binding to innate immune cells. An entirely different Hsp60-region, aa354-365 was found to bind lipopolysaccharide, thereby mediating the pro-inflammatory effects of Hsp60. Because of its immunoregulatory properties, Hsp60 has been proposed to act as intercellular danger signal, controlling innate and adaptive immune reactions.

p38-dependent enhancement of cytokine-induced nitric-oxide synthase gene expression by heat shock protein 70.

Heat shock protein (hsp) 70 protects cells against stress by means of its ability to chaperone denatured proteins and to modulate stress-activated signaling pathways. Because inflammatory processes are often accompanied by hsp expression and because stress and cytokines share several signaling pathways, we investigated the possibility that hsp70 might modulate the cellular response to cytokines. We found that stable cell clones overexpressing hsp70, or cells shortly after transfection with hsp70, produced 2 times more nitric oxide and inducible nitric-oxide synthase (iNOS) protein and mRNA in response to cytokines than control cells expressing undetectable amounts of hsp70. Since mitogen-activated protein kinases participate in the activation of iNOS by cytokines, we investigated whether hsp70 affected the activation of these signaling pathways. hsp70 overexpression led to a specific enhancement of the activation of the p38 pathway by cytokines, producing little or no effect on the activation of extracellular signal-regulated kinase or Jun N-terminal kinase. Blocking p38 activity with SB203580 totally abolished the enhancing effect of hsp70 on cytokine-induced endogenous iNOS mRNA accumulation or transcription of an iNOS promoter-driven luciferase gene, while having little effect on the cytokine response observed in control cells. We conclude that the p38 pathway acts as an enhancing factor in the activation of iNOS by cytokines and that hsp70 can modulate the cellular response to cytokines by acting on signaling elements upstream of p38.

Natural resistance of human beta cells toward nitric oxide is mediated by heat shock protein 70.

Human beta cells exhibit increased resistance against nitric oxide (NO) radicals as compared with rodent islet cells. Here we tested whether endogenous heat shock protein 70 (hsp70) accounts for the resistance of human cells. Stable transfection of the human beta cell line CM with an antisense hsp70 mRNA-expressing plasmid (ashsp70) caused selective suppression (>95%) of spontaneously expressed hsp70 but not of hsc70 or GRP75 protein. ashsp70 transfection abolished the resistance of CM cells to the NO donors (Z)-1- (2-(2-aminoethyl)-N-(2-ammonioethyl)amino)diazen-1-ium -1,2-diolate and sodium nitroprusside and increased the proportions of necrotic cells 3-5-fold (p < 0.05) and of apoptotic cells about 2-fold (p < 0.01). Re-induction of hsp70 expression by heat shock re-established resistance to NO toxicity. hsp70 did not exert its protective effect at the level of membrane lipid integrity because radical induced lipid peroxidation appeared independent of hsp70 expression. However, after NO exposure only hsp70-deficient cells showed significantly decreased mitochondrial activity, by 40-80% (p < 0.01). These results suggest a key role of hsp70 in the natural resistance of human beta cells against NO induced injury, by preserving mitochondrial function. These findings provide important implications for the development of beta cell protective strategies in type 1 diabetes and islet transplantation.

Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex.

Human heat shock protein 60 (hsp60) elicits a potent proinflammatory response in cells of the innate immune system and therefore has been proposed as a danger signal of stressed or damaged cells. We report here that macrophages of C3H/HeJ mice, carrying a mutant Toll-like-receptor (Tlr) 4 are nonresponsive to hsp60. Both the induction of TNF-alpha and NO formation were found dependent on a functional Tlr4 whereas stimulation of macrophages by CpG DNA was Tlr4 independent. We conclude that Tlr4 mediates hsp60 signaling. This is the first report of a putative endogenous ligand of the Tlr4 complex.

IL-18 inhibits diabetes development in nonobese diabetic mice by counterregulation of Th1-dependent destructive insulitis.

The development of type 1 diabetes in animal models is T cell and macrophage dependent. Islet inflammation begins as peripheral benign Th2 type insulitis and progresses to destructive Th1 type insulitis, which is driven by the innate immune system via secretion of IL-12 and IL-18. We now report that daily application of IL-18 to diabetes-prone female nonobese diabetic mice, starting at 10 wk of age, suppresses diabetes development (p < 0.001, 65% in sham-treated animals vs 33% in IL-18-treated animals by 140 days of age). In IL-18-treated animals, we detected significantly lower intraislet infiltration (p < 0.05) and concomitantly an impaired progression from Th2 insulitis to Th1-dependent insulitis, as evidenced from IFN-gamma and IL-10 mRNA levels in tissue. The deficient progression was probably due to lesser mRNA expression of the Th1 driving cytokines IL-12 and IL-18 by the innate immune system (p < 0.05). Furthermore, the mRNA expression of inducible NO synthase, a marker of destructive insulitis, was also not up-regulated in the IL-18-treated group. IL-18 did not exert its effect at the levels of islet cells. Cultivation of islets with IL-18 affected NO production or mitochondrial activity and did not protect from the toxicity mediated by IL-1beta, TNF-alpha, and IFN-gamma. In conclusion, we show for the first time that administration of IL-18, a mediator of the innate immune system, suppresses autoimmune diabetes in nonobese diabetic mice by targeting the Th1/Th2 balance of inflammatory immune reactivity in the pancreas.

Human 60-kDa heat-shock protein: a danger signal to the innate immune system.

Mammalian 60-kDa heat-shock protein (hsp60) is a key target of T cell and Ab responses in chronic inflammation or atherosclerosis. We show in this study that human hsp60 is also an Ag recognized by cells of the innate immune system, such as macrophages. Both mouse and human macrophages respond to contact with exogenous human hsp60 with rapid release of TNF-alpha; mouse macrophages in addition produce nitric oxide. The proinflammatory macrophage response is hsp60 dose dependent and similar in kinetics and extent to LPS stimulation. Human hsp60 was found to synergize with IFN-gamma in its proinflammatory activity. Finally, human hsp60 induces gene expression of the Th1-promoting cytokines IL-12 and IL-15. These findings identify autologous hsp60 as a danger signal for the innate immune system, with important implications for a role of local hsp60 expression/release in chronic Th1-dependent tissue inflammation.

Mice lacking the poly(ADP-ribose) polymerase gene are resistant to pancreatic beta-cell destruction and diabetes development induced by streptozocin.

Human type 1 diabetes results from the selective destruction of insulin-producing pancreatic beta cells during islet inflammation. Cytokines and reactive radicals released during this process contribute to beta-cell death. Here we show that mice with a disrupted gene coding for poly (ADP-ribose) polymerase (PARP-/- mice) are completely resistant to the development of diabetes induced by the beta-cell toxin streptozocin. The mice remained normoglycemic and maintained normal levels of total pancreatic insulin content and normal islet ultrastructure. Cultivated PARP-/- islet cells resisted streptozocin-induced lysis and maintained intracellular NAD+ levels. Our results identify NAD+ depletion caused by PARP activation as the dominant metabolic event in islet-cell destruction, and provide information for the development of strategies to prevent the progression or manifestation of the disease in individuals at risk of developing type 1 diabetes.

Nicotinamide in type 1 diabetes. Mechanism of action revisited.

Treatment with high doses of nicotinamide (niacinamide, vitamin B3) prevents or delays insulin-deficient diabetes in several animal models of type 1 diabetes and protects islet cells against cytotoxic actions in vitro. In recent-onset type 1 diabetes, nicotinamide administration improves beta-cell function, without significantly decreased insulin requirements. This review discusses the possible mechanism of action of nicotinamide in vivo. It is proposed that the key target of nicotinamide is the poly(ADP-ribose)polymerase (PARP), and to a lesser extent (mono)ADP-ribosyl transferases (ADPRTs). Suppression of PARP activity by nicotinamide not only decreases consumption of NAD+, the substrate of PARP, but also has major regulatory effects on gene expression, as shown for the major histocompatibility complex class II gene. In addition, PARP activity controls early steps of apoptosis. The possible suppression of ADPRTs by nicotinamide would also affect CD38, a membrane-bound external ADP-ribosyl transferase with potent immunoregulatory properties. Taken together, it is proposed that high doses of nicotinamide primarily affect ADP-ribosylation reactions in beta-cells as well as in immune cells and the endothelium. As a consequence, cell death pathways and gene expression patterns are modified, leading to improved beta-cell survival and an altered immunoregulatory balance.

Induction of tolerance in macrophages by cholera toxin B chain.

Model systems of human type 1 diabetes have revealed an important role of cellular immune reactions involving macrophages and T cells in the destruction of autologous insulin-producing pancreatic beta cells. Recently, the cholera toxin B chain (CTB) was found to suppress T cell-dependent autoimmune diseases including autoimmune diabetes of nonobese diabetic mice. Therefore, we tested the hypothesis that CTB exerts much of its immunomodulatory activity by targeting macrophages. These studies are reviewed here. Cells of the human monocyte line Mono Mac 6 were exposed to CTB and subsequently tested for proinflammatory immunoreactivity in response to challenge with endotoxin (LPS from Escherichia coli, 10 ng/ml for 5 h). Incubation of monocytes with CTB (10 microgram/ml) suppressed a later proinflammatory response to LPS as demonstrated by suppression of TNFalpha release from 6.7 +/- 0.7 ng/ml in cultures without CTB preexposure to 1.8 +/- 1.1 ng/ml in CTB-pretreated cells (p < 0.001). In contrast, the release of IL-10 remained inducible after CTB pretreatment. RT-PCR analysis showed that the suppression of TNFalpha production occurred at the level of mRNA formation. Control experiments excluded a role of possible contamination of CTB by endotoxin or the intact cholera toxin. Tolerance induction was maximal after 5 h of CTB exposure and persisted for 24 h. The suppressive effect of CTB was dose-dependent and no more recognizable at

Potent beta-cell protection in vitro by an isoquinolinone-derived PARP inhibitor.

Activation of the nuclear enzyme poly(ADP-ribose)polymerase (PARP) is a critical step in beta-cell death in response to exposure with free radicals or other DNA damaging agents. Nicotinamide, a B vitamin, exerts its beta-cell protective action primarily via its ability to block excessive PARP activity. We show here that the isoquinolinone derivative PD128763, a specific PARP inhibitor, provides protection from cell death in islet cells exposed in vitro to nitric oxide or oxygen radical generating compounds or to the beta-cell toxin streptozotocin, at concentrations 100 times less than required for nicotinamide. Furthermore, while the protective action of nicotinamide is rapidly lost after washing of islet cells, the effects of PD128763 are more long lasting. Both compounds had little capacity to rescue damaged islet cells from subsequent lysis. We conclude that the isoquinolinone derivative PD128763 is superior to nicotinamide in enhancing the resistance of beta-cells towards inflammatory attacks.

MHC class II-dependent abnormal reactivity toward bacterial superantigens in immune cells of NOD mice.

Superantigens have been implicated in the pathogenesis of type I diabetes and other immune-mediated diseases. We therefore tested the hypothesis of an abnormal reactivity of the immune system toward bacterial superantigens during the prediabetic phase. For this purpose, splenocytes from NOD (H-2g7) mice were exposed to two well-characterized superantigens: Staphylococcal aureus enterotoxin-B (SEB) and toxic shock syndrome toxin-1 (TSST-1). Cells from BALB/c (H-2d) and C57BL/6 (H-2b) mice as well as those from NON (H-2non) and NOR (H-2g7) mice were used as controls. After 72 h of co-culture with the superantigens or the mitogen concanavalin A (Con A), proliferative response and mitochondrial activity were determined. In the culture supernatants, the cytokines gamma-interferon (IFN-gamma) and interleukin 10 (IL-10) were measured. Striking similarities between NOD cells and major histocompatiblity complex (MHC)-identical NOR cells could be observed with regard to a low proliferative and mitochondrial response to SEB, accompanied by a normal response to TSST-1 and Con A, respectively. In addition, only NOD and NOR spleen cells were low producers of the T-helper 1 (Th1) cytokine IFN-gamma in response to SEB. Conversely, abnormally high IFN-gamma levels were induced by TSST-1 in NOD and NOR spleen cells. The cytokine response to Con A was also biased toward IFN-gamma in both NOD and NOR. Since IFN-gamma and IL-10 are crucial disease-promoting or -protecting mediators in prediabetic NOD mice, superantigens may affect pathogenesis by acting on the Th1/Th2 cytokine balance. The low responder status toward SEB in NOD spleen cells may be of pathogenetic relevance in view of recent findings that the insulin B-chain also interacts with the SEB binding site on MHC class II molecules. In conclusion, we show here that immune cells from mice with a diabetes-associated MHC type respond differently to common environmental superantigens than do immune cells from control strains.

Low stress response enhances vulnerability of islet cells in diabetes-prone BB rats.

In islet cells isolated from normal outbred Wistar rats, the known high vulnerability of islet cells toward oxygen radicals or nitric oxide can be abolished by inducing a stress response, such as by heat shock. We show here that islet cells from diabetes-prone BB rats are unable to mount such a protective response. Islet cells from diabetes-prone BB rats without recognizable insulitis were heat stressed. Subsequently, cells were exposed to nitric oxide, to oxygen radicals, or to the beta-cell toxin streptozotocin. While prior heat shock substantially increased the survival of toxin-treated Wistar rat islet cells, no protective stress response was noted for islet cells from diabetes-prone BB rats. Islet cells from diabetes-resistant BB rats were protected by heat stress to the same extent as Wistar rats. A survey of four additional major histocompatibility complex (MHC)-disparate rat strains confirmed the existence of a low and high responder type to stress. Parallel analysis of heat shock protein (hsp)70 induction by Western blot showed a low and high hsp70 response phenotype. A high hsp70 response coincided with a protective stress response. The presence (or absence) of a protective stress response correlated with the preservation (or loss) of intracellular NAD+ in toxin-treated islet cells. The lack of a protective stress response in islet cells from diabetes-prone BB rats, but not in diabetes-resistant BB rats, may promote beta-cell lysis and autoantigen release, and hence could be important for initiation or propagation of the disease process.

Metabolic activation of islet cells improves resistance against oxygen radicals or streptozocin, but not nitric oxide.

In the present study we investigated the effect of metabolic activation on the susceptibility of isolated rat pancreatic islet cells to the alkylating beta-cell toxin streptozocin (SZ), reactive oxygen intermediates (ROI), and nitric oxide (NO). The latter two represent physiologically occurring mediators involved in the autoimmune destruction of islet cells. ROI were generated by the enzyme xanthine oxidase, and NO was released from sodium nitroprusside. During 18 h of culture at a physiological glucose concentration (5 mmol/L), 75% of the islet cells were lysed by SZ, 81% by ROI, and 74% by NO, as determined by the trypan blue exclusion assay. Increasing concentrations of glucose or the nonnutrient stimulators theophylline and glibenclamide dose-dependently reduced SZ- and ROI-mediated islet cell lysis. In the presence of 29 mmol/L glucose, 5.5 mmol/L theophylline, or 10 micrograms/mL glibenclamide, SZ-induced lysis was reduced to 15%, 22%, or 15%, and ROI-induced lysis was reduced to 20%, 34%, or 15%, respectively. In contrast, stimulation by glucose, theophylline, or glibenclamide did not improve resistance against NO. The protection against SZ and ROI was associated with preserved mitochondrial activity, as determined by the ability of the islet cells to convert the tetrazolium salt 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide into its formazan. Elevation of the glucose concentration from 5.5 to 29 mmol/L increased the residual mitochondrial activity from 45% to 80% in SZ-exposed islet cells and from 21% to 78% in ROI-exposed cells. Conversely, the lack of protection against NO correlated with no preservation of mitochondrial activity in the presence of high concentrations of glucose, theophylline, or glibenclamide. In conclusion, our results show that metabolic stress does not render islet cells more susceptible to inflammatory insults in vitro. Rather, an increased mitochondrial energy supply improves the resistance against SZ and ROI, whereas the toxicity of NO was independent of islet cell activity.