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International audience; Chronic low grade inflammation is closely linked to obesity-associated insulin resistance. To examine how administration of the anti-inflammatory compound indomethacin, a general cyclooxygenase inhibitor, affected obesity development and insulin sensitivity, we fed obesity-prone male C57BL/6J mice a high fat/high sucrose (HF/HS) diet or a regular diet supplemented or not with indomethacin (±INDO) for 7 weeks. Development of obesity, insulin resistance, and glucose intolerance was monitored, and the effect of indomethacin on glucose-stimulated insulin secretion (GSIS) was measured in vivo and in vitro using MIN6 β-cells. We found that supplementation with indomethacin prevented HF/HS-induced obesity and diet-induced changes in systemic insulin sensitivity. Thus, HF/HS+INDO-fed mice remained insulin-sensitive. However, mice fed HF/HS+INDO exhibited pronounced glucose intolerance. Hepatic glucose output was significantly increased. Indomethacin had no effect on adipose tissue mass, glucose tolerance, or GSIS when included in a regular diet. Indomethacin administration to obese mice did not reduce adipose tissue mass, and the compensatory increase in GSIS observed in obese mice was not affected by treatment with indomethacin. We demonstrate that indomethacin did not inhibit GSIS per se, but activation of GPR40 in the presence of indomethacin inhibited glucose-dependent insulin secretion in MIN6 cells. We conclude that constitutive high hepatic glucose output combined with impaired GSIS in response to activation of GPR40-dependent signaling in the HF/HS+INDO-fed mice contributed to the impaired glucose clearance during a glucose challenge and that the resulting lower levels of plasma insulin prevented the obesogenic action of the HF/HS diet.

Summary Studies on isolated pancreatic islets suggest that neuromedin U (NMU), a brain and gastrointestinal peptide, acts as a decretin hormone, inhibiting glucose-stimulated insulin secretion. We investigated whether this effect could be reproduced in vivo and in isolated perfused rat pancreas. Unlike the incretin hormone, glucagon-like peptide 1 (GLP-1), intravenous NMU administration had no effects on blood glucose and plasma insulin and glucagon in vivo. Moreover, NMU neither changed insulin, glucagon, or somatostatin secretion from isolated perfused rat pancreas, nor affected GLP-1-stimulated insulin and somatostatin secretion. For NMU to act as a decretin hormone, its secretion should increase following glucose ingestion; however, glucose did not affect NMU secretion from isolated perfused rat small intestine, which contained extractable NMU. Furthermore, the two NMU receptors were not detected in endocrine rat or human pancreas. We conclude that NMU does not act as a decretin hormone in rats.

The recent molecular, biologic, and genetic understanding of the inflammasome has revolutionized the diagnosis of and therapy for the phenotypically heterogeneous group of rare oligogenic disorders, now recognized to have autoinflammatory origin. This article reviews the importance of inflammasome activation in the central and peripheral mechanisms underlying a common, multifactorial, lifestyle-related, and polygenetic disease (type 2 diabetes mellitus), and conceptualizes the notion that this health challenge should now be recognized to have an autoinflammatory cause. It is hoped that targeting these mechanisms will enable the introduction of novel therapies that attack the basic pathogenetic mechanisms of type 2 diabetes mellitus rather than the epiphenomena that are its consequences.

Disease is conventionally viewed as the chaotic inappropriate outcome of deranged tissue function resulting from aberrancies in cellular processes. Yet the patho-biology of cellular dysfunction and death encompasses a coordinated network no less sophisticated and regulated than maintenance of homeostatic balance. Cellular demise is far from passive subordination to stress but requires controlled coordination of energy-requiring activities including gene transcription and protein translation that determine the graded transition between defensive mechanisms, cell cycle regulation, dedifferentiation and ultimately to the activation of death programmes. In fact, most stressors stimulate both homeostasis and regeneration on one hand and impairment and destruction on the other, depending on the ambient circumstances. Here we illustrate this bimodal ambiguity in cell response by reviewing recent progress in our understanding of how the pancreatic β cell copes with inflammatory stress by changing gene transcription and protein translation by the differential and interconnected action of reactive oxygen and nitric oxide species, microRNAs and posttranslational protein modifications.

Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO2 phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated “TiSH”). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO2 pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~ 6600 unique phosphopeptides from 300 μg of peptides/condition (22 unique phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity > 94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of sample with high sensitivity and specificity.

The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas other RNA molecules show virtually no oxidation. The iron-storage disease hemochromatosis exhibits the most prominent general increase in RNA oxidation ever observed. Oxidation of RNA primarily leads to strand breaks and to oxidative base modifications. Oxidized mRNA is recognized by the ribosomes, but the oxidation results in ribosomal stalling and dysfunction, followed by decreased levels of functional protein as well as the production of truncated proteins that do not undergo proper folding and may result in protein aggregation within the cell. Ribosomal dysfunction may also signal apoptosis by p53-independent pathways. There are very few reports on interventions that reduce RNA oxidation, one interesting observation being a reduction in RNA oxidation by ingestion of raw olive oil. High urinary excretion of 8-oxo-guanosine, a biomarker for RNA oxidation, is highly predictive of death in newly diagnosed type 2 diabetics; this demonstrates the clinical relevance of RNA oxidation. Taken collectively the available data suggest that RNA oxidation is a contributing factor in several diseases such as diabetes, hemochromatosis, heart failure, and β-cell destruction. The mechanism involves free iron and hydrogen peroxide from mitochondrial dysfunction that together lead to RNA oxidation that in turn gives rise to truncated proteins that may cause aggregation. Thus RNA oxidation may well be an important novel contributing mechanism for several diseases.

Suitable analogs of d-mannoheptulose are currently considered as possible tools for the non-invasive imaging of pancreatic islet insulin-producing cells. Here, we examined whether (19)F-heptuloses could be used for non-invasive imaging of GLUT2-expressing cells. After 20 min incubation, the uptake of (19)F-heptuloses (25 mM) by rat hepatocytes, as assessed by (19)F NMR spectroscopy, ranged from 0.50 (1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose) to 0.25 (1,3-dideoxy-1,3-difluoro-d-mannoheptulose) and 0.13 (1-deoxy-1-fluoro-d-glucoheptulose, 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose) μmol per 3×10(6)cells. (19)F MRI experiments also allowed the detection of 1-deoxy-1-fluoro-d-mannoheptulose in rat hepatocytes. All three (19)F-mannoheptuloses cited above, as well as 7-deoxy-7-fluoro-d-mannoheptulose and 1-deoxy-1-fluoro-d-glucoheptulose inhibited insulin release evoked in rat isolated pancreatic islets by 10mM d-glucose to the same extent as that observed with an equivalent concentration (10mM) of d-mannoheptulose, while 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose (also 10mM) were less potent than d-mannoheptulose in inhibiting insulin release. The 1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose only marginally affected INS-1 cell viability. These findings are compatible with the view that selected (19)F-heptuloses may represent suitable tools for the non-invasive imaging of hepatocytes and insulin-producing cells by (19)F MRI.

Tumor necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine involved in the pathogenesis of several diseases including type 1 diabetes mellitus (T1DM). TNFalpha in combination with interleukin-1-beta (IL-1beta) and/or interferon-gamma (IFNgamma) induces specific destruction of the pancreatic insulin-producing beta cells. Suppressor of cytokine signalling-3 (SOCS-3) proteins regulate signalling induced by a number of cytokines including growth hormone, IFNgamma and IL-1beta which signals via very distinctive pathways. The objective of this study was to investigate the effect of SOCS-3 on TNFalpha-induced signalling in beta cells. We found that apoptosis induced by TNFalpha alone or in combination with IL-1beta was suppressed by expression of SOCS-3 in the beta cell line INSr3#2. SOCS-3 inhibited TNFalpha-induced phosphorylation of the mitogen activated protein kinases ERK1/2, p38 and JNK in INSr3#2 cells and in primary rat islets. Furthermore, SOCS-3 repressed TNFalpha-induced degradation of IkappaB, NFkappaB DNA binding and transcription of the NFkappaB-dependent MnSOD promoter. Finally, expression of Socs-3 mRNA was induced by TNFalpha in rat islets in a transient manner with maximum expression after 1-2h. The ability of SOCS-3 to regulate signalling induced by the three major pro-inflammatory cytokines involved in the pathogenesis of T1DM makes SOCS-3 an interesting therapeutic candidate for protection of the beta cell mass.

In models of type 1 diabetes, cytokines induce pancreatic beta-cell death by apoptosis. This process seems to be facilitated by a reduction in the amount of the islet-brain 1/JNK interacting protein 1 (IB1/JIP1), a JNK-scaffold with an anti-apoptotic effect. A point mutation S59N at the N terminus of the scaffold, which segregates in diabetic patients, has the functional consequence of sensitizing cells to apoptotic stimuli. Neither the mechanisms leading to IB1/JIP1 down-regulation by cytokines nor the mechanisms leading to the decreased capacity of the S59N mutation to protect cells from apoptosis are understood. Here, we show that IB1/JIP1 stability is modulated by intracellular calcium. The effect of calcium depends upon JNK activation, which primes the scaffold for ubiquitination-mediated degradation via the proteasome machinery. Furthermore, we observe that the S59N mutation decreases IB1/JIP1 stability by sensitizing IB1/JIP1 to calcium- and proteasome-dependent degradation. These data indicate that calcium influx initiated by cytokines mediates ubiquitination and degradation of IB1/JIP1 and may, therefore, provide a link between calcium influx and JNK-mediated apoptosis in pancreatic beta-cells.

The stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) is a central signal for interleukin-1beta (IL-1beta)-induced apoptosis in insulin-producing beta-cells. The cell-permeable peptide inhibitor of JNK (JNKI1), that introduces the JNK binding domain (JBD) of the scaffold protein islet-brain 1 (IB1) inside cells, effectively prevents beta-cell death caused by this cytokine. To define the molecular targets of JNK involved in cytokine-induced beta-cell apoptosis we investigated whether JNKI1 or stable expression of JBD affected the expression of selected pro- and anti-apoptotic genes induced in rat (RIN-5AH-T2B) and mouse (betaTC3) insulinoma cells exposed to IL-1beta. Inhibition of JNK significantly reduced phosphorylation of the specific JNK substrate c-Jun (p

L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, decreased IL-1 beta-induced nitrite release in rat islets and purified rat beta cells, nitrite formation and iNOS gene promoter activity in insulinoma cells, and iNOS mRNA expression in rat islets. The thiol depletor diethyl maleate (DEM) and an inhibitor of glutathione reductase 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced IL-1 beta-stimulated nitrite release in islets. We conclude that GSH regulates IL-1 beta-induced NO production in islets, purified beta cells and insulinoma cells by modulation of iNOS gene expression.

The molecules of the interleukin 1 (IL-1) system have been suggested to play a role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM), and polymorphisms in the genes encoding IL-1β ( IL1B ), the IL-1 Type 1 receptor ( IL1RTI ) and the IL-1 receptor antagonist ( IL1RN ) molecules have been associated with IDDM in case–control studies. It can be difficult to exclude selection biases in case–control studies leading to spurious association. This risk is eliminated when using the transmission disequilibrium test (TDT). Hence, by means of the TDT we have investigated four intragenic IL-1 gene-cluster polymorphisms, the IL1B AvaI , the IL1B TaqI , the IL1RTI PstI and the IL1RN 2 nd intron polymorphisms, for linkage and intra-familial association with IDDM in Danish IDDM multiplex family material comprising 245 families. We found no evidence for overall linkage or intra-familial association between any of the polymorphisms and IDDM. In addition, we did not find linkage between any of the polymorphisms and IDDM in HLA-DR3/4 heterozygous or HLA-non-DR3/4 heterozygous IDDM subjects, respectively. In conclusion, by means of intra-familial TDT analysis we found no linkage or intra-familial association between IDDM and the four IL-1 gene-cluster polymorphisms in Danish IDDM multiplex family material.

Interleukin-1beta (IL-1beta) is cytotoxic to rat pancreatic beta-cells by inhibiting glucose oxidation, causing DNA damage and inducing apoptosis. Nitric oxide (NO) is a necessary but not sufficient mediator of these effects. IL-1beta induced kinase activity toward Elk-1, activation transcription factor 2, c-Jun, and heat shock protein 25 in rat islets. By Western blotting with phosphospecific antibodies and by immunocomplex kinase assay, IL-1beta was shown to activate extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38) in islets and rat insulinoma cells. Specific ERK1/2 and p38 inhibitors individually reduced but in combination blocked IL-1beta-mediated islet NO synthesis, and reverse transcription-polymerase chain reaction of inducible NO synthase mRNA showed that ERK1/2 and p38 controlled IL-1beta-induced islet inducible NO synthase expression at the transcriptional level. Hyperosmolarity caused phosphorylation of Elk-1, activation transcription factor 2, and heat shock protein 25 and activation of ERK1/2 and p38 in islets comparable to that induced by IL-1beta but did not lead to NO synthesis. Inhibition of p38 but not of ERK1/2 attenuated IL-1beta-mediated inhibition of glucose-stimulated insulin release. We conclude that ERK1/2 and p38 activation is necessary but not sufficient for IL-1beta-mediated beta-cell NO synthesis and that p38 is involved in signaling of NO-independent effects of IL-1beta in beta-cells.

Substantial in vitro evidence suggests that nitric oxide may be a major mediator of interleukin 1 (IL-1) induced pancreatic beta-cell inhibition and destruction in the initial events leading to insulin-dependent diabetes mellitus. Using NG-nitro-L-arginine methyl ester, an inhibitor of both the constitutive and the cytokine inducible forms of nitric oxide synthase, and aminoguanidine, a preferential inhibitor of the inducible form of nitric oxide synthase, we investigated the impact of inhibiting nitric oxide production on food-intake, body weight and temperature, blood glucose, plasma insulin, glucagon, corticosterone and leukocyte- and differential-counts in normal rats injected once daily for 5 days with interleukin 1 beta (IL-1 beta) (0.8 microgram/rat = 4.0 micrograms/kg). Inhibition of both the constitutive and the inducible forms of nitric oxide synthase prevented IL-1 beta-induced fever, hyperglycaemia, hypoinsulinemia, and hyperglucagonemia, and partially prevented lymphopenia and neutrophilia, but had no effect on IL-1 beta-induced anorexia and changes in plasma corticosterone. Preferential inhibition of the inducible form of nitric oxide synthase using two daily injections of 5 mg/rat of aminoguanidine prevented IL-1 beta-induced hyperglycaemia and hypoinsulinaemia, and slightly reduced the pyrogenicity of IL-1 on 3 out of 5 days. Higher doses of aminoguanidine (100 mg/rat) prevented lymphopenia and neutrophilia. We conclude that nitric oxide produced by the inducible form of nitric oxide synthase, mediates the IL-1 beta-induced inhibition of insulin release and that the effect of IL-1 beta on temperature, pancreatic alpha-cells, and leukocyte differential counts seems to be mediated by nitric oxide produced by the constitutive form of nitric oxide synthase.

The variations in incidence rates of insulin-dependent diabetes mellitus (IDDM) in childhood within and between genetically very similar Scandinavian populations and the variations in incidence rates with time are difficult to explain. Epidemiological data show that the incidence of childhood IDDM may now be declining and suggest an inverse correlation between breast-feeding frequency and IDDM in childhood. Case-control data show that diabetic children were breast-fed for shorter periods of time than their healthy siblings and the population at large and that a smaller proportion of diabetic children were ever breast-fed. It is postulated that insufficient breast-feeding of genetically susceptible newborn infants may lead to beta-cell infection and IDDM later in life.