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Inflammatory β-cell failure contributes to type 1 and type 2 diabetes pathogenesis. Pro-inflammatory cytokines cause β-cell dysfunction and apoptosis, and lysine deacetylase inhibitors (KDACi) prevent β-cell failure in vitro and in vivo, in part by reducing NF-κB transcriptional activity. We investigated the hypothesis that the protective effect of KDACi involves transcriptional regulation of microRNAs (miRs), potential new targets in diabetes treatment. Insulin-producing INS1 cells were cultured with or without the broad-spectrum KDACi Givinostat, prior to exposure to the pro-inflammatory cytokines IL-1β and IFN-γ for 6 h or 24 h, and miR expression was profiled with miR array. Thirteen miRs (miR-7a-2-3p, miR-29c-3p, miR-96-5p, miR-101a-3p, miR-140-5p, miR-146a-5p, miR-146b-5p, miR-340-5p, miR-384-5p, miR-455-5p, miR-466b-2-3p, miR-652-5p, and miR-3584-5p) were regulated by both cytokines and Givinostat, and nine were examined by qRT-PCR. miR-146a-5p was strongly regulated by cytokines and KDACi and was analyzed further. miR-146a-5p expression was induced by cytokines in rat and human islets. Cytokine-induced miR-146a-5p expression was specific for INS1 and β-TC3 cells, whereas α-TC1 cells exhibited a higher basal expression. Transfection of INS1 cells with miR-146a-5p reduced cytokine signaling, including the activity of NF-κB and iNOS promoters, as well as NO production and protein levels of iNOS and its own direct targets TNF receptor associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1). miR-146a-5p was elevated in the pancreas of diabetes-prone BB-DP rats at diabetes onset, suggesting that miR-146a-5p could play a role in type 1 diabetes development. The miR array of cytokine-exposed INS1 cells rescued by KDACi revealed several other miRs potentially involved in cytokine-induced β-cell apoptosis, demonstrating the strength of this approach.

Obesity and the metabolic syndrome are characterized by chronic, low-grade inflammation mainly originating from expanding adipose tissue and resulting in inhibition of insulin signaling and disruption of glycemic control.Transgenic mice expressing human interleukin 37 (IL-37),an anti-inflammatory cytokine of the IL-1 family,are protected against metabolic syndrome when fed a high-fat diet (HFD) containing 45% fat. Here, we examined whether treatment with recombinant IL-37 ameliorates established insulin resistance and obesity-induced inflammation. WT mice were fed a HFD for 22 weeks and then treated daily with IL-37 (1 ug/mouse) during the last 2 weeks. Compared with vehicle only-treated mice, IL-37-treated mice exhibited reduced insulin in the plasma and had significant improvements in glucose tolerance and in insulin content of the islets.The IL-37 treatment also increased the levels of circulating IL-1 receptor antagonist. Cultured adipose tissues revealed that IL-37 treatment significantly decreases spontaneous secretions of IL-1β, tumor necrosis factor α (TNFα), and CXC motif chemokine ligand 1 (CXCL-1). We also fed mice a 60% fat diet with concomitant daily IL-37 for 2 weeks and observed decreased secretion of IL-1β, TNFα, and IL-6 and reduced intracellular levels of IL-1β in the liver and adipose tissue, along with improved plasma glucose clearance. Compared with vehicle treatment, these IL-37-treated mice had no apparent weight gain. In human adipose tissue cultures, the presence of 50 pM IL-37 reduced spontaneous release of TNF and 50% of lipopolysaccharide-induced TNFα. These findings indicate that IL-37's anti-inflammatory effects can ameliorate established metabolic disturbances during obesity.

Type 2 diabetes (T2D) arises when the pancreatic beta-cell fails to compensate for increased insulin needs due to insulin resistance. Glucolipotoxicity has been proposed to induce beta-cell dysfunction in T2D by formation of reactive oxygen species (ROS). Here we examined if modelling glucolipotoxic conditions by high glucose-high free fatty acid (FFA) exposure (GLT) regulate beta-cell iron transport, thereby increasing the cytosolic labile iron pool and iron-catalyzed ROS formation. We show that GLT -induced ROS production is regulated by an increased labile iron pool (LIP) associated with elevated expression of genes regulating iron import. Using pharmacological and transgenic approaches, we show that iron reduction and decreased iron import protects from GLT-induced ROS production, prevents impairment of the mitochondrial membrane potential, and inhibits apoptosis. This study identifies a novel pathway underlying GLT-induced apoptosis involving increased iron import, generation of hydroxyl radicals from hydrogen peroxide through the Fenton reaction in the cytosolic compartment associated with dissipation of the mitochondrial membrane potential and beta cell apoptosis.

A meta-analysis of the association of iron overload with gestational diabetes mellitus (GDM) may inform the health debate. We performed a meta-analysis investigating the association of iron biomarkers and dietary iron exposure with GDM. We identified 33 eligible studies (N = 44,110) published in 2001-2017. The standardized mean differences (SMD) in women who had GDM compared to pregnant women without were 0.25 mu g/dL (95% CI: 0.001-0.50) for iron, 1.54 ng/mL (0.56-2.53) for ferritin, 1.05% (0.02 to 2.08) for transferrin saturation, and 0.81 g/dL (0.40-1.22) for hemoglobin. Adjusted odds ratio for GDM were 1.58 (95% CI: 1.20-2.08) for ferritin, 1.30 (1.01-1.67) for hemoglobin, and 1.48 (1.29-1.69) for dietary heme intake. We did not find any differences in TIBC or transferrin concentration in women with and without GDM. We also did not find any association of increased transferrin receptor or increased intake of total dietary iron, non-heme iron or supplemental iron, with increased odds ratios for GDM. Considerable heterogeneity was present among the studies (0-99%), but no evidence of publication bias. Accumulating evidence suggests that circulating and dietary iron biomarkers among pregnant women are associated with GDM, but the results should be interpreted with caution due to the high heterogeneity of analyses. Randomized trials investigating the benefits of iron reduction in women at high risk for GDM are warranted.

Type 2 diabetes (T2D) arises when the pancreatic beta-cell fails to compensate for increased insulin needs due to insulin resistance. Glucolipotoxicity (GLT) has been proposed to induce beta-cell dysfunction in T2D by formation of reactive oxygen species (ROS). Here, we examined if modeling glucolipotoxic conditions by high glucose-high free fatty acid (FFA) exposure (GLT) regulates beta-cell iron transport, by increasing the cytosolic labile iron pool (LIP). In isolated mouse islets, the GLT-induced increase in the LIP catalyzed cytosolic ROS formation and induced apoptosis. We show that GLT-induced ROS production is regulated by an increased LIP associated with elevated expression of genes regulating iron import. Using pharmacological and transgenic approaches, we show that iron reduction and decreased iron import protects from GLT-induced ROS production, prevents impairment of the mitochondrial membrane potential (MMP) and inhibits apoptosis. This study identifies a novel pathway underlying GLT-induced apoptosis involving increased iron import, generation of hydroxyl radicals from hydrogen peroxide through the Fenton reaction in the cytosolic compartment associated with dissipation of the MMP and beta-cell apoptosis.

Interleukin 6 (IL-6) is a cytokine secreted from skeletal muscle in response to exercise which, based on animal and cell studies, has been suggested to contribute to glucose metabolism by increasing secretion of the incretin hormone glucagon-like peptide-1 (GLP-1) and affecting secretion of insulin and glucagon from the pancreatic islets. We investigated the effect of IL-6 on GLP-1 secretion in GLP-1 producing cells (GLUTag) and using the perfused mouse small intestine (harboring GLP-1 producing cells). Furthermore, the direct effect of IL-6 on insulin and glucagon secretion was studied using isolated perfused mouse pancreas. Incubating GLUTag cells with 1000 ng/mL of IL-6 for 2 h did not significantly increase secretion of GLP-1 whereas 10 mmol/L glucose (positive control) did. Similarly, IL-6 (100 ng/mL) had no effect on GLP-1 secretion from perfused mouse small intestine whereas bombesin (positive control) increased secretion. Finally, administering IL-6 (100 ng/mL) to perfused mouse pancreases did not significantly increase insulin or glucagon secretion regardless of perfusate glucose levels (3.5 vs. 12 mmol/L glucose). Acute effects of IL-6 therefore do not seem to include a stimulatory effect on GLP-1 secretion in mice.

BACKGROUND: It is unknown why increased plasma ferritin concentration predicts all-cause mortality. As low-grade inflammation and increased plasma ferritin concentration are associated with all-cause mortality, we hypothesized that increased plasma ferritin concentration is genetically associated with low-grade inflammation.METHODS: We investigated whether increased plasma ferritin concentration is associated with low-grade inflammation [i.e., increased concentrations of C-reactive protein (CRP) and complement component 3 (C3)] in 62537 individuals from the Danish general population. We also applied a Mendelian randomization approach, using the hemochromatosis genotype C282Y/C282Y as an instrument for increased plasma ferritin concentration, to assess causality.RESULTS: For a doubling in plasma ferritin concentration, the odds ratio (95% CI) for CRP ≥2 vs <2 mg/L was 1.12 (1.09-1.16), with a corresponding genetic estimate for C282Y/C282Y of 1.03 (1.01-1.06). For a doubling in plasma ferritin concentration, odds ratio (95% CI) for complement C3 >1.04 vs ≤1.04 g/L was 1.28 (1.21-1.35), and the corresponding genetic estimate for C282Y/C282Y was 1.06 (1.03-1.12). Mediation analyses showed that 74% (95% CI, 24-123) of the association of C282Y/C282Y with risk of increased CRP and 56% (17%-96%) of the association of C282Y/C282Y with risk of increased complement C3 were mediated through plasma ferritin concentration.CONCLUSIONS: Increased plasma ferritin concentration as a marker of increased iron concentration is associated observationally and genetically with low-grade inflammation, possibly indicating a causal relationship from increased ferritin to inflammation. However, as HFE may also play an immunological role indicating pleiotropy and as incomplete penetrance of C282Y/C282Y indicates buffering mechanisms, these weaknesses in the study design could bias the genetic estimates.

OBJECTIVE: Cardiovascular mortality risk remains high among patients with type 2 diabetes. Oxidative stress indicated by high urinary excretion of the biomarker for RNA oxidation, 8-oxo-7,8-dihydroguanosine (8-oxoGuo), is associated with an increased risk of death in newly diagnosed and treated patients. We assessed whether 8-oxoGuo is associated with specific cardiovascular and all-cause mortality risk.RESEARCH DESIGN AND METHODS: Urinary biomarkers for nucleic acid oxidation were measured in a cohort of patients with type 2 diabetes aged ≥60 years (n = 1,863), along with biochemical measurements, questionnaire findings, and Central Person Registry information to estimate the hazard ratios (HRs) for log2-transformed RNA oxidation using Cox regression.RESULTS: During the 5-year follow-up, 173 of 1,863 patients had died (9.3%), including 73 patients who died of cardiovascular disease (42.2%). Doubling of RNA oxidation was associated with an HR of all-cause mortality of 2.10 (95% CI 1.63-2.71; P < 0.001) and an HR of cardiovascular death of 1.82 (95% CI 1.20-2.77; P = 0.005) after multiple adjustments. The 5-year absolute risks (ARs) of all-cause mortality (AR 13.9 [95% CI 10.8-17.0] vs. AR 6.10 [95% CI 4.00-8.30]) and cardiovascular mortality (AR 5.49 [95% CI 3.44-7.55] vs. AR 3.16 [95% CI 1.59-4.73]) were approximately two times higher in the highest quartile of RNA oxidation than in the lowest quartile.CONCLUSIONS: We conclude that high RNA oxidation is associated with all-cause and cardiovascular mortality risk in patients with type 2 diabetes. Targeting oxidative stress via interventions with long-term follow-up may reveal the predictive potential of the biomarker 8-oxoGuo.

The relative contributions of the JNK subtypes in inflammatory β-cell failure and apoptosis are unclear. The JNK protein family consists of JNK1, JNK2, and JNK3 subtypes, encompassing many different isoforms. INS-1 cells express JNK1α1, JNK1α2, JNK1β1, JNK1β2, JNK2α1, JNK2α2, JNK3α1, and JNK3α2 mRNA isoform transcripts translating into 46 and 54 kDa isoform JNK proteins. Utilizing Lentiviral mediated expression of shRNAs against JNK1, JNK2, or JNK3 in insulin-producing INS-1 cells, we investigated the role of individual JNK subtypes in IL-1β-induced β-cell apoptosis. JNK1 knockdown prevented IL-1β-induced INS-1 cell apoptosis associated with decreased 46 kDa isoform JNK protein phosphorylation and attenuated Myc expression. Transient knockdown of Myc also prevented IL-1β-induced apoptosis as well as caspase 3 cleavage. JNK2 shRNA potentiated IL-1β-induced apoptosis and caspase 3 cleavage, whereas JNK3 shRNA did not affect IL-1β-induced β-cell death compared to nonsense shRNA expressing INS-1 cells. In conclusion, JNK1 mediates INS-1 cell death associated with increased Myc expression. These findings underline the importance of differentiated targeting of JNK subtypes in the development of inflammatory β-cell failure and destruction.

Glucagon-like Peptide-1 mimetics increase insulin secretion and reduces body weight in humans. In lean, healthy cats, short-term treatment has produced similar results, whereas the effect in obese cats or with extended duration of treatment is unknown. Here, prolonged (12 weeks) treatment with the Glucagon-like Peptide-1 mimetic, exenatide, was evaluated in 12 obese, but otherwise healthy, client-owned cats. Cats were randomized to exenatide (1.0 μg/kg) or placebo treatment twice daily for 12 weeks. The primary endpoint was changes in insulin concentration; the secondary endpoints were glucose homeostasis, body weight, body composition as measured by dual-energy x-ray absorptiometry and overall safety. An intravenous glucose tolerance test (1 g/kg body weight) was conducted at week 0 and week 12. Exenatide did not change the insulin concentration, plasma glucose concentration or glucose tolerance (P>0.05 for all). Exenatide tended to reduce body weight on continued normal feeding. Median relative weight loss after 12 weeks was 5.1% (range 1.7 to 8.4%) in the exenatide group versus 3.2% (range -5.3 to 5.7%) in the placebo group (P = 0.10). Body composition and adipokine levels were unaffected by exenatide (P>0.05). Twelve weeks of exenatide was well-tolerated, with only two cases of mild, self-limiting gastrointestinal signs and a single case of mild hypoglycemia. The long-term insulinotropic effect of exenatide appeared less pronounced in obese cats compared to previous short-term studies in lean cats. Further investigations are required to fully elucidate the effect on insulin secretion, glucose tolerance and body weight in obese cats.

As microRNAs (miRs) are gaining increasing attention as key regulators of cellular processes, expressional quantification is widely applied. However, in the processing of relatively quantified data, the importance of testing the stability of several reference mRNAs and/or miRs and choosing among these for normalization is often overlooked, potentially leading to biased results. Here, we have optimized the purification of miR-enriched total RNA from pancreatic insulin-producing INS-1 cells. Additionally, we optimized and analyzed miR expression by a qPCR-based microarray and by specific qPCR and tested the stability of candidate reference mRNAs and miRs. Hence, this study gives a widely applicable example on how to easily and systematically test and decide how to normalize miR quantification. We suggest that caution in the interpretation of miR quantification studies that do not comprise stability analysis should be exerted.

Dietary advice is the cornerstone in first-line treatment of metabolic diseases. Nutritional interventions directed at these clinical conditions mainly aim to (a) improve insulin resistance by reducing energy-dense macronutrient intake to obtain weight loss and (b) reduce fluctuations in insulin secretion through avoidance of rapidly absorbable carbohydrates. However, even in the majority of motivated patients selected for clinical trials, massive efforts using this approach have failed to achieve lasting efficacy. Less attention has been given to the role of micronutrients in metabolic diseases. Here, we review the evidence that highlights (a) the importance of iron in pancreatic beta-cell function and dysfunction in diabetes and (b) the integrative pathophysiological effects of tissue iron levels in the interactions among the beta cell, gut microbiome, hypothalamus, innate and adaptive immune systems, and insulin-sensitive tissues. We propose that clinical trials are warranted to clarify the impact of dietary or pharmacological iron reduction on the development of metabolic disorders.