High-fat diets lead to obesity, inflammation, and dysglycemia. 12-Lipoxygenase (12-LO) is activated by high-fat diets and catalyzes the oxygenation of cellular arachidonic acid to form proinflammatory intermediates. We hypothesized that 12-LO in the pancreatic islet is sufficient to cause dysglycemia in the setting of high-fat feeding. To test this, we generated pancreas-specific 12-LO knockout mice and studied their metabolic and molecular adaptations to high-fat diets. Whereas knockout mice and control littermates displayed identical weight gain, body fat distribution, and macrophage infiltration into fat, knockout mice exhibited greater adaptive islet hyperplasia, improved insulin secretion, and complete protection from dysglycemia. At the molecular level, 12-LO deletion resulted in increases in islet antioxidant enzymes Sod1 and Gpx1 in response to high-fat feeding. The absence or inhibition of 12-LO led to increases in nuclear Nrf2, a transcription factor responsible for activation of genes encoding antioxidant enzymes. Our data reveal a novel pathway in which islet 12-LO suppresses antioxidant enzymes and prevents the adaptive islet responses in the setting of high-fat diets.
The MAPKs are transducers of extracellular signals such as proinflammatory cytokines. In islet β-cells, cytokines acutely activate expression of the Nos2 gene encoding inducible nitric oxide synthase (iNOS), which ultimately impairs insulin release. Because iNOS production can also be regulated posttranscriptionally, we asked whether MAPKs participate in posttranscriptional regulatory events in β-cells and primary islets in response to cytokine signaling. We show that cytokines acutely reduce cellular oxygen consumption rate and impair aconitase activity. Inhibition of iNOS with l-NMMA or inhibition of Nos2 mRNA translation with GC7 [an inhibitor of eukaryotic translation initiation factor 5A (eIF5A) activity] reversed these defects, as did inhibition of p38 MAPK by PD169316. Although inhibition of p38 had no effect on the nuclear translocation of nuclear factor κB or the abundance of Nos2 transcripts during the immediate period after cytokine exposure, its inhibition or knockdown resulted in significant reduction in iNOS protein, a finding suggestive of a permissive role for p38 in Nos2 translation. Polyribosomal profiling experiments using INS-1 β-cells revealed that Nos2 mRNA remained associated with polyribosomes in the setting of p38 inhibition, in a manner similar to that seen with blockade of translational elongation by cycloheximide. Consistent with a role in translational elongation, p38 activity is required in part for the activation of the translational factor eIF5A by promoting its hypusination. Our results suggest a novel signaling pathway in β-cells in which p38 MAPK promotes translation elongation of Nos2 mRNA via regulation of eIF5A hypusination.
High-fat diets lead to obesity, inflammation, and dysglycemia. 12-Lipoxygenase (12-LO) is activated by high-fat diets and catalyzes the oxygenation of cellular arachidonic acid to form proinflammatory intermediates. We hypothesized that 12-LO in the pancreatic islet is sufficient to cause dysglycemia in the setting of high-fat feeding. To test this, we generated pancreas-specific 12-LO knockout mice and studied their metabolic and molecular adaptations to high-fat diets. Whereas knockout mice and control littermates displayed identical weight gain, body fat distribution, and macrophage infiltration into fat, knockout mice exhibited greater adaptive islet hyperplasia, improved insulin secretion, and complete protection from dysglycemia. At the molecular level, 12-LO deletion resulted in increases in islet antioxidant enzymes Sod1 and Gpx1 in response to high-fat feeding. The absence or inhibition of 12-LO led to increases in nuclear Nrf2, a transcription factor responsible for activation of genes encoding antioxidant enzymes. Our data reveal a novel pathway in which islet 12-LO suppresses antioxidant enzymes and prevents the adaptive islet responses in the setting of high-fat diets.
Chickens have a blood glucose level that is twice as high as that in most mammals and are regarded as an insulin resistant animal. We previously reported that the major insulin responsive glucose transporter gene, GLUT4, is deficient in broiler chickens, therefore insulin regulation of blood glucose level in chickens is not well understood. In the present study, we characterized gene expressions of GLUT, hexokinase (HK) and glycogen synthase (GS) in insulin-stimulated state in chicken skeletal muscles and cultured chicken myotubes. In a study in vivo, 3-week-old male chickens were injected with insulin (400μg/kg body weight) or 0.9% of NaCl. Skeletal muscles were collected at 1 and 3 h after insulin injection. Expressions of GLUT1, GLUT3, GLUT8, HK I, HK II, GS mRNA and 18S rRNA were determined by Real-Time PCR. GLUT1, GLUT3 and HK II mRNA expression were significantly increased at 3 h after insulin injection in skeletal muscle but no significant increase in GLUT8 and HKI mRNA expression was observed. In a study in vitro, myotubes derived from muscle satellite cells were incubated with serum-free medium for 1 or 3 h in presence or absence of insulin (0, 0.2, 1, 5 μg/ml). GLUT1 and HK II mRNA expression of cultured chicken myotubes at both 1 and 3 h after the incubation were lineally increased with an increase of insulin added to cultures. GLUT3 mRNA expression showed significantly increase at 3 h after incubation with insulin. No significant increase by insulin was observed in expression of HK1 mRNA. Although GLUT1 is not regarded as an insulin-responsive GLUT in mammals, our in vivo and in vitro studies show that insulin stimulates GLUT1 and HK II mRNA expressions in chicken muscles, suggesting that glucose uptake and glucose phosphorylation is regulated in species-specific manner in chicken skeletal muscles.
Abstract The translation factor eIF5A is the only protein known to contain the amino acid hypusine, which is formed posttranslationally. Hypusinated eIF5A is necessary for cellular proliferation and responses to extracellular stressors, and has been proposed as a target for pharmacologic therapy. Here, we provide the first comprehensive characterization of a novel polyclonal antibody (IU-88) that specifically recognizes the hypusinated eIF5A. IU-88 will be useful for the investigation of eIF5A biology and for the development of assays recognizing hypusinated eIF5A.
The maintenance of intracellular Ca(2+) homeostasis in the pancreatic β-cell is closely regulated by activity of the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) pump. Our data demonstrate a loss of β-cell SERCA2b expression in several models of type 2 diabetes including islets from db/db mice and cadaveric diabetic human islets. Treatment of 832/13 rat INS-1-derived cells with 25 mm glucose and the proinflammatory cytokine IL-1β led to a similar loss of SERCA2b expression, which was prevented by treatment with the peroxisome proliferator-activated receptor (PPAR)-γ agonist, pioglitazone. Pioglitazone was able to also protect against hyperglycemia and cytokine-induced elevations in cytosolic Ca(2+) levels, insulin-secretory defects, and cell death. To determine whether PPAR-γ was a direct transcriptional regulator of the SERCA2 gene, luciferase assays were performed and showed that a -259 bp region is sufficient to confer PPAR-γ transactivation; EMSA and chromatin immunoprecipitation experiments confirmed that PPAR-γ directly binds a PPAR response element in this proximal region. We next sought to characterize the mechanisms by which SERCA2b was down-regulated. INS-1 cells were exposed to high glucose and IL-1β in time course experiments. Within 2 h of exposure, activation of cyclin-dependent kinase 5 (CDK5) was observed and correlated with increased serine-273 phosphorylation of PPAR-γ and loss of SERCA2 protein expression, findings that were prevented by pioglitazone and roscovitine, a pharmacological inhibitor of CDK5. We conclude that pioglitazone modulates SERCA2b expression through direct transcriptional regulation of the gene and indirectly through prevention of CDK5-induced phosphorylation of PPAR-γ.
1. Glucose transporter (GLUT) proteins, one of which is the major insulin-responsive transporter GLUT4, play a crucial role in cellular glucose uptake and glucose homeostasis in mammals. The aim of this study was to identify the extent of mRNA expression of GLUT1, GLUT2, GLUT3 and GLUT8 in chickens intrinsically lacking GLUT4. 2. GLUT1 mRNA was detected in most tissues of 3-week-old broiler chickens, with the highest expression measured in brain and adipose tissue. GLUT2 was expressed only in the liver and kidney. GLUT3 was highly expressed in the brain. GLUT8 was expressed ubiquitously, with expression in kidney and adipose tissue relatively higher than that of other tissues. 3. Expression levels of GLUT isoforms 1, 3 and 8 in skeletal muscle tissue were very low compared to the other tissues tested. 4. [3H]Cytochalasin B binding assays on tissue from 3-week-old chickens showed that the number of cytochalasin B binding sites in skeletal muscle plasma membranes was higher than in liver plasma membranes. These results suggest that GLUT proteins and/or GLUT-like proteins that bind cytochalasin B are expressed in chicken skeletal muscles. 5. It is proposed that GLUT expression and glucose transport in chicken tissues are regulated in a manner different from that in mammals.
Deoxyhypusine synthase (DHS) catalyzes the post-translational formation of the amino acid hypusine. Hypusine is unique to the eukaryotic translational initiation factor 5A (eIF5A), and is required for its functions in mRNA shuttling, translational elongation, and stress granule formation. In recent studies, we showed that DHS promotes cytokine and ER stress signaling in the islet β cell and thereby contributes to its dysfunction in the setting of diabetes mellitus. Here, we review the evidence supporting a role for DHS (and hypusinated eIF5A) in cellular stress responses, and provide new data on the phenotype of DHS knockout mice. We show that homozygous knockout mice are embryonic lethal, but heterozygous knockout mice appear normal with no evidence of growth or metabolic deficiencies. Mouse embryonic fibroblasts from heterozygous knockout mice attenuate acute cytokine signaling, as evidenced by reduced production of inducible nitric oxide synthase, but show no statistically significant defects in proliferation or cell cycle progression. Our data are discussed with respect to the utility of sub-maximal inhibition of DHS in the setting of inflammatory states, such as diabetes mellitus.
Carbohydrate metabolism in chickens is character ized by hyperglycemia and insulin resistance compared to that observed in mammals. We previously reported that although gene of GLUT4 (an insulin-responsive glucose transporter ) is deficient in chickens (Seki et al., 2003), GLUT1 and GLUT8 are expressed, to a very small extent, in skeletal muscles (Kono et al., 2005). Fur thermore, it has been evidenced that glucose transpor t across the plasma membrane of skeletal muscles is stimulated by insulin injection in growing chicks (Tokushima et al., 2005). In the insulin-mediated glucose transport in mammals, peroxisome prolifer ator-activated receptor-gamma (PPAR ) plays a crucial role. The present study was under taken to assess the involvement of PPAR in the glucose toler ance and skeletal muscle glucose transport in chickens by using troglitazone, a PPAR agonist. Broiler chickens aged 1 to 2 week were orally administered with troglitazone (50 mg/kg body weight/day) 2 times a day for 17-22 days. Plasma glucose and NEFA concentr ations were decreased, to a small extent, by troglitazone administration for 22 days. In an oral glucose toler ance test, troglitazone suppressed an increase in plasma glucose concentr ations following glucose loading (2 g glucose/kg BW). A decrease in the plasma glucose concentration in insulin-injected (40 g/kg BW) chickens was par tly intensified by troglitazone administr ation for 22 days. Troglitazone administr ation for 17 days augmented insulin-mediated glucose transport, being determined by 2DG uptake, in skeletal muscles (extensor digitorum longus (EDL), pectoral superficialis and pectoral profundus) of chickens. These results suggest that PPAR is involved in the regulation of carbohydrate metabolism species-specific to chickens and troglitazone improves insulin resistance through modulation of skeletal muscle glucose transport.
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