Glutamate dehydrogenase activator BCH stimulating reductive amination prevents high fat/high fructose diet-induced steatohepatitis and hyperglycemia in C57BL/6J mice.

Individuals with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) induced by high calorie western diet are characterized by enhanced lipogenesis and gluconeogenesis in the liver. Stimulation of reductive amination may shift tricarboxylic acid cycle metabolism for lipogenesis and gluconeogenesis toward glutamate synthesis with increase of NAD+/NADH ratio and thus, ameliorate high calorie diet-induced fatty liver and hyperglycemia. Stimulation of reductive amination through glutamate dehydrogenase (GDH) activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) reduced both de novo lipogenesis and gluconeogenesis but increased the activities of sirtuins and AMP-activated kinase in primary hepatocytes. Long-term BCH treatment improved most metabolic alterations induced by high fat/high fructose (HF/HFr) diet in C57BL/6J mice. BCH prevented HF/HFr-induced fat accumulation and activation of stress/inflammation signals such as phospho-JNK, phospho-PERK, phospho-p38, and phospho-NFκB in liver tissues. Furthermore, BCH treatment reduced the expression levels of inflammatory cytokines such as TNF-α and IL-1ß in HF/HFr-fed mouse liver. BCH also reduced liver collagen and plasma levels of alanine transaminase and aspartate transaminase. On the other hand, BCH significantly improved fasting hyperglycemia and glucose tolerance in HF/HFr-fed mice. In conclusion, stimulation of reductive amination through GDH activation can be used as a strategy to prevent high calorie western diet-induced NAFLD and T2D.

TM-25659-Induced Activation of FGF21 Level Decreases Insulin Resistance and Inflammation in Skeletal Muscle via GCN2 Pathways.

The TAZ activator 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2'-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659) inhibits adipocyte differentiation by interacting with peroxisome proliferator-activated receptor gamma. TM-25659 was previously shown to decrease weight gain in a high fat (HF) diet-induced obesity (DIO) mouse model. However, the fundamental mechanisms underlying the effects of TM-25659 remain unknown. Therefore, we investigated the effects of TM-25659 on skeletal muscle functions in C2 myotubes and C57BL/6J mice. We studied the molecular mechanisms underlying the contribution of TM-25659 to palmitate (PA)-induced insulin resistance in C2 myotubes. TM-25659 improved PA-induced insulin resistance and inflammation in C2 myotubes. In addition, TM-25659 increased FGF21 mRNA expression, protein levels, and FGF21 secretion in C2 myotubes via activation of GCN2 pathways (GCN2-phosphoeIF2α-ATF4 and FGF21). This beneficial effect of TM-25659 was diminished by FGF21 siRNA. C57BL/6J mice were fed a HF diet for 30 weeks. The HF-diet group was randomly divided into two groups for the next 14 days: the HF-diet and HF-diet + TM-25659 groups. The HF diet + TM-25659-treated mice showed improvements in their fasting blood glucose levels, insulin sensitivity, insulin-stimulated Akt phosphorylation, and inflammation, but neither body weight nor food intake was affected. The HF diet + TM-25659-treated mice also exhibited increased expression of both FGF21 mRNA and protein. These data indicate that TM-25659 may be beneficial for treating insulin resistance by inducing FGF21 in models of PA-induced insulin resistance and HF diet-induced insulin resistance.

A compound (DW1182v) protecting high glucose/palmitate-induced glucolipotoxicity to INS-1 beta cells preserves islet integrity and improves hyperglycemia in obese db/db mouse.

Loss of beta cells is a pathogenic cause for the development of type 2 diabetes. High glucose/free fatty acid (HG/FFA)-induced glucolipotoxicity was thought to play a role in the beta cell loss. Thus, application of small molecules capable of preventing HG/FFA-induced glucolipotoxicty to beta cells could be an avenue for a therapeutic intervention for the development of type 2 diabetes. We screened a representative library supplied from Korean Chemical Bank for prevention of high glucose/palmitate (HG/PA)-induced viability reduction of INS-1 beta cells and were able to identify a new small molecule (DW1182v) with a function to protect HG/PA-induced glucolipotoxicity. The protective effect was specific to HG/PA-induced beta cell death since DW1182v did not protect streptozotocin- or cytokine-induced INS-1 cell death. The protective effect by DW1182v was likely due to the reduction of death-promoting endoplasmic reticulum (ER) stress responses such as phospho-C-Jun N-terminal kinase (JNK) and C/EBP homologous protein (CHOP). Treatment of obese diabetic db/db mice with DW1182v preserved islet integrity and thus increased insulin secretion and lowered blood glucose after glucose infusion. These results suggest that a small molecule protecting HG/PA-induced glucolipotoxicity to beta cells can be a new therapeutic candidate to prevent the development of type 2 diabetes.

Serum fibroblast growth factor 21 was elevated in subjects with type 2 diabetes mellitus and was associated with the presence of carotid artery plaques.

AIMS: Fibroblast growth factor 21 (FGF21) is an important regulator of glucose/lipid metabolism. Although there are studies examining the relationship between serum FGF21 levels and glucose homeostasis, the role of FGF21 remains unclear. The objective of this study was to examine whether serum FGF21 levels are associated with metabolic parameters in subjects with varying degrees of obesity and glucose tolerance and with complications in subjects with type2 diabetes mellitus (T2DM). METHODS: The study consisted of 213 subjects who were lean and had normal glucose tolerance (lean NGT), were overweight with NGT, had impaired glucose tolerance (IGT) or had T2DM. Serum FGF21 levels and their associations with the parameters of adiposity, glucose tolerance and the presence of diabetic complications were examined. RESULTS: The serum FGF21 levels in T2DM were higher than in lean NGT. Serum FGF21 levels showed a positive correlation with the urine albumin-to-creatinine ratio (ACR) in all subjects except for the T2DM subjects, who showed a correlation after adjustment of age, gender and body mass index. Moreover, the subjects with carotid artery plaque showed higher serum FGF21 levels than those without complications. CONCLUSION: Serum FGF21 levels were associated with the urine ACR and diabetic complications including carotid artery plaque.

ß-Cell-protective effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid as a glutamate dehydrogenase activator in db/db mice.

2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) is an activator of glutamate dehydrogenase (GDH), which is a mitochondrial enzyme with an important role in insulin secretion. We investigated the effect of BCH on the high-glucose (HG)-induced reduction in glucose-stimulated insulin secretion (GSIS), the HG/palmitate (PA)-induced reduction in insulin gene expression, and HG/PA-induced ß-cell death. We also studied whether long-term treatment with BCH lowers blood glucose and improves ß-cell integrity in db/db mice. We evaluated GSIS, insulin gene expression, and DNA fragmentation in INS-1 cells exposed to HG or HG/PA in the presence or absence of BCH. An in vivo study was performed in which 7-week-old diabetic db/db mice were treated with BCH (0.7  g/kg, n = 10) and placebo (n = 10) every other day for 6 weeks. After treatment, an intraperitoneal glucose tolerance test and immunohistological examinations were performed. Treatment with BCH blocked HG-induced GSIS inhibition and the HG/PA-induced reduction in insulin gene expression in INS-1 cells. In addition, BCH significantly reduced HG/PA-induced INS-1 cell death and phospho-JNK level. BCH treatment improved glucose tolerance and insulin secretion in db/db mice. BCH treatment also increased the ratio of insulin-positive ß-cells to total islet area (P < 0.05) and reduced the percentage of ß-cells expressing cleaved caspase 3 (P < 0.05). In conclusion, the GDH activator BCH improved glycemic control in db/db mice. This anti-diabetic effect may be associated with improved insulin secretion, preserved islet architecture, and reduced ß-cell apoptosis.

Capsaicin attenuates palmitate-induced expression of macrophage inflammatory protein 1 and interleukin 8 by increasing palmitate oxidation and reducing c-Jun activation in THP-1 (human acute monocytic leukemia cell) cells.

Capsaicin, a spicy component of hot peppers, has been shown to improve inflammatory disease and obesity. In this study, we tested the hypothesis that the anti-inflammatory activity of capsaicin can be used to improve free fatty acid (FFA)-induced inflammation by reducing gene expression of macrophage inflammatory protein 1 (MIP-1) and interleukin 8 (IL-8) in THP-1 (human acute monocytic leukemia cell) macrophages. To investigate whether capsaicin ameliorates palmitate-induced MIP-1 and IL-8 gene expressions, we treated THP-1 cells with palmitate in the presence or absence of capsaicin and measured MIP-1 and IL-8 by real-time polymerase chain reaction. To elucidate the mechanism by which capsaicin effects on palmitate-induced MIP-1 and IL-8 gene expressions, we performed immunoblotting with stress kinase-related antibodies and measured palmitate oxidation and palmitate oxidation-related gene expression. Palmitate and stearate but not the unsaturated FFA oleate significantly increased MIP-1 and IL-8 expressions in THP-1 macrophages. Treatment with capsaicin or FFA oxidation stimulators inhibited palmitate-induced MIP-1 and IL-8 expressions in THP-1 macrophages. Capsaicin increased the gene expression of carnitine palmitoyltransferase 1 and the ß-oxidation of palmitate. Furthermore, capsaicin significantly reduced palmitate-stimulated activation of c-Jun N-terminal kinase, c-Jun, and p38. Our data suggest that the attenuation of palmitate-induced MIP-1 and IL-8 gene expressions by capsaicin is associated with reduced activation of c-Jun N-terminal kinase, c-Jun, and p38 and preserved ß-oxidation activity.

Effect of sitagliptin plus metformin on ß-cell function, islet integrity and islet gene expression in Zucker diabetic fatty rats.

AIM: The combination of metformin and a dipeptidyl peptidase 4 (DPP-4) inhibitor has been shown to be an effective, safe, and well-tolerated treatment for type 2 diabetes. We evaluated ß-cell function and morphological changes in islets in Zucker diabetic fatty (ZDF) rats following combined therapy with sitagliptin and metformin and investigated the expression of potentially relevant genes using cDNA microarrays. METHODS: Nine-week-old ZDF rats were randomly divided into four treatment groups: no treatment (control); sitagliptin; metformin, and sitagliptin plus metformin. After 5 weeks of treatment, an oral glucose tolerance test was performed and plasma levels of active GLP-1 and islet morphology and gene expression were assessed. RESULTS: Combination therapy reduced fasting glucose and postprandial plasma glucose levels and increased active GLP-1 levels, compared with monotherapy. Combination therapy also increased insulin secretion, the proportion of small islets, and the intensity of insulin staining. Furthermore, it increased the expression of genes involved in cell survival and growth and downregulated apoptosis-associated genes, relative to monotherapy. CONCLUSIONS: Combination treatment with sitagliptin and metformin preserved ß-cell function and ß-cell integrity in ZDF rats. This may be associated with the transcriptional activation of anti-apoptotic and pro-survival genes, as well as the suppression of pro-apoptotic genes.

A rice dehydration-inducible SNF1-related protein kinase 2 phosphorylates an abscisic acid responsive element-binding factor and associates with ABA signaling.

By a differential cDNA screening technique, we have isolated a dehydration-inducible gene (designated OSRK1) that encodes a 41.8 kD protein kinase of SnRK2 family from Oryza sativa. The OSRK1 transcript level was undetectable in vegetative tissues, but significantly increased by hyperosmotic stress and Abscisic acid (ABA). To determine its biochemical properties, we expressed and isolated OSRK1 and its mutants as glutathione S-transferase fusion proteins in Escherichia coli. In vitro kinase assay showed that OSRK1 can phosphorylate itself and generic substrates as well. Interestingly, OSRK1 showed strong substrate preference for rice bZIP transcription factors and uncommon cofactor requirement for Mn(2+) over Mg(2+). By deletion of C-terminus 73 amino acids or mutations of Ser-158 and Thr-159 to aspartic acids (Asp) in the activation loop, the activity of OSRK1 was dramatically decreased. OSRK1 can transphosphorylate the inactive deletion protein. A rice family of abscisic acid-responsive element (ABRE) binding factor, OREB1 was phosphorylated in vitro by OSRK1 at multiple sites of different functional domains. MALDI-TOF analysis identified a phosphorylation site at Ser44 of OREB1 and mutation of the residue greatly decreased the substrate specificity for OSRK1. The recognition motif for OSRK1, RQSS is highly similar to the consensus substrate sequence of AMPK/SNF1 kinase family. We further showed that OSRK1 interacts with OREB1 in a yeast two-hybrid system and co-localized to nuclei by transient expression analysis of GFP-fused protein in onion epidermis. Finally, ectopic expression of OSRK1 in transgenic tobacco resulted in a reduced sensitivity to ABA in seed germination and root elongation. These findings suggest that OSRK1 is associated with ABA signaling, possibly through the phosphorylation of ABF family in vivo. The interaction between SnRK2 family kinases and ABF transcription factors may constitute an important part of cross-talk mechanism in the stress signaling networks in plants.