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.     

Involvement of Ca<Superscript>2+</Superscript>/calmodulin kinase II (CaMK II) in genistein-induced potentiation of leucine/glutamine-stimulated insulin secretion

Genistein has been reported to potentiate glucose-stimulated insulin secretion (GSIS). Inhibitory activity on tyrosine kinase or activation of protein kinase A (PKA) was shown to play a role in the genistein-induced potentiation effect on GSIS. The aim of the present study was to elucidate the mechanism of genistein-induced potentiation of insulin secretion. Genistein augmented insulin secretion in INS-1 cells stimulated by various energy-generating nutrients such as glucose, pyruvate, or leucine/glutamine (Leu/Gln), but not the secretion stimulated by depolarizing agents such as KCl and tolbutamide, or Ca²⁺ channel opener Bay K8644. Genistein at a concentration of 50 μM showed a maximum potentiation effect on Leu/Gln-stimulated insulin secretion, but this was not sufficient to inhibit the activity of tyrosine kinase. Inhibitor studies as well as immunoblotting analysis demonstrated that activation of PKA was little involved in genistein-induced potentiation of Leu/Gln-stimulated insulin secretion. On the other hand, all the inhibitors of Ca²⁺/calmodulin kinase II tested, significantly diminished genistein-induced potentiation. Genistein also elevated the levels of [Ca²⁺]_i and phospho-CaMK II. Furthermore, genistein augmented Leu/Gln-stimulated insulin secretion in CaMK II-overexpressing INS-1 cells. These data suggest that the activation of CaMK II played a role in genistein-induced potentiation of insulin secretion.     

Involvement of the TLR4 (Toll-like receptor4) signaling pathway in palmitate-induced INS-1 beta cell death

Fatty acid-induced cytotoxicity is believed to recapitulate lipotoxicity seen in obese type-2 diabetes, and, thus, contribute to beta cell loss in the disease. These studies were initiated to determine whether the Toll-like receptor (TLR) signaling pathway was involved in palmitate-induced beta cell death. Treatment of INS-1 beta cells with palmitate enhanced interaction between TLR and myeloid differentiation factor88 (MyD88). Concomitant with TLR/MyD88 interaction, the level of phospho-C-Jun N-terminal kinase (phospho-JNK) showed an increase; however, the level of inhibitory factor kappa B alpha (IκBα) showed a decrease. Gene knockdown of TLR4 prevented palmitate-induced INS-1 cell death, while knockdown of TLR2 did not. In addition, gene knockdown of TLR4 prevented palmitate-induced increase of phospho-JNK and decrease of IκBα. JNK inhibitor SP60125 significantly protected against palmitate-induced INS-1 cell death, while IκB kinase (IKK) inhibitor acetylsalicylate did not. These data suggest involvement of JNK activation through the TLR4 signaling pathway in palmitate-induced INS-1 beta cell death.     

Involvement of mitophagy in oncogenic K-Ras-induced transformation: overcoming a cellular energy deficit from glucose deficiency

Although mitochondrial impairment has often been implicated in carcinogenesis, the mechanisms of its development in cancer remain unknown. We report here that autophagy triggered by oncogenic K-Ras mediates functional loss of mitochondria during cell transformation to overcome an energy deficit resulting from glucose deficiency. When Rat2 cells were infected with a retrovirus harboring constitutively active K-Ras (V12) , mitochondrial respiration significantly declined in parallel with the acquisition of transformation characteristics. Decreased respiration was not related to mitochondrial biogenesis but was inversely associated with the increased formation of acidic vesicles enclosing mitochondria, during which autophagy-related proteins such as Beclin 1, Atg5, LC3-II and vacuolar ATPases were induced. Interestingly, blocking autophagy with conventional inhibitors (bafilomycin A, 3-methyladenin) and siRNA-mediated knockdown of autophagy-related genes recovered respiratory protein expression and respiratory activity; JNK was involved in these phenomena as an upstream regulator. The cells transformed by K-Ras (V12) maintained cellular ATP level mainly through glycolytic ATP production without induction of GLUT1, the low Km glucose transporter. Finally, K-Ras (V12) -triggered LC3-II formation was modulated by extracellular glucose levels, and LC3-II formation increased only in hepatocellular carcinoma tissues exhibiting low glucose uptake and increased K-Ras expression. Taken together, our observations suggest that mitochondrial functional loss may be mediated by oncogenic K-Ras-induced mitophagy during early tumorigenesis even in the absence of hypoxia, and that this mitophagic process may be an important strategy to overcome the cellular energy deficit triggered by insufficient glucose.     

Supplement of TCA cycle intermediates protects against high glucose/palmitate-induced INS-1 beta cell death

The aim of this study is to investigate the effect of mitochondrial metabolism on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death. Long-term treatment of INS-1 cells with HG/PA impaired energy-producing metabolism accompanying with depletion of TCA cycle intermediates. Whereas an inhibitor of carnitine palmitoyl transferase 1 augmented HG/PA-induced INS-1 cell death, stimulators of fatty acid oxidation protected the cells against the HG/PA-induced death. Furthermore, whereas mitochondrial pyruvate carboxylase inhibitor phenylacetic acid augmented HG/PA-induced INS-1 cell death, supplementation of TCA cycle metabolites including leucine/glutamine, methyl succinate/α-ketoisocaproic acid, dimethyl malate, and valeric acid or treatment with a glutamate dehydrogenase activator, aminobicyclo-heptane-2-carboxylic acid (BCH), significantly protected the cells against the HG/PA-induced death. In particular, the mitochondrial tricarboxylate carrier inhibitor, benzene tricarboxylate (BTA), also showed a strong protective effect on the HG/PA-induced INS-1 cell death. Knockdown of glutamate dehydrogenase or tricarboxylate carrier augmented or reduced the HG/PA-induced INS-1 cell death, respectively. Both BCH and BTA restored HG/PA-induced reduction of energy metabolism as well as depletion of TCA intermediates. These data suggest that depletion of the TCA cycle intermediate pool and impaired energy-producing metabolism may play a role in HG/PA-induced cytotoxicity to beta cells and thus, HG/PA-induced beta cell glucolipotoxicity can be protected by nutritional or pharmacological maneuver enhancing anaplerosis or reducing cataplerosis.     

Detection of differential proteomes associated with the development of type 2 diabetes in the Zucker rat model using the iTRAQ technique

Type 2 diabetes (T2D) is closely associated with obesity, and it arises when pancreatic β cells fail to achieve β cell compensation. However, the mechanism linking obesity, insulin resistance, and β cell failure in T2D is not fully understood. To explore this association, we carried out a differential proteomics study using the disease models of Zucker Fatty (ZF) and Zucker Diabetic Fatty (ZDF) rats as the rat models for obese/prediabetes and obese/diabetes, respectively. Differentially expressed islet proteins were identified among ZDF, ZF, and Zucker Lean (ZL, control rat) rats using three iTRAQ experiments, where three biological replicates and two technical replicates were examined to assess both the technical and biological reproducibilities. A total of 54 and 58 proteins were differentially expressed in ZDF versus ZL rats and in ZF versus ZL rats, respectively. Notably, the novel proteins involved in impaired insulin secretion (Scg2, Anxa2, and Rab10), mitochondrial dysfunction (Atp5b and Atp5l), extracellular matrix proteins (Lgal-1, Vim, and Fbn1), and microvascular ischemia (CPA1, CPA2, CPB, Cela2a, and Cela3b) were observed for the first time. With these novel proteins, our proteomics study could provide valuable clues for better understanding the underlying mechanisms associated with the dynamic transition of obesity to T2D.