Regulation of gut-derived resistin-like molecule beta expression by nutrients.

Resistin was initially identified as a protein, secreted by adipocytes, which inhibits insulin action and adipose differentiation. The three proteins homologous to resistin were identified and given the names resistin-like molecules (RELM) alpha, beta and gamma. Resistin and RELMalpha are abundantly expressed in adipose, but RELMbeta and RELMgamma are secreted mainly from the gut. Since nutrient composition greatly affects insulin sensitivity, we investigated the regulatory effects of various nutritional factors in food on the expressions of resistin family proteins. First, mice were given diets with different nutritional compositions (high-carbohydrate, high-protein and high-fat) for 2 weeks. RELMbeta mRNA expression in the intestines was markedly suppressed by the high-protein and high-carbohydrate diets, while slightly but not significantly upregulated by the high-fat diet. In the epididymal fat, resistin expression was unchanged, while RELMalpha expression was markedly decreased by the high-carbohydrate diet. Taking into consideration that humans have neither RELMalpha nor RELMgamma, our subsequent studies focused on RELMbeta expression. We used the human colon cancer cell line LS174T. Treatments with insulin and TNFalpha as well as stearic acid, a saturated free fatty acid, upregulated RELMbeta expression, while d-glucose downregulated RELMbeta. These results suggest RELMbeta expression to be regulated directly by nutrients such as glucose and saturated free fatty acids including stearic acid, as well as by hormones including insulin and TNFalpha. These regulations may play an important role in the nutrient-associated induction of insulin resistance.

Tau phosphorylation in the mouse brain during aversive conditioning.

Stress response is intimately involved in memory formation. Stress has been shown to cause reversible Alzheimer-like tau phosphorylation in the brain of experimental animals, but it is not known whether tau phoshorylation takes place during memory acquisition. As an initial investigation we chose contextual fear conditioning paradigm involving electric shocks, and studied tau phosphorylation in the hippocampus and a neighboring limbic region of the mouse brain. Quantitative immunoblot analyses of tissue extracts rapidly prepared from animals undergoing the conditioning showed statistically significant increases in the phosphorylation level at Thr231/Ser235 of tau in both tissues. The reaction reached statistical significance after 10 but not 3 shocks of 0.8mA. Ten shocks of 0.2mA were ineffective. Concurrent increases in phosphorylation of protein kinase TPKI/GSK3beta at Ser9 and of CaMKIIalpha at Thr286 were observed. These results suggest involvement of tau and TPKI/GSK3beta phosphorylation in an early phase of memory formation in the hippocampus and amygdala, raising a possibility that a dysregulation of tau phosphorylation may underlie memory impairment in incipient Alzheimer's disease.

Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity.

AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the beta1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the beta1-subunit of AMPK revealed amino acids 68-123 of the beta1-subunit to be sufficient for GDE binding. W100G and K128Q, both beta1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68-123 of the beta1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.