Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

We previously demonstrated that proto-oncogene Fyn decreased energy expenditure and increased metabolic phenotypes. Also Fyn decreased autophagy-mediated muscle mass by directly inhibiting LKB1 and stimulating STAT3 activities, respectively. AMPK, a downstream target of LKB1, was recently identified as a key molecule controlling autophagy. Here we identified that Fyn phosphorylates the α subunit of AMPK on Y436 and inhibits AMPK enzymatic activity without altering the assembly state of the AMPK heterotrimeric complex. As pro-inflammatory mediators are reported modulators of the autophagy processes, treatment with the pro-inflammatory cytokine TNFα resulted in 1) increased Fyn activity 2) stimulated Fyn-dependent AMPKα tyrosine phosphorylation and 3) decreased AICAR-dependent AMPK activation. Importantly, TNFα induced inhibition of autophagy was not observed when AMPKα was mutated on Y436. 4) These data demonstrate that Fyn plays an important role in relaying the effects of TNFα on autophagy and apoptosis via phosphorylation and inhibition of AMPK

Regulation of phosphorylase kinase by low concentrations of Ca ions upon muscle contraction: the connection between metabolism and muscle contraction and the connection between muscle physiology and Ca-dependent signal transduction

It had long been one of the crucial questions in muscle physiology how glycogenolysis is regulated in connection with muscle contraction, when we found the answer to this question in the last half of the 1960s. By that time, the two principal currents of muscle physiology, namely, the metabolic flow starting from glycogen and the mechanisms of muscle contraction, had already been clarified at the molecular level thanks to our senior researchers. Thus, the final question we had to answer was how to connect these two currents. We found that low concentrations of Ca ions (10−7–10−4 M) released from the sarcoplasmic reticulum for the regulation of muscle contraction simultaneously reversibly activate phosphorylase kinase, the enzyme regulating glycogenolysis. Moreover, we found that adenosine 3′,5′-monophosphate (cyclic AMP), which is already known to activate muscle phosphorylase kinase, is not effective in the absence of such concentrations of Ca ions. Thus, cyclic AMP is not effective by itself alone and only modifies the activation process in the presence of Ca ions (at that time, cyclic AMP-dependent protein kinase had not yet been identified). After a while, it turned out that our works have not only provided the solution to the above problem on muscle physiology, but have also been considered as the first report of Ca-dependent protein phosphorylation, which is one of the central problems in current cell biology. Phosphorylase kinase is the first protein kinase to phosphorylate a protein resulting in the change in the function of the phosphorylated protein, as shown by Krebs and Fischer. Our works further showed that this protein kinase is regulated in a Ca-dependent manner. Accordingly, our works introduced the concept of low concentrations of Ca ions, which were first identified as the regulatory substance of muscle contraction, to the vast field of Ca biology including signal transduction

Analysis of the spatial, temporal and tissue-specific transcription of γ-sarcoglycan gene using a transgenic mouse

AbstractTo evaluate the promoter function of the 5′-flanking sequence of mouse γ-sarcoglycan (γ-SG) gene in vivo, we generated transgenic mice harboring this sequence fused with enhanced green fluorescent protein reporter gene. The reporter expression was restricted in striated muscles and particularly strong in all myofibers in skeletal muscles. Using these mice, we examine the spatial and temporal transcriptional patterns of the γ-SG gene during mouse skeletal muscle development. The expression of basic helix loop helix transcriptional factors preceded that of the reporter. Differences between the expression of reporter and endogenous γ-SG genes in non-muscle tissues suggested the existence of additional promoter elements in the endogenous gene, and the analysis of endogenous mRNAs demonstrated the existence of a novel upstream exon and promoter active in non-muscle tissues

Dystrophin-associated protein A0 is a homologue of the Torpedo 87K protein

AbstractWe raised a monoclonal antibody, MA0, which reacts with A0, a 94-kDa rabbit skeletal muscle dystrophin-associated protein (DAP) bound to the syntrophin-binding domain of dystrophin. The antibody also reacted with the 62-kDa DAP which was moved to the locus close to β-syntrophins by 2-dimensional PAGE, but the DAP did not coincide with any known β-syntrophins. We have cloned a fragment of cDNA which codes the protein reacting with MA0 from a neonatal rabbit heart cDNA library. Based on the coincidence of cDNA sequences and the similarity in molecular mass, we concluded that the proteins reacting with MA0 are rabbit homologues of the Torpedo 87K protein

Continuous or Transient High Level of Glucose Exposure Differentially Increases Coronary Artery Endothelial Cell Proliferation and Human Colon Cancer Cell Proliferation

We studied effect of high glucose levels on coronary artery endothelial cell proliferation and human colon cancer cell proliferation. To examine the long-term effect of glucose exposure on cell growth, cells were cultured for 14 days in the absence or presence of 183 mg/dL D-glucose addition in the culture medium. Short effect of elevated glucose levels was examined by addition of 183 mg/dL D-glucose addition in the culture medium for just one hour per day followed by changing the culture to standard medium (5.5 mM D-glucose) during the next 23-hours period. Cell proliferation was estimated by 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT) assay and phosphor-Erk western blot analysis. We found that coronary artery endothelial cell proliferation was significantly increased in the culture medium with the acute one-hour addition of 183 mg/dL D-glucose compared to the absence or chronic presence of 183 mg/dL D-glucose addition in the culture medium. In contrast, colon cancer cell proliferation was significantly increased in the continuous presence of 183 mg/dL D-glucose addition in the culture medium compared to the acute one-hour addition of glucose. The extent of Erk2 phosphorylation paralleled with the relative changes in cellular proliferation in both cell types. Taken together, these results suggested that continuous or transient high level of glucose exposure differentially effects coronary artery endothelial and human colon cancer cell proliferation

Effect of carbohydrate counting using bolus calculators on glycemic control in type 1 diabetes patients during continuous subcutaneous insulin infusion

This study examined the long-term efficacy of insulin pump therapy for type 1 diabetes patients when performed using carbohydrate counting with bolus calculators for one year. Twenty-two type 1 diabetes patients who had just started continuous subcutaneous insulin infusion were examined and divided into 2 groups: one that was educated about carbohydrate counting using bolus calculators (n=14) and another that did not use bolus calculators (n=8). After one year, the hemoglobin A1c levels of the patient group that used bolus calculators decreased persistently and significantly (P=0.0297), while those of the other group did not. The body weight, total daily dose of insulin, and bolus percentage of both groups did not change. Carbohydrate counting using bolus calculators is necessary to achieve optimal and persistent glycemic control in patients undergoing continuous subcutaneous insulin infusio