Eijiro Yamada

Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS97xS99). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip–Syntaxin4 interaction.

Fyn-Dependent Regulation of Energy Expenditure and Body Weight Is Mediated by Tyrosine Phosphorylation of LKB1

Fyn null mice display reduced adiposity associated with increased fatty acid oxidation, energy expenditure, and activation of the AMP-dependent protein kinase (AMPK) in skeletal muscle and adipose tissue. The acute pharmacological inhibition of Fyn kinase activity with SU6656 in wild-type mice reproduces these metabolic effects and induced a specific reduction in fat mass with no change in lean mass. LKB1, the main upstream AMPK kinase (AMPKK) in peripheral tissues, was redistributed from the nucleus into the cytoplasm of cells treated with SU6656 and in cells expressing a kinase-deficient, but not a constitutively kinase-active, Fyn mutant. Moreover, Fyn kinase directly phosphorylated LKB1 on tyrosine 261 and 365 residues, and mutations of these sites resulted in LKB1 export into the cytoplasm and increased AMPK phosphorylation. These data demonstrate a crosstalk between Fyn tyrosine kinase and the AMPK energy-sensing pathway, through Fyn-dependent regulation of the AMPK upstream activator LKB1.

Mapping Autophagy on to Your Metabolic Radar

Autophagy, which literally translates into “eating ones own self,” is an evolutionarily conserved cellular recycling program that maintains “in-house” quality control by turning over cytoplasmic components within lysosomes (1). Although the discovery of lysosomes dates back to the 1950s through the electron microscopic work of Christian De Duve, recent years have seen a growing interest in autophagy research, and reports now link compromised autophagy to a wide array of common human pathologies, for instance, neurodegenerative disorders, metabolic alterations, microbial pathogenesis, and cancers, to mention just a few (2). These studies support the idea that the “housekeeping” role of autophagy, in fact, translates to key physiological functions. For instance, recycling of oxidized proteins and aged organelles through autophagic degradation protects against cellular toxicity and death (3). Recent findings now highlight roles for autophagy in mobilization of diverse cellular energy stores (4) and in adipocyte differentiation (5,6), thus presenting autophagy as an emerging player in the metabolic arena. As novel functions for autophagy continue to unfold, it becomes critical to be able to precisely monitor autophagy in diverse physiological systems. This article comments on the fundamental developments on roles for autophagy in metabolic regulation and discusses currently available methods to monitor autophagy. Mammalian cells exhibit three distinct forms of autophagy to deliver cytosolic cargo to the lysosomes, namely, macroautophagy, chaperone-mediated autophagy, and microautophagy (1). Traditionally, autophagy was considered a one-lane system for protein turnover and a mechanism for replenishing the intracellular amino acid pool during starvation. However, it is now becoming increasingly clear that autophagy, in particular macroautophagy, exhibits significant versatility in its ability to degrade mitochondria (mitophagy), endoplasmic reticulum (reticulophagy), ribosomes (ribophagy), and peroxisomes (pexophagy) (1). The second form of autophagy, chaperone-mediated autophagy, displays functional selectivity for the lysosomal targeting of specific soluble cytosolic proteins with the KFERQ signature …

Mouse Skeletal Muscle Fiber-Type-Specific Macroautophagy and Muscle Wasting Are Regulated by a Fyn/STAT3/Vps34 Signaling Pathway

Skeletal muscle atrophy induced by aging (sarcopenia), inactivity, and prolonged fasting states (starvation) is predominantly restricted to glycolytic type II muscle fibers and typical spares oxidative type I fibers. However, the mechanisms accounting for muscle fiber-type specificity of atrophy have remained enigmatic. In the current study, although the Fyn tyrosine kinase activated the mTORC1 signaling complex, it also induced marked atrophy of glycolytic fibers with relatively less effect on oxidative muscle fibers. This was due to inhibition of macroautophagy via an mTORC1-independent but STAT3-dependent reduction in Vps34 protein levels and decreased Vps34/p150/Beclin1/Atg14 complex 1. Physiologically, in the fed state endogenous Fyn kinase activity was increased in glycolytic but not oxidative skeletal muscle. In parallel, Y705-STAT3 phosphorylation increased with decreased Vps34 protein levels. Moreover, fed/starved regulation of Y705-STAT3 phosphorylation and Vps34 protein levels was prevented in skeletal muscle of Fyn null mice. These data demonstrate a Fyn/STAT3/Vps34 pathway that is responsible for fiber-type-specific regulation of macroautophagy and skeletal muscle atrophy.

CDK5-dependent Phosphorylation of the Rho Family GTPase TC10α Regulates Insulin-stimulated GLUT4 Translocation

Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10α on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10α was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10α) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10α but not T197A-TC10α and in parallel, T197D-TC10α but not T197A-TC10α depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10α in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10α through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10α cycling between lipid raft and non-lipid raft compartments.

The role of CHOP messenger RNA expression in the link between oxidative stress and apoptosis

Low expression of antioxidant enzymes makes pancreatic beta-cells susceptible to cell damage by oxidative stress. Pancreatic beta-cell loss caused by endoplasmic reticulum stress is associated with the onset of diabetes mellitus. The present studies were undertaken to investigate a possible involvement of proapoptotic gene CHOP in pancreatic beta-cells damage by oxidative stress. The induction of CHOP messenger RNA and apoptosis were investigated in betaHC-9 cells after the oxidative stress by hydrogen peroxide and ribose. Latter was examined after the suppression of CHOP by small interfering RNA. For in vivo study, the pancreatic beta-cells were examined in CHOP-knockout (KO) mice after multiple low-dose streptozotocin (MLDS) administration. In betaHC-9 cells, both hydrogen peroxide and ribose obviously increased apoptotic cells, accompanied with enhanced CHOP messenger RNA expression. However, the number of apoptotic cells by those stimulations was significantly reduced by the addition of small interfering RNA against CHOP. In vivo study also showed that CHOP-KO mice were less susceptible to diabetes after MLDS administration. Although the oxidative stress marker level was similar to that of MLDS-treated wild type, the pancreatic beta-cell area was maintained in CHOP-KO mice. The present studies showed that CHOP should be important in pancreatic beta-cell injury by oxidative stress and indicate that CHOP may play a role in the development of pancreatic beta-cell damage on the onset of diabetes mellitus.

Fyn deficiency promotes a preferential increase in subcutaneous adipose tissue mass and decreased visceral adipose tissue inflammation.

Previous studies have demonstrated that Fyn knockout (FynKO) mice on a standard chow diet display increased glucose clearance and whole-body insulin sensitivity associated with decreased adiposity resulting from increased fatty acid use and energy expenditure. Surprisingly, however, despite a similar extent of adipose tissue (AT) mass accumulation on a high-fat diet, the FynKO mice remained fully glucose tolerant and insulin sensitive. Physiologic analyses demonstrated that the FynKO mice had a combination of skewed AT expansion into the subcutaneous compartment rather than to the visceral depot, reduced AT inflammation associated with reduced T-cell and macrophage infiltration, and increased proportion of anti-inflammatory M2 macrophages. These data demonstrate that Fyn is an important regulator of whole-body integrative metabolism that coordinates AT expansion, inflammation, and insulin sensitivity in states of nutrient excess. These data further suggest that inhibition of Fyn function may provide a novel target to prevent AT inflammation, insulin resistance, and the dyslipidemia components of the metabolic syndrome.

Fyn kinase function in lipid utilization: a new upstream regulator of AMPK activity?

The balance of cellular energy levels in response to changes of nutrient availability, stress stimuli or exercise is a critical step in maintaining tissue and whole body homeostasis. Disruption of this balance is associated with various pathologies, including the metabolic syndrome. Recently, accumulating evidence has demonstrated that the AMP-activated protein kinase (AMPK) plays a central role in sensing changes in energy levels. The regulation of AMPK activity is currently the subject of significant investigation since this enzyme is a potential therapeutic target in both metabolic disorders and tumorigenesis. In this review, we present novel evidence of crosstalk between Fyn, one member of the Src kinase family, and AMPK.

Inhibition of Akt activity and calcium channel function coordinately drive cell-cell fusion in the BeWO choriocarcinoma placental cell line.

To establish a simple and quantitative live cell fusion assay for placental syncytialization, we generated stable GFP and dsRed expressing fusogenic BeWo cell lines. Fluorescent Activated Cell Sorting was shown to provide a quantitative determination of forskolin (cAMP-mediated) fusion in a time and concentration dependent manner consistent with the increased secretion of beta human chorionic gonadotrophin (β-HCG) and appearance of multi-nucleated cells. Analyses of the fusion process demonstrated that in addition to increased cAMP levels, simultaneous reduction of intracellular calcium and inhibition of Type 1 phosphatidylinositol 3 kinase (PI3K)/Akt signaling also resulted in cell fusion. Although individual blockade of calcium channel function or PI3K/Akt signaling was without effect, the combination with forskolin resulted in a potentiation of cell fusion. These data demonstrate syncytialization is a complex process that depends upon the regulation of distinct signaling inputs that function in concert with each other.

GNAS mutations in adrenal aldosterone-producing adenomas [Rapid Communication]

Mutations in GNAS, which encodes Gsα, have been documented in detail, particularly in human pituitary GH-secreting adenomas. Mutations have also recently been reported in adrenal cortisol-producing adenomas (CPAs), in addition to those in the PRKACA gene. However, mutations have not yet been examined in aldosterone-producing adenomas (APAs). Therefore, we herein investigated mutations in the GNAS gene in APAs. Two of the 15 (13%) CPAs with overt Cushings syndrome and one of the 9 (11%) CPAs with subclinical Cushings syndrome examined had the somatic mutations, p.R201S and p.R201C in the GNAS gene. We identified mutations in the GNAS gene (p.R201C) in 2 out of the 33 (6%) APAs tested, both of which showed autonomous cortisol secretion, while 24 APAs had mutations in the KCNJ5 gene (18 with p.G151R and 6 with p.L168R). These GNAS and KCNJ5 mutations were mutually exclusive in these adenomas. We herein demonstrated for the first time the presence of GNAS mutations in APAs, as well as in some cortisol-secreting adenomas. Our results suggest that these mutations, in addition to mutations in the KCNJ5 gene and other genes such as ATP1A1, ATP2B3 and CACNA1D, may be responsible for the tumorigenesis of APAs and CPAs with subclinical Cushings syndrome.