Human hexokinase: sequences of amino- and carboxyl-terminal halves are homologous

cDNA clones encoding human hexokinase have been isolated from an adult kidney library. Analysis of this 917 amino acid protein (Mr = 102,519) indicates that the sequences of the NH2- and COOH-terminal halves, corresponding to the regulatory and catalytic domains, respectively, are homologous; and that eukaryotic hexokinases evolved by duplication of a gene encoding a protein of 450 amino acids. The COOH-terminal half of the protein created by this gene duplication retained the glucose binding site and glucose phosphorylating activity while the substrate binding sites of the NH2-terminal half evolved into a new allosteric effector site.     

Transgenic rescue of adipocyte glucose-dependent insulinotropic polypeptide receptor expression restores high fat diet-induced body weight gain

The glucose-dependent insulinotropic polypeptide receptor (GIPr) has been implicated in high fat diet-induced obesity and is proposed as an anti-obesity target despite an uncertainty regarding the mechanism of action. To independently investigate the contribution of the insulinotropic effects and the direct effects on adipose tissue, we generated transgenic mice with targeted expression of the human GIPr to white adipose tissue or beta-cells, respectively. These mice were then cross-bred with the GIPr knock-out strain. The central findings of the study are that mice with GIPr expression targeted to adipose tissue have a similar high fat diet -induced body weight gain as control mice, significantly greater than the weight gain in mice with a general ablation of the receptor. Surprisingly, this difference was due to an increase in total lean body mass rather than a gain in total fat mass that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass, but it does not support a direct and independent role for the adipocyte or beta-cell GIPr in promoting adipogenesis.     

KATP channel knockout worsens myocardial calcium stress load in vivo and impairs recovery in stunned heart

Gene knockout of the KCNJ11-encoded Kir6.2 ATP-sensitive K(+) (K(ATP)) channel implicates this stress-response element in the safeguard of cardiac homeostasis under imposed demand. K(ATP) channels are abundant in ventricular sarcolemma, where subunit expression appears to vary between the sexes. A limitation, however, in establishing the full significance of K(ATP) channels in the intact organism has been the inability to monitor in vivo the contribution of the channel to intracellular calcium handling and the superimposed effect of sex that ultimately defines heart function. Here, in vivo manganese-enhanced cardiac magnetic resonance imaging revealed, under dobutamine stress, a significantly greater accumulation of calcium in both male and female K(ATP) channel knockout (Kir6.2-KO) mice compared with sex- and age-matched wild-type (WT) counterparts, with greatest calcium load in Kir6.2-KO females. This translated, poststress, into a sustained contracture manifested by reduced end-diastolic volumes in K(ATP) channel-deficient mice. In response to ischemia-induced stunning, male and female Kir6.2-KO hearts demonstrated accelerated time to contracture and increased peak contracture compared with WT. The outcome on reperfusion, in both male and female Kir6.2-KO hearts, was a transient reduction in systolic performance, measured as rate-pressure product compared with WT, with protracted increase in left ventricular end-diastolic pressure, exaggerated in female knockout hearts, despite comparable leakage of creatine kinase across groups. Kir6.2-KO hearts were rescued from diastolic dysfunction by agents that target alternative pathways of calcium handling. Thus K(ATP) channel deficit confers a greater susceptibility to calcium overload in vivo, accentuated in female hearts, impairing contractile recovery under various conditions of high metabolic demand.     

Terminal differentiation of keratinocytes was damaged in type 2 diabetic mice

Molecular Biology Reports - Although skin manifestations are common in diabetic patients, its characteristics are poorly identified. This study explored the differentiation process of keratinocytes...     

Induction of Macrophage-Like Immunosuppressive Cells from Mouse ES Cells That Contribute to Prolong Allogeneic Graft Survival

Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) as a donor resource for transplantation. However, immune suppression is still needed when the donor-recipient combination is allogeneic. Protection of ESCs-derived grafts from host immune response might be achieved thought the utilization of immunosuppressive cells generated from ESCs. In the present study, we show that a certain fraction of immunosuppressive cells can be generated from ESCs and help to suppress immune response against allogeneic grafts. ESCs-derived suppressor cells (ES-SCs) resembled macrophages in terms of cell surface molecule and gene expressions. Furthermore, gene expression analysis including microarray showed that ES-SCs have M1/M2 hybrid phenotype with high expression of genes correlated to immunosuppression of T cell response. Indeed, ES-SCs were effective to block allogeneic T cell proliferation in a nitric oxide-dependent manner, and prolonged the survival of ESCs-derived embryoid bodies or cardiomyocytes grafts transplanted into mouse kidney capsule. Thus, we consider the potential use of these ESCs-derived macrophage-like immunosuppressive cells as cellular therapies to promote long-term graft survival in future therapies.     

Identification and functional analysis of CBLB mutations in type 1 diabetes

Casitas B-lineage lymphoma b (Cblb) is a negative regulator of T-cell activation and dysfunction of Cblb in rats and mice results in autoimmunity. In particular, a nonsense mutation in Cblb has been identified in a rat model of autoimmune type 1 diabetes. To clarify the possible involvement of CBLB mutation in type 1 diabetes in humans, we performed mutation screening of CBLB and characterized functional properties of the mutations in Japanese subjects. Six missense mutations (A155V, F328L, N466D, K837R, T882A, and R968L) were identified in one diabetic subject each, excepting N466D. Of these mutations, F328L showed impaired suppression of T-cell activation and was a loss-of-function mutation. These data suggest that the F328L mutation is involved in the development of autoimmune diseases including type 1 diabetes, and also provide insight into the structure-function relationship of CBLB protein.     

Role of cadherin-mediated cell-cell adhesion in pancreatic exocrine-to-endocrine transdifferentiation

Although pancreatic exocrine acinar cells have the potential to transdifferentiate into pancreatic endocrine cells, the mechanisms are poorly understood. Here we report that intracellular signaling pathways, including those involving MAPK and phosphatidylinositol 3 (PI3)-kinase, are activated by enzymatic dissociation of pancreatic acinar cells and that spherical cell clusters are formed by cadherin-mediated cell-cell adhesion during transdifferentiation. Inhibition of PI3-kinase by LY294002 prevents spheroid formation by degrading E-cadherin and beta-catenin, blocking transdifferentiation into insulin-secreting cells. In addition, neutralizing antibody against E-cadherin suppresses the induction of genes characteristic of pancreatic beta-cells. We also show that loss of cadherin-mediated cell-cell adhesion induces and maintains a dedifferentiated state in isolated pancreatic acinar cells. Thus, disruption and remodeling of cadherin-mediated cell-cell adhesion is critical in pancreatic exocrine-to-endocrine transdifferentiation, in which the PI3-kinase pathway plays an essential role.     

Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation

Voltage-gated Ca2+ channels (VDCCs) are heteromultimeric proteins that mediate Ca2+ influx into cells upon membrane depolarization. These channels are involved in various cellular events, including gene expression, regulation of hormone secretion and synaptic transmission. Kir/Gem, Rad, Rem, and Rem2 belong to the RGK family of Ras-related small G proteins. RGK proteins interact with the beta-subunits and downregulate VDCC activity. Kir/Gem was proposed to prevent surface expression of functional Ca2+ channels, while for Rem2 the mechanism remains controversial. Here, we have analyzed the mechanism by which Rad and Rem regulate VDCC activity. We show that, similar to Kir/Gem and Rem2, 14-3-3 and CaM binding regulate the subcellular distribution of Rad and Rem, which both inhibit Ca2+ channel activity by preventing its expression on the cell surface. This function is regulated by calmodulin and 14-3-3 binding only for Rad and not for Rem. Interestingly, nuclear targeting of Rad and Rem can relocalize and sequester the beta-subunit to the nucleus, thus providing a novel mechanism for Ca2+ channel downregulation.