Bai Luo

Protein kinase Cα phosphorylates and negatively regulates diacylglycerol kinase ζ

Diacylglycerol kinase (DGK) terminates diacylglycerol (DAG) signaling by phosphorylating DAG to produce phosphatidic acid, which also has signaling properties. Thus, precise control of DGK activity is essential for proper signal transduction. We demonstrated previously that a peptide corresponding to the myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation site domain (PSD) in DGKζ was phosphorylated in vitro by an active fragment of protein kinase C (PKC). In the present study, we tested full-length DGKζ and found that PKCα phosphorylated DGKζ on serines within the MARCKS PSD in vitro and in vivo. DGKζ also coimmunoprecipitated with PKCα, suggesting that they reside in a regulated signaling complex. We then tested whether phosphorylation affected DAG kinase activity. We found that a mutant (DGKζ S/D) in which serines within the MARCKS PSD were altered to aspartates (to mimic phosphorylation) had lower activity compared with wild-type DGKζ or a control mutant (DGKζ S/N) in which the same serines were changed to asparagines. Furthermore, activation of PKCα by phorbol 12-myristate 13-acetate inhibited the activity of wild-type DGKζ, but not DGKζ S/D, in human embryonic kidney 293 cells. These results suggest that by phosphorylating the MARCKS PSD, PKCα attenuates DGKζ activity. Supporting this, we found that cells expressing DGKζ S/D had higher DAG levels and grew more rapidly compared with cells expressing DGKζ S/N that could not be phosphorylated. Taken together, these results indicate that PKCα phosphorylates DGKζ in cells, and this phosphorylation inhibits its kinase activity to remove cellular DAG, thereby affecting cell growth.

Iron Overload and Diabetes Risk: A Shift From Glucose to Fatty Acid Oxidation and Increased Hepatic Glucose Production in a Mouse Model of Hereditary Hemochromatosis

OBJECTIVE Excess tissue iron levels are a risk factor for diabetes, but the mechanisms underlying the association are incompletely understood. We previously published that mice and humans with a form of hereditary iron overload, hemochromatosis, exhibit loss of β-cell mass. This effect by itself is not sufficient, however, to fully explain the diabetes risk phenotype associated with all forms of iron overload. RESEARCH DESIGN AND METHODS We therefore examined glucose and fatty acid metabolism and hepatic glucose production in vivo and in vitro in a mouse model of hemochromatosis in which the gene most often mutated in the human disease, HFE, has been deleted (Hfe−/−). RESULTS Although Hfe−/− mice exhibit increased glucose uptake in skeletal muscle, glucose oxidation is decreased and the ratio of fatty acid to glucose oxidation is increased. On a high-fat diet, the Hfe−/− mice exhibit increased fatty acid oxidation and are hypermetabolic. The decreased glucose oxidation in skeletal muscle is due to decreased pyruvate dehydrogenase (PDH) enzyme activity related, in turn, to increased expression of PDH kinase 4 (pdk4). Increased substrate recycling to liver contributes to elevated hepatic glucose production in the Hfe−/− mice. CONCLUSIONS Increased hepatic glucose production and metabolic inflexibility, both of which are characteristics of type 2 diabetes, may contribute to the risk of diabetes with excessive tissue iron.

Protein Modification by O-Linked GlcNAc Reduces Angiogenesis by Inhibiting Akt Activity in Endothelial Cells

Objective—Glucose flux through the hexosamine biosynthesis pathway (HBP) has been implicated in the development of diabetic vascular complications. O-linked N-acetylglucosamine (O-GlcNAc) modification on protein is the major mechanism mediating the actions of the HBP. Impaired angiogenesis is well-recognized in diabetes; however, the mechanisms are not completely defined. Here, we investigated the role of protein O-GlcNAc modification in angiogenesis. Methods and Results—In a mouse aortic ring assay, elevated O-GlcNAc levels induced by high-fat diet, streptozotocin-induced diabetes, or in vitro glucosamine treatment were associated with impaired angiogenesis. In cultured human umbilical vein endothelial cells and EA.hy926 endothelial cells, glucosamine increased protein O-GlcNAc modification and inhibited cell migration and capillary-like structure formation. Conversely, removal of O-GlcNAc by adenoviral-mediated overexpression of O-GlcNAcase improved these steps of angiogenesis. Also, high concentrations of glucose reduced capillary-like structure formation of human umbilical vein endothelial cells. Akt was recognized by an O-GlcNAc specific lectin, and glucosamine increased the amounts of Akt protein in these lectin precipitates. Increased glycosylation paralleled reduced Akt activity in endothelial cells. Conclusion—These results suggest that elevated protein O-GlcNAc modification through the HBP impairs angiogenesis in endothelial cells, possibly by inhibiting Akt signaling.

Chronic Hexosamine Flux Stimulates Fatty Acid Oxidation by Activating AMP-activated Protein Kinase in Adipocytes *□

The hexosamine biosynthesis pathway (HBP) serves as a nutrient sensor and has been implicated in the development of type 2 diabetes. We previously demonstrated that fatty acid oxidation was enhanced in transgenic mouse adipocytes, wherein the rate-limiting enzyme of the HBP, glutamine:fructose-6-phosphate amidotransferase (GFA), was overexpressed. To explore the molecular mechanism of the HBP-induced fatty acid oxidation in adipocytes, we studied AMP-activated protein kinase (AMPK), an energy sensor that stimulates fatty acid oxidation by regulating acetyl-CoA carboxylase (ACC) activity. Phosphorylation and activity of AMPK were increased in transgenic fat pads and in 3T3L1 adipocytes treated with glucosamine to stimulate hexosamine flux. Glucosamine also stimulated phosphorylation of ACC and fatty acid oxidation in 3T3L1 adipocytes, and these stimulatory effects were diminished by adenovirus-mediated expression of a dominant negative AMPK in 3T3L1 adipocytes. Conversely, blocking the HBP with a GFA inhibitor reduced AMPK activity, ACC phosphorylation, and fatty acid oxidation. These changes are not explained by alterations in the cellular AMP/ATP ratio. Further demonstrating that AMPK is regulated by the HBP, we found that AMPK was recognized by succinylated wheat germ agglutinin, which specifically binds O-GlcNAc. The levels of AMPK in succinylated wheat germ agglutinin precipitates correlated with hexosamine flux in mouse fat pads and 3T3L1 adipocytes. Moreover, removal of O-GlcNAc by hexosaminidase reduced AMPK activity. We conclude that chronically high hexosamine flux stimulates fatty acid oxidation by activating AMPK in adipocytes, in part through O-linked glycosylation.

Regulation of Akt signaling by O-GlcNAc in euglycemia.

The hexosamine biosynthesis pathway (HBP) regulates the posttranslational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated that, in hyperglycemic conditions, excessive glucose flux through this pathway contributes to the development of insulin resistance. The role of the HBP in euglycemia, however, remains largely unknown. Here we investigated the effect of O-GlcNAc on hepatic Akt signaling at physiological concentrations of glucose. In HepG2 cells cultured in 5 mM glucose, removal of O-GlcNAc by adenoviral-mediated overexpression of O-GlcNAcase increased Akt activity and phosphorylation. We also observed that Akt was recognized by succinylated wheat germ agglutinin (sWGA), which specifically binds O-GlcNAc. Overexpression of O-GlcNAcase in HepG2 cells reduced the levels of Akt in sWGA precipitates. The increased Akt activity was accompanied by increased phosphorylation of Akt substrates and reduced mRNA for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK). The increased Akt activity was not a result of activation of its upstream activator phosphoinositide 3-kinase (PI 3-kinase). Further demonstrating Akt regulation by O-GlcNAc, we found that overexpression of O-GlcNAcase in the livers of euglycemic mice also significantly increased Akt activity, resulting in increased phosphorylation of downstream targets and decreased mRNA for glucose-6-phosphatase. Together, these data suggest that O-GlcNAc regulates Akt signaling in hepatic models under euglycemic conditions.

Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice

Excess iron is associated with hepatic damage and diabetes in humans, although the detailed molecular mechanisms are not known. To investigate how iron regulates glucose homeostasis, we fed C57BL/6J male mice with high‐iron (HI) diets (2 or 20 g Fe/kg chow). Mice fed an HI diet exhibited elevated AMP‐activated protein kinase (AMPK) activity and impaired insulin signaling in skeletal muscle and liver. Consistent with the increased AMPK activity, glucose uptake was enhanced in mice fed an HI diet. The effects of improved glucose tolerance induced by HI feeding were abolished in transgenic mice with expression of muscle specific dominant‐negative AMPK. Glucose output was suppressed in the liver of wild‐type mice fed an HI diet, due to decreased expression of gluconeogenic genes and decreased substrate (lactate) from peripheral glycolysis. Iron activated AMPK by increasing deacetylase and decreasing LKB1 acetylation, in turn stimulating the phosphorylation of LKB1 and AMPK. The effects of HI diet were abrogated by treatment of the mice with N‐acetyl cysteine, suggesting a redox‐dependent mechanism for increasing deacetylase activity. In addition, tissue from iron‐fed mice exhibited an elevated AMP/ATP ratio, further contributing to AMPK activation. In summary, a diet high in iron improves glucose tolerance by activating AMPK through mechanisms that include deacetylation.— Huang J., Simcox, J., Mitchell, T. C., Jones, D., Cox, J., Luo, B., Cooksey, R. C., Boros, L. G., McClain, D. A. Iron regulates glucose homeostasis in liver and muscle via AMP‐activated protein kinase in mice. FASEB J. 27, 2845–2854 (2013). www.fasebj.org

Increased Glucose Disposal and AMP-dependent Kinase Signaling in a Mouse Model of Hemochromatosis

Hereditary hemochromatosis is an inherited disorder of increased iron absorption that can result in cirrhosis, diabetes, and other morbidities. We have investigated the mechanisms underlying supranormal glucose tolerance despite decreased insulin secretion in a mouse model of hemochromatosis with deletion of the hemochromatosis gene (Hfe–/–). Hfe–/– mice on 129Sv or C57BL/6J backgrounds have decreased glucose excursions after challenge compared with controls. In the C57BL/6J/ Hfe–/–, for example, incremental area under the glucose curve is reduced 52% (p < 0.001) despite decreased serum insulin, and homeostasis model assessment insulin resistance is decreased 50% (p < 0.05). When studied by the euglycemic clamp technique 129Sv/Hfe–/– mice exhibit a 20% increase in glucose disposal (p < 0.05) at submaximal insulin but no increase at maximal insulin compared with wild types. [1,2-13C]d-glucose clearance from plasma is significantly increased in Hfe–/– mice (19%, p < 0.05), and lactate derived from glycolysis is elevated 5.1-fold in Hfe–/– mice (p < 0.0001). Basal but not insulin-stimulated glucose uptake is elevated in isolated soleus muscle from Hfe–/– mice (p < 0.03). Compared with controls Hfe–/– mice exhibit no differences in serum lipid, insulin, glucagon, or thyroid hormone levels; adiponectin levels are elevated 41% (p < 0.05), and the adiponectin message in adipocytes is increased 83% (p = 0.04). Insulin action measured by phosphorylation of Akt is not enhanced in muscle, but phosphorylation of AMP-dependent kinase is increased. We conclude that supranormal glucose tolerance in iron overload is characterized by increased glucose disposal that does not result from increased insulin action. Instead, the Hfe–/– mice demonstrate increased adiponectin levels and activation of AMP-dependent kinase.

Pleiotropic and Age-dependent Effects of Decreased Protein Modification by O-Linked N-Acetylglucosamine on Pancreatic β-Cell Function and Vascularization

The hexosamine biosynthesis pathway (HBP) regulates the post-translational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated increased flux through this pathway contributes to the development of β-cell dysfunction. The effect of decreased O-GlcNAc on the maintenance of normal β-cell function, however, is not well understood. We studied transgenic mice that over express β-N-acetylglucosaminidase (O-GlcNAcase), an enzyme that catalyzes the removal of O-GlcNAc from proteins, in the pancreatic β-cell under control of the rat insulin promoter. 3–4-Month-old O-GlcNAcase transgenic mice have higher glucose excursions with a concomitant decrease in circulating insulin levels, insulin mRNA levels, and total islet insulin content. In older (8–9-month-old) O-GlcNAcase transgenic mice glucose tolerance is no longer impaired. This is associated with increased serum insulin, islet insulin content, and insulin mRNA in the O-GlcNAcase transgenic mice. These improvements in β-cell function with aging are associated with increased angiogenesis and increased VEGF expression, with parallel increases in activation of Akt and expression of PGC1α. The biphasic effects as a function of age are consistent with published observations of mice with increased O-GlcNAc in islets and demonstrate that O-GlcNAc signaling exerts multiple effects on both insulin secretion and islet survival.