Donald A. McClain

Insulin signaling coordinately regulates cardiac size, metabolism, and contractile protein isoform expression

To investigate the role of insulin signaling on postnatal cardiac development, physiology, and cardiac metabolism, we generated mice with a cardiomyocyte-selective insulin receptor knockout (CIRKO) using cre/loxP recombination. Hearts of CIRKO mice were reduced in size by 20-30% due to reduced cardiomyocyte size and had persistent expression of the fetal beta-myosin heavy chain isoform. In CIRKO hearts, glucose transporter 1 (GLUT1) expression was reduced by about 50%, but there was a twofold increase in GLUT4 expression as well as increased rates of cardiac glucose uptake in vivo and increased glycolysis in isolated working hearts. Fatty acid oxidation rates were diminished as a result of reduced expression of enzymes that catalyze mitochondrial beta-oxidation. Although basal rates of glucose oxidation were reduced, insulin unexpectedly stimulated glucose oxidation and glycogenolysis in CIRKO hearts. Cardiac performance in vivo and in isolated hearts was mildly impaired. Thus, insulin signaling plays an important developmental role in regulating postnatal cardiac size, myosin isoform expression, and the switching of cardiac substrate utilization from glucose to fatty acids. Insulin may also modulate cardiac myocyte metabolism through paracrine mechanisms by activating insulin receptors in other cell types within the heart.

Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia

Insulin resistance and β cell toxicity are key features of type 2 diabetes. One leading hypothesis suggests that these abnormalities result from excessive flux of nutrients through the UDP–hexosamine biosynthetic pathway leading to “glucose toxicity.” How the products of the hexosamine pathway mediate these effects is not known. Here, we show that transgenic overexpression of an enzyme using UDP-GlcNAc to modify proteins with O-GlcNAc produces the type 2 diabetic phenotype. Even modest overexpression of an isoform of O-GlcNAc transferase, in muscle and fat, leads to insulin resistance and hyperleptinemia. These data support the proposal that O-linked GlcNAc transferase participates in a hexosamine-dependent signaling pathway that is linked to insulin resistance and leptin production.

Adipocyte iron regulates adiponectin and insulin sensitivity

Iron overload is associated with increased diabetes risk. We therefore investigated the effect of iron on adiponectin, an insulin-sensitizing adipokine that is decreased in diabetic patients. In humans, normal-range serum ferritin levels were inversely associated with adiponectin, independent of inflammation. Ferritin was increased and adiponectin was decreased in type 2 diabetic and in obese diabetic subjects compared with those in equally obese individuals without metabolic syndrome. Mice fed a high-iron diet and cultured adipocytes treated with iron exhibited decreased adiponectin mRNA and protein. We found that iron negatively regulated adiponectin transcription via FOXO1-mediated repression. Further, loss of the adipocyte iron export channel, ferroportin, in mice resulted in adipocyte iron loading, decreased adiponectin, and insulin resistance. Conversely, organismal iron overload and increased adipocyte ferroportin expression because of hemochromatosis are associated with decreased adipocyte iron, increased adiponectin, improved glucose tolerance, and increased insulin sensitivity. Phlebotomy of humans with impaired glucose tolerance and ferritin values in the highest quartile of normal increased adiponectin and improved glucose tolerance. These findings demonstrate a causal role for iron as a risk factor for metabolic syndrome and a role for adipocytes in modulating metabolism through adiponectin in response to iron stores.

Contribution of Insulin and Akt1 Signaling to Endothelial Nitric Oxide Synthase in the Regulation of Endothelial Function and Blood Pressure

Impaired insulin signaling via phosphatidylinositol 3-kinase/Akt to endothelial nitric oxide synthase (eNOS) in the vasculature has been postulated to lead to arterial dysfunction and hypertension in obesity and other insulin resistant states. To investigate this, we compared insulin signaling in the vasculature, endothelial function, and systemic blood pressure in mice fed a high-fat (HF) diet to mice with genetic ablation of insulin receptors in all vascular tissues (TTr-IR−/−) or mice with genetic ablation of Akt1 (Akt1−/−). HF mice developed obesity, impaired glucose tolerance, and elevated free fatty acids that was associated with endothelial dysfunction and hypertension. Basal and insulin-mediated phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in the vasculature was preserved, but basal and insulin-stimulated eNOS phosphorylation was abolished in vessels from HF versus lean mice. In contrast, basal vascular eNOS phosphorylation, endothelial function, and blood pressure were normal despite absent insulin-mediated eNOS phosphorylation in TTr-IR−/− mice and absent insulin-mediated eNOS phosphorylation via Akt1 in Akt1−/− mice. In cultured endothelial cells, 6 hours of incubation with palmitate attenuated basal and insulin-stimulated eNOS phosphorylation and NO production despite normal activation of extracellular signal-regulated kinase 1/2 and Akt. Moreover, incubation of isolated arteries with palmitate impaired endothelium-dependent but not vascular smooth muscle function. Collectively, these results indicate that lower arterial eNOS phosphorylation, hypertension, and vascular dysfunction following HF feeding do not result from defective upstream signaling via Akt, but from free fatty acid–mediated impairment of eNOS phosphorylation.

Insulin and glucosamine infusions increase O-linked N-acetyl-glucosamine in skeletal muscle proteins in vivo☆

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of serine/threonine residues of nuclear and cytoplasmic proteins. We determined whether insulin or coinfusion of glucosamine (GlcN) with insulin alters O-GlcNAc of skeletal muscle proteins. Three groups of conscious fasted rats received 6-hour infusions of either saline (BAS), insulin 18 mU/kg.min and saline (INS), or insulin and GlcN 30 micromol/kg.min (GLCN) during maintenance of normoglycemia. At 6 hours, the concentrations of muscle UDP-GlcNAc, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), glycogen, and N and O-linked GlcNAc (galactosyltransferase labeling followed by beta elimination) were measured in freeze-clamped abdominis muscle. Insulin increased whole-body glucose uptake from 49 +/- 5 to 239 +/- 8 micromol/kg.min (P < .001) and glycogen in abdominis muscle from 138 +/- 11 to 370 +/- 26 mmol/kg dry weight (P < .001). Insulin increased the amount of cytosolic N - and O-linked GlcNAc by 56% from 362 +/- 30 to 564 +/- 45 dpm/microg protein . 100 min (P < .02), and O-GlcNAc from 221 +/- 16 to 339 +/- 27 dpm/microg . 100 min (P < .02). Glycogen content was positively correlated with the amount of total (r = .90, P < .005) and O-linked GlcNAc in insulin-infused animals. Coinfusion of GlcN with insulin increased muscle UDP-GlcNAc about fourfold (100 +/- 6 nmol/g) compared with insulin (27 +/- 1, P < .001) or saline (25 +/- 1, P < .001) infusion. GlcN also decreased glucose uptake over 6 hours by 30% to 168 +/- 8 micromol/kg . min (P < .001 for GLCN v INS) and muscle glycogen to 292 +/- 24 mmol/kg dry weight (P < .05 for GLCN v INS). Both total (635 +/- 60 dpm/microg . 100 min, P < .002) and O-linked GlcNAc (375 +/- 36 dpm/microg . 100 min, P < .002) in the cytosol were significantly higher in GLCN rats (635 +/- 60 dpm/microg) versus BAS rats (P < .002). As in INS rats, muscle glycogen and O-GlcNAc were positively correlated in GLCN rats (r = .54, P < .05). Variation in total and O-linked GlcNAc in GLCN rats was due both to GlcN (P < .02) and to variation in the glycogen content (P < .005).

Glucose Deprivation Stimulates O-GlcNAc Modification of Proteins through Up-regulation of O-Linked N-Acetylglucosaminyltransferase

O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. Here we report on regulation of O-GlcNAcylation over a broad range of glucose concentrations. We have discovered a significant induction of O-GlcNAc modification of a limited number of proteins under conditions of glucose deprivation. Beginning 12 h after treatment, glucose-deprived human hepatocellular carcinoma (HepG2) cells demonstrate a 7.8-fold increase in total O-GlcNAc modification compared with cells cultured in normal glucose (5 mm; p = 0.008). Some of the targets of glucose deprivation-induced O-GlcNAcylation are distinct from those modified in response to high glucose (20 mm) or glucosamine (10 mm) treatment, suggesting differential targeting with glucose deprivation and glucose excess. O-GlcNAcylation of glycogen synthase is significantly increased with glucose deprivation, and this O-GlcNAc increase contributes to a 60% decrease (p = 0.004) in glycogen synthase activity. Increased O-GlcNAc modification is not mediated by increased UDP-GlcNAc, the rate-limiting substrate for O-GlcNAcylation. Rather, the mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (OGT) increases 3.4-fold within 6 h of glucose deprivation (p = 0.006). Within 12 h, OGT protein increases 1.7-fold (p = 0.01) compared with normal glucose-treated cells. In addition, 12-h glucose deprivation leads to a 49% decrease in O-GlcNAcase protein levels (p = 0.03). We conclude that increased O-GlcNAc modification stimulated by glucose deprivation results from increased OGT and decreased O-GlcNAcase levels and that these changes affect cell metabolism, thus inactivating glycogen synthase.

Oct1 loss of function induces a coordinate metabolic shift that opposes tumorigenicity

Cancer cells frequently undergo a shift from oxidative to glycolytic metabolism. Although there is interest in targeting metabolism as a form of cancer therapy, this area still remains in its infancy. Using cells, embryos and adult animals, we show here that loss of the widely expressed transcription factor Oct1 induces a coordinated metabolic shift: mitochondrial activity and amino acid oxidation are increased, while glucose metabolism is reduced. Altered expression of direct Oct1 targets encoding metabolic regulators provides a mechanistic underpinning to these results. We show that these metabolic changes directly oppose tumorigenicity. Collectively, our findings show that Oct1, the genes it regulates and the pathways these genes affect could be used as targets for new modes of cancer therapy.

Iron-Mediated Inhibition of Mitochondrial Manganese Uptake Mediates Mitochondrial Dysfunction in a Mouse Model of Hemochromatosis

Previous phenotyping of glucose homeostasis and insulin secretion in a mouse model of hereditary hemochromatosis (Hfe−/−) and iron overload suggested mitochondrial dysfunction. Mitochondria from Hfe−/− mouse liver exhibited decreased respiratory capacity and increased lipid peroxidation. Although the cytosol contained excess iron, Hfe−/− mitochondria contained normal iron but decreased copper, manganese, and zinc, associated with reduced activities of copper-dependent cytochrome c oxidase and manganese-dependent superoxide dismutase (MnSOD). The attenuation in MnSOD activity was due to substantial levels of unmetallated apoprotein. The oxidative damage in Hfe−/− mitochondria is due to diminished MnSOD activity, as manganese supplementation of Hfe−/− mice led to enhancement of MnSOD activity and suppressed lipid peroxidation. Manganese supplementation also resulted in improved insulin secretion and glucose tolerance associated with increased MnSOD activity and decreased lipid peroxidation in islets. These data suggest a novel mechanism of iron-induced cellular dysfunction, namely altered mitochondrial uptake of other metal ions.

Proposed Regulation of Gene Expression by Glucose in Rodent Heart

Background During pressure overload-induced hypertrophy, unloading-induced atrophy, and diabetes mellitus, the heart induces ‘fetal’ genes (e.g. myosin heavy chain β; mhcβ). Hypothesis We propose that altered glucose homeostasis within the cardiomyocyte acts as a central mechanism for the regulation of gene expression in response to environmental stresses. The evidence is as follows. Methods and Results Forced glucose uptake both ex vivo and in vivo results in mhc isoform switching. Restricting dietary glucose prevents mhc isoform switching in hearts of both GLUT1-Tg mice and rats subjected to pressure overload-induced hypertrophy. Thus, glucose availability correlates with mhc isoform switching under all conditions investigated. A potential mechanism by which glucose affects gene expression is through O-linked glycosylation of specific transcription factors. Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the flux generating step in UDP-N-acetylglucosamine biosynthesis, the rate determining metabolite in protein glycosylation. Ascending aortic constriction increased intracellular levels of UDP-N-acetylglucosamine, and the expression of gfat2, but not gfat1, in the rat heart. Conclusions Collectively, the results strongly suggest glucose-regulated gene expression in the heart, and the involvement of glucose metabolites in isoform switching of sarcomeric proteins characteristic for the fetal gene program.

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.