Inflammation in asthma, sepsis, transplant rejection, and many neurodegenerative diseases associates an up-regulation of NO synthesis with increased protein nitration at tyrosine. Nitration can cause protein dysfunction and is implicated in pathogenesis, but few proteins that appear nitrated in vivo have been identified. To understand how this modification impacts physiology and disease, we used a proteomic approach toward targets of protein nitration in both in vivo and cell culture inflammatory disease models. This approach identified more than 40 nitrotyrosine-immunopositive proteins, including 30 not previously identified, that became modified as a consequence of the inflammatory response. These targets include proteins involved in oxidative stress, apoptosis, ATP production, and other metabolic functions. Our approach provides a means toward obtaining a comprehensive view of the nitroproteome and promises to broaden understanding of how NO regulates cellular processes. Biologic nitration of protein tyrosine (to form 3-nitrotyrosine) is a phenomenon that is associated with over 50 diseases including transplant rejection, lung infection, central nervous system and ocular inflammation, shock, cancer, and neurological disorders (e.g., amylotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and stroke) (1-3). Protein nitration may be unique among posttranslational modifications in its dependency on reactivity of tyrosine residues in the protein target instead of specific sequence motifs or protein-protein interactions. Tyrosine nitration can cause both gain or loss of protein function (4). Nitric oxide (NO) synthases provide the biological precursor for nitrating agents that perform this modification in vivo. NO can form nitrating agents in a number of ways including reacting with superoxide to make peroxynitrite (HOONO) and through enzymatic oxidation of nitrite (the oxidation product of NO) to generate NO 2 ⅐ (1). To date all protein nitration targets have been identified exclusively by antibodies directed against nitrotyrosine. Although these antibodies have linked this modification to many diseases, very few proteins that appear nitrated in vivo have been identified (1, 3). This has restricted our understanding of what roles tyrosine nitration might play in normal physiology and disease and the selectivity, kinetics, and potential repair of the modification in vivo (5, 6). We are pursuing a proteomic approach to identify nitrated proteins in biological samples that utilizes a commercially available well characterized monoclonal antibody directed against nitrotyrosine (7). This antibody has indicated biological tyrosine nitration in numerous studies and was shown to recognize a variety of proteins subjected to chemical nitration, the nitrated tyrosine residues of which were confirmed later by mass spectrometric sequence analysis (8, 9). Here we identify over 40 different proteins that appear to undergo nitration during inflammatory challenge in vivo. Our results provide a platform to examine...

Long term administration of leptin decreases caloric intake and fat mass and improves glucose tolerance. Here we examine whether leptin acutely regulates peripheral and hepatic insulin action. Recombinant mouse leptin (0.3 mg/kg⅐h, Leptin ؉) or vehicle (Leptin ؊) were administered for 6 h to 4-month-old rats (n ‫؍‬ 20), and insulin (3 milliunits/kg⅐min) clamp studies were performed. During physiologic hyperinsulinemia (plasma insulin ϳ65 microunits/ml), the rates of whole body glucose uptake, glycolysis, and glycogen synthesis and the rates of 2-deoxyglucose uptake in individual tissues were similar in Leptin ؊ and Leptin ؉. Post-absorptive hepatic glucose production (HGP) was similar in the two groups. However, leptin enhanced insulin's inhibition of HGP (4.1 ؎ 0.7 and 6.2 ؎ 0.7 mg/kg⅐min; p < 0.05). The decreased HGP in the Leptin ؉ group was due to a marked suppression of hepatic glycogenolysis (0.7 ؎ 0.1 versus 4.1 ؎ 0.6 mg/kg⅐min, in Leptin ؉ versus Leptin ؊, respectively; p < 0.001), whereas the % contribution of gluconeogenesis to HGP was markedly increased (82 ؎ 3% versus 36 ؎ 4% in Leptin ؉ and Leptin ؊, respectively; p < 0.001). At the end of the 6-h leptin infusion, the hepatic abundance of glucokinase mRNA was decreased, whereas that of phosphoenolpyruvate carboxykinase mRNA was increased compared with Leptin ؊. We conclude that an acute increase in plasma leptin 1) enhances insulin's ability to inhibit HGP, 2) does not affect peripheral insulin action, and 3) induces a redistribution of intrahepatic glucose fluxes and changes in the gene expression of hepatic enzymes that closely resemble those of fasting.The recent discovery of the ob gene (1) and preliminary analysis of the properties of its product, leptin (2-7), have shed new light on the regulation of energy homeostasis (8). Since the most common alteration in energy balance, obesity, is tightly associated with insulin resistance, it has been proposed that leptin may play a role in carbohydrate metabolism and insulin action (8 -11). Indeed, recent work in cultured adipose cells (12, 13) and hepatocytes (9) has suggested that leptin may antagonize insulin action in these cells. Conversely, early work on the effect of exogenous leptin in ob/ob mice had demonstrated a marked decrease in both plasma insulin and glucose concentrations following prolonged administration of this protein (2,4,5,7,14,15). Since the decline in plasma glucose and insulin was greater in leptin-treated mice than in pair-fed control mice (2, 14), it has been proposed that leptin may directly or indirectly improve in vivo insulin action.However, it is well established that changes in food intake, body weight, fat mass and/or fat distribution similar to those associated with long term leptin administration can independently alter insulin action, particularly in insulin-resistant and obese animal models (16,17). Thus, it is presently unknown whether the short term administration of exogenous leptin modulates insulin's ability to promote glucose disposal and/or to regulate hepat...

.32) was linked to the ␣-skeletal actin gene promoter, express PEPCK-C in skeletal muscle (1-3 units/g). Breeding two founder lines together produced mice with an activity of PEPCK-C of 9 units/g of muscle (PEPCK-C mus mice). These mice were seven times more active in their cages than controls. On a mouse treadmill, PEPCK-C mus mice ran up to 6 km at a speed of 20 m/min, whereas controls stopped at 0.2 km. PEPCK-C mus mice had an enhanced exercise capacity, with a VO 2max of 156 ؎ 8.0 ml/kg/min, a maximal respiratory exchange ratio of 0.91 ؎ 0.03, and a blood lactate concentration of 3.7 ؎ 1.0 mM after running for 32 min at a 25°grade; the values for control animals were 112 ؎ 21 ml/kg/min, 0.99 ؎ 0.08, and 8.1 ؎ 5.0 mM respectively. The PEPCK-C mus mice ate 60% more than controls but had half the body weight and 10% the body fat as determined by magnetic resonance imaging. In addition, the number of mitochondria and the content of triglyceride in the skeletal muscle of PEPCK-C mus mice were greatly increased as compared with controls. PEPCK-C mus mice had an extended life span relative to control animals; mice up to an age of 2.5 years ran twice as fast as 6 -12-month-old control animals. We conclude that overexpression of PEPCK-C repatterns energy metabolism and leads to greater longevity. PEPCK-C2 is involved in gluconeogenesis in the liver and kidney cortex and in glyceroneogenesis in liver and white and brown adipose tissue (see Ref. 1 for a review). However, this enzyme is also present in a broad variety of mammalian tissues (2), including the small intestine, colon, mammary gland, adrenal gland, lung, and muscle; its metabolic role in these tissues remains obscure. To study the physiological function of PEPCK-C, the gene has been overexpressed or ablated in specific tissues of the mouse. When PEPCK-C was overexpressed in white adipose tissue, the mice had increased rates of glyceroneogenesis in their adipose tissue and became obese (3). In contrast, ablating the expression of PEPCK-C in adipose tissue resulted in mice with lipodystrophy (4). However, a systematic study involving other mammalian tissues where the enzyme has been detected has not been undertaken.We have overexpressed the gene for PEPCK-C in the skeletal muscle of transgenic mice to test the metabolic and physiological consequences. Skeletal muscle was selected as a target organ because there is no clear indication of the metabolic outcome of having a high activity of PEPCK-C in this tissue. Skeletal muscle does not synthesize and release glucose, although there have been reports over the years that the tissue can make glycogen de novo since both PEPCK-C and fructose-1-6-bisphosphatase activities have been found in skeletal muscle (5, 6). We have evidence from research ongoing in our laboratory 3 that glyceroneogenesis occurs in skeletal muscle. This pathway is an abbreviated version of gluconeogenesis, which involves the synthesis of glycerol-3-phosphate (used for triglyceride synthesis) from precursors other than glucose and glycerol. Howe...

Overproduction of glucose by the liver is the major cause of fasting hyperglycemia in both insulin-dependent and non-insulin-dependent diabetes mellitus. The distal enzymatic step in the process of glucose output is catalyzed by the glucose-6-phosphatase complex. We show here that 90% partially pancreatectomized diabetic rats have a >5-fold increase in the messenger RNA and a 3-4-fold increase in the protein level of the catalytic subunit of glucose-6-phosphatase in the liver. Normalization of the plasma glucose concentration in diabetic rats with either insulin or the glycosuric agent phlorizin normalized the hepatic glucose-6-phosphatase messenger RNA and protein within approximately 8 h. Conversely, phlorizin failed to decrease hepatic glucose-6-phosphatase gene expression in diabetic rats when the fall in the plasma glucose concentration was prevented by glucose infusion. These data indicate that in vivo gene expression of glucose-6-phosphatase in the diabetic liver is regulated by glucose independently from insulin, and thus prolonged hyperglycemia may result in overproduction of glucose via increased expression of this protein.

Hepatic gene expression of P-enolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (Glc-6-Pase) is regulated in response to changes in the availability of substrates, in particular glucose (Glc; Massillon, D., Barzilai, N., Chen, W., Hu, M., and Rossetti, L. (1996) J. Biol. Chem. 271, 9871-9874). We investigated the mechanism(s) in conscious rats. Hyperglycemia per se caused a rapid and marked increase in Glc-6-Pase mRNA abundance and protein levels. By contrast, hyperglycemia decreased the abundance of PEPCK mRNA. Importantly, inhibition of glucokinase activity by glucosamine infusion blunted both the stimulation of Glc-6-Pase and the inhibition of PEPCK gene expression by Glc, suggesting that an intrahepatic signal (metabolite) generated by the metabolism of glucose at or beyond Glc-6-P was responsible for the regulatory effect of Glc.The effect of Glc on the L-type pyruvate kinase gene is mediated by xylulose-5-P (Doiron, B., Cuif, M., Chen, R., and Kahn, A. (1996) J. Biol. Chem. 271, 5321-5324). Thus, we next investigated whether an isolated increase in the hepatic concentration of this metabolite can also reproduce the effects of Glc on Glc-6-Pase and PEPCK gene expression in vivo. Xylitol, which is directly converted to xylulose-5-P in the liver, was infused to raise the hepatic concentration of xylulose-5-P by ϳ3-fold. Xylitol infusion did not alter the levels of Glc-6-P and of fructose-2,6-biphosphate. However, it replicated the effects of hyperglycemia on Glc-6-Pase and PEPCK gene expression and resulted in a 75% increase in the in vivo flux through Glc-6-Pase (total glucose output).