We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFα and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.

AMP-activated protein kinase (AMPK) serves as an energy-sensing protein kinase that is activated by a variety of metabolic stresses that lower cellular energy levels. When activated, AMPK modulates a network of metabolic pathways that result in net increased substrate oxidation, generation of reduced nucleotide cofactors, and production of ATP. AMPK is activated by a high AMP:ATP ratio and phosphorylation on threonine 172 by an upstream kinase. Recent studies suggest that mechanisms that do not involve changes in adenine nucleotide levels can activate AMPK. Another sensor of the metabolic state of the cell is the NAD/NADH redox potential. To test whether the redox state might have an effect on AMPK activity, we examined the effect of ␤-NAD and NADH on this enzyme. The recombinant T172D-AMPK, which was mutated to mimic the phosphorylated state, was activated by ␤-NAD in a dose-dependent manner, whereas NADH inhibited its activity. We explored the effect of NADH on AMPK by systematically varying the concentrations of ATP, NADH, peptide substrate, and AMP. Based on our findings and established activation of AMPK by AMP, we proposed a model for the regulation by NADH. Key features of this model are as follows. (a) NADH has an apparent competitive behavior with respect to ATP and uncompetitive behavior with respect to AMP resulting in improved binding constant in the presence of AMP, and (b) the binding of the peptide is not significantly altered by NADH. In the absence of AMP, the binding constant of NADH becomes higher than physiologically relevant. We conclude that AMPK senses both components of cellular energy status, redox potential, and phosphorylation potential.The mammalian 5Ј-AMP-activated protein kinase (AMPK) 1 serves as an energy-sensing protein kinase. It is activated by a variety of metabolic stresses that lower cellular energy levels. These include exercise, nutrient starvation, ischemia/hypoxia, heat shock, and metabolic poisoning (1-4). AMPK is also activated by a number of pharmacological interventions. AICAR (5-aminoimidazole-4-carboxamide 1-b-D-ribonucleoside), which is converted to an AMP analog upon entering the cells, is a commonly used tool for activating AMPK (5). Leptin (6), adiponectin (7), metformin (8, 9), and rosiglitazone (8) activate AMPK through poorly characterized mechanisms that may not involve energy depletion. The activation of AMPK stimulates fatty acid oxidation, hexokinase activity, and uptake of glucose into skeletal muscle, which generates reduced nucleoside cofactors and ATP, and represses hepatic gluconeogenic and fatty acid synthesis enzymes, which consume them (10).AMPK is a heterotrimeric complex consisting of a catalytic (␣) subunit and two regulatory subunits (␤ and ␥) (11). The isoforms of all three subunits have been identified including two isoforms of the catalytic subunit, ␣ 1 and ␣ 2 (9), two of the regulatory subunits, ␤ 1 and ␤ 2 , and three of the regulatory subunits, ␥ 1 , ␥ 2 , and ␥ 3 (12). The formation of the trimeric complex is necessary for optimal kin...

LSN862 is a novel peroxisome proliferator-activated receptor (PPAR)alpha/gamma dual agonist with a unique in vitro profile that shows improvements on glucose and lipid levels in rodent models of type 2 diabetes and dyslipidemia. Data from in vitro binding, cotransfection, and cofactor recruitment assays characterize LSN862 as a high-affinity PPARgamma partial agonist with relatively less but significant PPARalpha agonist activity. Using these same assays, rosiglitazone was characterized as a high-affinity PPARgamma full agonist with no PPARalpha activity. When administered to Zucker diabetic fatty rats, LSN862 displayed significant glucose and triglyceride lowering and a significantly greater increase in adiponectin levels compared with rosiglitazone. Expression of genes involved in metabolic pathways in the liver and in two fat depots from compound-treated Zucker diabetic fatty rats was evaluated. Only LSN862 significantly elevated mRNA levels of pyruvate dehydrogenase kinase isozyme 4 and bifunctional enzyme in the liver and lipoprotein lipase in both fat depots. In contrast, both LSN862 and rosiglitazone decreased phosphoenol pyruvate carboxykinase in the liver and increased malic enzyme mRNA levels in the fat. In addition, LSN862 was examined in a second rodent model of type 2 diabetes, db/db mice. In this study, LSN862 demonstrated statistically better antidiabetic efficacy compared with rosiglitazone with an equivalent side effect profile. LSN862, rosiglitazone, and fenofibrate were each evaluated in the humanized apoA1 transgenic mouse. At the highest dose administered, LSN862 and fenofibrate reduced very low-density lipoprotein cholesterol, whereas, rosiglitazone increased very low-density lipoprotein cholesterol. LSN862, fenofibrate, and rosiglitazone produced maximal increases in high-density lipoprotein cholesterol of 65, 54, and 30%, respectively. These findings show that PPARgamma full agonist activity is not necessary to achieve potent and efficacious insulin-sensitizing benefits and demonstrate the therapeutic advantages of a PPARalpha/gamma dual agonist.

Obesity is associated with an increased risk for developing type 2 diabetes, insulin resistance, hypertension, dyslipidemia, cardiovascular disease, respiratory dysfunction, and certain forms of cancer. Insulin resistance in many type 2 diabetic patients is the result of increased visceral adiposity. To identify novel genes implicated in type 2 diabetes and/or obesity and to elucidate the molecular mechanisms underlying both diseases, we analyzed gene expression in omental fat from lean and obese nondiabetic subjects and obese type 2 diabetic patients using mRNA differential display and subtracted library techniques. After screening over 13,800 subtracted cDNA clones and 6,912 cDNA amplification products, we identified 2,078 cDNAs that showed potential differential expression in the omental fat of lean versus obese nondiabetic subjects versus obese type 2 diabetic patients. Data analysis showed that 70.7% of these clones corresponded to unknown genes (26.7% matched express sequence tags [ESTs]) and 29.3% corresponded to known genes. Reverse Northern and classic Northern analyses further confirmed that the expression of five of these cDNA clones was elevated in obese nondiabetic subjects and obese type 2 diabetic patients. Four candidate genes were further evaluated for tissue distribution, which showed expression primarily in adipose and skeletal muscle tissue, and chromosomal localization. We concluded that both mRNA differential display and subtracted cDNA libraries are powerful tools for identifying novel genes implicated in the pathogenesis of obesity and type 2 diabetes.