Methods Measurements of AMPK, ACC, and fatty acid oxidation in primary hepatocytes.Hepatocytes were isolated from male Sprague Dawley (SD) rats by collagenase digestion (18). For the AMPK assay, cells were seeded in six-well plates at 1.5 × 10 6 cells/well in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS, 100 nM insulin, 100 nM dexamethasone, and 5 µg/ml transferrin for 4 hours. Cells were then cultured in serum-free DMEM for 16 hours followed by treatment for 1 hour or 7 hours with control medium, 5-amino-imidazole carboxamide ribo-

Methods Measurements of AMPK, ACC, and fatty acid oxidation in primary hepatocytes.Hepatocytes were isolated from male Sprague Dawley (SD) rats by collagenase digestion (18). For the AMPK assay, cells were seeded in six-well plates at 1.5 × 10 6 cells/well in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS, 100 nM insulin, 100 nM dexamethasone, and 5 µg/ml transferrin for 4 hours. Cells were then cultured in serum-free DMEM for 16 hours followed by treatment for 1 hour or 7 hours with control medium, 5-amino-imidazole carboxamide ribo-

Adiponectin is an adipocyte-specific secretory protein that circulates in serum as a hexamer of relatively low molecular weight (LMW) and a larger multimeric structure of high molecular weight (HMW). Serum levels of the protein correlate with systemic insulin sensitivity. The full-length protein affects hepatic gluconeogenesis through improved insulin sensitivity, and a proteolytic fragment of adiponectin stimulates ␤ oxidation in muscle. Here, we show that the ratio, and not the absolute amounts, between these two oligomeric forms (HMW to LMW) is critical in determining insulin sensitivity. We define a new index, S A , that can be calculated as the ratio of HMW/(HMW ؉ LMW). db/db mice, despite similar total adiponectin levels, display decreased S A values compared with wild type littermates, as do type II diabetic patients compared with insulin-sensitive individuals. Furthermore, S A improves with peroxisome proliferator-activated receptor-␥ agonist treatment (thiazolidinedione; TZD) in mice and humans. We demonstrate that changes in S A in a number of type 2 diabetic cohorts serve as a quantitative indicator of improvements in insulin sensitivity obtained during TZD treatment, whereas changes in total serum adiponectin levels do not correlate well at the individual level. Acute alterations in S A (⌬S A ) are strongly correlated with improvements in hepatic insulin sensitivity and are less relevant as an indicator of improved muscle insulin sensitivity in response to TZD treatment, further underscoring the conclusions from previous clamp studies that suggested that the liver is the primary site of action for the full-length protein. These observations suggest that the HMW adiponectin complex is the active form of this protein, which we directly demonstrate in vivo by its ability to depress serum glucose levels in a dose-dependent manner.

Ceramides contribute to the lipotoxicity that underlies diabetes, hepatic steatosis, and heart disease. By genetically engineering mice, we deleted the enzyme dihydroceramide desaturase 1 (DES1), which normally inserts a conserved double bond into the backbone of ceramides and other predominant sphingolipids. Ablation of DES1 from whole animals or tissue-specific deletion in the liver and/or adipose tissue resolved hepatic steatosis and insulin resistance in mice caused by leptin deficiency or obesogenic diets. Mechanistic studies revealed ceramide actions that promoted lipid uptake and storage and impaired glucose utilization, none of which could be recapitulated by (dihydro)ceramides that lacked the critical double bond. These studies suggest that inhibition of DES1 may provide a means of treating hepatic steatosis and metabolic disorders.

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPAR␣, PPAR␦, and PPAR␥. PPAR␥ has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPAR␥ and PPAR␦ that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPAR␥ and PPAR␦ directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPAR␥ agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabetic db/db mice all PPAR␥ agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selective in vivo activation of PPAR␦ did not significantly affect these parameters. In vivo PPAR␣ activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPAR␥ and PPAR␦; 2) ligand-dependent activation of PPAR␦ involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPAR␥ activation (but not PPAR␦ or PPAR␣ activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPAR␥ agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPAR␣ activation is sufficient to affect triglyceride metabolism, PPAR␦ activation does not appear to modulate glucose or triglyceride levels.