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Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.

Activation of peroxisome proliferator-activated receptor (PPAR) alpha, delta, and gamma subtypes increases expression of genes involved in fatty acid transport and oxidation and alters adiposity in animal models of obesity and type-2 diabetes. PPARpan agonists which activate all three receptor subtypes have antidiabetic activity in animal models without the weight gain associated with selective PPARgamma agonists. Herein we report the effects of selective PPAR agonists (GW9578, a PPARalpha agonist, GW0742, a PPARdelta agonist, GW7845, a PPARgamma agonist), combination of PPARalpha and delta agonists, and PPARpan (PPARalpha/gamma/delta) activators (GW4148 or GW9135) on body weight (BW), body composition, food consumption, fatty acid oxidation, and serum chemistry of diet-induced obese AKR/J mice. PPARalpha or PPARdelta agonist treatment induced a slight decrease in fat mass (FM) while a PPARgamma agonist increased BW and FM commensurate with increased food consumption. The reduction in BW and food intake after cotreatment with PPARalpha and delta agonists appeared to be synergistic. GW4148, a PPARpan agonist, induced a significant and sustained reduction in BW and FM similar to an efficacious dose of rimonabant, an antiobesity compound. GW9135, a PPARpan agonist with weak activity at PPARdelta, induced weight loss initially followed by rebound weight gain reaching vehicle control levels by the end of the experiment. We conclude that PPARalpha and PPARdelta activations are critical to effective weight loss induction. These results suggest that the PPARpan compounds may be expected to maintain the beneficial insulin sensitization effects of a PPARgamma agonist while either maintaining weight or producing weight loss.

The peroxisome proliferator-activated receptors (PPARs) impart diverse cellular effects in biological systems. Because stellate cell activation during liver injury is associated with declining PPARgamma expression, we hypothesized that its expression is critical in stellate cell-mediated fibrogenesis. We therefore modulated its expression during liver injury in vivo. PPARgamma was depleted in rat livers by using an adenovirus-Cre recombinase system. PPARgamma was overexpressed by using an additional adenoviral vector (AdPPARgamma). Bile duct ligation was utilized to induce stellate cell activation and liver fibrosis in vivo; phenotypic effects (collagen I, smooth muscle alpha-actin, hydroxyproline content, etc.) were measured. PPARgamma mRNA levels decreased fivefold and PPARgamma protein was undetectable in stellate cells after culture-induced activation. During activation in vivo, collagen accumulation, assessed histomorphometrically and by hydroxyproline content, was significantly increased after PPARgamma depletion compared with controls (1.28 +/- 0.14 vs. 1.89 +/- 0.21 mg/g liver tissue, P < 0.03). In isolated stellate cells, AdPPARgamma overexpression resulted in significantly increased adiponectin mRNA expression and decreased collagen I and smooth muscle alpha-actin mRNA expression compared with controls. During in vivo fibrogenesis, rat livers exposed to AdPPARgamma had significantly less fibrosis than controls. Collagen I and smooth muscle alpha-actin mRNA expression were significantly reduced in AdPPARgamma-infected rats compared with controls (P < 0.05, n = 10). PPARgamma-deficient mice exhibited enhanced fibrogenesis after liver injury, whereas PPARgamma receptor overexpression in vivo attenuated stellate cell activation and fibrosis. The data highlight a critical role for PPARgamma during in vivo fibrogenesis and emphasize the importance of the PPARgamma pathway in stellate cells during liver injury.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists have been shown to have significant therapeutic benefits such as desirable glycemic control in type 2 diabetic patients; however, these agents may cause fluid retention in susceptible individuals. Since PPARgamma is expressed selectively in distal nephron epithelium, we studied the mechanism of PPARgamma agonist-induced fluid retention using male Sprague-Dawley rats treated with either vehicle or GI262570 (farglitazar), a potent PPARgamma agonist. GI262570 (20 mg/kg/day) induced a plasma volume expansion. The plasma volume expansion was accompanied by a small but significant decrease in plasma potassium concentration. Small but significant increases in plasma sodium and chloride concentrations were also observed. These changes in serum electrolytes suggested an activation of the renal mineralocorticoid response system; however, GI262570-treated rats had lower plasma levels of aldosterone compared with vehicle-treated controls. mRNA levels for a group of genes involved in distal nephron sodium and water absorption are changed in the kidney medulla with GI262570 treatment. In addition, due to a possible rebound effect on epithelial sodium channel (ENaC) activity, a low dose of amiloride did not prevent GI262570-induced fluid retention. On the contrary, the rebound effect after amiloride treatment potentiated GI262570-induced plasma volume expansion. This is at least partially due to a synergistic effect of GI262570 and the rebound from amiloride treatment on ENaCalpha expression. In summary, our current data suggest that GI262570 can increase water and sodium reabsorption in distal nephron by stimulating the ENaC and Na,K-ATPase system. This may be an important mechanism for PPARgamma agonist-induced fluid retention.

Elevated levels of the hormone resistin, which is secreted by fat cells, are proposed to cause insulin resistance and to serve as a link between obesity and type 2 diabetes. In this report we show that resistin expression is significantly decreased in the white adipose tissue of several different models of obesity including the ob/ob, db/db, tub/tub, and KKA(y) mice compared with their lean counterparts. Furthermore, in response to several different classes of antidiabetic peroxisome proliferator-activated receptor gamma agonists, adipose tissue resistin expression is increased in both ob/ob mice and Zucker diabetic fatty rats. These data demonstrate that experimental obesity in rodents is associated with severely defective resistin expression, and decreases in resistin expression are not required for the antidiabetic actions of peroxisome proliferator-activated receptor gamma agonists.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists, including the glitazone class of drugs, are insulin sensitizers that reduce glucose and lipid levels in patients with type 2 diabetes mellitus. To more fully understand the molecular mechanisms underlying their therapeutic actions, we have characterized the effects of the potent, tyrosine-based PPAR gamma ligand GW1929 on serum glucose and lipid parameters and gene expression in Zucker diabetic fatty rats. In time-course studies, GW1929 treatment decreased circulating FFA levels before reducing glucose and triglyceride levels. We used a comprehensive and unbiased messenger RNA profiling technique to identify genes regulated either directly or indirectly by PPAR gamma in epididymal white adipose tissue, interscapular brown adipose tissue, liver, and soleus skeletal muscle. PPAR gamma activation stimulated the expression of a large number of genes involved in lipogenesis and fatty acid metabolism in both white adipose tissue and brown adipose tissue. In muscle, PPAR gamma agonist treatment decreased the expression of pyruvate dehydrogenase kinase 4, which represses oxidative glucose metabolism, and also decreased the expression of genes involved in fatty acid transport and oxidation. These changes suggest a molecular basis for PPAR gamma-mediated increases in glucose utilization in muscle. In liver, PPAR gamma activation coordinately decreased the expression of genes involved in gluconeogenesis. We conclude from these studies that the antidiabetic actions of PPAR gamma agonists are probably the consequence of 1) their effects on FFA levels, and 2), their coordinate effects on gene expression in multiple insulin-sensitive tissues.

The discovery that peroxisome proliferator-activated receptor (PPAR)-gamma was the molecular target of the thiazolidinedione class of antidiabetic agents suggested a key role for PPAR-gamma in the regulation of carbohydrate and lipid metabolism. Through the use of high-throughput biochemical assays, GW1929, a novel N-aryl tyrosine activator of human PPAR-gamma, was identified. Chronic oral administration of GW1929 or troglitazone to Zucker diabetic fatty (ZDF) rats resulted in dose-dependent decreases in daily glucose, free fatty acid, and triglyceride exposure compared with pretreatment values, as well as significant decreases in glycosylated hemoglobin. Whole body insulin sensitivity, as determined by the euglycemic-hyperinsulinemic clamp technique, was significantly increased in treated animals. Comparison of the magnitude of glucose lowering as a function of serum drug concentrations showed that GW1929 was 2 orders of magnitude more potent than troglitazone in vivo. These data were consistent with the relative in vitro potencies of GW1929 and troglitazone. Isolated perfused pancreas studies performed at the end of the study confirmed that pancreata from vehicle-treated rats showed no increase in insulin secretion in response to a step change in glucose from 3 to 10 mmol/l. In contrast, pancreata from animals treated with GW1929 showed a first- and second-phase insulin secretion pattern. Consistent with the functional data from the perfusion experiments, animals treated with the PPAR-gamma agonist had more normal islet architecture with preserved insulin staining compared with vehicle-treated ZDF rats. This is the first demonstration of in vivo efficacy of a novel nonthiazolidinedione identified as a high-affinity ligand for human PPAR-gamma. The increased potency of GW1929 compared with troglitazone both in vitro and in vivo may translate into improved clinical efficacy when used as monotherapy in type 2 diabetic patients. In addition, the significant improvement in daily meal tolerance may impact cardiovascular risk factor management in these patients.

We have identified a novel series of antidiabetic N-(2-benzoylphenyl)-L-tyrosine derivatives which are potent, selective PPARgamma agonists. Through the use of in vitro PPARgamma binding and functional assays (2S)-3-(4-(benzyloxy)phenyl)-2-((1-methyl-3-oxo-3-phenylpropenyl)+ ++amin o)propionic acid (2) was identified as a structurally novel PPARgamma agonist. Structure-activity relationships identified the 2-aminobenzophenone moiety as a suitable isostere for the chemically labile enaminone moiety in compound 2, affording 2-((2-benzoylphenyl)amino)-3-(4-(benzyloxy)phenyl)propionic acid (9). Replacement of the benzyl group in 9 with substituents known to confer in vivo potency in the thiazolidinedione (TZD) class of antidiabetic agents provided a dramatic increase in the in vitro functional potency and affinity at PPARgamma, affording a series of potent and selective PPARgamma agonists exemplified by (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(methylpyridin-2-ylamino+ ++)ethoxy ]phenyl¿propionic acid (18), 3-¿4-[2-(benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propanoic acid (19), and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (20). Compounds 18 and 20 show potent antihyperglycemic and antihyperlipidemic activity when given orally in two rodent models of type 2 diabetes. In addition, these analogues are readily prepared in chiral nonracemic fashion from L-tyrosine and do not show a propensity to undergo racemization in vitro. The increased potency of these PPARgamma agonists relative to troglitazone may translate into superior clinical efficacy for the treatment of type 2 diabetes.

Bladder afferent nerve activity was recorded from the pelvic and hypogastric nerves of rats anesthetized with pentobarbital sodium. Bladder filling with isotonic NaCl at a rate of 250 microl/min excited multiunit afferent activity in the hypogastric nerve by 190 +/- 38% over background at a pressure of 30 mmHg, whereas 150 meq/l KCl at the same filling rate excited hypogastric nerve activity by 498 +/- 103% (P < 0.0001). This difference was localized to a group of chemosensitive fibers that are excited by bladder filling with KCl in a concentration-dependent fashion but are insensitive to bladder filling with NaCl. Bladder filling with 200 meq/l KCl at different filling rates caused a bursting pattern of discharge in these fibers at consistent bladder volumes but with a pressure threshold that increased proportionately with filling rate. Other hypogastric bladder afferent fibers were activated to a similar extent by NaCl and KCl solutions. Chemoreceptive bladder afferent fibers were rare in the pelvic nerve (1 of 15 units), and multiunit preparations showed comparable excitations during bladder filling with NaCl and KCl solutions. The bursting activation of bladder chemoreceptive afferent nerves in hypogastric nerves could signal noxious overdistension and/or inflammation of the bladder.

The hemodynamic effects of neuropeptide Y (NPY) were examined over a dose range of 0.03-30 nmol/kg, i.v., in anesthetized open-chest, ventilated dogs with and without ganglionic blockade. In normal (non-ganglion-blocked) animals, NPY produced significant, dose-dependent, and sustained (lasting 15-45 min) increases in mean arterial blood pressure and systemic vascular resistance (SVR) with a threshold dose of 0.3 nmol/kg and a maximum effective dose of 10 nmol/kg. Cardiac index (CI) decreased at doses > 1 nmol/kg, but stroke volume was not altered; heart rate (HR) decreased significantly at and above the 3 nmol/kg dose. No significant changes were observed in the left ventricular dP/dt (LVdP/dt) or the contractility index (LVdP/dt divided by systolic pressure). In ganglion-blocked animals, pressor and SVR responses to NPY were similar to those seen in normal animals but HR was not affected and a small but significant decrease in CI was seen only at the 30 nmol/kg. Furthermore, whereas LVdP/dt of ganglion-blocked dogs increased significantly at and above the 1 nmol/kg dose, the contractility index increased slightly only with the 10 and 30 nmol/kg doses. These data indicate that NPY produces sustained hypertension in dogs secondary to peripheral vasoconstriction, has a weak, direct positive inotropic action on the heart, and lacks chronotropic effects.

Defining the roles of the vasoconstrictor peptide neuropeptide Y (NPY) in the cardiovascular system is difficult due to lack of availability of specific NPY receptor antagonists. We report the in vivo NPY receptor blocking actions of a novel nonapeptide dimer, 1229U91 {(IleGluProDprTyrArgLeuArgTyrNH(2)(2)}, and describe its hemodynamic effects. In anesthetized normotensive rats, 1229U91 produced significant and dose-dependent reductions in NPY-reduced hemodynamic responses. 1229U91 (3-30 nmol/kg intravenously, i.v.) attenuated the pressor response (34 +/- 6-84 +/- 1%) and the increases in renal vascular resistance (RVR, 56 +/- 9-94 +/- 2%) produced by NPY (1 nmol/kg i.v.). Intravenous norepinephrine (NE)-induced hemodynamic responses were not altered by 1229U91. 1229U91 also produced dose-dependent inhibition of NPYinduced vasoconstrictor responses in anesthetized dogs and spontaneously hypertensive rats (SHR). These data demonstrate that 1229U91 is a selective NPY receptor antagonist. 1229U91 had no effect on resting hemodynamic variables in these preparations. In conscious SHR, 1229U91 did not produce significant changes in blood pressure (BP) or heart rate (HR) over a wide dose-range (15-1,500 nmol/kg i.v.). Lack of effect of the NPY receptor antagonist in SHR suggests that NPY does not contribute to the maintenance of BP in this hypertension model.

Acute left renal denervation in anesthetized volume-expanded rats produced an ipsilateral diuresis and natriuresis in 19 animals. A simultaneous decrease of glomerular filtration rate, p-aminohippurate clearance, urinary volume (P < 0.002), and percentage of filtered sodium excreted (4.0 +/- 0.6 (SE) vs. 1.9 +/- 0.4%, P < 0.003) occurred in the right innervated kidney in 10 rats. Prior denervation of the right kidney in the other nine rats prevented the renal hemodynamic changes and the fall of urinary volume and of sodium excretion (3.9 +/- 0.6 vs. 4.3 +/- 0.5%) by the right kidney after left renal denervation. Nerve traffic to the right kidney was measured in six other animals after left renal denervation and was found to increase to a mean value 33.8 +/- 6.3% above control levels (P < 0.007) 0-30 min after denervation, with a further significant increase to 66.2 +/- 16.1% above control levels (P < 0.025) 30-60 min after denervation. These results indicate that the functional changes in the right kidney after contralateral renal denervation in volume-expanded rats are caused by a reflex increase in nerve traffic to the right kidney, possibly as a consequence of an interruption of afferent nerve activity originating in the left kidney.

Acute unilateral renal denervation of control rats produced an ipsilateral diuresis (5.5 +/- 0.8 to 10.0 +/- 1.0 microliter/min, P less than 0.01) and natriuresis (579 +/- 202 to 2,668 +/- 225 neq/min, P less than 0.01) without a significant change in glomerular filtration rate or effective renal plasma flow. Inhibition of prostaglandin synthesis with indomethacin or meclofenamate (4 mg/kg iv) after acute unilateral denervation eliminated the diuresis (13.3 +/- 1.6 to 5.0 +/- 0.9 microliter/min, P less than 0.01) and attenuated the natriuresis (3,098 +/- 462 to 1,097 +/- 163 neq/min, P less than 0.01). Denervation diuresis and natriuresis were significantly impaired to the same extent when denervation was performed after inhibition of prostaglandin synthesis (3.2 +/- 0.3 to 4.9 +/- 0.4 microliter/min, NS; and 490 +/- 154 to 1,036 +/- 274 neq/min, P less than 0.05 vs. control, respectively). These results indicate that the natriuresis and diuresis seen after acute unilateral denervation in anesthetized rats are highly dependent upon prostaglandins and cannot be initiated or maintained when prostaglandin synthesis is impaired by indomethacin or meclofenamate.

Metabolism of dibutyryl cyclic AMP was studied by including the 3H- or C-labeled nucleotide (0.1 mM, 5 mumol) in the recirculating perfusate of the isolated rat kidney. Kidneys were perfused with nucleotide for 60 min. Dibutyryl cyclic AMP was almost completely cleared from the perfusate, about one-quarter as urinary excretion principally by probenecid-sensitive secretion and about one-half as metabolism beyond 3'-phosphate bond cleavage. The principal metabolite, N6-monobutyryl adenosine, accounted for one-third of added dibutyryl cyclic AMP. The remaining metabolites were ATP, ADP AMP, and N6-monobutyryl AMP. Dibutyryl cyclic AMP (0.1 or 1.0 mM) elevated renal ATP but did not alter uricogenesis. Both dibutyryl cyclic AMP and cyclic AMP at 0.2 mM produced similar activation and subcellular redistribution of renal protein kinase. N6-monobutyryl adenosine, unlike adenosine, had no effect on the renal activity of adenylate cyclase, low Km cyclic AMP phosphodiesterase, and protein kinase. Dibutyryl cyclic AMP is like exogenous cyclic AMP in that it penetrates the rat kidney, activates protein kinase, and is metabolized to ATP (R. Coulson, J. Biol. Chem. 251: 4958-4967, 1976), but is unlike cyclic AMP in its extent of secretion and metabolism to ATP and urate and in its formation of the unique metabolites N6-monobutyryl AMP and N6-monobutyryl adenosine.

Transport and metabolism of endogenous fatty acids was studied using an isolated rat kidney preparation. Results tended to confirm the inhibitory effects of probenecid on fatty acid uptake by the kidney but, at the concentration used, probenecid was ineffective in changing the distribution of 14C palmitate within tissue lipids. Alpha-bromopalmitate (alphaBP) (0.4 mM), an inhibitor of fatty acid oxidation, caused an initial enhancement of 14C palmitate uptake, a decrease in esterification into tissue phospholipids and an increase in 14C recovered as free fatty acid in the tissue. This increase had no inhibitory effect on entry of fatty acids. alpha BP had no effect on 14C octanoate uptake and appeared not to affect general metabolism. The results are consistent with the possibility that alphaBP interacts with a renal fatty acid binding protein.