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The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin's biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin's effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13 C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring f lux of 13 C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate f lux into 13 C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of longchain fatty acid synthesis from 13 C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13 C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13 C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13 C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.Leptin's ability to normalize food intake is mediated largely by receptors in the hypothalamus (1). The ob/ob mouse model of obesity and insulin resistance has an intact leptin receptor, but is leptin deficient owing to mutation of the ob gene (2). In the ob/ob mouse the lipid-depletion effects of chronic leptin treatment exceed that explained by reduced food intake and weight loss alone (3, 4). Discovery of leptin receptors in peripheral tissue (1, 5) raises questions about which of leptin's biological actions arise from direct effects of the hormone on extraneural tissues, and what intracellular mechanisms are responsible for leptin's effects on carbohydrate and lipid metabolism. Using nondestructive 13 C NMR methodology to follow the kinetics of intermediary metabolism, we define the action of leptin directly on liver. By monitoring flux through several multistep pathways simultaneously in the intact hepatocyte, 13 C NMR spectroscopy helped identify the metabolic pathways controlled; assays of enzymic activities supported the NMR findings. We found that leptin up-regulates hepatic glycogen synthesis in ob/ob mouse liver with both acute and chronic treatment. Simultaneously, we found that acut...

The triple quantum filtered 23Na NMR method is applied here to measure the effects of EIPA, a specific inhibitor of the Na+/H+ antiporter, on relative intracellular sodium concentrations in isolated working hearts at baseline, during ischemia, and at subsequent reperfusion. In analogy to the spectrophotometric isosbestic point, an approach is developed that defines a value of tau at which the effect of the relaxation times on the TQF signal intensities is minimized, and the signals are proportional to the sodium concentration for both ischemic and working hearts. EIPA at 1.5 microM significantly inhibited (P < 0.01) the influx of intracellular Na+ during 20 min of ischemia at 36.2 degrees C in this rat heart model. In parallel 31P NMR studies, EIPA had no effect on either the development of acidosis during ischemia or on the recovery of pHi during reperfusion despite its profound effect on intracellular Na+ influx. Thus, under our conditions the Na+/H+ antiporter did not play a critical role in the maintenance of intracellular pH. EIPA treatment resulted in improved recovery (P < 0.005) of mechanical function after 20 min of ischemia. [ATP] was higher in treated hearts during ischemia and reperfusion.

To assess the role of angiotensin II (AII) in the development of myocardial dysfunction during ischemia and reperfusion, the effects of either oral pretreatment with 1 mg/kg losartan or treatment with 4.5 mu M losartan in vitro were compared with effects measured in the respective placebo or in vitro control groups in an isolated rat working-heart model. Both groups treated with losartan showed significant improvement (p < 0.005) in functional recovery following 20 min of ischemia compared with the respective control groups. Coronary flow (CF) and cardiac output (CO) were also significantly increased during reperfusion in the drug treatment groups compared with controls (p < 0.05 to p < 0.001). The recovery of mechanical function, CO, and CF was significantly more rapid in hearts from rats treated orally with losartan than in hearts treated with losartan in vitro. As measured by 31P-nuclear magnetic resonance, the changes observed in ATP levels and in intracellular pH during ischemia and reperfusion were essentially the same under either treatment regimen. This article describes the initial observation of a significant reduction in myocardial dysfunction during reperfusion following 20 min of global ischemia in the isolated perfused heart as a result of acute AII AT1 receptor antagonism by losartan administered either directly in vitro or by oral pretreatment.

Four new azasteroid inhibitors of steroid 5 alpha-reductase were compared to the benchmark compound finasteride, each at a dose level of 1 mg/kg/day, as well to placebo and to castration, in seven groups of mature male beagle dogs with enlarged prostates. Prostate volumes were measured repetitively by a volume MRI method over 15 weeks of treatment. The study probed the obverse of the familiar relation between DHT and prostate growth, and provides the first documentation of a tight negative correlation between prostate regression and the prostatic concentration of DHT across a range of treatment regimens (r = -0.982). In this first direct comparison study of structure vs. in vivo activity for several azasteroids in the dog model of BPH, relative efficacy for induction of shrinkage of the dog prostate did not correlate at all with the inhibitor's relative activity against the dog 5 alpha-reductase in vitro. On the basis of the relative IC50 values it would not have been predicted that, at the dose tested, the analogue MK-434 (17 beta-benzoyl-4-aza-5 alpha-androst-1-en-3-one) was distinguished from the other inhibitors with respect to the induction of faster and more complete regression (69%) as well as greater reduction in prostatic DHT (95%), both of which approached the castrated dog levels of 75% prostatic shrinkage and > 98% reduction in DHT. Treatment with any one of the five azasteroids induced two- to five-fold increases in prostatic testosterone. However, total androgen was conserved at the placebo control level. Despite the differences noted, each azasteroid tested induced a highly significant decrease in prostatic volume that correlated tightly with a decreased prostatic DHT level in canine spontaneous BPH.