Leptin is currently believed to control body composition largely, if not entirely, via hypothalamic receptors that regulate food intake and thermogenesis. Here we demonstrate direct extraneural effects of leptin to deplete fat content of both adipocytes and nonadipocytes to levels far below those of pairfed controls. In cultured pancreatic islets, leptin lowered triglyceride (TG) content by preventing TG formation from free fatty acids (FFA) and by increasing FFA oxidation. In vivo hyperleptinemia, induced in normal rats by adenovirus gene transfer, depleted TG content in liver, skeletal muscle, and pancreas without increasing plasma FFA or ketones, suggesting intracellular oxidation. In islets of obese Zucker Diabetic Fatty rats with leptin receptor mutations, leptin had no effect in vivo or in vitro. The TG content was Ϸ20 times normal, and esterification capacity was increased 3-to 4-fold. Thus, in rats with normal leptin receptors but not in Zucker Diabetic Fatty rats, nonadipocytes and adipocytes esterify FFA, store them as TG, and later oxidize them intracellularly via an ''indirect pathway'' of intracellular fatty acid metabolism controlled by leptin. By maintaining insulin sensitivity and preventing islet lipotoxicity, this activity of leptin may prevent adipogenic diabetes.

The endoplasmic reticulum (ER) and mitochondria accumulate Ca2+ within their lumens to regulate numerous cell functions. However, determining the dynamics of intraorganellar Ca2+ has proven to be difficult. Here we describe a family of genetically encoded Ca2+ indicators, named calcium-measuring organelle-entrapped protein indicators (CEPIA), which can be utilized for intraorganellar Ca2+ imaging. CEPIA, which emit green, red or blue/green fluorescence, are engineered to bind Ca2+ at intraorganellar Ca2+ concentrations. They can be targeted to different organelles and may be used alongside other fluorescent molecular markers, expanding the range of cell functions that can be simultaneously analysed. The spatiotemporal resolution of CEPIA makes it possible to resolve Ca2+ import into individual mitochondria while simultaneously measuring ER and cytosolic Ca2+. We have used these imaging capabilities to reveal differential Ca2+ handling in individual mitochondria. CEPIA imaging is a useful new tool to further the understanding of organellar functions.

We have studied mechanisms by which leptin overexpression, which reduces body weight via anorexic and thermogenic actions, induces triglyceride depletion in adipocytes and nonadipocytes. Here we show that leptin alters in pancreatic islets the mRNA of the genes encoding enzymes of free fatty acid metabolism and uncoupling protein-2 (UCP-2). In animals infused with a recombinant adenovirus containing the leptin cDNA, the levels of mRNAs encoding enzymes of mitochondrial and peroxisomal oxidation rose 2-to 3-fold, whereas mRNA encoding an enzyme of esterification declined in islets from hyperleptinemic rats. Islet UCP-2 mRNA rose 6-fold. All in vivo changes occurred in vitro in normal islets cultured with recombinant leptin, indicating direct extraneural effects. Leptin overexpression increased UCP-2 mRNA by more than 10-fold in epididymal, retroperitoneal, and subcutaneous fat tissue of normal, but not of leptin-receptordefective obese rats. By directly regulating the expression of enzymes of free fatty acid metabolism and of UCP-2, leptin controls intracellular triglyceride content of certain nonadipocytes, as well as adipocytes.Overexpression of leptin, the adipocyte hormone that regulates body composition through its effects on food intake and energy metabolism (1-5), causes the rapid disappearance of all grossly visible body fat, usually within 1 week (6). In addition, the triglyceride (TG) content of nonadipocyte tissues such as the pancreatic islets is profoundly reduced (7). Because these changes in body fat are unaccompanied by an increase in plasma levels of free fatty acids (FFA) and ␤-hydroxybutyrate or by ketonuria, we have concluded that the TG must have undergone internal hydrolysis and oxidation within the individual cells (8). This conclusion is supported by in vitro studies showing that leptin lowers the TG content of isolated islets by reducing esterification and by increasing oxidation of FFA (8). It is also consistent with a report that leptin decreases the mRNA and activity of acetyl-CoA carboxylase (ACC) (9), which would increase mitochondrial oxidation of FFA by lowering its product, malonyl-CoA (10).In this study we examine possible mechanisms by which leptin overexpression reduces the TG content of nonadipocytes and adipocytes to unmeasurable levels. Islets are known to express leptin receptor isoforms (7,11,12). We therefore compared in pancreatic islets isolated from hyperleptinemic rats and from appropriate euleptinemic controls the mRNA levels of enzymes involved in esterification or oxidation of FFA. In addition, to explain the thermogenic effect of leptin, we examined in both islets and white adipose tissue the mRNA of uncoupling proteins (UCP)-1 and -2, proteins believed to play an important role in regulating thermogenesis (13). White adipocytes are also known to express leptin receptors (14). MATERIALS AND METHODSAnimals. Lean wild-type (ϩ͞ϩ) male Zucker diabetic fatty (ZDF) rats and obese homozygous ( fa͞fa) male ZDF rats were bred in our laboratory from [ZDF͞Drt-fa(F10)...

Sustained hyperleptinemia of 8 ng͞ml was induced for 28 days in normal Wistar rats by infusing a recombinant adenovirus containing the rat leptin cDNA (AdCMV-leptin). Hyperleptinemic rats exhibited a 30 -50% reduction in food intake and gained only 22 g over the experimental period versus 115-132 g in control animals that received saline infusions or a recombinant virus containing the ␤-galactosidase gene (AdCMV-␤Gal). Body fat was absent in hyperleptinemic rats, whereas control rats pair-fed to the hyperleptinemic rats retained Ϸ50% body fat. Further, plasma triglycerides and insulin levels were significantly lower in hyperleptinemic versus pair-fed controls, while fatty acid and glucose levels were similar in the two groups, suggestive of enhanced insulin sensitivity in the hyperleptinemic animals. Thus, despite equivalent reductions in food intake and weight gain in hyperleptinemic and pair-fed animals, identifiable fat tissue was completely ablated only in the former group, raising the possibility of a specific lipoatrophic activity for leptin.The obesity of ob͞ob mice is the result of leptin deficiency caused by a mutation in the ob gene (1), and it responds dramatically to injection therapy with recombinant leptin administered intraperitoneally (2-4). In normal lean rats the effect of high levels of leptin is less clear; large doses of the hormone were reported to reduce food intake and body weight, but the effect was less striking than in leptin-deficient ob͞ob mice (2, 4).To obtain a more complete assessment of the effects of chronic hyperleptinemia in normal animals, we have examined food intake and body weight in normal rats in which sustained 6-to 10-fold increases in plasma leptin levels were induced by recombinant adenovirus-mediated gene delivery. In hyperleptinemic rats studied for 28 days, we observed a 30% reduction in food intake and a virtual cessation of body weight gain, accompanied by disappearance of grossly identifiable adipose tissue. In marked contrast, only partial reduction of fat depots occurred in pair-fed controls despite a similar degree of weight loss.

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