Insulin resistance develops with ageing in humans and rodents. Here, we have studied the evolution of insulin sensitivity with ageing trying to discriminate the role of adiposity from that of ageing in this process. We performed oral glucose tolerance tests and determined overall and tissue-specific glucose utilization under euglycemic-hyperinsulinemic conditions in 3-, 8-, and 24-month-old rats fed ad libitum, and in 8-and 24-month-old rats after 3 months of calorie restriction. Body composition and adipocytederived cytokines such as leptin, resistin, and adiponectin were analyzed. Overall insulin sensitivity decreases with ageing. Calorie restriction improves global insulin sensitivity in 8-but not in 24-month-old rats. Insulin-stimulated glucose utilization in adipose tissues decreases in 8 months, while in oxidative muscles it reaches significance only in older rats. Calorie restriction restores adipose tissue insulin sensitivity only in 8-month-old rats and no changes are observed in muscles of 24-month-old rats. Resistin and leptin increase with ageing. Food restriction lowers resistin and increases adiponectin in 8-month-old rats and decreases leptin in both ages. Visceral and total fat increase with ageing and decrease after calorie restriction. We conclude that accretion of visceral fat plays a key role in the development of insulin resistance after sexual maturity, which is reversible by calorie restriction. With aging, accumulation of retroperitoneal and total body fat leads to impaired muscle glucose uptake and to a state of insulin resistance that is difficult to reverse.

L EPTIN, A CYTOKINE produced mainly by the white adipose tissue (WAT), is actively involved in the control of body weight and food intake. Dysregulations of leptin actions are associated with obesity, insulin resistance, and type 2 diabetes. These facts point to leptin and its actions as targets of study to clarify these metabolic disorders and identify potential therapeutic strategies. Several reports have shown that leptin regulates energy homeostasis by controlling peripheral lipid metabolism, and leptin administration reduces triacylglyceride (TAG) stores and promotes fatty acid (FA) oxidation in lean and adipose tissues (1-7). Therefore, it has been postulated that one of the roles of leptin is to reduce lipid accumulation in nonadipose tissues, preventing lipotoxicity (8).In the liver an acute iv leptin infusion decreases liver TAG secretion, increases hepatic FA oxidation and ketogenesis, and, as a result, decreases liver TAG levels (9). However, an acute intracerebroventricular (icv) leptin administration does not decrease liver TAG levels (9 -11). On the other hand, chronic icv leptin treatment decreases TAG content in liver and plasma compared with ad libitum fed controls (12), suggesting that leptin, acting at central level, plays an important role depleting TAG levels in this tissue.In WAT, the effect of leptin on lipid metabolism has not been fully characterized and remains controversial. Thus, adenovirus-induced hyperleptinemia increases the expression of enzymes of FA oxidation such as acyl-coenzyme A oxidase and carnitine palmitoyl transferase (CPT)-1, and depletes the TAG without a concomitant increase in the levels of circulating free FAs (3). This suggests that leptin favored intracellular FA oxidation in adipocytes. Furthermore, treatment of isolated rat adipocytes with leptin up-regulates the expression of acyl-coenzyme A oxidase, CPT-1, uncoupling protein 2, and peroxisome proliferator activated receptor (PPAR) ␣, all of which are involved in lipid oxidation (13). In addition, in vivo (14) and in vitro (15) studies indicated a paracrine/autocrine stimulation of lipolysis in rodent adi-

Leptin interacts with specific receptors in hypothalamic nuclei and modulates energy balance. Growing evidence has shown the association of obesity and hyperleptinaemia with non-insulin-dependent diabetes mellitus and insulin resistance. The aged Wistar rat shows peripheral insulin resistance in the absence of obesity and alterations of glucose homeostasis. However, it is not known whether, in these animals, the leptin action is altered. Here we studied the effect of ageing on plasma leptin concentration and the ability of hypothalamic nuclei to capture i.c.v.-injected digoxigenin-labelled leptin. Our data indicate that 24-month-old animals are hyperleptinaemic. However, daily food intake was greater in old animals, suggesting that they are leptin resistant. Leptin uptake in the hypothalamus was reduced in old rats. This uptake was a receptor-mediated process as demonstrated by displacement. Leptin accumulation in hypothalamic nuclei was partially colocalized with neuropeptide Y fibres. Immunohistochemical and western blot analyses showed a lower amount of the long form of leptin receptors in the hypothalamus of aged rats. Analysis by RT-PCR also demonstrated a decreased expression of leptin receptor mRNA in old animals. We conclude that the lower leptin uptake may be explained, at least in part, by a decreased amount of receptors in hypothalamic neurones of the aged rats.

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Obesity and type 2 diabetes are associated with insulin and leptin resistance, and increased ceramide contents in target tissues. Because the adipose tissue has become a central focus in these diseases, and leptin-induced increases in insulin sensitivity may be related to effects of leptin on lipid metabolism, we investigated herein whether central leptin was able to regulate total ceramide levels and the expression of enzymes involved in ceramide metabolism in rat white adipose tissue (WAT). After 7 d central leptin treatment, the total content of ceramides was analyzed by quantitative shotgun lipidomics mass spectrometry. The effects of leptin on the expression of several enzymes of the sphingolipid metabolism, sterol regulatory element binding protein (SREBP)-1c, and insulin-induced gene 1 (INSIG-1) in this tissue were studied. Total ceramide levels were also determined after surgical WAT denervation. Central leptin infusion significantly decreased both total ceramide content and the long-chain fatty acid ceramide species in WAT. Concomitant with these results, leptin decreased the mRNA levels of enzymes involved in de novo ceramide synthesis (SPT-1, LASS2, LASS4) and ceramide production from sphingomyelin (SMPD-1/2). The mRNA levels of enzymes of ceramide degradation (Asah1/2) and utilization (sphingomyelin synthase, ceramide kinase, glycosyl-ceramide synthase, GM3 synthase) were also down-regulated. Ceramide-lowering effects of central leptin were prevented by local autonomic nervous system denervation of WAT. Finally, central leptin treatment markedly increased INSIG-1 mRNA expression and impaired SREBP-1c activation in epididymal WAT. These observations indicate that in vivo central leptin, acting through the autonomic nervous system, regulates total ceramide levels and SREBP-1c proteolytic maturation in WAT, probably contributing to improve the overall insulin sensitivity.