Physiological response to long-term peripheral and central leptin infusion in lean and obese mice

Recent data have identified leptin as an afferent signal in a negative-feedback loop regulating the mass of the adipose tissue. High leptin levels are observed in obese humans and rodents, suggesting that, in some cases, obesity is the result of leptin insensitivity. This hypothesis was tested by comparing the response to peripherally and centrally administered leptin among lean and three obese strains of mice: diet-induced obese AKR/J, New Zealand Obese (NZO), and Ay. Subcutaneous leptin infusion to lean mice resulted in a dose-dependent loss of body weight at physiologic plasma levels. Chronic infusions of leptin intracerebroventricularly (i.c.v.) at doses of 3 ng/hr or greater resulted in complete depletion of visible adipose tissue, which was maintained throughout 30 days of continuous i.c.v. infusion. Direct measurement of energy balance indicated that leptin treatment did not increase total energy expenditure but prevented the decrease that follows reduced food intake. Diet-induced obese mice lost weight in response to peripheral leptin but were less sensitive than lean mice. NZO mice were unresponsive to peripheral leptin but were responsive to i.c.v. leptin. Ay mice did not respond to subcutaneous leptin and were 1/100 as sensitive to i.c.v. leptin. The decreased response to leptin in diet-induced obese, NZO, and Ay mice suggests that obesity in these strains is the result of leptin resistance. In NZO mice, leptin resistance may be the result of decreased transport of leptin into the cerebrospinal fluid, whereas in Ay mice, leptin resistance probably results from defects downstream of the leptin receptor in the hypothalamus.

Infusion of α-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb3; also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified α-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered α-gal A, (ii) to assess the pharmacokinetics of i.v.-administered α-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb3. α-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified α-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of α-gal A, nine of the 10 patients had significantly reduced Gb3 levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of α-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb3 burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.

Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1.

The Bcl-2 protein blocks programmed cell death (apoptosis) through an unknown mechanism. Previously we identified a Bcl-2 interacting protein BAG-1 that enhances the anti-apoptotic effects of Bcl-2. Like BAG-1, the serine/threonine protein kinase Raf-1 also can functionally cooperate with Bcl-2 in suppressing apoptosis. Here we show that Raf-1 and BAG-1 specifically interact in vitro and in yeast two-hybrid assays. Raf-1 and BAG-1 can also be coimmunoprecipitated from mammalian cells and from insect cells infected with recombinant baculoviruses encoding these proteins. Furthermore, bacterially-produced BAG-1 protein can increase the kinase activity of Raf-1 in vitro. BAG-1 also activates this mammalian kinase in yeast. These observations suggest that the Bcl-2 binding protein BAG-1 joins Ras and 14-3-3 proteins as potential activators of the kinase Raf-1.

Decreased food intake does not completely account for adiposity reduction after ob protein infusion.

The effects of recombinantly produced ob protein were compared to those of food restriction in normal lean and genetically obese mice. Ob protein infusion into ob/ob mice resulted in large decreases in body and fat-depot weight and food intake that persisted throughout the study. Smaller decreases in body and fat-depot weights were observed in vehicle-treated ob/ob mice that were fed the same amount of food as that consumed by ob protein-treated ob/ob mice (pair feeding). In lean mice, ob protein infusion significantly decreased body and fat-depot weights, while decreasing food intake to a much lesser extent than in ob/ob mice. Pair feeding of lean vehicle-treated mice to the intake of ob protein-treated mice did not reduce body fat-depot weights. The potent weight-, adipose-, and appetite-reducing effects exerted by the ob protein in ob protein-deficient mice (ob/ob) confirm hypotheses generated from early parabiotic studies that suggested the existence of a circulating satiety factor of adipose origin. Pair-feeding studies provide compelling evidence that the ob protein exerts adipose-reducing effects in excess of those induced by reductions in food intake.

Memory enhancement by intrahippocampal, intraamygdala, or intraentorhinal infusion of platelet-activating factor measured in an inhibitory avoidance task.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), which is thought to be a retrograde messenger in long-term potentiation (LTP), enhances glutamate release and LTP through an action on presynaptic nerve endings. The PAF antagonist BN 52021 blocks CA1 LTP in hippocampal slices, and, when infused into rat dorsal hippocampus pre- or posttraining, blocks retention of inhibitory avoidance. Here we report that memory is affected by pre- or posttraining infusion of the PAF analog 1-O-hexadecyl-2-N-methylcarbamoyl-sn-glycerol-3-phosphocholine (mc-PAF) into either rat dorsal hippocampus, amygdala, or entorhinal cortex. Male Wistar rats were implanted bilaterally with cannulae in these brain regions. After recovery from surgery, the animals were trained in step-down inhibitory avoidance or in a spatial habituation task and tested for retention 24 h later. mc-PAF (1.0 microgram per side) enhanced retention test performance of the two tasks when infused into the hippocampus before training without altering training session performance. In addition, mc-PAF enhanced retention test performance of the avoidance task when infused into (i) the hippocampus 0 but not 60 min after training; (ii) the amygdala immediately after training; and (iii) the entorhinal cortex 100 but not 0 or 300 min after training. In confirmation of previous findings, BN 52021 (0.5 microgram per side) was found to be amnestic for the avoidance task when infused into the hippocampus or the amygdala immediately but not 30 or more minutes after training or into the entorhinal cortex 100 but not 0 or 300 min after training. These findings support the hypothesis that memory involves PAF-regulated events, possibly LTP, generated at the time of training in hippocampus and amygdala and 100 min later in the entorhinal cortex.