Leptin deficiency results in a complex obesity phenotype comprising both hyperphagia and lowered metabolism. The hyperphagia results, at least in part, from the absence of induction by leptin of melanocyte stimulating hormone (MSH) secretion in the hypothalamus; the MSH normally then binds to melanocortin-4 receptor expressing neurons and inhibits food intake. The basis for the reduced metabolic rate has been unknown. Here we show that leptin administered to leptin-deficient (ob͞ob) mice results in a large increase in peripheral MSH levels; further, peripheral administration of an MSH analogue results in a reversal of their abnormally low metabolic rate, in an acceleration of weight loss during a fast, in partial restoration of thermoregulation in a cold challenge, and in inducing serum free fatty acid levels. These results support an important peripheral role for MSH in the integration of metabolism with appetite in response to perceived fat stores indicated by leptin levels.R ecent research has outlined a pathway for control of body weight (1-4): leptin, the product of the ob gene in mouse, is produced by adipocytes (5). It circulates to the hypothalamus where it binds to cells expressing the leptin receptor, the product of the db gene in mouse (6-9). Proopiomelanocortin (POMC) neurons are among the hypothalamic neurons expressing the leptin receptor (10). This leptin binding leads to the secretion of melanocyte stimulating hormone (MSH), which in turn binds to neurons expressing the melanocortin-4 receptor (MC4-R) (11); these neurons then suppress appetite (12)(13)(14). This outline is based on the phenotypes of spontaneous and induced mouse mutants (5,9,13,(15)(16)(17)(18)(19) as well as on the phenotype of homologous mutations in humans (20-24). These interpretations are in agreement that leptin is the signal from the fat stores (adipocytes) to the center, and further that MSH regulates appetite. However, there are significant aspects of the mutant phenotypes that suggest both a greater complexity of body weight homeostasis, specifically the integration of appetite and metabolism, and a factor from the central nervous system (CNS) to the periphery mediating this integration.First, pomc͞pomc mutants that completely lack POMC peptides, including MSH, show a phenotype of altered lipid metabolism in addition to hyperphagia. As the fat content of the diet increases, the mice gain weight out of proportion to their food intake (17). This shows a particular inability to use dietary fat for sustaining metabolic rate. And when these pomc͞pomc mutants are treated by peripheral administration of an ␣-MSH analog the mice lose weight and eat less, but the weight loss is much greater than the decrease in appetite (17). Again, this result is consistent with a role for MSH in mobilizing peripheral fat stores.Second, leptin-deficient mice (ob͞ob) show decreased metabolic rate (increased metabolic efficiency; ref. 25), which precedes the onset of obesity. Notably these mutants show: (i) weight gain when pair-fed with normal c...

No abstract

The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK ␣ domain and converts SK⌬59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK ␣ domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SK⌬59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SK⌬59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SK⌬59 does not protect plasmin from inhibition by ␣ 2 -antiplasmin. At the same time, SK⌬59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SK⌬59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl ؊ -deficient buffers. Taken together, these studies indicate that an intact ␣ domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact ␣ domain makes SK⌬59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by ␣ 2 -antiplasmin.

The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (␣, ␤, and ␥) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by ␣ 2 -antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase ␥؊domain, we examined the mechanism of action of a novel two-domain (␣,␤) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by ␣ 2 -antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase ␥-domain bound to the b-plasminogen⅐SUPA complex and significantly reduced these lag phases. The SUPA-b⅐plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b⅐plasmin complex also activated h-plasminogen but with a lower k cat (25-fold) and k cat /K m (7.9-fold) than SK. We conclude that a ␥-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase ␥-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.