Leptin and tumor necrosis factor-alpha induce the tyrosine phosphorylation of signal transducer and activator of transcription proteins in the hypothalamus of normal rats in vivo.

Tumor necrosis factor-alpha (TNFalpha) reduces food intake and participates in the regulation of energy homeostasis. However, TNFalpha signaling in the brain and the potential interaction with leptin have not been investigated to date. Here we studied the tyrosine phosphorylation of STAT (signal transducer and activator of transcription) proteins in the hypothalamus of normal rats after iv injection of recombinant murine leptin or TNFalpha or coinjection of both cytokines. Immunoblot analysis of hypothalamic lysates with a phospho-specific STAT3 antibody showed a 6- to 7-fold stimulation of STAT3 tyrosine phosphorylation in response to both leptin and TNFalpha. Importantly, when coinjecting both cytokines, a remarkable synergistic activation (24-fold increase in STAT3 phosphorylation) could be detected. No other STAT proteins (STAT1, STAT5) were activated by leptin, whereas TNFalpha injection resulted in a dose-dependent phosphorylation of hypothalamic STAT5. In contrast to its action in the brain, leptin was unable to produce STAT3 phosphorylation in the liver, either alone or in combination with TNFalpha. These data show that TNFalpha, independently of leptin, activates hypothalamic STAT signaling pathways and enhances leptin action at the level of STAT3. We therefore suggest that TNFalpha may represent a modulator of leptin action in the hypothalamus.

Leptin responsiveness of juvenile rats: proof of leptin function within the physiological range.

To bridge the gap between studies demonstrating leptin's role in protecting fat stores when food is scarce and other studies demonstrating the effects of treatment with leptin at doses that increase plasma levels to values found in overfed animals, we investigated whether leptin serves an adipostatic function within the normal range of free-feeding lean animals, i.e. within the very small range of endogenous plasma levels at which no leptin resistance occurs. For this purpose we applied recombinant leptin via mini-osmotic pumps to rats between 15 and 24 days of age and between 25 and 34 days of age and studied its dose-dependent effects on body mass and fat mass at plasma leptin concentrations extending down to the normal levels in lean animals. Using percentage change of fat mass (relative to that of saline-treated littermates) as the measure, a linear dose-response curve was found up to doses of 2 microg g(-1) day(-1), corresponding to plasma leptin concentrations between the normal physiological range and 50 ng ml(-1). In 15- to 24-day-old animals, analysis of the correlation (r = -0.89) between individual plasma concentrations and the corresponding leptin-induced changes of body fat content for a range extending down towards zero (i.e. towards the average fat content of the controls) yielded a zero value of 3.1 ng ml(-1), which was within the 2-4 ng ml(-1) range of plasma leptin concentrations found in the control pups. Likewise, regression analysis for the data from the 25- to 34-day-old pups (r = -0.88), for which the control range was 1-3 ng ml(-1), yielded a zero value of 1.9 ng ml(-1). We conclude that normal plasma leptin levels represent an adipostatic signal. Steady-state levels of plasma leptin in free-feeding lean animals thus provide a signal not only for protecting sufficiency but also for limiting increases of body fat stores.

Leptin suppresses semi-starvation induced hyperactivity in rats: implications for anorexia nervosa.

Semi-starvation induced hyperactivity (SIH) occurs in rodents upon caloric restriction. We hypothesized that SIH is triggered by the decline in leptin secretion associated with food restriction. To test this hypothesis, rats, which had established a stable level of activity, were treated with leptin or vehicle via implanted minipumps concomitantly to initiation of food restriction for 7 days. In a second experiment treatment was initiated after SIH had already set in. In contrast to the vehicle-treated rats, which increased their baseline activity level by 300%, the development of SIH was suppressed by leptin. Furthermore, leptin was able to stop SIH, after it had set in. These results underscore the assumed major role of leptin in the adaptation to semi-starvation. Because SIH has been viewed as a model for anorexia nervosa, we also assessed subjective ratings of motor restlessness in 30 patients with this eating disorder in the emaciated state associated with hypoleptinemia and after increments in leptin secretion brought upon by therapeutically induced weight gain. Hypoleptinemic patients ranked their motor restlessness higher than upon attainment of their maximal leptin level during inpatient treatment. Thus, hypoleptinemia might also contribute to the hyperactivity frequently associated with anorexia nervosa.

Upregulation of hepatic glucose 6-phosphatase gene expression in rats treated with an inhibitor of glucose-6-phosphate translocase.

The multicomponent hepatic glucose 6-phosphatase (Glc-6-Pase) system catalyzes the terminal step of hepatic glucose production and plays a key role in the regulation of blood glucose. We used the chlorogenic acid derivative S 3483, a reversible inhibitor of the glucose-6-phosphate (Glc-6-P) translocase component, to demonstrate for the first time upregulation of Glc-6-Pase expression in rat liver in vivo after inhibition of Glc-6-P translocase. In accordance with its mode of action, S 3483-treatment of overnight-fasted rats induced hypoglycemia and increased blood lactate, hepatic Glc-6-P, and glycogen. The metabolic changes were accompanied by rapid and marked increases in Glc-6-Pase mRNA (above 35-fold), protein (about 2-fold), and enzymatic activity (about 2-fold). Maximal mRNA levels were reached after 4 h of treatment. Glycemia, blood lactate, and Glc-6-Pase mRNA levels returned to control values, whereas Glc-6-P and glycogen levels decreased but were still elevated 2 h after S 3483 withdrawal. The capacity for Glc-6-P influx was only marginally increased after 8.5 h of treatment. Prevention of hypoglycemia by euglycemic clamp did not abolish the increase in Glc-6-Pase mRNA induced by S 3483 treatment. A similar pattern of hypoglycemia and possibly of associated counterregulatory responses elicited by treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinic acid could account for only a 2-fold induction of Glc-6-Pase mRNA. These findings suggest that the significant upregulation of Glc-6-Pase gene expression observed after treatment of rats in vivo with an inhibitor of Glc-6-P translocase is caused predominantly either by S 3483 per se or by the compound-induced changes of intracellular carbohydrate metabolism.

Leptin responsiveness and gene dosage for leptin receptor mutation (fa) in newborn rats.

To determine the degree to which the leptin receptor mutation (fa) influences the responsiveness to leptin during the first postnatal week, we injected recombinant leptin (600 pmol. g-1. day-1 sc from day 1 to day 7) into wild-type (+/+), heterozygous (+/fa), and fatty (fa/fa) rat pups. Growth and final body fat content of these leptin-treated pups were compared with those of saline-treated littermates of the same genotype. The body mass of the leptin-treated +/+ pups, but not that of the +/fa and fa/fa pups, increased more slowly than that of their respective controls, and fat content at day 7 was reduced by 37% in +/+ pups, by 22% in +/fa pups, but not at all in fa/fa pups. Plasma leptin remained excessively high throughout the day under this treatment, but a 30-fold lower leptin dose, causing only moderate changes of plasma leptin, still reduced the body fat of +/+ pups significantly. We conclude that leptin participates in the control of even the earliest stages of fat deposition and that the response to supraphysiological doses of leptin is markedly reduced in 1-wk-old pups with one fa allele and absent in pups with two fa alleles.

Chronic leptin treatment does not prevent the development of obesity in transgenic mice with brown fat deficiency.

With the exception of ob/ob mice, circulating plasma leptin is elevated in all other obese rodents as well as in obese humans, suggesting that leptin resistance rather than leptin deficiency is a characteristic feature of obesity. The exact molecular mechanisms leading to leptin resistance and the applicability of exogenous leptin to overcome resistance to the anorectic effect of the hormone, are insufficiently characterized. The aim of this study was to investigate whether chronic leptin administration could prevent the development of obesity and its associated disorders in transgenic mice with toxigene mediated ablation of brown adipose tissue (BAT). Daily injections of leptin were started at the age of 6 weeks, when body weight, food intake and plasma leptin levels of transgenics were not different from control mice. Over the next 6 weeks, leptin treated transgenics showed the same excessive body weight gain as transgenic mice injected with saline. Leptin treatment was furthermore not able to prevent the development of hyperphagia, hyperglycaemia, hyperinsulinaemia and hyperlipidaemia in transgenic mice. In contrast, control mice injected with leptin had significantly lower body weight, food intake and plasma triglycerides than those treated with saline. In summary, leptin treatment was not able to prevent the development of obesity and its associated abnormalities in transgenic mice with BAT deficiency. This data suggests that intact BAT function is of critical importance for leptin's effect on food intake and energy expenditure, and that primary dysfunction of BAT is associated with leptin resistance, even when hyperleptinaemia is not yet present.

Leptin impairs metabolic actions of insulin in isolated rat adipocytes.

Leptin is an adipocyte hormone involved in the regulation of energy homeostasis. Generally accepted biological effects of leptin are inhibition of food intake and stimulation of metabolic rate in ob/ob mice that are defective in the leptin gene. In contrast to these centrally mediated effects of leptin, we are reporting here on leptin effects on isolated rat adipocytes. Leptin impairs several metabolic actions of insulin, i.e. stimulation of glucose transport, glycogen synthase, lipogenesis, inhibition of isoproterenol-induced lipolysis, and protein kinase A activation, as well as stimulation of protein synthesis. Insulin effects were reduced by leptin (2 nM) with a half-life of about 8 h. At low leptin concentrations (<1 nM), the insulin sensitivity was reduced leading to a shift to the right in the dose-response curve. At higher concentrations the responsiveness was diminished, resulting in nearly complete inhibition of insulin effects at >30 nM leptin. The IC50 value of leptin was 3.1 +/- 1 nM after 15 h of preincubation of adipocytes in primary culture. The natural splice variant des-Gln49-leptin exhibited a significantly lower potency. Adipocytes regained full insulin sensitivity within a few hours after leptin removal. The stimulation of glucose transport by vanadate was not affected by leptin. These data show specific and potent impairment of insulin action by leptin in the physiological concentration range of both leptin and insulin, which may be related to the pathophysiology of insulin resistance in both non-insulin-dependent diabetes mellitus and obesity.

Leptin reduces juvenile fat stores by altering the circadian cycle of energy expenditure.

The recently identified hormone leptin (ob protein) secreted by white adipose tissue is widely thought to provide a feedback signal limiting fat storage by decreasing food intake. By artificially rearing leptin-treated and control littermates fed identical amounts of milk, however, we show here that lean suckling-age rats treated with recombinant murine leptin can reduce fat storage solely by increasing energy expenditure. Continuous measurements of core temperature and metabolic rate show that this increase is not uniform throughout the day but is especially prominent in the morning when rat pups usually conserve energy by entering a torpor-like state. Leptin's alleviation of hypometabolic, torpor-like states is thus not restricted to cases of impaired hormone production but seems instead to be a normal biological function independent of its effects on food intake.

Homocysteine-induced oxidative damage: mechanisms and possible roles in neurodegenerative and atherogenic processes.

Increased blood plasma concentrations of the sulphur amino acid homocysteine ("homocysteinemia") have been brought into context with neurodegenerative and arteriosclerotic symptoms and diseases. We recently reported on biochemical model reactions on the prooxidative activity of homocysteine including the desactivation of Na+/K(+)-ATPases and hemolysis of erythrocytes (Preibisch et al., 1993). In this communication we extend our model reactions including the oxidation of methionine, metabolization of pyridoxalphosphate and dihydroxyphenylalanine, desactivations of transaminases and peroxidation of low density lipoprotein.

Biochemical model reactions on the prooxidative activity of homocysteine.

The sulfur amino acid homocysteine has recently been addressed as marker for vessel damaging and atherosclerotic dispositions. The atherogenic index has been correlated with the one of cholesterol and is significantly higher in cholesterinemic as compared to normal lipidemic persons. In the present communication biochemical model reactions are presented indicating the prooxidative activity of homocysteine where a cooperative effect with the transition-metals copper and iron is indicated.

Unexpected translation initiation within the coding region of eukaryotic genes expressed in Escherichia coli.

When expressing several eukaryotic genes in Escherichia coli, we observed N-terminally truncated proteins which were attributed to translation initiation at downstream AUG codons. These AUG codons are located between 4 and 20 nucleotides 3' from sequences resembling bacterial SD elements. Although the presence of such downstream SD sequences is not sufficient for downstream initiation to occur, in two cases their removal abolishes synthesis of the truncated proteins. In one construct, a potential hairpin-loop structure is likely to inhibit translation initiation at the correct site and favor downstream initiation.

Cellular processing of pro-atrial natriuretic factor (pro-ANF): studies using an antiserum that selectively binds ANF-(99-126) after its cleavage from pro-ANF.

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a 15-17K prohormone, but circulates in plasma as a 3K, carboxy (C)-terminal fragment of the prohormone. The tissue location at which the cleavage of pro-ANF to its hormonal form occurs is unknown. In the present study, an immunological approach was taken to address this question. A polyclonal antiserum was generated which recognizes the hormonal form of ANF [ANF-(99-126)] only after its cleavage from the prohormone. This was accomplished by immunizing rabbits with a synthetic peptide corresponding to the seven amino (N)-terminal residues of ANF-(99-126) coupled to carrier protein via a C-terminal cysteine. This antiserum, anti-ANF-(99-105), demonstrated high affinity for ANF-(99-126) (IC50 = 170 pM), but displayed 100-fold less affinity for recombinant pro-ANF [ANF-(2-126)]. The N-terminal specificity of anti-ANF-(99-105) was evident by its failure to bind ANF-(103-126) at concentrations up to 100 nM. The specificity of anti-ANF-(99-105) for the hormonal form of ANF was examined by using thrombin to cleave pro-ANF and testing for the generation of anti-ANF-(99-105) immunoreactivity. Cleavage of atrial pro-ANF or 35S biosynthetically-labeled pro-ANF resulted in the production of immunoreactive material from the prohormone, whereas pro-ANF itself demonstrated no cross-reactivity with anti-ANF-(99-105). Anti-ANF-(99-105) could also recognize ANF released from the isolated perfused rat heart. When anti-ANF-(99-105) was used in immunohistochemical studies of rat atrial myocardium, no staining was observed in unfixed frozen sections. This suggests that proteolytic processing of pro-ANF is not an intracardiocytic event.

Structure and expression of the gene coding for the human serpin hLS2.

We have analyzed genomic clones encoding human leuserpin 2 (hLS2). The gene covers about 14.5 kilobases and consists of 5 exons and 4 introns. The genes coding for hLS2, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and rat angiotensinogen share an equivalent exon-intron structure and therefore constitute a distinct subgroup within the serpin gene family, which otherwise displays a highly variable exon-intron pattern. With the exception of a segment in the second exon, the sequence similarity of the genes coding for hLS2 and alpha 1-antitrypsin extends to all exons including one encoding the 5'-untranslated sequences. The implications of these findings with respect to the genesis of the amino-terminal heterogeneity in the serpin family are discussed.

Proatrial natriuretic factor is phosphorylated by rat cardiocytes in culture.

Proatrial natriuretic factor (proANF) is phosphorylated in primary cultures of neonatal rat cardiocytes. Rittenhouse et al. (Rittenhouse, J., Moberly, L., O'Donnell, M. E., Owen, N. E., and Marcus, F. (1986) J. Biol. Chem. 261, 7607-7610) observed that cyclic AMP-dependent protein kinase phosphorylated synthetic peptides related to atrial natriuretic factor (ANF) and that phosphorylated ANF peptides were more effective in stimulating Na/K/Cl cotransport in smooth muscle cells than nonphosphorylated forms. In our studies, rat cardiocytes in culture were incubated with [32P]orthophosphoric acid, and ANF-related peptides in cell extracts and culture media were isolated using antisera to ANF. Both atrial and ventricular cardiocytes contained and secreted phosphorylated proANF, a 126-amino acid precursor of ANF. Phosphorylated and nonphosphorylated isoforms of proANF were resolved by isoelectric focusing; approximately 35% of the proANF secreted by cardiocytes was phosphorylated. proANF is phosphorylated on a serine residue localized to a 42-amino acid tryptic fragment (proANF residues 26-67). We conclude that proANF is phosphorylated by rat cardiocytes but not within the portion of the molecule destined to become ANF (proANF residues 99-126). Phosphorylation may have a role in the cellular mechanisms of proANF storage and secretion or in the modulation of potential biological activities of the circulating amino-terminal portion of proANF.

Recombinational properties of the Saccharomyces cerevisiae FLP gene expressed in Escherichia coli.

The FLP gene from the 2-µm DNA of Saccharomyces cerevisiae is shown to be functionally expressed in Escherichia coli leading to site-specific intramolecular as well as intermolecular recombination between IR sequences. The expression was achieved under control of a low expression as well as a high expression E. coli promoter. The FLP gene was found to complement in trans a Flp(-) plasmid and promote its interconversion.By the use of a low Flp expression plasmid, it could be shown that the rate of interconversion of a Flp(-) plasmid by complementation in trans, was lower than that of a Flp(+) plasmid, suggesting that in addition to the IR sequences another cis-acting function exists.Expression of the FLP gene fused to the lac promoter in an in vitro system yielded two polypeptides with apparent molecular weights of 44,000 and 37,000. The 37,000 dalton polypeptide can also be produced from Flp(-) plasmids and is generated from a translation start within the FLP gene. The 44,000 dalton polypeptide is considered to represent the FLP gene product.