Margery Nicolson

Phenotype of the Obese Koletsky (f) Rat Due to Tyr763Stop Mutation in the Extracellular Domain of the Leptin Receptor (Lepr): Evidence for Deficient Plasma-to-CSF Transport of Leptin in Both the Zucker and Koletsky Obese Rat

The obese phenotypes of the diabetes (db) mouse and fatty (fa) rat are due to functional null mutations of the leptin receptor (Lepr). The recessive mutation in the Koletsky (f) obese rat maps to the same genetic intervals as db and fa and fails to complement the fa mutation. Comparison of the sequence of brain Lepr cDNA from +/+ and f/f animals reveals a T2349A transversion resulting in a Tyr763Stop nonsense mutation in the gene just before the transmembrane domain. Virtual absence of Lepr mRNA in whole brain from f/f animals is consistent with the presence of a null mutation. The predicted reduced cerebrospinal fluid (CSF) transport of leptin in both f/f and fa/fa mutants is reflected in the ∼10-fold lower ratio of CSF/plasma leptin concentration in the obese versus lean animals. However, equivalent CSF leptin concentration between lean and obese rats (fa/fa, f/f) indicates that leptin can enter the CSF through a non–Lepr-mediated mechanism, which may be saturated at normal physiological plasma leptin concentration.

Heterozygosity forLepob orLeprdb affects body composition and leptin homeostasis in adult mice

In an effort to understand the genetics of human obesity, we have studied the physiology and molecular genetics of rodent models with monogenetic forms of obesity including the leptin gene-defective ( Lepob / Lepob ) and leptin receptor gene-defective ( Leprdb / Leprdb ) mouse. In the experiments reported here, we investigated the effects of heterozygosity at Lepob and Leprdb on body composition and circulating leptin concentration in +/+, Leprdb /+, and Lepob /+ adult mice to identify possible gene dosage effects of these mutations that might elucidate their physiology. Adult mice heterozygous for the Lepob or Leprdb allele had equivalent fat mass and percentage body fat, which was increased 27-47% and 23-35%, respectively, relative to +/+ littermates. Plasma leptin concentrations adjusted for fat mass were 6.5 ng/ml in the Lepob /+, 9.6 ng/ml in the +/+, and 11.5 ng/ml in the Leprdb /+ mice. Sex had no effect on plasma leptin after controlling for fat mass. These data, and data from a small number of mice heterozygous at both Lepob and Leprdb (compound heterozygotes), suggest that leptin protein produced per mass of body fat is reduced in Lepob /+ mice and that body fat is increased in Lepob /+ mice until plasma leptin concentrations reach that of a normal +/+ mouse. The elevated plasma leptin concentration in the Leprdb /+ mice suggests that LEPR may mediate autocrine suppression of Lep expression. These results raise the possibility that human mutations that have even subtle effects on the leptin/leptin receptor system in either the homozygous or heterozygous state may have significant effects on adiposity.In an effort to understand the genetics of human obesity, we have studied the physiology and molecular genetics of rodent models with monogenetic forms of obesity including the leptin gene-defective (Lep(ob)/Lep(ob)) and leptin receptor gene-defective (Lep(rdb)/Lep(rdb)) mouse. In the experiments reported here, we investigated the effects of heterozygosity at Lep(ob) and Lep(rdb) on body composition and circulating leptin concentration in +/+, Lep(rdb)/+, and Lep(ob)/+ adult mice to identify possible gene dosage effects of these mutations that might elucidate their physiology. Adult mice heterozygous for the Lep(ob) or Lep(rdb) allele had equivalent fat mass and percentage body fat, which was increased 27-47% and 23-35%, respectively, relative to +/+ littermates. Plasma leptin concentrations adjusted for fat mass were 6.5 ng/ml in the Lep(ob)/+, 9.6 ng/ml in the +/+, and 11.5 ng/ml in the Lep(rdb)/+ mice. Sex had no effect on plasma leptin after controlling for fat mass. These data, and data from a small number of mice heterozygous at both Lep(ob) and Lep(rdb) (compound heterozygotes), suggest that leptin protein produced per mass of body fat is reduced in Lep(ob)/+ mice and that body fat is increased in Lep(ob)/+ mice until plasma leptin concentrations reach that of a normal +/+ mouse. The elevated plasma leptin concentration in the Lep(rdb)/+ mice suggests that LEPR may mediate autocrine suppression of Lep expression. These results raise the possibility that human mutations that have even subtle effects on the leptin/leptin receptor system in either the homozygous or heterozygous state may have significant effects on adiposity.

Effect of epidermal growth factor on ulcerative mucositis in hamsters that receive cancer chemotherapy.

Ulcerative mucositis is a common, bothersome, and dose-limiting complication of cancer chemotherapy. It has been hypothesized that mucosal susceptibility to the degenerative effects of stomatotoxic drugs is related to the renewal rate of the buccal epithelium. This study was undertaken to evaluate the effect of epidermal growth factor, a molecule known to stimulate epidermal cell division, on the course, frequency, and healing of ulcerative mucositis in an animal model. Golden Syrian hamsters were subjected to a standard mucositis-induction protocol with 5-fluorouracil. Osmotic pumps were implanted into a space between the retractor muscle and the platysma cervicale muscle, and delivered epidermal growth factor or placebo at a constant rate for 7 or 14 days. Epidermal growth factor increased oral mucosal breakdown in the face of antineoplastic therapy. The course and extent of mucositis was influenced by the timing of epidermal growth factor pump placement relative to the initiation of stomatotoxic therapy. These results support the hypothesis that the epithelial basal cell rate is one of the key elements in determining mucosal sensitivity to cancer chemotherapy.

Leptin levels and body fatness in children : Effects of gender, ethnicity, and sexual development

Leptin, a hormone secreted by adipocytes, is elevated in blood of obese adults. It is unknown whether the concentration is affected by gender, ethnicity, age, or stage of sexual maturation in children. We measured serum leptin levels in 183 children and 27 young adults using a double-antibody ELISA assay. Body fat mass (FM) and percent body fatness (%Fat) were determined by dual-energy x-ray absorptiometry. Correlations for serum leptin with FM,%Fat, and a body mass index were examined. Analyses of covariance were used to determine the effects of gender, ethnicity, and sexual maturation(Tanner stage). We found strong positive correlations (r = 0.56-0.88, p < 0.001) for serum leptin with body mass index,%Fat, and FM, which were gender-dependent (p < 0.001), but unaffected by ethnicity. At each Tanner stage, female subjects had higher serum leptin than male subjects (p < 0.001), and this difference remained significant (p < 0.001) when leptin was normalized for FM. For each gender, the mean leptin/FM ratios were relatively invariant during sexual maturation and no differences were observed between the oldest children(Tanner stage 5) and the young adults. The observation that female subjects have higher mean serum leptin and leptin/FM levels than male subjects at prepubertal ages may suggest that there are gender differences in leptin synthesis, clearance rates, bioactivity, and/or leptin transport.

NDF/heregulin stimulates the phosphorylation of Her3/erbB3

Her3/erbB3 has been identified as a third member of the epidermal growth factor receptor (EGFR) family [(1989) Proc. Natl. Acad. Sci. USA 86, 9193–9197; (1990) Proc. Natl. Acad. Sci. USA 87, 4905–4909]. The natural ligand for Her3 has not been identified. Although recently NDF has been proposed as a specific ligand for Her4 [(1993) Nature 366, 473–475; (1993) J. Biol. Chem. 268, 18407–18410], we report here that Her3 was phosphorylated on tyrosine not only in three breast carcinoma cell lines, MDAMB453, MDAMB468 and SKBR3, but also in Her3‐transfected CHO cells in response to NDF stimulation. In further studies, cells were reacted with 125I‐labeled NDF and then chemically crosslinked. Immunoprecipitation with anti‐Her3 revealed a dense high M W band, greater than 400 kDa. The results suggest that NDF may be a ligand of Her3 and induces receptor hetero‐oligomerization.

Quantitation of cytokine levels in skin graft donor site wound fluid

We quantified endogenous levels of multiple cytokines in skin graft donor site wounds in patients with small to moderate-sized burn injuries. Thirteen patients aged 11 months to 61 years with mean TBSA burn of 4 +/- 1 per cent underwent placement of occlusive wound dressings on partial skin thickness donor site wounds. Fluid was aspirated from beneath the dressing on postoperative day 1 and every subsequent 24 h until no further fluid could be obtained. Interleukin-1 alpha (IL-1) and tumour necrosis factor-alpha (TNF-alpha) levels were determined by enzyme-linked immunosorbent assay (ELISA). Epidermal growth factor (EGF), basic-fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were measured by an enzyme immunoassay (EIA). We found substantial levels of EGF and TNF-alpha in the donor site wound fluid in all 13 patients; detectable levels of bFGF in five patients; and elevated levels of IL-1 in three patients. There were no detectable levels of these cytokines in normal human serum. In contrast, there were no measurable levels of PDGF in any patients wound fluid; the mean level in serum was 1.5 ng/ml +/- 0.2 s.e.m. Studies of cytokines in the normal wound healing environment may help in the design of future therapies to augment wound healing.

Leptin Prevents Posthibernation Weight Gain But Does Not Reduce Energy Expenditure in Arctic Ground Squirrels

In mammals, leptin reduces energy intake and may increase energy expenditure as a means to maintain body weight and/or adiposity at an appropriate level. Hibernating mammals seasonally alter body mass, food intake, and body composition and, therefore, represent an attractive model for investigating the physiological regulation of changing body mass and adiposity. Previous experiments in our laboratory demonstrated that administration of mouse recombinant leptin reduces food intake and body weight in arctic ground squirrels during prehibernation fattening. In addition, leptin appeared to reduce metabolic efficiency (weight gain per unit of energy intake). This result suggests that reduced food intake alone may not account for the observed weight loss. Here, we describe the effect of a 3-week constant infusion of leptin given to posthibernation arctic ground squirrels on food consumption and energy expenditure. Mouse recombinant leptin (1 mg/ml) was administered through subcutaneously implanted mini-osmotic pumps (10 microliters/hr flow rate). Resting metabolic rate was monitored before and during the 3-week leptin administration period by indirect calorimetry. Body temperature and locomotory activity were monitored continuously by abdominal radiotransmitters. At the end of the leptin administration period, thermogenic capacity was evaluated by measuring brown fat uncoupling protein-1 mRNA and protein levels. Leptin administration resulted in reduced food intake and prevented posthibernation weight gain, but it did not alter any of the measured parameters of energy expenditure.

Relationship of the white blood cell count to body fat: role of leptin

The white blood cell (WBC) count is correlated to the amount of body fat in humans, but the mechanism for this association is unknown. Leptin, a 16 kD protein produced in adipocytes, circulates in humans in direct proportion to the amount and percentage of body fat. Recent evidence suggests that leptin and the leptin receptor are part of a novel pathway which stimulates haemopoiesis. This study was designed to test whether fasting plasma leptin concentrations contribute to the relationship between the WBC count and body fat. 117 Pima Indians with a wide range in body composition were studied. The WBC count was positively correlated with percentage of body fat (r = 0.44, P = 0.0001) and fasting plasma leptin concentration (r = 0.38, P = 0.0001). In multiple regression analyses, age, gender and percent body fat were significant independent determinants of the WBC count. After controlling for age and gender, percent body fat accounted for 23% of the variance in the WBC count (partial r = 0.48, P = 0.0001). In similar models which also included plasma leptin concentration, percent body fat remained significantly related to the WBC count, but only accounted for 7% of its variance (partial r = 0.27, P = 0.003). Based on these results, and the demonstration that leptin directly effects the stimulation of proliferation of haemopoietic stem cells in vitro, we hypothesize that the relation of the WBC count to percent body fat may be mediated, in part, through the effect of leptin.

Regulation of obese (ob) mRNA and Plasma Leptin Levels in Rhesus Monkeys EFFECTS OF INSULIN, BODY WEIGHT, AND NON-INSULIN-DEPENDENT DIABETES MELLITUS

We have cloned the rhesus monkey obese cDNA and have analyzed its expression in monkeys with a wide range of body weights (lean to very obese) and with or without non-insulin-dependent diabetes mellitus to examine the relationship of ob gene expression to obesity and non-insulin-dependent diabetes mellitus. The sequence of monkey ob protein, excluding the signal peptide, showed 91% identity with the human protein. We observed a significant correlation between the level of ob mRNA and body weight. We also found a significant relationship between ob mRNA and fasting plasma insulin concentration; however, insulin stimulation during a 100-140-min euglycemic/hyperinsulinemic clamp did not result in any changes in ob mRNA levels. Circulating levels of the ob gene product leptin were also significantly correlated with body weight. These results show that ob gene expression is related to body weight and is not acutely regulated by insulin.

Comparison of leptin protein levels in Prader-Willi syndrome and control individuals

Prader-Willi syndrome (PWS) is characterized by early childhood obesity, mental deficiency, hypogonadism, hypotonia, hypopigmentation, short stature, small hands and feet, and a characteristic face. It is the most common genetic cause of obesity and obesity is the most significant health problem for PWS patients. Ob protein (leptin), which is produced by adipose tissue, is thought to play a significant role in obesity; thus, unusually low plasma leptin levels, or relative loss of sensitivity to leptin in PWS subjects, could be an important factor in their obesity. We measured plasma leptin levels in 19 obese and 14 non-obese PWS patients [mean body mass index (BMI) 37.2 and 22.0, respectively] and compared these levels to those of 28 obese controls (mean BMI 35.5) and 16 non-obese control individuals (mean BMI 21.6). The mean plasma leptin concentration (ng/ml) for obese PWS subjects was 33.4 and 23.6 for non-obese PWS subjects. Obese control leptin was 36.2 ng/ml and non-obese control was 9.9. Among the control groups, leptin levels in females were significantly higher than those in males; the obese males and females had significantly higher leptin than their respective non-obese counterparts. These differences did not hold true for the PWS subjects. Leptin levels in obese PWS males and females were similar, and the same was true of the non-obese PWS males and females. The differences between obese and non-obese PWS subjects of both sexes were small and not significant. Comparing control groups with their PWS counterparts revealed no significant differences, with one exception: circulating plasma leptin levels in non-obese PWS males were nearly five times higher than in non-obese control males with similar BMI. This difference may reflect a more female pattern of fat distribution and hypogonadism, which are characteristic of PWS males. Leptin levels in PWS patients were not obviously correlated with the chromosome 15 finding seen in the patients.