Central leptin gene therapy corrects skeletal abnormalities in leptin-deficient ob/ob mice

Skeletal growth is tightly coupled to energy balance via complex and incompletely understood mechanisms. Leptin-deficient ob/ob mice are obese and develop multiple pathologies associated with the metabolic syndrome. Additionally, ob/ob mice have skeletal abnormalities. The objective of this study was to evaluate the effects of leptin deficiency and long duration selective central leptin repletion via recombinant adeno-associated virus-leptin (rAAV-lep) gene therapy on bone in growing ob/ob mice. The ob/ob mice were injected in the hypothalamus with either rAAV-lep or rAAV-GFP (control vector). Treated ob/ob and untreated wild-type (WT) mice were then maintained on a normal diet for 15 weeks. In a second experiment, similarly treated mice along with a group of pair-fed mice were maintained for 30 weeks. Leptin was not detected in blood of either rAAV-lep- or rAAV-GFP-treated mice although rAAV-lep-treated mice displayed leptin transgene expression in the hypothalamus. As expected, rAAV-lep normalized body weight and food intake. Compared to WT mice, rAAV-GFP-treated ob/ob mice had decreased femoral length (by 1.6 mm or 10%, P<0.001), decreased total femur bone volume (by 3.3 mm(3) or 19%, P<0.001), but increased cancellous bone volume in the distal femur (by 0.04 mm(3) or 60%, P<0.09) and lumbar vertebrae (by 0.26 mm(3) or 118%, P<0.001). Treatment with rAAV-lep rescued the ob/ob skeletal phenotype by increasing femoral length and total bone volume, and decreasing femoral and vertebral cancellous bone volume, so that at 15 weeks post-rAAV-lep injection the ob/ob mice no longer differed from WT mice. No further skeletal changes in either the femur or lumbar vertebra were observed at 30 weeks post-rAAV-lep administration. The results suggest that hypothalamic leptin functions as an essential permissive factor for normal bone growth.     

Leptin treatment markedly increased plasma adiponectin but barely decreased plasma resistin of ob/ob mice

Adiponectin (ApN) and leptin are two adipocytokines that control fuel homeostasis, body weight, and insulin sensitivity. Their interplay is still poorly studied. These hormones are either undetectable or decreased in obese, diabetic ob/ob mice. We examined the effects of leptin treatment on ApN gene expression, protein production, secretion, and circulating levels of ob/ob mice. We also briefly tackled the influence of this treatment on resistin, another adipocytokine involved in obesity-related insulin resistance. Leptin-treated (T) obese mice (continuous sc infusion for 6 days) were compared with untreated lean (L), untreated obese (O), and untreated pair-fed obese (PF) mice. Blood was collected throughout the study. At day 3 or day 6, fat pads were either directly analyzed (mRNA, ApN content) or cultured for up to 24 h (ApN secretion). The direct effect of leptin was also studied in 3T3-F442A adipocytes. Compared with L mice, ApN content of visceral or subcutaneous fat and ApN secretion by adipose explants were blunted in obese mice. Accordingly, plasma ApN levels of O mice were decreased by 50%. Leptin treatment of ob/ob mice increased ApN mRNAs, ApN content, and secretion from the visceral depot by 50-80%. Leptin also directly stimulated ApN mRNAs and secretion from 3T3-F442A adipocytes. After 6 days of treatment, plasma ApN of ob/ob mice increased 2.5-fold, a rise that did not occur in PF mice. Plasma resistin of T mice was barely decreased. Leptin treatment, but not mere calorie restriction, corrects plasma ApN in obese mice by restoring adipose tissue ApN concentrations and secretion, at least in part, via a direct stimulation of ApN gene expression. Such a treatment only minimally affects circulating resistin. ApN restoration could, in concert with leptin, contribute to the metabolic effects classically observed during leptin administration.     

Effects of leptin replacement on hypothalamic-pituitary growth hormone axis function and circulating ghrelin levels in ob/ob mice

Leptin-deficient obese mice (ob/ob) have decreased circulating growth hormone (GH) and pituitary GH and ghrelin receptor (GHS-R) mRNA levels, whereas hypothalamic GH-releasing hormone (GHRH) and somatostatin (SST) expression do not differ from lean controls. Given the fact that GH is suppressed in diet-induced obesity (a state of hyperleptinemia), it remains to be determined whether the absence of leptin contributes to changes in the GH axis of ob/ob mice. Therefore, to study the impact of leptin replacement on the hypothalamic-pituitary GH axis of ob/ob mice, leptin was infused for 7 days (sc), resulting in circulating leptin levels that were similar to wild-type controls (approximately 1 ng/ml). Leptin treatment reduced food intake, body weight, and circulating insulin while elevating circulating n-octanoyl ghrelin concentrations. Leptin treatment did not alter hypothalamic GHRH, SST, or GHS-R mRNA levels compared with vehicle-treated controls. However, leptin significantly increased pituitary GH and GHRH-R expression and tended to enhance circulating GH levels, but this latter effect did not reach statistical significance. In vitro, leptin (1 ng/ml, 24 h) did not affect pituitary GH, GHRH-R, or GHS-R mRNA but did enhance GH release. The in vivo effects of leptin on circulating hormone and pituitary mRNA levels were not replicated by pair feeding ob/ob mice to match the food intake of leptin-treated mice. However, leptin did prevent the fall in hypothalamic GHRH mRNA and circulating IGF-I levels observed in pair-fed mice. These results demonstrate that leptin replacement has positive effects on multiple levels of GH axis function in ob/ob mice.     

A role for leptin in brain development

Leptin, the product of the obese gene, is a circulating hormone involved in feeding behavior and energy homeostasis. Ob/ob mice which are leptin deficient have many phenotypic abnormalities including brains that are smaller in both weight and cortical volume. To this end, we monitored the effects of leptin administration on brain growth. Intraperitoneal administration of leptin for 2 weeks daily to 4-week-old ob/ob mice resulted in a maximal 10% increase in both wet and dry brain weights. This increase appears to be partially the result of increased cell number as indicated by a 19% increase in total brain DNA. In summary, our data suggest that the decreased brain size of the ob/ob mouse is due to a developmental defect that can be corrected upon leptin administration and therefore leptin plays a role in brain growth and development.     

Leptin：a multifunctional hormone

Leptin is the protein product encoded by the obese(ob) gene.It is a circulating hormone produced primarily by the adipose tissue.ob/ob mice with mutations of the gene encoding leptin become morbidly obese,infertile,hyperphagic,hypothermic,and diabetic.Since the cloning of leptin in 1994,our knowledge in body weight regulation and the role played by leptin has increased substantially.We now know that leptin signals through its receptor,OB-R,which is a member of the cytokine receptor superfamily.Leptin serves as an adiposity signal to inform the brain the adipose tissue mass in a negative feedback loop regulating food intake and energy expenditure.Leptin also plays important roles in angiogenesis,immune function,fertility,and bone formation.Humans with mutations in the gene encoding leptin are also morbidly obese and respond to leptin treatment,demonstrating that enhancing or inhibiting leptin‘s activities in vivo may have potential therapeutic benefits.     

A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts.

Leptin deficiency produces a phenotype of obesity, diabetes, and infertility in the ob/ob mouse. In humans, leptin deficiency occurs in some cases of congenital obesity and in lipodystrophic disorders characterized by reduced adipose tissue and insulin resistance. Cutaneous gene therapy is considered an attractive potential method to correct circulating protein deficiencies, since gene-transferred human keratinocytes can produce and secrete gene products with systemic action. However, no studies showing correction of a systemic defect have been reported. We report the successful correction of leptin deficiency using cutaneous gene therapy in the ob/ob mouse model. As a feasibility approach, skin explants from transgenic mice overexpressing leptin were grafted on immunodeficient ob/ob mice. One month later, recipient mice reached body weight values of lean animals. Other biochemical and clinical parameters were also normalized. In a second human gene therapy approach, a retroviral vector encoding both leptin and EGFP cDNAs was used to transduce HK and, epithelial grafts enriched in high leptin-producing HK were transplanted to immunosuppressed ob/ob mice. HK-derived leptin induced body weight reduction after a drop in blood glucose and food intake. Leptin replacement through genetically engineered HK grafts provides a valuable therapeutic alternative for permanent treatment of human leptin deficiency conditions.     

Decreased p110&alpha; catalytic activity accompanies increased myocyte apoptosis and cardiac hypertrophy in leptin deficient ob/ob mice

Disruption of leptin signaling has been associated with both obesity and heart failure. We recently demonstrated that leptin deficiency in ob/ob mice and leptin insensitivity in db/db mice leads to increased myocyte apoptosis and left ventricular (LV) hypertrophy. We showed that LV mass, while similar among young ob/ob, db/db, and WT (WT) mice, is significantly higher in old ob/ob and db/db versus WT. Ob/ob and db/db mice developed markedly increased rates of myocyte apoptosis by TUNEL and activated caspase 3 levels. An intriguing candidate for the study of obesity-associated cardiac hypertrophy and apoptosis is PI3K, which functions to regulate not only cell size but also maintains cell integrity through protection from apoptosis. Here we further show that ob/ob mice have decreased catalytic activity of PI3K (p110α) which is reversed with leptin treatment. Leptin repletion in ob/ob mice reduced both myocyte apoptosis and LV hypertrophy to WT levels. We have therefore concluded that normal leptin signaling is necessary to prevent age-related myocyte apoptosis and LV hypertrophy and that PI3K is a critical component of the leptin signaling axis. The decrease in p110α catalytic activity could explain the development of increased myocyte apoptosis and cardiac hypertrophy in these obese mouse models.     

Leptin attenuates gene expression for renal 25-hydroxyvitamin D3-1alpha-hydroxylase in mice via the long form of the leptin receptor

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We previously showed that leptin administration to leptin-deficient obese (ob/ob) mice suppressed mRNA expression and activity of renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). In leptin receptor-deficient (db/db) mice, we presently examined whether leptin affects 1alpha-hydroxylase expression in renal tubules through the active form of the leptin receptor (ObRb). Elevated serum concentrations of calcium and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in untreated ob/ob mice showed sharp reduction with leptin administration (4 mg/kg, i.p. every 12h for 2 days); no such reduction of elevation occurred in db/db mice. ObRb mRNA was expressed in kidney, brain, fat, lung, and bone in wild-type and ob/ob mice, but not db/db mice. The ob/ob and db/db mice showed large increases in renal 1alpha-hydroxylase mRNA expression and activity. Leptin administration (4 mg/kg) completely abrogated these increases in ob/ob but not db/db mice. Renal 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) mRNA synthesis also was greatly elevated in ob/ob and db/db mice; excesses decreased significantly with leptin administration in ob/ob mice, but increased in db/db mice. Renal tubular cells in primary culture expressed mRNAs including proximal tubules markers (1alpha-hydroxylase and megalin), parathyroid hormone receptor, and vitamin D receptor. Calcitonin receptor mRNA, synthesized mainly in distal tubules, was scant, indicating that most cultured cells were from proximal tubules. Cells did not express ObRb mRNA. Forskolin exposure at 10(-6)M for 3 or 6h significantly increased 1alpha-hydroxylase mRNA. Leptin at 10(-6)M did not change mRNA expression in either presence or absence of forskolin. Accordingly, leptin attenuates renal 1alpha-hydroxylase gene expression through ObRb. Furthermore, leptin appears to act indirectly on renal proximal tubules to regulate 1alpha-hydroxylase gene expression.     

Oleoyl-estrone lowers the body weight of both ob/ob and db/db mice.

Homozygous obese db/db (BKS-Lepr(db) and ob/ob (B6-Lep(ob)) mice were treated for 14 days with a continuous infusion of a fat emulsion (controls) or loaded with oleoyl-estrone at doses of 12.5 and 50 nmol/g x d using surgically inserted osmotic minipumps. Treatment with oleoyl-estrone resulted in a marked decrease in body weight in both strains, compared with the unchecked growth of controls. In db/db mice, plasma urea and insulin, as well as liver lipid decreased with treatment. In ob/ob mice, the effect on insulin was more marked, in parallel with higher plasma lipids pointing to increased fat mobilisation. The results suggest that oleoyl-estrone effects on body fat reserves and insulin resistance are not mediated by leptin, since ob/ob mice lack this hormone and in the db/db it is present but cannot induce effects because of defective leptin receptors; in both cases oleoyl-estrone treatment lowers body weight.     

Effects of acute leptin administration on the differences in proton leak rate in liver mitochondria from ob/ob mice compared to lean controls.

In this investigation, the effects on proton leak of leptin administration to ob/ob mice was measured for liver mitochondria. We and others have shown that proton leak is approximately 3 times greater in liver mitochondria from ob/ob mice compared to lean controls at any given membrane potential. The results are consistent with obese mammals having higher lean mass-specific metabolic rates compared to lean controls. The increase in proton leak rate at any given membrane potential cannot be explained by the presence of free fatty acids associated with mitochondria isolated from the obese animals. The difference in proton leak must therefore represent a real difference in inner membrane permeability. Acute leptin (OB protein) administration restores the liver mitochondrial proton leak rate of ob/ob mice to that of lean controls. There was no effect on proton leak rate of liver mitochondria from sham-treated ob/ob mice. These novel results indicate a role for leptin, either directly or indirectly, in regulating the efficiency of oxidative phosphorylation.