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.     

Identification of a physiological role for leptin in the regulation of ambulatory activity and wheel running in mice

Mechanisms regulating spontaneous physical activity remain poorly characterized despite evidence of influential genetic and acquired factors. We evaluated ambulatory activity and wheel running in leptin-deficient ob/ob mice and in wild-type mice rendered hypoleptinemic by fasting in both the presence and absence of subcutaneous leptin administration. In ob/ob mice, leptin treatment to plasma levels characteristic of wild-type mice acutely increased both ambulatory activity (by 4,000 ± 200 beam breaks/dark cycle, P < 0.05) and total energy expenditure (TEE; by 0.11 ± 0.01 kcal/h during the dark cycle, P < 0.05) in a dose-dependent manner and acutely increased wheel running (+350%, P < 0.05). Fasting potently increased ambulatory activity and wheel running in wild-type mice (AA: +25%, P < 0.05; wheel running: +80%, P < 0.05), and the effect of fasting was more pronounced in ob/ob mice (AA: +400%, P < 0.05; wheel running: +1,600%, P < 0.05). However, unlike what occurred in ad libitum-fed ob/ob mice, physiological leptin replacement attenuated or prevented fasting-induced increases of ambulatory activity and wheel running in both wild-type and ob/ob mice. Thus, plasma leptin is a physiological regulator of spontaneous physical activity, but the nature of leptin's effect on activity is dependent on food availability.     

Conjugated linoleic acid fails to worsen insulin resistance but induces hepatic steatosis in the presence of leptin in ob/ob mice

Conjugated linoleic acid (CLA) induces insulin resistance preceded by rapid depletion of the adipokines leptin and adiponectin, increased inflammation, and hepatic steatosis in mice. To determine the role of leptin in CLA-mediated insulin resistance and hepatic steatosis, recombinant leptin was coadministered with dietary CLA in ob/ob mice to control leptin levels and to, in effect, negate the leptin depletion effect of CLA. In a 2 x 2 factorial design, 6 week old male ob/ob mice were fed either a control diet or a diet supplemented with CLA and received daily intraperitoneal injections of either leptin or vehicle for 4 weeks. In the absence of leptin, CLA significantly depleted adiponectin and induced insulin resistance, but it did not increase hepatic triglyceride concentrations or adipose inflammation, marked by interleukin-6 and tumor necrosis factor-alpha mRNA expression. Insulin resistance, however, was accompanied by increased macrophage infiltration (F4/80 mRNA) in adipose tissue. In the presence of leptin, CLA depleted adiponectin but did not induce insulin resistance or macrophage infiltration. Despite this, CLA induced hepatic steatosis. In summary, CLA worsened insulin resistance without evidence of inflammation or hepatic steatosis in mice after 4 weeks. In the presence of leptin, CLA failed to worsen insulin resistance but induced hepatic steatosis in ob/ob mice.     

Increased leptin expression selectively in the hypothalamus suppresses inflammatory markers CRP and IL-6 in leptin-deficient diabetic obese mice

Low-grade systemic inflammation, as indicated by increased circulating levels of inflammatory markers CRP and IL-6, is linked to increased risks for cardiovascular diseases (CVD) and diabetes mellitus in obese subjects. Whereas hyperleptinemia in obesity are associated with increased CRP and IL-6 release, the hypothalamic versus peripheral site of leptin action has not been ascertained. The effects of increased leptin supply selectively in the hypothalamus by gene therapy on pro-inflammatory CRP and IL-6 levels and on markers of diabetes in the circulation of ob/ob mice displaying either age-related or dietary obesity were assessed. A recombinant adeno-associated viral vector encoding either green-fluorescent protein (control) or leptin gene was injected intracerebroventricularly. Five weeks later, one-half of each of the vector groups was switched to high-fat diet consumption and the other half continued to consume regular low-fat chow diet. Body weight and visceral white adipose tissue were drastically reduced and hyperinsulinemia and hyperglycemia were abrogated by leptin gene therapy, independent of the dietary fat content. The elevated plasma CRP and IL-6 levels seen in obese ob/ob mice receiving the control vector, regardless of the fat content of the diet, were markedly suppressed by increased hypothalamic leptin in both groups. The results show for the first time that leptin deficiency elevates and reinstatement of leptin selectively in the hypothalamus suppresses the release of pro-inflammatory biomarkers, a response likely to alleviate CVD associated with obesity.     

Modulation of circulating leptin levels by its soluble receptor

Leptin is an adipocyte-derived hormone with potent weight reducing effects. Genetically obese rodents with mutations of leptin or the leptin receptor are defective in leptin signaling and develop morbid obesity and diabetes. Interestingly, the levels of both leptin mRNA and protein are increased by up to 20-fold in these animals, suggesting the existence of a feedback mechanism controlling the amount of leptin in circulation. In this report, we attempted to determine whether the up-regulation of circulating leptin in Zucker Diabetic Fatty rats, which are nonresponsive to leptin due to a receptor point mutation, is entirely due to increased expression of leptin. We demonstrate that the high level of circulating leptin in these rats is attributable to at least two factors: increased leptin expression by the adipose tissue and delayed clearance of leptin from circulation due to binding to its soluble receptor. The latter conclusion was supported by three lines of evidence: 1) The soluble leptin receptor is up-regulated by about 20-fold in Zucker Diabetic Fatty rats; 2) Adenovirus-mediated overexpression of the soluble leptin receptor results in a similar -fold increase of circulating leptin; 3) In ob/ob mice, which have no endogenous leptin, exogenously administered leptin reaches a higher level when the soluble leptin receptor is overexpressed. The weight-reducing effect of leptin is enhanced in C57Bl/6 ob/ob mice with overexpression of the soluble leptin receptor. Soluble leptin receptor may be a significant factor determining the amount of total leptin in circulation.     

Partial leptin deficiency favors diet-induced obesity and related metabolic disorders in mice

Partial leptin deficiency is not uncommon in the general population. We hypothesized that leptin insufficiency could favor obesity, nonalcoholic steatohepatitis (NASH), and other metabolic abnormalities, particularly under high calorie intake. Thus, mice partially deficient in leptin (ob/+) and their wild-type (+/+) littermates were fed for 4 mo with a standard-calorie (SC) or a high-calorie (HC) diet. Some ob/+ mice fed the HC diet were also treated weekly with leptin. Our results showed that, when fed the SC diet, ob/+ mice did not present significant metabolic abnormalities except for elevated levels of plasma adiponectin. Under high-fat feeding, increased body fat mass, hepatic steatosis, higher plasma total cholesterol, and glucose intolerance were observed in +/+ mice, and these abnormalities were further enhanced in ob/+ mice. Furthermore, some metabolic disturbances, such as blunted plasma levels of leptin and adiponectin, reduced UCP1 expression in brown adipose tissue, increased plasma liver enzymes, beta-hydroxybutyrate and triglycerides, and slight insulin resistance, were observed only in ob/+ mice fed the HC diet. Whereas de novo fatty acid synthesis in liver was decreased in +/+ mice fed the HC diet, it was disinhibited in ob/+ mice along with the restoration of the expression of several lipogenic genes. Enhanced expression of several genes involved in fatty acid oxidation was also observed only in ob/+ animals. Leptin supplementation alleviated most of the metabolic abnormalities observed in ob/+ fed the HC diet. Hence, leptin insufficiency could increase the risk of obesity, NASH, glucose intolerance, and hyperlipidemia in a context of calorie overconsumption.     

Both leptin and leptin-receptor are essential for apolipoprotein M expression in vivo

It was previously shown that circulating levels of leptin and apolipoprotein M (apoM) correlate to each other. In this study, we examined whether plasma leptin and leptin-receptors are of importance for apoM expression in vivo. It was found that in both liver and kidney, expression of apoM was significantly lower in leptin deficient ob/ob mice and in leptin-receptor deficient db/db mice than in control mice. Furthermore, leptin administration (0.5 or 1.5 microg/g body weight) significantly increased plasma apoM levels and apoM mRNA levels in liver and in kidney in ob/ob mice. We conclude that both leptin and leptin-receptor are essential for the apoM expression, indicating that leptin is physiologically regulating apoM synthesis in vivo.     

Cloning the chicken leptin gene

Chicken is characterized by a relative insulin resistance and a physiological hyperglycemia (2g/L) and is also subjected to fattening. Fat deposits in chicken, as in mammals, are regulated by environmental and genetic factors. In mammals, leptin, an adipose cell-specific secreted protein has been characterized that is encoded by ob gene. Leptin regulates satiety through hypothalamic specific receptors, energy balance, energy efficiency and contributes to adaptation to starvation. The leptin gene has been characterized in various mammalian species, and the cloning and sequencing of the chicken leptin gene (ob gene) are reported. Using RT-PCR and primers flanking the coding region of the leptin gene selected from known mammalian sequences, we have successfully amplified a 600-bp fragment from chicken liver and adipose tissue total ARNs. The amplified fragment exhibits a similar size to that of the coding region of the mammalian leptin gene. The sequences of the coding region of chicken liver and adipose tissue are identical and presented 97%, 96% and 83% similarity to the mouse, rat and human sequences, respectively. Finally, this is the first report showing that leptin gene expression in chicken is not exclusively localized in adipose tissue but is also expressed in liver. The expression of leptin in liver may be associated with a key role of this organ in avian species in controlling lipogenesis.     

Diet-induced obesity causes severe but reversible leptin resistance in arcuate melanocortin neurons

Despite high leptin levels, most obese humans and rodents lack responsiveness to its appetite-suppressing effects. We demonstrate that leptin modulates NPY/AgRP and alpha-MSH secretion from the ARH of lean mice. High-fat diet-induced obese (DIO) mice have normal ObRb levels and increased SOCS-3 levels, but leptin fails to modulate peptide secretion and any element of the leptin signaling cascade. Despite this leptin resistance, the melanocortin system downstream of the ARH in DIO mice is over-responsive to melanocortin agonists, probably due to upregulation of MC4R. Lastly, we show that by decreasing the fat content of the mouse's diet, leptin responsiveness of NPY/AgRP and POMC neurons recovered simultaneously, with mice regaining normal leptin sensitivity and glycemic control. These results highlight the physiological importance of leptin sensing in the melanocortin circuits and show that their loss of leptin sensing likely contributes to the pathology of leptin resistance.     

Epitope mapping of secreted mouse leptin utilizing peripherally administered synthetic peptides

We have recently reported that intraperitoneal (i.p.) administration of synthetic peptide amides corresponding to amino acids 106-140 of mouse leptin significantly reduced food intake and body weight gain in female C57BL/6J ob/ob mice. These results suggested that leptin activity was localized in domains toward its C-terminus between residues 106-140. In the present study, 14 overlapping peptides encompassing the complete sequence of secreted mouse leptin were synthesized, and their effects on body weight and food intake in female C57BL/6 J ob/ob mice were assessed. When given as seven daily 1-mg i.p. injections, only peptides corresponding to amino acids 106-120, 116-130 and 126-140 caused significant reductions in body weight and food intake. These results confirmed our earlier study and suggest that in contrast to the domain encompassed by amino acids 106-140, the N-terminal of mouse leptin between amino acids 21-105 may not contain functional epitopes that can be utilized as lead compounds in the development of peripherally administered bioactive peptide analogs or nonpeptide mimetics of leptin, which may have potential usefulness in treatment of the energy imbalance associated with obesity.     

Defects in the leptin axis reduce abundance of the ABCG5-ABCG8 sterol transporter in liver

ABGG5 (G5) and ABCG8 (G8) are ABC half-transporters that dimerize within the endoplasmic reticulum, traffic to the cell surface, and mediate cholesterol excretion into bile. Mice harboring defects in the leptin axis (db/db and ob/ob) have reduced biliary cholesterol concentrations. Rapid weight loss brought about by administration of leptin or dietary restriction increases biliary cholesterol excretion. We hypothesized that the reduction in biliary cholesterol in mice harboring defects in the leptin axis is associated with a reduction in G5G8 transporters and that levels of the transporter would increase with leptin administration and dietary restriction. We examined mRNA and protein levels for G5 and G8 in db/db and ob/ob mice. In both models G5 and G8 protein levels were reduced. In ob/ob mice, both leptin administration and dietary restriction increased G5 and G8 protein and biliary cholesterol concentrations. Finally, we examined the effects of tauroursodeoxycholate, which has been shown to increase biliary cholesterol excretion and function as a molecular chaperone. Tauroursodeoxycholate increased G5 and G8 protein and biliary cholesterol concentrations in both wild-type and db/db mice. Our results indicate that the mechanism for reduced biliary cholesterol excretion in db/db and ob/ob mice involves reductions in G5 and G8 protein levels and that this may occur at the level of G5G8 heterodimer assembly within the endoplasmic reticulum.     

Obesity-induced upregulation of myocardial endothelin-1 expression is mediated by leptin

Several studies have shown that leptin, the product of the obese gene, may link obesity with cardiovascular diseases, and in particular with cardiac hypertrophy. In vitro studies suggest that the mechanism by which leptin causes cardiac hypertrophy involves the upregulation of endogenous endothelin-1 (ET-1), a potent vasoconstrictor and mitogen. Whether obesity-associated hyperleptinemia causes an increase in myocardial ET-1 expression in vivo remains unclear. To address this issue, we fed mice with a high-fat diet and analyzed serum levels of ET-1 and ET-1 mRNA in the heart. We found that in mice fed a high-fat diet, serum ET-1, myocardial ET-1, leptin and leptin receptor mRNA were all elevated. In contrast, in leptin-deficient obese (ob/ob) mice, both serum and myocardial ET-1 levels were not higher than in wild type mice. These findings suggest that upregulation of myocardial ET-1 by obesity is mediated by leptin.     

Effects of intranasal administration of a leptin-secreting Lactococcus lactis recombinant on food intake, body weight, and immune response of mice

Leptin is an adipocyte-derived pleiotropic hormone that modulates a large number of physiological functions, including control of body weight and regulation of the immune system. In this work, we show that a recombinant strain of the food-grade lactic acid bacterium Lactococcus lactis (LL-lep) can produce and efficiently secrete human leptin. The secreted leptin is a fully biologically active hormone, as demonstrated by its capacity to stimulate a STAT3 reporter gene in HEK293 cells transfected with the Ob-Rb leptin receptor. The immunomodulatory activity of leptin-secreting L. lactis was evaluated in vivo by coexpression with the human papillomavirus type 16 E7 protein. In C57BL/6 mice immunized intranasally with a recombinant L. lactis strain coproducing leptin and E7 antigen, the adaptive immune response was significantly higher than in mice immunized with recombinant L. lactis producing only E7 antigen, demonstrating adjuvanticity of leptin. We then analyzed the effects of intranasally administered LL-lep in obese ob/ob mice. We observed that daily administration of LL-lep to these mice significantly reduced body weight gain and food intake. These results demonstrate that leptin can be produced and secreted in an active form by L. lactis and that leptin-producing L. lactis regulates in vivo antigen-specific immune responses, as well as body weight and food consumption.     

Leptin selectively augments thymopoiesis in leptin deficiency and lipopolysaccharide-induced thymic atrophy

The thymus is a lymphoid organ that selects T cells for release to the peripheral immune system. Unfortunately, thymopoiesis is highly susceptible to damage by physiologic stressors and can contribute to immune deficiencies that occur in a variety of clinical settings. No treatment is currently available to protect the thymus from stress-induced involution. Leptin-deficient (ob/ob) mice have severe thymic atrophy and this finding suggests that this hormone is required for normal thymopoiesis. In this study, the ability of leptin to promote thymopoiesis in wild-type C57BL/6 and BALB/c mice, as well as in leptin-deficient (ob/ob) and endotoxin-stressed (Escherichia coli LPS) mice, was determined. Leptin administration induced weight loss and stimulated thymopoiesis in ob/ob mice, but did not stimulate thymopoiesis in wild-type C57BL/6 nor BALB/c mice. In endotoxin-stressed mice, however, leptin prevented LPS-induced thymus weight loss and stimulated TCRalpha gene rearrangement. Coadministration of leptin with LPS blunted endotoxin-induced systemic corticosterone response and production of proinflammatory cytokines. Thus, leptin has a selective thymostimulatory role in settings of leptin deficiency and endotoxin administration, and may be useful for protecting the thymus from damage and augmenting T cell reconstitution in these clinical states.     

Restoring leptin signaling reduces hyperlipidemia and improves vascular stiffness induced by chronic intermittent hypoxia

Chronic intermittent hypoxia (IH) during sleep can result from obstructive sleep apnea (OSA), a disorder that is particularly prevalent in obesity. OSA is associated with high levels of circulating leptin, cardiovascular dysfunction, and dyslipidemia. Relationships between leptin and cardiovascular function in OSA and chronic IH are poorly understood. We exposed lean wild-type (WT) and obese leptin-deficient ob/ob mice to IH for 4 wk, with and without leptin infusion, and measured cardiovascular indices including aortic vascular stiffness, endothelial function, cardiac myocyte morphology, and contractile properties. At baseline, ob/ob mice had decreased vascular compliance and endothelial function vs. WT mice. We found that 4 wk of IH decreased vascular compliance and endothelial relaxation responses to acetylcholine in both WT and leptin-deficient ob/ob animals. Recombinant leptin infusion in both strains restored IH-induced vascular abnormalities toward normoxic WT levels. Cardiac myocyte morphology and function were unaltered by IH. Serum cholesterol and triglyceride levels were significantly decreased by leptin treatment in IH mice, as was hepatic stearoyl-Coenzyme A desaturase 1 expression. Taken together, these data suggest that restoring normal leptin signaling can reduce vascular stiffness, increase endothelial relaxation, and correct dyslipidemia associated with IH.     

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.     

Effects of obesity on the relationship of leptin mRNA expression and adipocyte size in anatomically distinct fat depots in mice

In support of leptin's physiological role as humoral signal of fat mass, we have shown that adipocyte volume is a predominant determinant of leptin mRNA levels in anatomically distinct fat depots in lean young mice in the postabsorptive state. In this report, we investigated how obesity may affect the relationship between leptin mRNA levels and adipocyte volume in anatomically distinct fat depots in mice with genetic (Lep(ob)/Lep(ob) and A(y)/+), diet-induced, and aging-related obesity. In all of the obese mice examined, tissue leptin mRNA levels relative to the average adipocyte volume were lower in the perigonadal and/or retroperitoneal than in the inguinal fat depots and were lower than those of the lean young mice in the perigonadal fat depot. A close, positive correlation between leptin mRNA level and adipocyte volume was present from small to hypertrophic adipocytes within each perigonadal and inguinal fat pad in the obese mice, but the slopes of the regression lines relating leptin mRNA level to adipocyte volume were significantly lower in the perigonadal than in the inguinal fat pads of the same mice. These results suggest that obesity per se is associated with a decreased leptin gene expression per unit of fat mass in mice and that the positive correlation between leptin mRNA level and adipocyte volume is an intrinsic property of adipocytes that is not disrupted by adipocyte hypertrophy in obese mice.     

Luminal leptin activates mucin-secreting goblet cells in the large bowel

Leptin has been suggested to be involved in tissue injury and/or mucosal defence mechanisms. Here, we studied the effects of leptin on colonic mucus secretion and rat mucin 2 (rMuc2) expression. Wistar rats and ob/ob mice were used. Secretion of mucus was followed in vivo in the rat perfused colon model. Mucus secretion was quantified by ELISA, and rMuc2 mRNA levels were quantified by real-time RT PCR. The effects of leptin alone or in association with protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) inhibitors on mucin secreted by human mucus-secreting HT29-MTX cells were determined. Leptin was detected in the rat colonic lumen at substantial levels. Luminal perfusion of leptin stimulates mucus-secreting goblet cells in a dose-dependent manner in vivo in the rat. Leptin (10 nmol/l) increased mucus secretion by a factor of 3.5 and doubled rMuc2 mRNA levels in the colonic mucosa. There was no damage to mucosa 24 h after leptin, but the number of stained mucus cells significantly increased. Leptin-deficient ob/ob mice have abnormally dense mucus-filled goblet cells. In human colonic goblet-like HT29-MTX cells expressing leptin receptors, leptin increased mucin secretion by activating PKC- and PI3K-dependent pathways. This is the first demonstration that leptin, acting from the luminal side, controls the function of mucus-secreting goblet cells. Because the gel layer formed by mucus at the surface of the intestinal epithelium has a barrier function, our data may be relevant physiologically in defence mechanisms of the gastrointestinal tract.     

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.