Leptin Signaling in Adipose TissueNovelty and Significance
Role in Lipid Accumulation and Weight Gain
Rationale: The link between obesity, hyperleptinemia, and development of cardiovascular disease is not completely understood. Increases in leptin have been shown to impair leptin signaling via caveolin-1–dependent mechanisms. However, the role of hyperleptinemia versus impaired leptin signaling in adipose tissue is not known.
Objective: To determine the presence and significance of leptin-dependent increases in adipose tissue caveolin-1 expression in humans.
Methods and Results: We designed a longitudinal study to investigate the effects of increases in leptin on adipose tissue caveolin-1 expression during weight gain in humans. Ten volunteers underwent 8 weeks of overfeeding, during which they gained an average weight of 4.1±1.4 kg, with leptin increases from 7±3.8 to 12±5.7 ng/mL. Weight gain also resulted in changes in adipose tissue caveolin-1 expression, which correlated with increases in leptin (rho=0.79, P=0.01). In cultured human white preadipocytes, leptin increased caveolin-1 expression, which in turn impaired leptin cellular signaling. Functionally, leptin decreased lipid accumulation in differentiating human white preadipocytes, which was prevented by caveolin-1 overexpression. Further, leptin decreased perilipin and fatty acid synthase expression, which play an important role in lipid storage and biogenesis.
Conclusions: In healthy humans, increases in leptin, as seen with modest weight gain, may increase caveolin-1 expression in adipose tissue. Increased caveolin-1 expression in turn impairs leptin signaling and attenuates leptin-dependent lowering of intracellular lipid accumulation. Our study suggests a leptin-dependent feedback mechanism that may be essential to facilitate adipocyte lipid storage during weight gain.
Leptin is an important mediator of pathophysiological outcomes in obesity.1 Centrally, leptin plays an important role in maintaining energy homeostasis; therefore, the presence of high leptin in obesity has been suggested as evidence of resistance to central leptin actions. However, what is not clear is whether it is high leptin or impaired leptin signaling in peripheral tissue that contributes to development of obesity-related disorders.1–4 Because adipose tissue plays a role in development of metabolic and cardiovascular disease in obesity, the mechanistic role of leptin in adipose tissue during weight gain is of importance.5 Further, even though the presence of the leptin receptor on adipocytes is well-known, we have little understanding of the role of leptin in human adipose tissue.6 Several studies have indicated that leptin regulates lipid metabolism via modulation of lipid oxidation, lipid lysis, and lipid biogenesis, but these studies are limited to animals and nonadipose tissues.7 Therefore, to understand the implications of increases in leptin during weight gain in human adipose tissue, we investigated the role of leptin in regulation of lipid metabolism in cultured differentiating human preadipocytes. We tested the hypothesis that increases in leptin occurring with weight gain in humans would cause increased caveolin-1 expression in adipose tissue and, in turn, impair leptin signaling.
Detailed Methods are provided in the Online Supplement. Briefly, we recruited 10 healthy volunteers (7 men and 3 women) aged 23 to 36 years. The volunteers were overfed to increase weight gradually by 5% during an 8-week period. Measurements and adipose tissue biopsies were performed at baseline and after weight gain. The protocol was approved by the Institutional Review Board and informed consent was obtained. In vitro experiments were performed using human white preadipocytes (HWPs; PromoCell).
Effects of Overfeeding on Study Participants
The characteristics of the study participants at baseline and after weight gain are presented in Table. On average, the participants gained 4.1±1.4 kg during the 8-week period of overfeeding. The weight gain was a result of increased fat mass; lean mass did not change. Among the variables measured, only leptin increased significantly with weight gain.
The changes in adipose tissue caveolin-1 expression during weight gain were measured by Western blot analysis. Subjects with the highest leptin increases showed the greatest change in adipocyte caveolin-1 expression and subjects with relatively small increase in leptin showed decreases in caveolin-1 expression (Figure 1A). Also, subjects with smaller increases in leptin with weight gain had higher leptin and adipose tissue caveolin-1 expression at baseline. To test the predictors of adipose tissue caveolin-1 expression, we determined changes in caveolin-1 expression and its relationship with changes in other variables during weight gain (Online Table I). Changes in leptin significantly predicted the changes in caveolin-1 expression (rho=0.79; P=0.01; Figure 1B), suggesting that leptin regulates adipose tissue caveolin-1 expression in vivo. Because there was a very wide range of changes in leptin (8%–157% increase) with weight gain, there was a similarly variable response in caveolin-1 changes, and the group data from overall changes in adipose tissue caveolin-1 expression did not reach significance (Figure 1C). Additionally, caveolin-1 localization in adipocytes was determined by immunohistochemistry (Figure 1D).
Effects of Leptin and Caveolin-1 Expression
We examined the direct role of leptin on caveolin-1 expression using cultured HWPs and differentiated HWPs. Leptin increased caveolin-1 expression in a dose-dependent manner in both HWPs (P=0.05) and differentiated HWPs (P=0.01; Figure 2A and 2B).
Further, we sought to investigate the effect of increased caveolin-1 expression on leptin-dependent activation of cellular signaling pathways. To increase caveolin-1 expression, HWPs were infected with caveolin-1 encoding adenovirus (P<0.0001; Figure 2C) and treated with leptin (100 ng/mL). Notably, the caveolin-1 overexpressing cells (caveolin-1 encoding adenovirus–infected) showed increased basal activation of cellular signaling pathways along with impaired leptin-dependent activation of ERK1/2 (P<0.0001) and STAT3 (P<0.0001; Figure 2D and 2E). In addition, leptin receptor and caveolin-1 interaction was demonstrated using confocal imaging and immune precipitation (Online Figure I).
Effect of Leptin on Lipid Metabolism
To determine the implications of impaired adipose tissue leptin signaling, we first identified the effect of leptin in adipose tissue lipid metabolism. Differentiating HWPs in presence of leptin caused decreased lipid accumulation (P=0.006; Figure 3A and 3B). To examine the mechanisms through which leptin may decrease lipid content in differentiated HWPs, we investigated its role in regulation of key proteins involved in lipid metabolism. Perilipin is a protein present on the surface of the lipid droplet that serves as a protective coating, thereby facilitating lipid storage. Leptin decreased the transcription (P=0.02) and translation of perilipin in a concentration-dependent manner (P=0.01; Figure 3C and 3E). We also investigated the role of leptin in regulation of fatty acid synthase, which is an important enzyme involved in lipid biogenesis. There was a leptin concentration-dependent decrease in the expression of fatty acid synthase mRNA (P=0.03) and protein (P=0.016; Figure 3D and 3F). Additionally, increased caveolin-1 expression, via caveolin-1 encoding adenovirus infection, prevented leptin-dependent attenuation of lipid accumulation (P<0.0001; Figure 3G), and also prevented leptin-dependent decreases in perilipin and fatty acid synthase mRNA (Figure 3H and 3I).
The main finding of our study relates to the direct role of leptin in adipose tissue lipid metabolism and its implications in weight gain. Using a human weight gain model, we found that changes in leptin correlate with changes in adipose tissue caveolin-1 expression. To our knowledge, this is the first longitudinal study to examine and compare the changes in adipose tissue caveolin-1 expression with changes in leptin during weight gain in humans. Of note, increased caveolin-1 expression in obesity has been observed in a cross-sectional study in humans.8
Modest weight gain in our study subjects resulted in increases in serum leptin that ranged from 8% to 157% despite similar increases in weight. The increases in leptin with weight gain were negatively correlated with baseline body fat percentage (rho=−0.94; P<0.001) as well as baseline leptin levels (rho=−0.69; P=0.03). The subjects with smaller increases in leptin during weight gain did not show increases in adipose tissue caveolin-1 expression but had an elevated leptin and adipose tissue caveolin-1 expression at baseline, along with higher body fat percentages compared with those subjects in whom leptin and caveolin-1 expression increased with weight gain (body fat of 36%±3% vs 28.7%±2.4%). The greater level of body fat, leptin, and caveolin-1 at baseline in these “less responsive” subjects suggests that subjects with higher body fat percentages and leptin will have less of a leptin increase with further increases in body fat. Furthermore, there may be a level beyond which leptin is unable to further induce adipose tissue caveolin-1 expression in obese subjects. This “saturating” effect of leptin concentrations on caveolin-1 expression was also observed in our in vitro studies in which the increase in leptin from 100 ng/mL to 150 ng/mL induced little additional change in caveolin-1 expression (Figure 2B). Although it is a concern that the overall group changes in adipose tissue caveolin-1 expression did not reach significance during weight gain in our study, the lack of significance itself highlights the importance of the variability present in the physiological and pathological responses to obesity in the general population. Obesity has multifactorial etiologies, including heritable components such as epigenetic variations that could possibly further account for the variability in leptin and caveolin-1 response to weight gain.
Our study confirms the direct role of leptin in regulation of caveolin-1 expression in HWPs and differentiated HWPs. We also show that increased caveolin-1 expression impairs leptin-dependent activation of STAT3 and ERK1/2 pathways. In these experiments, the adenovirus-mediated increases in caveolin-1 expression were comparable with those seen in adipose tissue during weight gain, indicating that in obesity leptin–cellular signaling may be impaired in adipose tissue. Importantly, caveolin-1 overexpression in HWPs was associated with increased basal activation of these signaling pathways, which itself may contribute to dysfunctional adipose tissue along with prevention of extracellular stimuli from interacting with and regulating adipocyte function. Notably, the role of caveolin-1 in adipose tissue lipid metabolism has been demonstrated in caveolin-1–deficient mice that are resistant to diet-induced obesity despite being hyperphagic and manifest dyslipidemia along with adipocyte abnormalities.9 In these studies, Razani et al9 show that caveolin-1 deficiency prevents accumulation of lipids in the white adipose tissue. These findings are consistent with our conclusions that caveolin-1 plays an important role in modulating lipid accumulation during overfeeding.
Our findings suggest not only a role of leptin in adipose tissue but also changing dynamics during weight gain. The leptin-dependent attenuation of lipid accumulation in differentiating HWPs is consistent with previous studies and indicates the role of a caveolin-1–dependent leptin feedback mechanism in preventing antilipogenic effects of leptin.7,10 The development of impaired leptin signaling in adipose tissue during weight gain therefore would allow safe storage of excess energy as lipid in adipose tissue.11 Further, because adipose tissue is the main contributor to systemic leptin levels, we speculate that the cells of adipose tissue would be exposed to higher leptin levels and impaired leptin signaling may develop before other critical nonadipose tissue cells, which may provide benefit by preventing lipotoxicity with short-term modest weight gain. However, additional studies are needed before such conclusions can be drawn.
Leptin acts via the leptin receptor, which is present on cells of liver, kidney, pancreas, muscle, heart, and the vasculature. We previously have shown similar leptin–caveolin-1 interactions in vascular endothelial cells; therefore, our findings do not appear to be confined to adipose tissue.12 If our results also hold true for these other cell types, then the role of leptin in lowering intracellular lipid accumulation via decreasing perilipin and fatty acid synthase expression suggests an antiatherogenic mechanism through which leptin may prevent lipotoxicity in these cells. Several studies in animals have shown that leptin decreases in lipid accumulation in cells of liver, heart, and vasculature,7,13 and leptin resistance is associated with increased lipid accumulation in the liver.14 Alternatively, our findings that peripheral leptin signaling may be impaired in obesity indicates a mechanism through which leptin resistance, but not hyperleptinemia, may be proatherogenic in these tissues, and therapeutics aimed at eliminating leptin resistance may improve pathophysiological outcomes in obesity.
The strength of our study is in the unique longitudinal approach in humans, combined with an in vitro component, which allows investigation into the autocrine role of leptin and weight gain. However, the study was limited to defining the relationship between leptin and adipose tissue caveolin-1 expression. Future studies aimed at investigating the effects of leptin in other peripheral organs, through which leptin may contribute to metabolic and cardiovascular diseases in obesity, are needed. This is important, especially in light of studies aimed at investigating the therapeutic effect of leptin administration in the treatment of diabetes.15,16
In summary, modest weight gain in healthy humans results in proportionate changes in leptin and caveolin-1 expression, consistent with in vitro findings of a cause-and-effect relationship. In adipose tissue, increased caveolin-1 expression, in turn, impairs leptin signaling, which provides an advantage during early stages of weight gain in that the adipocytes can serve as a “reservoir” for increased lipid accumulation in the presence of hyperleptinemia.
Sources of Funding
P.S. is supported by American Heart Association 11SDG7260046, and National Institutes of Health (NIH) grants DK81014, HL087214, and HL065176. V.K.S. is supported by NIH grants HL73211, HL087214, DK81014, and HL065176. M.D.J. is supported by NIH grants DK45343 and DK40484. This publication was made possible by funding from the National Center for Research Resources (1UL1 RR024150). Its contents are solely the responsibility of the authors and do not represent the official view of National Center for Research Resources or NIH.
In May 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.0 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.273656/-/DC1.
Non-standard Abbreviations and Acronyms
- adenovirus encoding caveolin-1
- human white preadipocyte
- Received May 11, 2012.
- Revision received June 18, 2012.
- Accepted June 22, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Increased cardiovascular risk in obesity is mediated, in part, by the expansion of adipose tissue and elevated levels of adipokines, including leptin.
Although the central role of leptin in energy homeostasis is well-known, its effects on peripheral cells such as adipocytes are unclear.
In cultured vascular endothelial cells, high levels of leptin increase caveolin-1 expression, which in turn impairs leptin signaling.
What New Information Does This Article Contribute?
Leptin decreases the accumulation of lipids in adipocytes.
In humans, increases in leptin seen with modest weight gain could increase adipose tissue caveolin-1 expression.
Increased caveolin-1 expression in adipose tissue could impair leptin-dependent activation of signaling pathways and allow the storage of lipids in differentiating preadipocytes.
The relative contribution of hyperleptinemia and peripheral tissue leptin resistance to the development of obesity-related disorders remains unclear. We investigated the autocrine role of leptin in adipose tissue and its changing dynamics with weight gain. Our data suggest that increases in leptin, as seen with modest weight gain in humans, increases adipose tissue caveolin-1 expression and impairs leptin-dependent cellular signaling. For the first time to our knowledge, we show that leptin acts directly on differentiating preadipocytes to lower lipid accumulation by decreasing the expression of key proteins involved in lipid biogenesis and storage. Thus, impairment of adipose tissue leptin signal could be beneficial during the early stages of weight gain because this would facilitate safe lipid storage in adipose tissue. However, in established obesity leptin resistance, but not hyperleptinemia, could contribute to lipid accumulation and lipotoxicity in peripheral tissues such as liver, heart, and vasculature. Further studies are needed to investigate the effects of leptin in peripheral tissues. The development of strategies to eliminate leptin resistance in obesity could be of potential clinical benefit in the treatment of obesity and related disorders.