Abstract
Rationale
The link between obesity, hyperleptinemia, and development of cardiovascular disease are 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 eight 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 (HWP), leptin increased caveolin-1 expression which in turn impaired leptin cellular signaling. Functionally, leptin decreased lipid accumulation in differentiating HWP 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 intra-cellular lipid accumulation. Our study suggests a leptin-dependent feedback mechanism which may be essential to facilitate adipocyte lipid storage during weight gain.
Keywords: Leptin, weight gain, caveolin-1, impaired signaling, lipid accumulation
INTRODUCTION
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 non-adipose 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.
METHODS
Detailed methods are provided in the online supplement. Briefly, we recruited 10 healthy volunteers (7 men and 3 women) aged 23–36 years. The volunteers were overfed to increase weight gradually by 5 % in an 8 week period. Measurements and adipose tissue biopsies were obtained at baseline and after weight gain. The protocol was approved by the Institutional Review Board and informed consent was obtained. In-vitro experiments were done using human white preadipocytes (HWP) (PromoCell, Germany).
RESULTS
Effects of overfeeding on study participants
The characteristics of the study participants at baseline and after weight gain are presented in Table 1. 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.
TABLE 1.
Effects of overfeeding on study participants
| Variable | Baseline | Weight gain |
|---|---|---|
| Total body weight (kg) | 71.6 ± 14.5 | 75.7 ± 15.2* |
| Total body fat mass (kg) | 22 ± 7.9 | 26.2 ± 7.9* |
| Total body lean mass (kg) | 46.6 ± 8.9 | 46.5 ± 8.8 |
| BMI (kg/m2) | 23.3 ± 3.7 | 24.6 ± 3.9* |
| Cholesterol (mg/dl) | 169 ± 20 | 168 ± 37 |
| HDL (mg/dl) | 46 ±12 | 42 ± 9 |
| Triglycerides (mg/dl) | 86 ± 27 | 91 ± 51 |
| Insulin (μU/ml) | 5.2 ± 3.2 | 6.1 ± 2.6 |
| Leptin (ng/ml) | 7 ± 3.8 | 12 ± 5.7* |
| Adiponectin (ng/ml) | 8816 ± 4787 | 10081 ± 5539 |
| Glucose (mg/dl) | 93 ± 5 | 94 ± 5 |
N=10 (7M /3F), values are Mean ± SD,
is p<0.05 determined by paired Wilcoxon Signed-Rank test; BMI: Body mass index; HDL: High density lipoprotein.
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 a 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. Since 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).
Figure 1. Adipose tissue caveolin-1 expression during weight gain in humans.
Western blots (A) showing the range of changes in caveolin-1 expression in the subjects with high (#1) and low (#2) leptin changes during weight gain. Graph (B) showing significant correlation between changes in caveolin-1 expression and leptin during weight gain (n=10). Open square represent data from men, and closed circle represent data from women. Graph (C) showing changes in adipose tissue caveolin-1 expression during weight gain Data presented as Mean ± SD (n=10). 1: Baseline, 2: Weight gain. Confocal images (D) showing localization of caveolin-1 (red) in adipocytes (Perilipin, green: adipocyte specific marker).
Effects of leptin and caveolin-1 expression
We examined the direct role of leptin on caveolin-1 expression using cultured HWP and dHWP. Leptin increased caveolin-1 expression in a dose dependent manner in both HWP (p = 0.05) and dHWP (p = 0.01, Figure 2A, B).
Figure 2. Leptin increases caveolin-1 protein expression which impairs leptin-dependent activation of cellular signaling pathways.
Western blot and graph showing leptin concentration dependent increases in caveolin-1 expression in human white preadipocytes (HWP) (A) and differentiated HWP (B). HWP were infected with adenovirus encoding caveolin-1 or control (Null). Western blot and graph showing increased caveolin-1 expression (C), and impaired leptin-dependent ERK activation (D), and STAT3 activation (E) in cells infected with Ad-cav-1. Data presented as Mean ± SEM (n=4). * is p < 0.05 compared to control (0 min) in each group and # is p < 0.05 compared to same time point in the two groups.
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, HWP were infected with caveolin-1 encoding adenovirus (Ad-Cav-1) (p < 0.0001) (Figure 2C) and treated with leptin (100 ng/ml). Notably, the caveolin-1 overexpressing cells (Ad-cav-1 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, E). 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 HWP in presence of leptin caused decreased lipid accumulation (p = 0.006) (Figure 3A, B). To examine the mechanisms through which leptin may decrease lipid content in dHWP, 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 which 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) (Figure3C, E). We also investigated the role of leptin in regulation of fatty acid synthase (FASN) which is an important enzyme involved in lipid biogenesis. There was a leptin-concentration dependent decrease in the expression of FASN mRNA (p = 0.03) and protein (p = 0.016) (Figure 3D, F). Additionally, increased caveolin-1 expression, via Ad-cav-1 infection, prevented leptin-dependent attenuation of lipid accumulation (p <0.0001) (Figure 3G), and also prevented leptin-dependent decreases in perilipin and FASN mRNA (Figure 3H, I).
Figure 3. Leptin decreases lipid accumulation in differentiating HWP.
Images (A), and graph (B) showing the effect of leptin treatment on lipid accumulation in on differentiating HWP. Graph showing leptin dependent reduction in perilipin (C) and fatty acid synthase (FASN) (D) mRNA. Western blot and graph showing leptin concentration dependent decreases in perilipin (E) and FASN (F) protein. Graphs showing the effect of caveolin-1 overexpression on leptin dependent decreases in lipid accumulation (G), perilipin (H) and FASN (I) mRNA. Data presented as Mean ± SEM (n=4). * is p<0.05 determined by Wilcoxon rank sum test compared to control (0 ng/ml) leptin experiment.
DISCUSSION
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 which 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 to 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 where the increase in leptin from 100 ng/ml to150 ng/ml induced little additional change in caveolin-1 expression (Figure 2B). While 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 physiologic and pathologic responses to obesity in the general population. Obesity has multifactorial etiologies, including heritable components such as epigenetic variations which 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 HWP and dHWP. 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 to 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 HWP was associated with increased basal activation of these signaling pathways which may itself contribute to dysfunctional adipose tissue along with preventing extra-cellular 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 which are resistant to diet-induced obesity despite being hyperphagic, and manifest dyslipidemia along with adipocyte abnormalities.9 In these studies, Razani et. al. 9 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 changing dynamics during weight gain. The leptin-dependent attenuation of lipid accumulation in differentiating HWP is consistent with previous studies and indicates the role of a caveolin-1-dependent leptin feedback mechanism in preventing anti-lipogenic effects of leptin.7, 10 The development of impaired leptin signaling in adipose tissue during weight gain would therefore allow safe storage of excess energy as lipid in adipose tissue.11 Further, as 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 may develop impaired leptin signaling before other critical non-adipose 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 have previously 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, leptin’ s role in lowering intracellular lipid accumulation via decreasing perilipin and fatty acid synthase expression, suggests an anti-atherogenic mechanism though which leptin may prevent lipotoxicity in these cells. Indeed, 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 though which leptin resistance, and 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 lies 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.
Supplementary Material
Novelty and Significance.
What Is Known?
Increased cardiovascular risk in obesity is mediated, in partby 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 that 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, 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 as this would facilitate safe lipid storage in adipose tissue. However, in established obesity, leptin resistance and not hyperleptinemia would 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.
Acknowledgments
SOURCES OF FUNDING
PS is supported by American Heart Association 11SDG7260046, European Union (EU) grant CZ.1.05/1.1.00/02.0123, NIH grants DK81014, HL087214,; VKS is supported by EU grant CZ.1.05/1.1.00/02.0123, NIH Grants HL73211, HL087214, DK81014. MDJ is supported by NIH grants DK45343, DK40484. This publication was made possible by grant from the National Center for Research Resources (NCRR) (1UL1 RR024150). Its contents are solely the responsibility of the authors and do not represent the official view of NCRR or NIH.
Non-standard Abbreviations
- HWP
Human white preadipocytes
- dHWP
differentiated human white preadipocytes
- Ad-Cav-1
Adenovirus encoding caveolin-1
- FASN
Fatty acid synthase
Footnotes
DISCLOSURES
None
References
- 1.Sweeney G. Cardiovascular effects of leptin. Nature reviews. 2010;7:22–29. doi: 10.1038/nrcardio.2009.224. [DOI] [PubMed] [Google Scholar]
- 2.Mark AL, Correia ML, Rahmouni K, Haynes WG. Loss of leptin actions in obesity: Two concepts with cardiovascular implications. Clin Exp Hypertens. 2004;26:629–636. doi: 10.1081/ceh-200031948. [DOI] [PubMed] [Google Scholar]
- 3.Devaraj S, Torok N. Leptin: The missing link between obesity and heart disease? Atherosclerosis. 2011;217:322–323. doi: 10.1016/j.atherosclerosis.2011.04.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Martin SS, Qasim A, Reilly MP. Leptin resistance: A possible interface of inflammation and metabolism in obesity-related cardiovascular disease. Journal of the American College of Cardiology. 2008;52:1201–1210. doi: 10.1016/j.jacc.2008.05.060. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.DeClercq V, Taylor C, Zahradka P. Adipose tissue: The link between obesity and cardiovascular disease. Cardiovascular & hematological disorders drug targets. 2008;8:228–237. doi: 10.2174/187152908785849080. [DOI] [PubMed] [Google Scholar]
- 6.Bornstein SR, Abu-Asab M, Glasow A, Path G, Hauner H, Tsokos M, Chrousos GP, Scherbaum WA. Immunohistochemical and ultrastructural localization of leptin and leptin receptor in human white adipose tissue and differentiating human adipose cells in primary culture. Diabetes. 2000;49:532–538. doi: 10.2337/diabetes.49.4.532. [DOI] [PubMed] [Google Scholar]
- 7.Ceddia RB. Direct metabolic regulation in skeletal muscle and fat tissue by leptin: Implications for glucose and fatty acids homeostasis. International journal of obesity. 2005;29:1175–1183. doi: 10.1038/sj.ijo.0803025. [DOI] [PubMed] [Google Scholar]
- 8.Catalan V, Gomez-Ambrosi J, Rodriguez A, Silva C, Rotellar F, Gil MJ, Cienfuegos JA, Salvador J, Fruhbeck G. Expression of caveolin-1 in human adipose tissue is upregulated in obesity and obesity-associated type 2 diabetes mellitus and related to inflammation. Clinical endocrinology. 2008;68:213–219. doi: 10.1111/j.1365-2265.2007.03021.x. [DOI] [PubMed] [Google Scholar]
- 9.Razani B, Combs TP, Wang XB, Frank PG, Park DS, Russell RG, Li M, Tang B, Jelicks LA, Scherer PE, Lisanti MP. Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities. The Journal of biological chemistry. 2002;277:8635–8647. doi: 10.1074/jbc.M110970200. [DOI] [PubMed] [Google Scholar]
- 10.Wang MY, Orci L, Ravazzola M, Unger RH. Fat storage in adipocytes requires inactivation of leptin’s paracrine activity: Implications for treatment of human obesity. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:18011–18016. doi: 10.1073/pnas.0509001102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Unger RH, Scherer PE. Gluttony, sloth and the metabolic syndrome: A roadmap to lipotoxicity. Trends Endocrinol Metab. 2010;21:345–352. doi: 10.1016/j.tem.2010.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Singh P, Peterson TE, Sert-Kuniyoshi FH, Jensen MD, Somers VK. Leptin upregulates caveolin-1 expression: Implications for development of atherosclerosis. Atherosclerosis. 2011;217:499–502. doi: 10.1016/j.atherosclerosis.2010.10.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Jiang L, Wang Q, Yu Y, Zhao F, Huang P, Zeng R, Qi RZ, Li W, Liu Y. Leptin contributes to the adaptive responses of mice to high-fat diet intake through suppressing the lipogenic pathway. PLoS One. 2009;4:e6884. doi: 10.1371/journal.pone.0006884. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Fishman S, Muzumdar RH, Atzmon G, Ma X, Yang X, Einstein FH, Barzilai N. Resistance to leptin action is the major determinant of hepatic triglyceride accumulation in vivo. Faseb J. 2007;21:53–60. doi: 10.1096/fj.06-6557com. [DOI] [PubMed] [Google Scholar]
- 15.Wang MY, Chen L, Clark GO, Lee Y, Stevens RD, Ilkayeva OR, Wenner BR, Bain JR, Charron MJ, Newgard CB, Unger RH. Leptin therapy in insulin-deficient type i diabetes. Proceedings of the National Academy of Sciences of the United States of America. 2010;107:4813–4819. doi: 10.1073/pnas.0909422107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Mantzoros CS, Magkos F, Brinkoetter M, Sienkiewicz E, Dardeno TA, Kim SY, Hamnvik OP, Koniaris A. Leptin in human physiology and pathophysiology. American journal of physiology Endocrinology and metabolism. 2011;301:E567–584. doi: 10.1152/ajpendo.00315.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.