International Journal of Obesity (2016) 40, 266–274 © 2016 Macmillan Publishers Limited All rights reserved 0307-0565/16 www.nature.com/ijo ORIGINAL ARTICLE Differential effects of leptin on adiponectin expression with weight gain versus obesity P Singh1, P Sharma1, KR Sahakyan1, DE Davison1, FH Sert-Kuniyoshi1, A Romero-Corral1,2, JM Swain3,4, MD Jensen5, F Lopez-Jimenez1, T Kara1,6 and VK Somers1 BACKGROUND/OBJECTIVE: Adiponectin exerts beneficial effects by reducing inflammation and improving lipid metabolism and insulin sensitivity. Although the adiponectin level is lower in obese individuals, whether weight gain reduces adiponectin expression in humans is controversial. We sought to investigate the role of weight gain, and consequent changes in leptin, on altering adiponectin expression in humans. METHODS/RESULTS: Forty-four normal-weight healthy subjects were recruited (mean age 29 years; 14 women) and randomized to either gain 5% of body weight by 8 weeks of overfeeding (n = 34) or maintain weight (n = 10). Modest weight gain of 3.8 ± 1.2 kg resulted in increased adiponectin level (P = 0.03), whereas weight maintenance resulted in no changes in adiponectin. Further, changes in adiponectin correlated positively with changes in leptin (P = 0.0085). In-vitro experiments using differentiated human white preadipocytes showed that leptin increased adiponectin mRNA and protein expression, whereas a leptin antagonist had opposite effects. To understand the role of leptin in established obesity, we compared adipose tissue samples obtained from normal-weight versus obese subjects. We noted, first, that leptin activated cellular signaling pathways and increased adiponectin mRNA in the adipose tissue from normal-weight participants, but did not do so in the adipose tissue from obese participants. Second, we noted that obese subjects had increased caveolin-1 expression, which attenuates leptin-dependent increases in adiponectin. CONCLUSIONS: Modest weight gain in healthy individuals is associated with increases in adiponectin levels, which correlate positively with changes in leptin. In vitro, leptin induces adiponectin expression, which is attenuated by increased caveolin-1 expression. In addition, the adipose tissue from obese subjects shows increased caveolin-1 expression and impaired leptin signaling. This leptin signal impairment may prevent concordant increases in adiponectin levels in obese subjects despite their high levels of leptin. Therefore, impaired leptin signaling may contribute to low adiponectin expression in obesity and may provide a target for increasing adiponectin expression, hence improving insulin sensitivity and cardio-metabolic profile in obesity. International Journal of Obesity (2016) 40, 266–274; doi:10.1038/ijo.2015.181 INTRODUCTION receptor-deficient db/db mice, is characterized by low adiponectin 4,14 Adiponectin is an adipokine with profound anti-atherogenic, expression. In addition, supplementation of leptin in ob/ob 15 anti-inflammatory and insulin-sensitizing properties.1–3 Paradoxi- mice results in increased adiponectin expression. Importantly, cally, it is the only adipokine that is decreased in obesity.4–6 This increases in adiponectin expression on leptin administration were 16 negative association between obesity and adiponectin raises the observed before weight loss. Furthermore, adipocyte-selective possibility that weight gain decreases adiponectin expression. reduction of leptin receptor expression diminishes adiponectin However, the effect of weight gain on adiponectin expression in expression, suggesting that leptin may be directly signaling healthy humans is controversial and the molecular mechanisms adipocytes to induce adiponectin expression.17 Similarly, in underlying decreased adiponectin expression in human obesity leptin-deficient conditions in humans, such as lipodystrophy, remain largely unknown.7–11 On the other hand, cross-sectional decreased expression of both leptin and adiponectin is seen,18 population-based studies have shown the presence of higher and leptin treatment in leptin-deficient adults increases adipo- adiponectin levels in the metabolically healthy obese population, nectin expression.19 However, the role of leptin in regulation of which provides support for the potential role of adiponectin in adiponectin in humans is unclear, as cross-sectional population disassociating obesity per se from cardiometabolic dysfunction.12 studies show a negative correlation between leptin and Indeed, increasing adiponectin expression is being targeted as a adiponectin.20,21 Therefore, we designed a study to first examine mechanism to improve insulin sensitivity and decrease cardiovas- the effect of weight gain on adiponectin expression in normal- cular risk in the obese population.13 weight healthy subjects and, second, to investigate the role of Several lines of evidence suggest that leptin, an adipokine leptin in regulation of adiponectin. We measured adiponectin increased in obesity, may regulate adiponectin expression. expression in normal-weight healthy humans, at baseline and after The absence of leptin, as seen in leptin-deficient ob/ob and leptin overfeeding-induced weight gain versus weight maintenance 1Division of Cardiovascular Disease, Mayo Clinic, Rochester, MN, USA; 2Division of Cardiology, Einstein Medical Center, Philadelphia, PA, USA; 3Department of Surgery, Mayo Clinic, Rochester, MN, USA; 4Scottsdale Healthcare Bariatric Center, University of Arizona College of Medicine, Phoenix, AZ, USA; 5Endocrinology Research Unit, Mayo Clinic, Rochester, MN, USA and 6ICRC–Department of Cardiovascular Diseases, St. Anne’s University Hospital Brno, Brno, Czech Republic. Correspondence: Dr P Singh, Division of Cardiovascular Diseases, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. E-mail: [email protected] Received 12 March 2015; revised 12 August 2015; accepted 19 August 2015; accepted article preview online 16 September 2015; advance online publication, 13 October 2015 Leptin induces adiponectin expression P Singh et al 267 (controls). We also explored the role of leptin in regulating antagonize central leptin-dependent changes in appetite, weight and adiponectin expression in vitro and extended our studies to hypertension has been previously demonstrated.26–28 include cross-sectional ex-vivo studies in normal-weight versus To determine the cell signaling pathway through which leptin obese subjects, so as to determine novel molecular mechanisms modulates adiponectin mRNA, we treated differentiated HWP with ERK that may have a role in decreasing adiponectin expression in inhibitor (PD98059, 30 μM, Sigma), AMPK inhibitor (Insolution AMPK established obesity. We hypothesized that leptin upregulates inhibitor Compund C, 20 μM, EMD Chemicals Inc., Gibbstown, NJ, USA), adiponectin expression, and that the decreased adiponectin AKT inhibitor IV (1 μM, EMD Chemicals Inc.) and signal transducer and activator of transcription 3 (STAT3) inhibitor VII (3 μM, EMD Chemicals Inc.) expression in established obesity is secondary to an impairment − 1 of leptin signaling. for 30 min before and during 6 h of leptin (100 ng ml ) treatment. The effect of pathway inhibition on adiponectin mRNA was determined by reverse-transcriptase PCR analysis. We also examined the ability of leptin- − − MATERIALS AND METHODS antagonist (1.5 μgml 1) to prevent leptin-dependent (100 ng ml 1) activation of ERK and STAT3 pathways in differentiated HWP. Longitudinal weight gain study To determine the effect of caveolin-1 overexpression on leptin- We used a longitudinal overfeeding study in humans to determine the dependent increases in adiponectin mRNA, differentiated HWPs were 22,23 effects of weight gain on adiponectin expression. Forty-four healthy infected with adenovirus encoding caveolin-1 gene or control null adults (30 men and 14 women) aged 29 ± 6 years, who were sedentary and adenovirus at an multiplicity of infection of 100 (Vector BioLabs, free of any chronic diseases such as diabetes, hypertension and Philadelphia, PA, USA). These cells were then treated with leptin (10 and dyslipidemia were recruited to participate in the overfeeding protocol. − 100 ng ml 1) for 6 h before the determination of adiponectin mRNA Tobacco users and shift workers were excluded. The study was conducted at the Mayo Clinic Center for Translational Science Activities Clinical transcripts by reverse-transcriptase PCR. Research Unit and the protocol was approved by the Institutional Review Board. Informed written consent was obtained from all participants. Ex-vivo adipose tissue studies Findings from this study relating to endothelial dysfunction, heart rate variability and adipose tissue changes have been published To understand the dynamics of direct leptin action in established obesity, elsewhere.22–25 All the subjects in whom leptin and adiponectin data we examined the ability of leptin to activate cellular signaling pathways were available at both baseline and after weight gain were included to test and increase adiponectin mRNA expression in human adipose tissue from the hypothesis. a cross-sectional population comprising of normal-weight and obese The details of the longitudinal weight gain model have been described individuals. Seventeen adults (8 men and 9 women) aged 20–67 years previously.22,23 Briefly, after a 3-day period during which calories required were recruited to participate in these studies. The protocol was approved to maintain weight were estimated, subjects were randomized 4:1 to either by the