oa Depot-Specific Differences in the Effects of IGF-1, HGF and VEGF on Human Preadipocytes Proliferation and Differentiation

Introduction

Accumulating evidence suggests that adipose tissue is a dynamic organ that undergoes expansion and contraction in response to energy demands and obesity-associated tissue injury. The dynamic nature of the adipose tissue is achieved via changes in both size and number of mature adipocytes through adipocyte hypertrophy and preadipocytes proliferation and differentiation respectively. These changes are mediated through various growth factors and cytokines secreted by cells within the adipose tissues (Spalding et al. 2008). Hypertrophied adipocytes secrete cytokines such as IL-6 that can trigger macrophage infiltration and secretion of IL-1β, TNFα and IL-8 (Bjorntorp 1974; Kobashi et al. 2009), causing impairment of preadipocytes differentiation and induction of insulin resistance (Gustafson et al. 2007; Veilleux et al. 2009). TNFα-stimulates HGF release from fibroblasts and monocytes within the adipose tissue serving as a potent mitogenic factor (Bell et al. 2008). Mature adipocytes secrete IGF-I that plays a major role in preadipocyte differentiation (Bluher et al. 2005). Other growth factors such as VEGF were suggested to regulate the balance between osteoblast and adipocyte differentiation from pre-preadipocytes (mesenchymal stem cells) (Liu et al. 2012). Preadipocytes exhibit unique depot-specific characteristics that persist in expanded in vitro such as different size, lipoprotein binding, fatty acid transfer, protein secretion and response to insulin and lipolytic agents (Tchkonia et al. 2013). Cultured human abdominal subcutaneous preadipocytes (SC) accumulate more fat and exhibit a greater expression of adipogenic transcription factor than omental preadipocytes (OM) (Tchkonia et al. 2005). SC adipocytes serve as the first optimal fat storage choice (Tchkonia et al. 2013). Obesity causes impairment in SC fat storing capacity as a result of reduction in the number of differentiating preadipocytes and hypertrophy of mature adipocytes (Isakson et al. 2009; Spalding et al. 2008). This leads to expansion of visceral fat, including OM depot, with ectopic fat accumulation in the liver, skeletal muscle and heart (Okuno et al. 1998; Tchkonia et al. 2010). Visceral and ectopic fat accumulation results in further augmentation of insulin resistance in these tissues (Petersen and Shulman 2006), which is associated with a depot-dependent (OM > SC) IL-6 release in vivo and ex vivo (Fried et al. 1998; Mohamed-Ali et al. 1997).

Methods

In this study we compared the secretion of IL-6, IL-1β, TNFα and IL-8 in 15 paired subcutaneous (SC) and omental (OM) preadipocytes cultures expanded from stromal-vascular fraction (SVF), isolated from morbidly obese patients (8 males and 7 females) undergoing weight reduction surgery at Hamad Medical Corporation (HMC). We further evaluated the effect of IGF-1, HGF and VEGF on preadipocytes proliferation and differentiation in paired SC and OM preadipocytes cultures isolated from 3 randomly selected female subjects matched for age and BMI. For this purpose, SVF-derived cells (passage 1–2) were grown in stromal medium overnight then incubated in differentiation medium for 7 days, followed by 12 days in maintenance medium as previously described (Lee et al. 2012). Accumulated levels of secreted IL-6, IL-1β, TNFα and IL-8 in the last four days of differentiation were measured in media supernatants using Inflammatory Cytokine Human Magnetic 5-Plex (Life Technologies) according to manufacturer's instructions and assessed by Luminex Flexmap 3D using xPONENT® software. For experiments investigating the effect of growth factors on preadipocytes proliferation and differentiation, cells were grown as above in the absence or presence of 200 ng/ml IGF-1, 100 ng/ml HGF or 50 ng/ml VEGF for the entire differentiation and maintenance periods (once every 3–4 days). To assess proliferation and differentiation capacity, cells were fixed with 4% formalin for 10 min, stained with DAPI and subsequently with Lipidtox (Life Technologies) for 20 min. Total number of nuclei (DAPI, indicator of proliferation) and differentiated adipocytes (Lipidtox positive cells) were scored in 20 fields per well by ArrayScan XTI (Thermo Scientific) using automated spot detection module. Differentiation capacity was assessed by calculating the ratio of Lipidtox positive cells/total number of stained nuclei and presented as a percentage (adipogenic capacity). All protocols were approved by Institutional Research Boards of ADLQ and HMC (SCH-ADL-070, SCH-JOINT-111).

Results

Levels of secreted IL-6, IL-1β and IL-8 were significantly higher in OM preadipocytes compared to their SC-derived counterparts (Fig. 1), whereas secreted TNFα levels were below the level of detection. Compared to their age and BMI matched males counterpart, SC preadipocytes from females exhibited significantly higher levels of secreted IL-6 by 49.2% (p = 0.05) (Table 1) with no significant differences in other measured cytokines in either tissue. Treatment of SC and OM preadipocytes with IGF1, HGF and VEGF increased subcutaneous preadipocytes proliferation by 20% (n = 3, p ≤ 0.01) but had no significant effect on omental preadipocytes (Fig. 2). In contrast, IGF1 increased omental preadipocyte differentiation by 50% (p = 0.02), whereas neither HGF nor VEGF exhibited significant effect on subcutaneous or omental preadipocytes differentiation (Fig. 3).

Discussion and Conclusion

Our data suggest that elevated levels of secreted IL-6, IL-1β and IL-8 in OM compared to SC expanded cultures confirm the greater inflammatory nature of OM-expanded in vitro cultures shown previously in vivo and ex vivo (Fain 2006; Fried et al. 1998; Maury et al. 2007). The greater IL-6 secretion in female SC preadipocytes may suggest a role of sex steroid hormones (estrogen and androgen), but mechanisms for depot-specific differences remain poorly understood (Lee et al. 2013). Previous data has shown that preadipocytes are potent sources of growth factors such as VEGF, HGF, and IGF-1 in response to inflammatory mediators via a p38 MAPK-dependent mechanism (Wang et al. 2006). Investigation of the function of these growth factors on proliferation and differentiation of SC and OM expanded cultures confirmed the mitogenic nature of all these growth factors in SC but not in OM preadipocytes, while only IGF-1 enhanced differentiation of OM preadipocytes. These depot-specific differences in preadipocyte proliferation and differentiation in response to various mitogenic and adipogenic factors may be explained by their different cellular composition and physiological role (Lee et al. 2013). The molecular mechanisms underlying these differences and their impact on metabolic syndrome remain elusive. The contribution of secreted cytokines and growth factors on depot-specific differences and metabolic complications associated with central obesity may shed some light on these mechanisms.

Acknowledgment

This research was sponsored by Qatar National Research Fund (QNRF), Grant number NPRP6-235-1-048.

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