The role of melanocortins and cytokines in human adipose tissue and adipocytes

During the last decades obesity has become a major health problem in developed countries, and more recently also in the developing countries it is seriously affecting parts of the populations. Since obesity is an important risk factor for various, partly life-threatening diseases, including heart diseases, stroke, diabetes type 2, atherosclerosis or some type of cancers, the elucidation of the molecular as well as integrated causation of obesity is urgently needed. With this general aim and in close collaboration with the St. Claraspital we wanted to address some crucial unsolved questions of obesity. The melanocortin system is critical in the regulation of energy homeostasis and feeding. It includes the melanocortins, i.e. peptide hormones derived post-transcriptionally from the POMC gene product (e.g. α-MSH, β-MSH, γ-MSH, ACTH), as well as the five melanocortin receptors (MC1-R, MC2-R, MC3-R, MC4-R, MC5-R) through which the melanocortins signal into target cells. It has been shown that α-MSH produced in the hypothalamus (arcuate nucleus) reveals a potent anorectic effect via the MC4-R, also present in this brain region. Furthermore, MC4-R mutations are the most common monogenic cause of morbid obesity in humans to date. Laparoscopic gastric banding has been shown to efficiently reduce excessive body weight. However, some patients develop complications (e.g. insufficient weight loss) after the operation, which necessitate a re-operation. We intended to verify in the patient group of the St. Claraspital whether patients requiring re-operation have a higher MC4-R mutation/polymorphism rate. If so, the sequencing of MC4-R prior to the operation could be used as prediction marker for the outcome of the operation. Therefore, the complete MC4-R gene of 37 patients that developed complications after the operation was sequenced. In 95% of the patients we found a normal, unmutated MC4-R gene. However, one novel silent mutation Ile198Ile (C594T) and one polymorphism Ile251Leu (A1144C) were found. This polymorphism had previously been shown to lead to a fully functional receptor. To summarize, we could not confirm the observation previously published that MC4-R defects are associated with a higher complication rate following laparoscopic gastric banding. Since α-MSH also circulates at low levels in the bloodstream, and its level is elevated in the obese state, there might be a potential role of the melanocortin system in the periphery. Possibly, there even exists a direct feedback loop of the melanocortins from the adipose tissue to the brain or vice versa. However, the importance of the melanocortin system in obesity, the expression of the melanocortin receptors in human adipose tissue has never been clearly investigated. Therefore, we analyzed the expression of the five melanocortin receptors, and of POMC, together with additional obesity-relevant genes (AgRP, leptin, leptin receptor, UCP-1) in human subcutaneous versus omental adipose tissue. Furthermore, we compared the expression levels in the obese to normal-weight subjects. Of the five melanocortin receptor subtypes, only MC1-R mRNA was substantially expressed and its expression was slightly elevated in the obese subject group. Since we obtained no POMC mRNA transcripts, an auto/paracrine action of α-MSH is doubtful. Fluorescent immunohistochemistry for detection of the MC1-R in human adipose tissue revealed high protein expression on macrophages and to a lesser extent on adipocytes. Human MSC-derived adipocytes were used as in vitro model to analyze the functionality of the MC1-R. Thereby, the cAMP production was dose-dependently increased upon stimulation with the potent MC1-R agonist NDP-MSH, suggesting that MC1-R in human adipocytes are functional. Furthermore, we tried to elucidate the function of MC1-R in undifferentiated MSCs as well as in adipocytes derived from these cells. We found a significant anti-proliferative effect of NDP-MSH on undifferentiated MSCs. This finding implies a role of α-MSH in regulating the de novo buildup of fat cells and subsequently the development of obesity. However, in adipocytes we were unable to find an effect of NDP-MSH on lipolysis, metabolic rate and inflammation. In the last years the concept has emerged that obesity is characterized by a chronic mild inflammation. Several cytokines associated with inflammation are elevated in obesity, which may lead to the well known co-morbidities of obesity (e.g. hypertension, atherosclerosis, diabetes type 2). Nevertheless, it is still controversial which cell types in the adipose tissue secrete which cytokines. We analyzed the protein secretion and mRNA expression of the cytokines TNF-α, IL- 6, IL-8 and IL-10 in human adipose tissue and in adipocytes, which were either derived from preadipocytes or MSCs. Whereas the adipose tissue secreted all four cytokines into the medium, in the supernatants from adipocytes no TNF-α and IL-10 was detectable (even upon stimulation with highest endotoxin (LPS) concentrations). Adipocytes secreted IL-6 and IL-8 in large quantities. Further investigations on the mRNA expression of cytokines revealed also high expression rates for IL-6 and IL-8. In contrast, TNF-α was expressed only transiently and at low levels after inducing of inflammation with LPS. When we analyzed co-cultures of macrophages isolated from buffy coats, either stimulated or unstimulated with LPS, together with adipocytes, we found substantial amounts of TNF-α protein. We obtained much more TNF-α from the LPSstimulated cultures. Moreover, when we investigated the biological effects of exogenously administered cytokines on adipocytes, only TNF-α showed an increase in lipolysis, glucose uptake and IL-6 mRNA expression. To summarize, IL-6 and IL-8 are secreted from adipocytes. Whereas adipocytes express no IL-10, TNF-α is only transiently and weakly expressed. Nevertheless, the adipocytes respond to exogenous TNF-α. Thus, TNF-α secreted from adipose tissue seems to be derived from cells of the stromovascular fraction (probably macrophages). In addition we found further evidence for a cross-talk between macrophages and adipocytes, which results in an elevated inflammation state. Thus, elimination of this macrophage-adipocyte crosstalk might be a future target in order to prevent augmented inflammation in the obese state, and probably avoiding the developing of the co-morbidities of obesity. To conclude, these studies elucidated the expression of melanocortin receptors in human adipose tissue and adipocytes. We found MC1-R to be expressed in the adipose tissue on macrophages and adipocytes. Further analyzes confirmed the presence of the protein and the functionality of the MC1-R on adipocytes. Whereas MC1-R seems to play role in the regulation of proliferation in undifferentiated MSCs, in adipocytes we found no effect on lipolysis, metabolic rate and inflammation. Further investigations on cytokines in the adipose tissue revealed a very weak TNF-α production, suggesting that TNF-α in the adipose tissue is derived from macrophages rather than from adipocytes. However, human adipocytes respond to administration of exogenous TNF-α, indicating the presence of a cross-talk between adipocytes and macrophages in the adipose tissue. With this work another tessera could be obtained that can be placed on the huge mosaic called the pathophysiology of obesity. A mosaic, which hopefully will be once completed in the future.