Regulation of the Fibrosis and Angiogenesis Promoter SPARC/Osteonectin in Human Adipose Tissue by Weight Change, Leptin, Insulin, and Glucose

OBJECTIVE

Matricellular Secreted Protein, Acidic and Rich in Cysteine (SPARC), originally discovered in bone as osteonectin, is a mediator of collagen deposition and promotes fibrosis. Adipose tissue collagen has recently been found to be linked with metabolic dysregulation. Therefore, we tested the hypothesis that SPARC in human adipose tissue is influenced by glucose metabolism and adipokines.

RESEARCH DESIGN AND METHODS

Serum and adipose tissue biopsies were obtained from morbidly obese nondiabetic subjects undergoing bariatric surgery and lean control subjects for analysis of metabolic markers, SPARC, and various cytokines (RT-PCR). Additionally, 24 obese subjects underwent a very-low-calorie diet of 1,883 kJ (450 kcal)/day for 16 weeks and serial subcutaneous-abdominal-adipose tissue (SCAT) biopsies (weight loss: 28 ± 3.7 kg). Another six lean subjects underwent fast-food–based hyperalimentation for 4 weeks (weight gain: 7.2 ± 1.6 kg). Finally, visceral adipose tissue explants were cultured with recombinant leptin, insulin, and glucose, and SPARC mRNA and protein expression determined by Western blot analyses.

RESULTS

SPARC expression in human adipose tissue correlated with fat mass and was higher in SCAT. Weight loss induced by very-low-calorie diet lowered SPARC expression by 33% and increased by 30% in adipose tissue of subjects gaining weight after a fast-food diet. SPARC expression was correlated with leptin independent of fat mass and correlated with homeostasis model assessment–insulin resistance. In vitro experiments showed that leptin and insulin potently increased SPARC production dose dependently in visceral adipose tissue explants, while glucose decreased SPARC protein.

CONCLUSIONS

Our data suggest that SPARC expression is predominant in subcutaneous fat and its expression and secretion in adipose tissue are influenced by fat mass, leptin, insulin, and glucose. The profibrotic effects of SPARC may contribute to metabolic dysregulation in obesity.Secreted Protein, Acidic and Rich in Cysteine (SPARC), a 34-kDa matricellular glycoprotein, is also known as osteonectin and BM-40. It was initially found to be secreted from bone (1), but SPARC is expressed in most tissues and was the first extracellular matrix protein described in adipose tissue (2,3).SPARC is a multifunctional protein: it is involved in osteogenesis, angiogenesis, wound healing, tumorigenesis, and the pathogenesis of fibrosis involving the kidney (4,5) and liver (6). SPARC also contributes to collagen fibril formation in the dermis, and SPARC knockout mice have reduced collagen content in the dermis (7). More recent evidence suggests that fibrosis in adipose tissue impairs metabolic function and reduces the capacity of fat expansion (3). SPARC is secreted from human adipose tissue and is predominantly derived from adipocytes, where it has a role in adipocyte differentiation, adipogenesis, and adipose tissue hyperplasia (2,8); however, it is unknown whether SPARC contributes to the pathogenesis of insulin resistance or the metabolic syndrome. The aim of this study was therefore to study 1) the depot-specific expression of SPARC, 2) the association of SPARC with markers of insulin resistance, 3) the association of SPARC adipose tissue expression with the adipokines leptin and adiponectin, and 4) the effect of weight loss and weight gain on adipose tissue SPARC expression.We determined the adipose tissue depot expression in visceral fat (VAT) and subcutaneous abdominal adipose tissue (SCAT) in lean and morbidly obese subjects in association with adipokine levels and markers of insulin resistance and explored the gene expression of SPARC in a longitudinal intervention study involving obese subjects treated with a very-low-calorie diet (VLCD) to induce weight loss. Cell culture studies confirmed the regulation of SPARC by insulin, glucose, and leptin.     

Implications of Resveratrol in Obesity and Insulin Resistance: A State-of-the-Art Review

Background: Resveratrol is a polyphenol chemical that naturally occurs in many plant-based dietary products, most notably, red wine. Discovered in 1939, widespread interest in the potential health benefits of resveratrol emerged in the 1970s in response to epidemiological data on the cardioprotective effects of wine. Objective: To explore the background of resveratrol (including its origins, stability, and metabolism), the metabolic effects of resveratrol and its mechanisms of action, and a potential future role of dietary resveratrol in the lifestyle management of obesity. Data sources: We performed a narrative review, based on relevant articles written in English from a Pubmed search, using the following search terms: “resveratrol”, “obesity”, “Diabetes Mellitus”, and “insulin sensitivity”. Results: Following its ingestion, resveratrol undergoes extensive metabolism. This includes conjugation (with sulfate and glucuronate) within enterocytes, hydrolyzation and reduction within the gut through the action of the microbiota (with the formation of metabolites such as dihydroresveratrol), and enterohepatic circulation via the bile. Ex vivo studies on adipose tissue reveal that resveratrol inhibits adipogenesis and prevents the accumulation of triglycerides through effects on the expression of Peroxisome Proliferator-activated Receptor γ (PPARγ) and sirtuin 1, respectively. Furthermore, resveratrol induces anti-inflammatory effects, supported by data from animal-based studies. Limited data from human-based studies reveal that resveratrol improves insulin sensitivity and fasting glucose levels in patients with Type 2 Diabetes Mellitus and may improve inflammatory status in human obesity. Although numerous mechanisms may underlie the metabolic benefits of resveratrol, evidence supports a role in its interaction with the gut microbiota and modulation of protein targets, including sirtuins and proteins related to nitric oxide, insulin, and nuclear hormone receptors (such as PPARγ). Conclusions: Despite much interest, there remain important unanswered questions regarding its optimal dosage (and how this may differ between and within individuals), and possible benefits within the general population, including the potential for weight-loss and improved metabolic function. Future studies should properly address these important questions before we can advocate the widespread adoption of dietary resveratrol supplementation.     

Raised serum, adipocyte, and adipose tissue retinol-binding protein 4 in overweight women with polycystic ovary syndrome: effects of gonadal and adrenal steroids

CONTEXT: Polycystic ovary syndrome (PCOS) is associated with insulin resistance and obesity. Recent studies have shown that serum retinol-binding protein 4 (RBP4) levels increase with obesity. Currently, no data exist on the relative expression of RBP4 in either serum or adipose tissue of PCOS women. OBJECTIVES: mRNA expression of RBP4 from sc and omental (om) adipose tissue and sc adipocytes in overweight PCOS women were compared with matched controls; RBP4 protein in adipose tissue and serum RBP4 levels were also assessed. Additionally, we studied the effects of testosterone, 17beta-estradiol, androstenedione, and dehydroepiandrosterone sulfate on RBP4 expression in adipose tissue explants. DESIGN: Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of RBP4. Biochemical measurements were also performed. RESULTS: Compared with controls, there was significant up-regulation of RBP4 mRNA in sc (P < 0.05) and om (P < 0.01) adipose tissue as well as isolated sc adipocytes (P < 0.01) of PCOS women. In addition to elevated serum RBP4 levels in PCOS women (P < 0.05), RBP4 protein levels were significantly greater in sc and om adipose tissue of PCOS women (P < 0.05 and P < 0.05, respectively). Furthermore, in human sc and om adipose tissue explants, 17beta-estradiol significantly increased RBP4 mRNA expression, protein levels, and secretion into the culture media (P < 0.05). CONCLUSIONS: The precise reason for elevated levels of RBP4 in overweight PCOS women is unknown, but it appears that 17beta-estradiol may play a role in their regulation in adipose tissue.     

Das polyzystische Ovarsyndrom

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting women in the reproductive age and is a major cause of anovulation, hyperandrogenism and infertility. Since obesity and insulin resistance are predominant features of women with PCOS, a variety of metabolic disturbances are associated. There is a marked increase in the risk of developing type-2 diabetes in these patients and a majority of women with PCOS will subsequently harbour an enhanced cardiovascular risk.     

Differential responses of corticotropin-releasing hormone receptor type 1 variants to protein kinase C phosphorylation

Corticotropin-releasing hormone (CRH) regulates diverse biological functions in mammals, through activation of two types of specific G protein-coupled receptors that are expressed as multiple mRNA spliced variants. In most cells, the type 1alpha CRH receptor (CRH-R1alpha) preferentially activates the G(s)-adenylyl cyclase signaling cascade. CRH-R1alpha-mediated signaling activity is impaired by insertion of 29 amino acids in the first intracellular loop, a sequence modification that is characteristic of the human-specific CRH-R1beta variant. In various tissues, CRH signaling events are regulated by protein kinase C (PKC). The CRH receptors contain multiple putative PKC phosphorylation sites that represent potential targets. To investigate this, we expressed recombinant CRH-R1alpha or CRH-R1beta in human embryonic kidney 293 cells and analyzed signaling events after PKC activation. Agonist (oxytocin) or phorbol 12-myristate 13-acetate-induced activation of PKC led to phosphorylation of both CRH-R1 variants. However, CRH-R1alpha and CRH-R1beta exhibited different functional responses to PKC-induced phosphorylation, with only the CRH-R1beta susceptible to cAMP signaling desensitization. This was associated with a significant decrease of accessible CRH-R1beta receptors expressed on the cell surface. Both CRH-R1 variants were susceptible to homologous desensitization and internalization following treatment with CRH; however, PKC activation increased internalization of CRH-R1beta but not CRH-R1alpha in a beta-arrestin-independent manner. Our findings indicate that CRH-R1alpha and -R1beta exhibit differential responses to PKC-induced phosphorylation, and this might represent an important mechanism for functional regulation of CRH signaling in target cells.     

Human RBP4 adipose tissue expression is gender specific and influenced by leptin

Objective The role of retinol‐binding protein‐4 (RBP4) in human insulin resistance remains controversial, which may in part be explained by a gender‐specific secretion of RBP4 in adipose tissue (AT). The aim of the study was to determine gender‐specific depot expression of RBP4 and to identify metabolic parameters and cytokines/adipokines associated with RBP4. Research Design and Methods The study is an ex ‐ vivo prospective analysis of paired AT‐samples from 22 men and 26 women of similar age [men: 43·4 ± 13 (mean ± SD)years, women: 44·1 ± 12 years], BMI (men: 41·9 ± 18kg/m², women: 38·4 ± 11kg/m²) and homeostasis model assessment of insulin resistance taken during elective surgery and ex‐vivo culture using visceral‐AT (VAT)‐explants (n = 10). Plasma RBP4 and cytokines were measured by ELISA and mRNA expression in AT by real‐time PCR. VAT‐explants were cultured with recombinant leptin and insulin and RBP4 determined by western blot analyses. Results Overall subcutaneous AT (SCAT)‐RBP4 mRNA expression was higher than VAT‐expression [3·1 ± 0·26 signal units (SU; mean ± SE) vs 1·79 ± 0·18 SU, n = 48, P < 0·0001], but neither correlated with circulating RBP4. SCAT‐RBP4 expression was higher in women and correlated with BMI (r = ?0·5, P = 0·009) and fat mass (r = ?0·5, P = 0·002). VAT‐RBP4 correlated positively with GLUT‐4 expression and adiponectin in men only (r = 0·54, P = 0·03 and r = 0·64, P < 0·002, respectively) when correcting for age and fat mass. Multiple regression determined leptin AT‐expression as a positive predictor of AT‐RBP4 in women (SCAT: β = 0·50, P = 0·002; VAT: β = 0·58, P = 0·003) and adiponectin for VAT‐RBP4 in men (β = 0·69; P = 0·001). AT‐RBP4 mRNA expression showed no relation with insulin resistance. Leptin stimulated RBP‐4 secretion ex‐vivo, whilst insulin did not affect RBP4. Conclusion AT‐derived RBP4‐mRNA expression is gender specific and regulated by leptin. Circulating RBP4 levels appear to be independent of AT‐RBP4 secretion.     

PCOS remains a diagnosis of exclusion: a concise review of key endocrinopathies to exclude

Polycystic ovarian syndrome (PCOS) is a heterogenous disorder associated with clinical, endocrine and ultrasonographic features that can also be encountered in a number of other diseases. It has traditionally been suggested that prolactin excess, enzymatic steroidogenic abnormalities and thyroid disorders need to be excluded before a diagnosis of PCOS is made. However, there is paucity of data regarding the prevalence of PCOS phenotype in some of these disorders, whereas other endocrine diseases that exhibit PCOS‐like features may elude diagnosis and proper management if not considered. This article reviews the data of currently included entities that exhibit a PCOS phenotype and those that potentially need to be looked for, and attempts to identify specific features that distinguish them from idiopathic PCOS.     

Protective Actions of Globular and Full-Length Adiponectin on Human Endothelial Cells: Novel Insights into Adiponectin-Induced Angiogenesis

Background/Aims: Adiponectin levels are decreased in diabetes and atherosclerosis. Coexisting hyperglycaemia and systemic inflammation predisposes to dysregulated angiogenesis and vascular disease. We investigated the effect of globular adiponectin (gAd) and full-length adiponectin (fAd) on angiogenesis and pro-angiogenic molecules, i.e. matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF), in human microvascular endothelial cells (HMEC-1). Methods: Angiogenesis was assessed by studying capillary tube formation in HMEC-1 on growth factor-reduced Matrigel. Endothelial cell migration assay was performed in a modified Boyden chamber. Results: Endothelial cell proliferation, in vitro migration and angiogenesis were significantly increased by gAd (mediated by AdipoR1, AMPK-Akt pathways), and gAd significantly increased MMP-2, MMP-9 and VEGF expression levels. The effect of gAd on VEGF appears to be mediated by AdipoR1, whilst the effect of gAd on MMP-2 and MMP-9 appears to be mediated by AdipoR1 and AdipoR2. Only endothelial cell proliferation was significantly increased by fAd in human microvascular endothelial cells and appears to be mediated by AdipoR2. No significant effects on MMP-2, MMP-9 and VEGF were observed. Importantly, gAd decreased glucose and C-reactive protein-induced angiogenesis with a concomitant reduction in MMP-2, MMP-9 and VEGF in HMEC-1 cells. Conclusion: We report novel insights into the mechanisms of adiponectin on angiogenesis.     

Effects of insulin and glucocorticoids on the leptin system are mediated through free leptin

OBJECTIVE: Insulin and glucocorticoids are known to increase total leptin levels. However, the effects of insulin and glucocorticoids on the components of the leptin system - free leptin (FL), bound leptin (BL) and soluble leptin receptor (SR) - have not been elucidated. The aim of this study was to determine if there is a differential effect of insulin and glucocorticoids on the leptin system. MATERIAL AND METHODS: In the first of two studies (study 1), we measured free leptin (FL), bound leptin (BL), a soluble leptin receptor (SR) and insulin, by specific RIA methods, in six healthy subjects on a control day, and subsequently during a hyperinsulinaemic euglycaemic clamp study. In the second study (study 2) we measured the same parameters in six healthy subjects, before (day 1) and during administration of dexamethasone over 3 consecutive days. RESULTS: In study 1, on the control day FL levels rose over the 16 h monitoring period (P = 0.057) and SR levels declined (P < 0.001), but there was no change of BL levels. Even after accounting for diurnal variation, FL levels increased even more substantially over 12 h of insulin infusion than observed on the control day (P < 0.001). In study 2, mean FL concentration doubled from day 1 to day 2 (P = 0.01) and remained elevated subsequently. In contrast to FL, BL and SR levels remained unchanged during the study. Fasting insulin levels (pmol/l) increased from day 1 to day 2, but this rise only approached significance on day 4 (P = 0.05). CONCLUSION: We conclude that insulin and dexamethasone increase free leptin levels, but do not change the concentrations of bound leptin and soluble leptin receptor. Furthermore, the dexamethasone-induced rise in leptin levels is (at least partially) independent of the effects of glucocorticoid-induced hyperinsulinaemia.