Growth arrest-specific 6 modulates adiponectin expression and insulin resistance in adipose tissue

Abstract

Aims/introduction: Obesity is characterized by disturbed adipocytokine expression and insulin resistance in adipocytes. Growth arrest-specific 6 (GAS6) is a gene encoding the Gas6 protein, which is expressed in fibroblasts, and its related signaling might be associated with adipose tissue inflammation, glucose intolerance and insulin resistance. The aim of this study was to investigate the associations among Gas6, adipocytokines and insulin resistance in adipocytes.

Materials and methods: Mature Simpson Golabi Behmel Syndrome adipocytes were treated with high levels of insulin to mimic insulin resistance, and were examined for the expressions of Gas6, cytokines and adipocytokines from preadipocytes in differentiation. In an animal study, high-fat diet-induced obese mice were used to verify the Gas6 expression in vitro.

Results: During the differentiation of adipocytes, the expression of Gas6 gradually decreased, and was obviously downregulated with adipocyte inflammation and insulin resistance. Gas6 levels were found to be in proportion to the expression of adiponectin, which has been regarded as closely relevant to improved insulin sensitivity after metformin treatment. Similar results were also confirmed in the animal study.

Conclusions: Our results suggest that Gas6 might modulate the expression of adiponectin, and might therefore be associated with insulin resistance in adipose tissues.

Keywords: Adipocyte; Adiponectin; Gas6.

Figure 1

The morphology and expression of insulin resistance markers during adipocytes differentiation. (a) Preadipocyte Simpson Golabi Behmel Syndrome cells were induced to differentiate for the indicated times, and the morphology of day‐0 (D0), day‐7 (D7), day‐14 (D14) and day‐16 (D16) adipocytes (magnification: ×10 and ×40). The quantification of lipid content by Oil Red O staining. (b) Insulin resistance markers (insulin receptor substrate [IRS], fatty acid synthase [FASN], tumor necrosis factor‐α [TNF‐α], peroxisome proliferator‐activated receptor gamma [PPAR‐γ]) expression during differentiation of adipocyte by real‐time polymerase chain reaction. Data are presented as the mean ± standard deviation. All P for trend <0.01 and *P < 0.05. mRNA, messenger ribonucleic acid.

Figure 2

Growth arrest‐specific 6 (Gas6) expression during the differentiation of adipocyte. (a) The messenger (ribonucleic acid) and (b) protein of Gas6 expression during the differentiation of adipocytes by real‐time polymerase chain reaction and western blot. (c) The secretion of Gas6 proteins during the differentiation of adipocytes by enzyme‐linked immunosorbent assay. Data are presented as the mean ± standard deviation. All P for trend <0.01 and *P < 0.05.

Figure 3

Growth arrest‐specific 6 (Gas6) modulated the expression of adiponectin in adipocytes. (a) The efficiency of Gas6 knockdown in day‐7 (D7) adipocytes. (b) The adipocytokines gene expression (leptin, adiponectin, resistin and retinol‐binding protein 4 [RBP4]) after knockdown Gas6 in D7 adipocytes by real‐time polymerase chain reaction. (c) The adiponectin expression after knockdown Gas6 in D7 adipocytes by enzyme‐linked immunosorbent assay. Data are presented as the mean ± standard deviation. *P < 0.05. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; mRNA, messenger ribonucleic acid.

Figure 4

The expression of growth arrest‐specific 6 (Gas6) and adiponectin in insulin‐resistant obese mice. Mice fed a normal diet (ND; 10% of calories from fat) or high‐fat diet (HF; 60% of calories from fat) for 16 weeks, and blood samples were collected for measurement of blood glucose and insulin. (a) Bodyweight, fasting blood glucose, homeostasis model of assessment of insulin resistance and triglycerides levels are shown. (b) Immunohistochemistry images of Gas6 and adiponectin in adipocyte tissue (magnification: ×40). Data are presented as the mean ± standard deviation. *P < 0.05; n = 10 animals per group.

Figure 5

Metformin ameliorated insulin resistance by enhancing the expression of growth arrest‐specific 6 (Gas6). Using thiazolidinedione (TZD) treatment day‐16 (D16) adipocytes treated with metformin (2 mmol/L) for 6 h. (a) The protein of Gas6, phospho‐adenosine monophosphate‐activated protein kinase (p‐AMPK), adenosine monophosphate‐activated protein kinase (AMPK), phospho‐insulin receptor substrate‐1 (p‐IRS1[307]), insulin receptor substrate‐1(IRS1), phospho‐protein kinase B (p‐Akt) and protein kinase B (Akt) expression by western blot. (b) The adipocytokine gene expressions (adiponectin, leptin, resistin and retinol‐binding protein 4 [RBP4]) by real‐time polymerase chain reaction. (c) The adiponectin expression after metformin treatment in day‐16 (D16) adipocyte by enzyme‐linked immunosorbent assay. Data were presented as the mean ± standard deviation. *P < 0.05. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.