Salt Induces Adipogenesis/Lipogenesis and Inflammatory Adipocytokines Secretion in Adipocytes

Abstract

It is well known that high salt intake is associated with cardiovascular diseases including hypertension. However, the research on the mechanism of obesity due to high salt intake is rare. To evaluate the roles of salt on obesity prevalence, the gene expression of adipogenesis/lipogenesis and adipocytokines secretion according to adipocyte dysfunction were investigated in salt-loading adipocytes. High salt dose-dependently increased the expression of adipogenic/lipogenic genes, such as PPAR-γ, C/EBPα, SREBP1c, ACC, FAS, and aP2, but decreased the gene of lipolysis like AMPK, ultimately resulting in fat accumulation. With SIK-2 and Na⁺/K⁺-ATPase activation, salt increased the metabolites involved in the renin-angiotensin-aldosterone system (RAAS) such as ADD1, CYP11β2, and MCR. Increasing insulin dependent insulin receptor substrate (IRS)-signaling, resulting in the insulin resistance, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and Akt-mTOR were activated but AMPK(Thr¹⁷²) was depressed in salt-loading adipocytes. The expression of pro-inflammatory adipocytokines, TNFα, MCP-1, COX-2, IL-17A, IL-6, leptin, and leptin to adiponectin ratio (LAR) were dose-dependently increased by salt treatment. Using the inhibitors of MAPK/ERK, U0126, we found that the crosstalk among the signaling pathways of MAPK/ERK, Akt-mTOR, and the inflammatory adipogenesis can be the possible mechanism of salt-linked obesity. The possibilities of whether the defense mechanisms against high dose of intracellular salts provoke signaling for adipocytes differentiation or interact with surrounding tissues through other pathways will be explored in future research.

Keywords: Akt-mTOR; MAPK/ERK; adipogenesis; inflammatory cytokines; obesity; salt.

Figure 1

Effect of different levels of salts on cell viability, the expression of SIK2 and Na⁺/K⁺-APTase and insulin-related renin-angiotensin-aldosterone system (RAAS) signaling. 3T3-L1 preadipocytes were treated with various doses of salt (0 to 400 mM) during the differentiation period (from day 0 to day 7). Cell viability was measured by the CCK-8 kit in both of preadipocytes (A-a) and adipocytes (A-b). Protein expression of SIK2 (B) and Na⁺/K⁺ -ATPase (C) in salt-loading adipocytes were measured by western blotting. The major metabolites of RAAS, ADD1, CYP11β2, Ang II, and mineralocorticoid receptor (MCR), were analyzed by western blotting (D). Data are expressed as mean ± SE of three independent experiments or more. * p < 0.05, ** p < 0.01, *** p < 0.001, significant difference vs control.

Figure 2

Effect of different levels of salts on TG accumulation, gene expression related to adipogenesis and lipolysis in adipocytes. Using confocal immunofluorescent analysis of 3T3-L1 adipocytes, lipid droplets have been labeled with BODIPY^® 493/503 (green), and fluorescent DNA dye (blue). The fluorescence intensity ratio shows the concentration dependence of salt induced lipid accumulation normalized to the number of cells. (A-a) Triglyceride levels in culture supernatant of salt treated 3T3-L1 adipocytes. (A-b) The nuclear active form of SREBP-1c was increased, but cytoplasmic SREBP-1c protein was significantly depressed by high salt treatment of 100 mM. (B) Protein expression for PPARɣ and C/EBPα and transcription factors of aP2 related to adipogenesis were detected by immunoblotting assay. (C) Messenger RNA of lipogenesis genes, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), were measured by rt-PCR. (D) Total- and phospho-AMPK^Thr172 were analyzed by western blotting. (E) Data are expressed as mean ± SE of three independent experiments or more. * p < 0.05, ** p < 0.01, *** p < 0.001, significant difference vs control.

Figure 3

Salt modulates the production of pro-inflammatory cytokines and insulin resistance in adipocytes. Adipocytes were pretreated with concentrations of salt (0 to 100 mM) during differentiation of adipocytes. Pro-inflammatory cytokines were detected by western blotting (A-a), ELISA (A-b), and flow cytometry (B-a,-b). The leptin and adiponectin, and leptin/adiponectin ratio (LAR) were analyzed by western blotting (C) and semi-quantitative rt-PCR (D). Cell lysates were immunoblotted with antibodies against the insulin signaling such as IRS-1, phospho-IRS-1 (Ser^636/639), AS160, and phospho-AS160 (Thr⁶⁴²). (E) Data are expressed as mean ±SE of three independent experiments or more. * p < 0.05, ** p < 0.01, *** p < 0.001, significant difference vs control.

Figure 3

Salt modulates the production of pro-inflammatory cytokines and insulin resistance in adipocytes. Adipocytes were pretreated with concentrations of salt (0 to 100 mM) during differentiation of adipocytes. Pro-inflammatory cytokines were detected by western blotting (A-a), ELISA (A-b), and flow cytometry (B-a,-b). The leptin and adiponectin, and leptin/adiponectin ratio (LAR) were analyzed by western blotting (C) and semi-quantitative rt-PCR (D). Cell lysates were immunoblotted with antibodies against the insulin signaling such as IRS-1, phospho-IRS-1 (Ser^636/639), AS160, and phospho-AS160 (Thr⁶⁴²). (E) Data are expressed as mean ±SE of three independent experiments or more. * p < 0.05, ** p < 0.01, *** p < 0.001, significant difference vs control.

Figure 4

Salt regulates MAPK/ERKs and Akt-mTOR pathways. The ratio of phospho-ERK vs total ERK, phospho-p38 vs total p38 and phospho-JNK vs total JNK proteins were measured by immunoblotting. (A) U0126, inhibitor of MAPK and ERK, depressed the salt-induced up-regulation of p-ERK/ERK1/2, and PPARɣ. (B) Post-confluent 3T3-L1 preadipocytes were preincubated by MEK/ERK inhibitor for 24 h prior to induction of differentiation. The ratio of phospho-Akt^Ser473/Atk protein levels expression were increased by increasing salt treatment, but phosphorylation at Akt^Ser308 was not involved. The inhibitor of MAPK/ERK, U0126, declined the salt-induced Akt activation and mTOR protein expression. (C,D). Data are expressed as mean ± SE of three independent experiments or more. * p < 0.05, ** p < 0.01, *** p < 0.001, significant difference vs control.