High-salt intake negatively regulates fat deposition in mouse

Abstract

High-salt (HS) intake contributes to hypertension and cardiopathy, but the effect of HS on fat deposition is controversial. Feed intake, fat mass, the percentage of abdominal fat, heat production, rate of oxygen consumption and the respiratory exchange ratio of mice on a HS diet were significantly decreased (P < 0.01 or 0.05) compared with mice on a normal-salt (NS) diet. An in vitro experiment with differentiating pre-adipocytes showed reduced fat deposition in the presence of high concentrations of NaCl (>0.05 M). Abdominal fat mRNA profiles and protein measurements showed that 5 known genes involved in lipolysis were up-regulated significantly and 9 genes related to lipogenesis were down-regulated in HS mice. Abundant genes and some proteins (ATP2a1, AGT, and ANGPTL4) related to calcium ion metabolism or the renin-angiotensin system (RAS) were differentially expressed between HS and NS mice. Of special interest, CREB1 phosphorylation (S133 and S142), a key factor involved in calcium signaling and other pathways, was up-regulated in HS mice. By IPA analysis, a network mediated by calcium was established providing the molecular mechanisms underlying the negative effect of HS on fat deposition.

Figure 1

Change in fat deposition in adipose tissue. Mice were fed a HS (4% NaCl) or a NS (0.4% NaCl) diet or pair-fed (PF) with NS at the level of HS consumed for 8 wk. (A) live weight; (B) AFP (% of liveweight); (C) abdominal fat mass measured; and (D) pictured. Data are means ± SEM, n = 10. *P < 0.05; **P < 0.01.

Figure 2

Changes in feed intake and body metabolism. Feed intake and body metabolism were measured after mice were fed a HS (4% NaCl) or NS (0.4% NaCl) diet for 8 wk. (A) Feed intake (g), (B) heat production (kcal/hr), (C) VO₂ (oxygen consumption, ml/kg/hr), and (D) respiratory exchange rate (RER). Data are means ± SEM, n = 8. *P < 0.05; **P < 0.01.

Figure 3

The effect of NaCl on lipid content in 3T3-L1 cells. Cells were examined, 10 d after inducing differentiation, in media containing 0, 0.0005 M, 0.005 M, 0.05 M, or 0.5 M additional NaCl. (A) The lipid content significantly decreased with 0.05 M and 0.5 M additional NaCl compared with controls. Data are means ± SEM, n = 6. *P < 0.05; **P < 0.01. (B) Morphological changes and lipid deposition induced by 0.5 M additional NaCl (200×).

Figure 4

Technical validation of digital gene expression profile results by q-PCR. The r value (r = 0.932) indicates Spearman’s Correlation between the 2 methods.

Figure 5

Changes in mRNA abundance of genes related to feed intake and lipid metabolism in adipose tissue after mice were fed 8 wk with HS and NS. (A) mRNA levels of lep, which is involved in food intake, was down-regulated. (B) mRNA levels of genes, involved in lipogenesis were down-regulated. (C) mRNA levels of genes involved in lipolysis or carbohydrate utilization were up-regulated. Data are means ± SEM, n = 3. *P < 0.05; **P < 0.01.

Figure 6

Sodium ion concentration in serum from mice fed HS and NS diets. The sodium ion concentration in serum was significantly higher (P < 0.01) in HS mice. Data are means ± SEM, n = 6, **P < 0.01.

Figure 7

Up-regulated expression of genes related to sodium, potassium and calcium ion metabolism, and protein level of ATP2a1 in adipose tissue after mice were fed for 8 wk with HS. (A) mRNA levels of genes related to sodium ion metabolism were up-regulated. (B) mRNA levels of genes related to potassium ion metabolism were up-regulated. (C) mRNA levels of some genes related to calcium ion and lipid metabolism were up-regulated. (D) The protein content of ATP2a1 was increased. Data are means ± SEM, n = 3. *P < 0.05; **P < 0.01.

Figure 8

The mechanistic regulatory network, based on calcium ion and DEGs identified between HS group and NS group, by IPA software.

Figure 9

Changes in the mRNA and protein levels of genes involved in RAS signaling in adipose tissue from feeding a HS diet. (A) Transcript abundance of genes, which are jointly involved in RAS and lipid metabolism, were up-regulated. (B) The protein contents of AGT and ANGPTL4 in abdominal fat (AF) were increased. (C) Transcript abundance of genes, which are jointly involved in RAS and lipid metabolism, were down-regulated. Data are means ± SEM, n = 3. *P < 0.05; **P < 0.01.

Figure 10

Phosphorylation of CREB1 protein (A) and the regulatory network based on calcium ion, CREB1 and DEGs by IPA software (B).