Acute-phase serum amyloid A as a marker of insulin resistance in mice

Abstract

Acute-phase serum amyloid A (A-SAA) was shown recently to correlate with obesity and insulin resistance in humans. However, the mechanisms linking obesity-associated inflammation and elevated plasma A-SAA to insulin resistance are poorly understood. Using high-fat diet- (HFD-) fed mice, we found that plasma A-SAA was increased early upon HFD feeding and was tightly associated with systemic insulin resistance. Plasma A-SAA elevation was due to induction of Saa1 and Saa2 expression in liver but not in adipose tissue. In adipose tissue Saa3 was the predominant isoform and the earliest inflammatory marker induced, suggesting it is important for initiation of adipose tissue inflammation. To assess the potential impact of A-SAA on adipose tissue insulin resistance, we treated 3T3-L1 adipocytes with recombinant A-SAA. Intriguingly, physiological levels of A-SAA caused alterations in gene expression closely resembling those observed in HFD-fed mice. Proinflammatory genes (Ccl2, Saa3) were induced while genes critical for insulin sensitivity (Irs1, Adipoq, Glut4) were down-regulated. Our data identify HFD-fed mice as a suitable model to study A-SAA as a biomarker and a novel possible mediator of insulin resistance.

Figure 1

(a) Fasting plasma insulin, (b) glucose, (c) adiponectin, and (d) A-SAA levels in mice fed either control diet (Chow), control diet for 15 weeks, and HFD for one week (1 w HFD), and HFD for 16 weeks (16 w HFD), respectively. Bar graphs are presented as mean ±SEM (n = 5–8), statistics:*P < .05 versus Chow, **P < .01 versus Chow, ^(a) P < .05 versus 1 w HFD, ^(b) P < .01 versus 1 w HFD; Mann-Whitney U-Test was used for insulin (7/8 measurements in Chow group were below detection level, 56 pmol/L); two-tailed Student's t-test for all other measurements.

Figure 2

(a), (b) Diet-induced expression of SAA isoforms and (c), (d) SAA inducers in (a), (c) liver and (b), (d) adipose tissue, quantified by real-time quantitative PCR. Data are mean ±SEM normalized to control animals (fold over Chow). Number of animals (n), see Table 1. Statistics:*P < .05 versus Chow, **P < .01 versus Chow, ^(a) P < .05 versus 1 w HFD, ^(b) P < .01 versus 1 w HFD; two-tailed Student's t-test.

Figure 3

(a) Diet-induced expression of inflammatory genes in liver and (b) adipose tissue, quantified by real-time quantitative PCR. Data are mean ±SEM normalized to control animals (fold over Chow). Number of animals (n), see Table 2. Statistics:*P < .05 versus Chow, **P < .01 versus Chow, ^(a) P < .05 versus 1 w HFD, ^(b) P < .01 versus 1 w HFD, two-tailed Student's t-test.

Figure 4

(a) Diet-induced expression of genes related to insulin sensitivity in liver and (b) adipose tissue, quantified by real-time quantitative PCR. Data are mean ±SEM normalized to control animals (fold over Chow). Number of animals (n), see Table 2. Statistics:*P < .05 versus Chow, **P < .01 versus Chow, ^(a) P < 0.05 versus 1 w HFD, ^(b) P < .01 versus 1 w HFD, two-tailed Student's t-test.

Figure 5

Effects of recombinant A-SAA and TNFα on gene expression in differentiated 3T3-L1 adipocytes. (a) Dose-dependent induction of Saa3 and Ccl2 by recombinant A-SAA. Differentiated 3T3-L1 adipocytes were washed with PBS and starved overnight in DMEM containing 0.1% BSA. Subsequently, cells were treated for 24 hours with various concentrations of recombinant A-SAA (0, 0.125 1.25, 12.5, 25 μg/mL). (b) Regulation of signaling genes by A-SAA and TNFα: Differentiated 3T3-L1 adipocytes were treated as described under (a) and subsequently incubated with A-SAA (12.5 μg/mL, closed bars) and TNFα (10 ng/mL, hatched bars) for 24 hours. (c) Effect of polymyxin B (PMB) on induction of Saa3 and Ccl2. PMB at 10 μg/mL was added alone or in combination with lipopolysaccharide (LPS, 2.5 ng/mL) and A-SAA (1.25 μg/mL), respectively. Experimental conditions were as described under (a). Copy numbers are relative to one copy of TBP mRNA. Representative data from at least three independent experiments are shown. Data are mean ± standard deviation relative to untreated controls.