Acute-phase serum amyloid A: an inflammatory adipokine and potential link between obesity and its metabolic complications

Abstract

Background: Obesity is associated with low-grade chronic inflammation, and serum markers of inflammation are independent risk factors for cardiovascular disease (CVD). However, the molecular and cellular mechanisms that link obesity to chronic inflammation and CVD are poorly understood.

Methods and findings: Acute-phase serum amyloid A (A-SAA) mRNA levels, and A-SAA adipose secretion and serum levels were measured in obese and nonobese individuals, obese participants who underwent weight-loss, and persons treated with the insulin sensitizer rosiglitazone. Inflammation-eliciting activity of A-SAA was investigated in human adipose stromal vascular cells, coronary vascular endothelial cells and a murine monocyte cell line. We demonstrate that A-SAA was highly and selectively expressed in human adipocytes. Moreover, A-SAA mRNA levels and A-SAA secretion from adipose tissue were significantly correlated with body mass index (r = 0.47; p = 0.028 and r = 0.80; p = 0.0002, respectively). Serum A-SAA levels decreased significantly after weight loss in obese participants (p = 0.006), as well as in those treated with rosiglitazone (p = 0.033). The magnitude of the improvement in insulin sensitivity after weight loss was significantly correlated with decreases in serum A-SAA (r = -0.74; p = 0.034). SAA treatment of vascular endothelial cells and monocytes markedly increased the production of inflammatory cytokines, e.g., interleukin (IL)-6, IL-8, tumor necrosis factor alpha, and monocyte chemoattractant protein-1. In addition, SAA increased basal lipolysis in adipose tissue culture by 47%.

Conclusions: A-SAA is a proinflammatory and lipolytic adipokine in humans. The increased expression of A-SAA by adipocytes in obesity suggests that it may play a critical role in local and systemic inflammation and free fatty acid production and could be a direct link between obesity and its comorbidities, such as insulin resistance and atherosclerosis. Accordingly, improvements in systemic inflammation and insulin resistance with weight loss and rosiglitazone therapy may in part be mediated by decreases in adipocyte A-SAA production.

Figure 1. Tissue-Restricted Expression of A-SAA mRNA

(A) Representative semiquantitative RT-PCR analysis of A-SAA and β-actin mRNA in SVCs and adipocytes fractionated from human omental (O) and subcutaneous (S) adipose tissues. (B and C) Northern analyses of multiple tissue blots from the human and mouse, respectively. For all Northern analyses, 15 μg of total RNA from the indicated tissues were electrophoresed, blotted onto a nylon membrane, and hybridized with a radiolabeled human (B) or murine (C) SAA2 cDNA probe, which detects both SAA1 and SAA2 (upper gels). Equality of RNA loadings was estimated by ethidium bromide staining (lower gels). Comparison of A-SAA expression was made in five independent participants (B, right). Epi, epididymal; SubQ, subcutaneous

Figure 2. Circulating A-SAA Levels are Positively Correlated with BMI

A-SAA levels were measured in plasma of normal human participants who were divided into lean (BMI < 25 kg m ⁻², n = 54), overweight (BMI 25–30 kg m ⁻², n = 49) and obese (BMI ≥ 30 kg m ⁻², n = 31) groups. Data are expressed as mean ± SEM (ln-transformed for analysis, back-transformed for presentation), adjusted for age, sex, and family structure. * p = 0.013 versus lean group.

Figure 3. Adipose A-SAA Gene Expression and Secretion Are Increased with BMI

Adipose A-SAA mRNA levels, measured by quantitative real-time RT-PCR (top) and A-SAA release by adipose tissue (middle), were significantly correlated with BMI. Furthermore, adipose A-SAA mRNA levels were increased with the adipocyte size (bottom). Dotted lines indicate 95% confidence intervals.

Figure 4. Reductions of Serum A-SAA and Fat Mass are Correlated

Correlation between changes in serum A-SAA levels and changes in body fat mass before and after weight loss. Dotted lines indicate 95% confidence intervals.

Figure 5. Rosiglitazone Reduces Serum A-SAA Levels and Adipose A-SAA Production in Humans

Serum A-SAA ( n = 8) (top) and adipose secretion of A-SAA ex vivo ( n = 7) (bottom) were measured in nondiabetic participants before and after 3 mo of rosiglitazone treatment. The data are plotted with lines connecting the A-SAA levels of each individual. Serum A-SAA and adipose secretion of A-SAA (one symbol for same person of both studies) were significantly decreased by rosiglitazone ( p = 0.033 and p = 0.034, respectively; paired t-test after log-transformation).

Figure 6. Rosiglitazone Directly Suppresses A-SAA Production in Adipose Tissue

Human adipose tissue explants were incubated in cell culture medium 199 (basal) or medium with insulin (Ins, 7 nM) and dexamethasone (Dex, 25 nM) in the presence or absence of rosiglitazone (Rosi, 1 μM) for 48 h. A-SAA production between 24 and 48 h was measured and corrected for tissue weight. Data are expressed as mean ± SEM, n = 3 independent experiments. * p = 0.03, ** p = 0.002, two sample t-test after log-transformation.

Figure 7. SAA Is a Potent Proinflammatory Mediator

Human coronary artery endothelial cells (HCAECs, A), adipose stromal vascular cells (SVCs, B) and mouse RAW264 monocytes (C) were treated with vehicle (PBS, white bar), low (0.47 μg ml ⁻¹, hatched bar), or high (2.34 μg ml ⁻¹, black bar) concentrations of recombinant human SAA for 8 h in serum-free medium. Cell-free supernatants were then assayed for cytokines. Data are expressed as mean ± SEM from n = 3–5 independent experiments. Statistical significance (* p < 0.05; ** p < 0.01; two sample t-test) was observed between the SAA-treated groups and vehicle.

Figure 8. SAA Stimulates Lipolysis

Adipose tissues (eight subcutaneous and one omental) were cultured in the presence or absence of SAA (2.34 μg ml ⁻¹) for 24 h. SAA treatment increased lipolysis by 47% ± 11% as assessed by measurement of glycerol accumulation in the culture medium. Data are expressed as mean ± SEM (log-transformed for analysis, back-transformed for presentation). * p = 0.001, n = 9.

Figure 9. Schematic Diagram of Proposed Pathophysiological Role of Adipocyte-Derived A-SAA in Human Obesity

A-SAA secreted from adipocytes acts locally on adipose SVCs to stimulate cytokine release and in adipocytes to stimulate lipolysis, increasing FFA release and decreasing insulin sensitivity in adipocytes, and possibly contributing to systemic dyslipidemia. In addition, A-SAA secretion by adipocytes into the circulation stimulates cytokine production at more distant sites, including in endothelial cells and monocytes, resulting in endothelial dysfunction, monocyte infiltration, accelerated atherosclerosis, and possibly insulin resistance in muscle and liver. A-SAA-stimulated lipolysis increases circulating FFA concentrations, further contributing to insulin resistance in muscle and liver. Finally, A-SAA incorporation into HDL accelerates its degradation and impairs its function, resulting in decreased HDL and accelerated atherosclerosis.