Conceived and designed the experiments: AF PF SC. Performed the experiments: AF PF SC. Analyzed the data: AF PF SC. Contributed reagents/materials/analysis tools: AF PF SC. Wrote the paper: AF PF SC.
Aurelie Faty and Stephane Commans had their salary paid by GlaxoSmithKline. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all Plos ONE policies on sharing data and materials.
The acute phase response (APR) is characterized by alterations in lipid and glucose metabolism leading to an increased delivery of energy substrates. In adipocytes, there is a coordinated decrease in Free Fatty acids (FFAs) and glucose storage, in addition to an increase in FFAs mobilization. Serum Amyloid A (SAA) is an acute phase protein mainly associated with High Density Lipoproteins (HDL). We hypothesized that enrichment of HDL with SAA, during the APR, could be implicated in the metabolic changes occurring in adipocytes.
Besides its well-characterized role in cholesterol metabolism, SAA has direct metabolic effects on human adipocytes. These metabolic changes could be at least partly responsible for alterations of adipocyte metabolism observed during the APR as well as during pathophysiological conditions such as obesity and conditions leading to insulin resistant states.
The acute phase response (APR) induced during infection or inflammation, is an early and highly complex reaction of the host, which protects it from further injury. The APR is characterized by an increased resting energy expenditure, extensive protein and fat catabolism, negative nitrogen balance, hyperglycemia and hypertriglyceridemia
Serum Amyloid A (SAA) is one of the major acute-phase proteins predominantly produced by the liver
We thus hypothesized that saaHDL, through SAA, could play a major role in the alteration of adipocyte metabolism, providing a molecular link between APR or low grade inflammatory disorders and associated lipid and glucose metabolism abnormalities.
Human recombinant SAA was purchased from PeproTech (Rocky Hill, NJ) and corresponds to human apoSAA1. Human HDL, SAA-enriched HDL and BAY 11–7082 were from Calbiochem. SAA content of SAA-enriched HDL was analyzed by SDS-PAGE and found to be in the range of 8–10% of total proteins. SAA and SAA-enriched HDL endotoxin content was assessed using the LAL assay from Lonza and found to be less that 0.1 ng per μg (0.1 EU/μg). [γ-32P]ATP was from Amersham Life Sciences. Primary antibodies for perilipin were from Progen Biotechnik. All other primary antibodies and MEK1/2 inhibitor PD95059 were from Cell Signaling. Secondary antibodies were obtained from Rockland. All other chemicals were from Sigma, including SB203580, SP600125 and H-89 inhibitors. For western-blots, molecular weights were calculated by interpolation from known standards (Bio-rad, München, Germany).
Adipocyte differentiation of Multipotent Adipose-Derived Stem cells isolated from human adipose tissue (hMADS) was performed as described previously
Subcutaneous abdominal adipose tissue was obtained from nondiabetic subjects who underwent plastic abdominal surgery at the Department of General Surgery, St Louis hospital, Paris, France. Samples were collected with the approval of the St Louis Ethics Committee and all subjects gave their written consent. Subjects on endocrine therapy or antihypertensive therapy and patients with malignant diseases were excluded. Mature adipocytes were isolated by the flotation method following 1h of collagenase (Roche) digestion and several washes. 3.106 ells in DMEM 4.5 g/L glucose with L-Glutamine, sodium pyruvate, 1 g/L BSA were used within 24 h.
Cytotoxicity was measured with the Lactate Dehydrogenase Assay (Cayman). Lipolysis was evaluated by measuring glycerol release from adipocytes using a Glycerol Colorimetric kit (RANDOX®). Adipocytes were incubated in DMEM/F12 supplemented with 2 g/L low endotoxin BSA. Secreted adipokines and cytokines were measured using ELISA kits according to the manufacturers' instructions (human MCP-1 and IL-6 kits: Bender Medsystems; human IL-8, adiponectin and leptin).
Total RNA was isolated using ABIPRISM 6100® (Applied Biosystems), except for isolated mature adipocytes for which Total RNA Isolation Kit (Macherey Nagel) was used. Levels of mRNA were assessed by RTqPCR as described previously
Upon stimulation with SAA (10 µg/mL), hMADS adipocytes cultured in 6-well plates were rinsed briefly with ice-cold PBS. The cells were scraped in 300 µL lysis buffer (Cell Signaling). The lysates were passed through a 25-gauge needle and centrifuged at 20,000×
Results are shown as means ± S.D. Statistical significance was determined using the Student's
As already shown in human and porcine adipocytes, we found that SAA and SAA-enriched HDL (saaHDL) were able to stimulate lipolysis by a mechanism involving the phosphorylation of HSL (results not shown).
One important aspect of adipocyte function is the secretion of a number of anti- or proinflammatory molecules. We then examined the effects of both SAA and saaHDL on cytokine and chemokine secretion in hMADS adipocytes following a 24 h treatment. SAA dose-dependently induced MCP-1, IL-6 and IL-8 secretion (
. hMADS adipocytes were cultured in presence or absence of human recombinant SAA (1, 3, 10 and 30 µg/mL), humans HDL and saaHDL (12.5, 25, 50 and 100 µg/mL) for 24 h. Upon treatment, the supernatants were recovered and secreted concentrations of MCP-1 (Panels A and B), IL-6 (Panels C and D) and IL-8 (Panels E and F) were measured by ELISA. Data are expressed as mean ± SD from n = 3–4 independent experiments. Statistical significance: *
In order to rule out the contribution of a potential SAA endotoxin contamination to the stimulation of cytokines and chemokines secreted by hMADS adipocytes, we have tested the effect of SAA in the presence of a TLR-4 signaling inhibitor, TAK-242
Fully differentiated hMADS adipocytes were preincubated with or without 2 µM TAK-242 inhibitor for 1 h. Upon preincubation, cells were treated in the presence or absence of recombinant human SAA (1, 3 and 10 µg/mL) or LPS (1, 3 and 10 ng/mL) for 24 h. At the end of the treatment period, the supernatants were recovered and secreted MCP-1 (Panels A and B) and IL-6 (Panels C and D) were measured by ELISA. Data are expressed as mean ± SD from 3 independent experiments.
A 24 h SAA treatment did not modify adiponectin secretion (data not shown). However, upon 72 h treatment we observed a dose-dependent decrease in adiponectin secretion (up to 55% inhibition at 30 µg/mL) (
hMADS adipocytes were cultured in the presence or absence of human recombinant SAA (1, 3, 10 and 30 µg/mL), humans HDL and saaHDL (12.5, 25, 50 and 100 µg/mL) for 3 days following which secreted adiponectin (Panels A and B) and leptin (Panels C and D) were measured by ELISA. Data are expressed as mean ± SD from 3 independent experiments. Statistical significance: *
To determine whether SAA-mediated alterations of adipocyte metabolism were concomitant with changes in adipogenic gene expression, we examined the effect of SAA on the expression of two adipocyte transcription factors involved in adipocyte differentiation, Peroxisome Proliferator Activated Receptor-gamma 2 (PPARγ2, and CCAAT/Enhancer Binding Protein alpha (C/EBPα and one involved in lipid synthesis, Sterol Regulatory Element Binding Protein-1c (SREBP-1c). We also measured the expression of several of their target genes. Dose-dependent decreases in the expression of these three genes were apparent after a 24 h treatment of hMADS with SAA (
hMADS adipocytes were cultured in the presence or absence of human recombinant SAA (1, 3, 10 and 30 µg/mL) for 24 h. Upon treatment, total RNA was extracted and expression levels of various genes were analyzed by RTqPCR. Panel A: three important transcription factor (PPARγ2, C/EBPα and SREBP-1c). Panel B: lipogenic genes and adiponectin. Panel C: inflammation related proteins. The mRNA levels, normalized to LRP10 RNA expression, were determined relative to untreated control cells. Data are expressed as mean ± SD from n = 3–5 independent experiments. Statistical significance: *
We then addressed the potential intracellular signalling pathways involved in the metabolic and gene alterations induced by SAA. Since SAA induces NF-κB and MAPK signaling pathways in immune cells, we examined these early signaling events by treating hMADS adipocytes with SAA (10 µg/mL) for up to 1 h. In order to determine whether SAA activates the NF-κB pathway, an electromobility shift assay was performed using nuclear extracts of hMADs adipocytes treated with SAA or with TNFα as a positive control. SAA stimulated the translocation of the NF-κB complex in the nuclear compartment as early as 15 min in a way very similar to TNFα (
To determine whether the induction of proinflammatory factors is a consequence of the activation of these signaling pathways, we used SB203580, a p38 inhibitor, PD98059, a MEK1/2 inhibitor, SP600125, a JNK inhibitor and BAY11-7082, an inhibitor of IκB-alpha phosphorylation. Only BAY11-7082 (10 µM), a JNK inhibitor was able to inhibit IL-8 secretion induced by SAA, IL-6 secretion (data not shown) and MCP-1expression (
While BAY11-7082 blunted SAA-induced MCP-1 gene expression (
In order to confirm the effects of SAA in human adipocytes, we have analysed the effects of SAA on freshly isolated mature human adipocytes from subcutaneous adipose tissue following a 24 h treatment. A low dose (1 µg/mL) of SAA resulted in a maximal induction of IL-6 secretion (
Isolated human adipocytes were prepared as described in “
In this study, we demonstrate that beside its effects on cholesterol metabolism and transport
During APR, there is a decrease in the capacity of adipose tissue to store FFAs. Our data suggests that the decrease in lipid content is a consequence of several effects of SAA on adipocytes such as 1) decrease in gene expression of transcription factors important for adipocyte storage such as PPARγ2, C/EBPαand SREBP-1c; 2) decrease in gene expression of the main enzymes involved in lipogenesis like G3PDH and FAS; 3) increase in lipolysis through activation of the ERK pathway and HSL as previously described also in other models
Given that SAA is a pro-inflammatory cytokine on immune cells, we studied the adipokines secretion profile of human adipocytes
In humans, SAA1 is mainly produced by the liver during the APR, while in obesity adipocytes contribute to the plasma SAA levels
We previously reported that SAA is a player in the dialogue between hypertrophied adipocytes and macrophages through its regulation of adipocyte cholesterol efflux
This is the first study to address the early SAA intracellular signaling pathways in human adipocytes which demonstrates that SAA activates NF-κB and MAPK signaling pathways (p44/42, p38 and p46-JNK) in these cells. Furthermore, BAY-117082, a NF-κB pathway inhibitor, completely blunted SAA-induced inflammation while PD98059, an ERK pathway inhibitor blunted SAA-induced lipolysis. A majority of studies has shown that SAA signals through a seven transmembrane G-coupled receptor FPRL1 leading to NF-κB and MAPK pathways activation whilst other reports suggest that SAA could also bind and signal through Scavenger Receptor class B type I (SR-BI/CLA-1) at least for MAPK activation
We have extended these results to mature human adipocytes freshly isolated from subcutaneous adipose tissue. In mature human adipocytes, a low dose of SAA (1 µg/mL) was able to confer a maximal induction of inflammation as well as a maximal repression of the transcription factors (PPARγ2, C/EBPα and SREBP-1c) suggesting higher SAA receptor expression.
In summary, our data suggest that SAA could be at least partly responsible for the metabolic changes of the adipose tissue during the APR. At the molecular level, SAA represses the gene expression of PPARγ2, C/EBPα and SREBP-1c, three important transcription factors. In addition, SAA activates the NF-κB pathway leading to the induction of inflammation. These effects could translate into a phenotype characterized by a coordinated decrease in the storage of FFAs and an increase in FFAs mobilization and at least partly explain the reduced insulin efficiency concomitant with APR. Our findings also reveal that in conditions of low grade inflammation such as obesity, SAA could participate to the metabolic phenotype characterized by adipose tissue inflammation, insulin resistance and fatty acid overflow from adipocytes.
Forward and reverse oligonucleotides used for Real Time quantitative PCR.
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We thank Dr Fabienne Foufelle, Julie Lesieur and Dr Olivier Bourron (INSERM, UMR-S 872) for their help with the isolation of mature human adipocytes from subcutaneous adipose tissue and Isabelle Hainault (INSERM, UMR-S 872) for technical contributions, Dr Stéphane Huet, Dr Edwige Nicodème and Dr Akanksha Gangar (GSK, Metabolic Pathways CEDD) for critical reading of the manuscript, and Valérie Baudet (GSK, Metabolic Pathways CEDD) for technical contributions.