C1q-dependent axis mediating inflammation in adipose tissue after chronic ethanol feeding to mice - PubMed (original) (raw)

Identification of a cytochrome P4502E1/Bid/C1q-dependent axis mediating inflammation in adipose tissue after chronic ethanol feeding to mice

Becky M Sebastian et al. J Biol Chem. 2011.

Abstract

Chronic, heavy alcohol exposure results in inflammation in adipose tissue, insulin resistance, and liver injury. Here we have identified a CYP2E1/Bid/C1q-dependent pathway that is activated in response to chronic ethanol and is required for the development of inflammation in adipose tissue. Ethanol feeding for 25 days to wild-type (C57BL/6J) mice increased expression of multiple markers of adipose tissue inflammation relative to pair-fed controls independent of increased body weight or adipocyte size. Ethanol feeding increased the expression of CYP2E1 in adipocytes, but not stromal vascular cells, in adipose tissue and Cyp2e1(-/-) mice were protected from adipose tissue inflammation in response to ethanol. Ethanol feeding also increased the number of TUNEL-positive nuclei in adipose tissue of wild-type mice but not in Cyp2e1(-/-) or Bid (-/-) mice. Apoptosis contributed to adipose inflammation, as the expression of multiple inflammatory markers was decreased in mice lacking the Bid-dependent apoptotic pathway. The complement protein C1q binds to apoptotic cells, facilitating their clearance and activating complement. Making use of C1q-deficient mice, we found that activation of complement via C1q provided the critical link between CYP2E1/Bid-dependent apoptosis and onset of adipose tissue inflammation in response to chronic ethanol. In summary, chronic ethanol increases CYP2E1 activity in adipose, leading to Bid-mediated apoptosis and activation of complement via C1q, finally resulting in adipose tissue inflammation. Taken together, these data identify a novel mechanism for the development of adipose tissue inflammation that likely contributes to the pathophysiological effects of ethanol.

PubMed Disclaimer

Figures

FIGURE 1.

Inflammatory markers in adipose tissue after ethanol feeding. A, crown-like structures were detected in hematoxylin- and eosin-stained adipose tissue after chronic ethanol feeding for 25d. Images are representative of at least four mice per diet group. B–D, expression of mRNA for cytokines and chemokines (B), inflammatory cell markers (C), and factors in the complement pathway (D) were measured by quantitative real-time-PCR and normalized to 18 S in adipose tissue collected from pair- or ethanol-fed mice at 18 or 25 days. Values represent the means ± S.E., n = 3–6. *, p < 0.04 compared with pair-fed.

FIGURE 2.

Cytochrome P450 2E1 and adipose tissue inflammation and apoptosis after chronic ethanol feeding. Wild-type and _Cyp2e1_−/− mice were allowed free access to ethanol containing diets for up to 25 days or pair-fed control diets. A, quantity of immunoreactive CYP2E1 protein was measured by Western blot in adipose tissue. HSC70 was used as a loading control. Values with different superscripts are significantly different from each other, p < 0.05, n = 4–7. B, the enzymatic activity of CYP2E1 was measured in microsomes isolated from adipose tissue (n = 4). *, p < 0.05 compared with pair-fed. C, the quantity of immunoreactive CYP2E1 protein was measured by Western blot in adipocytes and stromal vascular cells (SVC) isolated from adipose tissue. Images are representative of at least four mice per treatment group. P, pair; E, ethanol. D, expression of mRNA for inflammatory markers was measured as in Fig. 1. Data are expressed as -fold over pair-fed of the same genotype; there were no differences in expression between genotypes in pair-fed mice (data not shown). Values represent the means ± S.E. n = 3–7. *, p < 0.05 compared with pair-fed. E, TUNEL-positive nuclei (red) were detected in adipose tissue from wild-type and _Cyp2e1_−/− mice. Nuclei were labeled with DAPI (blue). TUNEL-positive nuclei were counted and expressed as the percent of DAPI positive nuclei (WT pair-fed, 4.2 ± 0.4; ethanol-fed, 21.2 ± 5.4 (p < 0.05 compared with wild-type pair-fed mice); _Cyp2e1_−/− pair-fed, 1.4 ± 0.6; ethanol-fed, 1.5 ± 1.0, values represent the means ± S.E., n = 3–6).

FIGURE 3.

Apoptosis in adipose tissue after chronic ethanol feeding; role of Bid. Wild-type and _Bid_−/− mice were allowed free access to ethanol-containing diets for up to 25 days or pair-fed control diets. A, TUNEL-positive nuclei (red) were detected in adipose tissue from wild-type and _Bid_−/− mice. Nuclei were labeled with DAPI (blue). TUNEL-positive nuclei were counted and expressed as percent of DAPI positive nuclei (WT pair-fed, 4.3 ± 1.5; ethanol-fed, 19.8 ± 5.9 (p < 0.05 compared with wild-type pair-fed mice); _Bid_−/− pair-fed, 1.6 ± 1.6; ethanol-fed, 7.3 ± 4.5, values represent the means ± S.E., n = 4). B, expression of mRNA was measured as in Fig. 1. Data are expressed as -fold over pair-fed of the same genotype; there were no differences in expression between genotypes in pair-fed mice (data not shown). Values represent the means ± S.E. n = 3–7. Values with different superscripts are significantly different from each other, p < 0.05 compared with pair-fed, except for C5aR, where p < 0.06.

FIGURE 4.

Activation of complement in adipose tissue during ethanol feeding to mice. A, quantity of immunoreactive C3d in adipose tissue lysates was measured by Western blot analysis in wild-type mice fed ethanol diets for up to 25 days or pair-fed control diets for 25 days. HSC70 was used as a loading control. Values represent the means ± S.E., n = 4–8. *, p < 0.02 compared with pair-fed. B, quantity of immunoreactive C3d in plasma was measured by Western blot from pair- or ethanol-fed wild-type mice at 18 and 25 days. Immunoreactive C3d is expressed relative to the quantity of plasma loaded onto the gels. Values represent the means ± S.E., n = 8–10. *, p < 0.01, compared with pair-fed. C and D, immunoreactive C3b (red) was detected by immunohistochemistry in paraffin-embedded section of adipose from pair (P)- and ethanol (E)-fed wild-type (C) or _cyp2e1_−/− or _Bid_−/− mice (D). DAPI (blue) was used to visualize nuclei. Chow-fed _C3_−/− mice (C) were used as a negative control. Images are representative of at least four mice per diet group.

FIGURE 5.

C1qa, but not MBLa/c or Cfd, contributes to chronic ethanol-induced adipose inflammation. Shown is expression of mRNA after 25 days of ethanol or pair feeding in adipose tissue from wild-type or knock-out mice deficient in specific pathways of complement activation (A and C) or triple-knock out (MBLa/c/_Cfd_−/−) mice expressing only the classical pathway of complement activation (D). A, C, and D, mRNA was measured by quantitative real-time-PCR and normalized to 18 S. Data are expressed as -fold over pair-fed of the same genotype; there were no differences in expression between genotypes in pair-fed mice (data not shown). Values represent the means ± S.E., n = 3–6. *, p < 0.05 compared with pair-fed in each genotype. B, TUNEL-positive nuclei (red) were detected in adipose tissue from wild-type and _C1qa_−/− mice. Nuclei were labeled with DAPI (blue). TUNEL-positive nuclei were counted and expressed as percent of DAPI positive nuclei (wild-type pair-fed, 0 ± 0; ethanol-fed. 21.0 ± 5.1 (values represent means ± S.E., n = 4); _C1qa_−/− pair-fed, 4.9 ± 1.0; ethanol-fed, 29.0 ± 7.1 (values represent means ± S.E., n = 4).

FIGURE 6.

Schematic diagram illustrating the role of CYP2E1, Bid-dependent apoptosis, and complement activation in the development of adipose tissue inflammation during chronic ethanol feeding to mice. Chronic ethanol feeding induces the expression of CYP2E1 in adipocytes. CYP2E1 then leads to increased expression of TNF-α, which in turn activates Bid-dependent apoptosis of adipocytes. C1q, a component of the classical pathway of complement, binds to cell surface markers on apoptotic adipocytes, resulting in complement activation. Complement activation products, interacting with the anaphylatoxin receptors, C3a receptor and C5a receptor, further increase cytokine and chemokine production in adipose. The cumulative result of this CYP2E1 → Bid-dependent apoptosis → C1q-mediated complement activation pathway is the development of adipose tissue inflammation.

References

1. Tilg H., Moschen A. R. (2006) Nat. Rev. Immunol. 6, 772–783 -PubMed
1. Tilg H., Diehl A. M. (2000) New Engl. J. Med. 343, 1467–1476 -PubMed
1. Kang L., Sebastian B. M., Pritchard M. T., Pratt B. T., Previs S. F., Nagy L. E. (2007) Alcohol Clin. Exp. Res. 31, 1581–1588 -PubMed
1. Chen X., Sebastian B. M., Tang H., McMullen M. M., Axhemi A., Jacobsen D. W., Nagy L. E. (2009) Hepatology 49, 1554–1562 -PMC -PubMed
1. Kang L., Chen X., Sebastian B. M., Pratt B. T., Bederman I. R., Alexander J. C., Previs S. F., Nagy L. E. (2007) J. Biol. Chem. 282, 28465–28473 -PubMed

Identification of a cytochrome P4502E1/Bid/C1q-dependent axis mediating inflammation in adipose tissue after chronic ethanol feeding to mice - PubMed (original) (raw)