Palmitate-Induced Apoptosis in Cultured Bovine Retinal Pericytes

Roles of NAD(P)H Oxidase, Oxidant Stress, and Ceramide

  1. Jose M. Cacicedo,
  2. Sunun Benjachareowong,
  3. Eva Chou,
  4. Neil B. Ruderman and
  5. Yasuo Ido
  1. Department of Medicine and Section of Endocrinology, Diabetes and Metabolism Research Unit, Boston University School of Medicine, Boston, Massachusetts
  1. Address correspondence and reprint requests to Dr. Neil B. Ruderman, Diabetes and Metabolism Research Unit, Department of Medicine and Section of Endocrinology, Boston University School of Medicine, 650 Albany St., 8th Floor, Room 820, Boston, MA 02118. E-mail: nrude{at}bumc.bu.edu

Abstract

Apoptosis of pericytes (PCs) is an early event in diabetic retinopathy. It is generally thought to be a consequence of sustained hyperglycemia. In keeping with this, long-term (>7 days) incubation of cultured PCs in a high-glucose media has been shown to increase apoptosis. We examine here whether the saturated free fatty acid palmitate, the concentration of which is often elevated in diabetes, has similar effects on cultured PCs. Incubation with 0.4 mmol/l palmitate for 24 h induced both oxidant stress and apoptosis, as evidenced by a sixfold increase in DCF fluorescence and a twofold increase in caspase-3 activation, respectively. NAD(P)H oxidase appeared to be involved in these responses, since overexpression of dominant-negative subunits of NAD(P)H oxidase, such as phox47(DN), diminished oxidant stress, and phox67(DN) and N-17 RAC1(DN) prevented the increase in caspase-3 activity. Likewise, overexpression of vRAC, a constitutively active RAC1, increased caspase-3 activity to the same extent as palmitate alone. The effects of vRAC and palmitate were not additive. In parallel with the increases in oxidative stress, the redox-sensitive transcription factor nuclear factor-κB (NF-κB) was activated in cells incubated with 0.4 mmol/l palmitate. Furthermore, inhibition of NF-κB activation by various means inhibited caspase-3 activation. Finally, incubation with palmitate increased the cellular content of ceramide, a molecule linked to apoptosis and increases in oxidative stress and NF-κB activation in other cells. In keeping with such a role, in PCs both coincubation with fumonisin B1 (a ceramide synthase inhibitor) and overexpression of ceramidase I reversed the proapoptotic effect of palmitate. On the other hand, they increased rather than decreased DCF fluorescence. In conclusion, the results suggest that palmitate-induced apoptosis in PCs is associated with activation of NAD(P)H oxidase and NF-κB and an increase in ceramide. The precise interactions between these molecules in causing apoptosis and the importance of oxidant stress as a contributory factor remain to be determined.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Accepted March 17, 2005.
    • Received January 7, 2005.
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