Palmitate-Induced Apoptosis in Cultured Bovine Retinal Pericytes

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-B (NF-B) was activated in cells incubated with 0.4 mmol/l palmitate. Furthermore, inhibition of NF-B 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-B 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-B 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.

Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes

“…2A, columns 6 and 8, respectively), and these unsaturated fatty acids also reversed the palmitate-induced apoptosis ( Fig. 2A, columns 7 and 9, respectively), consistent with previous reports in other cell types (4,7,30). However, when these cells were subsequently exposed to a low-serum (1%) medium, we found a greater cell loss in cultures pretreated with the fatty acid mixture than in nonpretreated cultures (Fig.…”

The role of fatty acids in the development and progression of nonalcoholic fatty liver disease

“…The mechanisms by which they elicit these protective effects are unclear, although reductions in the magnitude of steatosis, oxidative stress and inflammatory pathway activation appear to be involved. Since long chain saturated fatty acids promote oxidative stress and activate inflammatory pathways in cells and tissues not exposed to alcohol [29,37,46,[98][99][100], it seems likely that the presence of alcohol alters metabolism of specific fatty acids within tissues. Subsequent studies that directly compare the effect of saturated fatty acids in models of alcoholic-and nonalcoholic fatty liver disease are needed to address these discrepancies.…”

“…Ceramide accumulation, which can occur via enhanced de novo synthesis using palmitate or increased sphingomyelin breakdown, has been linked to both insulin resistance and apoptosis [42][43][44][45]. In pancreatic β cells and bovine retinal pericytes, saturated fatty acids not only increase ceramide levels, but inhibition of ceramide production prevents saturated fatty acid-induced apoptosis [26,46]. To determine the role of ceramide in saturated fatty acid-mediated apoptosis in liver cells, we incubated H4IIE cells with palmitate in the absence or presence of the ceramide synthetase inhibitor fumonisin B 1 [34].…”

RNASET2 is required for ROS propagation during oxidative stress-mediated cell death

“…[5][6][7][8][9] Compensatory incorporation of lipids into new membrane synthesis or triglyceride stores are likely to be initially protective, 10,11 but ultimately prove maladaptive because of the deleterious consequences of altered membrane composition on organelle function, 12 and because lipids may ultimately be mobilized from inert pools during prolonged exposure. 13 Similarly, whereas engagement of the endoplasmic reticulum (ER) stress machinery or generation of reactive oxygen species (ROS) can serve adaptive or productive signaling functions in response to lipid overload, extreme ER and oxidative stress engage cell death pathways.…”