Treatment of fetal rat hepatocytes with transforming growth factor beta (TGF‐ß) is followed by apoptotic cell death. Analysis of radical oxygen species (ROS) content and mitochondrial transmembrane potential (Δψ _m ), using specific fluorescent probes in FACScan and confocal microscopy, showed that TGF‐ß mediates ROS production that precedes the loss of Δψ _m , the release of cytochrome c, and the activation of caspase 3. TGF‐ß induces a decrease in the protein and mRNA levels of bcl‐x _L , an antiapoptotic member of the Bcl‐2 family. In contrast, there is no change in the expression and/or translocation of Bax, a proapoptotic member of the same family. EGF maintains Bcl‐x _L , preventing Δψ _m collapse and release of cytochrome c. The presence of radical scavengers blocks the decrease in bcl‐x _L levels, Δψ _m collapse, cytochrome c release, and activation of caspase 3; in contrast, the presence of glutathione synthesis inhibitors such as BSO accentuated the effect. The incubation of fetal hepatocytes in the presence of ter‐butyl‐hydroperoxide alone produces a decrease in bcl‐x _L . These results indicate that during the apoptosis mediated by TGF‐ß in fetal hepatocytes, ROS may be responsible for the decrease in bcl‐x _L mRNA levels that precedes the loss of Δψ _m , the release of cytochrome c , and the activation of caspase 3, culminating in cell death.—Herrera, B., Alvarez, A. M., Sanchez, A., Fernandez, M., Roncero, C., Benito, M., Fabregat, I. Reactive oxygen species (ROS) mediates the mitochondrial‐dependent apoptosis induced by transforming growth factor ß in fetal hepatocytes. FASEB J. 15, 741‐751 (2001)

Type 2 diabetes mellitus is a progressive disease that is characterized by the appearance of insulin resistance. The term insulin resistance is very wide and could affect different proteins involved in insulin signaling, as well as other mechanisms. In this review, we have analyzed the main molecular mechanisms that could be involved in the connection between type 2 diabetes and neurodegeneration, in general, and more specifically with the appearance of Alzheimer’s disease. We have studied, in more detail, the different processes involved, such as inflammation, endoplasmic reticulum stress, autophagy, and mitochondrial dysfunction.

Transforming growth factor-β (TGF-β), a growth regulator of fetal hepatocytes in primary culture, also regulates death of these cells. Dose-response analysis showed that the TGF-β concentration needed to induce hepatocyte death (2.5 ng/ml) was 5 times that needed to inhibit growth in these cells (0.5 ng/ml). In response to TGF-β, hepatocytes induced DNA fragmentation and the appearance of nuclei with a DNA content lower than 2C (diploid content), typical of a programmed cell death model. TGF-β-induced apoptosis in fetal hepatocytes was preceded by an induction of reactive oxygen species production and a decrease in the glutathione intracellular content, indicating that this factor induces oxidative stress in fetal hepatocytes. Studies performed to analyze levels of c-fos mRNA, a gene whose expression is modulated by redox state, demonstrated that only high, apoptotic concentrations of TGF-β (2.5 ng/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of tert-butyl hydroperoxide. Gel mobility shift assays showed that the c-fos-induced expression was coincident with an increase in AP-1 activity. Finally, cell death induced by TGF-β in fetal hepatocytes was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-β in these cells. In summary, the results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-β in fetal hepatocytes.

Activity of the sympathetic nervous system is an important factor involved in the pathogenesis of insulin resistance and associated metabolic and vascular abnormalities. In this study, we investigate the molecular basis of cross-talk between β₃-adrenergic and insulin signaling systems in mouse brown adipocytes immortalized by SV40 T infection. Insulin-induced tyrosine phosphorylation of the insulin receptor, insulin receptor substrate 1 (IRS-1), and IRS-2 was reduced by prestimulation of β₃-adrenergic receptors (CL316243). Similarly, insulin-induced IRS-1-associated and phosphotyrosine-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity, but not IRS-2-associated PI 3-kinase activity, was reduced by β₃-adrenergic prestimulation. Furthermore, insulin-stimulated activation of Akt, but not mitogen-activated protein kinase, was diminished. Insulin-induced glucose uptake was completely inhibited by β₃-adrenergic prestimulation. These effects appear to be protein kinase A-dependent. Furthermore inhibition of protein kinase C restored the β₃-receptor-mediated reductions in insulin-induced IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity. Together, these findings indicate cross-talk between adrenergic and insulin signaling pathways. This interaction is protein kinase A-dependent and, at least in part, protein kinase C-dependent, and could play an important role in the pathogenesis of insulin resistance associated with sympathetic overactivity and regulation of brown fat metabolism.

P38α mitogen-activated protein (MAP) kinase is a broadly expressed signaling molecule that participates in the regulation of cellular responses to stress as well as in the control of proliferation and survival of many cell types. We have used cell lines derived from p38α knockout mice to study the role of this signaling pathway in the regulation of apoptosis. Here, we show that cardiomyocytes and fibroblasts lacking p38α are more resistant to apoptosis induced by different stimuli. The reduced apoptosis of p38α-deficient cells correlates with decreased expression of the mitochondrial proapoptotic protein Bax and the apoptosis-inducing receptor Fas/CD-95. Cells lacking p38α also have increased extracellular signal-regulated kinase (ERKs) MAP kinase activity, and the up-regulation of this survival pathway seems to be at least partially responsible for the reduced levels of apoptosis in the absence of p38α. Phosphorylation of the transcription factor STAT3 on Ser-727, mediated by the extracellular signal-regulated kinase MAP kinase pathway, may contribute to the decrease in both Bax and Fas expression in p38α-/- cells. Thus, p38α seems to sensitize cells to apoptosis via both up-regulation of proapoptotic proteins and down-regulation of survival pathways.

Several translational studies have identified the differential role between saturated and unsaturated fatty acids at cardiovascular level. However, the molecular mechanisms that support the protective role of oleate in cardiovascular cells are poorly known. For these reasons, we studied the protective role of oleate in the insulin resistance and in the atherosclerotic process at cellular level such as in cardiomyocytes (CMs), vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). The effect of oleate in the cardiovascular insulin resistance, vascular dysfunction, inflammation, proliferation and apoptosis of VSMCs were analyzed by Western blot, qRT-PCR, BrdU incorporation and cell cycle analysis. Palmitate induced insulin resistance. However, oleate not only did not induce cardiovascular insulin resistance but also had a protective effect against insulin resistance induced by palmitate or TNFα. One mechanism involved might be the prevention by oleate of JNK-1/2 or NF-κB activation in response to TNF-α or palmitate. Oleate reduced MCP-1 and ICAM-1 and increased eNOS expression induced by proinflammatory cytokines in ECs. Furthermore, oleate impaired the proliferation induced by TNF-α, angiotensin II or palmitate and the apoptosis induced by TNF-α or thapsigargin in VSMCs. Our data suggest a differential role between oleate and palmitate and support the concept of the cardioprotector role of oleate as the main lipid component of virgin olive oil. Thus, oleate protects against cardiovascular insulin resistance, improves endothelial dysfunction in response to proinflammatory signals and finally, reduces proliferation and apoptosis in VSMCs that may contribute to an ameliorated atherosclerotic process and plaque stability.