Inhibition of phorbol ester-induced AP-1-DNA binding, c-Jun protein and c-jun mRNA by dietary energy restriction is reversed by adrenalectomy in SENCAR mouse epidermis.

The aim of this study was to determine the effects of 40% dietary energy restriction (DER) relative to ad libitum feeding on AP-1-DNA binding and expression of c-Jun protein and c-jun mRNA in SENCAR mouse skin treated with acetone or 12-O-tetradecanoylphorbol 13-acetate (TPA). The role of the glucocorticoid hormone corticosterone (CCS) was investigated by adding CCS or vehicle control to the drinking water of adrenalectomized mice. AP-1-DNA binding, measured by electrophoretic mobility shift assay, showed that TPA treatment for 4 h increased AP-1-DNA binding by 2-fold over acetone controls (P < 0.05) and that DER reduced basal and TPA-induced AP-1-DNA binding in comparison with ad libitum fed groups in sham-operated mice (P < 0.05). TPA treatment increased c-Jun protein levels in control fed mice (4-fold) and in DER mice (2-fold) over basal levels 4 h post-treatment (P < 0.05). Analyzed over all groups, DER reduced c-Jun protein levels (P < 0.01) and this effect was reversed by adrenalectomy. TPA induction of c-jun mRNA was also reduced by DER compared with ad libitum fed mice (P < 0.05). Adrenalectomy and CCS supplementation demonstrated that the effects of DER on AP-1-DNA binding were mediated in part by CCS. Measurement of blood plasma CCS concentrations showed that: (i) DER increased CCS 5-fold over ad libitum fed mice in sham-operated animals (P < 0.05); (ii) adrenalectomy decreased CCS over sham-operated mice (P < 0.05); (iii) TPA treatment had no effect on CCS. Blood plasma IGF-I concentrations were unaffected by CCS modulation or TPA treatment but were decreased by DER compared with ad libitum fed mice (P < 0.05). Thus, dietary energy restriction may inhibit cancer mechanistically by reducing overall AP-1 transcription through a process that is mediated in part by glucocorticoid hormones.

Dietary energy restriction inhibits ERK but not JNK or p38 activity in the epidermis of SENCAR mice.

Ongoing studies in our laboratory have demonstrated that dietary energy restriction (DER) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 transcription factor binding to DNA in the epidermis of SENCAR mice. To dissect the specific signal transduction pathways through which DER inhibits the AP-1:DNA binding, we analyzed the activities of three major MAP kinases that lead to the induction of AP-1. The changes in ERK1 and ERK2 protein expression and phosphorylation were further characterized by western blot analysis. Female SENCAR mice were pre-fed ad libitum (AL) or 40% DER diet for 8-10 weeks. The kinase activities in mouse epidermis were determined by immune complex kinase assays at 0.5, 1, 4, or 6 h following treatment with 3.2 nmol TPA to the shaved dorsal backs. ERK activity at 1 h post-TPA treatment was nearly 5-fold (P< 0.005) above basal levels in AL mice while the increase was abolished in DER mice. The TPA-induced ERK activity in AL mice was accompanied by increased phosphorylation of ERK1 and ERK2 (P< 0.05), which was abrogated in DER mice. In addition, DER mice exhibited reduced expression of total ERK1 and ERK2 and higher proportions of ERK1 and ERK2 phosphorylation in comparison with AL mice (P<0.05). JNK activity was decreased at 1 and 6 h but increased at 4 h (P<0.05) post-TPA treatment. TPA did not change p38 kinase activity at the time points tested. Neither JNK nor p38 activity was altered by DER. Taken together, our results indicated for the first time that DER blocked the TPA stimulation of ERK activity and suggested that the inhibition of TPA-induced AP-1 activity by DER is likely through inhibition of ERK but not JNK or p38 kinase pathway.

Dietary energy restriction in the SENCAR mouse: elevation of glucocorticoid hormone levels but no change in distribution of glucocorticoid receptor in epidermal cells.

The purpose of this study was to demonstrate that dietary energy restriction elevates plasma glucocorticoid hormone (GCH) levels while maintaining a circadian profile. Furthermore, we indirectly measured the effect of energy restriction on receptor activation in epidermis by determining the cellular localization of receptor protein in control-fed and energy-restricted (ER) mice. SENCAR mice were maintained on an ad libitum control diet or an ER diet that provided 60% of the total energy consumed by control-fed mice. Plasma corticosterone levels were determined by radioimmunoassay. Glucocorticoid receptor (GR) protein levels in epidermal lysates were measured by western blotting. Electron microscopy was used to identify gold-conjugated immunoreactive GR in epidermal cells of the skin in control and ER mice. Plasma corticosterone levels in ER mice were significantly increased 10 times over the levels in control mice at 0700 h, significantly increased two times over control levels at 1600 h, and not different from controls at 2300 h in the circadian cycle. The total amount of epidermal GR protein in ER mice was 140% (95% confidence interval, 104-169%) of that in controls at the early time point, not different at the midpoint, and 60% (95% confidence interval, 48-79%) of that in controls at the late time point. The distribution of gold-conjugated GR in the cytoplasmic and nuclear compartments of epidermal cells was similar in control and ER mice. Thus, we showed that dietary ER increased the level of plasma GCH without abolishing diurnal variation. However, an increase in ligand activation in epidermal cells, as indicated by nuclear localization of GR protein, was not supported by the results of this study.

Dietary energy restriction-induced modulation of protein kinase C zeta isozyme in the hamster pancreas.

Dietary restriction in experimental animals enhances life span, delays disease, inhibits immunological perturbations, and ameliorates cancer. Protein kinase C (PKC) isozymes mediate signals generated by hormones, growth factors, and neurotransmitters for cell proliferation and differentiation. The results of our study showed that a C-terminally directed anti-PKC zeta antibody detected an 81-kDa band in the pancreases of control and energy-restricted hamsters. Syrian golden hamsters were fed energy-restricted diets formulated such that the hamsters received 90% (10% energy restriction (ER)), 80% (20% ER), or 60% (40% ER) of the total energy consumed by control hamsters, with the energy reduced proportionally from fat and carbohydrate. ER decreased PKC zeta isozyme levels by 40-75% in hamsters fed 10, 20, and 40% ER diets for 8 wk. PKC zeta isozyme expression was decreased by 75-80% in hamsters fed ER diets for 15 wk. Although ER caused significant decreases in PKC zeta isozyme levels compared with those of control hamsters at both time points, the relative differences in PKC zeta levels between the dietary ER groups (10, 20, and 40%) were small and not significant. A significant decrease in the body weights of ER animals compared with those of controls was observed at both time points. No differences in tomato lectin and phytohemagglutinin reactivity were observed between control animals and animals fed 10, 20, and 40% ER diets. Furthermore, the cellular expression of PKC zeta in the hamster pancreas did not differ among hamsters fed the various ER diets. These observations may be important for understanding not only the role of dietary ER in pancreatic cancers but also PKC zeta signal transduction mechanisms in normal pancreatic physiology.