We have previously demonstrated sex-specific stimulation of creatine kinase specific activity (CK) in bone cells both in vivo and in vitro, in primary culture cells derived from rat and human bone and in established human bone-derived cell lines. We found that the female-derived cell line, SaOS-2, responded to 17 beta-estradiol (E2) by increased CK specific activity. The effects of E2 on the CK activity in SaOS-2 cells was inhibited by 100-fold excess of 4-hydroxytamoxifen (Tam) as well as by the other antiestrogen, ICI 164,384. Tam by itself had some stimulatory effect whereas ICI 164,384 showed no estrogenic activity. We also demonstrated the estrogenic-like effect of another anti-estrogen, raloxifene (Ral), which is agonist only in the SaOS-2 osteoblast-like cells but not in the human endometrial, Ishikawa cell line. Ishikawa cells respond to E2 and to Tam by increased CK activity. In both osteoblasts and endometrial cell lines, Ral and Tam were inhibitory in the presence of E2. The effects of E2 on SaOS-2 cells are at least partially mediated by the estrogen receptor (ER) at the level of transcription as demonstrated by transient transfection experiments using the human creatine kinase promoter chloramphenicol acetyltransferase in these cells. Pretreatment of SaOS-2 with calcitropic hormones, either 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or human parathyroid hormone (1-34) (hPTH(1-34)) increased the stimulation of CK by E2 by 40-60% relative to E2 alone and significantly increased the sensitivity of the cells to E2 by lowering the effective hormonal dose needed for stimulation of CK by E2 by 100-fold. This stimulatory effect of pretreatment of the cells with 1,25(OH)2D3 was due to a 2.5-fold increase in the level of ER expression as measured directly by enzyme immunoassay in the SaOS-2/1 subline. The increase in the responsiveness to E2 by hPTH(1-34) was not due to an increase in ER level in the cells. We can conclude that in cell cultures as in vivo, Ral shows different effects depending on the cell type, namely estrogenic-like activity in skeletal cells but not in uterine cells. We can also conclude that as with rat-derived cells, in bone cells derived from human bone 1,25(OH)2D3 increased the sensitivity to E2 due to an increase in the number of ER in the cells, whereas PTH(1-34) augmented the response to E2 without increasing ER, by another, as yet unknown, mechanism. These studies suggest that the treatment of pathological bone disorders may be improved by combined hormone therapy.

Others reported that rats fed a high-fructose diet for 6 months were leptin resistant. We tested peripheral and/or central leptin responses in rats fed fructose for shorter time periods. Rats fed a diet containing 60% energy (% kcal) fructose and 10% kcal fat diet for 21 days had the same serum triglycerides (TG), gained less weight than controls, decreased their food intake and weight gain in response to central injections of 0.5 or 1.0 ug leptin, but were resistant to an i.p. injection of 2.0 mg leptin/kg. An i.p. injection of 1 mg leptin/kg increased phosphorylation of hypothalamic signal transducer and activator of transcription 3 (PSTAT3) implying resistance was not a failure of leptin to cross the blood brain barrier. The effects of dietary fructose were compared with those of dietary fat. Rats fed a 10% kcal fructose and 30% kcal fat diet for 39 days were leptin resistant whereas rats fed a 40% kcal fructose and 30% kcal fat diet responded to i.p. leptin. Another monosaccharide, glucose, replicated the effects of fructose in the 30% kcal fat diet. Surprisingly, none of the rats showed a reliable response to third ventricle leptin and peripheral leptin failed to stimulate hypothalamic PSTAT3 although it did increase PSTAT3 in the brainstem of rats fed the 40% kcal fructose or glucose diets. Thus a high-fructose, low-fat diet induces peripheral leptin resistance in less than 4 weeks, but high dietary concentrations of fructose or glucose prevent peripheral leptin resistance in rats fed a high-fat diet.

No abstract

Previously we showed that a 60% kcal fructose, and 10% kcal fat diet induces leptin resistance in rats. All rats fed the 60% kcal fructose diet were thinner than those fed a diet with 17% fructose and 10% fat (LFruc/LF), therefore, we tested leptin responsiveness in rats fed either a 40% kcal fructose and 30% kcal fat (MFruc/HF) or the LFruc/LF diet. An i.p injection of 2.0 mg/kg leptin inhibited food intake and weight gain of both MFruc/HF and LFruc/LF rats after days 7, 14, and 21 on diet. On day 36, leptin inhibited 14‐hour energy intake only in LFruc/LF fed rats. To determine whether changes in leptin responsiveness were due to fructose specifically or increased dietary monosaccharide, a second study tested leptin responsiveness in rats fed a 50% kcal glucose and 30% kcal fat diet (HGluc/HF), MFruc/HF or LFruc/HF diet. After 10.5 weeks, i.p. injections of 2.0 mg/kg leptin inhibited 14 hour weight gain and food intake in MFruc/HF and HGluc/HF groups but not LFruc/HF rats. These data suggest that dietary fat and carbohydrate have independent effects on leptin responsiveness and monosaccharides may reverse dietary fat‐induced leptin resistance. Supported by NIH grant DK053903.