Inflammation plays a central role in the development and progression of coronary heart disease (CHD). The sex hormones estrogen and testosterone have been shown to modify the inflammatory response by influencing cytokine expression in human macrophages obtained from younger individuals. The effect of these hormones on the expression of proinflammatory markers in macrophages obtained from a CHD age-relevant population has not been studied. Human monocyte-derived macrophages (HMDMs) were obtained from healthy normolipidemic men and postmenopausal women (age 50-70 years), and cultured in autologous serum along with both physiological and supraphysiological concentrations of estrogen or testosterone. HMDMs were stimulated with oxidized low-density lipoproteins, and the expression of the cytokines tumor necrosis factor a (TNF-a or TNF), interleukin (IL)6, and IL-1b (IL1B) and of the acute-phase protein C-reactive protein (CRP) was measured. Both physiological and supraphysiological concentrations of testosterone reduced the expression and secretion of TNF-a and reduced the expression of IL-1b, but did not affect the expression of IL6 or CRP. Estrogen did not modify the expression of TNF-a, IL6, and IL-1b. Estrogen caused a variable response in CRP expression that was positively associated with the plasma small dense LDL-cholesterol concentration of the donors. There were no gender differences in any of the observed effects. Our results indicate that testosterone may exert anti-inflammatory effects by reducing macrophage TNF-a expression, while the effects of estrogen on macrophage CRP expression may depend upon the extracellular lipid environment.

Golden-Syrian hamsters have been used as an animal model to assess diet-induced atherosclerosis since the early 1980s. Advantages appeared to include a low rate of endogenous cholesterol synthesis, receptor-mediated uptake of LDL cholesterol, cholesteryl ester transfer protein activity, hepatic apoB-100 and intestinal apoB-48 secretion, and uptake of the majority of LDL cholesterol via the LDL receptor pathway. Early work suggested hamsters fed high cholesterol and saturated fat diets responded similarly to humans in terms of lipoprotein metabolism and aortic lesion morphology. Recent work has not consistently replicated these findings. Reviewed was the literature related to controlled hamster feeding studies that assessed the effect of strain, background diet (non-purified, semi-purified) and dietary perturbation (cholesterol and/or fat) on plasma lipoprotein profiles and atherosclerotic lesion formation. F1B hamsters fed a non-purified cholesterol/fat-supplemented diet had more atherogenic lipoprotein profiles (nHDL-C > HDL-C) than other hamster strains or hamsters fed cholesterol/fat-supplemented semi-purified diets. However, fat type; saturated (SFA), monounsaturated or n-6 polyunsaturated (PUFA) had less of an effect on plasma lipoprotein concentrations. Cholesterol- and fish oil-supplemented semi-purified diets yielded highly variable results when compared to SFA or n-6 PUFA, which were antithetical to responses observed in humans. Dietary cholesterol and fat resulted in inconsistent effects on aortic lipid accumulation. No hamster strain was reported to consistently develop lesions regardless of background diet, dietary cholesterol or dietary fat type amount. In conclusion, at this time the Golden-Syrian hamster does not appear to be a useful model to determine the mechanism(s) of diet-induced development of atherosclerotic lesions.

The beta-catenin signaling pathway is dysregulated in most cases of colon cancer resulting in an accumulation of nuclear beta-catenin and increased transcription of genes involved in tumor progression. This study examines the effect of retinol on beta-catenin protein levels in three all-trans retinoic acid (ATRA)-resistant human colon cancer cell lines: HCT-116, WiDr, and SW620. Each cell line was treated with increasing concentrations of retinol for 24 or 48 h. Retinol reduced beta-catenin protein levels and increased ubiquitinated beta-catenin in all cell lines. Treatment with the proteasomal inhibitor MG132 blocked the retinol-induced decrease in beta-catenin indicating retinol decreases beta-catenin by increasing proteasomal degradation. Multiple pathways direct beta-catenin to the proteasome for degradation including a p53/Siah-1/adenomatous polyposis coli (APC), a Wnt/glycogen synthase kinase-3beta/APC, and a retinoid "X" receptor (RXR)-mediated pathway. Due to mutations in beta-catenin (HCT-116), APC (SW620), and p53 (WiDr), only the RXR-mediated pathway remains functional in each cell line. To determine if RXRs facilitate beta-catenin degradation, cells were treated with the RXR pan-antagonist, PA452, or transfected with RXRalpha small interfering RNA (siRNA). The RXR pan-antagonist and RXRalpha siRNA reduced the ability of retinol to decrease beta-catenin protein levels. Nuclear beta-catenin induces gene transcription via interaction with T cell factor/lymphoid enhancer factor (TCF/LEF) proteins. Retinol treatment decreased the transcription of a TOPFlash reporter construct and mRNA levels of the endogenous beta-catenin target genes, cyclin D1 and c-myc. These results indicate that retinol may reduce colon cancer cell growth by increasing the proteasomal degradation of beta-catenin via a mechanism potentially involving RXR.

The lower susceptibility of palmitoleic acid (16:1) to oxidation compared to PUFA may confer functional advantages with respect to finding acceptable alternatives to partially hydrogenated fats, but limited data are available on its effect on cardiovascular risk factors. This study investigated the effect of diets (10% fat, 0.1% cholesterol, wt:wt) enriched with macadamia [monounsaturated fatty acid (MUFA)16:1], palm (SFA,16:0), canola (MUFA,18:1), or safflower (PUFA,18:2) oils on lipoprotein profiles and aortic cholesterol accumulation in F1B Golden Syrian hamsters (n = 16/group). After 12 wk, 8 hamsters in each group were killed (phase 1). The remaining hamsters fed palm oil were changed to a diet containing coconut oil, while hamsters in the other diet groups continued on their original diets for an additional 6 wk (phase 2). With minor exceptions, the time course and dietary SFA source did not alter the study outcomes. Macadamia oil-fed hamsters had lower non-HDL cholesterol and triglyceride concentrations compared with the palm and coconut oil-fed hamsters and higher HDL-cholesterol compared with the coconut, canola, and safflower oil-fed hamsters. The aortic cholesterol concentration was not affected by dietary fat type. The hepatic cholesterol concentration was higher in the unsaturated compared with the saturated oil-fed hamsters. RBC membrane and aortic cholesteryl ester, triglyceride, and phospholipid fatty acid profiles reflected that of the dietary oil. These data suggest that an oil relatively high in palmitoleic acid does not adversely affect plasma lipoprotein profiles or aortic cholesterol accumulation and was similar to other unsaturated fatty acid-rich oils.

Retinol (vitamin A) is thought to exert its effects through the actions of its metabolite, all-trans-retinoic acid (ATRA), on gene transcription mediated by retinoic acid receptors (RAR) and retinoic acid response elements (RARE). However, retinoic acid resistance limits the chemotherapeutic potential of ATRA. We examined the ability of retinol to inhibit the growth of ATRA-sensitive (HCT-15) and ATRA-resistant (HCT-116, SW620, and WiDR) human colon cancer cell lines. Retinol inhibited cell growth in a dose-responsive manner. Retinol was not metabolized to ATRA or any bioactive retinoid in two of the cell lines examined. HCT-116 and WiDR cells converted a small amount of retinol to ATRA; however, this amount of ATRA was unable to inhibit cell growth. To show that retinol was not inducing RARE-mediated transcription, each cell line was transfected with pRARE-chloramphenicol acetyltransferase (CAT) and treated with ATRA and retinol. Although treatment with ATRA increased CAT activity 5-fold in ATRAsensitive cells, retinol treatment did not increase CAT activity in any cell line examined. To show that growth inhibition due to retinol was ATRA, RAR, and RARE independent, a pan-RAR antagonist was used to block RAR signaling. Retinol-induced growth inhibition was not alleviated by the RAR antagonist in any cell line, but the antagonist alleviated ATRA-induced growth inhibition of HCT-15 cells. Retinol did not induce apoptosis, differentiation or necrosis, but affected cell cycle progression. Our data show that retinol acts through a novel, RAR-independent mechanism to inhibit colon cancer cell growth. (Cancer Res 2005; 65(21): 9923-33)

Our objective was to compare the effects of a low-carbohydrate diet to a high-carbohydrate/calorie-restricted diet on weight loss, hormones, and transplanted colon tumor growth. Eighty male C57BL/6 mice consumed a diet-induced obesity regimen (DIO) ad libitum for 7 weeks. From Weeks 8 to 14, the mice consumed a 1) DIO diet ad libitum (HF); 2) low-carbohydrate diet ad libitum (LC); 3) high-carbohydrate diet ad libitum (HC); or 4) HC calorie restricted diet (HC-CR). MC38 cells were injected at Week 15. At the time of injection, the HC-CR group displayed the lowest body weight (25.5 +/- 0.57 g), serum insulin-like growth factor I (IGF-I; 135 +/- 56.0 ng/ml), and leptin (1.0 +/- 0.3 ng/ml) levels. This group also exhibited the longest time to palpable tumor (20.1 +/- 0.9 days). Compared to the HF group, the HC group exhibited lower body weight (39.4 +/- 1.4 vs. 32.9 +/- 0.7 g, respectively), IGF-I (604 +/- 44.2 vs. 243.4 +/- 88.9 ng/ml, respectively), and leptin (15.6 +/- 2.2 vs. 7.0 +/- 0.7 ng/ml, respectively) levels but similar tumor growth. IGF-I levels were lower in the LC group (320.0 +/- 39.9 ng/ml) than the HF group, but tumor growth did not differ. These data suggest LC diets do not slow colon tumor growth in obese mice.

Retinol utilizes a retinoid X receptor (RXR)-mediated degradation pathway to decrease beta-catenin protein in all-trans retinoic acid (ATRA)-resistant human colon cancer cells. In this study, we examined interactions between RXRalpha and beta-catenin in ATRA-resistant human colon cancer cells treated with retinol. Retinol treatment triggers relocation of beta-catenin and RXRalpha proteins. Cells treated with retinol for 8 and 24 h displayed increased cytosolic but decreased nuclear beta-catenin and RXRalpha. Retinol treatment increased beta-catenin and RXRalpha protein interaction. Previously, we showed that 24 h of retinol treatment increased RXRalpha protein. Here we show this increase in RXRalpha levels is due to increased RXRalpha messenger RNA. Treatment with 48 h with retinol decreased RXRalpha protein levels. Last, by transfecting HCT-116 cells with a RXRalpha construct lacking the activation function-1 and DNA binding domains, we show RXRalpha and beta-catenin binding is required for proteosomal degradation of beta-catenin. These results suggest retinol induces RXRalpha and beta-catenin binding and transport to the cytosol where they are proteasomally degraded.

Estrogen and testosterone are thought to modulate coronary heart disease (CHD) risk. To examine how these hormones affect human macrophage cholesterol transport, a key factor in atherogenesis, we obtained monocytes from healthy male and postmenopausal female donors (age 50–70 y). Cells were allowed to differentiate in autologous serum. Human monocyte-derived macrophages (HMDMs) were exposed to estrogen, testosterone, or vehicle, during differentiation. Cells were cholesterol-enriched with oxidized LDL (oxLDL) in the presence of treatment. Cell cholesterol mass, efflux, and the expression of proteins involved in HMDM cholesterol transport were examined. Estrogen significantly reduced cholesteryl ester content in both female and male HMDMs while having no measurable effect on cholesterol efflux. Testosterone did not affect cholesterol content or efflux. Both hormones significantly but modestly affected the gene expression of several proteins involved in HMDM transport, yet these effects did not translate into significant changes in protein expression. In THP-1 macrophages, the effect of estrogen on cholesteryl ester content was more potent in unloaded macrophages and was estrogen receptor-dependent. A trend for a reduction in non-oxidized LDL uptake by estrogen was observed and was also found to be dependent upon ER activation. Our data indicate that estrogen, but not testosterone, reduces cholesteryl ester accumulation in HMDMs obtained from a CHD-age relevant population, independent of changes in the expression of proteins important to macrophage cholesterol transport. In THP-1 cells, this effect is reduced in the presence of oxLDL indicating that a pro-atherogenic lipoprotein milieu is an important variable in sex hormone modulation of CHD.