Diet composition influences the effect of high fat diets on bone in growing male mice.

The effect of diet-induced obesity on bone in rodents is variable, with bone mass increases, decreases, and no impact reported. The goal of this study was to evaluate whether the composition of obesogenic diet may influence bone independent of its effect on body weight. As proof-of-principle, we used a mouse model to compare the skeletal effects of a commonly used high fat 'Western' diet and a modified high fat diet. The modified high fat diet included ground English walnut and was isocaloric for macronutrients, but differed in fatty acid composition and contained nutrients (e.g. polyphenols) not present in the standard 'Western' diet. Eight-week-old mice were randomized into 1 of 3 dietary treatments (n = 8/group): (1) low fat control diet (LF; 10 % kcal fat); (2) high fat 'Western' diet (HF; 46 % kcal fat as soybean oil and lard); or (3) modified high fat diet supplemented with ground walnuts (HF + walnut; 46 % kcal fat as soybean oil, lard, and walnut) and maintained on their respective diets for 9 weeks. Bone response in femur was then evaluated using dual energy x-ray absorptiometry, microcomputed tomography, and histomorphometry. Consumption of both obesogenic diets resulted in increased weight gain but differed in impact on bone and bone marrow adiposity in distal femur metaphysis. Mice consuming the high fat 'Western' diet exhibited a tendency for lower cancellous bone volume fraction and connectivity density, and had lower osteoblast-lined bone perimeter (an index of bone formation) and higher bone marrow adiposity than low fat controls. Mice fed the modified high fat diet did not differ from mice fed control (low fat) diet in cancellous bone microarchitecture, or osteoblast-lined bone perimeter, and exhibited lower bone marrow adiposity compared to mice fed the 'Western' diet. This proof-of-principal study demonstrates that two obesogenic diets, similar in macronutrient distribution and induction of weight gain, can have different effects on cancellous bone in distal femur metaphysis. Because the composition of the diets used to induce obesity in rodents does not recapitulate a common human diet, our finding challenges the translatability of rodent studies evaluating the impact of diet-induced obesity on bone.

Perspective: Challenges and Future Directions in Clinical Research with Nuts and Berries.

Consumption of nuts and berries are considered part of a healthy eating pattern. Nuts and berries contain a complex nutrient profile consisting of essential vitamins and minerals, fiber, polyunsaturated fatty acids, and phenolics in quantities that improve physiological outcomes. The spectrum of health outcomes that may be impacted by the consumptions of nuts and berries includes cardiovascular, gut microbiome, and cognitive, among others. Recently, new insights regarding the bioactive compounds found in both nuts and berries have reinforced their role for use in precision nutrition efforts. However, challenges exist that can affect the generalizability of outcomes from clinical studies, including inconsistency in study designs, homogeneity of test populations, variability in test products and control foods, and assessing realistic portion sizes. Future research centered on precision nutrition and multi-omics technologies will yield new insights. These and other topics such as funding streams and perceived risk-of-bias were explored at an international nutrition conference focused on the role of nuts and berries in clinical nutrition. Successes, challenges, and future directions with these foods are presented here.

Consumption of Antioxidant-Rich "Cerrado" Cashew Pseudofruit Affects Hepatic Gene Expression in Obese C57BL/6J High Fat-Fed Mice.

The pseudofruit of A. othonianum Rizzini, "Cerrado" cashew pulp, has been described as rich in flavonoids, phenolic compounds, and vitamin C. The objective of this work was to evaluate the beneficial health effects seen with the addition of "Cerrado" cashew pulp (CP) to an obesogenic high fat diet provided to C57BL/6J male mice. In week 9, the HF-fed group had a significantly higher baseline glucose concentration than the LF- or HF+CP-fed groups. In RNAseq analysis, 4669 of 5520 genes were found to be differentially expressed. Among the genes most upregulated with the ingestion of the CP compared to HF were Ph1da1, SLc6a9, Clec4f, and Ica1 which are related to glucose homeostasis; Mt2 that may be involved steroid biosynthetic process; and Ciart which has a role in the regulation of circadian rhythm. Although "Cerrado" CP intake did not cause changes in the food intake or body weight of fed mice with HF diet, carbohydrate metabolism appeared to be improved based on the observed changes in gene expression.

Intake of an enological oak tannin powder alters hepatic gene express patterns indicative of a reduction of inflammation in male mice fed an obesogenic diet.

Hydrolysable tannins, mainly gallotannins and ellagitannins, either extracted directly from oak or as a part of lyophilized extracts from finished wine, have been associated with antioxidant and anti-inflammatory properties that may benefit human health. In this work we hypothesized that a commercially available oak tannin powder provided to C57BL/6J male mice fed a western-style obesogenic diet for 10 weeks would significantly alter hepatic gene expression patterns as determined by RNA sequencing. Over two-thousand genes were uniquely expressed between three different diet groups. Among the 25 canonical pathways that were significantly regulated, intake of oak powder reduced the TNF-alpha/NF-κB, complement activation, IL-5, and Type II interferon signaling; these significant reductions are consistent with a reduction in chronic systemic inflammation associated with consumption of a commercially prepared enological oak tannin.

Intake of Watermelon and Watermelon Byproducts in Male Mice Fed a Western-Style Obesogenic Diet Alters Hepatic Gene Expression Patterns, as Determined by RNA Sequencing.

BACKGROUND: Consumption of watermelon has been associated with beneficial effects on metabolism, including reductions in systolic blood pressure, improved fasting blood glucose levels, and changes in hepatic metabolite accumulation. OBJECTIVES: In the present study, we investigated the impact of consumption of watermelon flesh (WF), watermelon rind (WR), and watermelon skin (WS) on hepatic gene expression patterns in an obesogenic mouse model. METHODS: Hepatic RNA was isolated and RNA sequencing was performed following a 10-week feeding trial during which C57BL/6 J mice were provided either a low-fat diet (LF), high-fat diet (HF; controls), or HF plus either WS, WR, or WF. Bioinformatic approaches were used to determine changes in the canonical pathways and gene expression levels for lipid- and xenobiotic-regulating nuclear hormone receptors and other related transcription factors, including the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), farnesyl X receptor, peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor gamma, liver X receptor, pregnane X receptor, and nuclear factor erythroid 2-related factor 2. RESULTS: There were 9394 genes that had unchanged expression levels between all 5 diet groups, and 247, 58, and 34 genes were uniquely expressed in the WF, WR, and WS groups, respectively. The relative levels of mRNAs regulated by AhR, CAR, and PPARα were upregulated in mice in the WF group, as compared to the HF control mice; in comparison, mRNAs regulated mainly by CAR were upregulated in mice in the WR and WS groups, compared to those in the HF control group. CONCLUSIONS: At modest levels of intake reflective of typical human consumption, mice in the WF, WS, and WR groups exhibited hepatic gene expression profiles that were altered when compared to mice in the HF control group. Several of these changes involve genes regulated by ligand-responsive transcription factors implicated in xenobiotic and lipid metabolisms, suggesting that the modulation of these transcription factors occurred in response to the consumption of WS, WR, and WF. Some of these changes are likely due to nuclear hormone receptor-mediated changes involved in lipid and xenobiotic metabolisms.

Metabolic Syndrome Is Reduced in C57BL/6J Mice Fed High-Fat Diets Supplemented with Oak Tannins.

BACKGROUND: Wine aged in oak barrels will incorporate polyphenols inherent in the staves, suggesting that wine stored in these wooden containers will introduce oak compounds into the human body after consumption. OBJECTIVE: The purpose of the present study is to test whether consumption of these oak compounds could favorably influence metabolism in mice fed an obesogenic diet. METHODS: C57BL/6  male mice (n = 8) were fed diets for 10 wk as follows: low-fat (LF), high-fat (HF), and HF containing 0.17% of oak tannin (HF+OT). A second 10-wk study was completed; mice were provided LF, HF, and HF diets supplemented with 7.0% of concentrates made from oaked wine (HF+OWC) or unoaked wine (HF+UWC). Physiological parameters were measured during the feeding trial and serum markers and hepatic gene expression measured from samples obtained at necropsy. RESULTS: Intake of HF+OT significantly reduced body-weight gain (18.4 ± 1.2 g in HF vs. 13.2 ± 1.4 g in HF+OT, P < 0.05). Serum resistin concentrations were lower in HF+OT mice compared with HF mice (301 ± 10.1 pg/mL in HF+OT vs. 374 ± 10.9 pg/mL in HF; P < 0.05). Hepatic lipid accumulation and expression of glutathione-S-transferase-m2 (Gstm2) and NAD(P)H:quinone oxidoreductase (Nqo1) mRNAs were significantly decreased in HF+OT compared with HF mice (P < 0.05). When compared with HF-fed mice, intake of both OWC and UWC decreased body-weight gain (P < 0.05), with no significant impact on food consumption. Fasting glucose concentrations, serum insulin, and hepatic lipid accumulation were reduced in HF+OWC-fed mice compared with HF+UWC-fed mice (P < 0.05). Furthermore, hepatic glutathione-S-transferase-a1 (Gsta1) mRNA levels were significantly reduced in OWC-supplemented (0.25 ± 0.08) compared with UWC-supplemented (1.71 ± 0.24) mice (P < 0.05). CONCLUSIONS: In this mouse model of metabolic disease, intake of OTs and a concentrate made from an oaked wine had a potent impact on alleviating HF-induced metabolic syndrome. Thus, intake of OTs, provided passively in oaked wine or as a dietary supplement, may act as an agent to attenuate the markers of metabolic syndrome.

Consuming Diet Supplemented with Either Red Wheat Bran or Soy Extract Changes Glucose and Insulin Levels in Female Obese Zucker Rats.

Type 2 diabetes mellitus is characterized by the inability to regulate blood glucose levels due to insulin resistance, resulting in hyperglycemia and hyperinsulinemia. Research has shown that consuming soy and fiber may protect against type 2 diabetes mellitus. We performed a study to determine whether supplementing diet with soy extract (0.5% weight of diet) or fiber (as red wheat bran; 11.4% weight of diet) would decrease serum insulin and blood glucose levels in a pre-diabetic/metabolic syndrome animal model. In our study, female obese Zucker rats were fed either a control diet (n = 8) or control diet supplemented with either soy extract (n = 7) or red wheat bran (n = 8) for seven weeks. Compared to rats consuming control diet, rats fed treatment diets had significantly lower (p-value < 0.05) fasting serum insulin (control = 19.34±1.6; soy extract = 11.1±1.54; red wheat bran = 12.4±1.11) and homeostatic model assessment of insulin resistance values (control = 2.16±0.22; soy extract = 1.22±0.21; red wheat bran = 1.54±0.16). Non-fasted blood glucose was also significantly lower (p-value < 0.05) in rats fed treatment diets compared to rats consuming control diet at weeks four (control = 102.63±5.67; soy extract = 80.14±2.13; red wheat bran = 82.63±3.16), six (control = 129.5±10.83; soy extract = 89.14±2.48; red wheat bran = 98.13±3.54), and seven (control = 122.25±8.95; soy extract = 89.14±4.52; red wheat bran = 84.75±4.15). Daily intake of soy extract and red wheat bran may protect against type 2 diabetes mellitus by maintaining normal glucose homeostasis.

Intake of Watermelon or Its Byproducts Alters Glucose Metabolism, the Microbiome, and Hepatic Proinflammatory Metabolites in High-Fat-Fed Male C57BL/6 J Mice.

BACKGROUND: Watermelon intake has demonstrated effects on blood pressure regulation along with other health benefits. OBJECTIVE: We hypothesized that intake of whole watermelon and products made from watermelon rind (WR) and watermelon skin (WS) would remediate metabolic complications in C57BL/6 J male mice fed a diet modeling a Western-style diet. METHODS: Ten-week-old male C57BL/6 J mice were provided either a low-fat (LF) diet [10% fat (by energy), 8% sucrose (by energy) and no added cholesterol], a high-fat (HF) diet [45% fat (by energy), 20% kcal sucrose (by energy), and 1% (w/w) cholesterol], or an HF diet plus WS, WR, or watermelon flesh (WF) for 10 wk. Dried WF was provided at 8% of total energy (equivalent to 2 servings/d) and watermelon skin and rind were added at 2.25% (w/w, dry weight of additives) of diet. Animals were provided experimental diets ad libitum. Body weights, food intake, and glucose tolerance were determined. Serum insulin, inflammatory markers, microbiome, and the relative hepatic concentrations of 709 biochemicals were measured postmortem. RESULTS: The final body weight of the LF control group was significantly lower than that of the HF-fed control group (32.8 ± 0.9 g compared with 43.0 ± 1.7 g, P ≤ 0.05). Mice in treatment groups fed HF supplemented with watermelon products had final body weights similar to those of the HF-fed control mice. Serum insulin concentrations were reduced by ∼40% in mice fed an HF diet with WR supplementation compared with mice fed an HF diet alone (P ≤ 0.05). Depending on the individual species or group, microbiome populations changed significantly. Supplementation with WF resulted in a return to the basal hepatic concentrations of monohydroxy fatty acids and eicosanoids observed in LF-fed mice (P ≤ 0.05). CONCLUSIONS: In obese male mice, supplementation with each of the watermelon products to an HF diet improved fasting blood glucose, circulating serum insulin concentrations, and changes in hepatic metabolite accumulation. At a modest level of supplementation to an HF diet, fiber-rich additives made from WR and WS further improved glucose metabolism and energy efficiency and shifted the microbiome composition.

Genistein and daidzein decrease food intake and body weight gain in mice, and alter LXR signaling in vivo and in vitro.

The study is designed to determine whether consumption of the soy isoflavones, genistein and daidzein, differentially influence metabolic syndrome, and to further investigate the involvement of Liver X Receptor (LXR) regulation. C57BL/6J mice were fed diets as follows: low fat diet (LF), western-style diet (WD), and WD containing 0.16% (w/w) of genistein (WD + G) or daidzein (WD + D) for 10 weeks. Intake of WD + G and WD + D produced a robust decrease in body weight gain by 40% and 19%, respectively (p < 0.05). Genistein reduced energy intake by 26%, and daidzein decreased energy intake by 8% (p < 0.05). A glucose tolerance test indicated that genistein consumption significantly decreased the incremental areas under the curve (AUC) from 60-120 min, compared to WD-fed mice. Gene array profiling of hepatic mRNA, and cell studies utilizing transiently-transfected HepG2 cells and mouse embryonic fibroblast cells devoid of or expressing LXRα, indicate that genistein and daidzein induce LXR-mediated pathways. In summary, addition of genistein, compared to daidzein, to a western-style diet, more profoundly decreased food intake, body weight gain, while both appear to regulate LXR-mediated pathways.

Gene Expression Patterns Are Altered in Athymic Mice and Metabolic Syndrome Factors Are Reduced in C57BL/6J Mice Fed High-Fat Diets Supplemented with Soy Isoflavones.

Soy isoflavones exert beneficial health effects; however, their potential to ameliorate conditions associated with the metabolic syndrome (MetS) has not been studied in detail. In vitro and in vivo models were used to determine the effect of isoflavones on lipid metabolism, inflammation, and oxidative stress. In nude mice, consumption of Novasoy (NS) increased cholesterol and lipid metabolism gene expression, including Scd-1 (27.7-fold), Cyp4a14 (35.2-fold), and Cyp4a10 (9.5-fold), and reduced anti-inflammatory genes, including Cebpd (16.4-fold). A high-fat (HF) diet containing 0.4% (w/w) NS for 10 weeks significantly reduced percent weight gain (74.6 ± 2.5 vs 68.6 ± 3.5%) and hepatic lipid accumulation (20 ± 1.2 vs 27 ± 1.5%), compared to HF alone (p < 0.05) in C57BL/6J mice. NS also increased lipid oxidation and antioxidant gene expression while decreasing inflammatory cytokines. In vitro analysis in HepG2 cells revealed that genistein dose-dependently decreases oleic acid-induced lipid accumulation. Soy isoflavones may ameliorate symptoms associated with MetS via anti-inflammatory, antioxidant, and hypolipidemic modulation.

Improvements in Metabolic Health with Consumption of Ellagic Acid and Subsequent Conversion into Urolithins: Evidence and Mechanisms.

Ellagic acid (EA) is a naturally occurring polyphenol found in some fruits and nuts, including berries, pomegranates, grapes, and walnuts. EA has been investigated extensively because of its antiproliferative action in some cancers, along with its anti-inflammatory effects. A growing body of evidence suggests that the intake of EA is effective in attenuating obesity and ameliorating obesity-mediated metabolic complications, such as insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, and atherosclerosis. In this review, we summarize how intake of EA regulates lipid metabolism in vitro and in vivo, and delineate the potential mechanisms of action of EA on obesity-mediated metabolic complications. We also discuss EA as an epigenetic effector, as well as a modulator of the gut microbiome, suggesting that EA may exert a broader spectrum of health benefits than has been demonstrated to date. Therefore, this review aims to suggest the potential metabolic benefits of consumption of EA-containing fruits and nuts against obesity-associated health conditions.

Consumption of Walnuts in Combination with Other Whole Foods Produces Physiologic, Metabolic, and Gene Expression Changes in Obese C57BL/6J High-Fat-Fed Male Mice.

BACKGROUND: Although a reductionist approach has sought to understand the roles of individual nutrients and biochemicals in foods, it has become apparent that there can be differences when studying food components in isolation or within the natural matrix of a whole food. OBJECTIVE: The objective of this study was to determine the ability of whole-food intake to modulate the development of obesity and other metabolic dysfunction in mice fed a high-fat (HF), Western-style obesogenic diet. To test the hypothesis that an n-3 (ω-3) polyunsaturated fatty acid-rich food could synergize with other, largely polyphenol-rich foods by producing greater reductions in metabolic disease conditions, the intake of English walnuts was evaluated in combination with 9 other whole foods. METHODS: Eight-week-old male C57Bl/6J mice were fed low-fat (LF; 10% fat) and HF control diets, along with an HF diet with 8.6% (wt:wt) added walnuts for 9 wk. The HF control diet contained 46% fat with added sucrose (10.9%, wt:wt) and cholesterol (1%, wt:wt); the added sucrose and cholesterol were not present in the LF diet. Other groups were provided the walnut diet with a second whole food-raspberries, apples, cranberries, tart cherries, broccoli sprouts, olive oil, soy protein, or green tea. All of the energy-containing whole foods were added at an energy level equivalent to 1.5 servings/d. Body weights, food intake, and glucose tolerance were determined. Postmortem, serum lipids and inflammatory markers, hepatic fat, gene expression, and the relative concentrations of 594 biochemicals were measured. RESULTS: The addition of walnuts with either raspberries, apples, or green tea reduced glucose area under the curve compared with the HF diet alone (-93%, -64%, and -54%, respectively, P < 0.05). Compared with HF-fed mice, mice fed walnuts with either broccoli sprouts or green tea (-49% and -61%, respectively, P < 0.05) had reduced hepatic fat concentrations. There were differences in global gene expression patterns related to whole-food content, with many examples of differences in LF- and HF-fed mice, HF- and walnut-fed mice, and mice fed walnuts and walnuts plus other foods. The mean ± SEM increase in relative hepatic concentrations of the n-3 fatty acids α-linolenic acid, eicosapentanoic acid, and docosapentanoic acid in all walnut-fed groups was 124% ± 13%, 159% ± 11%, and 114% ± 10%, respectively (P < 0.0001), compared with LF- and HF-fed mice not consuming walnuts. CONCLUSIONS: In obese male mice, walnut consumption with an HF Western-style diet caused changes in hepatic fat concentrations, gene expression patterns, and fatty acid concentrations. The addition of a second whole food in combination with walnuts produced other changes in metabolite concentrations and gene expression patterns and other physiologic markers. Importantly, these substantial changes occurred in mice fed typical amounts of intake, representing only 1.5 servings each food/d.

Consumption of Quercetin and Quercetin-Containing Apple and Cherry Extracts Affects Blood Glucose Concentration, Hepatic Metabolism, and Gene Expression Patterns in Obese C57BL/6J High Fat-Fed Mice.

BACKGROUND: Intake of polyphenols and polyphenol-rich fruit extracts has been shown to reduce markers of inflammation, diabetes, and hepatic complications that result from the consumption of a high-fat (HF) diet. OBJECTIVE: The objective of this study was to determine whether mice fed polyphenol-rich apple peel extract (AE), cherry extract (CE), and quercetin, a phytochemical abundant in fruits including apples and cherries, would modulate the harmful effects of adiposity on blood glucose regulation, endocrine concentrations, and hepatic metabolism in HF-fed C57BL/6J male mice. METHODS: Groups of 8-wk-old mice (n = 8 each) were fed 5 diets for 10 wk, including low-fat (LF; 10% of total energy) and HF (60% of total energy) control diets and 3 HF diets containing polyphenol-rich AE, CE, and quercetin (0.2% wt:wt). Also, an in vitro study used HepG2 cells exposed to quercetin (0-100 µmol/L) to determine whether intracellular lipid accumulation could be modulated by this phytochemical. RESULTS: Mice fed the HF control diet consumed 36% more energy, gained 14 g more body weight, and had ∼50% elevated blood glucose concentrations (all P < 0.05) than did LF-fed mice. Mice fed HF diets containing AE, CE, or quercetin became as obese as HF-fed mice, but had significantly lower blood glucose concentrations after food deprivation (-36%, -22%, -22%, respectively; P < 0.05). Concentrations of serum C-reactive protein were reduced 29% in quercetin-fed mice compared with HF-fed controls (P < 0.05). A qualitative evaluation of liver tissue sections suggested that fruit phytochemicals may reduce hepatic lipid accumulation. A quantitative analysis of lipid accumulation in HepG2 cells demonstrated a dose-dependent decrease in lipid content in cells treated with 0-100 µmol quercetin/L (P < 0.05). CONCLUSIONS: In mice, consumption of AE, CE, or quercetin appears to modulate some of the harmful effects associated with the consumption of an obesogenic HF diet. Furthermore, in a cell culture model, quercetin was shown to reduce intracellular lipid accumulation in a dose-dependent fashion.

Ellagic acid modulates lipid accumulation in primary human adipocytes and human hepatoma Huh7 cells via discrete mechanisms.

Previously, we have reported that consumption of a muscadine grape phytochemical powder (MGP) decreased lipid accumulation in high-fat fed mice. The aim of this study was to identify the responsible polyphenolic constituents and elucidate the underlying mechanisms. In mice, MGP supplementation significantly reduced visceral fat mass as well as adipocyte size. To determine whether MGP affects adipogenesis or hypertrophic lipid accumulation, we used a human adipogenic stem cell (hASCs) model. Among the MGP, ellagic acid (EA) was identified as a potent negative regulator of adipogenesis of hASCs. In addition, EA substantially decreased the conversion of [(3)H]-acetyl CoA into fatty acids (FAs), suggesting that EA inhibits de novo synthesis of FA in mature adipocytes. Similarly, MGP supplementation significantly decreased hepatic triglyceride (TG) levels. The TG-lowering effects of EA were confirmed in human hepatoma Huh7 cells. EA reduced [(3)H]-oleic acid esterification into [(3)H]-TG as well as the de novo synthesis of FA from [(3)H]-acetyl CoA in Huh7 cells. Intriguingly, EA also increased oxygen consumption rate and ß-oxidation-related gene expression. Taken together, EA attenuated new fat cell formation and FA biosynthesis in adipose tissue, while it reduced the synthesis of TG and FA and increased FA oxidation in the liver. These results suggest that EA exerts unique lipid-lowering effects both in adipose tissue and liver via discrete mechanisms.

Muscadine grape (Vitis rotundifolia) and wine phytochemicals prevented obesity-associated metabolic complications in C57BL/6J mice.

The objective of this study was to determine the effects of muscadine grape or wine (cv. Noble) phytochemicals on obesity and associated metabolic complications. Muscadine grape or wine phytochemicals were extracted using Amberlite FPX66 resin. Male C57BL/6J mice were given a low-fat diet (LF, 10% kcal fat), high-fat diet (HF, 60% kcal fat), HF + 0.4% muscadine grape phytochemicals (HF+MGP), or HF + 0.4% muscadine wine phytochemicals (HF+MWP) for 15 weeks. At 7 weeks, mice fed HF+MGP had significantly decreased body weights by 12% compared to HF controls. Dietary MGP or MWP supplementation reduced plasma content of free fatty acids, triglycerides, and cholesterol in obese mice. Inflammation was alleviated, and activity of glutathione peroxidase was enhanced. Consumption of MGP or MWP improved insulin sensitivity and glucose control in mice. Thus, consumption of muscadine grape and wine phytochemicals in the diet may help to prevent obesity-related metabolic complications.

Daidzein and the daidzein metabolite, equol, enhance adipocyte differentiation and PPARgamma transcriptional activity.

Dietary soy isoflavones have been shown to favorably alter the metabolic phenotypes associated with Type 2 diabetes. However, the identification of direct targets and the underlying molecular mechanisms by which soy isoflaovones exert antidiabetic effects remain elusive. Since the insulin-sensitizing effects of thiazolidinediones, antidiabetic drugs, are mediated through activation of peroxisome proliferators-activated receptor gamma (PPARgamma), we examined the effects of daidzein and the daidzein metabolite, equol, on adipocyte differentiation and PPARgamma activation. In 3T3-L1 cells, daidzein enhanced adipocyte differentiation and PPARgamma expression in a dose-dependent manner. Daidzein also dose-dependently increased insulin-stimulated glucose uptake and the relative abundance of insulin-responsive glucose transporter 4 (GLUT4) and insulin receptor substrate 1 (IRS-1) mRNA. In C3H10T1/2 cells, both daidzein and equol at 1 micromol/L and higher significantly increased adipocyte differentiation and insulin-stimulated glucose uptake. Furthermore, daidzein and equol up-regulated PPARgamma-mediated transcriptional activity, and daidzein restored the PPARgamma antagonist-induced inhibition of aP2 and GLUT4 mRNA levels. Our results indicate that daidzein enhances insulin-stimulated glucose uptake in adipocytes by increasing the expression of GLUT4 and IRS-1 via the activation of PPARgamma. These data further support the recent findings that favorable effects of dietary soy isoflavones may be attributable to daidzein and its metabolite equol.

Human CYP3A4 and murine Cyp3A11 are regulated by equol and genistein via the pregnane X receptor in a species-specific manner.

Pregnane X receptor (PXR) is an important component of the body's adaptive defense system responsible for the elimination of various toxic xenobiotics. PXR activation by endogenous and exogenous chemicals, including steroids, antibiotics, bile acids, and herbal compounds, results in induction of drug metabolism. We investigated the ability of the isoflavones genistein, daidzein, and the daidzein metabolite equol to activate human and mouse PXR in vitro using cell-based transient transfection studies and primary hepatocytes and in vivo in a mouse model. In transient transfection assays, the isoflavones genistein and daidzein activate full-length, wild-type mouse PXR, but not a mutant form, with genistein being the most potent. In contrast, equol was a more potent activator of human PXR than genistein or daidzein. In a mammalian 2-hybrid assay, isoflavones induced recruitment of the coactivator steroid receptor coactivator 1 to PXR. When tested against the native human Cytochrome P450 3A4 (CYP3A4) promoter, equol was the more potent activator and treatment of human hepatocytes with equol increased CYP3A4 mRNA and immunoreactive protein expression. Treatment of wild-type, but not PXR(-/-), mouse hepatocytes showed that genistein and daidzein induced the expression of Cytochrome P450 3A11 (Cyp3A11) mRNA, whereas equol had no effect. Cyp3A11 mRNA was also induced in vivo in mice fed a soy protein-containing diet. The results presented herein demonstrate that there is a species-specific difference in the activation of PXR by isoflavones and equol.

Human and murine hepatic sterol-12-alpha-hydroxylase and other xenobiotic metabolism mRNA are upregulated by soy isoflavones.

The transport and metabolism of xenobiotics is controlled by the drug transporters and drug-metabolizing enzymes in the liver and small intestine. Expression of these genes is 1 factor affecting the half-life of drugs and xenobiotics. Isoflavone-containing soyfood products and supplements are promoted to treat several different health conditions, including improvement of blood lipid profiles. Because relatively high isoflavone intake may be possible via use of supplements, we tested the hypothesis that isoflavones regulate the expression of genes critical to drug transport and metabolism. Using a gene array screening method, 2 drug transporters, Multidrug restistant-1 and Multidrug-related protein-2; 3 phase I enzymes, cytochrome 1A1, 3A4, and 8B1; and 2 phase II enzymes, carbohydrate sulfotransferase-5 and glutathione-sulfotransferase-2, were upregulated 3-fold or more of the initial expression levels in primary human hepatocytes exposed to soy isoflavones for 48 h. Isoflavone-related induction of 12-alpha-hydroxylase (CYP8B1) was further studied in other in vitro and murine in vivo models. Transfection studies suggest that isoflavones may act as a weak activating ligand for hepatocyte nuclear factor 4alpha, which in turn may activate the transcription of CYP8B1. The action of soy isoflavones on CYP8B1 may increase the conversion of cholesterol into bile acids and enhance synthesis of cholic acid. These isoflavone-induced changes in gene expression may help explain how isoflavones modulate cholesterol metabolism.

Soy protein and isoflavones influence adiposity and development of metabolic syndrome in the obese male ZDF rat.

BACKGROUND/AIMS: Previously, we demonstrated that soy protein ameliorates the diabetic phenotype in several rodent models of obesity and metabolic syndrome (MS). This study was designed to further elucidate factors related to adiposity, glycemic control, and renal function in male Zucker Diabetic Fatty (ZDF/Lepr(fa)) rats. METHODS: Animals were randomly assigned to one of four diets: control, casein (C); low isoflavone (LIS) soy protein; high isoflavone (HIS) soy protein, or casein + rosiglitazone (CR) for 11 weeks. At sacrifice, physiological, biochemical, and molecular parameters were determined. RESULTS: Body weight and total adiposity were higher in LIS and CR diet groups despite lower food intake. Additionally, these animals exhibited differential regulation of adipose-specific proteins (PPAR-gamma and GLUT4) and enzyme activity (FAS and GPDH). HIS-fed animals had reduced total and liver adiposity. Glycemic control was prolonged in both soy-based and rosiglitazone (RGZ) groups. Renal dysfunction was significantly reduced in soy-fed and RGZ-treated rodents as demonstrated by lower levels of proteinuria and dilated tubules with proteinaceous casts. CONCLUSION: Collectively, these data provide evidence that soy protein with low or high isoflavone content may have therapeutic significance in reducing severity of diabetes, MS, and renal disease as demonstrated in this preclinical model.

Dietary isoflavone supplementation modulates lipid metabolism via PPARalpha-dependent and -independent mechanisms.

Intake of soy protein has been associated with improvements in lipid metabolism, with much attention being focused on the serum cholesterol-lowering property of soy. The component or components of soy that are responsible for improvements in lipid metabolism have been investigated and their specific actions debated. One component, the isoflavones, has been shown to have weak estrogenic activity, and recently, several research groups have suggested that isoflavones are activating peroxisome proliferator-activated receptors (PPARs). The three different isoforms of PPARs (alpha, gamma, and delta) have overlapping tissue distributions and functions associated with lipid metabolism. The goal of the present study was to investigate the hypothesis that the effect of isoflavones is mediated through the PPARalpha receptor. Male and female 129/Sv mice were obtained, including both wild-type and genetically altered PPARalpha knockout mice. Groups of mice were fed high-fat atherogenic diets containing soy protein +/- isoflavones and PPARalpha agonist fenofibrate for 6 wk. At the end of 6 wk, serum and tissue lipid levels were measured along with hepatic gene expression. Most notably, serum triglycerides were reduced by isoflavone consumption. Compared with intake of a low-isoflavone basal diet, isoflavone intake reduced serum triglyceride levels by 36 and 52% in female and male wild-type mice, respectively, compared with 55 and 52% in fenofibrate-treated mice. Isoflavones also improved serum triglyceride levels in knockout mice, whereas fenofibrate did not, suggesting that two different regulatory mechanisms may be affected by isoflavone intake. Isoflavone intake resembled action of fenofibrate on PPARalpha-regulated gene expression, although less robustly compared with fenofibrate. We suggest that, at the levels consumed in this study, isoflavone intake is altering lipid metabolism in a manner consistent with activation of PPARalpha and also via a PPARalpha-independent mechanism as well.