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.

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.

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.

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.

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.

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.

Noradrenergic activity in the paraventricular nucleus and macronutrient choice during early dark onset by zinc deficient rats.

Carbohydrate is a preferred macronutrient of rats during the early dark phase and associated with an increase in norepinephrine (NE) and neuropeptide Y (NPY) in the paraventricular nucleus (PVN). Macronutrient choice is altered during zinc deficiency (ZD). The relationship between NE activity in the PVN and macronutrient choice during early dark was evaluated in rats fed zinc adequate (ZA) or ZD diet for 14 days. Total caloric intake was similar for ZA and ZD groups (-20 kJ) but ZA rats selected 63 +/- 5% of calories as carbohydrate while ZD rats selected 53 +/- 5% of their calories from protein (p < 0.01). Pair-fed (PF) rats selected 62 +/- 5% of calories as carbohydrate. Noradrenergic activity was lower (p < 0.01) in ZD and PF compared to ZA. The association between increased NE in the PVN at dark onset and selection of carbohydrate is not supported by the present results.

Regulation of gene transcription by botanicals: novel regulatory mechanisms.

Early investigations of gene regulation revealed that nutrients could modulate gene expression, an example being the discovery of metal-regulated gene transcription ( 11, 19, 44). Only more recently have we focused on the ability of non-nutritional botanicals or functional food components to affect gene expression at the transcriptional level. Significant findings include the discovery that hyperforin is an active ingredient of the herbal remedy St. John's wort, and activates gene transcription of cytochrome p450-3A4, causing significant botanical-drug interactions. Recently, the lipid-regulating peroxisome proliferator-activated receptors have been studied as receptors activated by soy isoflavones, perhaps explaining the lipid-lowering effect of soy intake. Epigallocatechin gallate has been shown to be an inhibitor of the protealytic activity of the proteasome; this inhibition has a significant implication for cell proliferation and the stability of transcription factors in the nucleus. Very recently, the effects of botanicals have been studied as activators of sirtuins, important deacetylation enzymes that have been shown to enhance lifespan in a variety of organisms. Sirtuins have been implicated in the lifespan-enhancing effect of caloric restriction. Originally presumed to act mainly on compaction or accessibility of DNA, recent evidence shows important activity of sirtuins as controllers of transcriptional coactivator availability. This review focuses on novel mechanisms by which botanical products regulate cell function via gene transcription. Investigating these newly appreciated mechanisms will assist with the characterization and clarification of specific effects of botanicals on gene expression.

Molecular mechanisms of action of the soy isoflavones includes activation of promiscuous nuclear receptors. A review.

Consumption of soy has been demonstrated to reduce circulating cholesterol levels, most notably reducing low-density lipoprotein (LDL) cholesterol levels in hypercholesterolemic individuals. The component or components that might be responsible for this effect is still a matter of debate or controversy among many researchers. Candidate agents include an activity of soy protein itself, bioactive peptides produced during the digestive process, or the soy isoflavones. Although soy intake may provide other health benefits including preventative or remediative effects on cancer, osteoporosis and symptoms of menopause, this review will focus on isoflavones as agents affecting lipid metabolism. Isoflavones were first discovered as a bioactive agent disrupting estrogen action in female sheep, thereby earning the often-used term 'phytoestrogens'. Subsequent work confirmed the ability of isoflavones to bind to estrogen receptors. Along with the cholesterol-lowering effect of soy intake, research that is more recent has pointed to a beneficial antidiabetic effect of soy intake, perhaps mediated by soy isoflavones. The two common categories of antidiabetic drugs acting on nuclear receptors known as peroxisome proliferator activated receptors (PPARs) are the fibrates and glitazones. We and others have recently asked the research question 'do the soy isoflavones have activities as either "phytofibrates" or "phytoglitazones"?' Such an activity should be able to be confirmed both in vivo and in vitro. In both the in vivo and in vitro cases, this action has indeed been confirmed. Further work suggests a possible action of isoflavones similar to the nonestrogenic ligands that bind the estrogen-related receptors (ERRs). Recently, these receptors have been demonstrated to contribute to lipolytic processes. Finally, evaluation of receptor activation studies suggests that thyroid receptor activation may provide additional clues explaining the metabolic action of isoflavones. The recent advances in the discovery and evaluation of the promiscuous nuclear receptors that bind many different chemical ligands should prove to help explain some of the biological effects of soy isoflavones and other phytochemicals.

Soy isoflavones affect sterol regulatory element binding proteins (SREBPs) and SREBP-regulated genes in HepG2 cells.

Soy intake reduces cholesterol levels. However, both the identity of the soy component or components that contribute to this reduction and the cellular mechanism producing this reduction are unknown. Soy consists of protein, lipids, fiber, and phytochemicals including isoflavones. We propose that the isoflavone component of soy mediates this effect, at least in part, by affecting cellular sterol homeostasis. We investigated the effects of an isoflavone-containing soy extract and the individual isoflavones on the maturation of the sterol regulatory element binding proteins (SREBP) and the expression of SRE-regulated genes controlling lipid metabolism. We found a corresponding increase in the mature form of SREBP-2 in both soy extract- and isoflavone-treated HepG2 cells, whereas there was no significant change in the levels of SREBP-1. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase protein and HMG CoA synthase mRNA levels also increased. When HepG2 cells were transiently transfected with HMG CoA synthase and LDL receptor reporter plasmids there was an increase in expression in response to soy extract or isoflavone treatment from both of these promoters, but this induction was blunted in the presence of sterols (P < 0.05). The mechanism responsible for this effect may be via a statin-like inhibition of HMG CoA reductase enzyme activity or by enhanced SREBP processing via the SREBP cleavage activating protein. We hypothesize that maturation of SREBP and induction of SRE-regulated genes produce an increase in surface LDL receptor expression that increases the clearance of plasma cholesterol, thus decreasing plasma cholesterol levels.

Hepatic cytochrome p450-2A and phosphoribosylpyrophosphate synthetase-associated protein mRNA are induced in gerbils after consumption of isoflavone-containing protein.

Soy intake reduces cholesterol levels, but neither the exact component in soy causing this reduction nor the mechanism by which cholesterol is reduced is known with certainty. In this study, a genetic screen was performed to identify hepatic mRNA in gerbils regulated by soy or soy isoflavones. Gerbils were fed casein, an alcohol-washed soy-based diet (containing low levels of isoflavones), and the soy-based diet supplemented with an isoflavone-containing soy extract. After feeding for 28 d, gerbils were killed, hepatic RNA was isolated, and genes that were differentially expressed in any of the three dietary conditions were identified. Fifteen different mRNA were originally selected, including two mRNA that were studied further and shown to be highly regulated. Messenger RNA levels for both cytochrome P450-2A and phosphoribosylpyrophosphate synthetase-associated protein were up-regulated in a dose-dependent manner when soy replaced casein in the diet at 0, 33, 67 and 100% of original casein levels. A subsequent experiment used purified amino acid mixtures resembling the percentage amino acid composition of soy and casein to ensure that isoflavone-free protein sources could be tested. Using these mixtures, a 2 x 2 x 2 design tested: natural vs. synthetic protein sources, casein- vs. soy-based diets, and isoflavone extract-supplemented or supplement-free diets. This design demonstrated that these two mRNA were again significantly up-regulated more than twofold (P < 0.05) in gerbils fed all diets containing isoflavones. Induction of these two mRNA by soy may be due to the aryl hydrocarbon receptor element in the promoter region of both genes.

Zinc status affects neurotransmitter activity in the paraventricular nucleus of rats.

Alterations in neurochemical activity in the paraventricular nucleus (PVN) of the hypothalamus may account for decreased intake of zinc-deficient diets. Male Sprague-Dawley rats were fed zinc-deficient (ZD) or zinc-adequate (ZA) diet for 14 d before samples of extracellular fluid in the PVN were collected by microdialysis or push-pull perfusion. A third set of rats was pair-fed (PF) an amount of ZA diet equal to the intake of ZD rats. Samples were collected over a 2-h period spanning the transition from light to dark. All rats then consumed the zinc adequate diet ad libitum for 3 d before a second set of samples was collected. The increase in extracellular norepineprhrine (NE) during h 1 of the dark period to 147 +/- 13% of baseline (P < 0.05) was apparent only in ZA rats at d 14. After the 3-d repletion period, the increase in NE at dark onset occurred in all three groups. An increase in extracellular neuropeptide Y (NPY) at dark onset to 174 +/- 32% of baseline in rats fed ZA (P < 0.01) was measured in all three groups at both d 14 and 17. Basal NPY concentrations were significantly elevated in PF rats on d 14 (7.45 +/- 2.01 vs. 0.58 +/- 0.23 pmol/L, P = 0.01) and returned to ZA levels by d 17. The activities of the NE and NPY systems in the PVN were altered in rats fed a zinc-deficient diet; however, it is unclear whether the disruption in the NE and NPY neural systems in the PVN results in the altered feeding behavior accompanying zinc deficiency.