Leucine and Sildenafil Combination Therapy Reduces Body Weight and Metformin Enhances the Effect at Low Dose: A Randomized Controlled Trial.

BACKGROUND: This study evaluated the potential of activating the fuel-sensing enzymes Adenine monophosphate (AMP)-activated protein kinase and the deacetylase sirtuin1, to promote weight loss. We tested the efficacy of a fixed dose combination of the amino acid leucine and 2 well-characterized agents with established safety profiles to modulate energy metabolism and facilitate weight loss. STUDY QUESTION: Will a combination of l-leucine with low-dose metformin and sildenafil produce a novel synergistic interaction that reduces body weight? STUDY DESIGN: We conducted a 24-week randomized controlled trial evaluating the effect on weight loss of leucine 1.1 g and sildenafil 1.0 mg or 4.0 mg, with and without metformin 500 mg (Leu/Sil 1.0, Leu/Sil 4.0, Leu/Met/Sil 1.0, and Leu/Met/Sil 4.0 twice/day). We enrolled 267 participants who were 18-65 years of age without diabetes and with the body mass index (BMI) of 30-45 kg/m2. MEASURES AND OUTCOMES: The primary endpoint was percentage weight change after 24 weeks. Adverse events were evaluated. The primary analysis was performed using the perprotocol population analysis of covariance estimation. Subgroup analyses of patients residing above certain threshold limits at baseline and in populations at increased risk of obesity were assessed post-hoc as exploratory end points. RESULTS: Placebo-adjusted mean bodyweight reductions in the Leu/Met/Sil 1.0, Leu/Met/Sil 4.0, and Leu/Sil 4.0 groups were -1.99%, -1.69%, and -1.67% (P = 0.015, 0.035, and 0.036, respectively). The most common adverse events were gastrointestinal-related and occurred in the metformin-treated groups consistent with metformin treatment. In African Americans, Leu/Met/Sil 1.0 produced 5.4% mean weight loss. In participants with BMI <40 kg/m2 treated with Leu/Met/Sil 1.0, the weight loss increased to 2.84%, particularly in participants with baseline insulin ≥12mU/L (3.5%). CONCLUSIONS: Leu/Met/Sil 1.0 and 4.0 and Leu/Sil 4.0 reduced body weight, but Leu/Met/Sil 1.0 was associated with robust weight loss in African Americans, and individuals with BMI 30-39.9 kg/m2, especially participants with hyperinsulinemia.

Biphasic Effect of Sildenafil on Energy Sensing is Mediated by Phosphodiesterases 2 and 3 in Adipocytes and Hepatocytes.

Sirt1 (Sirtuin 1), AMPK (AMP-activated protein kinase), and eNOS (endothelial nitric oxide synthase) modulate hepatic energy metabolism and inflammation and play a major role in the development of NASH. Cyclic nucleotide phosphodiesterases (PDEs) play an important role in signal transduction by modulating intracellular levels of cyclic nucleotides. We previously found the PDE5 inhibitor sildenafil to synergize with leucine and leucine-metformin combinations in preclinical studies of NASH and obesity. However, efficacy is diminished at higher sildenafil concentrations. Herein, we have successfully modeled the U-shaped sildenafil dose-response in vitro and utilized this model to assess potential mechanisms of this dose-response relationship. Adipocytes and liver cells were treated with leucine (0.5 mM) and different concentrations of sildenafil (1 nM to 100 µM). cAMP, cGMP, and P-AMPK protein expression were used to demonstrate the biphasic response for increasing concentrations of sildenafil. The reversal with higher sildenafil levels was blunted by PDE2 inhibition. These data indicate that sildenafil-mediated increases in cGMP inhibits PDE3 at lower concentrations, which increases cAMP. However, further increases in cGMP from higher sildenafil concentrations activate PDE2 and consequently decrease cAMP, which demonstrates crosstalk between cAMP and cGMP via PDE2, PDE3, and PDE5. These changes in cAMP concentration are further reflected in downstream effects, including AMPK activation.

A combination of probiotics and whey proteins enhances anti-obesity effects of calcium and dairy products during nutritional energy restriction in aP2-agouti transgenic mice.

Lactobacillus rhamnosus GG, Lactobacillus paracasei TMC0409, Streptococcus thermophilus TMC1543 and whey proteins were used to prepare fermented milk. For the experiment aP2- agouti transgenic mice were pre-treated with a high-sucrose/high-fat diet for 6 weeks to induce obesity. The obese mice were fed a diet containing 1·2% Ca and either non-fat dried milk (NFDM) or probiotic-fermented milk (PFM) with nutritional energy restriction for 6 weeks. The animals were examined after the treatment for changes in body weight, fat pad weight, fatty acid synthase (FAS) activity, lypolysis, the expression levels of genes related to lipid metabolism, insulin sensitivity in adipocytes and skeletal muscle and the presence of biomarkers for oxidative and inflammatory stress in plasma. It was found that the PFM diet significantly reduced body weight, fat accumulation, and adipocyte FAS activity, and increased adipocyte lipolysis as compared with the effects of the NFDM diet (P<0·05). The adipose tissue gene expression of 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) was significantly suppressed in mice that were fed PFM as compared with those that were fed NFDM (P<0·05). PFM caused a greater up-regulation of skeletal muscle PPARα, PPARδ, uncoupling protein 3 (UCP3) and GLUT4 expression and a significant decrease in the plasma concentration of insulin, malondialdehyde, TNF-α, monocyte chemotactic protein-1 and C-reactive protein as compared with the effects of NFDM (P<0·05). Fermentation of milk with selected probiotics and supplementation of milk with whey proteins may thus enhance anti-obesity effects of Ca and dairy products by the suppression of adipose tissue lipogenesis, activation of fat oxidation in skeletal muscle and reduction of oxidative and inflammatory stress.

Modulation of Energy Sensing by Leucine Synergy with Natural Sirtuin Activators: Effects on Health Span.

Sirt1 and 5' adenosine monophosphate-activated protein kinase (AMPK) are energy-sensing systems that work cooperatively and regulate mitochondrial biogenesis and fuel metabolism, and mediate, in part, the salutary effects of caloric restriction on lifespan and healthspan. We have shown that leucine activates Sirt1 and enables synergy with sirtuin co-activators. Resveratrol is a widely recognized activator of Sirt1; however, poor bioavailability and rapid metabolism limit effective clinical translation of promising animal data. However, we found that combining low resveratrol doses with leucine increased skeletal muscle and adipocyte Sirt1 activity, mitochondrial biogenesis and fatty acid oxidation; these effects result in increased lifespan and marked reductions in insulin resistance, inflammatory markers, body weight, and visceral adiposity in preclinical models. To translate these data to humans, we assessed the effects of resveratrol (50 mg)/leucine (1.11 g) on glucose dynamics in a 4-week placebo-controlled trial of 36 prediabetic subjects. Leucine-resveratrol reduced insulin resistance (homeostatic model assessment for insulin resistance) 33% with corresponding reductions in glucose and insulin area under the curve in oral glucose tolerance tests. We extended these concepts in preclinical studies using both direct Sirt1 activators and Sirt1 pathway activators. Low-dose (10 nM) NAD+ precursors (nicotinic acid, nicotinamide mononucleotide, and nicotinamide riboside) synergized with leucine to increase Sirt1 activity in adipocytes, hepatocytes, and muscle cells (30-100%, P < .01) and lifespan in Caenorhabditis elegans (25%, P = .025) and to significantly regress atherosclerotic lesion size and macrophage infiltration in a mouse model of atherosclerosis. Thus, synergistic activation of Sirt1 using leucine and a co-activator exerts pleiotropic effects impacting cardiometabolic endpoints.

Effect of a Leucine/Pyridoxine Nutraceutical on Caloric Intake and Body Composition of Obese Dogs Losing Weight.

The aim of this 29-week randomized, positively and negatively controlled study was to investigate whether a nutraceutical containing 1 g leucine and 13 mg pyridoxine can enhance weight loss while maintaining lean muscle mass in obese dogs. Twenty-four healthy, 2-year-old beagles were initially divided into obesification (n = 18) or ideal body weight groups (n = 6). After obesification, the 18 dogs were divided into three weight loss groups and fed one of the following over 12 weeks: nutraceutical with canned adult diet (CAD; ObN), placebo with CAD (ObP), or a canned therapeutic weight loss diet (WLD). Dogs in the ideal body weight (IBW) group were fed maintenance calorie requirements with CAD over 12 weeks. Based on MANOVA, ObN and WLD lost similar amounts of total weight (3.6 ± 0.9 vs. 4.4 ± 1.1 kg, respectively) and fat mass (3.1 ± 0.6 vs. 3.9 ± 0.8 kg, respectively) after 12 weeks of treatment, and more than ObP (1.1 ± 1.2 kg weight; 0.9 ± 1.0 kg fat; p < 0.0001). These data show the nutraceutical is a promising option for successful weight loss in dogs. Maintenance levels of CAD were able to induce weight loss without risk of hypo- or anorexia, or the need to switch diets or restrict energy intake.

Lipid metabolism predicts changes in body composition during energy restriction in overweight humans.

Dietary weight loss regimens could be more effective by selectively targeting adipose while sparing lean mass (LM) if predictive information about individuals' lipid metabolic responses to an intervention were available. The objective of this study was to examine the relationships among changes in 4 anthropometric outcomes, weight, waist circumference (WC), percent body fat (BF), and percent LM, and comprehensive circulating lipid metabolites in response to energy reduction in overweight participants. This was a cohort study (n = 46) from a larger multi-center (n = 105) weight loss trial. We used stepwise regression to examine relationships among baseline plasma fatty acids of 7 lipid classes, biochemical metabolites, and diet to explain the variance of 4 anthropometric outcomes after intervention. No predictor variables explained the variance in the percent change in body weight. The circulating concentration of FFA 18:1(n-9) at baseline explained 31% of the variance in percent change of WC, with adjustment for energy intake at 12 wk. Circulating concentrations of phosphatidylcholine 18:0 and FFA 18:1(n-9) at baseline together explained 33% of the variance in percent LM change. The circulating concentration of phosphatidylcholine 18:0 at baseline explained 23% of the variance in the change in percent BF. This study determined relationships among comprehensive and quantitative measurements of complex lipid metabolites and metabolic outcomes as changes in body composition. Measurements of plasma circulating metabolites explained 20-30% of the variance in changes in body composition after a weight loss intervention. Thus, circulating lipids reflect lipid metabolism in relation to changes in body composition.

Can a dairy-rich diet be effective in long-term weight control of young children?

OBJECTIVE: To determine the long-term effect of a randomized controlled trial of a dairy-rich diet on generalized and abdominal obesity, as well as on the components of the metabolic syndrome, among obese prepubescent children. METHODS: This trial was conducted among a population-based sample of 120 obese prepubescent children who were randomly assigned to 3 groups of equal number. In addition to attending 6 consecutive monthly family-centered education sessions about healthy lifestyle, an isocaloric dairy-rich diet (>800 mg ca/d) was recommended to the children of one group (DR: dairy-rich diet), the second group was placed on a caloric-restricted regimen (ER: energy-restricted), and the third group received no additional recommendation (C: controls). The groups were then followed-up twice a year for 3 years. RESULTS: The mean age of the children was 5.6 +/- 0.5 years. Of 120 participants, 95 (75%) completed the study; the DR group had the highest retention rate. In all groups, body mass index-standard deviation score (BMI-SDS) and waist circumference decreased significantly after the 6-month trial, but had a sustained significant rise during the follow-up period to the end of the study; however, in the DR group, this rise was significantly lower than in the 2 other groups. After the 6-month trial, in all groups, serum triglycerides (TG) and insulin levels decreased, and serum high-density lipoprotein cholesterol (HDL-C) level and homeostasis model assessment of insulin resistance (HOMA-R) increased. In the DR group, the TG, insulin and HOMA-R levels remained significantly lower than baseline until the 12-month follow-up. CONCLUSIONS: We suggest that in addition to lifestyle changes, an isocaloric diet rich in dairy products may be a well-accepted regimen and can be a safe and practical strategy for weight control in young, overweight children.

Effects of dairy products on intracellular calcium and blood pressure in adults with essential hypertension.

BACKGROUND: Consumption of dairy foods has been associated with lower blood pressure in certain populations. OBJECTIVE: This study examined the effects of dairy foods on blood pressure (BP) and intracellular calcium ((Ca)(i)) and the dependence of BP changes on changes in (Ca)(i). DESIGN: Twenty-three stage 1 hypertensive adults were fed the following 3 experimental diets (5 wk each) in a randomized cross-over design study; a dairy-rich, high fruits and vegetables diet (D-FandV; 30% fat, 7% saturated fat (SFA), 3.4 servings/d dairy), a high fruits and vegetables diet (FandV; 30% fat, 7% SFA, 0.4 servings/d dairy), and an average Western diet (control; 36% fat, 15% SFA, 0.4 servings/d dairy). Systolic (SBP) and diastolic (DBP) BP, calcium regulatory hormones, and erythrocyte (Ca)(i) were determined. RESULTS: SBP and DBP were significantly reduced by approximately 2 mm Hg following both D-F&V and F&V diets vs. the control (P < 0.05). The D-F&V diet significantly lowered 1,25-dihydroxyvitaminD compared with the F&V and control diets (P < 0.01). Serum calcium, parathyroid hormone, calcitonin, and renin activity were unchanged. The D-F&V diet lowered (Ca)(i) vs. the other two diets (P < 0.01), and this change correlated with the fall in DBP (r = 0.52, P < 0.05). Subjects who responded to the D-F&V diet by significantly reducing (Ca)(i) exhibited significantly greater net decreases in DBP on the D-F&V vs. the F&V (-2.8 +/- 1.0 mm Hg) and control diets (-5.4 +/-1.0 mm Hg; diet x group interaction, P < 0.02). CONCLUSION: Consumption of dairy foods beneficially affects (Ca)(i), resulting in improved BP in a subgroup defined by (Ca)(i) response.

Proposed role of calcium and dairy food components in weight management and metabolic health.

Dietary calcium and dairy foods have demonstrated an antiobesity effect in animal studies, observational and population studies, and randomized clinical trials. Moreover, there is a strong theoretical framework to explain the effects of dietary calcium on energy metabolism. The supporting mechanisms include dietary calcium-correcting suboptimal calcium intakes, thereby preventing the endocrine response (parathyroid hormone [PTH] and calcitriol), which favors adipocyte energy storage and inhibits adipocyte loss via apoptosis. Dietary calcium appears to further promote energy loss via formation of calcium soaps in the gastrointestinal tract and thereby modestly reduces net energy absorption. Dietary calcium appears to be responsible for approximately 50% of the antiobesity bioactivity of dairy foods. The additional dairy bioactivity has not been fully identified, but is primarily localized in whey protein. The major components are the angiotensin-converting enzyme (ACE) inhibitor activity of whey proteins and the high concentration of leucine in whey. This high leucine content appears to be primarily responsible for the repartitioning of dietary energy from adipose tissue to skeletal muscle during weight loss, resulting in greater preservation of skeletal muscle and accelerated loss of adipose tissue during negative energy balance. Finally, high-calcium diets suppress obesity-induced oxidative and inflammatory stress independently from its role in modulating adiposity; these effects are similarly augmented by other dairy food components. However, the number of randomized clinical trials conducted is still modest, and a small number have not confirmed significant effects in weight management. Thus, the protective effects of dairy foods against obesity and its comorbidities are promising, but warrant further large-scale studies.

[Role of whey protein and whey components in weight management and energy metabolism].

An anti-obesity effect of dietary calcium and dairy foods is evident from animal studies, observational and population studies, and randomized clinical trials. Dietary calcium appears to be responsible for 50% of the anti-obesity bioactivity of dairy. The additional dairy bioactivity has not been fully identified, but is localized primarily in whey, the anti-obesity effect appears to result from calcium, high proportion of branched chain amino acids, and specific bioactive whey-derived peptides. This article presents recent research progress on the role of whey protein and whey components in weight management and energy metabolism.

Randomized Controlled Trial of a Leucine-Metformin-Sildenafil Combination (NS-0200) on Weight and Metabolic Parameters.

OBJECTIVE: Leucine was previously demonstrated to allosterically activate mammalian sirtuin 1 and synergize with other sirtuin 1/AMP-activated protein kinase/nitric oxide pathway activators to modulate energy metabolism. The objective of this study was to evaluate the effects of a triple combination of leucine, metformin, and sildenafil (NS-0200) on body weight and obesity comorbidities in a phase 2 randomized trial. METHODS: A total of 91 subjects with obesity were randomized to placebo, low dose (1.1 g leucine/0.5 g metformin/0.5 mg sildenafil), or high dose (1.1 g leucine/0.5 g metformin/1.0 mg sildenafil) twice daily for 16 weeks. Seventy subjects completed the trial and met all a priori compliance criteria. Hypertensive (n = 35) and hypertriglyceridemic (n = 22) subcohorts were also analyzed. RESULTS: NS-0200 dose-responsively reduced weight; high dose reduced weight by 2.4 and 5.0 kg in the full and high-triglyceride cohorts, respectively (P < 0.0001). High-dose NS-0200 treatment also decreased blood pressure (-5.5 mm Hg diastolic pressure; P = 0.011), with greater effects among hypertensive subjects. NS-0200 also significantly reduced triglycerides and hemoglobin A1c. Significant improvement in ≥ 2 comorbidities was exhibited by 54% of subjects in the high-dose arm versus 5% of placebo subjects (P = 0.0009). Treatment-emergent adverse events did not significantly differ among groups. CONCLUSIONS: These data support further study of NS-0200 as a therapy for obesity and associated comorbidities.

Dietary calcium and dairy products modulate oxidative and inflammatory stress in mice and humans.

We have recently shown 1alpha,25-dihydroxycholecalciferol increased oxidative stress and inflammatory stress in vitro, whereas suppression of 1alpha,25-dihydroxycholecalciferol with dietary calcium (Ca) decreased oxidative and inflammatory stress in vivo. However, dairy products contains additional factors, such as angiotensin-converting enzyme inhibitors, which may further suppress oxidative and inflammatory stress. Accordingly, this study was designed to study the effects of the short-term (3 wk) basal suboptimal Ca (0.4%), high-Ca (1.2% from CaCO(3)), and high-dairy (1.2% Ca from milk) obesigenic diets on oxidative and inflammatory stress in adipocyte fatty acid-binding protein-agouti transgenic mice. Adipose tissue reactive oxygen species (ROS) production and NADPH oxidase mRNA and plasma malondialdehyde (MDA) were reduced by the high-Ca diet (P < 0.001) compared with the basal diet and ROS and MDA were further decreased by the high-dairy diet (P < 0.001). The high-Ca and -dairy diets also resulted in suppression of adipose tissue tumor necrosis factor alpha and interleukin (IL)-6 mRNA (P = 0.001) compared with the basal diet, whereas an inverse pattern was noted for adiponectin and IL-15 mRNA (P = 0.002). Consequently, we conducted a follow-up evaluation of adiponectin and C-reactive protein (CRP) in archival samples from 2 previous clinical trials conducted in obese men and women. Twenty-four weeks of feeding a high-dairy eucaloric diet and hypocaloric diet resulted in an 11 (P < 0.03) and 29% (P < 0.01) decrease in CRP, respectively (post-test vs. pre-test), whereas there was no significant change in the low-dairy groups. Adiponectin decreased by 8% in subjects fed the eucaloric high-dairy diet (P = 0.003) and 18% in those fed the hypocaloric high-dairy diet (P < 0.05). These data demonstrate that dietary Ca suppresses adipose tissue oxidative and inflammatory stress.

Calcitriol and calcium regulate cytokine production and adipocyte-macrophage cross-talk.

OBJECTIVE: The objective of this study was to investigate the effects of calcitriol on adipocyte and macrophage cytokine expression as well as release and on adipocyte-macrophage cross-talk in local modulation of inflammation. RESEARCH PROCEDURES AND RESULTS: We investigated calcitriol modulation of the expression of macrophage inhibitory factor (MIF) and macrophage surface-specific protein CD14, two key factors in regulating macrophage function and survival, in differentiated human adipocytes. Calcitriol significantly increased MIF and CD14 expression by 59% and 33%, respectively, while calcium-channel antagonism with nifedipine completely reversed these effects, indicating that calcitriol stimulates MIF and CD14 expression via a calcium-dependent mechanism. Similar results were also found in cultured 3T3-L1 adipocytes; in addition, calcitriol also up-regulated macrophage colony-stimulating factor, macrophage inflammatory protein, interleukin-6 (IL-6) as well as monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes and stimulated tumor necrosis factor as well as IL-6 expression in RAW 264 macrophages. These effects were blocked by either a calcium-channel antagonist (nifedipine) or a mitochondrial uncoupler (dinitrophenol). Moreover, co-culture of 3T3-L1 adipocytes with RAW 264 macrophages significantly increased the expression and production of multiple inflammatory cytokines in response to calcitriol in both cell types. CONCLUSIONS: These data demonstrate that calcitriol regulates local inflammation via modulating the interaction between adipocytes and macrophages as well as regulating inflammatory cytokine production in each cell type via calcium-dependent and mitochondrial uncoupling-dependent mechanisms. These data provide further mechanistic explanation for our recent observations that suppression of calcitriol by dietary calcium reduces inflammatory cytokine expression and oxidative stress in adipose tissue.

Leucine and calcium regulate fat metabolism and energy partitioning in murine adipocytes and muscle cells.

Dietary calcium modulation of adiposity is mediated, in part, by suppression of calcitriol, while the additional effect of dairy products is mediated by additional components; these include the high concentration of leucine, a key factor in the regulation of muscle protein turnover. We investigated the effect of leucine, calcitriol and calcium on energy metabolism in murine adipocytes and muscle cells and on energy partitioning between adipocytes and skeletal muscle. Leucine induced a marked increase in fatty acid oxidation in C2C12 muscle cells (P<0.001) and decreased FAS expression by 66% (P<0.001) in 3T3-L1 adipocytes. Calcitriol decreased muscle cell fatty acid oxidation by 37% (P<0.001) and increased adipocyte FAS gene expression by threefold (P<0.05); these effects were partially reversed by either leucine or calcium channel antagonism with nifedipine. Co-culture of muscle cells with adipocytes or incubation with 48-h adipocyte conditioned medium decreased muscle fatty acid oxidation by 62% (P<0.001), but treating adipocytes with leucine and/or nifedipine attenuated this effect. Leucine, nifedipine and calcitriol also modulated adiponectin production and thereby exerted additional indirect effects on fatty acid oxidation in C2C12 myotubes. Adiponectin increased IL-15 and IL-6 release by myotubes and partially reversed the inhibitory effects of calcitriol. Comparable effects of leucine, calcitriol and adiponectin were found in myotubes treated with conditioned medium derived from adipocytes or co-cultured with adipocytes. These data suggest that leucine and nifedipine promote energy partitioning from adipocytes to muscle cells, resulting in decreased energy storage in adipocytes and increasing fatty acid utilization in muscle.

Leucine-nicotinic acid synergy stimulates AMPK/Sirt1 signaling and regulates lipid metabolism and lifespan in Caenorhabditis elegans, and hyperlipidemia and atherosclerosis in mice.

BACKGROUND/AIMS: Nicotinic acid (NA), a lipid-lowering drug, serves as a source of NAD+, the cofactor for Sirt1. Leucine (Leu) stimulates the AMPK/Sirt1 axis and amplifies the effects of other AMPK/Sirt1 activating compounds. Therefore, we tested the interactive effects of leucine and low dose NA on AMPK/Sirt1 signaling and downstream effects of lipid metabolism in cell culture, C. elegans and mice. METHODS: LDL-receptor knockout mice were fed an atherogenic Western diet supplemented with leucine (24 g/kg diet) and sub-therapeutic NA combinations (50 mg/kg diet and 250 mg/kg diet) or low therapeutic NA (1000 mg/kg diet) for 8 weeks to evaluate markers of hyperlipidemia and atherosclerosis. RESULTS: NA-Leu increased P-AMPK and Sirt1 in adipocytes and myotubes. In C. elegans, NA-Leu increased P-AMPK and DAF-16 (FOXO), reduced lipid accumulation and increased median survival under mild oxidative stress conditions. In the mice, NA-Leu reduced total cholesterol, cholesterol esters, plasma triglycerides, atherosclerotic lesion size, lipid area, and aortic macrophage infiltration, similar to the therapeutic NA dose. CONCLUSION: Leu amplifies the effects of NA on lipid metabolism, hyperlipidemia and atherosclerosis in mice, at least in part by activation of the AMPK/Sirt1 axis. This combination may be a potential therapeutic alternative for hyperlipidemia and atherosclerosis.

Erratum: Leucine-nicotinic acid synergy stimulates AMPK/Sirt1 signaling and regulates lipid metabolism and lifespan in Caenorhabditis elegans, and hyperlipidemia and atherosclerosis in mice.

[This corrects the article on p. 33 in vol. 7, PMID: 28533928.].

Prostaglandin E(2) protects intestinal tumors from nonsteroidal anti-inflammatory drug-induced regression in Apc(Min/+) mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are antitumorigenic in humans as well as in animal models of intestinal neoplasia, such as the adenomatous polyposis coli (Min/+) (Apc(Min/+)) mouse. NSAIDs inhibit cyclooxygenase (COX) isozymes, which are responsible for the committed step in prostaglandin biosynthesis, and this has been considered the primary mechanism by which NSAIDs exert their antitumorigenic effects. However, mounting evidence suggests the existence of COX-independent mechanisms. In the present study, we attempted to clarify this issue by treating Apc(Min/+) mice bearing established tumors with NSAIDs (piroxicam and sulindac, 0.5 and 0.6 mg/mouse/day, respectively) for 6 days and concomitantly bypassing COX inhibition by treatment with the E prostaglandin (EP) receptor agonists 16,16-dimethyl-prostaglandin E(2) (PGE(2)) and 17-phenyl-trinor-PGE(2) (10 microg each, three times daily) administered via gavage and/or i.p. routes. Treatment with piroxicam and sulindac resulted in 95% and 52% fewer tumors, respectively, and a higher ratio of apoptosis:mitosis in tumors from sulindac-treated mice as compared with controls. These effects were attenuated by concomitant EP receptor agonist treatment, suggesting PGE(2) is important in the maintenance of tumor integrity. Immunological sequestration of PGE(2) with an anti-PGE(2) monoclonal antibody likewise resulted in 33% fewer tumors in Apc(Min/+) mice relative to untreated controls, additionally substantiating a role for PGE(2) in tumorigenesis. The EP receptor subtype EP1 mediates the effects of PGE(2) by increasing intracellular calcium levels ([Ca(2+)](i)), whereas antagonism of EP1 has been shown to attenuate tumorigenesis in Apc(Min/+) mice. We demonstrate that [Ca(2+)](i) is significantly elevated in tumors of Apc(Min/+) mice relative to the adjacent normal-appearing mucosa. Furthermore, treatment with piroxicam results in significantly lower [Ca(2+)](i) in tumors, and this effect is attenuated by concomitant treatment with the EP1/EP3 receptor agonist 17-phenyl-trinor-PGE(2). Overall, our results suggest that NSAIDs exert their antitumorigenic effects, in part, via interference with PGE(2) biosynthesis, and these effects may be mediated through changes in intracellular calcium levels.

Activation of the AMPK/Sirt1 pathway by a leucine-metformin combination increases insulin sensitivity in skeletal muscle, and stimulates glucose and lipid metabolism and increases life span in Caenorhabditis elegans.

BACKGROUND: We have previously shown leucine (Leu) to activate Sirt1 by lowering its KM for NAD+, thereby amplifying the effects of other sirtuin activators and improving insulin sensitivity. Metformin (Met) converges on this pathway both indirectly (via AMPK) and by direct activation of Sirt1, and we recently found Leu to synergize with Met to improve insulin sensitivity and glycemic control while achieving ~80% dose-reduction in diet-induced obese mice. Accordingly, we sought here to define the mechanism of this interaction. METHODS: Muscle cells C2C12 and liver cells HepG2 were used to test the effect of Met-Leu on Sirt1 activation. Caenorhabditis elegans was used for glucose utilization and life span studies. RESULTS: Leu (0.5mmol/L)+Met (50-100µmol/L) synergistically activated Sirt1 (p<0.001) at low (≤100µmol/L) NAD+ levels while Met exerted no independent effect. This was associated with an increase in AMPK and ACC, phosphorylation, and increased fatty acid oxidation, which was prevented by AMPK or Sirt inhibition or silencing. Met-Leu also increased P-IRS1/IRS1 and P-AKT/AKT and in insulin-independent glucose disposal in myotubes (~50%, p<0.002) evident within 30 min as well as a 60% reduction in insulin EC50. In addition, in HepG2 liver cells nuclear CREB regulated transcription coactivator 2 (CRTC2) protein expression and phosphorylation of glycogen synthase was decreased, while glycogen synthase kinase phosphorylation was increased indicating decreased gluconeogenesis and glycogen synthesis. We utilized C. elegans to assess the metabolic consequences of this interaction. Exposure to high glucose impaired glucose utilization and shortened life span by ~25%, while addition of Leu+Met to high glucose worms increased median and maximal life span by 29 and 15%, respectively (p=0.023), restored normal glucose utilization and increased fat oxidation ~two-fold (p<0.005), while metformin exerted no independent effect at any concentration (0.1-0.5mmol/L). CONCLUSION: Thus, Leu and Met synergize to enable Sirt1 activation at low NAD+ concentrations (typical of energy replete states). Sirt1 and AMPK activations are required for Met-Leu's full action, which result in improvements in energy metabolism and insulin sensitivity.

A Combination of Leucine, Metformin, and Sildenafil Treats Nonalcoholic Fatty Liver Disease and Steatohepatitis in Mice.

Sirt1, AMPK, and eNOS modulate hepatic energy metabolism and inflammation and are key players in the development of NASH. L-leucine, an allosteric Sirt1 activator, synergizes with low doses of metformin or sildenafil on the AMPK-eNOS-Sirt1 pathway to reverse mild NAFLD in preclinical mouse models. Here we tested a possible multicomponent synergy to yield greater therapeutic efficacy in NAFLD/NASH. Liver cells and macrophages or an atherogenic diet induced NASH mouse model was treated with two-way and three-way combinations. The three-way combination Sild-Met-Leu increased hepatic fatty acid oxidation and reduced lipogenic gene expression and inflammatory marker in vitro. In mice, Sild-Met-Leu reduced the diet induced increases of ALT, TGFß, PAI-1, IL1ß, and TNFα, hepatic collagen expression, and nearly completely reversed hepatocyte ballooning and triglyceride accumulation, while all two-way combinations had only modest effects. Therefore, these data provide preclinical evidence for therapeutic efficacy of Sild-Met-Leu in the treatment of NAFLD and NASH.

Role of uncoupling protein 2 (UCP2) expression and 1alpha, 25-dihydroxyvitamin D3 in modulating adipocyte apoptosis.

We previously found that 1alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2-D3] modulates adipocyte lipid metabolism via a Ca2+-dependent mechanism and inhibits adipocyte UCP2 expression, indicating that the anti-obesity effects of dietary calcium are mediated by suppression of 1alpha, 25-(OH)2-D3 levels. However, because UCP2 reduces mitochondrial potential, we have evaluated the roles of UCP2, mitochondrial uncoupling, and 1alpha, 25-(OH)2-D3 in adipocyte apoptosis. Overexpressing UCP2 in 3T3-L1 cells induced marked reductions in mitochondrial potential (Deltapsi) and ATP production (P<0.01), increases in the expression of caspases (P<0.05), and a decrease in Bcl-2/Bax expression ratio (P<0.01). Physiological doses of 1alpha, 25-(OH)2-D3 (0.1-10 nM) restored mitochondrial Deltapsi in LI-UCP2 cells and protected against UCP2 overexpression-induced apoptosis (P<0.01), whereas a high dose (100 nM) stimulated apoptosis in 3T3-L1 and L1-UCP2 cells (P<0.05). 1alpha, 25-(OH)2-D3 stimulated cytosolic Ca2+ dose-dependently in both 3T3-L1 and L1-UCP2 cells. However, physiological doses suppressed mitochondrial Ca2+ levels by approximately 50% whereas the high dose increased mitochondrial Ca2+ by 25% (P<0.05); this explains stimulation of apoptosis by the high dose of 1alpha, 25-(OH)2-D3. Using high-calcium diets to suppress 1alpha, 25-(OH)2-D3 stimulated adipose tissue apoptosis in aP2 transgenic mice (P<0.01), suggesting that increasing dietary calcium stimulates adipose apoptosis and thereby further contributes to an anti-obesity effect of dietary calcium.