A Dietary Mixture Containing Fish Oil, Resveratrol, Lycopene, Catechins, and Vitamins E and C Reduces Atherosclerosis in Transgenic Mice123

Chronic inflammation and proatherogenic lipids are important risk factors of cardiovascular disease (CVD). Specific dietary constituents such as polyphenols and fish oils may improve cardiovascular risk factors and may have a beneficial effect on disease outcomes. We hypothesized that the intake of an antiinflammatory dietary mixture (AIDM) containing resveratrol, lycopene, catechin, vitamins E and C, and fish oil would reduce inflammatory risk factors, proatherogenic lipids, and endpoint atherosclerosis. AIDM was evaluated in an inflammation model, male human C-reactive protein (CRP) transgenic mice, and an atherosclerosis model, female ApoE*3Leiden transgenic mice. Two groups of male human-CRP transgenic mice were fed AIDM [0.567% (wt:wt) powder and 0.933% (wt:wt oil)] or placebo for 6 wk. The effects of AIDM on basal and IL-1β–stimulated CRP expression were investigated. AIDM reduced cytokine-induced human CRP and fibrinogen expression in human-CRP transgenic mice. In the atherosclerosis study, 2 groups of female ApoE*3Leiden transgenic mice were fed an atherogenic diet supplemented with AIDM [0.567% (wt:wt) powder and 0.933% (wt:wt oil)] or placebo for 16 wk. AIDM strongly reduced plasma cholesterol, TG, and serum amyloid A concentrations compared with placebo. Importantly, long-term treatment of ApoE*3Leiden mice with AIDM markedly reduced the development of atherosclerosis by 96% compared with placebo. The effect on atherosclerosis was paralleled by a reduced expression of the vascular inflammation markers and adhesion molecules inter-cellular adhesion molecule-1 and E-selectin. Dietary supplementation of AIDM improves lipid and inflammatory risk factors of CVD and strongly reduces atherosclerotic lesion development in female transgenic mice

Propionic acid and not caproic acid, attenuates nonalcoholic steatohepatitis and improves (cerebro) vascular functions in obese Ldlr^−/−.Leiden mice

The obesity epidemic increases the interest to elucidate impact of short-chain fatty acids on metabolism, obesity, and the brain. We investigated the effects of propionic acid (PA) and caproic acid (CA) on metabolic risk factors, liver and adipose tissue pathology, brain function, structure (by MRI), and gene expression, during obesity development in Ldlr−/−.Leiden mice. Ldlr−/−.Leiden mice received 16 weeks either a high-fat diet (HFD) to induce obesity, or chow as reference group. Next, obese HFD-fed mice were treated 12 weeks with (a) HFD + CA (CA), (b) HFD + PA (PA), or (c) a HFD-control group. PA reduced the body weight and systolic blood pressure, lowered fasting insulin levels, and reduced HFD-induced liver macrovesicular steatosis, hypertrophy, inflammation, and collagen content. PA increased the amount of glucose transporter type 1-positive cerebral blood vessels, reverted cerebral vasoreactivity, and HFD-induced effects in microstructural gray and white matter integrity of optic tract, and somatosensory and visual cortex. PA and CA also reverted HFD-induced effects in functional connectivity between visual and auditory cortex. However, PA mice were more anxious in open field, and showed reduced activity of synaptogenesis and glutamate regulators in hippocampus. Therefore, PA treatment should be used with caution even though positive metabolic, (cerebro) vascular, and brain structural and functional effects were observed.</p

Therapeutic intervention with anti-complement component 5 antibody does not reduce nash but does attenuate atherosclerosis and mif concentrations in ldlr-/-.Leiden mice

Background: Chronic inflammation is an important driver in the progression of nonalcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in innate immunity, has been implicated in both diseases. However, the potential therapeutic value of complement inhibition in the ongoing disease remains unclear. Methods: After 20 weeks of high-fat diet (HFD) feeding, obese Ldlr-/-.Leiden mice were treated twice a week with an established anti-C5 antibody (BB5.1) or vehicle control. A separate group of mice was kept on a chow diet as a healthy reference. After 12 weeks of treatment, NASH was analyzed histopathologically, and genome-wide hepatic gene expression was analyzed by next-generation sequencing and pathway analysis. Atherosclerotic lesion area and severity were quantified histopathologically in the aortic roots. Results: Anti-C5 treatment considerably reduced complement system activity in plasma and MAC deposition in the liver but did not affect NASH. Anti-C5 did, however, reduce the development of atherosclerosis, limiting the total lesion size and severity independently of an effect on plasma cholesterol but with reductions in oxidized LDL (oxLDL) and macrophage migration inhibitory factor (MIF). Conclusion: We show, for the first time, that treatment with an anti-C5 antibody in advanced stages of NASH is not sufficient to reduce the disease, while therapeutic intervention against established atherosclerosis is beneficial to limit further progression

Intervention with isoleucine or valine corrects hyperinsulinemia and reduces intrahepatic diacylglycerols, liver steatosis, and inflammation in Ldlr−/−.Leiden mice with manifest obesity-associated NASH

Non-alcoholic steatohepatitis (NASH) is associated with a disturbed metabolism in liver, insulin resistance, and excessive accumulation of ectopic fat. Branched-chain amino acids (BCAAs) may beneficially modulate hepatic lipids, however, it remains unclear whether individual BCAAs can attenuate already established NASH and associated oxidative-inflammatory stress. After a 26 weeks run-in on fast food diet (FFD), obese Ldlr−/−.Leiden mice were treated for another 12 weeks with either valine or isoleucine (3% of FFD) and then compared to FFD controls. Valine and isoleucine did not affect obesity, dyslipidemia, gut permeability, or fecal fatty acid excretion, but significantly reduced hyperinsulinemia. Valine and isoleucine reduced ALT, CK18-M30, and liver steatosis with a particularly pronounced suppression of the microvesicular component (−61% by valine and −71% by isoleucine). Both BCAAs decreased intrahepatic diacylglycerols and 4-hydroxynonenal immunoreactivity, a marker for oxidative stress-induced lipid peroxidation. Functional genomics analysis demonstrated that valine and isoleucine affected BCAA metabolism genes, deactivated master regulators of anabolic pathways related to steatosis (e.g., SREBPF1), and activated master regulators of mitochondrial biogenesis (e.g., PPARGC1A) and lipid catabolism (e.g., ACOX1, AMPK). This correction of critical metabolic pathways on gene expression level was accompanied by a significant decrease in histological liver inflammation, and suppression of FFD-stimulated cytokine and chemokine proteins KC/CXCL1, MCP-1/CCL2, and MIP-2/CXCL2 and their pathways. In conclusion, dietary intervention with either valine or isoleucine corrected liver diacylglycerols, gene expression of multiple metabolic processes, and reduced NASH histology with profound hepatoprotective effects on oxidative stress and inflammatory proteins

Heat-Inactivated Akkermansia muciniphila Improves Gut Permeability but Does Not Prevent Development of Non-Alcoholic Steatohepatitis in Diet-Induced Obese Ldlr−/−.Leiden Mice

The development of non-alcoholic steatohepatitis (NASH) has been associated with alterations in gut microbiota composition and reduced gut barrier function. Akkermansia muciniphila is a gut microbe that is thought to have health-promoting properties, including the ability to improve gut barrier function and host metabolism, both when administered live and after heat-inactivation. We questioned whether heat-inactivated A. muciniphila may reduce NASH development. Ldlr−/−.Leiden mice, a translational, diet-induced model for NASH, were fed a NASH-inducing high-fat diet (HFD) supplemented with heat-inactivated A. muciniphila. After 28 weeks, effects of the treatment on obesity and associated metabolic dysfunction in the gut (microbiota composition and permeability), adipose tissue, and liver were studied relative to an untreated HFD control. Treatment with heat-inactivated A. muciniphila did not affect body weight or adiposity and had no effect on plasma lipids, blood glucose, or plasma insulin. Heat-inactivated A. muciniphila had some minor effects on mucosal microbiota composition in ileum and colon and improved gut barrier function, as assessed by an in vivo functional gut permeability test. Epidydimal white adipose tissue (WAT) hypertrophy and inflammation were not affected, but heat-inactivated A. muciniphila did reduce hypertrophy in the mesenteric WAT which is in close proximity to the intestine. Heat-inactivated A. muciniphila did not affect the development of NASH or associated fibrosis in the liver and did not affect circulating bile acids or markers of liver fibrosis, but did reduce PRO-C4, a type IV collagen synthesis marker, which may be associated with gut integrity. In conclusion, despite beneficial effects in the gut and mesenteric adipose tissue, heat-inactivated A. muciniphila did not affect the development of NASH and fibrosis in a chronic disease setting that mimics clinically relevant disease stages

Replacement of Dietary Saturated Fat by PUFA-Rich Pumpkin Seed Oil Attenuates Non-Alcoholic Fatty Liver Disease and Atherosclerosis Development, with Additional Health Effects of Virgin over Refined Oil.

As dietary saturated fatty acids are associated with metabolic and cardiovascular disease, a potentially interesting strategy to reduce disease risk is modification of the quality of fat consumed. Vegetable oils represent an attractive target for intervention, as they largely determine the intake of dietary fats. Furthermore, besides potential health effects conferred by the type of fatty acids in a vegetable oil, other minor components (e.g. phytochemicals) may also have health benefits. Here, we investigated the potential long-term health effects of isocaloric substitution of dietary fat (i.e. partial replacement of saturated by unsaturated fats), as well as putative additional effects of phytochemicals present in unrefined (virgin) oil on development of non-alcoholic fatty liver disease (NAFLD) and associated atherosclerosis. For this, we used pumpkin seed oil, because it is high in unsaturated fatty acids and a rich source of phytochemicals.ApoE*3Leiden mice were fed a Western-type diet (CON) containing cocoa butter (15% w/w) and cholesterol (1% w/w) for 20 weeks to induce risk factors and disease endpoints. In separate groups, cocoa butter was replaced by refined (REF) or virgin (VIR) pumpkin seed oil (comparable in fatty acid composition, but different in phytochemical content).Both oils improved dyslipidaemia, with decreased (V)LDL-cholesterol and triglyceride levels in comparison with CON, and additional cholesterol-lowering effects of VIR over REF. While REF did not affect plasma inflammatory markers, VIR reduced circulating serum amyloid A and soluble vascular adhesion molecule-1. NAFLD and atherosclerosis development was modestly reduced in REF, and VIR strongly decreased liver steatosis and inflammation as well as atherosclerotic lesion area and severity.Overall, we show that an isocaloric switch from a diet rich in saturated fat to a diet rich in unsaturated fat can attenuate NAFLD and atherosclerosis development. Phytochemical-rich virgin pumpkin seed oil exerts additional anti-inflammatory effects resulting in more pronounced health effects

Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice

The prevalence of dyslipidemia and obesity resulting from excess energy intake and physical inactivity is increasing. The liver plays a pivotal role in systemic lipid homeostasis. Effective, natural dietary interventions that lower plasma lipids and promote liver health are needed. Our goal was to determine the effect of dietary sphingolipids on plasma lipids and liver steatosis. APOE*3Leiden mice were fed a Western-type diet supplemented with different sphingolipids. Body cholesterol and triacylglycerol metabolism as well as hepatic lipid concentrations and lipid-related gene expression were determined. Dietary sphingolipids dose-dependently lowered both plasma cholesterol and triacylglycerol in APOE*3Leiden mice; 1% phytosphingosine (PS) reduced plasma cholesterol and triacylglycerol by 57% and 58%, respectively. PS decreased the absorption of dietary cholesterol and free fatty acids by 50% and 40%, respectively, whereas intestinal triacylglycerol lipolysis was not affected. PS increased hepatic VLDL-triacylglycerol production by 20%, whereas plasma lipolysis was not affected. PS increased the hepatic uptake of VLDL remnants by 60%. Hepatic messenger RNA concentrations indicated enhanced hepatic lipid synthesis and VLDL and LDL uptake. The net result of these changes was a strong decrease in plasma cholesterol and triacylglycerol. The livers of 1% PS-fed mice were less pale, 22% lighter, and contained 61% less cholesteryl ester and 56% less triacylglycerol than livers of control mice. Furthermore, markers of liver inflammation (serum amyloid A) and liver damage (alanine aminotransferase) decreased by 74% and 79%, respectively, in PS-fed mice. Sphingolipids lower plasma cholesterol and triacylglycerol and protect the liver from fat- and cholesterol-induced steatosi

Broadening the spectrum of loss-of-function variants in NPR-C-related extreme tall stature

CONTEXT: Natriuretic peptide receptor-C (NPR-C, encoded by NPR3) belongs to a family of cell membrane–integral proteins implicated in various physiological processes, including longitudinal bone growth. NPR-C acts as a clearance receptor of natriuretic peptides, including C-type natriuretic peptide (CNP), that stimulate the cGMP-forming guanylyl cyclase-coupled receptors NPR-A and NPR-B. Pathogenic variants in CNP, NPR2, and NPR3 may cause a tall stature phenotype associated with macrodactyly of the halluces and epiphyseal dysplasia. OBJECTIVE: Here we report on a boy with 2 novel biallelic inactivating variants of NPR3. METHODS: History and clinical characteristics were collected. Biochemical indices of natriuretic peptide clearance and in vitro cellular localization of NPR-C were studied to investigate causality of the identified variants. RESULTS: We identified 2 novel compound heterozygous NPR3 variants c.943G>A p.(Ala315Thr) and c.1294A>T p.(Ile432Phe) in a boy with tall stature and macrodactyly of the halluces. In silico analysis indicated decreased stability of NPR-C, presumably resulting in increased degradation or trafficking defects. Compared to other patients with NPR-C loss-of-function, the phenotype seemed to be milder: pseudo-epiphyses in hands and feet were absent, biochemical features were less severe, and there was some co-localization of p.(Ile432Phe) NPR-C with the cell membrane, as opposed to complete cytoplasmic retention. CONCLUSION: With this report on a boy with tall stature and macrodactyly of the halluces we further broaden the genotypic and phenotypic spectrum of NPR-C-related tall stature

Combined Treatment with L-Carnitine and Nicotinamide Riboside Improves Hepatic Metabolism and Attenuates Obesity and Liver Steatosis

Obesity characterized by adiposity and ectopic fat accumulation is associated with the development of non-alcoholic fatty liver disease (NAFLD). Treatments that stimulate lipid utilization may prevent the development of obesity and comorbidities. This study evaluated the potential anti-obesogenic hepatoprotective effects of combined treatment with L-carnitine and nicotinamide riboside, i.e., components that can enhance fatty acid transfer across the inner mitochondrial membrane and increase nicotinamide adenine nucleotide (NAD+) levels, which are necessary for β-oxidation and the TCA cycle, respectively. Ldlr -/-.Leiden mice were treated with high-fat diet (HFD) supplemented with L-carnitine (LC; 0.4% w/w), nicotinamide riboside (NR; 0.3% w/w) or both (COMBI) for 21 weeks. L-carnitine plasma levels were reduced by HFD and normalized by LC. NR supplementation raised its plasma metabolite levels demonstrating effective delivery. Although food intake and ambulatory activity were comparable in all groups, COMBI treatment significantly attenuated HFD-induced body weight gain, fat mass gain (-17%) and hepatic steatosis (-22%). Also, NR and COMBI reduced hepatic 4-hydroxynonenal adducts. Upstream-regulator gene analysis demonstrated that COMBI reversed detrimental effects of HFD on liver metabolism pathways and associated regulators, e.g., ACOX, SCAP, SREBF, PPARGC1B, and INSR. Combination treatment with LC and NR exerts protective effects on metabolic pathways and constitutes a new approach to attenuate HFD-induced obesity and NAFLD.</p