In vitro Raman imaging of human macrophages: Impact of eicosapentaenoic acid on the hydrolysis of cholesterol esters in lipid droplets.

Atherosclerosis - a cardiovascular disease and the primary cause of morbidity and mortality in industrialized countries - is linked to the existence of atherosclerotic plaques characterized by cholesterol-laden macrophages called foam cells. In these cells, cholesterol esters associated with triglycerides form lipid droplets (LD). The only way to remove this excess cholesterol is to promote free cholesterol efflux from macrophages to specific acceptors. It has been shown recently that eicosapentaenoic acid (EPA) reduces efflux on cholesterol-loaded THP-1 macrophages in vitro due to decreased cholesterol esters hydrolysis. These in vitro observations could reflect EPA's difficulty in facilitating in vivo the antiatherogenic process of cholesterol efflux within advanced atherosclerotic plaques. This work aims to study in vitro the impact of EPA on cholesterol esters hydrolysis in the LD of human THP-1 macrophages using vibrational Raman microspectroscopy. For this, we used deuterated EPA and recorded spectral images at the cell scale after different hydrolysis times. RESULTS: showed that EPA is involved in forming triglycerides and phospholipids of LD. Hydrolysis kinetics slowed down after 24 h, triglycerides increased, and the intensity of the characteristic bands linked to deuteration decreased. The size of LD without hydrolysis (H0) is higher than that after 24 h (H1) or 48 h (H2) of hydrolysis. The size decrease is sharper when going from H0 to H1 than from H1 to H2. Principal component analysis illustrated data' projection according to the cellular compartment, the hydrolysis time, and the supplementation of the medium.

Homocysteine Metabolism Pathway Is Involved in the Control of Glucose Homeostasis: A Cystathionine Beta Synthase Deficiency Study in Mouse.

Cystathionine beta synthase (CBS) catalyzes the first step of the transsulfuration pathway from homocysteine to cystathionine, and its deficiency leads to hyperhomocysteinemia (HHcy) in humans and rodents. To date, scarce information is available about the HHcy effect on insulin secretion, and the link between CBS activity and the setting of type 2 diabetes is still unknown. We aimed to decipher the consequences of an inborn defect in CBS on glucose homeostasis in mice. We used a mouse model heterozygous for CBS (CBS+/-) that presented a mild HHcy. Other groups were supplemented with methionine in drinking water to increase the mild to intermediate HHcy, and were submitted to a high-fat diet (HFD). We measured the food intake, body weight gain, body composition, glucose homeostasis, plasma homocysteine level, and CBS activity. We evidenced a defect in the stimulated insulin secretion in CBS+/- mice with mild and intermediate HHcy, while mice with intermediate HHcy under HFD presented an improvement in insulin sensitivity that compensated for the decreased insulin secretion and permitted them to maintain a glucose tolerance similar to the CBS+/+ mice. Islets isolated from CBS+/- mice maintained their ability to respond to the elevated glucose levels, and we showed that a lower parasympathetic tone could, at least in part, be responsible for the insulin secretion defect. Our results emphasize the important role of Hcy metabolic enzymes in insulin secretion and overall glucose homeostasis.

Eicosapentaenoic acid membrane incorporation stimulates ABCA1-mediated cholesterol efflux from human THP-1 macrophages.

A high intake in polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) (C20:5 n-3), is cardioprotective. Dietary PUFAs incorporate into membrane phospholipids, which may modify the function of membrane proteins. We investigated the consequences of the membrane incorporation of several PUFAs on the key antiatherogenic ABCA1-mediated cholesterol efflux pathway. Human THP-1 macrophages were incubated with EPA, arachidonic acid (AA) (C20:4 n-6) or docosahexaenoic acid (DHA) (C22:6 n-3) for a long time to mimic a chronic exposure. EPA 70 µM, but not AA 50 µM or DHA 15 µM, increased ABCA1-mediated cholesterol efflux to apolipoprotein (apo) AI by 28% without altering aqueous diffusion. No variation in ABCA1 expression or localization was observed after EPA treatment. EPA incorporation did not affect the phenotype of THP-1 macrophages. The membrane phospholipids composition of EPA cells displayed higher levels of both EPA and its elongation product docosapentaenoic acid, which was associated with drastic lower levels of AA. Treatment by EPA increased the ATPase activity of the transporter, likely through a PKA-dependent mechanism. Eicosanoids were not involved in the stimulated ABCA1-mediated cholesterol efflux from EPA-enriched macrophages. In addition, EPA supplementation increased the apo AI binding capacity from macrophages by 38%. Moreover, the increased apo AI binding in EPA-enriched macrophages can be competed. In conclusion, EPA membrane incorporation increased ABCA1 functionality in cholesterol-normal human THP-1 macrophages, likely through a combination of different mechanisms. This beneficial in vitro effect may partly contribute to the cardioprotective effect of a diet enriched with EPA highlighted by several recent clinical trials.

Aldosterone-Related Myocardial Extracellular Matrix Expansion in Hypertension in Humans: A Proof-of-Concept Study by Cardiac Magnetic Resonance.

OBJECTIVES: This study sought to assess the respective effects of aldosterone and blood pressure (BP) levels on myocardial fibrosis in humans. BACKGROUND: Experimentally, aldosterone promotes left ventricular (LV) hypertrophy, and interstitial myocardial fibrosis in the presence of high salt intake. METHODS: The study included 20 patients with primary aldosteronism (PA) (high aldosterone and high BP), 20 patients with essential hypertension (HTN) (average aldosterone and high BP), 20 patients with secondary aldosteronism due to Bartter/Gitelman (BG) syndrome (high aldosterone and normal BP), and 20 healthy subjects (HS) (normal aldosterone and normal BP). Participants in each group were of similar age and sex distributions, and asymptomatic. Cardiac magnetic resonance including cine and T1 mapping was performed blind to the study group to quantify global LV mass index, as well as intracellular mass index and extracellular mass index considered as a measure of myocardial fibrosis in vivo. RESULTS: Median plasma aldosterone concentration was as follows: PA = 709 pmol/l (interquartile range [IQR]: 430 to 918 pmol/l); HTN = 197 pmol/l (IQR: 121 to 345 pmol/l); BG = 297 pmol/l (IQR: 180 to 428 pmol/l); and HS = 105 pmol/l (IQR: 85 to 227 pmol/l). Systolic BP was as follows: PA = 147 ± 15 mm Hg; HTN = 133 ± 19 mm Hg; BG = 116 ± 9 mm Hg; and HS = 117 ± 12 mm Hg. LV end-diastolic volume showed underloading in BG and overloading in patients with PA (63 ± 13 ml/m2 vs. 82 ± 15 ml/m2; p < 0.0001). Intracellular mass index increased with BP across groups (BG: 36 [IQR: 29 to 41]; HS: 40 [IQR: 36 to 46]; HTN: 51 [IQR: 42 to 54]; PA: 50 [IQR: 46 to 67]; p < 0.0001). Extracellular mass index was similar in BG, HS, and HTN (16 [IQR: 12 to 20]; 15 [IQR: 11 to 18]; and 14 [IQR: 12 to 17], respectively) but 30% higher in PA (21 [IQR: 18 to 29]; p < 0.0001) remaining significant after adjustment for mean BP. CONCLUSIONS: Only primary pathological aldosterone excess combined with high BP increased both extracellular myocardial matrix and intracellular mass. Secondary aldosterone excess with normal BP did not affect extracellular myocardial matrix. (Study of Myocardial Interstitial Fibrosis in Hyperaldosteronism; NCT02938910).

Molecular Rescue of Dyrk1A Overexpression Alterations in Mice with Fontup® Dietary Supplement: Role of Green Tea Catechins.

Epigallocatechin gallate (EGCG) is an inhibitor of DYRK1A, a serine/threonine kinase considered to be a major contributor of cognitive dysfunctions in Down syndrome (DS). Two clinical trials in adult patients with DS have shown the safety and efficacy to improve cognitive phenotypes using commercial green tea extract containing EGCG (45% content). In the present study, we performed a preclinical study using FontUp®, a new nutritional supplement with a chocolate taste specifically formulated for the nutritional needs of patients with DS and enriched with a standardized amount of EGCG in young mice overexpressing Dyrk1A (TgBACDyrk1A). This preparation is differential with previous one used, because its green tea extract has been purified to up 94% EGCG of total catechins. We analyzed the in vitro effect of green tea catechins not only for EGCG, but for others residually contained in FontUp®, on DYRK1A kinase activity. Like EGCG, epicatechin gallate was a noncompetitive inhibitor against ATP, molecular docking computations confirming these results. Oral FontUp® normalized brain and plasma biomarkers deregulated in TgBACDyrk1A, without negative effect on liver and cardiac functions. We compared the bioavailability of EGCG in plasma and brain of mice and have demonstrated that EGCG had well crossed the blood-brain barrier.

Contrasting effects of membrane enrichment with polyunsaturated fatty acids on phospholipid composition and cholesterol efflux from cholesterol-loaded J774 mouse or primary human macrophages.

A high consumption of polyunsaturated fatty acids (PUFAs), particularly n-3 PUFAs, is atheroprotective. PUFAs incorporation into membrane phospholipids alters the functionality of membrane proteins. We studied the consequences of the in vitro supplementation of several PUFAs on the FA profiles and on ABCA1-dependent cholesterol efflux capacities from cholesterol-loaded macrophages. Arachidonic acid (AA, C20:4 n-6) and, to a lesser extent, eicosapentaenoic acid (EPA, C20:5 n-3), dose-dependently impaired cholesterol efflux from cholesterol-loaded J774 mouse macrophages without alterations in ABCA1 expression, whereas docosahexaenoic acid (DHA, C22:6 n-3) had no impact. AA cells exhibited higher proportions of arachidonic acid and adrenic acid (C22:4 n-6), its elongation product. EPA cells exhibited slightly higher proportions of EPA associated with much higher proportions of docosapentaenoic acid (C22:5 n-3), its elongation product and with lower proportions of AA. Conversely, both EPA and DHA and, to a lesser extent, AA decreased cholesterol efflux from cholesterol-loaded primary human macrophages (HMDM). The differences observed in FA profiles after PUFA supplementations were different from those observed for the J774 cells. In conclusion, we are the first to report that AA and EPA, but not DHA, have deleterious effects on the cardioprotective ABCA1 cholesterol efflux pathway from J774 foam cells. Moreover, the membrane incorporation of PUFAs does not have the same impact on cholesterol efflux from murine (J774) or human (HMDM) cholesterol-loaded macrophages. This finding emphasizes the key role of the cellular model in cholesterol efflux studies and may partly explain the heterogeneous literature data on the impact of PUFAs on cholesterol efflux.

Investigation of lipid modifications in J774 macrophages by vibrational spectroscopies after eicosapentaenoic acid membrane incorporation in unloaded and cholesterol-loaded cells.

Atherosclerosis is an inflammatory disease of the arterial wall caused by the formation of an atheroma plaque in the vessel wall. The uptake of modified LDL lipoproteins by sub-endothelial macrophages induces the latter's transformation into foam cells, which is the key step of atheroma plaque formation. The modifications of neutral lipids caused by foam cells formation are marked by the appearance of lipid droplets. Polyunsaturated fatty acids (PUFAs) incorporation into membrane phospholipids (PL) modifies their composition, which may influence membrane protein functions. The incorporation of eicosapentaenoic acid (EPA) reduces the anti-atherogenic ABCA1 (ATP Binding Cassette transporter A1) pathway and induces PLs modifications. In order to study lipids directly in the cell environment, a comparative study is conducted by vibrational spectroscopies on murine macrophages J774, loaded or not with cholesterol, which were enriched or not with eicosapentaenoic acid (EPA). The study enabled to identify changes in the spectral signature after cells enrichment with fatty acid (FA) relying only on chemometric analysis without deuterium labelling. Results highlighted spectral changes in the regions attributed to lipids associated to triglycerides, phospholipids and cholesterol in both Raman and IR.

Homocysteine-lowering gene therapy rescues signaling pathways in brain of mice with intermediate hyperhomocysteinemia.

Hyperhomocysteinemia due to cystathionine beta synthase (CBS) deficiency is associated with diverse cognitive dysfunction. Considering the role of the serine/threonine kinase DYRK1A, not only in developmental defects with life-long structural and functional consequences, but also in multiple neurodegenerative diseases, its protein expression and kinase activity has been analyzed in brain of heterozygous CBS deficient mice and found to be increased. We previously demonstrated that specific liver treatment with an adenovirus expressing Dyrk1A normalizes hepatic DYRK1A level and decreases hyperhomocysteinemia in mice with moderate to intermediate hyperhomocysteinemia. We here use a hepatocyte-specific recombinant adeno-associated viral (AAV) serotype 8-mediated DYRK1A gene therapy (AAV2/8-DYRK1A) to analyze the effect of hepatic Dyrk1A gene transfer on some altered molecular mechanisms in brain of mice with intermediate hyperhomocysteinemia. Our selective hepatic treatment alleviates altered DYRK1A protein level and signaling pathways in brain of mice, the MAPK/ERK and PI3K/Akt pathways initiated by receptor tyrosine kinase, the BDNF dependent TrkB pathway, and NFkB pathway. These results demonstrate the positive effect of AAV2/8-DYRK1A gene transfer on neuropathological and inflammatory processes in brain of mice with intermediate hyperhomocysteinemia.

Role of myocardial collagen degradation and fibrosis in right ventricle dysfunction in transposition of the great arteries after atrial switch.

BACKGROUND: Heart failure is a serious event in patients with transposition of the great arteries (D-TGA) after atrial redirection surgery. We aimed to determine the association between myocardial fibrosis and systolic and diastolic systemic right ventricle (sRV) dysfunction. METHODS: Diastolic and systolic function of sRV was prospectively assessed using echocardiography and cardiac magnetic resonance imaging (CMR) in 48 patients with atrially switched D-TGA and 26 healthy subjects. Diastolic function of the subaortic ventricle was assessed by echocardiography Doppler and DTI. In CMR, ejection fraction of sRV and wall stress defined as the product of the systolic blood pressure and volume/mass ratio were assessed. Fibrosis extent within sRV myocardium was evaluated using gadolinium-enhanced magnetic resonance and serum collagen turnover biomarkers. RESULTS: Late gadolinium enhancement (LGE) was found in 35% of D-TGA patients, and the collagen degradation biomarker pro-MMP1:TIMP1 ratio was significantly increased in D-TGA patients compared to healthy subjects (1.0 × 10-2vs. 2.5 × 10-2, p = 0.04). Increase in sRV wall stress was significantly associated with LGE (p = 0.01) and pro-MMP1:TIMP1 ratio (r = 0.77, p < 0.01). After adjustment for age, sex, BMI, blood pressure and cardiac treatment, pro-MMP1:TIMP1 ratio was the strongest determinant of sRVEF (R2 = 0.85, p < 0.01). Pro-MMP1:TIMP1 ratio was also significantly correlated with the early diastolic filling parameter E/E' (r = 0.53, p = 0.02), but this was not anymore the case after adjustment. CONCLUSIONS: Diastolic and systolic sRV dysfunction is related to myocardial collagen degradation and fibrosis. Research in medical therapies that reduce systemic sRV afterload and limit collagen degradation is warranted in this setting.

Eicosapentaenoic acid membrane incorporation impairs cholesterol efflux from cholesterol-loaded human macrophages by reducing the cholesteryl ester mobilization from lipid droplets.

A diet containing a high n-3/n-6 polyunsaturated fatty acids (PUFA) ratio has cardioprotective properties. PUFAs incorporation into membranes influences the function of membrane proteins. We investigated the impact of the membrane incorporation of PUFAs, especially eicosapentaenoic acid (EPA) (C20:5 n-3), on the anti-atherogenic cholesterol efflux pathways. We used cholesteryl esters (CE)-loaded human monocyte-derived macrophages (HMDM) to mimic foam cells exposed to the FAs for a long period of time to ensure their incorporation into cellular membranes. Phospholipid fraction of EPA cells exhibited high levels of EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which was associated with a decreased level of arachidonic acid (AA) (C20:4 n-6). EPA 70µM reduced ABCA1-mediated cholesterol efflux to apolipoprotein (apo) AI by 30% without any alteration in ABCA1 expression. The other tested PUFAs, DPA, docosahexaenoic acid (DHA) (C22:6 n-3), and AA, were also able to reduce ABCA1 functionality while the monounsaturated oleic FA slightly decreased efflux and the saturated palmitic FA had no impact. Moreover, EPA also reduced cholesterol efflux to HDL mediated by the Cla-1 and ABCG1 pathways. EPA incorporation did not hinder efflux in free cholesterol-loaded HMDM and did not promote esterification of cholesterol. Conversely, EPA reduced the neutral hydrolysis of cytoplasmic CE by 24%. The reduced CE hydrolysis was likely attributed to the increase in cellular TG contents and/or the decrease in apo E secretion after EPA treatment. In conclusion, EPA membrane incorporation reduces cholesterol efflux in human foam cells by reducing the cholesteryl ester mobilization from lipid droplets.

Conjugation of squalene to gemcitabine as unique approach exploiting endogenous lipoproteins for drug delivery.

Once introduced in the organism, the interaction of nanoparticles with various biomolecules strongly impacts their fate. Here we show that nanoparticles made of the squalene derivative of gemcitabine (SQGem) interact with lipoproteins (LPs), indirectly enabling the targeting of cancer cells with high LP receptors expression. In vitro and in vivo experiments reveal preeminent affinity of the squalene-gemcitabine bioconjugates towards LP particles with the highest cholesterol content and in silico simulations further display their incorporation into the hydrophobic core of LPs. To the best of our knowledge, the use of squalene to induce drug insertion into LPs for indirect cancer cell targeting is a novel concept in drug delivery. Interestingly, not only SQGem but also other squalene derivatives interact similarly with lipoproteins while such interaction is not observed with liposomes. The conjugation to squalene represents a versatile platform that would enable efficient drug delivery by simply exploiting endogenous lipoproteins.

Effect of lyophilized prune extract on hyperhomocysteinemia in mice.

Altered homocysteine metabolism defined as hyperhomocysteinemia is implicated as pathogenic factor in several cardiovascular diseases and atherosclerosis. The purpose of this study was to investigate the efficacy of prune extract, a good source of phenolic antioxidants, on lowering plasma homocysteine level in male hyperhomocysteinemic mice from average weight of 28 g. The administration of lyophilized prune extract was carried out by intraperitoneal injection one day preceding and one hour before sacrifice of mice. Prune extract decreased significantly plasma homocysteine level, correlated with an increased activity of S-adenosylhomocysteine (SAH) hydrolase and NAD(P)H: quinone oxydoreductase-1 activities. Our results suggest a beneficial effect of prune extract on hyperhomocysteinemia with reduction of homocysteine level by its conversion on to SAH by S-adenosylhomocysteine hydrolase, which is activated by NAD+, a by-product of NAD(P)H: quinone oxydo reductase-1.

Effect of cadmium administration in hyperhomocysteinemic mice due to cystathionine beta synthase deficiency.

Homocysteine, a sulfur-containing amino acid formed during the metabolism of methionine, is commonly slightly elevated in the plasma of the general population. Additionally, we previously found that cystathionine beta synthase-deficient mice, a murine model of hyperhomocysteinemia, exhibit altered activities of xenobiotic metabolizing enzymes (XME), which dispose of foreign chemicals, in the liver. Thus, hyperhomocysteinemia may result in susceptibility to xenobiotics like cadmium, a heavy-metal toxicant found in drinking water, atmospheric air, and food. Consequently, we exposed hyperhomocysteinemic mice to cadmium via their drinking water for one month to analyze the combined effects of hyperhomocysteinemia and cadmium exposure in liver. No difference in plasma homocysteine level was found after cadmium administration in control and hyperhomocysteinemic mice, but the glutathione level was significantly lower in exposed hyperhomocysteinemic mice compared to control mice, reflecting oxidative stress. We therefore analyzed the effect of Cd administration on hepatic XMEs known to be dysregulated in hyperhomocysteinemic mice: paraoxonase 1, a phase I XME, and NAD(P)H: quinone oxidoreductase, a phase II XME. Cadmium exposure negatively affected activity of paraoxonase 1, a calcium-dependent enzyme. Thus, we analyzed another calcium-dependent enzyme known to be dysregulated in liver of hyperhomocysteinemic mice, calpain, which was also significantly lower after cadmium administration. A comparison of the calculated affinities of cadmium docking versus calcium redocking suggested that cadmium ions may inhibit enzymatic activities by preventing the binding of calcium ions. Moreover, the increased NAD(P)H: quinone oxidoreductase activity observed after cadmium administration could indicate the presence of protective mechanisms in liver of mice. In conclusion, although cadmium administration had no effect on plasma homocysteine level, its effects on plasma glutathionine level suggest a susceptibility to cadmium in the condition of hyperhomocysteinemia, which could be countered by an increased NAD(P)H: quinone oxidoreductase activity.

Impact of Dyrk1A level on alcohol metabolism.

Alcoholic liver diseases arise from complex phenotypes involving many genetic factors. It is quite common to find hyperhomocysteinemia in chronic alcoholic liver diseases, mainly due to deregulation of hepatic homocysteine metabolism. Dyrk1A, involved in homocysteine metabolism at different crossroads, is decreased in liver of hyperhomocysteinemic mice. Here, we hypothesized that Dyrk1A contributes to alcohol-induced hepatic impairment in mice. Control, hyperhomocysteinemic and mice overexpressing Dyrk1A were fed using a Lieber-DeCarli liquid diet with or without ethanol (5% v/v ethanol) for one month, and liver histological examination and liver biochemical function tests were performed. Plasma alanine aminotransferase and homocysteine levels were significantly decreased in mice overexpressing Dyrk1A compared to control mice with or without alcohol administration. On the contrary, the mean plasma alanine aminotransferase and homocysteine levels were significantly higher in hyperhomocysteinemic mice than that of control mice after alcohol administration. Paraoxonase 1 and CYP2E1, two phase I xenobiotic metabolizing enzymes, were found increased in the three groups of mice after alcohol administration. However, NQO1, a phase II enzyme, was only found increased in hyperhomocysteinemic mice after alcohol exposure, suggesting a greater effect of alcohol in liver of hyperhomocysteinemic mice. We observed positive correlations between hepatic alcohol dehydrogenase activity, Dyrk1A and ADH4 protein levels. Importantly, a deleterious effect of alcohol consumption on hepatic Dyrk1A protein level was found. Our study reveals on the one hand a role of Dyrk1A in ethanol metabolism and on the other hand a deleterious effect of alcohol administration on hepatic Dyrk1A level.

Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages.

A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70µM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages.

Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia.

Hyperhomocysteinemia results from hepatic metabolism dysfunction and is characterized by a high plasma homocysteine level, which is also an independent risk factor for cardiovascular disease. Elevated levels of homocysteine in plasma lead to hepatic lesions and abnormal lipid metabolism. Therefore, lowering homocysteine levels might offer therapeutic benefits. Recently, we were able to lower plasma homocysteine levels in mice with moderate hyperhomocysteinemia using an adenoviral construct designed to restrict the expression of DYRK1A, a serine/threonine kinase involved in methionine metabolism (and therefore homocysteine production), to hepatocytes. Here, we aimed to extend our previous findings by analyzing the effect of hepatocyte-specific Dyrk1a gene transfer on intermediate hyperhomocysteinemia and its associated hepatic toxicity and liver dysfunction. Commensurate with decreased plasma homocysteine and alanine aminotransferase levels, targeted hepatic expression of DYRK1A in mice with intermediate hyperhomocysteinemia resulted in elevated plasma paraoxonase-1 and lecithin:cholesterol acyltransferase activities and apolipoprotein A-I levels. It also rescued hepatic apolipoprotein E, J, and D levels. Further, Akt/GSK3/cyclin D1 signaling pathways in the liver of treated mice were altered, which may help prevent homocysteine-induced cell cycle dysfunction. DYRK1A gene therapy could be useful in the treatment of hyperhomocysteinemia in populations, such as end-stage renal disease patients, who are unresponsive to B-complex vitamin therapy.

Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise.

Hyperhomocysteinemia resulting from cystathionine beta synthase (CBS) deficiency can produce cognitive dysfunction. We recently found that CBS-deficient mice exhibit increased expression of the serine/threonine kinase dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) in the brain. When dysregulated, DYRK1A contributes to the neurodegeneration, neuronal death, and loss of function observed in neurodegenerative diseases. However, brain plasticity can be improved by interventions like enriched environment combined with voluntary exercise (EE/VE). The present study sought to assess the effects of EE/VE on molecular mechanisms linked to DYRK1A overexpression in the brain of CBS-deficient mice. EE/VE was applied to 3-month-old female CBS-deficient mice for 1 month. Without intervention, CBS-deficient mice exhibited increased DYRK1A and decreased brain-derived neurotrophic factor (BDNF) levels in the cortex and hippocampus. However, EE/VE rescued these altered DYRK1A and BDNF levels in the hippocampus of CBS-deficient mice. We conclude that exercise combined with enriched environment can restore the altered molecular mechanisms in the brain of CBS-deficient mice.

High-protein-low-carbohydrate diet: deleterious metabolic and cardiovascular effects depend on age.

High-protein-low-carbohydrate (HP-LC) diets have become widespread. Yet their deleterious consequences, especially on glucose metabolism and arteries, have already been underlined. Our previous study (2) has already shown glucose intolerance with major arterial dysfunction in very old mice subjected to an HP-LC diet. The hypothesis of this work was that this diet had an age-dependent deleterious metabolic and cardiovascular outcome. Two groups of mice, young and adult (3 and 6 mo old), were subjected for 12 wk to a standard or to an HP-LC diet. Glucose and lipid metabolism was studied. The cardiovascular system was explored from the functional stage with Doppler-echography to the molecular stage (arterial reactivity, mRNA, immunohistochemistry). Young mice did not exhibit any significant metabolic modification, whereas adult mice presented marked glucose intolerance associated with an increase in resistin and triglyceride levels. These metabolic disturbances were responsible for cardiovascular damages only in adult mice, with decreased aortic distensibility and left ventricle dysfunction. These seemed to be the consequence of arterial dysfunctions. Mesenteric arteries were the worst affected with a major oxidative stress, whereas aorta function seemed to be maintained with an appreciable role of cyclooxygenase-2 to preserve endothelial function. This study highlights for the first time the age-dependent deleterious effects of an HP-LC diet on metabolism, with glucose intolerance and lipid disorders and vascular (especially microvessels) and cardiac functions. This work shows that HP-LC lead to equivalent cardiovascular alterations, as observed in very old age, and underlines the danger of such diet.

Dual effects of resveratrol on arterial damage induced by insulin resistance in aged mice.

Aging leads to increased insulin resistance and arterial dysfunction, with oxidative stress playing an important role. This study explored the metabolic and arterial effects of a chronic treatment with resveratrol, an antioxidant polyphenol compound that has been shown to restore insulin sensitivity and decrease oxidative stress, in old mice with or without a high-protein diet renutrition care. High-protein diet tended to increase insulin resistance and atheromatous risk. Resveratrol improved insulin sensitivity in old mice fed standard diet by decreasing homeostasis model of assessment-insulin resistance and resistin levels. However, resveratrol did not improve insulin resistance status in old mice receiving the high-protein diet. In contrast, resveratrol exhibited deleterious effects by increasing inflammation state and superoxide production and diminishing aortic distensibility. In conclusion, we demonstrate that resveratrol has beneficial or deleterious effects on insulin sensitivity and arterial function, depending on nutritional status in our models.

One-carbon cycle alterations induced by Dyrk1a dosage.

Hyperhomocysteinemia due to cystathionine beta synthase deficiency confers diverse clinical manifestations. It is characterized by elevated plasma homocysteine levels, a common amino acid metabolized by remethylation to methionine or transsulfuration to cysteine. We recently found a relationship between hepatic Dyrk1A protein expression, a serine/threonine kinase involved in signal transduction in biological processes, hepatic S-adenosylhomocysteine activity, and plasma homocysteine levels. We aimed to study whether there is also a relationship between Dyrk1a and cystathionine beta synthase activity. We used different murine models carrying altered gene coy numbers for Dyrk1a, and found a decreased cystathionine beta synthase activity in the liver of mice under-expressing Dyrk1a, and an increased in liver of mice over-expressing Dyrk1a. For each model, a positive correlation was found between cystathionine beta synthase activity and Dyrk1a protein expression in the liver of mice, which was confirmed in a non-modified genetic context. The positive correlation found between liver Dyrk1a protein expression and CBS activity in modified and non-modified genetic context strengthens the role of this kinase in one carbon metabolism.