Sequence Determinants for Regulated Degradation of Yeast 3-Hydroxy-3-Methylglutaryl-CoA Reductase, an Integral Endoplasmic Reticulum Membrane Protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27–54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.

Study of the 3-Hydroxy Eicosanoyl-Coenzyme A Dehydratase and (E)-2,3 Enoyl-Coenzyme A Reductase Involved in Acyl-Coenzyme A Elongation in Etiolated Leek Seedlings1

(R,S)-[1-14C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-14C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-14C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-14C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-14C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-14C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-14C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg−1 h−1, which is the highest ever reported for a plant system.

3-Methylcrotonyl-Coenzyme A Carboxylase Is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants1

3-Methylcrotonyl-coenzyme A carboxylase (MCCase) is a mitochondrial biotin-containing enzyme whose metabolic function is not well understood in plants. In soybean (Glycine max) seedlings the organ-specific and developmentally induced changes in MCCase expression are regulated by mechanisms that control the accumulation of MCCase mRNA and the activity of the enzyme. During soybean cotyledon development, when seed-storage proteins are degraded, leucine (Leu) accumulation peaks transiently at 8 d after planting. The coincidence between peak MCCase expression and the decline in Leu content provides correlative evidence that MCCase is involved in the mitochondrial catabolism of Leu. Direct evidence for this conclusion was obtained from radiotracer metabolic studies using extracts from isolated mitochondria. These experiments traced the metabolic fate of [U-14C]Leu and NaH14CO3, the latter of which was incorporated into methylglutaconyl-coenzyme A (CoA) via MCCase. These studies directly demonstrate that plant mitochondria can catabolize Leu via the following scheme: Leu → α-ketoisocaproate → isovaleryl-CoA → 3-methylcrotonyl-CoA → 3-methylglutaconyl-CoA → 3-hydroxy-3-methylglutaryl-CoA → acetoacetate + acetyl-CoA. These findings demonstrate for the first time, to our knowledge, that the enzymes responsible for Leu catabolism are present in plant mitochondria. We conclude that a primary metabolic role of MCCase in plants is the catabolism of Leu.

(+)-Abscisic Acid Metabolism, 3-Ketoacyl-Coenzyme A Synthase Gene Expression, and Very-Long-Chain Monounsaturated Fatty Acid Biosynthesis in Brassica napus Embryos1

Microspore-derived embryos of Brassica napus cv Reston were used to examine the effects of exogenous (+)-abscisic acid (ABA) and related compounds on the accumulation of very-long-chain monounsaturated fatty acids (VLCMFAs), VLCMFA elongase complex activity, and induction of the 3-ketoacyl-coenzyme A synthase (KCS) gene encoding the condensing enzyme of the VLCMFA elongation system. Of the concentrations tested, (+)-ABA at 10 μm showed the strongest effect. Maximum activity of the elongase complex, observed 6 h after 10 μm (+)-ABA treatment, was 60% higher than that of the untreated embryos at 24 h. The transcript of the KCS gene was induced by 10 μm (+)-ABA within 1 h and further increased up to 6 h. The VLCMFAs eicosenoic acid (20:1) and erucoic acid (22:1) increased by 1.5- to 2-fold in embryos treated with (+)-ABA for 72 h. Also, (+)-8′-methylene ABA, which is metabolized more slowly than ABA, had a stronger ABA-like effect on the KCS gene transcription, elongase complex activity (28% higher), and level of VLCMFAs (25–30% higher) than ABA. After 24 h approximately 60% of the added (+)-[3H]ABA (10 μm) was metabolized, yielding labeled phaseic and dihydrophaseic acid. This study demonstrates that (+)-ABA promotes VLCMFA biosynthesis via increased expression of the KCS gene and that reducing ABA catabolism would increase VLCMFAs in microspore-derived embryos.

Effects of brominated flame retardants and brominated dioxins on steroidogenesis in H295R human adrenocortical carcinoma cell line

Brominated flame retardants (BFRs) and brominated dioxins are emerging persistent organic pollutants that are ubiquitous in the environment and can be accumulated by wildlife and humans. These chemicals can disturb endocrine function. Recent studies have demonstrated that one of the mechanisms of endocrine disruption by chemicals is modulation of steroidogenic gene expression or enzyme activities. In this study, an in vitro assay based on the H295R human adrenocortical carcinoma cell line, which possesses most key genes or enzymes involved in steroidogenesis, was used to examine the effects of five bromophenols, two polybrominated biphenyls (PBBs 77 and 169), 2,3,7,8-tetrabromodibenzo-p-dioxin, and 2,3,7,8-tetrabromodibenzofuran on the expression of 10 key steroidogenic genes. The H295R cells were exposed to various BFR concentrations for 48 h, and the expression of specific genescytochrome P450 (CYP11A, CYP11B2, CYP17, CYP19, and CYP21), 3 beta-hydroxysteroid dehydrogenase (3PHSD2), 17 beta-hydroxysteroid dehydrogenase (17 beta HSD1 and 17 beta HSD4), steroidogenic acute regulatory protein (StAR), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-was quantitatively measured using real-time polymerase chain reaction. Cell viability was not affected at the doses tested. Most of the genes were either up- or down-regulated, to some extent, by BFR exposure. Among the genes tested, 3PHSD2 was the most markedly up-regulated, with a range of magnitude from 1.6- to 20-fold. The results demonstrate that bromophenol, bromobiphenyls, and bromodibenzo-p-dioxin/furan are able to modulate steroidogenic gene expression, which may lead to endocrine disruption.

Molecular and cellular aspects of the SREBP pathway

The SREBP (sterol response element binding proteins) transcription factors are central to regulating de novo biosynthesis of cholesterol and fatty acids. The SREBPs are regulated by retention or escape from the ER to the Golgi where they are proteolytically cleaved into active forms. The SREBP cleavage activating protein (SCAP) and the INSIG proteins are essential in this regulatory process. The aim of this thesis is to further characterise the molecular and cellular aspects surrounding regulation of SREBP processing. SREBP and SCAP are known to interact via their carboxy-terminal regulatory domains (CTDs) but this interaction is poorly characterised. Significant steps were achieved in this thesis towards specific mapping of the interaction site. These included cloning and over expression and partial purification of tagged SREBP1 and SREBP2 CTDs and probing of a SCAP peptide array with the CTDs. Results from the SREBP2 probing were difficult to interpret due to insolubility issues with the protein, however, probing with SREBP1 revealed five potential binding sites which were detected reproducibly. Further research is necessary to overcome SREBP2 insolubility issues and to confirm the identified SREBP1 interaction site(s) on SCAP. INSIG1 has a central role in regulating SREBP processing and in regulating stability of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a rate limiting enzyme in cholesterol biosynthesis. There are two protein isoforms of human INSIG1 produced through the use of two in-frame alternative start sites. Bioinformatic analysis indicated that the presence of two in-frame start sites within the 5-prime region of INSIG1 mRNA is highly conserved and that production of two isoforms of INSIG1is likely a conserved event. Functional differences between these two isoforms were explored. No difference in either the regulation of SREBP processing or HMGCR degradation between the INSIG1 isoforms was observed and the functional significance of the two isoforms is as yet unclear. The final part of this thesis focused on enhancing the cytotoxicity of statins by targeted inhibition of SREBP processing by oxysterols. Statins have significant potential as anti-cancer agents as they inhibit the activity of HMGCR leading to a deficiency in mevalonate which is essential for cell survival. The levels of HMGCR fluctuate widely due to cholesterol feedback of SREBP processing. The relationship between sterol feedback and statin mediated cell death was investigated in depth in HeLa cells. Down regulation of SREBP processing by sterols significantly enhanced the efficacy of statin mediated cell death. Investigation of sterol feedback in additional cancer cell lines showed that sterol feedback was absent in cell lines A- 498, DU-145, MCF-7 and MeWo but was present in cell lines HT-29, HepG2 and KYSE-70. In the latter inhibition of SREBP processing using oxysterols significantly enhanced statin cytotoxicity. The results indicate that this approach is valid to enhance statin cytotoxicity in cancer cells, but may be limited by deregulation of SREBP processing and off target effects of statins, which were observed for some of the cancer cell lines screened.

Étude du polymorphisme rs3846662 de l’HMGCR dans le cerveau et le système périphérique

Dans cette thèse, l’impact du polymorphisme rs3846662 sur l’épissage alternatif de la 3-hydroxy-3-méthylglutaryl coenzyme A réductase (HMGCR) a été investigué in vivo, chez des patients atteints d’hypercholestérolémie familiale (HF) ou de maladie d’Alzheimer (MA). Le premier manuscrit adresse la problématique de la normalisation de la quantification relative des ARNm par PCR quantitative. Les découvertes présentées dans ce manuscrit nous ont permis de déterminer avec un haut niveau de confiance les gènes de référence à utiliser pour la quantification relative des niveaux d’ARNm de l’HMGCR dans des échantillons de sang (troisième manuscrit) et de tissus cérébraux post-mortem (quatrième manuscrit). Dans le deuxième manuscrit, nous démontrons grâce à l’emploi de trois cohortes de patients distinctes, soit la population canadienne française du Québec et les deux populations nord américaines « Alzheimer’s Disease Cooperative Study (ADCS) » et « Alzheimer’s Disease Neuroimaging Initiative (ADNI) », que le génotype AA au locus rs3846662 confère à ces porteurs une protection considérable contre la MA. Les femmes porteuses de ce génotype voient leur risque de MA diminuer de près de 50% et l’âge d’apparition de leurs premiers symptômes retarder de 3.6 ans. Les porteurs de l’allèle à risque APOE4 voient pour leur part leurs niveaux de plaques séniles et dégénérescences neurofibrillaires diminuer significativement en présence du génotype AA. Enfin, les individus atteints de déficit cognitif léger et porteurs à la fois de l’allèle APOE4 et du génotype protecteur AA voient leur risque de convertir vers la MA chuter de 76 à 27%. Dans le troisième manuscrit, nous constatons que les individus atteints d’HF et porteurs du génotype AA ont, contrairement au modèle établi chez les gens normaux, des niveaux plus élevés de cholestérol total et de LDL-C avant traitement comparativement aux porteurs de l’allèle G. Le fait que cette association n’est observée que chez les non porteurs de l’APOE4 et que les femmes porteuses du génotype AA présentent à la fois une augmentation des niveaux d’ARNm totaux et une résistance aux traitements par statines, nous indique que ce génotype influencerait non seulement l’épissage alternatif, mais également la transcription de l’HMGCR. Comme une revue exhaustive de la littérature ne révèle aucune étude abondant dans ce sens, nos résultats suggèrent l’existence de joueurs encore inconnus qui viennent influencer la relation entre le génotype AA, l’épissage alternatif et les niveaux d’ARNm de l’HMGCR. Dans le quatrième manuscrit, l’absence d’associations entre le génotype AA et les niveaux d’ARNm Δ13 ou de protéines HMGCR nous suggère fortement que ce polymorphisme est non fonctionnel dans le SNC affecté par la MA. Une étude approfondie de la littérature nous a permis d’étayer cette hypothèse puisque les niveaux de HNRNPA1, la ribonucléoprotéine influencée par l’allèle au locus rs3846662, sont considérablement réduits dans la MA et le vieillissement. Il est donc proposé que les effets protecteurs contre la MA associés au génotype AA soient le résultat d’une action indirecte sur le processus physiopathologique.

alpha-tocopherol affects androgen metabolism in male rat

The Alpha-Tocopherol Beta-Carotene Cancer Prevention Study has provided the first evidence implicating vitamin E in hormone synthesis. The effect of vitamin E on stereoidogenesis in testes and adrenal glands was assessed in growing rats using Affymetrix gene-chip technology. Dietary supplementation of rats with vitamin E (60 mg/kg feed) for a period of 429 days caused a significant repression of genes encoding for proteins centrally involved in the uptake (low-density lipoprotein receptor) and de novo synthesis (for example, 7-dehydrocholesterol reductase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, isopentenyl-diphosphate delta-isomerase, and farnesyl pyrophosphate synthetase) of cholesterol, the precursor of all steroid hormones. The present investigation indicates that dietary vitamin E may induce changes in stereoidogenesis by affecting cholesterol homeostasis.

A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in an hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main precursor of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homestasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

Effect of supplementation of two sources and two levels of copper on lipid metabolism in Nellore beef cattle

This study was conducted with 35 Nellore beef cattle to determine the effect of supplementation of two levels and two copper sources (organic and inorganic) on metabolism of lipids and cholesterol of meat. The five treatments used were: Control: without copper supplementation, 110 or 140: 10 or 40 mg/kg DM (as Cu sulfate), O10 or O40: 10 or 40 mg/kg DM (as Cu proteinate). In general, the copper supplementation changed the fatty acid profile of meat (p < 0.05), with a higher proportion of unsaturated fatty acids and reduction of saturated fatty acids. There was no effect of supplementation on blood cholesterol and triglycerides, however; in general, there was a reduction in cholesterol concentration in the L dorsi (p < 0.05) compared to the control treatment through the reduction (p < 0.05) in the concentrations of GSH and GSH/GSSG ratio. The Cu supplementation did have an influence on metabolism of lipids. The production of healthier meat is beneficial to public health by reducing the risk of cardiovascular disease. (C) 2012 Elsevier Ltd. All rights reserved.

Association between diet and polymorphisms in individuals with statin-controlled dyslipidaemia grouped according to oxidative stress biomarkers

The objective of this study was to investigate whether differences in diet and in single-nucleotide polymorphisms (SNPs) found in paraoxonase-1 (PON-1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), cholesterol ester transfer protein (CETP) and apolipoprotein E (APOE) genes, are associated with oxidative stress biomarkers and consequently with susceptibility of low-density cholesterol (LDL) to oxidation. A multivariate approach was applied to a group of 55 patients according to three biomarkers: plasma antioxidant activity, malondialdehyde and oxidized LDL (oxLDL) concentrations. Individuals classified in Cluster III showed the worst prognoses in terms of antioxidant activity and oxidative status. Individuals classified in Cluster I presented the lowest oxidative status, while individuals grouped in Cluster II presented the highest levels of antioxidant activity. No difference in nutrient intake was observed among the clusters. Significantly higher gamma- and delta-tocopherol concentrations were observed in those individuals with the highest levels of antioxidant activity. No single linear regression was statistically significant, suggesting that mutant alleles of the SNPs selected did not contribute to the differences observed in oxidative stress response. Although not statistically significant, the p value of the APO E coefficient for oxLDL response was 0.096, indicating that patients who carry the TT allele of the APO E gene tend to present lower plasma oxLDL concentrations. Therefore, the differences in oxidative stress levels observed in this study could not be attributed to diet or to the variant alleles of PON-1, CETP, HMGCR or APO E. This data supports the influence of gamma-tocopherol and delta-tocopherol on antioxidant activity, and highlights the need for further studies investigating APO E alleles and LDL oxidation.

Coordinated gene expression in adipose tissue and liver differs between cows with high or low NEFA concentrations in early lactation

Dairy cows with high and low plasma non-esterified fatty acid (NEFA) concentrations in early lactation were compared for plasma parameters and mRNA expression of genes in liver and subcutaneous adipose tissue. The study involved 16 multiparous dairy cows with a plasma NEFA concentration of >500 mumol/l [n = 8, high NEFA (HNEFA)] and <140 mumol/l [n = 8, low NEFA (LNEFA)] in the first week post-partum (pp). Blood samples, adipose and liver tissues were collected on day 1 (+1d) and at week 3 pp (+3wk). Blood plasma was assayed for concentrations of metabolites and hormones. Subcutaneous adipose and liver tissues were analysed for mRNA abundance by real-time qRT-PCR encoding parameters related to lipid metabolism. Results showed that mean daily milk yield and milk fat quantity were higher in HNEFA than in LNEFA cows (p < 0.01), and the NEB was more negative in HNEFA than in LNEFA in +3wk too (p < 0.05). HNEFA cows had slightly lower (p < 0.1) insulin concentrations than LNEFA cows across the study period, and the body condition score decreased more from +1d to +3wk in HNEFA than in LNEFA (p = 0.09). The mRNA abundance of genes in the liver related to fatty acid oxidation (carnitine palmitoyltransferase 2 and very long chain acyl-coenzyme A dehydrogenase) and ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2) were lower in HNEFA than in LNEFA cows. No differences between the two groups were observed for mRNA expression of genes in adipose tissue. The number of calculated significant correlation coefficients (moderately strong) between parameters in the liver and in adipose tissue was nearly similar on +1d, and higher for HNEFA compared with LNEFA cows in +3wk. In conclusion, dairy cows with high compared with low plasma NEFA concentrations in early lactation show differentially synchronized mRNA expression of genes in adipose tissue and liver in +3wk that suggests a different orchestrated homeorhetic regulation of lipid metabolism.

Statins induce regulatory T cell recruitment via a CCL1 dependent pathway

The statins, a group of inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, are reported to influence a variety of immune system activities through 3-hydroxy-3-methylglutaryl coenzyme A reductase-dependent and -independent mechanisms. How statin treatment regulates immune system function in vivo nonetheless remains to be fully defined. We analyzed the immunomodulatory effects of lovastatin in a Candida albicans-induced delayed-type hypersensitivity reaction in mice. In this model, lovastatin administration reduced the acute inflammatory response elicited by C. albicans challenge. This anti-inflammatory activity of lovastatin was associated with a shift from a Th1 to a Th2 immune response, as well as an increase in the percentage of regulatory T cells at the inflammation site and in the regional draining lymph node. The lovastatin-induced increase in regulatory T cells in the inflamed skin was dependent on expression of CCL1, a chemokine that is locally up-regulated by statin administration. The anti-inflammatory effect of lovastatin was abrogated in CCL1-deficient mice. These results suggest that local regulation of chemokine expression may be an important process in statin-induced modulation of the immune system.

Variation in hepatic regulation of metabolism during the dry period and in early lactation in dairy cows

The purpose of this study was to investigate variations in hepatic regulation of metabolism during the dry period, after parturition, and in early lactation in dairy cows. For this evaluation, cows were divided into 2 groups based on the plasma concentration of beta-hydroxybutyric acid (BHBA) in wk 4 postpartum (PP; group HB, BHBA >0.75 mmol/L; group LB, BHBA <0.75 mmol/L, respectively). Liver biopsies were obtained from 28 cows at drying off (mean 59 +/- 8 d antepartum), on d 1, and in wk 4 and 14 PP. Blood samples were collected every 2 wk during this entire period. Liver samples were analyzed for mRNA abundance of genes related to carbohydrate metabolism (pyruvate carboxylase, PC; phosphoenolpyruvate carboxykinase, PEPCK; citrate synthase, CS), fatty acid biosynthesis (ATP citrate lyase, ACLY) and oxidation (acyl-CoA synthetase long-chain, ACSL; carnitine palmitoyltransferase 1A, CPT 1A; carnitine palmitoyltransferase 2, CPT 2; acyl-coenzyme A dehydrogenase very long chain, ACADVL), cholesterol biosynthesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, HMGCS1), ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2, HMGCS2), and of genes encoding the transcription factors peroxisome proliferator-activated receptor alpha (PPARalpha), peroxisome proliferator-activated receptor gamma (PPARgamma), and sterol regulatory element binding factor 1 (SREBF1). Blood plasma was assayed for concentrations of glucose, BHBA, nonesterified fatty acids, cholesterol, triglycerides, insulin, insulin-like growth factor-I, and thyroid hormones. In both groups, plasma parameters followed a pattern usually observed in dairy cows. However, changes were moderate and the energy balance in cows turned positive in wk 7 PP for both groups. Additionally, the energy balance and milk yield were similar for both groups after parturition onwards. Significant group effects were found at drying off, when plasma concentrations of triglycerides were higher in LB than in HB, and in wk 4 PP, when plasma concentrations of glucose and IGF-I were lower in HB than in LB. Similarly, moderate changes in mRNA expression of hepatic genes between the different time points were observed, although HB cows showed more adaptive performance than LB cows based on changes in mRNA expression of PEPCKc, PEPCKm, CS, CPT 1A, CPT 2, and PPARalpha. Part of the variation measured in this study was explained by parity. Significant Spearman rank correlation coefficients between the variables were not similar at each time point and were not similar between the groups at each time point, suggesting that metabolic regulation differs between cows. In conclusion, metabolic regulation in dairy cows is a dynamic system, and differs obviously between cows at different metabolic stages related to parturition.

HMG-CoA Reductase Inhibition Promotes Neurological Recovery, Peri-Lesional Tissue Remodeling, and Contralesional Pyramidal Tract Plasticity after Focal Cerebral Ischemia

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors are widely used for secondary stroke prevention. Besides their lipid-lowering activity, pleiotropic effects on neuronal survival, angiogenesis, and neurogenesis have been described. In view of these observations, we were interested whether HMG-CoA reductase inhibition in the post-acute stroke phase promotes neurological recovery, peri-lesional, and contralesional neuronal plasticity. We examined effects of the HMG-CoA reductase inhibitor rosuvastatin (0.2 or 2.0 mg/kg/day i.c.v.), administered starting 3 days after 30 min of middle cerebral artery occlusion for 30 days. Here, we show that rosuvastatin treatment significantly increased the grip strength and motor coordination of animals, promoted exploration behavior, and reduced anxiety. It was associated with structural remodeling of peri-lesional brain tissue, reflected by increased neuronal survival, enhanced capillary density, and reduced striatal and corpus callosum atrophy. Increased sprouting of contralesional pyramidal tract fibers crossing the midline in order to innervate the ipsilesional red nucleus was noticed in rosuvastatin compared with vehicle-treated mice, as shown by anterograde tract tracing experiments. Western blot analysis revealed that the abundance of HMG-CoA reductase was increased in the contralesional hemisphere at 14 and 28 days post-ischemia. Our data support the idea that HMG-CoA reductase inhibition promotes brain remodeling and plasticity far beyond the acute stroke phase, resulting in neurological recovery.