Post-ischemic protein restriction induces sustained neuroprotection, neurological recovery, brain remodeling, and gut microbiota rebalancing.

BACKGROUND: Moderate dietary protein restriction confers neuroprotection when applied before ischemic stroke. How a moderately protein-reduced diet influences stroke recovery when administered after stroke, is a clinically relevant question. This question has not yet been investigated. METHODS: Male C57BL6/J mice were exposed to transient intraluminal middle cerebral artery occlusion. Immediately after the stroke, mice were randomized to two normocaloric diets: a moderately protein-reduced diet containing 8% protein (PRD) or normal diet containing 20% protein (ND). Post-stroke neurological deficits were evaluated by a comprehensive test battery. Antioxidant and neuroinflammatory responses in the brain and liver were evaluated by Western blot and RTqPCR. Stroke-induced brain injury, microvascular integrity, glial responses, and neuroplasticity were assessed by immunohistochemistry. Fecal microbiota analysis was performed using 16S ribosomal RNA amplicon sequencing. RESULTS: We show that PRD reduces brain infarct volume after three days and enhances neurological and, specifically, motor-coordination recovery over six weeks in stroke mice. The recovery-promoting effects of PRD were associated with increased antioxidant responses and reduced neuroinflammation. Histochemical studies revealed that PRD increased long-term neuronal survival, increased peri-infarct microvascular density, reduced microglia/macrophage accumulation, increased contralesional pyramidal tract plasticity, and reduced brain atrophy. Fecal microbiota analysis showed reduced bacterial richness and diversity in ischemic mice on ND starting at 7 dpi. PRD restored bacterial richness and diversity at these time points. CONCLUSION: Moderate dietary protein restriction initiated post-ischemic stroke induces neurological recovery, brain remodeling, and neuroplasticity in mice by mechanisms involving antiinflammation and, in the post-acute phase, commensal gut microbiota rebalancing.

Hypocaloric Diet Initiated Post-Ischemia Provides Long-Term Neuroprotection and Promotes Peri-Infarct Brain Remodeling by Regulating Metabolic and Survival-Promoting Proteins.

Calorie restriction confers post-ischemic neuroprotection, when administered in a defined time window before ischemic stroke. How a hypocaloric diet influences stroke recovery when initiated after stroke has not been investigated. Male C57BL6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. Immediately post-ischemia, mice were randomized to two groups receiving moderately hypocaloric (2286 kcal/kg food) or normocaloric (3518 kcal/kg) diets ad libitum. Animals were sacrificed at 3 or 56 days post-ischemia (dpi). Besides increased low density lipoprotein at 3 days and reduced alanine aminotransferase and increased urea at 56 days, no alterations of plasma markers were found in ischemic mice on hypocaloric diet. Body weight mildly decreased over 56 dpi by 7.4%. Hypocaloric diet reduced infarct volume in the acute stroke phase at 3 dpi and decreased brain atrophy, increased neuronal survival and brain capillary density in peri-infarct striatum and reduced motor coordination impairment in tight rope tests in the post-acute stroke phase over up to 56 dpi. The abundance of brain-derived neurotrophic factor, the NAD-dependent deacetylase and longevity protein sirtuin-1, the anti-oxidant glutathione peroxidase-3, and the ammonium detoxifier glutamine synthetase in the peri-infarct brain tissue was increased by hypocaloric diet. This study shows that a moderately hypocaloric diet that is initiated after stroke confers long-term neuroprotection and promotes peri-infarct brain remodeling.

9-PAHSA Prevents Mitochondrial Dysfunction and Increases the Viability of Steatotic Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is quickly becoming the most common liver disease worldwide. Within the NAFLD spectrum, patients with nonalcoholic steatohepatitis (NASH) are at the highest risk of developing cirrhosis and disease progression to hepatocellular carcinoma. To date, therapeutic options for NASH patients have been ineffective, and therefore, new options are urgently needed. Hence, a model system to develop new therapeutic interventions is needed. Here, we introduce two new in vitro models of steatosis induction in HepG2 cells and primary murine hepatocytes. We used a recently discovered novel class of bioactive anti-inflammatory lipids called branched fatty acid esters of hydroxyl fatty acids. Among these bioactive lipids, palmitic-acid-9-hydroxy-stearic-acid (9-PAHSA) is the most promising as a representative nondrug therapy based on dietary supplements or nutritional modifications. In this study, we show a therapeutic effect of 9-PAHSA on lipotoxicity in steatotic primary hepatocytes and HepG2 cells. This could be shown be increased viability and decreased steatosis. Furthermore, we could demonstrate a preventive effect in HepG2 cells. The outcome of 9-PAHSA administration is both preventative and therapeutically effective for hepatocytes with limited damage. In conclusion, bioactive lipids like 9-PAHSA offer new hope for prevention or treatment in patients with fatty liver and steatosis.

Neuroprotection Induced by Energy and Protein-Energy Undernutrition Is Phase-Dependent After Focal Cerebral Ischemia in Mice.

Malnutrition predisposes to poor stroke outcome. In animal models, undernutrition protected against ischemic injury in some, but not in other studies. In view of diverse stroke models and food restriction paradigms, the consequences of undernutrition are poorly understood. Herein, we exposed mice to energy-reduced and protein-energy-reduced diets for 7-30 days and subsequently induced intraluminal middle cerebral artery occlusion. Undernutrition phase dependently influenced ischemic injury. Short-lasting 7 days of protein-energy undernutrition, but not energy undernutrition, decreased post-ischemic brain leukocyte infiltration and microglial activation and reduced brain Il-1ß mRNA, but did not protect against ischemic injury. Fourteen days of energy and protein-energy undernutrition, on the other hand, reduced ischemic injury despite absence of anti-inflammatory effects. Anti-oxidant genes (Sod-1, Sod-2, and Cat mRNAs) were regulated in the liver and, to a lesser extent, the ischemic brain, indicating an adapted, compensated stage. Conversely, 30 days of energy and protein-energy undernutrition caused progressive animal exhaustion associated with post-ischemic hypoperfusion, rise of metabolic markers (Sirt-1 and Glut-1 mRNAs, Sirt-1 protein) in the ischemic brain, and reregulation of pro- and anti-oxidant markers (now also Nox-4 and Gpx-3 mRNAs) in the liver. In the latter condition, no neuroprotection was noted. Our study suggests an adaptation of metabolic systems that provides neuroprotection in a circumscribed time window.

Moderate Protein Restriction Protects Against Focal Cerebral Ischemia in Mice by Mechanisms Involving Anti-inflammatory and Anti-oxidant Responses.

Food composition influences stroke risk, but its effects on ischemic injury and neurological deficits are poorly examined. While severe reduction of protein content was found to aggravate neurological impairment and brain injury as a consequence of combined energy-protein malnutrition, moderate protein restriction not resulting in energy deprivation was recently suggested to protect against perinatal hypoxia-ischemia. Male C57BL6/j mice were exposed to moderate protein restriction by providing a normocaloric diet containing 8% protein (control: 20% protein) for 7, 14, or 30 days. Intraluminal middle cerebral artery occlusion was then induced. Mice were sacrificed 24 h later. Irrespective of the duration of food modification (that is, 7-30 days), protein restriction reduced neurological impairment of ischemic mice revealed by a global and focal deficit score. Prolonged protein restriction over 30 days also reduced infarct volume, brain edema, and blood-brain barrier permeability and increased the survival of NeuN+ neurons in the core of the stroke (i.e., striatum). Neuroprotection by prolonged protein restriction went along with reduced brain infiltration of CD45+ leukocytes and reduced expression of inducible NO synthase and interleukin-1ß. As potential mechanisms, increased levels of the NAD-dependent deacetylase sirtuin-1 and anti-oxidant glutathione peroxidase-3 were noted in ischemic brain tissue. Irrespective of the protein restriction duration, a shift from pro-oxidant oxidative stress markers (NADPH oxidase-4) to anti-oxidant markers (superoxide dismutase-1/2, glutathione peroxidase-3 and catalase) was found in the liver. Moderate protein restriction protects against ischemia in the adult brain. Accordingly, dietary modifications may be efficacious strategies promoting stroke outcome.

Effects of Undaria pinnatifida, Himanthalia elongata and Porphyra umbilicalis extracts on in vitro α-glucosidase activity and glucose diffusion.

BACKGROUND: Seaweeds are good sources of dietary fibre, which can influence glucose uptake and glycemic control. OBJECTIVE: To investigate and compare the in vitro inhibitory activity of different extracts from Undaria pinnatifida (Wakame), Himanthalia elongata (Sea spaghetti) and Porphyra umbilicalis (Nori) on α-glucosidase activity and glucose diffusion. METHODS: The in vitro effects Chloroform-, ethanol- and water-soluble extracts of the three algae were assayed on α- glucosidase activity and glucose diffusion through membrane. Principal Components Analysis (PCA) was applied to identify patterns in the data and to discriminate which extract will show the most proper effect. RESULTS: Only water extracts of Sea spaghetti possessed significant in vitro inhibitory effects on α-glucosidase activity (26.2% less mmol/L glucose production than control, p < 0.05) at 75 min. PCA distinguished Sea spaghetti effects, supporting that soluble fibre and polyphenols were involved. After 6 h, Ethanol-Sea spaghetti and water-Wakame extracts exerted the highest inhibitory effects on glucose diffusion (65.0% and 60.2% vs control, respectively). This extracts displayed the lowest slopes for glucose diffusion-time lineal adjustments (68.2% and 62.8% vs control, respectively). CONCLUSIONS: The seaweed hypoglycemic effects appear multi-faceted and not necessarily concatenated. According to present results, ethanol and water extracts of Sea spaghetti, and water extracts of Wakame could be useful for the development of functional foods with specific hypoglycemic properties.

Antecedentes: Las algas son importante fuente alimentaria de fibra dietética y puede influir sobre la absorción de glucosa y el control glucémico. Objetivo: Evaluar y comparar in vitro los efectos de diferentes extractos de las algas Undaria pinnatifida (Wakame), Himanthalia elongata (Espagueti de mar) y Porphyra umbilicalis (Nori) sobre la actividad enzimática -glucosidasa y la difusión de glucosa. Métodos: Se estudiaron los efectos de los extractos clorofórmicos, etánólicos y acuosos de las tres algas sobre la actividad -glucosidasa y la difusión de glucosa a través de una membrana de diálisis. Se aplicó a los resultados un análisis de los componentes principales (PCA) para identificar posibles patrones de composición y seleccionar el extracto que mejores propiedades posea. Resultados: El extracto acuoso de Espagueti de mar inhibió de forma significativa la actividad -glucosidasa (26,2% menos liberación de glucosa, p < 0,05). El PCA sugiere que la fibra soluble y los polifenoles son los responsables de tal efecto. Respecto a la difusión de glucosa, el extracto etanólico de Espagueti de mar y el acuoso de Wakame mostraron un mayor efecto inhibidor después de 6 horas (65% y 60,2% vs control, respectivamente) y las menores pendientes en los ajustes lineales difusión de glucosa- tiempo (68,2% y 62,8% vs control respectivamente). Conclusiones: Los resultados de los diferentes extractos sugieren que los efectos hipogluceminates de las algas son variados y no están necesariamente concatenados. Los extractos acuosos y etanólicos de Espagueti de mar y los acuosos de Wakame parecen los más adecuados para el desarrollo de alimentos funcionales con propiedades hipoglucemiantes.

Protective effects of sea spaghetti-enriched restructured pork against dietary cholesterol: effects on arylesterase and lipoprotein profile and composition of growing rats.

There is a general assumption that seaweeds are hypocholesterolemics and antioxidants. However, controversial results suggest specific properties for each individual alga. This study aims to assess the effect of including Sea Spaghetti alga (S) in a restructured-pork (RP) diet, both enriched and not enriched with dietary cholesterol, on arylesterase (AE) activity and lipoprotein concentration and composition of Wistar rats. Four groups of 10 growing male Wistar rats were each fed a mix of 85% AIN-93M diet and 15% freeze-dried RP for 5 weeks. The control group (C) consumed control RP-C; the S group consumed RP-S with 5% seaweeds; the Chol-C group consumed the C diet but enriched with cholesterol (2.43%) and cholic acid (0.49%); the Chol-S group consumed the S diet but enriched with cholesterol and cholic acid. AE activity was five times higher (P<.01) in S compared with C rats, but three times lower in Chol-S compared with Chol-C rats (P<.01). The Chol-C diet induced hypercholesterolemia but reduced triglycerides (TG), giving rise to the presence of very low-density lipoprotein (VLDL) that was enriched in cholesterol. The Chol-S diet partially blocked (P<.001) the hypercholesterolemic induction of the Chol-C diet, and reduced TG levels (P<.05) with respect to S rats. The cholesterol supplementation increased total cholesterol, VLDL-cholesterol, and intermediate-density lipoprotein+LDL-cholesterol (IDL+LDL)-cholesterol (P<.001) in Chol-C rats, but the effect was lower in the Chol-S diet. In conclusion, RP-S increases the antioxidant capacity within a noncholesterol enriched diet while improving the lipoprotein profile within a cholesterol-enriched diet.

Nori- and sea spaghetti- but not wakame-restructured pork decrease the hypercholesterolemic and liver proapototic short-term effects of high-dietary cholesterol consumption.

Restructured pork (RP) enriched in Seaweeds are potential functional foods. The antiapoptotic and hypocholesterolemic effects of consuming cholesterol enriched diets containing Wakame-RP (CW), Nori-RP (CN) and Sea Spaghetti (CS) were tested in a 1-wk study. Groups of six rats per group were fed a mix of 85% AIN-93M rodent-diet containing cholesterol and cholic acid as a cholesterol rising agent plus 15% RP containing alga. These diets were compared to control-RP diets enriched or not in cholesterol (CC and C, respectively). After 1-wk, cholesterol feeding significantly increased liver apoptosis markers which were significantly reduced by CS (cellular cycle DNA, caspase-3, and cytochrome c), CN (caspase-3 and cytochrome c) and CW (caspase-3) diets. CN and CS diets significantly blocked the cholesterolaemic rising effect observed in the CC group but no protective effect was observed in the CW group. Differences in seaweed composition added to RP appear responsible for blocking or not the proapoptotic and hypercholesterolemic effects of high cholesterol-RP consumption; thus, any generalization on seaweed effects or food containing seaweeds must be avoided. Although present results are worthy, future studies are demanded to ascertain the utility of consuming algal-RP as part of usual diets.

La incorporación de algas, ricas en fibra y compuestos asociados, a reestructurados de carne de cerdo (R) resulta en derivados cárnicos potencialmente funcionales. En este trabajo se estudian los efectos antiapoptóticos e hipocolesterolemiantes de dietas en las que se incluyen agentes hipercolesterolemiantes y R enriquecidos en diferentes algas, Wakame (RW), Nori-R (RN) y Espagueti de Mar (RE). Durante una semana grupos de seis ratas cada uno recibieron una mezcla constituida por 85% de dieta AIN- 93M para roedores suplementada con colesterol y ácido cólico, como agente hipercolesterolemiante, y 15% de RW, RN o RE. Estas dietas fueron comparadas con otras a las que se incorporó R control y a las que se añadió o no el agente hipercolesterolemiante. Después de 1 semana de tratamiento el incremento de marcadores de apoptosis hepática observado en el lote control con colesterol se redujo por el consumo de las dietas RE (DNA ciclo celular, caspasa-3, y citocromo c), RN (caspasa-3, y citocromo c) and RW (caspasa-3). Sólo las dietas con RN and RE bloquearon significativamente la inducción hipercolesterolemiante de la dieta control enriquecida en colesterol. Teniendo en cuenta las diferencias observadas entre los lotes respecto a sus efectos hipocolesterolémicos y antiapoptóticos, debe evitarse cualquier generalización sobre el consumo de algas y en particular de carnes conteniendo algas. Aunque los resultados son relevantes, deben realizarse estudios futuros para determinar la utilidad del consumo de estos R enriquecidos en algas dentro de dietas habituales.

Effects of seaweed-restructured pork diets enriched or not with cholesterol on rat cholesterolaemia and liver damage.

Seaweed enriched-restructured pork (RP) is a potential functional food. However, indications of adverse effects associated with herbal medications, which include among others liver failure, toxic hepatitis, and death have been reported. Cholesterol feeding produces hepatomegalia and fat liver infiltration. The effect of seaweed-RP diet, cholesterol-enriched or not, on plasma cholesterol, liver damage markers, structure, and cytochrome CYP4A-1 were evaluated after 5 wk. Eight rat groups were fed a mix of 85% AIN-93M rodent-diet plus 15% RP. The Cholesterol-control (CC), Cholesterol-Wakame (CW), Cholesterol-Nori (CN) and Cholesterol-Sea Spaghetti (CS) groups respectively consumed similar diets to control (C), Wakame (W), Nori (N), and Sea Spaghetti (S) but as part of hypercholesterolaemic diets. CN and CS significantly blocked the hypercholesterolaemic effect observed in CC group. After 5-wk, N and S diets increased the CYP4A-1 expression. However, seaweed-RPs were unable to reduce the histological liver alterations observed in CC group. Larger and more abundant hepatocellular alterations were found in CS and CN rats suggesting that the hypocholesterolaemic effects of these seaweed-RPs seem to be a two-edged sword as they increased liver damage. Future studies are needed to understand the involved mechanisms.

Effects of restructured pork containing Himanthalia elongata on adipose tissue lipogenic and lipolytic enzyme expression of normo- and hypercholesterolemic rats.

BACKGROUND: Inclusion of seaweeds in restructured pork (RP) would improve meat health properties. AIM: The aim was to determine the effects of RP containing Himanthalia elongata on growth, organ weight, cholesterolemia, and expression of lipoprotein lipase (LPL), acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), and hormone-sensitive lipase (HSL) in adipose tissue of Wistar rats. METHODS: The control group (C) consumed control RP, and the seaweed group (SS) consumed RP with 5% Himanthalia elongata. Animals on cholesterol-enriched diets (CholC, CholSS) consumed their corresponding basal diets with supplementary cholesterol (2.43%) plus cholic acid (0.49%) as hypercholesterolemic agents. RESULTS: Seaweed inclusion did not modify cholesterolemia but reduced (p < 0.01) the hypercholesterolemic effect of dietary cholesterol plus cholic acid. CholC rats presented lower (p < 0.05) adipose fat and weight than C rats. Cholesterol feeding affected HSL, ACC, and FAS expression (p < 0.05), while alga consumption influenced HSL and ACC (p < 0.05). LPL was not significantly affected by both factors. CholSS rats showed decreased HSL and FAS (p < 0.05) but increased ACC (p < 0.05) expression compared with CholC rats. CONCLUSION: Himanthalia RP partially blocked the dietary hypercholesterolemic effect and changed the lipogenic/lipolytic enzyme expression, reducing the wasting effect of hypercholesterolemia on adipose tissue in rats.