Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase.

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.

Effect of Hyperbaric Oxygen Therapy (HBOT) on Insulin Resistance Associated with Abdominal Obesity in Wistar Rats with Dietary Sucrose-Induced Metabolic Syndrome.

Metabolic syndrome (MS) is a combination of risk factors related to the development of mainly type 2 diabetes mellitus, cardiovascular disease (CVD) and nonalcoholic fatty liver disease (NAFLD). Its prevalence has increased worldwide, and healthcare systems will face major challenges in addressing this problem. The aim of this work was to evaluate the effect of hyperbaric oxygen therapy (HBOT) on insulin resistance (IR) and obesity associated with MS in Wistar rats. The experimental design consisted of three groups of sucrose-induced MS rats: the MS group that consumed sucrose (MS-Suc; n=5), the MS group that ingested sucrose and HBOT (MS-Suc-HBOT; n=5), the MS group that did not consume sucrose and that received HBOT (MS-HBOT; n=5) and the control group. The rats received HBOT for 20 d at 2.4 atmospheres absolute (ATA) for 60 min. Subsequently, the rats were euthanized, and body fat weight, serum biochemical parameters and microscopic analysis of adipose tissue were determined. Rats with hyperoxia had decreased body weight, adipose tissue hypertrophy, and abdominal and epididymal fat. Likewise, markers of insulin resistance (glucose, insulin and HOMA-IR), biochemical parameters of dyslipidemia (cholesterol and triglycerides) and nonalcoholic fatty liver (AST and ALT) decreased; in contrast, compared to the control group, HBOT increased the 1/HOMA-IR, HOMA-ßCell and McAuley indexes, which were related to the improvement in insulin sensitivity (p<0.05; p<0.01). HBOT showed beneficial effects in the treatment of IR and obesity associated with sucrose-induced metabolic syndrome in Wistar rats.

Effect of CLA supplementation on factors related to vascular dysfunction in arteries of orchidectomized rats.

The vascular endothelium is a monolayer of flat epithelial cells located between the circulating blood and the underlying connective tissue. It conveys key functions that when impaired, lead to endothelial dysfunction. This condition is responsible for the pathogenesis of vascular diseases. The cardioprotective effect of sex hormones is widely known; hence, a murine orchidectomized model has been employed to study the effects caused by their deficiency. In the search for approaches to maintain vascular health, the effect of dietary fatty acids as CLA on cardiovascular diseases has been studied. Some proven beneficial properties of CLA are antioxidant, antiatherogenic and anti-inflammatory. Our objective was to evaluate the effect of a diet supplemented with 1.8 % (w/w) of CLA, administered during eight weeks, on the amount of cholesterol oxidation products (COPs) produced by orchidectomy and on factors related to vascular dysfunction in the aorta and the mesenteric arteries. The diet with CLA prevented the increase in prostanoids formation and maintained the normal physiological conditions of NO and antioxidant activity. In addition, it prevented the increase in cholesterol and COPs at the vascular wall. CLA-supplemented diet prevented the orchidectomy-induced alterations on prostanoids, NO and COPs and also improved the antioxidant activity. These findings could contribute to understand the mechanisms of actions of CLA involved in the prevention of cardiovascular diseases.

Bioactive compounds from Prosthechea karwinskii decrease obesity, insulin resistance, pro-inflammatory status, and cardiovascular risk in Wistar rats with metabolic syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: The orchid Prosthechea karwinskii is a species endemic to Mexico, which is used in traditional medicine to lower glucose levels in patients with diabetes, and to treat inflammation-related problems. Recent studies have shown that this orchids can reduce glucose, cholesterol, and triglyceride levels in Wistar rats, which were previously induced to have metabolic syndrome (MS). AIM OF THE STUDY: To evaluate the effect of P. karwinskii leaves extract on the components of metabolic syndrome: obesity, insulin resistance, pro-inflammatory status, and cardiovascular risk in a Wistar rat model, and to identify the bioactive compounds in the extract. MATERIALS AND METHODS: UPLC-ESI-qTOF-MS/MS was used to identify the compounds present in the extract. MS was induced in Wistar rats through administration of a 40% sucrose diet for 20 weeks. The rats were divided into five groups that received different treatments for 4 weeks: one group without any treatment, one group receiving metformin (200 mg/kg p.o.), and three groups receiving different doses of P. karwinskii leaves extract (100, 200, and 300 mg/kg p.o.). The animals' body weights were recorded weekly, and at the end of the experiment, they were sacrificed after fasting for 18 h to determine the levels of glucose, insulin, insulin resistance index, total cholesterol, triglycerides, and adiponectin in the serum, as well as levels of TNF-α and HS-CRP in the serum and liver homogenates. The abdominal and pericardial fat weights were also recorded. RESULTS: The main bioactive compounds of the extract were quinic acid, neochlorogenic acid, chlorogenic acid, rutin, kaempferol-3-o-ß-rutinoside, and embelin, known to exhibit MS-related bioactivity. Oral administration of P. karwinskii leaves extract at a dose of 300 mg/kg decreased weight gain, abdominal and pericardial fat deposits, and insulin resistance. At the end of the treatment, levels of triglycerides, TNF-α, HS-CRP, and adiponectin returned to levels similar to normal. CONCLUSION: P. karwinskii extract (300 mg/kg) had an anti-obesity effect, decreased insulin resistance, pro-inflammatory status, and cardiovascular risk in rats with induced MS by increasing adiponectin levels and decreasing TNF-α and HS-CRP levels. The compounds identified in the extract could be responsible for these effects, acting alone or in synergy, as several compounds in the extract are known to have MS-related bioactivity. The foliar extract of P. karwinskii has potential as an effective alternative to a cocktail of drugs used to treat problems associated with MS.

Effect of Curcumin Nanoemulsions Stabilized with MAG and DAG-MCFAs in a Fructose-Induced Hepatic Steatosis Rat Model.

Current changes in diet, characterized by an increase in the intake of sweetened beverages, are heavily related to metabolic disorders such as non-alcoholic fatty liver. This condition can produce simple steatosis and, in worse cases, potentially result in steatohepatitis, fibrosis, and cirrhosis, comparable to the damage caused by the consumption of more or less 20-30 g of alcohol per day. The main objective of this research was to evaluate the effect of curcumin (Curcuma longa) nanoemulsions, using mono- and diacylglycerides medium chain fatty acids as stabilizers in an in vivo hepatic steatosis rat model. Pathology was induced by providing 30% fructose intake in the drinking water. Globule sizes under 200 nm that were stable for 4 weeks were obtained; curcumin encapsulated in the nanoemulsion was >70%. The results revealed an improvement regarding body and liver weight in the animals treated with curcumin nanoemulsions. A decrease in total cholesterol, LDL, AST/ALT, and HDL in serum was observed; however, no apparent improvement regarding serum glucose or triacylglycerides values was noted. Histological analysis showed a significant decrease in the extent of steatosis, inflammation, and brown adipose tissue in the treated animals.

Curcumin Nanoemulsions Stabilized with Modified Phosphatidylcholine on Skin Carcinogenesis Protocol.

BACKGROUND: Cancer is one of the main causes of death by disease; several alternative treatments have been developed to counteract this condition. Curcumin (diferuloylmethane), extracted from the rhizome of Curcuma longa, has antioxidant, anti-inflammatory, and anti-cancer properties; however, it has low water solubility and poor intestinal absorption. Carrier systems, such as nanoemulsions, can increase the bioavailability of lipophilic bioactive compounds. OBJECTIVE: To evaluate the effect of curcumin nanoemulsions prepared with lecithin modified with medium-chain fatty acids as an emulsifier, on the expression of the Cdk4, Ccne2, Casp8 and Cldn4 genes involved in the carcinogenesis process in K14E6 transgenic mice. METHODS: The emulsifier was prepared by interesterification of medium-chain fatty acids, pure lecithin, and immobilized phospholipase-1 on Duolite A568. An Ultraturrax homogenizer and a Branson Ultrasonic processor were used for the preparation of nano-emulsions, and a Zetasizer evaluated the particle size. qRT-PCR analysis was performed to quantify the cancer-related genes expressed in the K14E6 mice. The development and evolution of skin carcinogenesis were assessed through histological analysis to compare cell morphology. RESULTS: Ca 59% of the MCFA were incorporated via esterification into the PC within 12 hours of the reaction. An emulsifier yield used to formulate the NE of 86% was achieved. Nanoemulsions with a particle size of 44 nm were obtained. The curcumin nano-emulsion group had a 91.81% decrease in the tumorigenesis index and a reduction in tumor area of 89.95% compared to the sick group. Histological analysis showed that the group administered with free curcumin developed a microinvasive squamous cell carcinoma, as opposed to the group with nanoemulsion which presented only a slight inflammation. In gene expression, only a significant difference in Cdk4 was observed in the nanoemulsion group.

Cd36 gene expression in adipose and hepatic tissue mediates the lipids accumulation in liver of obese rats with sucrose-induced hepatic steatosis.

Obesity is considered a global epidemic and is mainly associated with the development of diabetes, cardiovascular diseases and Non-Alcoholic Fatty Liver (NAFLD). The pathogenesis between obesity and hepatic steatosis is partially known, but could involve differentiated or tissue-specific participation of the expression of Cd36 mRNA that codes for a receptor which is a transporter of free fatty acids (FFA) in different tissues, favoring the lipids storage. This relative expression was evaluated in adipose and liver tissue in rats with steatosis after consumption of sucrose for 30 and 40 weeks. Ten Wistar rats were divided into two experimental groups (St-30 and St-40), which received a standard diet plus 30 % sucrose in their water intake. These rats showed a significant increase in abdominal fat, serum biochemical determinations, HOMA-IR; as well as, changes in adipocytes size and mild portal hepatitis and grade 2 hepatic steatosis. The relative expression of Cd36 mRNA increased in liver tissue after 30 (4.5-fold) and 40 (8.5-fold) weeks of sucrose ingestión but no in adipose tissue; with respect to control group (P < 0.05). This expression was associated with a significant increase in the levles of sCD36 in serum, which is indicator of the presence of the FFA transporter in the hepatocyte membrane causing lipids accumulation. The above shows the link between the adipose and hepatic tissue for the accumulation of steatotic fat in the liver through time, mediated by the relative expression of cd36 mRNA that encodes for the FFA transporter.

Comparative Effect between Sardine Oil and Fish Oil Rich in Omega-3 Fatty Acids on Hypertension and the Membrane Composition of Adipocytes in SHR Rats.

Omega-3 polyunsaturated fatty acids, have an important role in reducing hypertriglyceridemia, these acids decrease the mortality for Coronary Heart Disease. Very important is the relationship between fatty acid biosynthesis and distribution in organs and tissues involved in insulin resistance and hypertension due to its role in the production of vasoactive eicosanoids and their effects on insulin sensitivity; which is estimated with the HOMA-IR index, which relates the physiological and metabolic behavior of glucose and insulin in the body. The aim of this project was to compare the effect of sardine oil and omega-3 oils rich in polyunsaturated fatty acids: EPA (≈30%) and DHA (≈50%) administered for 6 to 8 wk respectively; on the lipid composition of the plasma membrane of epididymal adipocytes in spontaneously hypertensive rats (SHR) and their relation to obesity, insulin resistance and hypertension. The administration of omega-3 enriched oil significantly decreased the HOMA criteria as an insulin resistance indicator compared to the sardine oil.

Tissue Changes in the Development of Fatty Liver by Chronic Ingestion of Sucrose Associated with Obesity and Dyslipidemia in Rats.

A diet high in sucrose, which is a common food constituent, induces obesity and non- alcoholic fatty liver (NFLD) caused by high caloric intake; however, it is important to investigate those sequential changes in the hepatic parenchyma related to sugar consumption which are associated to obesity and dyslipidemia. We analyzed the effects of long-term sucrose intake on fatty liver development, by the administration of 30% sucrose in drinking water in healthy Wistar rats during 30 weeks. Serum variables, body fat index, caloric intake and microscopic examination of liver tissue were monitored. In the first week, grade 1 steatosis was observed with ballooned hepatocytes, with a caloric intake of 125 ± 1.90 kcal / day / 100 g of body weight; together with a gain of 71% in abdominal fat with respect to the control group and dyslipidemia. During the 10 to 20 weeks period, steatosis grade 2 with noticeable inflammation (steatohepatitis), polymorphic cells and ballooned hepatocytes were evident. After 10 weeks, the caloric intake was 72.9 ± 5.99 kcal / day / 100 g of body weight with 199% of gain in abdominal fat in SUC groups with respect control group (p < 0.01) and moderate dyslipidemia; while after 20 weeks, the caloric intake was 61.6 ± 4.65 kcal / day / 100 g of body weight with 208% of gain in abdominal fat and also moderate dyslipidemia. After 30 weeks steatosis grade 3 with marked inflammation (steatohepatitis), periportal fibrosis, globose and fat-filled hepatocytes were observed, with a caloric intake of 52.3 ± 3.05 kcal / day / 100 g of body weight and 232% of gain in abdominal fat that was related to severe dyslipidemia. In conclusion, the sequential changes in the development of NAFLD were associated with the ingestion of sucrose and obesity since the first week of administration.

Alteration of some inflammatory biomarkers by dietary oxysterols in rats.

Oxysterols are structurally similar to cholesterol, but are characterized by one or more additional oxygen-containing functional groups. These compounds are implicated in inflammation given their ability to cause irreversible damage to vascular cells. The aim of this study was to study the alteration of some inflammatory biomarkers in Wistar rats in response to dietary oxysterols. Eighteen rats were randomly divided into three groups of six rats each. A standard diet supplemented with 1% (w/w) pure cholesterol (Chol group) or 1% (w/w) of an oxidized cholesterol mixture (COPs group) was fed for 8 weeks. Blood serum was separated; abdominal, pericardial, and epididymal adipose tissue was removed carefully. The COPs subjects exhibited significant increase in blood pressure and serum triacylgycerols as well as increased body fat index and pericardic, abdominal, and epididymal adipose tissue. These effects were accompanied by elevated circulating levels of plasma high-sensitivity C-reactive protein, tumor necrosis factor alpha, and resistin. We suggest that dietary oxysterols have an important pro-inflammatory effect.

Dietary n-3 polyunsaturated fatty acids modify fatty acid composition in hepatic and abdominal adipose tissue of sucrose-induced obese rats.

The fatty acid profile of hepatocytes and adipocytes is determined by the composition of the dietary lipids. It remains unclear which fatty acid components contribute to the development or reduction of insulin resistance. The present work examined the fatty acid composition of both tissues in sucrose-induced obese rats receiving fish oil to determine whether the effect of dietary (n-3) polyunsaturated fatty acids (PUFAs) on the reversion of metabolic syndrome in these rats is associated to changes in the fatty acid composition of hepatocyte and adipocyte membrane lipids. Animals with metabolic syndrome were divided into a corn-canola oil diet group and a fish oil diet group, and tissues fatty acids composition were analyzed after 6 weeks of dietary treatment. Fatty acid profiles of the total membrane lipids were modified by the fatty acid composition of the diets fed to rats. N-3 PUFAs levels in animals receiving the fish oil diet plus sucrose in drinking water were significantly higher than in animals under corn-canola oil diets. It is concluded that in sucrose-induced obese rats, consumption of dietary fish oil had beneficial effects on the metabolic syndrome and that such effects would be conditioned by the changes in the n-3 PUFAs composition in hepatic and adipose tissues because they alter membrane properties and modify the type of substrates available for the production of active lipid metabolites acting on insulin resistance and obesity.

Effect of conjugated linoleic acid on body fat, tumor necrosis factor alpha and resistin secretion in spontaneously hypertensive rats.

Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivaties of linoleic acid found mainly in beef and dairy products. CLA has been reported to reduce body fat, as well as to possess anticarcinogenic, antiatherogenic and procatabolic activities in animals. The objective of this study was to evaluate the effect of CLA supplementation to spontaneously hypertensive rats (SHR) on body fat, biochemical parameters of serum related tumor necrosis factor alpha (TNF-alpha) and resistin secretion. Thirty rats were divided in three groups, the first group of spontaneously hypertensive rats received a standard diet (V-SHR group, n=10), a second group of SHR was fed 1.5% of conjugated linoleic acid (CLA-SHR group, n=10) and the third was the control, non-hypertensive group (KW, n=10) also on a standard diet including 7.5% of sunflower oil during eight weeks. After CLA diet administration, spontaneously hypertensive rats showed a significant reduction in blood pressure, serum glucose, cholesterol and triacylglycerols, together with reduction of index of body fat, pericardic, abdominal and epididymal adipose tissue. These effects were accompanied by a decrease in the secretion of TNF-alpha and resistin.

Biochemical and histopathological effects of dietary oxidized cholesterol in rats.

Cholesterol oxidation products (COPs) have been associated with the genesis of chronic degenerative diseases, such as atherosclerosis. The purpose of this work was to study the histological changes by toxic effects of dietary COPs in liver and kidney. Five-week-old male Wistar albino rats were randomly divided into three groups of 10 rats each. Standard rat chow was supplemented with either 1% (w/w) pure cholesterol or 1% oxidized cholesterol and fed to the rats for 8 weeks. Control animals were fed standard rat chow. At the end of the treatment period, the serum lipid profile was determined. The aorta, liver and kidneys were excised immediately, frozen with liquid nitrogen, and held at -70 degrees C. The histological study was carried out using conventional hematoxylin-eosin staining, and histochemical red oil 'O' was applied. COPs were analyzed by gas chromatography. Intake of dietary COPs altered biochemical parameters involved in lipid metabolism associated with atherogenesis in rats: total cholesterol, triacylglycerols and low density lipoproteins in serum. COPs detected in the liver and kidneys modified the organ original structure, caused an inflammatory process and promoted atherogenesis and atrophy of the tissue.

Induction of Cd36 expression elicited by fish oil PUFA in spontaneously hypertensive rats.

Cd36 is an integral membrane glycoprotein expressed on the surface of cells active in fatty acid metabolism (adipocytes, muscle cells, platelets, monocytes, heart and intestine cells). This protein plays diverse functions including uptake of long-chain fatty acids and oxidized low-density lipoproteins. A recent report demonstrates that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridemia in spontaneously hypertensive rats (SHRs). Cd36 is a tightly regulated protein whose expression is modulated through peroxisome proliferator-activated receptor (PPAR) transcription factors, by conditions that alter lipid metabolism such as diabetes mellitus and high-fat feeding. The purpose of this study was to evaluate the effect of dietary fish oil, rich in n-3 polyunsaturated fatty acids (PUFAs), on metabolic parameters and on the expression levels of Cd36 in adipose tissue in the SHR. Spontaneously hypertensive rats showed lower Cd36 mRNA levels when compared to Kyoto-Wistar (KW) rats (control). After 6 weeks of fish oil (FO) administration, this group of SHRs (FO-SHR) presented increased levels of Cd36 mRNA, concomitantly with decreased insulin, free fatty acids (FFAs), triglycerides, cholesterol, LDL, HDL, total lipids and blood pressure, in comparison to control rats that received a corn-canola oil diet. The study confirmed the beneficial effects of fish oil administration on the metabolic syndrome, suggesting that the induction of Cd36 expression could be one of the molecular mechanisms elicited by fish oil PUFAs.