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Imbalance in the n-6 polyunsaturated fatty acids (PUFA) and n-3 PUFA in the Western diet may increase the risk of nonalcoholic fatty liver disease (NAFLD). This study investigates the impact of substitution of linoleic acid with α-linolenic acid (ALA) or long chain (LC) n-3 PUFA and hence decreasing n-6:n-3 fatty acid ratio on high fat, high fructose (HFHF) diet induced nonalcoholic steatohepatitis (NASH). Male Sprague-Dawley rats were divided into four groups and fed control diet, HFHF diet (n-6:n-3 ratio of 200), HFHF diet with ALA (n-6:n-3 ratio of 2) or HFHF diet with LC n-3 PUFA (n-6:n-3 ratio of 5) for 24 weeks. Rats fed HFHF diet with n-6:n-3 ratio of 200 resulted in hepatic steatosis, induced glucose intolerance, insulin resistance and oxidative stress accompanied by increase in markers of inflammation, plasma lipids and aminotransferase levels. Histopathological examination of liver further confirmed the establishment of NASH. ALA and LC n-3 PUFA supplementation prevented hepatic steatosis and dyslipidemia by inhibiting lipogenesis and increasing insulin sensitivity. Furthermore, n-3 PUFA supplementation attenuated hepatic oxidative stress by restoring antioxidant status, decreased inflammation and preserved hepatic architecture. These finding suggest that decreasing n-6:n-3 ratio prevented HFHF induced NASH by attenuating oxidative stress and inflammation.
Nonalcoholic fatty liver disease (NAFLD) is the chronic liver disease of unknown etiology and is more common in affluent countries, affecting both adults and children. Clinically, it is defined as the fat accumulation in the hepatocytes (≥5% of the total weight) in the absence of excessive alcohol consumption (>20 g/d for women and >30 g/d for men)1. NAFLD is characterized by ectopic fat accumulation in hepatocytes (steatosis), which is relatively benign. However, approximately 20% of the individuals with simple steatosis develop nonalcoholic steatohepatitis (NASH) which may further progress into liver fibrosis, cirrhosis and eventually hepatocellular carcinoma2,3. By 2020, NASH has been projected to become the leading cause of liver transplantation, overtaking hepatitis C virus infection related transplantation in Western countries4. NAFLD is now considered as multisystem disease and associated with a range of chronic diseases such as cardiovascular disease, chronic kidney disease, type 2 diabetes and colorectal cancer5. It is estimated that, 20–30% of the general adult population is affected by NAFLD in Western countries6. The prevalence of NAFLD in India ranges from 16–32% in general population with higher prevalence in those with obesity, overweight and diabetes7,8. Recently, a "multiple parallel-hit" hypothesis has been proposed to explain the development and progression of NAFLD, which suggests that multiple insults such as insulin resistance, mitochondrial dysfunction, endotoxemia, adipocytokines, and gut microbiota synergistically act together resulting in immune cell activation leading to hepatic injury and cell death9.
Despite high prevalence of NAFLD, currently there is no FDA approved drug specifically for the treatment of NAFLD10. Hence, lifestyle modifications which include increased physical activity and proper diet are the only options for the management of NAFLD. Consumption of Western diet which are high calorie diets rich in processed sugars (fructose or sucrose) and fat has been implicated in the recent increase in the prevalence of NAFLD11,12,13. Furthermore, Kechagias et al. demonstrated that healthy subjects consuming fast food based diet for 4 weeks induced fatty liver, insulin resistance and increased aminotransferase levels14. Although Western diets are known to induce NAFLD, it is not clear how the individual components of the Western diet influence the development/progression of NAFLD. In this context, our recent study demonstrated that in a series of high energy diets mimicking Western diet, the type of dietary fat determines the development of advanced form of NAFLD15. Fructose in combination with saturated fatty acids (SFA) or trans fatty acids induced hepatic steatosis of comparable degree, whereas fructose in combination with trans fatty acids induced NASH with fibrosis15.
The polyunsaturated fatty acids (PUFA), linoleic acid (LA, 18:2 n-6) and α-linolenic acid (ALA, 18:3 n-3) are the short chain precursors that are converted into biologically active long chain (LC) PUFA such as arachidonic acid (20:4 n-6), eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) respectively. The n-6 and n-3 PUFA are not interconvertible and are metabolically and functionally distinct16. Generally, the eicosanoids derived from n-6 PUFA are proinflammatory, whereas those derived from n-3 PUFA are anti-inflammatory. The PUFA composition of the cell membrane is determined by the dietary levels of n-6 and n-3 PUFA and their ratio. High intake of n-6 PUFA create proinflammatory milieu, which in turn, may affect the development or progression of several diet related chronic diseases including NAFLD17. In the present Western diet, the level of n-6 PUFA is significantly higher than the n-3 PUFA, which resulted in a shift of n-6:n-3 ratio to >10:1 from the traditional 2:118. In fact, a study evaluating dietary pattern in patients with NASH, revealed high intake of n-6 PUFA and n-6:n-3 ratio thereby implicated in promoting NASH19. Clinical studies reported altered PUFA metabolism with reduced LC PUFA content in the liver of NASH patients suggesting unbalanced n-6 and n-3 PUFA in the pathogenesis of NASH20,21,22. Several animal studies showed that LC n-3 PUFA supplementation confer protection against the development of NAFLD in diet induced models of NAFLD23,24,25. Furthermore, transgenic mice engineered to express FAT-1 gene encoding n-3 fatty acid desaturase, which is capable of converting n-6 PUFA to LC n-3 PUFA prevents high fat26 and Western diet27 induced NAFLD by restoring LC n-3 PUFA. Nevertheless, clinical trials with LC n-3 PUFA supplementation in patients with NAFLD showed mixed results28. The failure of some of the clinical trials could be due to difference in the dosage of LC n-3 PUFA, duration of the trial, poor compliance or difference in the intake of other fatty acids. To our knowledge, there is no information about the level of n-3 PUFA (particularly in relation to n-6 PUFA) needed to prevent NAFLD. We hypothesize that imbalance in the dietary n-6:n-3 ratio in the Western diet is likely to induce NASH and substitution of n-6 PUFA with n-3 PUFA may prevent the development of NASH. Therefore, the objective of the present study was to investigate the impact of substituting n-6 PUFA (LA) with n-3 PUFA (ALA or LC n-3 PUFA) on the development of high fat, high fructose (HFHF) diet induced NASH. Blends of peanut oil, palmolein and linseed oil/fish oil were used to create a series of diets with differing levels of n-3 PUFA and ratios of n-6:n-3 fatty acids. The glucose metabolism, plasma lipid profile and adipocytokines, hepatic lipid content and histopathological changes were studied. In addition, the impact of n-3 PUFA supplementation on hepatic oxidative stress and expression of genes related to lipid metabolism and inflammation were also studied to understand the possible molecular mechanism by which n-3 PUFA supplementation prevents NASH.
Materials and Methods
Ethical approval
The present study was conducted as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India. The research protocol was approved by institutional animal ethical committee of National Institute of Nutrition, Hyderabad, India (Approval ID-P4/IAEC/NIN/2012/4/AI/SD rats).
Animal experiment and study design
Thirty-two weanling male Sprague-Dawley rats were acquired from animal house facility of National Institute of Nutrition, Hyderabad, India. Animals were housed individually in polypropylene cages with constantly regulated temperature (21 ± 1 °C) and light controlled housing conditions (12:12 h light/dark cycle). Rats were randomly divided into four groups (n = 8 per group) and fed starch/fructose - casein based synthetic diet. In the control group, starch was used as the source of carbohydrate, whereas, in the experimental groups, fructose was used as the source of carbohydrate. The n-6/n-3 PUFA ratio was altered by blending of peanut oil, palmolein and linseed oil (source of ALA) to get the ratio of 200 and 2 or by blending of peanut oil, palmolein and fish oil (source of EPA and DHA) to get the ratio of 5. The n-6:n-3 PUFA ratios of 2 and 5 were selected based on the results of our earlier studies on dietary PUFA and insulin resistance29,30. The various experimental groups were as follows: a control group with n-6/n-3 PUFA ratio of 200 (CON-200) and experimental groups with varying n-6/n-3 PUFA ratios of 200 (HFHF-200), 2 (HFHF-2) and 5 (HFHF-5). The diet composition was (% by weight) 50% carbohydrates, 20% casein, 20% oil, 5% cellulose, 1% vitamin mixture, 3.5% mineral mixture, 0.3% L-cysteine and 0.2% choline chloride. The salt and vitamin mixtures were prepared as per AIN-93 recommendations31. The HFHF diets also contain 1.5% cholesterol and 0.2% sodium cholate. All the diets were isocaloric, isolipidic and differed only in type of carbohydrate and n-6/n-3 PUFA ratio. Carbohydrates provided 44% of calories; fats provided 39% of calories and proteins provided 17% of calories. The total SFA, monounsaturated fatty acids (MUFA) and PUFA were similar in all the groups and n-6/n-3 ratio was altered by substituting n-6 PUFA with n-3 PUFA. Fatty acid analyses of the diets (Table 1) were performed by gas chromatography32. The diets were prepared once weekly by adding the oils to the base mixture containing other nutrients and stored at 4 °C before use. The oil and base mixture were stored at 4 °C until preparation of the diet. Fish oil (MAXEPA, Merck) was kept under an atmosphere of nitrogen and stored at −20 °C. All the diets were supplemented with α-tocopherol (0.015 g/kg diet) to prevent oxidation. Rats received food and drinking water ad libitum. Body weight was measured weekly and food consumption was monitored daily. At the end of 24 week, rats were fasted overnight and blood samples were collected from retro-orbital plexus in EDTA coated vials. Blood was centrifuged at 4,000 rpm for 15 min. at 4 °C and plasma was stored at −80 °C for subsequent determination of biochemical parameters. Rats were euthanized by CO2 asphyxiation. Liver, kidney, retroperitoneal fat (RP), epididymal fat (EP) and mesenteric fat (MS) were excised quickly and rinsed with saline. The organs were weighed, snap frozen in liquid nitrogen and stored at −80 °C for further analysis. A small piece of liver was stored in RNA Later (Sigma-Aldrich) for gene expression analysis and another portion of the liver was immediately immersed in neutral buffered 10% formalin for histopathological examination. Adiposity index was computed as the sum of visceral adipose tissue (RP + EP + MS) weights and expressed as a percentage of total body weight.
Intraperitoneal glucose tolerance test
The intraperitoneal glucose tolerance test (IPGTT) was performed as described previously15. Briefly, rats were fasted overnight and fasting blood sample was taken from retro-orbital plexus. The rats were injected intraperitoneally with a glucose solution (1 g/kg body weight). Blood samples were drawn at 15, 30, 60 and 120 min. intervals after the glucose injection and glucose levels were estimated by enzymatic kit method. Glucose and insulin areas under the curve (AUC) values were calculated using the trapezoidal rule.
Plasma biochemistry
Glucose, triglycerides, total cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the plasma were estimated by kit method (Biosystems, Barcelona, Spain). Plasma insulin was measured using rat insulin ELISA kit (Mercodia AB, Uppsala, Sweden) according to the manufacturer's instructions. Homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated from fasting plasma glucose and insulin to estimate insulin resistance. The adiponectin (Invitrogen, California, USA), leptin (EMD Millipore, Billerica, MA, USA) and resistin (BioVendor, Czech Republic) levels in the plasma were estimated by commercial ELISA kits.
Determination of liver triglyceride and cholesterol contents
Liver tissue (50–100 mg) was homogenized in 5% aqueous Nonidet P-40 (NP-40) solution to solubilize the lipids15,33. Intrahepatic triglycerides and cholesterol were assayed using enzymatic kit (Biosystems, Barcelona, Spain).
Assessment of lipid peroxidation, activity of antioxidant enzymes and glutathione levels in the liver
Liver lipid peroxidation in the homogenate was estimated by measuring malondialdehyde using thiobarbituric acid reactive substances (TBARS) assay34. Catalase activity was measured according to the method of Aebi35. Glutathione peroxidase (GPX) activity was determined according to Flohe and Gunzler36. The total superoxide dismutase (SOD) activity was measured following the method of McCord and Fridovich37. Reduced glutathione content was measured by the Anderson method38. Total protein content of the liver homogenate was determined by Lowry's method39.
Quantification of liver phospholipid fatty acid composition
Liver total lipids were extracted according to Bligh and Dyer method40. To analyze liver phospholipid fatty acid composition, neutral lipids were separated from phospholipids as described previously15. Phospholipid fatty acid profile was quantified by gas chromatography as described32.
Liver gene expression studies by quantitative real time PCR
RNA extraction and cDNA synthesis were performed as described previously15. Quantitative PCR was performed by using SYBR Green Master mix kit (Takara, Otsu, Shiga, Japan) in Bio-rad CFX96 system (Bio-rad Laboratories, Hercules, California, USA) with β-actin as the control gene. Primers were obtained from Sigma - Aldrich and the specific primer sequences used in this study are listed in Supplementary Table S1. Relative expression of each gene was calculated by the 2–(ΔΔCt) method41.
Histological assessment of liver
Liver samples fixed in formalin were embedded in paraffin wax and cut into 5 μm sections for hematoxylin and eosin (H&E) staining. Hepatic steatosis and fibrosis were assessed by Oil Red O staining and Masson's trichrome staining respectively. NAFLD activity score (NAS) was calculated based on the individual scores of steatosis (0–3 points), lobular inflammation (0–3 points) and hepatocyte ballooning (0–2 points) in a blinded manner to assess the severity of NAFLD42. A score of >4 was considered as NASH.
Statistical analysis
All the data were presented as mean ± SD. The differences between the groups were analyzed by one-way analysis of variance (ANOVA) with least significant difference (LSD) post hoc test using SPSS 16.0 software package. Significance was considered to be at P < 0.05.
General characteristics
Food intake and body weight gain were comparable between the groups (Table 2). Compared to control diet (CON-200), the HFHF diet (HFHF-200) increased liver and kidney weights 1.4 (P < 0.001) and 1.2 (P < 0.001) folds respectively. However, substitution of n-6 PUFA with n-3 PUFA in HFHF fed rats (HFHF-2 and HFHF-5) significantly decreased liver weight without altering kidney weight. The weights of retroperitoneal and mesenteric fat pads were increased by 70% (P < 0.001) and 30% (P = 0.045) respectively in rats fed diet with HFHF compared to control diet fed rats. Accordingly, the calculated values of adiposity index were increased by 38% (P = 0.003) in rats fed diet with HFHF. Substitution of n-6 PUFA with n-3 PUFA normalized the fat pad weights and adiposity index.
Substituting LA with ALA or LC n-3 PUFA improved HFHF induced alteration in fasting metabolic parameters
Compared to control group, the HFHF feeding increased plasma triglycerides and cholesterol levels by 39% (P = 0.011) and 47% (P < 0.001) respectively (Table 3). Substitution of n-6 PUFA with n-3 PUFA normalized the plasma lipid levels. Fasting plasma glucose was increased (13%, P = 0.011) in rats fed diet with HFHF and substitution of n-6 PUFA with n-3 PUFA normalized the level. Rats fed the HFHF diet had higher fasting plasma insulin level (by 1.9 fold, P < 0.001) and HOMA-IR (by 2.1 fold, P < 0.001) than those fed control diet. In HFHF fed rats, substituting n-6 PUFA with n-3 PUFA decreased (HFHF-2) or normalized (HFHF-5) the plasma insulin level and HOMA-IR. Rats fed the HFHF diet had lower plasma adiponectin (P < 0.001) and higher leptin (P = 0.027) compared to control diet fed rats. Substitution of n-6 PUFA with n-3 PUFA in HFHF diet normalized the leptin level without altering the adiponectin level. Furthermore, adiponectin:leptin ratio (P = 0.004) was lower in rats fed the HFHF diet compared to control diet fed rats and substitution of n-6 PUFA with n-3 PUFA normalized the ratio. Although plasma resistin level was not altered by HFHF diet, substitution of LA with ALA or LC n-3 PUFA in HFHF diet decreased the resistin levels by 15% (P = 0.033) and 30% (P < 0.001) respectively.
Substituting LA with ALA or LC n-3 PUFA prevented the HFHF induced glucose intolerance and insulin resistance
As depicted in the Fig. 1, HFHF feeding induced glucose intolerance as evidenced by high glucose excursion and AUC of glucose (23%, P = 0.006) in response to a glucose load in the IPGTT. In contrast, substitution of n-6 PUFA with n-3 PUFA prevented the HFHF induced glucose intolerance as evidenced by decreased glucose excursion and AUC of glucose (Fig. 1a). Along with impaired glucose intolerance, HFHF feeding induced insulinemia as evidenced by increased insulin AUC (60%, P = 0.023) after a glucose load and substitution of n-6 PUFA with n-3 PUFA corrected the HFHF induced insulinemia (Fig. 1b).
Substituting LA with ALA or LC n-3 PUFA mitigated HFHF induced hepatomegaly and liver injury
As shown in Fig. 2, compared with the control group, the livers of the rats fed diet with HFHF were grossly enlarged and pale in color (Fig. 2a) combined with accumulation of triglycerides and cholesterol (Fig. 2b,c). Nevertheless, substitution of n-6 PUFA with n-3 PUFA reduced hepatomegaly and lipid accumulation. The activities of plasma ALT and AST, which are the markers of hepatic injury, were increased by 3.2 fold (P < 0.001) and 2.8 fold (P < 0.001) respectively in the rats fed diet with HFHF, compared to control diet fed rats (Fig. 2d,e). Substitution of n-6 PUFA with n-3 PUFA normalized the aminotransferase activity.
Substituting LA with ALA or LC n-3 PUFA attenuate hepatic oxidative stress and increase antioxidant enzyme activity and glutathione level in HFHF fed rat
Liver TBARS, which is the marker of oxidative stress was increased by 1.8 fold (P < 0.001) in HFHF fed rats compared with the rats fed control diet (Fig. 3a). In HFHF fed rats, substituting n-6 PUFA with n-3 PUFA decreased (HFHF-2) or normalized (HFHF-5) the TBARS level. In addition, catalase, superoxide dismutase activities and glutathione level were decreased by 1.4 fold (P < 0.001), 1.2 fold (P = 0.032) and 1.5 fold (P < 0.001) respectively in the rats fed HFHF diet when compared with rats fed control diet (Fig. 3b–e). However, substitution of n-6 PUFA with n-3 PUFA restored the antioxidant enzyme activities and glutathione levels in the liver.
Substituting LA with ALA or LC n-3 PUFA restored the hepatic expression of genes involved in lipid metabolism and proinflammatory cytokines in HFHF fed rat
To understand the molecular mechanisms by which n-3 PUFA supplementation protects the HFHF induced development of NASH, the mRNA expression levels of genes associated with lipid metabolism, inflammation and fibrosis were analyzed. As shown in Fig. 4, HFHF feeding upregulated the expression of SREBP-1c (2.9 fold, P = 0.002), the key transcriptional regulator of de novo lipogenesis and its target genes SCD-1 (6.3 fold, P = 0.023) and ACC α (2.2 fold, P = 0.005), whereas, the expression of other lipogenic genes, ChREBP and PPAR γ were not altered (Fig. 4a). The mRNA levels of PPAR α, ACOX-2 and CPT-1, which are involved in fatty acid oxidation, were not altered by feeding HFHF diet. Substitution of n-6 PUFA with n-3 PUFA in HFHF diet normalized the mRNA levels of SREBP-1c, SCD-1 and ACC α. The HFHF feeding induced inflammation as shown by increased hepatic mRNA expression level of proinflammatory cytokines, TNFα, IL-1β and IL-6 by 3.2 fold (P = 0.001), 5.1 fold (P = 0.004) and 5.8 fold (P = 0.016) respectively (Fig. 4b). Substitution of n-6 PUFA with n-3 PUFA in HFHF diet normalized the proinflammatory cytokine expression levels. Interestingly, substitution of n-6 PUFA with n-3 PUFA in HFHF diet upregulated the mRNA expression level of HO-1 (Fig. 4b). Neither HFHF feeding nor n-3 PUFA supplementation altered the mRNA expression level of anti-inflammatory cytokine (IL-10) and fibrogenic marker (collagen α1) (Fig. 4c).
Substituting LA with ALA or LC n-3 PUFA attenuated liver pathology in HFHF induced NASH
Representative histological finding and NAS in the livers of the rat fed various diets are shown in Fig. 5. The liver H&E staining of control rat showed normal parenchyma and architecture without any evidence of steatosis or inflammation. In contrast, the HFHF feeding induced histological features of NASH with microvesicular and macrovesicular steatosis, infiltration of inflammatory cells and hepatocyte ballooning (Fig. 5a), leading to 5.8 fold (P < 0.001) increased NAS (Fig. 5c). Oil red O staining showed extensive lipid deposition in hepatocytes from rats fed HFHF diet (Fig. 5b). Masson trichrome staining showed no evidence of fibrosis. Substitution of n-6 PUFA with n-3 PUFA in HFHF diet reduced fat deposition, inflammation and ballooning, resulting in normalization of NAS (Fig. 5a–c).
Liver phospholipid fatty acid profile
In line with the liver SCD-1 mRNA expression level, the data on phospholipid fatty acid composition showed that compared to control, HFHF feeding increased the levels of C16:1 and C18:1 with consequent increase in the C16:1/C16:0 (2.3 fold, P < 0.001) and C18:1/C18:0 (1.4 fold, P = 0.003) ratios which are the estimated index of SCD-1 activity (Table 4). However, substitution of n-6 PUFA with n-3 PUFA normalized the ratios. Total SFA, MUFA and PUFA did not differ among the groups. Decreasing n-6:n-3 ratio in HFHF group significantly altered the n-6 and n-3 PUFA composition in phospholipids. Substituting n-6 PUFA with n-3 PUFA in the diet was associated with decrease in the proportion of LC n-6 PUFA (C20:4 n-6, C22:4 n-6 and C22:5 n-6) and increase in the proportion of LC n-3 PUFA (C20:5 n-3, C22:5 n-3 and C22:6 n-3) in the liver phospholipids. Compared to ALA supplementation (HFHF-2), LC n-3 PUFA supplementation (HFHF-5) increased the incorporation of C20:5 n-3 and C22:6 n-3 to a greater extent.
High levels of n-6 PUFA in the Western diet could potentiate inflammatory response and may induce NASH. In the present study by mimicking typical Western diet, we investigated the impact of substitution of n-6 PUFA with n-3 PUFA (varying n-6:n-3 ratio) on the development of NASH. The n-6:n-3 ratios were chosen on the basis of our previous studies, wherein we have investigated the impact of varying n-6:n-3 ratios of 200, 50, 10, 5 and 2, representing wide range of n-6 and n-3 PUFA in the present human diet, on sucrose induced insulin resistance29,30. The results of the aforementioned studies demonstrated that compared to n-6:n-3 ratios of 200, 50 and 10, substitution of LA with ALA (n-6:n-3 ratio of 2) or LC n-3 PUFA (n-6:n-3 ratio of 5) prevented sucrose induced insulin resistance and dyslipidemia. Hence, n-6:n-3 ratios of 200 (representing high n-6 PUFA and n-3 PUFA deficient), 2 and 5 were selected. The results of the study, for the first time, demonstrated that partial replacement of LA with ALA (n-6: n-3 ratio of 2) or LC n-3 PUFA (n-6:n-3 ratio of 5) attenuated Western diet induced NASH. The protective effect of n-3 PUFA supplementation include attenuation of insulin resistance and glucose intolerance, optimization of plasma and liver lipid levels, mitigation of hepatic oxidative stress and inflammation, and improvements of aminotransferase activities and histological score.
It is established that NAFLD is the hepatic manifestation of metabolic syndrome and insulin resistance may play a key role in the development and progression of NAFLD43. However, whether insulin resistance is the cause or consequence of NAFLD is yet to be established44. Elevated levels of aminotransferase, which is the marker of liver injury has been reported in NASH subjects with insulin resistance, suggesting that insulin resistance may be involved in the progression of NAFLD45,46. Increased visceral adiposity by facilitating adipose tissue lipolysis and ectopic fat accumulation induces insulin resistance. Although obesity is the major risk factor for NAFLD, it is not rare in individuals with normal BMI particularly in Asian population47. Several animal studies demonstrated that both LC n-3 PUFA and ALA prevented high fat or high sucrose diet induced insulin resistance by reducing visceral adiposity48,49. Our earlier studies also showed that substitution of linoleic acid with ALA (n-6:n-3 ratio of 2) or LC n-3 PUFA (n-6:n-3 ratio of 5) prevented sucrose induced insulin resistance by increasing peripheral insulin sensitivity29,30. In the present study, rats fed HFHF diet increased visceral adiposity without altering the body weight. Also, substitution of n-6 PUFA with n-3 PUFA in HFHF diet effectively prevented the insulin resistance and glucose intolerance. The protective effect of n-3 PUFA supplementation was associated with decrease in visceral adiposity.
High level of fructose and n-3 PUFA deficiency in the Western diet has been implicated in the development of NAFLD50. Hepatic lipogenesis is modulated by SREBP-1c, which is the key transcription factor of de novo lipogenesis and regulates downstream genes such as SCD-1, ACC and FAS. SREBP-1c plays a crucial role in the development of NAFLD51,52. Indeed, Yamada et al. demonstrated that in patients with NASH, the gene expression of hepatic SREBP-1, SCD-1, FAS and PPARγ were enhanced53. In addition, C18:1/C18:0 ratio was associated with the steatosis score whereas C16:1/C16:0 ratio was associated with lobular inflammation score53. Several studies have shown that the fructose component of the Western diet is responsible for the de novo lipogenesis by up regulating SREBP-1c54. It is well documented that dietary n-3 PUFA prevents hepatic steatosis by down regulating SREBP-1c and up regulating PPAR-α which regulates genes involved in fatty acid oxidation55,56. In the present study, HFHF feeding induced hepatic steatosis by up regulating the hepatic mRNA expression of SREBP-1c and SCD-1 without altering PPAR-α expression. Additionally, the desaturation index C16:1/C16:0 and C18:1/C18:0, which is used to estimate the SCD-1 activity was significantly increased which further supports the involvement of SCD-1 in hepatic steatosis. Substitution of n-6 PUFA with n-3 PUFA (ALA or LC n-3 PUFA) in HFHF diet prevented hepatic steatosis by down regulating the expression of SREBP-1c and SCD-1. Decreased C18:1/C18:0 and C16:1/C16:0 ratios in liver by n-3 PUFA supplementation further confirms the anti-steatotic and anti-inflammatory role of n-3 PUFA. Studies have shown that among the LC n-3 PUFA, DHA is the potent regulator of SREBP-1c, whereas EPA is PPAR- α activator55. The observed anti-steatotic effect of ALA supplementation could be due to its conversion to DHA and EPA. Recently, Monteiro et al. demonstrated that using delta-5 and delta-6 knockout mice, ALA supplementation prevents hepatic lipogenesis and the development of steatosis57, suggesting that ALA could bring about its anti-steatotic effect which is independent of its conversion to LC n-3 PUFA.
Besides significant reduction in hepatic steatosis, n-3 PUFA supplementation also prevents HFHF induced dyslipidemia. Several animal and human studies suggest that dietary n-3 PUFA (ALA and LC n-3 PUFA) decrease the risk factors associated with metabolic syndrome including dyslipidemia58,59. It is worthy to mention that both ALA and LC n-3 PUFA are equally effective in preventing HFHF induced dyslipidemia.
According to multiple hit hypothesis of NAFLD pathogenesis, oxidative stress is considered as a key contributor to the transition from simple steatosis to NASH and fibrosis60. The excessive production of reactive oxygen species due to mitochondrial dysfunction causes lipid peroxidation which in turn induces inflammation and activation of stellate cells leading to fibrogenesis. Indeed, elevated levels of markers of oxidative stress and lipid peroxidation have been reported in patients with NASH60. In the present study, HFHF feeding induced oxidative stress as evidenced by increase in liver TBARS and decrease in antioxidant enzyme activity (catalase and SOD) and GSH. Due to high degree of unsaturation, there is a concern that n-3 PUFA particularly LC n-3 PUFA may induce oxidative stress and lipid peroxidation. However, emerging evidence indicate that although high levels of LC n-3 PUFA induce oxidative stress, moderate/appropriate dose of n-3 PUFA exerts antioxidant effect61. In fact, a recent study showed that LC n-3 PUFA supplementation decrease hepatic oxidative stress and triglyceride content in high fat diet induced fatty liver25. In the present study, both ALA and LC n-3 PUFA supplementation prevented the HFHF induced hepatic oxidative stress by improving the antioxidant status through restoring the antioxidant enzyme activities and GSH level. HO-1 is an inducible antioxidant enzyme and plays an important role in the cytoprotection and tissue injury62. Interestingly, n-3 PUFA supplementation up regulated the mRNA expression of HO-1 although HFHF feeding per se did not alter its expression. Recent in vitro studies have shown that LC n-3 PUFA prevents oxidative stress by up regulating HO-1 through activation of nuclear factor erythroid 2 related factor (Nrf-2)63. Further studies are necessary to understand the molecular mechanism by which LC n-3 PUFA modulates the antioxidant enzymes particularly in NAFLD. Oxidative stress triggers inflammatory process by activating redox - sensitive transcriptional factor, NF-κB thereby causing necroinflammation leading to NASH. Rats fed HFHF diet showed up regulation of proinflammatory cytokines with the development of NASH and n-3 PUFA supplementation has been shown to prevent NASH by suppressing the inflammatory response as evidenced by down regulation of proinflammatory cytokines. Tapia et al. showed the LC n-3 PUFA supplementation prevents high fat induced hepatic steatosis and inflammation by down regulating NF-κB23. Furthermore, a recent randomized clinical trial demonstrated that combined ALA and LC n-3 PUFA supplementation in NASH patients decreased plasma lipids with improvements in liver histology64. In addition to down regulating the proinflammatory cytokines, the anti-inflammatory effect of n-3 PUFA supplementation may also be mediated by resolvins and protectins derived from EPA and DHA which are known to resolve the inflammation65.
Dysregulation of adipocytokines by orchestrating proinflammatory and insulin resistance state may contribute to the development and progression of NAFLD. Leptin is known to induce insulin resistance, hepatic steatosis and has proinflammatory role and also contributes to fibrosis, whereas, adiponectin has anti-inflammatory and insulin sensitizing effect. Several clinical studies demonstrated that circulating leptin, TNF-α and IL-6 were significantly higher in patients with NAFLD/NASH, conversely adiponectin levels were significantly reduced in NAFLD/NASH patients66. However, the role of resistin which is proinflammatory in NAFLD is inconclusive. Lemoite et al. showed that in patients with NAFLD the adiponectin:leptin ratio is inversely related to the severity of NAFLD and proposed as predictive factor of NASH67. There is evidence that LC n-3 PUFA modulates the adipocytokines by increasing circulatory adiponectin and decreasing leptin levels68. Our study showed that both ALA and LC n-3 PUFA supplementation decreased plasma leptin and resistin levels and increased adiponectin:leptin ratio, suggesting that by correcting adipocytokine imbalance, n-3 PUFA ameliorates Western diet induced NASH.
ALA undergoes series of complicated desaturation and chain elongation pathway and gets converted into biologically active LC n-3 PUFA. High intake of LA and hence high LA:ALA ratio could inhibit the conversion of ALA. Studies have suggested that substituting LA with ALA would be the most appropriate way of optimizing the conversion of ALA to LC n-3 PUFA69. In the present study, substituting LA with ALA (n-6: n-3 ratio of 2) has been shown to increase the incorporation of LC n-3 PUFA at the expense of LC n-6 PUFA in liver phospholipids, suggesting the competitive interaction and inhibition of the desaturation and elongation of LA to LC n-6 PUFA and preferential conversion of ALA to LC n-3 PUFA.
In conclusion, the results of the present study demonstrated that substitution of dietary linoleic acid with α-linolenic acid (n-6:n-3 ratio of 2) or LC n-3 PUFA (n-6:n-3 ratio of 5) protects against the development of Western diet induced NASH as evidenced by improved liver histology, decreased liver and plasma lipids and reduced plasma aminotransferase levels. The protective effect of n-3 PUFA supplementation was attributed to the marked reduction in hepatic oxidative stress and proinflammatory cytokines. The present study also highlights the importance of balancing the n-6 and n-3 PUFA in the diet through suitable blending of vegetable oils for the prevention and management of diet related chronic diseases including NAFLD.
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This work was supported by Grants in aid (5/4/3-7/TF/2011/NCD-II) from Indian Council of Medical Research, Government of India to AI. JS was supported by a fellowship from Indian Council of Medical Research, Government of India.
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Jeyapal, S., Kona, S.R., Mullapudi, S.V. et al. Substitution of linoleic acid with α-linolenic acid or long chain n-3 polyunsaturated fatty acid prevents Western diet induced nonalcoholic steatohepatitis. Sci Rep 8, 10953 (2018). https://doi.org/10.1038/s41598-018-29222-y
DOI: https://doi.org/10.1038/s41598-018-29222-y
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i think it's weird time to ask about new features, as all main discussions are about Scaler 2, which is only available to the beta testers… at least for 1 month
i've been asked to add 1 minute in the middle of an already pretty finished track,
without changing everything but keeping listeners interested, this for a dance show.
( but this situation can generalize to clients coming with a ref track etc… )
there's only rhythmic chords and rhythm, no melodic part.
my 4 chords progression is something like :
CM / Em / Gm / Dm
there is many ways to approach that, but i will mainly >
find a "melody" ( top voice leading), than play with inversions and adding tension to match it using always same 4 chords.
maybe playing with a few substitutions to add interest at some point ( if pertinent )
or just laying out all sort of options and do trial-and-error to find what sounds good…
but usually this is a long process, as my harmonic beginning and end are already there,
there is a lot of possibility, but only a few will work …
So my question is >
is there a way to show all possible subs / replacement for a chord ( including borrowing possibilities, modal interchange, chromatic mediants, tritone subs… )
on one page ( pad view ) ?
And to sort them, for exemple by common notes / closeness to the original,
but also by top or bass note ?


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Memorandum Circular No. 89
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Memorandum Circular No. 78
July 9, 2015
Upon the Recommendation of the National Disaster Risk Reduction and Management Council (NDRRMC) and in View of the Inclement Weather Brought About By the Southwest Monsoon, Enhanced By Typhoon "Falcon," Work in Government Offices in the National Capital Region, Is Hereby Suspended Starting 3:00 P.M. Today, 09 July 2015.
Memorandum Circular No. 77
July 6, 2015
Upon the Recommendation of the National Disaster Risk Reduction and Management Council (NDRRMC) and in View of the Inclement Weather Brought About By Typhoon "Egay," Work in Government Offices and Classes in All Levels, Both Public and Private, in the National Capital Region, Are Hereby Suspended Today, 06 July 2015.
Memorandum Circular No. 76
April 15, 2015
Urging All Government Departments, Bureaus, Offices, Agencies, Including Government-Owned and Controlled Corporations, Government Financial Institutions, State Universities and Colleges, and Local Government Units to Implement the Integrity Management Program (Imp) Through the Establishment of Integrity Management Systems (Ims) and Adopt the Imp Handbook For This Purpose
Memorandum Circular No. 75
February 9, 2015
Authorizing All Government Agencies and Instrumentalities Including Government-Owned Or -Controlled Corporations (GOCCS) and Encouraging Local Government Units (Lgus) to Extend Support and Participation to the Kabisig Philippine Government Expo and Trade Fair 2015
The Lawphil Project - Arellano Law Foundation, Inc.


text:
Just an idea to help people optimise / troubleshoot any streaming problems, but could Infuse display the protocol level it has negotiated with the source server when establishing the connection/share. Would not need to take up much screen space at all. Very discreet
Although it should be possible to do it on the server side. Unfortunately I have got all kind of information, at least for SMB on my NAS (running Linux) but I couldn't manage o see the SMB version for the connections.


text:
Custom-made solid wood for all your projects
Already a customer ?
New customer?Register
In stock - Estimated delivery between 21/02 and 24/02
Origin Portugal
Manufacturing Medium Density Fiberboard (MDF): panel made using wood fibres that are integrally dyed with natural organic colouring agents and a special, non-toxic resin.
Dimensional tolerances
Thickness: +/-0.3 mm
Dimensions: +/- 2 mm
Fire classification
Class D-s2, d0
M3 classification according to standard En 13501
(mildly flammable)
Other technical characteristics
Valchromat E05 is a mass-dyed wood fiber panel with formaldehyde emission ≤ 0.05 ppm.
The fibers are impregnated with organic dyes and chemically bonded to each other by a special resin which gives Valchromat unique physico-mechanical characteristics.
Valchromat is an MDF.HLS, according to the EN 622-5 standard.
The Valchromat panel, due to the use of organic dyes and the natural variation in the color of the wood, has differences in hue. This variation can be observed on the same face, between faces of the same panel, between different productions or thicknesses.
Valchromat is a water-repellent panel, supplied unfinished, in which the application of varnish, wax or oil is essential.
Whenever the panel is used in wet areas, such as kitchen worktops and bathrooms, it should be protected with varnish and the faces and fields should be well sealed.
The Valchromat panel should not be used in areas where there is direct contact with water, such as kitchen worktop sink cutouts and shower cubicles.
The Valchromat panel must be applied with a finish, in order to protect the surface, maintaining its natural appearance.
The finish can be done with varnishes, waxes or oils.
Any suitable varnish for wood can be applied over Valchromat. Two-component acrylic varnishes are widely used, as they do not yellow over time. When the finish is made with varnish, the first layer to be applied is a primer, also called "insulator" or "pore filler". After the first layer has dried, the surfaces are sanded with a fine grain of 320, in order to eliminate the raised pores of the wood. Then, a finishing varnish is applied in one or two coats, depending on the manufacturer's prescription. Between coats, surfaces are sanded with 320 fine grit. It is recommended that the applied primer and clearcoat be from the same manufacturer so that there is no incompatibility between them.
Waxes or oils are usually applied in a single coat to previously prepared surfaces. These types of finishes should not be applied to panels that will be installed in humid environments, such as kitchens and bathrooms. Avoid linseed oil type oils.
Average score :
3 avis


text:
"Most of the current systems are not well matched to the needs of future conflicts, which DARPA anticipates being much less permissive, very dynamic and characterized by a higher level of threats, contested electromagnetic spectrum and relocatable targets," DARPA said.
As USA Today reports, a DARPA budget released this month shows an increase in spending on drone development. For the 2015 fiscal year, which begins Oct. 1, DARPA will spend $15 million on the CODE program, up from $8 million this current year.
Read more: http://www.usatoday.com/story/nation/2014/03/28/pentagon-drones-collaborative-operations-denied-environment/7003605


text:
Central blotter: Man smoking weed with friends doesn't realize his car had been stolen
While a man was at a party in the 1100 block of Sea Pines Way, someone stole his 2000 Nissan Altima. The man told police he was smoking pot with his friends and last saw his car around midnight. The keys were left inside it. He came out at 2 a.m. and saw the car was missing. Because he thought his friends had moved his car, he continued smoking pot with his friends and did not report the missing vehicle right away.
More Palm Beach County and Treasure Coast police blotters
A laptop was stolen from a residence in the 8000 block of Ambach Way in Hypoluxo. The victim told police she was gone for about five minutes. The front door was left unlocked because it was broken.
Palm Beach County Booking Blotter: See who's been arrested
Someone threw a piece of concrete and broke a window of a residence in the 700 block of Ridge Road.
Crime in Palm Beach County: Latest news, booking blotter, more
A wine vendor working at a store in the 1400 block of South Dixie Highway set down his cellphone and left his display stand to talk to his boss. When he returned, the phone was gone. A review of surveillance video showed an older man take the phone.
Wanted fugitives in Palm Beach County
A woman in the 3200 block of Turtle Cove told police she posted a photo of snow on the ground in Michigan, where she was about to travel to see family. A former high school classmate posted four troubling comments below the photo, making threats to kill her and her family. She said she has since blocked the former classmate from her Facebook account. She wanted police to document the incident.
An asset protection employee at a store in the 500 block of South Rosemary Avenue observed two juveniles handing off clothes to each other. One of the youths took all the clothing and headed to the exit without stopping to pay for the items. He was detained and his friend ran off. The stolen merchandise included shirts, socks, hats and a jacket, with a total value of $382. The juvenile was arrested and taken to the Juvenile Detention Center.
A man placed $121.59 worth of merchandise into his waistband and tried walking out of a store in the 1500 block of Palm Beach Lakes Boulevard without paying for the items. He was detained and arrested. He admitted taking the drill bits, switchblade and nut driver to support his heroin addiction. He was given a notice to appear in court and released at the scene with a trespass warning.
A man left his work van with the keys in the ignition parked in front of a residence in the 400 block of 17th Avenue South. He came outside to see the van driving off.
A burglar entered a residence in the 1100 block of South C Street by breaking a bathroom window. The only thing missing from the home was change taken from a bedroom drawer.
A stereo was taken from an apartment in the 1300 block of South Federal Highway by someone who pried open the front door.
A TV was stolen from a residence in the 400 block of South J Street.
Someone broke a window on a vehicle in the 1600 block of 17th Avenue North and removed a wallet. Contents included credit cards, a driver's license and $45.
An unlocked vehicle in the 200 block of North E Street was ransacked. Keys to a workplace were stolen.
An iPod, sunglasses and an umbrella were taken from a vehicle parked in the 1700 block of 22nd Avenue North.
Clothing, a wallet and a 9 mm Smith & Wesson handgun were removed from a vehicle parked in the 1800 block of Pierce Drive. The victim said he believed he locked his vehicle, but there was no sign of forced entry.
A woman in the 600 block of Latona Avenue took pity on a former neighbor and let her spend the night. In the morning, the woman awoke to find her guest gone, along with a video gaming system, controllers and about 100 games. She told a deputy she had known the former neighbor for about a year, but did not know her last name. She said the neighbor had asked her to cash a check for $175 for her, and she agreed and deposited the check into an ATM. Attempts by the deputy to contact the former neighbor by phone were unsuccessful.
An antiques dealer was in the process of moving some of his merchandise from one storage unit to another in the 1500 block of North Dixie Highway. He left a wooden side table valued at $1,450 next to the elevator. When he returned, the table was gone. A witness said a woman driving a minivan had taken the table and a vase.
Two checks and a computer tablet were taken during a break-in at a residence in the 1100 block of Grandview Circle.
A woman in the 200 block of Bobwhite Road was getting ready for work and opened her garage door. When she went back into the house, she saw a teen riding off on her bicycle. A deputy spoke to a woman walking her dog in the neighborhood, who told him someone had left a bike in her driveway that matched the description of the stolen bike. A man on the same block reported that someone tried to steal his bicycle, but abandoned it because it had a flat tire.
A man was arrested after he stole ink packets with a total value of $175.94 from a store in the 9900 block of Belvedere Road.
A man was given a trespass warning from a business in the 1300 block of Royal Palm Beach Boulevard after he argued with the manager. On a previous occasion the man had written on the bathroom mirrors and tip jar, because the TV in the seating area wasn't working.
A man at a gas station in the 1100 block of Royal Palm Beach Boulevard told a deputy that a woman in her mid-40s reached into his vehicle and stole his wallet. He said she appeared to be a "crack head." She ran off toward a nearby gas station.
A man in the 1100 block of Basswood Place was cleaning his pickup when he realized his Smith & Wesson .380 semi-automatic pistol was missing. He was unable to pinpoint when the theft occurred.
Someone rummaged through a pickup parked in the 1500 block of Wyndcliff Drive, but nothing appeared to be missing.
A golf cart was stolen from the 14400 block of Pierson Road. The cart had been chained to a wooden fence, which was broken by the thief. The value of the golf cart was $5,000.
Someone broke off an exterior hose valve stem, causing water to run constantly from the exterior of a residence in the 10600 block of Versailles Boulevard.
A vehicle struck a mailbox in the 15400 block of Meadow Wood Drive. Damage to the mailbox was estimated at $450.
A picnic table was damaged at a park in the 1000 block of Goldenrod Road. Charring on the table top was evident. Damage was about $300. The table was removed from the park.
A burglar cut the wires to the alarm system of a trailer at a business in the 15500 block of Okeechobee Boulevard, then stole a laptop computer, $400 and a large quantity of vitamins. Damage to the alarm system was estimated to be $500.
Someone broke into a trailer at a residence in the 14500 block of 85th Road North and stole two motorcycles, two helmets, six motorcycle jerseys and six pairs of pants.
A lock latch on a wooden shed was tampered with at a property in the 13100 block of 40th Street North. Nothing was taken from the shed.
A backhoe was damaged overnight at a construction site in the 15800 block of Southern Boulevard. Damage was estimated at $500. It was believed someone was trying to start up the backhoe in order to remove heavy objects placed in front of cargo containers to stop burglaries.
Two men got into a fight at a family party in the 400 block of Jennings Avenue. Police arrived just after midnight to quell the disturbance. Neither of the combatants would provide details of the incident. Officers noticed one man had been drinking. His eyes were bloodshot, his speech slurred and his balance impaired. He began yelling at the other man, continuing the altercation. The commotion was disturbing the neighborhood, so police arrested him. He was taken to the county jail.
During a traffic stop on South Jog Road and Cresthaven Boulevard, a passenger was arrested when a police officer found marijuana on her seat and a bag of pot on the ground next to her door. She was taken to the county jail.
An asset protection associate at a store in the 6200 block of Forest Hill Boulevard saw a man select a CD player and stuff it down the front of his pants. The man then headed for the door. He was detained and the $41.13 CD was recovered. Because the man had more than two prior convictions for petit theft, he was arrested on a felony petit theft charge and taken to the county jail.
A customer at a store in the 6200 block of Forest Hill Boulevard put a cellphone charger and memory card into his shorts, then exited the store. He was stopped and the items, with a total value of $34.82, were recovered. The man was arrested and released with a notice to appear in court.


text:
Just Andersen
Vase en metal patiné de Just Andersen, Suède
Just Andersen
The other side of the vase
Just Andersen
Side view
Just Andersen
the inside of the vase, showing traces of use
Just Andersen
Markon the bottom of the base
Just Andersen flower vase in patinated disco metal
Just Andersen (1884-1943), a good art deco flower vase in disco metal, height 16 cm, width 18 cm, depth 9,5 cm marked and with model number D20 on the base. Traces of use, some scratching, inside with chalk residue.
Period : 1920-1940
Size : Medium
Price : 150,00 €
Contact us about this item.
We can offer you various tailor-made solutions for shipping, please contact us if you require a shipping quote. Within Belgium, insofar as we do not have to rely on third parties and / or rental of transport material, deliveries are free from a purchase of € 1000. Deliveries within Ghent are free but subject to the limitations of the circulation plan. Deliveries always take place on the ground floor.
Contact us about this item.


text:
Zoom SGH-6 Shotgun Microphone Capsule for H5 and H6 Recorders
The SGH-6 from Zoom offers a shotgun microphone solution for use with the H5 and H6 handheld recorders. This capsule employs three internal microphones to achieve a frequency-independent, super-cardioid polar pattern. The SGH-6 is an appropriate choice for electronic news gathering, film, video, and other applications that call for tight, highly directional shotgun miking.
- Use with the H5 and H6 Recorders
- Directional Shotgun Mic
- Three Internal Microphones
- Built-In Preamp
- Super-Cardioid Polar Pattern
- Designed for Film, ENG and More
- Hairy Windscreen Included
- Microphone Type
- Shotgun
- Polar Pattern
- Super-cardioid
- Preamplifier
In The Box
6 and 12 Month Special Financing* Available
On purchases of $299 or more. Online through Klarna, available at checkout.


text:
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