The Effect of a Western Diet on Hepatic Autophagy in Age Accelerated SAMP8 Mice

Non-alcoholic steatohepatitis (NASH) is characterized as a dysregulation of hepatic lipid metabolism and a chronic inflammatory state. It is hypothesized the link between lipid dysregulation and inflammation may be due in part to defective hepatic autophagy and reduced mitochondrial capacity to oxidize fatty acids. It remains to be determined; however, the effects of a Western diet on hepatic autophagy and mitochondrial function during aging. PURPOSE: The purpose of this study was to determine the effect of a high-fat high fructose diet (HFF) on markers of hepatic autophagy and mitochondrial function in an age accelerated mouse model. METHODS: Twenty week old, male and female, SAMP8 mice (n=49) were randomly assigned, matching for gender, to either a standard chow (SC) or HFF (45% fat, 24% fructose) diet for 32 weeks. Liver tissue was analyzed for mRNA expression of autophagic (BNIP3, Beclin 1, p62, and Atg7) and mitochondrial (PGC1α and COXIV) genes. Differences between gender and dietary groups were identified by a 2 x 2 ANOVA and statistical significance was set at p\u3c0.05. RESULTS: Following 32 weeks of feeding, male mice fed the HFF diet were significantly heavier than male mice in the SC group (31.6 g vs 26.5 g; p=0.001); however, no difference was observed between diet groups for female mice. The HFF diet resulted in higher autophagic activity as observed by Beclin 1 (+36%; p=0.001) and BNIP3 (+40%; P=0.003) expression. Despite the higher autophagic activity, p62 was higher (+31%; p\u3c0.001) in the HFF compared to the SC group, suggesting impaired autophagic flux. In addition, mitochondrial COXIV expression was elevated (+43%; P\u3c0.001) in the HFF group compared to the SC group suggesting increased β-oxidation. Overall, the expression of all autophagic and mitochondrial markers was higher in male compared to female mice; however, both sexes responded similarly to the HFF diet. CONCLUSION: Despite the higher expression of autophagic and mitochondrial genes, elevated expression of p62 suggests an impaired autophagic flux in age accelerated mice following a Western diet

Dietary Enrichment of Fish-Oils Attenuates Diet-Induced Obesity and Hepatic Steatosis

Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of excess hepatic fat, exceeding 5% of total liver mass. NAFLD is present in one-third of Americans and up to 90% in those who are obese. NAFLD develops largely in part to consumption of a Western diet, defined as 40-60% kcal from saturated fats; however, a diet rich in fish-oils may prevent and reverse the development of steatosis. PURPOSE: To determine the effects of fish oils on the development of NAFLD. METHODS: C57BL/6 (n=91) mice were randomly assigned to four dietary groups for 32-weeks: 10% lard (LFL), 10% fish-oil (LFFO), 41% lard (HFL), or 41% fish-oil (HFFO) diet. Significant differences (p\u3c0.05) between groups were identified by a one-way ANOVA. RESULTS: When compared to HFFO, mice in the HFL group saw an greater (Table 1) body mass and net glucose AUC by 13% (p\u3c0.001) and 24% (p=0.08), respectively. No significant difference was observed between LFL and LFFO for body mass, net glucose AUC or HOMA-IR. This is interesting given no significant difference was observed between groups for the mean weekly caloric intake. HFFO mice showed an 86% lower (p\u3c0.001) total hepatic lipid and 4.8-fold lower (p\u3c0.001) hepatic triglyceride concentration when compared to HFL. HFFO mice also saw a 32% lower (p\u3c0.001) total hepatic cholesterol when compared to HFL. There was no significant difference in total hepatic lipids between LFL and LFFO. CONCLUSION: Despite for no significant difference in caloric intake between high-fat diet groups, consumption of a high-fat diet rich in fish-oils prevented dietary induced obesity, insulin resistance and hepatic steatosis. These results suggest that a diet rich in fish-oils have preventative effects on the development of NAFLD

Fish-oils Increase BAMBI Expression to Protect Against Fibrotic Activity in LPS Stimulated Hepatic Tissue

Non-alcoholic steatohepatitis (NASH), defined as excess hepatic lipid and chronic inflammation, provides an environment prone for the development of hepatic fibrosis. Recent evidence suggests that the antifibrotic protein BAMBI (BMP-Activin membrane bound inhibitor) is downregulated in the presence of inflammation, and may be central to the development of fibrosis. Diets rich in omega-3 (w-3) fatty acids are known to provide anti-inflammatory effects; however, the effects of w-3 fatty acids on hepatic fibrosis are not well-established. PURPOSE: To determine the effects of fish-oils on the hepatic fibrosis signaling cascade, following 32-weeks of high-fat feeding in a LPS-induced model of NASH. METHODS: Male C57BL/6 mice were randomly assigned to one of four diets for 32 weeks (n=9/group): low-fat lard based (LFL, 10% kcal fat), low-fat fish-oil based (LFFO, 10% kcal fat), high-fat lard based (HFL, 41% kcal fat), or high-fat fish-oil based (HFFO, 41% kcal fat). Following in situ LPS stimulation, liver mRNA expression of CD14, TLR4, MyD88, BAMBI, and TGF-β1 was quantified using quantitative RT-PCR. Differences between diets were identified using a one-way ANOVA with statistical significance set at p\u3c0.05. RESULTS: Following LPS stimulation, CD14 was increased 2.5 fold (p=0.020) in HFFO when compared to HFL. Despite the increase in CD14, TLR4 showed no difference between groups. In contrast, MyD88 was 2.8 fold greater (p\u3c0.001) in HFL compared to HFFO. In comparison to untreated tissue, BAMBI was 1.7 fold (p=0.017) higher in the HFFO LPS-stimulated tissue, which best explained the 1-fold (p=0.004) lower expression of TGF-β1 in HFFO when compared to HFL post-LPS stimulation. CONCLUSION: Despite the increase in extracellular LPS signaling receptor CD14, the consumption of fish-oils produced a protective intracellular response as observed by an increase in BAMBI and decrease in TGF-β1. These results suggest that a diet high in w-3 fatty acids may protect against the development of hepatic fibrosi

Evidence for a Role of Endocannabinoids, Astrocytes and p38 Phosphorylation in the Resolution of Postoperative Pain

An alarming portion of patients develop persistent or chronic pain following surgical procedures, but the mechanisms underlying the transition from acute to chronic pain states are not fully understood. In general, endocannabinoids (ECBs) inhibit nociceptive processing by stimulating cannabinoid receptors type 1 (CB(1)) and type 2 (CB(2)). We have previously shown that intrathecal administration of a CB(2) receptor agonist reverses both surgical incision-induced behavioral hypersensitivity and associated over-expression of spinal glial markers. We therefore hypothesized that endocannabinoid signaling promotes the resolution of acute postoperative pain by modulating pro-inflammatory signaling in spinal cord glial cells.To test this hypothesis, rats receiving paw incision surgery were used as a model of acute postoperative pain that spontaneously resolves. We first characterized the concentration of ECBs and localization of CB(1) and CB(2) receptors in the spinal cord following paw incision. We then administered concomitant CB(1) and CB(2) receptor antagonists/inverse agonists (AM281 and AM630, 1 mg x kg(-1) each, i.p.) during the acute phase of paw incision-induced mechanical allodynia and evaluated the expression of glial cell markers and phosphorylated p38 (a MAPK associated with inflammation) in the lumbar dorsal horn. Dual blockade of CB(1) and CB(2) receptor signaling prevented the resolution of postoperative allodynia and resulted in persistent over-expression of spinal Glial Fibrillary Acidic Protein (GFAP, an astrocytic marker) and phospho-p38 in astrocytes. We provide evidence for the functional significance of these astrocytic changes by demonstrating that intrathecal administration of propentofylline (50 microg, i.t.) attenuated both persistent behavioral hypersensitivity and over-expression of GFAP and phospho-p38 in antagonist-treated animals.Our results demonstrate that endocannabinoid signaling via CB(1) and CB(2) receptors is necessary for the resolution of paw incision-induced behavioral hypersensitivity and for the limitation of pro-inflammatory signaling in astrocytes following surgical insult. Our findings suggest that therapeutic strategies designed to enhance endocannabinoid signaling may prevent patients from developing persistent or chronic pain states following surgery

Diet and Sex Differences Induce Unique Alterations of Markers for Blood Brain Barrier Integrity in Age-Accelerated Mice

The role of diet on brain health has received significant attention, with the Western diet (WD) contributing to cerebrovascular alterations and neurodegenerative disease. The blood-brain barrier (BBB) may play a particularly important role as it forms the interface between the peripheral circulation and the central nervous system. The WD has been shown to negatively impact the BBB. Whether there are sex specific differences with diet on BBB integrity remains unclear. PURPOSE: To determine the effect of diet and sex on the mRNA expression of markers of BBB integrity in an age-accelerated mouse model. METHODS: Male and female Senescence Accelerated Mouse-Prone 8 (SAMP8) mice were randomly assigned to a standard diet (SD) or WD formula for a 32-week period, matched for sex, ending at 12-months of age (n=10-14/group). At 12-months of age, cortical brain tissue was evaluated for the expression of mRNA for targets associated with BBB integrity (Cldn-1, Cldn-3, Cldn-5, Cldn-12, F11r, Lsr, Msfd2a, Ocln, Tjp) using quantitative RT-PCR. A two-way ANOVA was used to identify whether mRNA expression of these targets differed with sex, diet, and their interaction. RESULTS: A significant (pCONCLUSION: Overall, female mice presented with higher expression of mRNA markers for BBB integrity, which may be a protective factor. Furthermore, mice fed the WD had lower mRNA expression of markers of BBB integrity suggesting that a Western diet may accelerate the pathogenesis of the disease state