A Designer Synbiotic Attenuates Chronic-Binge Ethanol-Induced Gut-Liver Injury in Mice.

Gut dysbiosis and altered short-chain fatty acids are associated with ethanol-induced liver injury. SCFA are fermentation byproducts of the gut microbiota known to have many beneficial biological effects. We tested if a designer synbiotic could protect against ethanol-induced gut-liver injury. C57BL/6 female mice were exposed to chronic-binge ethanol feeding consisting of ethanol (5% vol/vol) for 10 days, followed by a single gavage (5 g/kg body weight) 6 h before euthanasia. A group of mice also received oral supplementation daily with a designer synbiotic, and another group received fecal slurry (FS); control animals received saline. Control mice were isocalorically substituted maltose dextran for ethanol over the entire exposure period. Ethanol exposure reduced expression of tight junction proteins in the proximal colon and induced hepatocyte injury and steatosis. Synbiotic supplementation not only mitigated losses in tight junction protein expression, but also prevented ethanol-induced steatosis and hepatocyte injury. Ethanol exposure also increased hepatic inflammation and oxidative stress, which was also attenuated by synbiotic supplementation. Mice receiving FS were not protected from ethanol-induced liver injury or steatosis. Results were associated with luminal SCFA levels and SCFA transporter expression in the proximal colon and liver. These results indicate supplementation with a designer synbiotic is effective in attenuating chronic-binge ethanol-induced gut-liver injury and steatosis in mice, and highlight the beneficial effects of the gut microbial fermentation byproducts.

Successful detection, expression and purification of the alternatively spliced truncated Sm14 antigen of an Egyptian strain of Schistosoma mansoni.

Schistosoma mansoni causes intestinal schistosomiasis, a disease that is prevalent in several regions worldwide. To date, a protective vaccine against S. mansoni is still lacking. Several promising antigens have been discovered and evaluated for vaccine protection, such as Sm14 and Sm28GST. In this short communication, we report the successful detection of an alternatively spliced truncated form of Sm14 which was highly expressed in an Egyptian strain of S. mansoni. This truncated Sm14 (TrSm14) protein was formerly reported to be practically non-existent and its complementary DNA (cDNA) was thought to be 'a rare misprocessing of mRNA precursor'. Our finding demonstrates that there is inter-strain variation in the S. mansoni transcriptome and subsequently in the role/function of the expressed proteins. We expressed TrSm14 successfully in Escherichia coli as a fusion protein with the schistosomal antigen Sm28GST. The fusion protein was purified using metal affinity chromatography and was found to be reactive with serum from S. mansoni-infected patients. This suggests a possible diagnostic value for this protein in detection of anti-schistosomal antibodies. In addition, this fusion protein could offer a potential bivalent vaccine candidate against S. mansoni that is worthy of further investigation.

Apolipoprotein E isoforms disrupt long-chain fatty acid distribution in the plasma, the liver and the adipose tissue of mice.

Evidences suggest that omega-3 fatty acid (n-3 PUFA) metabolism is imbalanced in apolipoprotein E epsilon 4 isoform carriers (APOE4). This study aimed to investigate APOE genotype-dependant modulation of FA profiles, protein and enzyme important to fatty acid (FA) metabolism in the adipose tissue, the liver and the plasma using human APOE-targeted replacement mouse-model (N=37). FA transport (FATP) and binding (FABP) protein levels in tissues and concentrations of liver carnitine palmitoyltransferase 1 (CPT1) were performed. N-3 PUFA concentration was >45% lower in the adipose tissue and liver of APOE4 mice compared to APOE3 mice. In APOE4 mice, there were higher levels of FATP and FABP in the liver and higher FATP in the adipose tissue compared to APOE2 mice. There was a trend towards higher CPT1 concentrations in APOE4 mice compared to APOE3 mice. Therefore, since APOE-isoform differences were not always in line with the unbalanced n-3 PUFA profiles in organs, other proteins may be involved in maintaining n-3 PUFA homeostasis in mice with different APOE-isoforms.

Role of fatty acid transport protein 4 in oleic acid-induced glucagon-like peptide-1 secretion from murine intestinal L cells.

The antidiabetic intestinal L cell hormone glucagon-like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion and inhibits gastric emptying. GLP-1 secretion is stimulated by luminal oleic acid (OA), which crosses the cell membrane by an unknown mechanism. We hypothesized that L cell fatty acid transport proteins (FATPs) are essential for OA-induced GLP-1 release. Therefore, the murine GLUTag L cell model was used for immunoblotting, [(3)H]OA uptake assay, and GLP-1 secretion assay as determined by radioimmunoassay following treatment with OA ± phloretin, sulfo-N-succinimidyl oleate, or siRNA against FATP4. FATP4(-/-) and cluster-of-differentiation 36 (CD36)(-/-) mice received intraileal OA, and plasma GLP-1 was measured by sandwich immunoassay. GLUTag cells were found to express CD36, FATP1, FATP3, and FATP4. The cells demonstrated specific (3)H[OA] uptake that was dose-dependently inhibited by 500 and 1,000 µM unlabeled OA (P < 0.001). Cell viability was not altered by treatment with OA. Phloretin and sulfo-N-succinimidyl oleate, inhibitors of protein-mediated transport and CD36, respectively, also decreased [(3)H]OA uptake, as did knockdown of FATP4 by siRNA transfection (P < 0.05-0.001). OA dose-dependently increased GLP-1 secretion at 500 and 1,000 µM (P < 0.001), whereas phloretin, sulfo-N-succinimidyl oleate, and FATP4 knockdown decreased this response (P < 0.05-0.01). FATP4(-/-) mice displayed lower plasma GLP-1 at 60 min in response to intraileal OA (P < 0.05), whereas, unexpectedly, CD36(-/-) mice displayed higher basal GLP-1 levels (P < 0.01) but a normal response to intraileal OA. Together, these findings demonstrate a key role for FATP4 in OA-induced GLP-1 secretion from the murine L cell in vitro and in vivo, whereas the precise role of CD36 remains unclear.

Diagnosis in bile acid-CoA: amino acid N-acyltransferase deficiency.

Cholate-CoA ligase (CCL) and bile acid-CoA: amino acid N-acyltransferase (BAAT) sequentially mediate bile-acid amidation. Defects can cause intrahepatic cholestasis. Distinction has required gene sequencing. We assessed potential clinical utility of immunostaining of liver for CCL and BAAT. Using commercially available antibodies against BAAT and CCL, we immunostained liver from an infant with jaundice, deficiency of amidated bile acids, and transcription-terminating mutation in BAAT. CCL was normally expressed. BAAT expression was not detected. Immunostaining may facilitate diagnosis in bile-acid amidation defects.

Fatty acid- and cholesterol transporter protein expression along the human intestinal tract.

BACKGROUND: Protein distribution profiles along the human intestinal tract of transporters involved in the absorption of cholesterol and long-chain fatty acids (LCFA) have been scarcely evaluated. METHODOLOGY/PRINCIPAL FINDINGS: In post-mortem samples from 11 subjects, intestinal transporter distribution profiles were determined via Western Blot. Differences in transporter protein levels were statistically tested using ANOVA and Tukey's Post Hoc comparisons. Levels in all segments were expressed relative to those in duodenum. Except for ABCG5 and FATP4, levels (mean+/-SEM) were the highest in the ileum. For ABCA1, ileal levels (1.80+/-0.26) differed significantly from those in duodenum (P = 0.049) and proximal colon (0.92+/-0.14; P = 0.029). ABCG8 levels in ileum (1.91+/-0.30) differed from those in duodenum (P = 0.041) and distal colon (0.84+/-0.22; P = 0.010) and jejunum (1.64+/-0.26) tended to be higher than distal colon (0.84+/-0.22; P = 0.087). Ileal NPC1L1 levels (2.56+/-0.51) differed from duodenum levels (P = 0.019) and from distal colon (1.09+/-0.22; P = 0.030). There was also a trend (P = 0.098) for higher jejunal (2.23+/-0.37) than duodenal NPC1L1 levels. The levels of ABCG5 did not correlate with those of ABCG8. FAT/CD36 levels in ileum (2.03+/-0.42) differed from those in duodenum (P = 0.017), and proximal and distal colon (0.89+/-0.13 and 0.97+/-0.15 respectively; P = 0.011 and P = 0.014). FABPpm levels in ileum (1.04+/-0.13) differed from proximal (0.64+/-0.07; P = 0.026) and distal colon (0.66+/-0.09; P = 0.037). CONCLUSIONS/SIGNIFICANCE: The distribution profiles showed a bell-shape pattern along the GI-tract with the highest levels in ileum for ABCA1, ABCG8, NPC1L1, FATCD36 and FABPm, suggesting a prominent role for ileum in transporter-mediated uptake of cholesterol and LCFAs.

Preparing for migration? The effects of photoperiod and exercise on muscle oxidative enzymes, lipid transporters, and phospholipids in white-crowned sparrows.

The extreme energetic demands of avian migration result in various physiological changes that can be observed during the migratory period. However, the degree to which birds alter muscle physiology in advance of migration has been poorly studied. We studied the effects of "migratory" photoperiod and exercise on metabolic enzymes, fatty acid transporter mRNA expression, and muscle phospholipid fatty acid composition in captive white-crowned sparrows (Zonotrichia leucophrys). Ten sparrows were held on short photoperiod (8L:16D) for 58 d then switched to long days (16L:8D) for 3 wk before sampling. Increased nightly activity indicated that the birds were indeed in migratory condition. Another 13 birds were held on short days during the entire experiment, and a subset (5) were exercised for 1 h every other day for the last 2 wk. "Migratory" photoperiod did not change the activities of citrate synthase, carnitine palmitoyl transferase, and 3-hydroxyacyl-CoA dehydrogenase or the expression of FAT/CD36, FABPpm, and H-FABP mRNA in pectoralis muscle, suggesting that these cannot be increased in advance of migratory flight. Docosahexaenoic acid increased in pectoralis muscle phospholipids with exercise but was negatively correlated with catabolic enzyme activity, indicating that the presence of this fatty acid may not aid migratory performance as suggested by other studies.

FATP1 localizes to mitochondria and enhances pyruvate dehydrogenase activity in skeletal myotubes.

Fatty acid transport protein 1 (FATP1) has been previously immunolocalized in intracellular compartments. Here we show that FATP1 localizes to the mitochondria in cultured myotubes, by immunoblots of subcellular fractions and immunocytology of the fusion protein FATP1-GFP. FATP1 strongly stimulates CO(2) production from glucose whereas nonmitochondrial metabolism of glucose is only slightly enhanced. FATP1 raises the activity and activates the pyruvate dehydrogenase (PDH) complex and the pyruvate decarboxylase PDH-E1 catalytic subunit, without changing E2, E3BP or E1alpha and increasing E1beta protein content. These data reveals the localization and points to a regulatory function of FATP1 in myotube mitochondria.

Fatty acid transporter levels and palmitate oxidation rate correlate with ejection fraction in the infarcted rat heart.

OBJECTIVES: Cardiac fatty acid uptake occurs predominantly via sarcolemmal transporter proteins; fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm) and fatty acid transporter proteins (FATP) 1 and 6. We hypothesised that levels of the fatty acid transporters would be reduced in the chronically infarcted rat heart, in parallel with reduced dependence on fatty acid utilisation. METHODS AND RESULTS: In vivo left ventricular ejection fractions, measured using echocardiography, were 36% lower in rats six months after coronary artery ligation than in sham-operated control rats. In isolated, perfused, infarcted hearts, 3H-palmitate oxidation was 30% lower, and correlated with in vivo ejection fractions. As myocardial lipid incorporation was also reduced by 25%, total palmitate utilisation was 29% lower in the infarcted rat heart. The protein levels of the cardiac fatty acid transporters were reduced in the infarcted rat heart; FAT/CD36 by 36%, FABPpm by 12%, FATP6 by 21% and FATP1 by 26%, and the cytosolic fatty acid binding protein (cFABP) was 47% lower than in sham-operated rat hearts. Fatty acid transporter levels correlated with both palmitate oxidation rates and cardiac ejection fractions. CONCLUSIONS: Reductions in fatty acid oxidation and lipid incorporation rates were accompanied by downregulation of the cardiac fatty acid transporters. The metabolic shift away from fatty acid utilisation was proportional to the degree of functional impairment in the chronically infarcted rat heart.

Viability of the isolated soleus muscle during long-term incubation.

Skeletal muscle metabolism has been examined in perfused hindlimb muscles and in isolated muscle preparations. While long-term viability of the fast-twitch epitrochlearis has been documented with respect to glucose transport, it appears that long-term incubated soleus muscles are less stable when incubated ex vivo for many hours. Therefore, in the present study, we have examined whether the isolated soleus muscle remains metabolically viable for up to 18 h with respect to maintaining ATP and phosphocreatine (PCr) concentrations, carbohydrate and fatty-acid metabolism, insulin signalling, and protein expression. Soleus muscles were incubated in well-oxygenated Medium 199 (M199) supplemented with low concentrations of insulin (14.3 microU/mL) for 0, 6, 12, and 18 h. During this incubating period the concentrations of ATP and PCr were stable, indicating that oxygenation and substrate supply were being maintained. In addition, the concentrations of proglycogen and macroglycogen were not altered, whereas an increase (+30%) in intramuscular triacylglycerol concentration was observed at the end of 18 h of incubation (p < 0.05). Complex molecular processes in the long-term incubated muscles were also stable. This was shown by maintenance of basal as well as insulin-stimulated rates of 3-O-methyl glucose transport, and by the maintenance of protein expression of the glucose transporter GLUT4 and the fatty acid transporters FAT/CD36 and FABPpm. In addition, the insulin-stimulated translocation of GLUT4 to the plasma membrane, which involves a complex signalling cascade, was fully preserved. In conclusion, in well-oxygenated soleus muscles maintained in M199 supplemented with extremely low concentrations of insulin, ATP and PCr concentrations, carbohydrate and fatty acid metabolism, insulin signalling, and protein expression were stably maintained for up to 18 h. This provides for opportunities to examine muscle metabolic function under very highly controlled conditions.

Increased malonyl-CoA levels in muscle from obese and type 2 diabetic subjects lead to decreased fatty acid oxidation and increased lipogenesis; thiazolidinedione treatment reverses these defects.

Increased accumulation of fatty acids and their derivatives can impair insulin-stimulated glucose disposal by skeletal muscle. To characterize the nature of the defects in lipid metabolism and to evaluate the effects of thiazolidinedione treatment, we analyzed the levels of triacylglycerol, long-chain fatty acyl-coA, malonyl-CoA, fatty acid oxidation, AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), malonyl-CoA decarboxylase, and fatty acid transport proteins in muscle biopsies from nondiabetic lean, obese, and type 2 subjects before and after an euglycemic-hyperinsulinemic clamp as well as pre-and post-3-month rosiglitazone treatment. We observed that low AMPK and high ACC activities resulted in elevation of malonyl-CoA levels and lower fatty acid oxidation rates. These conditions, along with the basal higher expression levels of fatty acid transporters, led accumulation of long-chain fatty acyl-coA and triacylglycerol in insulin-resistant muscle. During the insulin infusion, muscle fatty acid oxidation was reduced to a greater extent in the lean compared with the insulin-resistant subjects. In contrast, isolated muscle mitochondria from the type 2 subjects exhibited a greater rate of fatty acid oxidation compared with the lean group. All of these abnormalities in the type 2 diabetic group were reversed by rosiglitazone treatment. In conclusion, these studies have shown that elevated malonyl-CoA levels and decreased fatty acid oxidation are key abnormalities in insulin-resistant muscle, and, in type 2 diabetic patients, thiazolidinedione treatment can reverse these abnormalities.