Markers Of Fatty Acid Oxidation Research Articles

Role of ACOT1 in hepatic lipid trafficking (821.6)

Peroxisome proliferator‐activated regulator alpha (PPARα) is a major regulator of fasting lipid metabolism. PPARα induces transcription of cytosolic Acyl‐CoA Thioesterase 1 (ACOT1), which hydrolyzes acyl‐CoAs into long‐chain free fatty acids and Coenzyme A. However, the physiological role of ACOT1 in lipid metabolism is not known. Thus, the objective of this research was to determine the contribution of hepatic ACOT1 to lipid metabolism and signaling. To test our objectives, we injected C57/Bl6 mice with an adenovirus harboring shRNA targeted to ACOT1 or a scrambled control. One week post‐injection, mice were fasted overnight and sacrificed. Compared to control mice, ACOT1 knockdown resulted in an increase in serum β‐hydroxybutyrate, a marker of fatty acid oxidation, and an increase in serum triglyceride, indicating a possible increase in very‐low density lipoprotein secretion. These changes were not accompanied by a significant change in liver triglyceride. In addition, genes involved in mitochondrial biogenesis were decreased in response to ACOT1 knockdown suggesting that ACOT1 alters cell signaling to influence gene expression. In summary, these results show that ACOT1 plays an important role in regulating hepatic lipid metabolism and may serve an important novel role in trafficking of lipids to influence cell signaling.

Muscle quality and metabolomics analysis of young and old subjects (1026.9)

Sarcopenia, with declines in muscle mass and function, represents an independent risk factor for frailty, disability and mortality in older adults. Underlying changes are not clearly defined but may include muscle atrophy and myopathy. Contributing factors include declines in sex hormones, loss of protein synthesis capacity, neuromuscular integrity, lower physical activity, inflammation and oxidative stress. To understand the impact of aging on systemic metabolism, global metabolomic analysis of 10 serum samples/group were performed in young women (YW) (29.1 ± 4.2 y, 24.2 ± 2.7 BMI), older women (OW) (74.6 ± 6.4 y, 21.2± 2.5 BMI) and older men (75.2 ± 9.8 y, 24.5 ± 2.2 BMI). Fatty acid oxidation markers were higher in older subjects. Acylcarnitines (oleoyl‐, octanoyl‐ and acetyl‐) were elevated in OW compared to YW but did not differ between OW and men. Markers of renal function and steroid hormones followed similar patterns. Acetoacetate was higher in OW compared to older men and glycerol was elevated in OW compared to both groups. EPA, DPA, and DHA, were higher in older women compared to YW but did not differ from older men. Metabolites indicate a decline in renal function and steroid hormones in OW; however, indicators of elevated mitochondrial β‐oxidation were higher in older subjects. Ongoing analyses will evaluate the relationship between these metabolite changes to body composition and muscle performance.

High-fat diet from perilla oil induces insulin resistance despite lower serum lipids and increases hepatic fatty acid oxidation in rats

BackgroundThe purpose of this study is to investigate the effects of a high-fat diet from perilla oil on serum lipids, hepatic lipid metabolism and insulin sensitivity.MethodsMale Sprague–Dawley (SD) rats were fed either a control (CT) diet or a diet high in perilla oil (HP). After 16 weeks of feeding, the serum lipids were measured, and the gene expressions involved in hepatic fatty acid oxidation and synthesis were determined. In addition, hepatic fat deposition was detected, and insulin sensitivity was evaluated by means of euglycemic-hyperinsulinemic clamp.ResultsCompared with the rats in the CT group, the HP-feeding significantly decreased the levels of triglyceride (TG), total cholesterol (TCH) and HDL-cholesterol (HDL-c). HP-feeding did not change the levels of LDL-cholesterol (LDL-c), free fatty acid (FFA), intrahepatic lipids or body weight. Moreover, the HP-feeding dramatically increased the mRNA expressions of fatty acid oxidation markers (PPAR-alpha, CPT1A) and fatty acid synthesis markers (SREBP-1, FASN and ACC) in the liver. The HP-feeding induced increased protein levels of CPT1A, while reducing the protein levels of FASN and ACC in the liver. However, the glucose infusion rate significantly increased in the HP group compared with the CT group.ConclusionsOur data show that, in rats, excessive perilla oil intake may significantly lower serum lipids, strengthen hepatic fatty acid oxidation, and inhibit hepatic fatty acid synthesis, but at the same time may also lead to insulin resistance.

A diet high in α-linolenic acid and monounsaturated fatty acids attenuates hepatic steatosis and alters hepatic phospholipid fatty acid profile in diet-induced obese rats

This study investigated the efficacy of the plant-based n−3 fatty acid, α-linolenic acid (ALA), a dietary precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), for modulating hepatic steatosis. Rats were fed high fat (55% energy) diets containing high oleic canola oil, canola oil, a canola/flax oil blend (C/F, 3:1), safflower oil, soybean oil, or lard. After 12 weeks, C/F and weight-matched (WM) groups had 20% less liver lipid. Body mass, liver weight, glucose and lipid metabolism, inflammation and molecular markers of fatty acid oxidation, synthesis, desaturation and elongation did not account for this effect. The C/F group had the highest total n−3 and EPA in hepatic phospholipids (PL), as well as one of the highest DHA and lowest arachidonic acid (n−6) concentrations. In conclusion, the C/F diet with the highest content of the plant-based n−3 ALA attenuated hepatic steatosis and altered the hepatic PL fatty acid profile.

GPR119 regulates genetic markers of fatty acid oxidation in cultured skeletal muscle myotubes

Gene knockout and agonist studies indicate that activation of the G protein-coupled receptor, GPR119, protects against diet-induced obesity and insulin resistance. It is not known if GPR119 activation in skeletal muscle mediates these effects. To address this uncertainty, we measured GPR119 expression in skeletal muscle and determined the effects of PSN632408, a GPR119 agonist, on the expression of genes and proteins required for fatty acid and glucose oxidation in cultured myotubes. GPR119 expression was readily detected in rat skeletal muscle and mRNAs were induced by 12weeks of high-fat feeding. Treatment of cultured mouse C2C12 myotubes with 5μM PSN632408 or 0.5mM palmitate reduced expression of mRNAs encoding fatty acid oxidation genes to similar extents. More so, treatment with PSN632408 decreased AMPKα (Thr172 phosphorylation) activity in the absence of palmitate and ACC (Ser79 phosphorylation) activity in the presence of palmitate. In human primary myotubes PSN632408 decreased expression of PDK4 and AMPKα2 mRNA in myotubes derived from obese donors. These data suggest GPR119 activation in skeletal muscle may impair fatty acid and glucose oxidation.

Long-Term Bezafibrate Treatment Improves Skin and Spleen Phenotypes of the mtDNA Mutator Mouse

Pharmacological agents, such as bezafibrate, that activate peroxisome proliferator-activated receptors (PPARs) and PPAR γ coactivator-1α (PGC-1α) pathways have been shown to improve mitochondrial function and energy metabolism. The mitochondrial DNA (mtDNA) mutator mouse is a mouse model of aging that harbors a proofreading-deficient mtDNA polymerase γ. These mice develop many features of premature aging including hair loss, anemia, osteoporosis, sarcopenia and decreased lifespan. They also have increased mtDNA mutations and marked mitochondrial dysfunction. We found that mutator mice treated with bezafibrate for 8-months had delayed hair loss and improved skin and spleen aging-like phenotypes. Although we observed an increase in markers of fatty acid oxidation in these tissues, we did not detect a generalized increase in mitochondrial markers. On the other hand, there were no improvements in muscle function or lifespan of the mutator mouse, which we attributed to the rodent-specific hepatomegaly associated with fibrate treatment. These results showed that despite its secondary effects in rodent’s liver, bezafibrate was able to improve some of the aging phenotypes in the mutator mouse. Because the associated hepatomegaly is not observed in primates, long-term bezafibrate treatment in humans could have beneficial effects on tissues undergoing chronic bioenergetic-related degeneration.

Retinaldehyde Dehydrogenase 1 Coordinates Hepatic Gluconeogenesis and Lipid Metabolism

Recent data link vitamin A and its retinoid metabolites to the regulation of adipogenesis, insulin sensitivity, and glucose homeostasis. Retinoid metabolism is tightly controlled by an enzymatic network in which retinaldehyde dehydrogenases (Aldh1-3) are the rate-limiting enzymes that convert retinaldehyde to retinoic acid. Aldh1a1-deficient mice are protected from diet-induced obesity and hence diabetes. Here we investigated whether Aldh1a1 and the retinoid axis regulate hepatic glucose and lipid metabolism independent of adiposity. The impact of Aldh1a1 and the retinoid pathway on glucose homeostasis and lipid metabolism was analyzed in hepatocytes in vitro and in chow-fed, weight-matched Aldh1a1-deficient vs. wild-type (WT) mice in vivo. Aldh1a1-deficient mice displayed significantly decreased fasting glucose concentrations compared with WT controls as a result of attenuated hepatic glucose production. Expression of key gluconeogenic enzymes as well as the activity of Forkhead box O1 was decreased in Aldh1a1-deficient vs. WT livers. In vitro, retinoid or cAMP agonist stimulation markedly induced gluconeogenesis in WT but not Aldh1a1-deficient primary hepatocytes. Aldh1a1 deficiency increased AMP-activated protein kinase α activity, decreased expression of lipogenic targets of AMP-activated protein kinase α and significantly attenuated hepatic triacylglycerol synthesis. In metabolic cage studies, lean Aldh1a1-deficient mice manifested enhanced oxygen consumption and reduced respiratory quotient vs. WT controls, consistent with increased expression of fatty acid oxidation markers in skeletal muscle. Taken together, this work establishes a role for retinoid metabolism in glucose homeostasis in vivo and for Aldh1a1 as a novel determinant of gluconeogenesis and lipid metabolism independent of adiposity.

Immunohistochemical Detection of Polyunsaturated Fatty Acid Oxidation Markers in Acetaminophen-Induced Liver Injury in Rats

To clarfity whether polyunsaturated fatty acid (PUFA) oxidation is involved in the mechanism of acetaminophen (APAP)-induced apoptotic cell death, the production and localization of PUFA oxidation markers N(ε)-propanoyl-modified lysine, N(ε)-hexanoyl-modified lysine, 4-hydroxyhexenal-modified histidine and crotonaldehyde-modified lysine were evaluated in the development of APAP-induced liver injury. The immunoexpression of these markers in the liver was examined up to 24 hr post-APAP intraperitoneal injection in rats (1 g/kg body weight). The histopathological changes in the liver appeared 3 hr after APAP injection and became exacerbated with time. Proapoptotic protein Bax immunoreactivity was first detected in the degenerative hepatocytes 3 hr after the injection and areas positively immunostained for Bax reached a peak level at 6 hr, and then decreased at 12 and 24 hr. There was a significant increase in the TUNEL-positive rate at 12 and 24 hr. Immunohistological expression of all these oxidation markers was first detected in the degenerative hepatocytes 3 hr after the injection, and earlier than the occurrence of hepatocyte apoptosis. Immunohistochemical expression of these markers were observed in almost all degenerative hepatocytes 3-24 hr after APAP injection. Areas positively immunostained for these markers reached a peak level at 6 hr, and then decreased at 12 and 24 hr. The results thus suggest that the generation of PUFA oxidation markers may be the signature of early events preceding the induction of liver cell apoptosis and thus useful for early detection of oxidative stress-related liver cell injury.

Skeletal muscle fatty acid oxidation is not directly associated with AMPK or ACC2 phosphorylation

Rescue of palmitate-induced insulin resistance has been linked with improvements in fatty acid oxidation, but importantly, not always with concurrently altered AMPK or ACC2 phosphorylation. Therefore, we examined the interrelationships among AMPK, ACC2, and fatty acid oxidation under 12 controlled conditions in isolated muscle. Incubation of soleus muscle (0-12 h) did not alter fatty acid oxidation, but did increase AMPK and ACC2 phosphorylation (24%-30%). Muscle incubation with palmitate (2mmol·L(-1)) inhibited palmitate oxidation (∼55%), but paradoxically, this was associated with increased AMPK and ACC2 phosphorylation (∼50%). Addition of an AMPK activator (thujone) to control (no palmitate) muscle increased AMPK and ACC2 phosphorylation (∼25%) but did not alter palmitate oxidation. Addition of AMPK inhibitors, compound C (50µmol·L(-1)) or adenine 9-β-d-arabinofuranoside (Ara; 2.5mmol·L(-1)), to thujone-treated muscles (no palmitate) did not alter palmiate oxidation but reduced AMPK phosphorylation (32%-42%), while ACC2 phosphorylation remained above basal level (+14%-18%). Finally, in palmitate-treated muscle, thujone increased AMPK (+100%) and ACC2 phosphorylation (+52%) and restored palmitate oxidation. Compound C or Ara, administered along with thujone in palmitate-treated muscle, only partly blunted palmitate oxidation recovery despite inhibiting AMPK phosphorylation (-22%), although ACC2 phosphorylation remained upregulated (+33%). Among these experiments, AMPK phosphorylation and ACC2 phosphorylation were positively correlated. However, AMPK phosphorylation was not correlated with palmitate oxidation, and unexpectedly, palmitate oxidation was negatively correlated with ACC2 phosphorylation. Our study, in accordance with a growing body of evidence, indicates that neither AMPK phosphorylation nor ACC2 phosphorylation is by itself an appropriate marker of fatty acid oxidation, and further serves to question their regulatory role.

Dietary Capsaicin Attenuates Metabolic Dysregulation in Genetically Obese Diabetic Mice

Metabolic dysregulation (e.g., hyperglycemia, hyperinsulinemia, hyperlipidemia, etc.) is a hallmark of obesity-related diseases such as insulin resistance, type 2 diabetes, and fatty liver disease. In this study, we assessed whether dietary capsaicin attenuated the metabolic dysregulation in genetically obese diabetic KKAy mice, which have severe diabetic phenotypes. Male KKAy mice fed a high-fat diet for 2 weeks received a 0.015% capsaicin supplement for a further 3 weeks and were compared with nonsupplemented controls. Dietary capsaicin markedly decreased fasting glucose/insulin and triglyceride levels in the plasma and/or liver, as well as expression of inflammatory adipocytokine genes (e.g., monocyte chemoattractant protein-1 and interleukin-6) and macrophage infiltration. At the same time expression of the adiponectin gene/protein and its receptor, AdipoR2, increased in adipose tissue and/or plasma, accompanied by increased activation of hepatic AMP-activated protein kinase, a marker of fatty acid oxidation. These findings suggest that dietary capsaicin reduces metabolic dysregulation in obese/diabetic KKAy mice by enhancing expression of adiponectin and its receptor. Capsaicin may be useful as a dietary factor for reducing obesity-related metabolic dysregulation.

Adipose triglyceride lipase expression in human adipose tissue and muscle. Role in insulin resistance and response to training and pioglitazone

Adipose triglyceride lipase (ATGL) catalyzes the first step in adipocyte and muscle triglyceride hydrolysis, and comparative gene identification–58 (CGI-58) is an essential cofactor. We studied the expression of ATGL and CGI-58 in human adipose and muscle and examined correlations with markers of muscle fatty acid oxidation. Nondiabetic volunteers were studied. Subjects with impaired glucose tolerance were treated with pioglitazone or metformin for 10 weeks. Subjects with normal glucose tolerance underwent a 12-week training program. We examined changes in ATGL and CGI-58 with obesity and insulin resistance, and effects of exercise and pioglitazone. Adipose triglyceride lipase messenger RNA (mRNA) expression showed no correlation with either body mass index or insulin sensitivity index in either adipose or muscle. However, adipose ATGL protein levels were inversely correlated with body mass index ( r = −0.64, P < .02) and positively correlated with insulin sensitivity index ( r = 0.67, P < .02). In muscle, ATGL mRNA demonstrated a strong positive relationship with carnitine palmitoyltransferase I mRNA ( r = 0.82, P < .0001) and the adiponectin receptors AdipoR1 mRNA ( r = 0.71, P < .0001) and AdipoR2 mRNA ( r = 0.74, P < .0001). Muscle CGI-58 mRNA was inversely correlated with intramyocellular triglyceride in both type 1 ( r = −0.35, P < .05) and type 2 ( r = −0.40, P < .05) fibers. Exercise training resulted in increased muscle ATGL, and pioglitazone increased adipose ATGL by 31% ( P < .05). Pioglitazone also increased ATGL in adipocytes. Adipose ATGL protein is decreased with insulin resistance and obesity; and muscle ATGL mRNA is associated with markers of fatty acid oxidation in muscle, as is CGI-58. The regulation of ATGL and CGI-58 has important implications for the control of lipotoxicity.

Eicosapentaenoic Acid Prevents and Reverses Insulin Resistance in High-Fat Diet-Induced Obese Mice via Modulation of Adipose Tissue Inflammation1–3

We investigated the effects of eicosapentaenoic acid (EPA) on prevention (P) and reversal (R) of high saturated-fat (HF) diet-induced obesity and glucose-insulin homeostasis. Male C57BL/6J mice were fed low-fat (LF; 10% energy from fat), HF (45% energy from fat), or a HF-EPA-P (45% energy from fat; 36 g/kg EPA) diet for 11 wk. A 4th group was initially fed HF for 6 wk followed by the HF-EPA-R diet for 5 wk. As expected, mice fed the HF diet developed obesity and glucose intolerance. In contrast, mice fed the HF-EPA-P diet maintained normal glucose tolerance despite weight gain compared with the LF group. Whereas the HF group developed hyperglycemia and hyperinsulinemia, both HF-EPA groups (P and R) exhibited normal glycemia and insulinemia. Further, plasma adiponectin concentration was lower in the HF group but was comparable in the LF and HF-EPA groups, suggesting a role of EPA in preventing and improving insulin resistance induced by HF feeding. Further analysis of adipose tissue adipokine levels and proteomic studies in cultured adipocytes indicated that dietary EPA supplementation of HF diets was associated with reduced adipose inflammation and lipogenesis and elevated markers of fatty acid oxidation. In C57BL/6J mice, EPA minimized saturated fat-induced insulin resistance and this is in part mediated by its effects on fatty acid oxidation and inflammation.

Chronic AMP-activated protein kinase activation and a high-fat diet have an additive effect on mitochondria in rat skeletal muscle

Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMP-activated protein kinase (AMPK), calcium, and circulating free fatty acids (FFAs). Chronic treatment with either 5-aminoimidazole-4-carboxamide riboside (AICAR), a chemical activator of AMPK, or increasing circulating FFAs with a high-fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high-fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high-fat diet (HF), AICAR injections (AICAR), or a high-fat diet and AICAR injections (HF + AICAR) for 6 wk. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on long-chain acyl-CoA dehydrogenase, cytochrome c, and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein, as well as on citrate synthase and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD) activity. In contrast, no additive effect was noted in the soleus, and in the red quadriceps only beta-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high-fat diet-induced posttranscriptional increase in PGC-1alpha protein and AMPK-mediated increase in PGC-1alpha protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high-fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.

Carnitine and Choline Supplementation with Exercise Alter Carnitine Profiles, Biochemical Markers of Fat Metabolism and Serum Leptin Concentration in Healthy Women

We sought to determine the effects of supplementary choline, carnitine and a combination of the two with or without exercise on serum and urinary carnitine and biochemical markers of fatty acid oxidation in healthy humans. Nineteen women were placed in three groups: 1) placebo, choline or carnitine preloading period of 1 wk followed by 2) supplementation with choline plus carnitine during wk 2-wk 3 and 3) all groups exercised in wk 3. Although there were no changes in the placebo group, serum and urinary carnitine decreased in the choline-supplemented group during wk 1. Introduction of carnitine to the choline group restored serum and urinary carnitine. Serum and urinary carnitine increased during wk 1 in the carnitine-supplemented group and, although the introduction of choline to this group depressed serum and urinary carnitine, they remained significantly greater than control. Serum beta-hydroxybutyrate and serum as well as urinary acetylcarnitine were elevated by the supplements. A mild exercise regimen increased the concentration of serum beta-hydroxybutyrate, and serum and urinary acylcarnitines; it also decreased serum leptin concentrations in all groups. The effects of supplements were sustained until wk 2 after cessation of choline plus carnitine supplementation and exercise. We conclude that the choline-induced decrease in serum and urinary carnitine is buffered by carnitine preloading, and these supplements shift tissue partitioning of carnitine that favors fat mobilization, incomplete oxidation of fatty acids and disposal of their carbons in urine as acylcarnitines in humans.

Shift of anaerobic to aerobic metabolism in the rats acclimatized to hypoxi

1. 1. Metabolic acclimatization by repeated exposure to a simulated altitude of 4000, 5000 and 6000 m for 2 hr per day throughout 2 to 11 days was evaluated by the increased formation of ketone bodies as a marker of fatty acid oxidation and the decreased production of lactate and uric acid, the indicators of anaerobic metabolism in rats exposed to an altitude of 8000 m. 2. 2. Pre-exposure of rats to an altitude of 5000 m and over caused an acclimatization to hypoxia. The rise of the altitude to which rats were pre-exposed reduced the period until the acquisition of metabolic acclimatization. 3. 3. Acclimatized rats showed an increased activity of mitochondrial glutamate dehydrogenase without changes in glycolytic enzyme activity in skeletal muscle, heart and liver. 4. 4. Acclimatization to high altitude hypoxia is concluded to involve a shift of the anaerobic glycolysis to aerobic metabolism by the increase in the oxidative enzymes.