Mitochondrial Fatty Acid Oxidation Research Articles

Long-chain dicarboxylic acids play a critical role in inducing peroxisomal β-oxidation and hepatic triacylglycerol accumulation

Recent studies provide evidence that peroxisomal β-oxidation negatively regulates mitochondrial fatty acid oxidation, and induction of peroxisomal β-oxidation causes hepatic lipid accumulation. However, whether there exists a triggering mechanism inducing peroxisomal β-oxidation is not clear. Long-chain dicarboxylic acids (LCDAs) are the product of mono fatty acids subjected to ω-oxidation, and both fatty acid ω-oxidation and peroxisomal β-oxidation are induced under ketogenic conditions, indicating there might be a crosstalk between. Here, we revealed that administration of LCDAs strongly induces peroxisomal fatty acid β-oxidation and causes hepatic steatosis in mice through the metabolites acetyl-CoA and hydrogen peroxide. Under ketogenic conditions, upregulation of fatty acid ω-oxidation resulted in increased generation of LCDAs and induction of peroxisomal β-oxidation, which causes hepatic accumulation of lipid droplets in animals. Inhibition of fatty acid ω-oxidation reduced LCDA formation and significantly lowered peroxisomal β-oxidation and improved hepatic steatosis. Our results suggest that endogenous LCDAs act as triggering molecules inducing peroxisomal β-oxidation and hepatic triacylglycerol deposition. Targeting fatty acid ω-oxidation might be an effective pathway in treating fatty liver and related metabolic diseases through regulating peroxisomal β-oxidation.

Flaxseed Reduces Cancer Risk by Altering Bioenergetic Pathways in Liver: Connecting SAM Biosynthesis to Cellular Energy.

This article illustrates how dietary flaxseed can be used to reduce cancer risk, specifically by attenuating obesity, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). We utilize a targeted metabolomics dataset in combination with a reanalysis of past work to investigate the "metabo-bioenergetic" adaptations that occur in White Leghorn laying hens while consuming dietary flaxseed. Recently, we revealed how the anti-vitamin B6 effects of flaxseed augment one-carbon metabolism in a manner that accelerates S-adenosylmethionine (SAM) biosynthesis. Researchers recently showed that accelerated SAM biosynthesis activates the cell's master energy sensor, AMP-activated protein kinase (AMPK). Our paper provides evidence that flaxseed upregulates mitochondrial fatty acid oxidation and glycolysis in liver, concomitant with the attenuation of lipogenesis and polyamine biosynthesis. Defatted flaxseed likely functions as a metformin homologue by upregulating hepatic glucose uptake and pyruvate flux through the pyruvate dehydrogenase complex (PDC) in laying hens. In contrast, whole flaxseed appears to attenuate liver steatosis and body mass by modifying mitochondrial fatty acid oxidation and lipogenesis. Several acylcarnitine moieties indicate Randle cycle adaptations that protect mitochondria from metabolic overload when hens consume flaxseed. We also discuss a paradoxical finding whereby flaxseed induces the highest glycated hemoglobin percentage (HbA1c%) ever recorded in birds, and we suspect that hyperglycemia is not the cause. In conclusion, flaxseed modifies bioenergetic pathways to attenuate the risk of obesity, type 2 diabetes, and NAFLD, possibly downstream of SAM biosynthesis. These findings, if reproducible in humans, can be used to lower cancer risk within the general population.

Medium-chain fatty acid oxidation is independent of l-carnitine in liver and kidney but not in heart and skeletal muscle.

Medium-chain fatty acid (MCFA) consumption confers a wide range of health benefits that are highly distinct from long-chain fatty acids (LCFAs). A major difference between the metabolism of LCFAs compared with MCFAs is that mitochondrial LCFA oxidation depends on the carnitine shuttle, whereas MCFA mitochondrial oxidation is not. Although MCFAs are said to range from 6 to 14 carbons long based on physicochemical properties in vitro, the biological cut-off length of acyl chains that can bypass the carnitine shuttle in different mammalian tissues is unknown. To define the range of acyl chain length that can be oxidized in the mitochondria independent of carnitine, we determined the oxidative metabolism of free fatty acids (FFAs) from 6 to 18 carbons long in the liver, kidney, heart, and skeletal muscle. The liver oxidized FFAs 6 to 14 carbons long, whereas the kidney oxidized FFAs from 6 to 10 carbons in length. Heart and skeletal muscle were unable to oxidize FFAs of any chain length. These data show that while the liver and kidney can oxidize MCFAs in the free form, the heart and skeletal muscle require carnitine for the oxidative metabolism of MCFAs. Together these data demonstrate that MCFA oxidation independent of carnitine is tissue-specific.NEW & NOTEWORTHY This work demonstrates that the traditional concept of mitochondrial medium-chain fatty acid oxidation as unregulated and independent of carnitine applies only to liver metabolism, and to kidney to a lesser extent, but not the heart or skeletal muscle. Thus, the benefits of dietary medium-chain fatty acids are set by liver metabolic activity and peripheral tissues are unlikely to receive direct benefits from medium-chain fatty acid metabolism, but rather metabolic byproducts of liver's medium-chain oxidative metabolism.

Pathophysiology of the right ventricle in health and disease: an update.

The contribution of the right ventricle (RV) to cardiac output is negligible in normal resting conditions when pressures in the pulmonary circulation are low. However, the RV becomes relevant in healthy subjects during exercise and definitely so in patients with increased pulmonary artery pressures both at rest and during exercise. The adaptation of RV function to loading rests basically on an increased contractility. This is assessed by RV end-systolic elastance (Ees) to match afterload assessed by arterial elastance (Ea). The system has reserve as the Ees/Ea ratio or its imaging surrogate ejection fraction has to decrease by more than half, before the RV undergoes an increase in dimensions with eventual increase in filling pressures and systemic congestion. RV-arterial uncoupling is accompanied by an increase in diastolic elastance. Measurements of RV systolic function but also of diastolic function predict outcome in any cause pulmonary hypertension and heart failure with or without preserved left ventricular ejection fraction. Pathobiological changes in the overloaded RV include a combination of myocardial fibre hypertrophy, fibrosis and capillary rarefaction, a titin phosphorylation-related displacement of myofibril tension-length relationships to higher pressures, a metabolic shift from mitochondrial free fatty acid oxidation to cytoplasmic glycolysis, toxic lipid accumulation, and activation of apoptotic and inflammatory signalling pathways. Treatment of RV failure rests on the relief of excessive loading.

Fatty acid oxidation organizes mitochondrial supercomplexes to sustain astrocytic ROS and cognition

Having direct access to brain vasculature, astrocytes can take up available blood nutrients and metabolize them to fulfil their own energy needs and deliver metabolic intermediates to local synapses1,2. These glial cells should be, therefore, metabolically adaptable to swap different substrates. However, in vitro and in vivo studies consistently show that astrocytes are primarily glycolytic3–7, suggesting glucose is their main metabolic precursor. Notably, transcriptomic data8,9 and in vitro10 studies reveal that mouse astrocytes are capable of mitochondrially oxidizing fatty acids and that they can detoxify excess neuronal-derived fatty acids in disease models11,12. Still, the factual metabolic advantage of fatty acid use by astrocytes and its physiological impact on higher-order cerebral functions remain unknown. Here, we show that knockout of carnitine-palmitoyl transferase-1A (CPT1A)—a key enzyme of mitochondrial fatty acid oxidation—in adult mouse astrocytes causes cognitive impairment. Mechanistically, decreased fatty acid oxidation rewired astrocytic pyruvate metabolism to facilitate electron flux through a super-assembled mitochondrial respiratory chain, resulting in attenuation of reactive oxygen species formation. Thus, astrocytes naturally metabolize fatty acids to preserve the mitochondrial respiratory chain in an energetically inefficient disassembled conformation that secures signalling reactive oxygen species and sustains cognitive performance.

Effects of myonectin on porcine intramuscular adipocyte differentiation and exogenous free fatty acid utilization

As an important factor secreted by skeletal muscle, myonectin can regulate lipid metabolism and energy metabolism, but its role in the utilization of peripheral free fatty acids (FFAs) by porcine intramuscular fat cells remains to be further investigated. In this study, porcine intramuscular adipocytes were treated with recombinant myonectin and palmitic acid (PA), either alone or in combination, and then were examined for their uptake of exogenous FFAs, intracellular lipid synthesis and catabolism, and mitochondrial oxidation of fatty acids. The results showed that myonectin decreased the area of lipid droplets in intramuscular adipocytes (p < 0.05) and significantly increased (p < 0.05) the expression levels of hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL). Moreover, myonectin can up-regulate the expression of p38 mitogen-activated protein kinase (p38 MAPK). Myonectin significantly promoted the uptake of peripheral FFAs (p < 0.01), improved (p < 0.05) the expression of fatty transport protein 1 (FATP1) and fatty acid binding protein 4 (FABP4) in intramuscular adipocytes. Myonectin also significantly increased (p < 0.05) the expression levels of fatty acid oxidation markers: transcription factor (TFAM), uncoupling protein-2 (UCP2) and oxidative respiratory chain marker protein complex I (NADH-CoQ) in mitochondria of intramuscular adipocytes. In summary, myonectin promoted the absorption, transport, and oxidative metabolism of exogenous FFAs in mitochondria, thereby inhibiting lipid deposition in porcine intramuscular adipocytes.

Low Fasting Concentrations of Glucagon in Patients with Very Long-Chain Acyl-CoA Dehydrogenase Deficiency.

(1) Background: Deficiencies of mitochondrial fatty acid oxidation (FAO) define a subgroup of inborn errors of metabolism, with medium-chain acyl-CoA dehydrogenase deficiency (MCAD) and very long-chain acyl-CoA dehydrogenase deficiency (VLCAD) being two of the most common. Hypoketotic hypoglycemia is a feared clinical complication and the treatment focuses on avoiding hypoglycemia. In contrast, carnitine uptake deficiency (CUD) is treated as a mild disease without significant effects on FAO. Impaired FAO has experimentally been shown to impair glucagon secretion. Glucagon is an important glucose-mobilizing hormone. If and how glucagon is affected in patients with VLCAD or MCAD remains unknown. (2) Methods: A cross-sectional study was performed with plasma hormone concentrations quantified after four hours of fasting. Patients with VLCAD (n = 10), MCAD (n = 7) and CUD (n = 6) were included. (3) Results: The groups were similar in age, sex, weight, and height. The glucagon and insulin levels were significantly lower in the VLCAD group compared to the CUD group (p < 0.05, respectively). The patients with CUD had glucagon concentrations similar to the normative data. No significant differences were seen in GLP-1, glicentin, glucose, amino acids, or NEFAs. (4) Conclusions: Low fasting concentrations of glucagon are present in patients with VLCAD and cannot be explained by altered stimuli in plasma.

1630-P: Reduced Fatty Acid Oxidation Does Not Prevent Intralipid-Induced Peripheral Insulin Resistance in Humans

Mitochondrial handling of excess lipid substrate is thought to be an important determinant of insulin resistance. Evidence from animal models support insulin signaling defects resulting from either intra-cellular lipid accumulation or by mitochondrial dysfunction and altered fatty acid oxidation (FAO) flux. To explore the relationship between FAO and insulin sensitivity, we measured the change in peripheral glucose disposal (Rd) during a 5-hour hyperinsulinemic euglycemic clamp with co-infusion of a control glycerol solution versus a 20% intralipid solution in 18 subjects with an inherited defect in FAO (FAOD; including Carnitine Palmitoyltransferase 2, Very long-chain, medium-chain or long-chain 3-hydroxy AcylcoA Dehydrogenase Deficiencies) and 18 age, sex, and weight-matched normal healthy controls. Rd was similar between groups during the glycerol control clamp. Intramyocellular lipid (IMCL) content as measured by MRS increased after the 5 hour intralipid infusion in controls but not in participants with FAOD. Plasma free fatty acids during intralipid infusion were higher in FAOD compared to controls suggesting infused lipid remained in circulation. Muscle fiber type and expression of lipid transporters CD36 and FATP1 were similar in biopsy samples between groups and cannot explain decreased lipid uptake into muscle among FAOD subjects. However, intralipid suppressed Rd equally in subjects with FAOD and controls inducing peripheral insulin resistance in both groups. Impaired FAO decreased lipid accumulation in the muscle during intralipid infusion, but despite lower IMCL, subjects with FAOD developed peripheral insulin resistance similar to controls. Increased IMCL is not essential for, nor is reduced FAO flux protective against, the development of peripheral insulin resistance during intralipid infusion. Disclosure M.B.Gillingham: None. J.Q.Purnell: Advisory Panel; Boehringer Ingelheim International GmbH, Novo Nordisk. A.N.Gregor: None. O.Varlamov: None. J.J.Robino: None. C.E.Mccurdy: None. K.T.Greyslak: None. D.Choi: None. E.Baetscher: Consultant; Biogen. W.Rooney: Consultant; Ultragenix, Research Support; Revalesio. Funding National Institutes of Health (DK102813)

L-Carnitine improves mechanical responses of cardiomyocytes and restores Ca2+ homeostasis during aging.

L-Carnitine (β-hydroxy-γ-trimethylaminobutyric acid, LC) is a crucial molecule for the mitochondrial oxidation of fatty acids. It facilitates the transport of long-chain fatty acids into the mitochondrial matrix. The reduction in LC levels during the aging process has been linked to numerous cardiovascular disorders, including contractility dysfunction, and disrupted intracellular Ca2+ homeostasis. The aim of this study was to examine the effects of long-term (7months) LC administration on cardiomyocyte contraction and intracellular Ca2+ transients ([Ca2+]i) in aging rats. Male albino Wistar rats were randomly assigned to either the control or LC-treated groups. LC (50mg/kg body weight/day) was dissolved in distilled water and orally administered for a period of 7months. The control group received distilled water alone. Subsequently, ventricular single cardiomyocytes were isolated, and the contractility and Ca2+ transients were recorded in aging (18months) rats. This study demonstrates, for the first time, a novel inotropic effect of long-term LC treatment on rat ventricular cardiomyocyte contraction. LC increased cardiomyocyte cell shortening and resting sarcomere length. Furthermore, LC supplementation led to a reduction in resting [Ca2+]i level and an increase in the amplitude of [Ca2+]i transients, indicative of enhanced contraction. Consistent with these results, decay time of Ca2+ transients also decreased significantly in the LC-treated group. The long-term administration of LC may help restore the Ca2+ homeostasis altered during aging and could be used as a cardioprotective medication in cases where myocyte contractility is diminished.

#5666 FATTY ACID BINDING PROTEINS (FABPS) INHIBITION IMPAIRS METABOLIC HOMEOSTASIS IN RENAL TUBULAR CELLS

Abstract Background and Aims The fatty acid binding proteins family of lipid chaperones (FABPs) mediate lipid trafficking and lipid-mediated metabolic pathways. Despite inhibition of induced FABPs may be beneficial in kidney damage, their enrolment in renal metabolic homeostasis remains elusive. Therefore, we approached this issue in cultured renal tubular cells. Method FABP mRNA and protein expression were evaluated in C57/BL6 mice subjected to folic-acid kidney injury. Mitochondrial redox status, proliferation, death rate, and fatty acid oxidation (FAO) were studied in renal tubular cells in conditions of overall FABP inhibition with BMS309403. In these conditions, lipid trafficking was addressed by oil red content and pulse-chase assays using the fluorescent lipid RedC12. Delivery of RedC12 within LD or mitochondria was followed with BODIPY493/503 and Mitotracker and confocal microscopy. Free and esterified RedC12 species were assessed by thin-layer chromatography (TLC). The impact of ATGL lipase activity on cellular behaviour and bioenergetics were assessed by diethylumbelliferyl phosphate (DEUP)-mediated inhibition. Results Kidneys from mice with AKI exhibited early lowered FABPs tubular expression. In serum-depleted murine tubular cells, pharmacological FABPs inhibition with BM309403 (BMS) led to sudden necrosis, whereas, in non-depleted cells, BMS eventually leads to apoptosis, increased the mRNA expression of proinflammatory cytokines and oxidative stress-responsive genes, and restrained proliferation. BMS also rapidly decreased ATP levels consistent with FAO inhibition and induced adaptive AMPK phosphorylation/activation and FAO gene program (but not glycolytic gene expression). BMS also mediates mitochondrial depolarization, mitochondrial oxidative stress and decrease of mitochondria number. Serum-depleted cells revealed lipid droplet (LD) voiding and RedC12 delivery to mitochondria. By contrast, in BMS-treated cells growing in serum, despite the lowered ATP content and transcriptional FAO upregulation, RedC12 neither localized with LDs nor with mitochondria, suggesting a low-energy state by impairment of both lipid transit and FAO. Accordingly, TLC assessment did not show lipid breakdown-derived products, which otherwise did appear in serum-depleted cells doing FAO. Mechanistically, ATGL inhibition protects from general ROS production, LD voiding and apoptosis. Conclusion In brief, FABP inhibition disrupts tubular energetic status and favours cell stress. Thus, protecting FABP activity could help to preserve tubular metabolic homeostasis during early kidney damage.

Patulin alleviates hepatic lipid accumulation by regulating lipogenesis and mitochondrial respiration

AimsThe aim of this study is to evaluate the effects of patulin on hepatic lipid metabolism and mitochondrial oxidative function and elucidate the underlying molecular mechanisms. Main methodsThe effects of patulin on hepatic lipid accumulation were evaluated in free fatty acid-treated AML12 or HepG2 cells through oil red O staining, triglyceride assay, real-time polymerase chain reaction, and western blotting. Alteration of mitochondrial oxidative capacity by patulin treatment was determined using Seahorse analysis to measure the oxygen consumption rate. Key findingsThe increased amounts of lipid droplets induced by free fatty acids were significantly reduced by patulin treatment. Patulin markedly activated the CaMKII/AMP-activated protein kinase (AMPK)/proliferator-activated receptor-γ coactivator (PGC)-1α signaling pathway in hepatocytes, reduced the expression of sterol regulatory element binding protein 1c (SREBP-1c) and lipogenic genes, and increased the expression of genes related to mitochondrial fatty acid oxidation. In addition, patulin treatment enhanced the mitochondrial consumption rate and increased the expression of mitochondrial oxidative phosphorylation proteins in HepG2 hepatocytes. The effects of patulin on anti-lipid accumulation; SREBP-1c, PGC-1α, and carnitine palmitoyltransferase 1 expression; and mitochondrial oxidative capacity were significantly prevented by compound C, an AMPK inhibitor. SignificancePatulin is a potent inducer of the AMPK pathway, and AMPK-mediated mitochondrial activation is required for the efficacy of patulin to inhibit hepatic lipid accumulation. This study is the first to report that patulin is a promising bioactive compound that prevents the development and worsening of fatty liver diseases, including non-alcoholic fatty liver disease, by improving mitochondrial quality and lipid metabolism.

Exercise intervention improves mitochondrial quality in non-alcoholic fatty liver disease zebrafish.

Recent reports indicate that mitochondrial quality decreases during non-alcoholic fatty liver disease (NAFLD) progression, and targeting the mitochondria may be a possible treatment for NAFLD. Exercise can effectively slow NAFLD progression or treat NAFLD. However, the effect of exercise on mitochondrial quality in NAFLD has not yet been established. In the present study, we fed zebrafish a high-fat diet to model NAFLD, and subjected the zebrafish to swimming exercise. After 12 weeks, swimming exercise significantly reduced high-fat diet-induced liver injury, and reduced inflammation and fibrosis markers. Swimming exercise improved mitochondrial morphology and dynamics, inducing upregulation of optic atrophy 1(OPA1), dynamin related protein 1 (DRP1), and mitofusin 2 (MFN2) protein expression. Swimming exercise also activated mitochondrial biogenesis via the sirtuin 1 (SIRT1)/ AMP-activated protein kinase (AMPK)/ PPARgamma coactivator 1 alpha (PGC1α) pathway, and improved the mRNA expression of genes related to mitochondrial fatty acid oxidation and oxidative phosphorylation. Furthermore, we find that mitophagy was suppressed in NAFLD zebrafish liver with the decreased numbers of mitophagosomes, the inhibition of PTEN-induced kinase 1 (PINK1) - parkinRBR E3 ubiquitin protein ligase (PARKIN) pathway and upregulation of sequestosome 1 (P62) expression. Notably, swimming exercise partially recovered number of mitophagosomes, which was associated with upregulated PARKIN expression and decreased p62 expression. These results demonstrate that swimming exercise could alleviate the effects of NAFLD on the mitochondria, suggesting that exercise may be beneficial for treating NAFLD.

Insoluble Dietary Fiber from Soybean Residue (Okara) Exerts Anti-Obesity Effects by Promoting Hepatic Mitochondrial Fatty Acid Oxidation.

Numerous investigations have shown that insoluble dietary fiber (IDF) has a potentially positive effect on obesity due to a high-fat diet (HFD). Our previous findings based on proteomic data revealed that high-purity IDF from soybean residue (okara) (HPSIDF) prevented obesity by regulating hepatic fatty acid synthesis and degradation pathways, while its intervention mechanism is uncharted. Consequently, the goal of this work is to find out the potential regulatory mechanisms of HPSIDF on hepatic fatty acid oxidation by determining changes in fatty acid oxidation-related enzymes in mitochondria and peroxisomes, the production of oxidation intermediates and final products, the composition and content of fatty acids, and the expression levels of fatty acid oxidation-related proteins in mice fed with HFD. We found that supplementation with HPSIDF significantly ameliorated body weight gain, fat accumulation, dyslipidemia, and hepatic steatosis caused by HFD. Importantly, HPSIDF intervention promotes medium- and long-chain fatty acid oxidation in hepatic mitochondria by improving the contents of acyl-coenzyme A oxidase 1 (ACOX1), malonyl coenzyme A (Malonyl CoA), acetyl coenzyme A synthase (ACS), acetyl coenzyme A carboxylase (ACC), and carnitine palmitoyl transferase-1 (CPT-1). Moreover, HPSIDF effectively regulated the expression levels of proteins involved with hepatic fatty acid β-oxidation. Our study indicated that HPSIDF treatment prevents obesity by promoting hepatic mitochondrial fatty acid oxidation.

Maternal elevated inflammation impairs placental fatty acids β-oxidation in women with gestational diabetes mellitus.

An adverse proinflammatory milieu contributes to abnormal cellular energy metabolism response. Gestational diabetes mellitus (GDM) is closely related to an altered maternal inflammatory status. However, its role on lipid metabolism regulation in human placenta has not yet been assessed. The aim of this study was to examine the impact of maternal circulating inflammatory mediators ([TNF]-α, [IL]-6, and Leptin) on placental fatty acid metabolism in GDM pregnancies. Fasting maternal blood and placental tissues were collected at term deliveries from 37 pregnant women (17 control and 20 GDM). Molecular approach techniques as radiolabeled lipid tracers, ELISAs, immunohistochemistry and multianalyte immunoassay quantitative analysis, were used to quantify serum inflammatory factors' levels, to measure lipid metabolic parameters in placental villous samples (mitochondrial fatty acid oxidation [FAO] rate and lipid content [Triglycerides]), and to analyze their possible relationships. The effect of potential candidate cytokines on fatty acid metabolism in ex vivo placental explants culture following C-section a term was also examined. Maternal serum IL-6, TNF-α and leptin levels were significantly increased in GDM patients compared with control pregnant women (9,9±4,5 vs. 3,00±1,7; 4,5±2,8 vs. 2,1±1,3; and 10026,7±5628,8 vs. 5360,2±2499,9 pg/ml, respectively). Placental FAO capacity was significantly diminished (~30%; p<0.01), whereas triglyceride levels were three-fold higher (p<0.01) in full-term GDM placentas. Uniquely the maternal IL-6 levels showed an inverse and positive correlation with the ability to oxidize fatty acids and triglyceride amount in placenta, respectively (r= -0,602, p=0.005; r= 0,707, p=0.001). Additionally, an inverse correlation between placental FAO and triglycerides was also found (r=-0.683; p=0.001). Interestingly, we ex vivo demonstrated by using placental explant cultures that a prolonged exposure with IL-6 (10 ng/mL) resulted in a decline in the fatty acid oxidation rate (~25%; p=0.001), along to acute increase (2-fold times) in triglycerides accumulation (p=0.001), and in lipid neutral and lipid droplets deposits. Enhanced maternal proinflammatory cytokines levels (essentially IL-6) is closely associated with an altered placental fatty acid metabolism in pregnancies with GDM, which may interfere with adequate delivery of maternal fat across the placenta to the fetus.

A ketogenic diet substantially reshapes the human metabolome

Western dietary habits (WD) have been shown to promote chronic inflammation, which favors the development of many of today's non-communicable diseases. Recently, ketogenic diets (KD) have emerged as an immune-regulating countermeasure for WD-induced metaflammation. To date, beneficial effects of KD have been solely attributed to the production and metabolism of ketone bodies. Given the drastic change in nutrient composition during KD, it is reasonable to assume that there are widespread changes in the human metabolome also contributing to the impact of KD on human immunity. The current study was conducted to gain insight into the changes of the human metabolic fingerprint associated with KD. This could allow to identify metabolites that may contribute to the overall positive effects on human immunity, but also help to recognize potential health risks of KD. We conducted a prospective nutritional intervention study enrolling 40 healthy volunteers to perform a three-week ad-libitum KD. Prior to the start and at the end of the nutritional intervention serum metabolites were quantified, untargeted mass spectrometric metabolome analyses and urine analyses of the tryptophan pathway were performed. KD led to a marked reduction of insulin (-21.45%±6.44%, p=0.0038) and c-peptide levels (-19.29%±5.45%, p=0.0002) without compromising fasting blood glucose. Serum triglyceride concentration decreased accordingly (-13.67%±5.77%, p=0.0247), whereas cholesterol parameters remained unchanged. LC-MS/MS-based untargeted metabolomic analyses revealed a profound shift of the human metabolism towards mitochondrial fatty acid oxidation, comprising highly elevated levels of free fatty acids and acylcarnitines. The serum amino acid (AA) composition was rearranged with lower abundance of glucogenic AA and an increase of BCAA. Furthermore, an increase of anti-inflammatory fatty acids eicosatetraenoic acid (p<0.0001) and docosahexaenoic acid (p=0.0002) was detected. Urine analyses confirmed higher utilization of carnitines, indicated by lower carnitine excretion (-62.61%±18.11%, p=0.0047) and revealed changes to the tryptophan pathway depicting reduced quinolinic acid (-13.46%±6.12%, p=0.0478) and elevated kynurenic acid concentrations (+10.70%±4.25%, p=0.0269). A KD fundamentally changes the human metabolome even after a short period of only three weeks. Besides a rapid metabolic switch to ketone body production and utilization, improved insulin and triglyceride levels and an increase in metabolites that mediate anti-inflammation and mitochondrial protection occurred. Importantly, no metabolic risk factors were identified. Thus, a ketogenic diet could be considered as a safe preventive and therapeutic immunometabolic tool in modern medicine. German Clinical Trials Register; DRKS-ID: DRKS00027992 (www.drks.de).

Increased acylcarnitine ratio indices in newborn screening for carnitine-acylcarnitine translocase deficiency shows increased sensitivity and reduced false-positivity.

Carnitine-acylcarnitine translocase (CACT) deficiency is a rare autosomal recessive metabolic disorder of mitochondrial long-chain fatty acid oxidation. Newborn screening via tandem mass spectrometry (MS/MS) technology enables early diagnosis. However, previous analyses of MS/MS data of patients showed that some results were misdiagnosed because they did not show typical acylcarnitine profiles of CACT deficiency. This study aimed to identify additional indices to assist the diagnosis of CACT deficiency. To evaluate the acylcarnitine profile and the acylcarnitine ratios of individuals with CACT deficiency, the MS/MS data of 15 patients diagnosed via genetic testing were retrospectively analysed. The sensitivity and false-positive rates of primary acylcarnitine markers and ratio indices were validated using the data from 28,261 newborns and 53 false-positive cases. Additionally, the MS/MS data of 20 newborns carrying the c.199-10T>G mutation in SLC25A20 and 40 normal controls were compared to verify whether the carriers had abnormal acylcarnitine concentrations. The acylcarnitine profiles from 15 patients were classified into three categories using C12, C14, C16, C18, C16:1, C18:1, and C18:2 as the primary diagnostic markers. The first category represented a typical profile (P1-P6). The second category for patients P7 and P8 showed a significant decrease in the C0 level and a normal concentration of long-chain acylcarnitines. The third category for patients P9-P15 showed the presence of interfering acylcarnitines. The second and third categories may have been misdiagnosed. An acylcarnitine ratio analysis showed that C14/C3, C16/C2, C16/C3, C18/C3, C16:1/C3, and C16:1-OH/C3 were significantly increased in all 15 patients. The verification of 28,261 newborn screening results showed that the false-positive rate of ratios, except for (C16 + C18)/C0, was lower than that of acylcarnitine indices (0.02-0.08% vs. 0.16-0.88%). None of the single long-chain acylcarnitines could separate patients from the false-positive cases; however, all ratios produced good discrimination between the two groups. Based on the primary acylcarnitine markers alone, CACT deficiency can be misdiagnosed in newborn screening. The ratios of the primary markers (C16 + C18:1)/C2, C16/C2, C16:1/C3, and C16:1-OH/C3 can facilitate the diagnosis of CACT deficiency, thereby increasing sensitivity and reducing false-positivity.

Gut microbial metabolite delta - valerobetaine (VB) influences energy metabolism by altering fat utilization and satiety-related hormones in mice

There is mounting evidence that the gut microbiota exerts profound influences on systemic homeostasis and metabolism, including effects on host energy metabolism, fatty acid oxidation, and obesity. However, we know little about how the microbiota mechanistically influences these aspects of host biology. Previously, we used mass spectrometry-based metabolomics platforms to demonstrate remarkable differences in the metabolite composition in the hepatic mitochondria of germ-free (GF) and conventional mice and identified Δ-valerobetaine (VB) as a key discriminative molecule. VB was undetectable in GF mice but present in conventional mice. We showed that VB is produced by specific bacterial species and inhibited mitochondrial fatty acid oxidation by suppression of the carnitine shuttle. VB administration to GF and conventional mice increased visceral fat mass and exacerbated hepatic steatosis with a Western-style diet but not a chow control diet and can thus function as a microbiota-derived obesogen. Here we show that VB impacts the physiological aspects of energy metabolism. VB reduces the Oxygen consumption rate (OCR) in HEPG2 cells and alters mitochondria-related protein markers (PGC1α, AMPKα, TFAM) suggesting changes in VB can alter the function of mitochondria. To evaluate whole animal consequences, we utilized the CLAMS-HC metabolic cage system to measure the respiratory exchange ratio (RER) in conventional mice. We found that western diet-fed animals treated with VB (IP 50mg/kg) for a week significantly increased RER compared to control groups on the same diet, whereas there was no impact of VB on regular chow diet-fed animals. Higher RER indicates the usage of glucose rather than the usage of fat, thus shifting the resources to produce energy and enhancing the accumulation/retention of adipose tissue. We also found a significant increase in food intake among VB-treated animals regardless of diet. We further investigated the impact of VB on energy-regulating hormones from serum and found a significant alteration in leptin and ghrelin levels. These data suggest VB alters energy utilization in a diet-dependent fashion possibly through regulating energy satiety hormones. Manipulation of VB levels by changing the intake of dietary precursors, or with VB-producing bacterial taxa, may provide therapeutic approaches to disorders of energy/lipid metabolism. This project has been funded by U.S. NIH R01 AI64462. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loss of APOO (MIC26) aggravates obesity-related whitening of brown adipose tissue via PPARα-mediated functional interplay between mitochondria and peroxisomes

BackgroundMitochondrial dysfunction and aberrant structure in adipose tissue occur in obesity and obesity-linked brown adipose tissue (BAT) whitening; however, whether this aberrant architecture contributes to or is the result of obesity is unknown. Apolipoprotein O (APOO) is a constitutive protein of the mitochondrial cristae organizing system complex. This study aimed to characterize the physiological consequences of APOO deficiency in vivo. MethodsAPOO expression was analyzed in different human and murine adipose depots, and mice lacking APOO in adipocytes (ApooACKO) are developed to examine the metabolic consequences of adipocyte-specific APOO ablation in vitro and in vivo. ResultsResults showed that APOO expression is reduced in BAT from both diet-induced and leptin-deficient obese mice. APOO-knockout mice showed increased adiposity, BAT dysfunction and whitening, reduced non-shivering thermogenesis, and blunted responses to cold stimuli. APOO deficiency disrupted mitochondrial structure in brown adipocytes and impaired oxidative phosphorylation, thereby inducing a shift from oxidative to glycolytic metabolism, increasing lipogenic enzyme levels and BAT whitening. APOO inactivation inhibited thermogenesis in BAT by reducing mitochondrial long-chain fatty acid oxidation. It also disturbed peroxisomal biogenesis and very long-chain fatty acid oxidation via peroxisome proliferator-activated receptor α. ConclusionsAltogether, APOO deficiency in adipocytes aggravates BAT whitening and diet-induced obesity; thus, APOO could be a therapeutic target for obesity.

Mitochondrial fusion induced by transforming growth factor-β1 serves as a switch that governs the metabolic reprogramming during differentiation of regulatory T cells

Although metabolic reprogramming during the differentiation of regulatory T cells (Treg cells) has been extensively studied, the molecular switch to alter energy metabolism remains undefined. The present study explores the critical role of mitochondrial dynamics in the reprogramming and consequent generation of Treg cells. The results showed that during Treg cell differentiation, mitochondrial fusion but not fission led to elevation of oxygen consumption rate values, facilitation of metabolic reprogramming, and increase of number of Treg cells and expression of Foxp3 in vitro and in vivo. Mechanistically, mitochondrial fusion favored fatty acid oxidation but restricted glycolysis in Treg cells through down-regulating the expression of HIF-1α. Transforming growth factor-β1 (TGF-β1) played a crucial role in the induction of mitochondrial fusion, which activated Smad2/3, promoted the expression of PGC-1α and therefore facilitated the expression of mitochondrial fusion proteins. In conclusion, during Treg cell differentiation, TGF-β1 promotes PGC-1α-mediated mitochondrial fusion, which drives metabolic reprogramming from glycolysis to fatty acid oxidation via suppressing HIF-1α expression, and therefore favors the generation of Treg cells. The signals and proteins involved in mitochondrial fusion are potential therapeutic targets for Treg cell-related diseases.

Implantation of CPT1AM-expressing adipocytes reduces obesity and glucose intolerance in mice

Obesity and its associated metabolic comorbidities are a rising global health and social issue, with novel therapeutic approaches urgently needed. Adipose tissue plays a key role in the regulation of energy balance and adipose tissue-derived mesenchymal stem cells (AT-MSCs) have gained great interest in cell therapy. Carnitine palmitoyltransferase 1A (CPT1A) is the gatekeeper enzyme for mitochondrial fatty acid oxidation. Here, we aimed to generate adipocytes expressing a constitutively active CPT1A form (CPT1AM) that can improve the obese phenotype in mice after their implantation. AT-MSCs were differentiated into mature adipocytes, subjected to lentivirus-mediated expression of CPT1AM or the GFP control, and subcutaneously implanted into mice fed a high-fat diet (HFD). CPT1AM-implanted mice showed lower body weight, hepatic steatosis and serum insulin and cholesterol levels alongside improved glucose tolerance. HFD-induced increases in adipose tissue hypertrophy, fibrosis, inflammation, endoplasmic reticulum stress and apoptosis were reduced in CPT1AM-implanted mice. In addition, the expression of mitochondrial respiratory chain complexes was enhanced in the adipose tissue of CPT1AM-implanted mice. Our results demonstrate that implantation of CPT1AM-expressing AT-MSC-derived adipocytes into HFD-fed mice improves the obese metabolic phenotype, supporting the future clinical use of this ex vivo gene therapy approach.