Free Fatty Acid Metabolism Research Articles

A novel tracer for in vivo optical imaging of fatty acid metabolism in the heart and brown adipose tissue

Multiplexed imaging is essential for the evaluation of substrate utilization in metabolically active organs, such as the heart and brown adipose tissue (BAT), where substrate preference changes in pathophysiologic states. Optical imaging provides a useful platform because of its low cost, high throughput and intrinsic ability to perform composite readouts. However, the paucity of probes available for in vivo use has limited optical methods to image substrate metabolism. Here, we present a novel near-infrared (NIR) free fatty acid (FFA) tracer suitable for in vivo imaging of deep tissues such as the heart. Using click chemistry, Alexa Fluor 647 DIBO Alkyne was conjugated to palmitic acid. Mice injected with 0.05 nmol/g bodyweight of the conjugate (AlexaFFA) were subjected to conditions known to increase FFA uptake in the heart (fasting) and BAT [cold exposure and injection with the β3 adrenergic agonist CL 316, 243(CL)]. Organs were subsequently imaged both ex vivo and in vivo to quantify AlexaFFA uptake. The blood kinetics of AlexaFFA followed a two-compartment model with an initial fast compartment half-life of 0.14 h and a subsequent slow compartment half-life of 5.2 h, consistent with reversible protein binding. Ex vivo fluorescence imaging after overnight cold exposure and fasting produced a significant increase in AlexaFFA uptake in the heart (58 ± 12%) and BAT (278 ± 19%) compared to warm/fed animals. In vivo imaging of the heart and BAT after exposure to CL and fasting showed a significant increase in AlexaFFA uptake in the heart (48 ± 20%) and BAT (40 ± 10%) compared to saline-injected/fed mice. We present a novel near-infrared FFA tracer, AlexaFFA, that is suitable for in vivo quantification of FFA metabolism and can be applied in the context of a low cost, high throughput, and multiplexed optical imaging platform.

Metformin alleviates hyperuricaemia-induced serum FFA elevation and insulin resistance by inhibiting adipocyte hypertrophy and reversing suppressed white adipose tissue beiging.

Hyperuricaemia (HUA) significantly increases the risk of metabolic syndrome and is strongly associated with the increased prevalence of high serum free fatty acids (FFAs) and insulin resistance. However, the underlying mechanisms are not well established, especially the effect of uric acid (UA) on adipose tissue, a vital organ in regulating whole-body energy and FFA homeostasis. In the present study, we noticed that adipocytes from the white adipose tissue of patients with HUA were hypertrophied and had decreased UCP1 expression. To test the effects of UA on adipose tissue, we built both in vitro and in vivo HUA models and elucidated that a high level of UA could induce hypertrophy of adipocytes, inhibit their hyperplasia and reduce their beige-like characteristics. According to mRNA-sequencing analysis, UA significantly decreased the expression of leptin in adipocytes, which was closely related to fatty acid metabolism and the AMPK signalling pathway, as indicated by KEGG pathway analysis. Moreover, lowering UA using benzbromarone (a uricosuric agent) or metformin-induced activation of AMPK expression significantly attenuated UA-induced FFA metabolism impairment and adipose beiging suppression, which subsequently alleviated serum FFA elevation and insulin resistance in HUA mice. Taken together, these observations confirm that UA is involved in the aetiology of metabolic abnormalities in adipose tissue by regulating leptin-AMPK pathway, and metformin could lessen HUA-induced serum FFA elevation and insulin resistance by improving adipose tissue function via AMPK activation. Therefore, metformin could represent a novel treatment strategy for HUA-related metabolic disorders.

Sleep-Disordered Breathing and Free Fatty Acid Metabolism

Sleep-disordered breathing (SDB) is independently associated with insulin resistance, glucose intolerance, and type 2 diabetes mellitus. However, data on whether SDB alters the metabolism of free fatty acids (FFAs) are lacking. The primary objective of the current study was to characterize alterations in FFA metabolism across the spectrum of SDB severity. The study sample included 118 participants with and without SDB who underwent full-montage polysomnography, the frequently sampled IV glucose tolerance test (FSIGTT), and body composition measurements including determination of percent body fat. Parameters of lipolysis suppression, time to FFA nadir, and FFA rebound after an IV glucose challenge were derived using a mathematical model. Multivariable regression analyses were used to characterize the independent associations between SDB severity and parameters of FFA metabolism. SDB severity, as assessed by the apnea-hypopnea index, was associated with adipocyte insulin resistance, a decrease in the glucose- and insulin-mediated suppression of lipolysis, a longer duration to reach a nadir in FFA levels during the FSIGTT, and a sluggish rebound in FFA levels after suppression. Severity of SDB-related hypoxemia was independently associated with adipocyte insulin resistance and the time to reach the FFA nadir during the FSIGTT. Finally, a higher percentage of stage N3 sleep was positively associated with greater suppression of lipolysis and a faster rebound in the FFA levels during the FSIGTT. Independent of adiposity, SDB is associated with impairments in FFA metabolism, which may contribute to the development of glucose intolerance and type 2 diabetes in SDB.

233-LB: Spinal Cord Injury Inhibited-FGF21 Signaling Is Associated with Dysregulation of Metabolic Gene Expression in Mouse Liver and Adipose Tissue

Spinal cord injury (SCI) causes deleterious changes in the metabolic profile due to muscle atrophy and sedentary lifestyle. SCI is associated with increased risk for cardiovascular disease, liver disorders and type 2 diabetes mellitus (T2DM). Understanding of the mechanisms for these disorders in those with SCI is poor. Fibroblast growth factor 21 (FGF-21), a hepatic hormone, is reported to have beneficial effects on glucose/lipid metabolism in animal models and human subjects with T2DM. However, the expression levels and the potential effect of FGF-21 on SCI related metabolic dysfunction have not been studied. To address this issue, we compared FGF-21 serum and hepatic mRNA levels in sham and SCI mice fed control or high fat diet (HFD) at 84d after SCI. SCI reduced serum FGF-21 levels by 60% and hepatic FGF-21 mRNA levels by 50% compared to sham mice; levels were further suppressed by HFD. These changes were associated with elevated serum levels of alanine aminotransferase, a liver damage marker. In adipose tissue (AT), SCI decreased mRNA expression of FGF-21 receptor (FGFR1), β-Klotho, adiponectin, PPARγ, and leptin. In addition, SCI reduced IRS1 and Glut4 mRNA levels in AT, indicating impaired insulin signaling. Moreover, elevated levels of TNFα in AT and CD11b (a macrophage marker) in the liver were observed, implying a proinflammatory state after SCI. Finally, post-SCI mice fed a HFD had elevated free fatty acid (FFA) levels in serum and liver compared to sham-mice fed a HFD, suggesting defective FFA uptake and metabolism. These data suggest that reduced FGF-21 signaling after SCI may, at least in part, be contributing to the metabolic dysfunction after SCI, and that FGF-21 may be a candidate for the treatment of SCI-related metabolic dysfunction. Disclosure X. Liu: None. L. Harlow: None. Z. Graham: None. J.F. Yarrow: None. K. Cusi: Consultant; Self; Allergan plc., AstraZeneca, Bristol-Myers Squibb, Genentech, Inc., Gilead Sciences, Inc., Merck & Co., Inc. Research Support; Self; Cirius Therapeutics, Echosens, Eli Lilly and Company, Inventiva Pharma, Janssen Pharmaceuticals, Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Poxel SA, Zydus Pharmaceuticals, Inc. W.A. Bauman: None. C.P. Cardozo: None. Funding U.S. Department of Veterans Affairs (B9212C, B2020C)

Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia colifadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2 In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenesIMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

The Important Role of STAT3 in Chronic Lymphocytic Leukaemia Biology.

Signal transducer and activator of transcription (STAT) proteins are cytoplasmic transcription factors that transmit the signal of cytokines, hormones and growth factors. STAT proteins control fundamental cellular processes including survival, proliferation and differentiation. Inappropriate activation of STATs might contribute to cellular transformation and leukaemogenesis. About 70% of all solid and haematological tumours exhibit aberrant STAT3 expression and/or activation, highlighting its essential role in tumourigenesis. Aberrant STAT3 activation has been found in several solid tumours and haematologic malignancies. Importantly, constitutive activation of STAT proteins has been found in several leukaemias including acute myeloid leukaemia, acute promyelocytic leukaemia, acute lymphoblastic leukaemia, chronic myeloid leukaemia and chronic lymphocytic leukaemia (CLL). Constitutively activated STAT3 plays an important role in CLL biology. CLL cells harbour constitutive phosphorylation on S727 and acetylation on K685 and transient phosphorylation on Y705 residues. Moreover, STAT3 messenger RNA expression is significantly higher in CLL cells compared to healthy B-lymphocytes. Interestingly, STAT3 inhibition was disclosed as an important by-product of ibrutinib treatment in CLL patients. The purpose of this review is to describe the consequences of STAT3 dysregulation in CLL cells. Here, we discuss aberrantly modified processes by STAT3 activation in CLL cells such as proliferation, apoptosis, B cell receptor signalling, cytokine secretion, immune checkpoint regulation, microRNA regulation, free fatty acid metabolism and electron transport chain in the mitochondria.

Mitochondrial Translocator Protein (TSPO) Prevents Heart Failure by Increasing Cardiac Utilization of Fatty Acids

Introduction: For more than two decades, the peripheral mitochondrial translocator protein (TSPO) was considered to play a major role in mPTP formation, cholesterol transport, and steroidogenesis. However, recent genetic studies have questioned these previously established functions of TSPO, and therefore, the exact molecular mechanism of TSPO action remains unclear. We have previously demonstrated that TSPO expression was significantly upregulated in heart failure (HF) induced by transverse aortic constriction (TAC) in mice, and cardiac-specific conditional knockout (KO) of TSPO limited the development of pressure overload induced HF (Thai, Sci Reports, 2018, 8(1):16213). However, the exact mechanism of this protection has not been elucidated. Objective: Determine the mechanism of TSPO KO protection against pressure-overload HF. Methods and Results: We examined protein profiles in hearts from 4 experimental groups (WT sham, KO sham, WT TAC and KO TAC) using liquid chromatography-mass spectrophotometry. Our data revealed major changes in NADH:ubiquinone oxidoreductase subunit AB1 of the mitochondrial complex I (NDUFAB1), mitochondrial acyl-CoA binding protein (ACBP), mitochondrial long-chain specific acyl-CoA dehydrogenase (ACADL), and mitochondrial D-2-hydroxyglutarate dehydrogenase between KO and WT animals, with more modest changes in cytoskeletal and collagen associated proteins. Since NDUFAB1 and ACBP participate in free fatty acids (FFA) synthesis and ACADL is an important enzyme in FFA oxidation (FAO) pathway, we tested whether TSPO modulates FFA biosynthesis and oxidation.We measured changes in FAO in WT and TSPO KO mice under sham and TAC conditions and found that TSPO KO significantly increased fatty acid consumption and oxidation. Conclusion: Since HF is characterized by a shift in metabolism from FFA to less efficient glycolysis, thereby resulting in energetic failure, contractile dysfunction and cell death, the recovery of FFA metabolism by TSPO inhibition may be an effective drug target for HF.

Astaxanthin stimulates mitochondrial biogenesis in insulin resistant muscle via activation of AMPK pathway.

BackgroundSkeletal muscle is mainly responsible for insulin‐stimulated glucose disposal. Dysfunction in skeletal muscle metabolism especially during obesity contributes to the insulin resistance. Astaxanthin (AX), a natural antioxidant, has been shown to ameliorate hepatic insulin resistance in obese mice. However, its effects in skeletal muscle are poorly understood. The current study aimed to investigate the molecular target of AX in ameliorating skeletal muscle insulin resistance.MethodsWe fed 6‐week‐old male C57BL/6J mice with normal chow (NC) or NC supplemented with AX (NC+AX) and high‐fat‐diet (HFD) or HFD supplemented with AX for 24 weeks. We determined the effect of AX on various parameters including insulin sensitivity, glucose uptake, inflammation, kinase signaling, gene expression, and mitochondrial function in muscle. We also determined energy metabolism in intact C2C12 cells treated with AX using the Seahorse XFe96 Extracellular Flux Analyzer and assessed the effect of AX on mitochondrial oxidative phosphorylation and mitochondrial biogenesis.ResultsAX‐treated HFD mice showed improved metabolic status with significant reduction in blood glucose, serum total triglycerides, and cholesterol (p< 0.05). AX‐treated HFD mice also showed improved glucose metabolism by enhancing glucose incorporation into peripheral target tissues, such as the skeletal muscle, rather than by suppressing gluconeogenesis in the liver as shown by hyperinsulinemic–euglycemic clamp study. AX activated AMPK in the skeletal muscle of the HFD mice and upregulated the expressions of transcriptional factors and coactivator, thereby inducing mitochondrial remodeling, including increased mitochondrial oxidative phosphorylation component and free fatty acid metabolism. We also assessed the effects of AX on mitochondrial biogenesis in the siRNA‐mediated AMPK‐depleted C2C12 cells and showed that the effect of AX was lost in the genetically AMPK‐depleted C2C12 cells. Collectively, AX treatment (i) significantly ameliorated insulin resistance and glucose intolerance through regulation of AMPK activation in the muscle, (ii) stimulated mitochondrial biogenesis in the muscle, (iii) enhanced exercise tolerance and exercise‐induced fatty acid metabolism, and (iv) exerted antiinflammatory effects via its antioxidant activity in adipose tissue.ConclusionsWe concluded that AX treatment stimulated mitochondrial biogenesis and significantly ameliorated insulin resistance through activation of AMPK pathway in the skeletal muscle.

Pathogenetic aspects of the effects of the bacterial overgrowth syndrome on metabolism of free fatty acids in patients with chronic diffusive liver diseases

Objective — to investigate blood serum levels of free fatty acids of patients with chronic diffusive liver diseases (CDLD) depending on the etiology and microbiocenosis disturbances.Materials and methods. The study involved 98 patients with CDLD aged 59.2 ± 2.4 years. Depending on the etiological factors of liver injury, they were divided into five groups: group I consisted of 14 patients with non‑alcoholic fatty liver disease (NAFLD); group II — 31 patients with non‑alcoholic steatohepatitis (NASH); III — 12 persons with chronic viral hepatitis associated with C virus (CHC); IV — 24 patients with alcoholic liver disease (ALD); V — 17 patients with toxic hepatitis (TH). The control group included 16 practically healthy subjects. The diagnosis of bacterial overgrowth syndrome (BOS) was based on the glucose breath test results with the use of «Gastro» Gastrolyzer (Bedfont Scientific Ltd, UK). Blood serum levels of free fatty acids (FFAs) and their fractions were measured with gas chromatography.Results. The mean level of exhaled hydrogen in all groups was higher than normal, thus confirming the BOS in patients with CDLD. Thus index was significantly higher in patients with ALD (10.9 ± 1.6 ppm) and TH (13.1 ± 2.8 ppm) in comparison with subjects with NAFLD (6.6 ± 1.0 ppm; р &lt; 0.05). The FFA spectrum in blood serum revealed multidirectional changes depending on the etiological factor. The significant increase of FFA levels vs control groups has been established in patients with NAFLD, NASH and ALD, and patients with CHC and TH demonstrated a tendency to its reduction when compared with the control group.Conclusions. The changes in the small intestine microflora was observed in 64.3 % of CDLD patients, predominantly at alcoholic liver disease (79.2 %), and more are at NAFLD (57.1 %). The serum FFA levels were increased in patients with NAFLD, NASH and ALD at both BOS presence and absence, the most intensive it was at SOP absence.

Metformin Alleviates Hyperuricemia-Induced Serum Free Fatty Acid Elevation and Insulin Resistance by Improving Adipose Tissue Function

Background: Hyperuricemia (HUA) significantly increases risk of metabolic syndrome, and is strongly associated with the increased prevalence of high serum free fatty acids (FFA) and insulin resistance. However, the detailed mechanism remains unclear. Adipose tissue plays a crucial role in FFA metabolism. In this study, we aimed to explore the effects and underlying mechanism of uric acid (UA) on the metabolic function of adipocytes. Further, we investigated whether the administration of metformin could alleviate HUA-related metabolic disorders Methods: We performed H&E and immunohistochemistry to analyze adipose tissues of normal and HUA patients. Then mRNA microarray analysis was used to compare mRNA expression profiles of 3T3-L1 cells with and without UA. HUA mice were treated with UA lowering agent (benzbromarone) and metformin, respectively, and then epididymal white adipose tissue (eWAT) were isolated for in vitro experiment. Fundings: UA induced adipocyte hypertrophy, inhibited hyperplasia and reduced its beige-like characteristics. The mRNA microarray data indicated that UA significantly decreased the expression of leptin, which was closely related to FFA metabolism and 5-AMP-activated protein kinase (AMPK) signaling pathway according to KEGG pathway analysis. Moreover, benzbromarone and metformin induced activation of AMPK expression significantly attenuated UA-induced FFA metabolism impairment and adipose beiging depression, which subsequently alleviated serum FFA elevation and insulin resistance in HUA mice. Interpretation: UA is involved in the etiology of serum FFA elevation and insulin resistance by impairing adipose tissue function, which is mediated by leptin-AMPK pathway. AMPK activation by metformin represents a novel potential treatment strategy for HUA-related metabolic disorders. Funding Statement: This project was supported by grants from National Nature Scientific Foundation of China (No.81830012, No.81670297). Doctoral Fund of Ministry of Education of Heilongjiang Province (LBH-Z17180) and Harbin Medical University Science Innovation Foundation (YJSKYCX2018-91HYD) also provided additional support. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Clinical sampling was approved by the Ethical Committee of First Affiliated Hospital of Harbin Medical University. The Animal Care and Use Committee of the First Affiliated Hospital of Harbin Medical University approved all protocols.

β-cell function, incretin response, and insulin sensitivity of glucose and fat metabolism in obese youth: Relationship to OGTT-time-to-glucose-peak.

In adults, the time-to-glucose-peak at or after 30 minutes during an oral glucose tolerance test (OGTT) identifies physiologically distinct groups with differences in insulin sensitivity, β-cell function and risk for type 2 diabetes. In obese non-diabetic adolescents, we investigated if the OGTT-time-to-glucose-peak also reflects incretin and free fatty acid (FFA) responses besides insulin sensitivity and β-cell function, measured by the clamp. Obese adolescents (n = 278) were categorized according to their OGTT-time-to-glucose-peak by Early-peak (at 30 minutes) vs Late-peak (>30 minutes) groups. Body composition, visceral adipose tissue, oral disposition index and OGTT-area under the curve (AUC) were examined. A subset of 102 participants had both hyperinsulinemic-euglycemic and hyperglycemic clamps to measure in vivo insulin sensitivity, insulin secretion, and β-cell function relative to insulin sensitivity. Compared with the Early-peak group, the Late-peak group had impaired β-cell function relative to insulin sensitivity, lower glucose-dependent insulinotropic polypeptide-AUC, and higher FFA-AUC despite higher insulin- and C-peptide-AUC. They also had lower hepatic and peripheral insulin sensitivity despite similar percent body fat and visceral adipose tissue, and had higher prevalence of impaired glucose tolerance (all P < .05). In obese non-diabetic youth, those with a Late-peak vs an Early-peak glucose during an OGTT showed diminished β-cell function, blunted incretin secretion, and lower insulin sensitivity of glucose and FFA metabolism. It remains to be determined if Late-peak glucose predicts the future development of type 2 diabetes in these high-risk youth.

Plin5/p-Plin5 Guards Diabetic CMECs by Regulating FFAs Metabolism Bidirectionally.

Background Hyper-free fatty acidemia (HFFA) impairs cardiac capillaries, as well as type 2 diabetes mellitus (T2DM). Perilipin 5 (Plin5) maintains metabolic balance of free fatty acids (FFAs) in high oxidative tissues via the states of nonphosphorylation and phosphorylation. However, when facing to T2DM-HFFA, Plin5's role in cardiac microvascular endothelial cells (CMECs) is not defined. Methods In mice of WT or Plin5−/−, T2DM models were rendered by high-fat diet combined with intraperitoneal injection of streptozocin. CMECs isolated from left ventricles were incubated with high glucose (HG) and high FFAs (HFFAs). Plin5 phosphorylation was stimulated by isoproterenol. Plin5 expression was knocked down by small interfering RNA (siRNA). We determined cardiac function by small animal ultrasound, apoptotic rate by flow cytometry, microvessel quantity by immunohistochemistry, microvascular integrity by scanning electron microscopy, intracellular FFAs by spectrophotometry, lipid droplets (LDs) by Nile red staining, mRNAs by quantitative real-time polymerase chain reaction, proteins by western blots, nitric oxide (NO) and reactive oxygen species (ROS) by fluorescent dye staining and enzyme-linked immunosorbent assay kits. Results In CMECs, HFFAs aggravated cell injury induced by HG and activated Plin5 expression. In mice with T2DM-HFFA, Plin5 deficiency reduced number of cardiac capillaries, worsened structural incompleteness, and enhanced diastolic dysfunction. Moreover, in CMECs treated with HG-HFFAs, both ablation and phosphorylation of Plin5 reduced LDs content, increased intracellular FFAs, stimulated mitochondrial β-oxidation, added ROS generation, and reduced the expression and activity of endothelial nitric oxide synthase (eNOS), eventually leading to increased apoptotic rate and decreased NO content, all of which were reversed by N-acetyl-L-cysteine. Conclusion Plin5 preserves lipid balance and cell survival in diabetic CMECs by regulating FFAs metabolism bidirectionally via the states of nonphosphorylation and phosphorylation.

P121918F-FDG perfusion-metabolism mismatch 3 days after acute myocardial infarction predicts worse outcome: molecular glucose steel phenomenon visualized by hybrid PET-MRI images

Abstract Background The disturbance of cardiac energy substrate metabolism has a decisive role in the pathogenesis of adverse cardiac remodeling and heart failure. Under normoxic conditions, the free fatty acid metabolism is the predominant pathway by providing the highest energy per substrate molecule. Severe myocardial hypoxia leads to a switch from beta-oxidation to glucose metabolism to increase the ATP production per oxygen molecule. This metabolic disorder appears as perfusion-metabolism mismatch in 18F-fluorodeoxyglucose (18F-FDG) PET images, as increased 18F-FDG uptake in the under-perfused hypoxic myocardial area. Purpose The aim of our study was to evaluate the simultaneous perfusion, metabolism and function of the ischemic heart by hybrid 18F-FDG-PET-cMRI with late enhancement images in a translation animal model of heart failure. Methods Under general anesthesia, closed chest reperfused acute myocardial infarction (AMI) was induced in 36 domestic pigs by 90 min occlusion of the mid left anterior descending artery with a percutaneous intracoronary balloon, followed by reperfusion. Three days and 1 month after AMI, after 12h fasting, 18F-FDG-PET-cMRI were performed by using standardized acquisition protocols (n=30). Cardiac functional parameter, such as ejection fraction (EF), end-diastolic volume (EDV), infarct size, and mean tracer uptake of the infarcted area were quantitatively assessed. Six animals were euthanized after the 3-day 18F-FDG-PET-cMRI images to elaborate the differences in gene expression patterns in animals with perfusion-metabolic mismatch by using next generation sequencing (NGS) and pathway network analyses. Results Eight (group Mismatch) of the 30 animals (group Match) with 1-month follow-up showed high 18F-FDG uptake in the infarcted area (perfusion-metabolism mismatch) at the 3-day 18F-FDG-PET-cMRI-LE images (Figure). The animals in the Mismatch group had significantly lower EF at 3 days (34±8.8 vs 42±3%) and at the 1-month follow-up (35.8±6 vs 43±6.6%) and larger infarct size at day 3 (26.6±6.6 vs 22.1±4.4%) and 1 month (28±5.4 vs 20.3±4.3%) with higher EDV at 1 month. Mean tracer uptake of the infarcted area was significantly reduced in the Mismatch group at 1 month (56±23.1 vs 64.7±13.2%). NGS revealed downregulation of the cholesterol metabolism pathway, and upregulation of carbohydrate derivative catabolism pathway with highly activated innate immune system and genes responsible for cytokine activation in the infarcted area 3 days post-AMI in the Mismatch group, which explains the paradox high 18F-FDG tracer uptake in the infarction zone. Accordingly, the high energy demand of the severe hypoxic area led to “glucose steel phenomenon” at the molecular level, subtracting the 18F-FDG from the normally perfused non-ischemic myocardial regions. Conclusions 18F-FDG-glucose perfusion-metabolism mismatch early after AMI visualized by hybrid 18F-FDG-PET-MRI images predict development of LV adverse remodeling. Acknowledgement/Funding Fibrotarget EU Grant Nr 602904

Levocarnitine does not impair chemotherapy cytotoxicity against acute lymphoblastic leukemia

Asparaginase (ASNase) is an integral part of pediatric induction chemotherapy that has also been shown to improve adult survival rates; however, pegylated (PEG)-ASNase induces severe hepatotoxicity in this population. Recent case reports describe the incorporation of levocarnitine (LC) supplementation into PEG-ASNase-containing induction regimens to prevent or treat hepatotoxicity. Because LC facilitates the metabolism of free fatty acids (FFA), a primary fuel source for ALL cells, LC could potentially interfere with ALL chemotherapy efficacy. To test this, we employed in vitro and in vivo models of ALL. We show in vitro that LC supplementation does not impact cytotoxicity from vincristine, daunorubicin, dexamethasone, or ASNase on human ALL cells nor lead to an increase in ALL cell metabolic rate. In vivo, we demonstrate LC does not impair PEG-ASNase monotherapy in mice with syngeneic ALL. Together, our findings show that LC supplementation is a safe strategy to prevent/reverse ASNase-induced toxicities in preclinical models.

Co-expression network analysis identified hub genes critical to triglyceride and free fatty acid metabolism as key regulators of age-related vascular dysfunction in mice.

Background: Aging has often been linked to age-related vascular disorders. The elucidation of the putative genes and pathways underlying vascular aging likely provides useful insights into vascular diseases at advanced ages. Transcriptional regulatory network analysis is the key to describing genetic interactions between molecular regulators and their target gene transcriptionally changed during vascular aging.Results: A total of 469 differentially expressed genes were parsed into 6 modules. Among the incorporated sample traits, the most significant module related to vascular aging was associated with triglyceride and enriched with biological terms like proteolysis, blood circulation, and circulatory system process. The module associated with triglyceride was preserved in an independent microarray dataset, indicating the robustness of the identified vascular aging-related subnetwork. Additionally, Enpp5, Fez1, Kif1a, F3, H2-Q7, and their interacting miRNAs mmu-miR-449a, mmu-miR-449c, mmu-miR-34c, mmu-miR-34b-5p, mmu-miR-15a, and mmu-let-7, exhibited the most connectivity with external lipid-related traits. Transcriptional alterations of the hub genes Enpp5, Fez1, Kif1a, and F3, and the interacting microRNAs mmu-miR-34c, mmu-miR-34b-5p, mmu-let-7, mmu-miR-449a, and mmu-miR-449c were confirmed.Conclusion: Our findings demonstrate that triglyceride and free fatty acid-related genes are key regulators of age-related vascular dysfunction in mice and show that the hub genes for Enpp5, Fez1, Kif1a, and F3 as well as their interacting miRNAs mmu-miR-34c, mmu-miR-34b-5p, mmu-let-7, mmu-miR-449a, and mmu-miR-449c, could serve as potential biomarkers in vascular aging.Methods: The microarray gene expression profiles of aorta samples from 6-month old mice (n=6) and 20-month old mice (n=6) were processed to identify nominal differentially expressed genes. These nominal differentially expressed genes were subjected to a weighted gene co-expression network analysis. A network-driven integrative analysis with microRNAs and transcription factors was performed to define significant modules and underlying regulatory pathways associated with vascular aging, and module preservation test was conducted to validate the age-related modules based on an independent microarray gene expression dataset in mice aorta samples including three 32-week old wild-type mice (around 6-month old) and three 78-week old wild-type mice (around 20-month old). Gene ontology and protein-protein interaction analyses were conducted to determine the hub genes as potential biomarkers in the progress of vascular aging. The hub genes were further validated with quantitative real-time polymerase chain reaction in aorta samples from 20 young (6-month old) mice and 20 old (20-month old) mice.

Effect of Substrate on Adult Human Cardiomyocytes Contractility

Prior in vivo studies have reported that the development of heart failure is accompanied by a transition from the normal preferential metabolism of free fatty acids (FFA) to increases in glucose utilization and even ketone bodies, which normally provide a modest contribution to energy balance. However, the functional significance of the altered substrate utilization in the failing heart is poorly understood. Recognizing that nearly all prior studies examining isolated cardiomyocyte physiology have used glucose as the sole metabolic substrate, we initiated studies to examine the impact of alternative metabolic substrates on contractility in isolated human cardiomyocytes. We employed freshly isolated adult human left ventricular cardiomyocytes from non-failing hearts obtained from organ donors (NF, N=11 hearts, N=110 cells) and failing hearts obtained from transplant recipients (HF, N=13 hearts, n=130 cells). Cardiomyocytes were resuspended in a conventional 5mM Glucose Tyrode solution with alternative substrates (Glucose, FFA, R-3-Hydroxybutyrate (3-OHB) or Mix (Glucose + FFA + 3-OHB)). Myocytes were field stimulated at 1 Hz and sarcomere length, fractional shortening, contraction velocity and relaxation velocity were measured using a video-based sarcomere length detection system (IonOptix Corp). Studies using isolated cardiac myocyte contractility as readout confirm that myocytes from NF human hearts are omnivorous: high levels of myocyte fractional shortening (FS) can be achieved under unstressed conditions (1 Hz, unloaded) with any substrate (FSGlucose: 0.132±0.012; FSFFA: 0.143±0.013; FS3OHB: 0.134±0.014; FSMIX: 0.155±0.020). In the failing heart, glucose alone is insufficient to produce normal unstressed myocyte fractional shortening (FSGlucose: 0.088±0.009***, p

996-P: Therapeutic Effect of a Novel Long-Acting GLP-1/GIP/Glucagon Triple Agonist (HM15211) in a Dyslipidemia Animal Model

Dyslipidemia is a well-established risk factor for cardiovascular disease. Although an efficacy has been demonstrated for several drugs including HMGCR inhibitor and PCSK9 inhibitor, overall outcome was still in doubt due to heterogeneity of this disease. Thus, targeting multiple pathways may provide promising efficacy in broad patient population. Previously, we showed that a long-acting GLP-1/GIP/Glucagon triple agonist, HM15211, not only provided efficient weight loss, but also improved lipid profiles in DIO mice. Of note, unlike current drugs possessing a single target, HM15211 could affect multiple pathways in lipid metabolism, suggesting HM15211 as a potential therapeutic option for dyslipidemia. Here, we investigated the effect of HM15211 on dyslipidemia and its mode of action (MoA). In high fructose-fed hamsters, HM15211 showed greater LDL lowering (-69.3% vs. Veh.) than evolocumab, PCSK9 Ab (-17.9% vs. Veh.). To elucidate the responsible MoA, HepG2 cells were treated with HM15211, and significantly increased LDLR expression (4.1-fold for HM15211, 2.3-fold for evolocumab vs. CTL) was observed. Consistently, the amount of LDL uptake (3.6-fold for HM15211, 2.8-fold for evolocumab vs. CTL) indicated enhanced LDL clearance by HM15211 via increasing LDLR expression even greater than evolocumab. To further identify the HM15211 target, the activity status of HMGCR, the key enzyme for cholesterol synthesis, was evaluated. Surprisingly, HM15211 progressively induced phosphorylation and degradation of HMGCR, thereby inhibiting its enzymatic activity. In case of free fatty acid (FFA) metabolism, HM15211 led to a favorable gene expression signature associated with β-oxidation and enhanced β-oxidation. Increased ApoA1 may explain, at least in part, the increase of HDL by HM15211. In conclusion, we propose that by targeting multiple steps essential for LDL and FFA metabolism, HM15211 could provide therapeutic benefits for dyslipidemia patients. Disclosure J. Choi: None. H. Jo: None. J. Kim: None. S. Lee: Employee; Self; Hanmi Pharm. Co., Ltd. S. Lee: Employee; Self; Hanmi Pharm. Co., Ltd. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd.

Circulating miR‐122‐5p: A Novel Exerkine in Exercise‐induced Angiogenesis

Physical exercise effectively boosts angiogenesis in skeletal muscles, but the mechanisms remain poorly understood. Our previous study showed that 4‐week swimming training (1.5 h×2/d, 7 d/wk×4 weeks) significantly promoted angiogenesis in skeletal muscle. And we had found the expression of several circulating miRNAs profoundly changed. Among which, miR‐122‐5p, a liver specific microRNA, promoted endothelial proliferation significantly. Here we further determined the role of circulating miR‐122‐5p in exercise‐induced angiogenesis. Exercised mice (running wheel training, 1.5 h/d ×9 d) exhibited elevated miR‐122‐5p in serum and aorta (2.132±0.294, 3.275±0.819 fold of Sedentary, p&lt;0.05), concomitant with increased capillary density in the quadriceps marked with fluorescent CD31 (3.562±0.354 fold of Sedentary, p&lt;0.01). Whereas, tail vein injection of miR‐122‐5p antagomir partially decreased the exercise‐induced VEGF expression and CD31 staining in quadriceps, suggesting that elevated circulating mir‐122‐5p partially contributes to the exercise‐induced angiogenesis. In vitro study on primary aortic endothelial cells (ECs) transfected with miR‐122‐5p mimic showed increased tube numbers, migration distance and relative sprouting distance, accompanied with increased expression of p‐ERK/ERK and VEGF. Mechanistically, miR‐122‐5p mimic increased the expression of CD36, CPT1a and CPT1b together with the uptake of fatty acid assessed by BODIPY fluorescence intensity in ECs, suggesting an increased free fatty acid (FFA) metabolism. Transfection of CD36 siRNA blunted the pro‐angiogenic effect of miR‐122‐5p. In conclusion, our findings demonstrate that exercise‐induced proangiogenic effects are partly attributed to elevated circulating miR‐122‐5p via increasing endothelial FFA metabolism.Support or Funding InformationThis study was supported by the grants from the National Natural Science Foundation of China (Grant No. 816770253)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Modest Weight Gain On Circulating Free Fatty Acids

BackgroundIncreases in circulating free‐fatty acids (FFAs) contribute to obesity‐associated cardiometabolic pathology and may be considered a surrogate marker for adipose tissue dysfunction. However, alterations in FFA metabolism resulting from modest weight gain have not been previously evaluated.ObjectiveExamine the changes in FFAs during modest weight gain in healthy individuals and to investigate the relationship between regional fat depots.Study DesignWe evaluated the effects of overfeeding in 8 healthy, non‐obese participants (4 men; age 29 ± 5.7 years; BMI: 25.1 ± 2.1 kg/m2). FFA concentrations were measured using liquid chromatography/mass spectrometry under fasting conditions and during oral‐glucose tolerance test (OGTT) at baseline and after 8 weeks of weight gain. Prior to the baseline and weight gain visits, subjects consumed isocaloric macro‐nutrient stable diet for three days (50% carbohydrate, 35% fat and 15% protein). Body composition and regional fat depots were assessed using dual‐energy X‐ray absorptiometry and abdominal computed tomography scans.ResultsOverfeeding was accompanied by 3.9 ± 1.6 kg weight gain (p=0.01) resulting from 2.9 ± 0.9 kg (p=0.01) increase in fat mass and 1.0 ± 0.9 kg (p=0.04) increase in fat‐free mass. Compared to baseline, fasting FFA (439 ± 162 μM vs. 368 ± 123 μM, p=0.55) and area under the curve (AUC) for FFA during OGTT (23319 ± 11686 μM. min−1 vs. 20495 ± 4847 μM.min−1, p=0.55) did not change with weight gain. However, at 120 min. of the OGTT FFA were significantly greater after weight gain (60 ± 18 μM vs. 77 ± 34 μM, p=0.04). We found a negative relationship between relative changes in visceral fat area and FFA at 120 min of glucose challenge (rho=−0.76, p=0.03). Furthermore, relative changes in fasting FFA and AUC for FFA during OGTT with weight gain were associated with baseline total fat mass (fasting FFA: rho=0.76, p=0.03; AUC FFA: rho=0.81; p=0.02) and baseline abdominal fat area (fasting FFA: rho=0.76, p=0.03; AUC FFA: rho=0.79; p=0.02).ConclusionsModest weight gain does not alter fasting FFA or overall FFA response to oral glucose challenge. However, our data suggest development of early stages of adipose tissue insulin resistance which is apparent via lesser suppression of FFA at 120 min. of OGTT. Furthermore, the relationships between fasting FFA, AUC‐FFA, and baseline fat mass suggest that individuals with high baseline adiposity may be more susceptible to develop adipose tissue insulin resistance with weight gain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Exercise on Fatty Acid Metabolism and Adipokine Secretion in Adipose Tissue.

Increased physical activity is an optimal way to maintain a good health. During exercise, triacylglycerols, an energy reservoir in adipose tissue, are hydrolyzed to free fatty acids (FAs) which are then released to the circulation, providing a fuel for working muscles. Thus, regular physical activity leads to a reduction of adipose tissue mass and improves metabolism. However, the reduction of lipid reservoir is also associated with many other interesting changes in adipose tissue FA metabolism. For example, a prolonged exercise contributes to a decrease in lipoprotein lipase activity and resultant reduction of FA uptake. This results in the improvement of mitochondrial function and upregulation of enzymes involved in the metabolism of polyunsaturated fatty acids. The exercise-induced changes in adipocyte metabolism are associated with modifications of FA composition. The modifications are adipose tissue depot-specific and follow different patterns in visceral and subcutaneous adipose tissue. Moreover, exercise affects adipokine release from adipose tissue, and thus, may mitigate inflammation and improve insulin sensitivity. Another consequence of exercise is the recently described phenomenon of adipose tissue “beiging,” i.e., a switch from energy-storing white adipocyte phenotype to thermogenic FA oxidizing beige adipocytes. This process is regulated by myokines released during the exercise. In this review, we summarize published evidence for the exercise-related changes in FA metabolism and adipokine release in adipose tissue, and their potential contribution to beneficial cardiovascular and metabolic effects of physical activity.