Hiroaki Sunaga

Stearoyl-CoA Desaturase-1 (SCD1) Augments Saturated Fatty Acid-Induced Lipid Accumulation and Inhibits Apoptosis in Cardiac Myocytes

Mismatch between the uptake and utilization of long-chain fatty acids in the myocardium leads to abnormally high intracellular fatty acid concentration, which ultimately induces myocardial dysfunction. Stearoyl-Coenzyme A desaturase-1 (SCD1) is a rate-limiting enzyme that converts saturated fatty acids (SFAs) to monounsaturated fatty acids. Previous studies have shown that SCD1-deficinent mice are protected from insulin resistance and diet-induced obesity; however, the role of SCD1 in the heart remains to be determined. We examined the expression of SCD1 in obese rat hearts induced by a sucrose-rich diet for 3 months. We also examined the effect of SCD1 on myocardial energy metabolism and apoptotic cell death in neonatal rat cardiac myocytes in the presence of SFAs. Here we showed that the expression of SCD1 increases 3.6-fold without measurable change in the expression of lipogenic genes in the heart of rats fed a high-sucrose diet. Forced SCD1 expression augmented palmitic acid-induced lipid accumulation, but attenuated excess fatty acid oxidation and restored reduced glucose oxidation. Of importance, SCD1 substantially inhibited SFA-induced caspase 3 activation, ceramide synthesis, diacylglycerol synthesis, apoptotic cell death, and mitochondrial reactive oxygen species (ROS) generation. Experiments using SCD1 siRNA confirmed these observations. Furthermore, we showed that exposure of cardiac myocytes to glucose and insulin induced SCD1 expression. Our results indicate that SCD1 is highly regulated by a metabolic syndrome component in the heart, and such induction of SCD1 serves to alleviate SFA-induced adverse fatty acid catabolism, and eventually to prevent SFAs-induced apoptosis.

Deranged fatty acid composition causes pulmonary fibrosis in Elovl6 -deficient mice

Despite the established role of alveolar type II epithelial cells for the maintenance of pulmonary function, little is known about the deregulation of lipid composition in the pathogenesis of pulmonary fibrosis. The elongation of long-chain fatty acids family member 6 (Elovl6) is a rate-limiting enzyme catalysing the elongation of saturated and monounsaturated fatty acids. Here we show that Elovl6 expression is significantly downregulated after an intratracheal instillation of bleomycin (BLM) and in human lung with idiopathic pulmonary fibrosis. Elovl6-deficient (Elovl6⁻/⁻) mice treated with BLM exhibit severe fibroproliferative response and derangement of fatty acid profile compared with wild-type mice. Furthermore, Elovl6 knockdown induces a change in fatty acid composition similar to that in Elovl6⁻/⁻ mice, resulting in induction of apoptosis, TGF-β1 expression and reactive oxygen species generation. Our findings demonstrate a previously unappreciated role for Elovl6 in the regulation of lung homeostasis, and in pathogenesis and exacerbation of BLM-induced pulmonary fibrosis.

CD36 is indispensable for thermogenesis under conditions of fasting and cold stress

Hypothermia can occur during fasting when thermoregulatory mechanisms, involving fatty acid (FA) utilization, are disturbed. CD36/FA translocase is a membrane protein which facilitates membrane transport of long-chain FA in the FA consuming heart, skeletal muscle (SkM) and adipose tissues. It also accelerates uptake of triglyceride-rich lipoprotein by brown adipose tissue (BAT) in a cold environment. In mice deficient for CD36 (CD36(-/-) mice), FA uptake is markedly reduced with a compensatory increase in glucose uptake in the heart and SkM, resulting in lower levels of blood glucose especially during fasting. However, the role of CD36 in thermogenic activity during fasting remains to be determined. In fasted CD36(-/-) mice, body temperature drastically decreased shortly after cold exposure. The hypothermia was accompanied by a marked reduction in blood glucose and in stores of triacylglycerols in BAT and of glycogen in glycolytic SkM. Biodistribution analysis using the FA analogue (125)I-BMIPP and the glucose analogue (18)F-FDG revealed that uptake of FA and glucose was severely impaired in BAT and glycolytic SkM in cold-exposed CD36(-/-) mice. Further, induction of the genes of thermogenesis in BAT was blunted in fasted CD36(-/-) mice after cold exposure. These findings strongly suggest that CD36(-/-) mice exhibit pronounced hypothermia after fasting due to depletion of energy storage in BAT and glycolytic SkM and to reduced supply of energy substrates to these tissues. Our study underscores the importance of CD36 for nutrient homeostasis to survive potentially life-threatening challenges, such as cold and starvation.

Fatty Acid Binding Protein 4 and 5 Play a Crucial Role in Thermogenesis under the Conditions of Fasting and Cold Stress

Hypothermia is rapidly induced during cold exposure when thermoregulatory mechanisms, including fatty acid (FA) utilization, are disturbed. FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipose tissues and macrophages, have been identified as key molecules in the pathogenesis of overnutrition-related diseases, such as insulin resistance and atherosclerosis. We have recently shown that FABP4/5 are prominently expressed in capillary endothelial cells in the heart and skeletal muscle and play a crucial role in FA utilization in these tissues. However, the role of FABP4/5 in thermogenesis remains to be determined. In this study, we showed that thermogenesis is severely impaired in mice lacking both FABP4 and FABP5 (DKO mice), as manifested shortly after cold exposure during fasting. In DKO mice, the storage of both triacylglycerol in brown adipose tissue (BAT) and glycogen in skeletal muscle (SkM) was nearly depleted after fasting, and a biodistribution analysis using 125I-BMIPP revealed that non-esterified FAs (NEFAs) are not efficiently taken up by BAT despite the robustly elevated levels of serum NEFAs. In addition to the severe hypoglycemia observed in DKO mice during fasting, cold exposure did not induce the uptake of glucose analogue 18F-FDG by BAT. These findings strongly suggest that DKO mice exhibit pronounced hypothermia after fasting due to the depletion of energy storage in BAT and SkM and the reduced supply of energy substrates to these tissues. In conclusion, FABP4/5 play an indispensable role in thermogenesis in BAT and SkM. Our study underscores the importance of FABP4/5 for overcoming life-threatening environments, such as cold and starvation.

Pressure mediated hypertrophy and mechanical stretch up-regulate expression of the long form of leptin receptor (ob-Rb) in rat cardiac myocytes

BackgroundHyperleptinemia is known to participate in cardiac hypertrophy and hypertension, but the relationship between pressure overload and leptin is poorly understood. We therefore examined the expression of leptin (ob) and the leptin receptor (ob-R) in the pressure-overloaded rat heart. We also examined gene expressions in culture cardiac myocytes to clarify which hypertension-related stimulus induces these genes.ResultsPressure overload was produced by ligation of the rat abdominal aorta, and ob and ob-R isoform mRNAs were measured using a real-time polymerase chain reaction (PCR). We also measured these gene expressions in neonatal rat cardiac myocytes treated with angiotensin II (ANGII), endothelin-1 (ET-1), or cyclic mechanical stretch. Leptin and the long form of the leptin receptor (ob-Rb) gene were significantly increased 4 weeks after banding, but expression of the short form of the leptin receptor (ob-Ra) was unchanged. ob-Rb protein expression was also detected by immunohistochemistry in hypertrophied cardiac myocytes after banding. Meanwhile, plasma leptin concentrations were not different between the control and banding groups. In cultured myocytes, ANGII and ET-1 increased only ob mRNA expression. However, mechanical stretch activated both ob and ob-Rb mRNA expression in a time-dependent manner, but ob-Ra mRNA was unchanged by any stress.ConclusionsWe first demonstrated that both pressure mediated hypertrophy and mechanical stretch up-regulate ob-Rb gene expression in heart and cardiac myocytes, which are thought to be important for leptin action in cardiac myocytes. These results suggest a new local mechanism by which leptin affects cardiac remodeling in pressure-overloaded hearts.

Independent and incremental prognostic value of novel cardiac biomarkers in chronic hemodialysis patients

UNLABELLED End-stage renal disease is a major clinical and public health problem, and cardiovascular disease accounts for half of the mortality in hemodialysis patients. An existing mortality risk score (AROii score) or N-terminal pro-brain natriuretic peptide (NT-proBNP) level have modest predictive power, but there is room for improvement. There are emerging cardiac biomarkers (soluble isoforms of ST2 [sST2], galectin-3 [Gal-3]), and uremic toxicity (indoxyl sulfate). We sought to determine whether these biomarkers predict cardiovascular outcomes in hemodialysis patients and have incremental prognostic value over the clinical score and NT-proBNP level. METHODS A total of 423 hemodialysis patients were prospectively followed up for primary (all-cause death) and secondary end points (a composite of all-cause death or cerebrocardiovascular events). RESULTS During a mean follow-up of 2.1 ± 0.4 years, there were 48 all-cause deaths and 78 composite outcomes. Soluble isoforms of ST2, Gal-3, and NT-proBNP were associated with all-cause deaths but indoxyl sulfate was not in both log-rank test and receiver operating characteristic analysis. Both sST2 and Gal-3 had independent and incremental prognostic value for both outcomes over the AROii score and NT-proBNP. Although adding sST2 did not reclassify over the model-based AROii score and NT-proBNP for all-cause death, further addition of Gal-3 did. Subgroup analyses of patients with left ventricular ejection fraction measurement (n = 301) corroborated these results, where the 2 biomarkers remained independent and incremental for both all-cause death and composite outcome after adjusting for the risk score and the ejection fraction. CONCLUSIONS Both sST2 and Gal-3 had independent and incremental prognostic values over NT-proBNP and an established risk score in patients with hemodialysis. Assessment of sST2 and Gal-3 further enhances risk stratification.

Early increase in serum fatty acid binding protein 4 levels in patients with acute myocardial infarction

Background: Acute myocardial infarction (AMI) induces marked activation of the sympathetic nervous system. Fatty acid binding protein 4 (FABP4) is not only an intracellular protein, but also a secreted adipokine that contributes to obesity-related metabolic complications. Here, we examined the role of serum FABP4 as a pathophysiological marker in patients with AMI. Methods and results: We studied 106 patients presenting to the emergency unit with a final diagnosis of AMI, including 12 patients resuscitated from out-of-hospital cardiac arrest (OHCA) caused by ventricular fibrillation. FABP4 levels peaked on admission or just after percutaneous coronary intervention and declined thereafter. Regression analysis revealed no significant correlation between peak FABP4 and peak cardiac troponin T determined by Roche high-sensitive assays (hs-TnT). Notably, FABP4 levels were particularly elevated in AMI patients who were resuscitated from OHCA (median 130.2 ng/mL, interquartile range (IQR) 51.8–243.9 ng/mL) compared with those without OHCA (median 26.1 ng/ml, IQR 17.1–43.4 ng/mL), while hs-TnT levels on admission were not associated with OHCA. Immunohistochemistry of the human heart revealed that FABP4 is abundantly present in adipocytes within myocardial tissue and epicardial adipose tissue. An in vitro study using cultured adipocytes showed that FABP4 is released through a β3-adrenergic receptor (AR)-mediated mechanism. Conclusions: FABP4 levels were significantly elevated during the early hours after the onset of AMI and were robustly increased in OHCA survivors. Together with the finding that FABP4 is released from adipocytes via β3-AR-mediated lipolysis, our data provide a novel hypothesis that serum FABP4 may represent the adrenergic overdrive that accompanies acute cardiovascular disease, including AMI.

Robust suppression of cardiac energy catabolism with marked accumulation of energy substrates during lipopolysaccharide-induced cardiac dysfunction in mice

BACKGROUND Myocardial contractile dysfunction in sepsis has been attributed mainly to increased inflammatory cytokines, insulin resistance, and impaired oxidative phosphorylation of fatty acids (FAs). However, precise molecular mechanisms underlying the cardiac dysfunction in sepsis remain to be determined. We previously reported major shift in myocardial energy substrates from FAs to glucose, and increased hepatic ketogenesis in mice lacking fatty acid-binding protein 4 (FABP4) and FABP5 (DKO). PURPOSE We sought to determine whether a shift of energy substrates from FAs to glucose and increased availability of ketone bodies are beneficial or detrimental to cardiac function under the septic condition. METHODS Lipopolysaccharide (LPS, 10mg/kg) was intraperitoneally injected into wild-type (WT) and DKO mice. Twelve hours after injection, cardiac function was assessed by echocardiography and serum and hearts were collected for further analyses. RESULTS Cardiac contractile function was more deteriorated by LPS injection in DKO mice than WT mice despite comparable changes in pro-inflammatory cytokine production. LPS injection reduced myocardial uptake of FA tracer by 30% in both types of mice, while uptake of the glucose tracer did not significantly change in either group of mice in sepsis. Storage of glycogen and triacylglycerol in hearts was remarkably increased by LPS injection in both mice. Metabolome analysis revealed that LPS-induced suppression of pool size in the TCA cycle was more enhanced in DKO hearts. A tracing study with 13C6-glucose further revealed that LPS injection substantially reduced glucose-derived metabolites in the TCA cycle and related amino acids in DKO hearts. Consistent with these findings, glucose oxidation in vitro was similarly and markedly reduced in both mice. Serum concentration of β-hydroxybutyrate and cardiac expression of genes associated with ketolysis were reduced in septic mice. CONCLUSIONS Our study demonstrated that LPS-induced cardiac contractile dysfunction is associated with the robust suppression of catabolism of energy substrates including FAs, glucose and ketone bodies and accumulation of glycogen and triacylglycerol in the heart. Thus, a fuel shift from FAs to glucose and/or ketone bodies may be detrimental rather than protective under septic conditions.

Association Between Circulating Ketone Bodies and Worse Outcomes in Hemodialysis Patients

Background Cardiovascular disease is the leading cause of morbidity and mortality in patients receiving hemodialysis. Systemic metabolic perturbation is one of the hallmark abnormalities in patients at high cardiovascular risk. We sought to determine the relationship between circulating ketone body and clinical outcomes in patients with prevalent hemodialysis. Methods and Results We retrospectively assessed the relationship between serum β‐hydroxybutyrate (βOHB), the most abundant ketone body in the circulation, and prognosis in 405 stable hemodialysis patients. During a mean follow‐up of 3.2±0.9 years, there were 54 major adverse cardiovascular events (defined as cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, and hospitalization attributed to heart failure) and 67 all‐cause deaths. Major adverse cardiovascular events rates increased from 11.1 per 1000 person‐years in the lowest βOHB quintile (<89 μmol/L) to 80.1 per 1000 person‐years in the highest quintile (>409 μmol/L). After adjusting for demographic characteristics, coronary artery disease, and atrial fibrillation, the highest βOHB quintile was associated with increased risk of major adverse cardiovascular events compared with the lowest quintile (hazard ratio, 10.2; 95% confidence interval [3.35–44.0]; P<0.001). Increased quintiles of βOHB were independently and incrementally associated with major adverse cardiovascular events over the model based on an established risk score (the second Analyzing Data, Recognizing Excellence and Optimizing Outcomes cohort score) and N‐terminal pro‐B‐type natriuretic peptide (chi square 39.9 versus 21.7; P<0.001; c‐statistics, 0.713). Sensitivity analyses also confirmed the robustness of association between βOHB and all‐cause death. Conclusions Increased serum βOHB levels were independently associated with cardiovascular events and all‐cause death in patients receiving hemodialysis. These results highlight the need for future studies to understand the mechanisms underlying these observations.

Elongation of Long‐Chain Fatty Acid Family Member 6 (Elovl6)‐Driven Fatty Acid Metabolism Regulates Vascular Smooth Muscle Cell Phenotype Through AMP‐Activated Protein Kinase/Krüppel‐Like Factor 4 (AMPK/KLF4) Signaling

Background Fatty acids constitute the critical components of cell structure and function, and dysregulation of fatty acid composition may exert diverging vascular effects including proliferation, migration, and differentiation of vascular smooth muscle cells (VSMCs). However, direct evidence for this hypothesis has been lacking. We investigated the role of elongation of long‐chain fatty acid member 6 (Elovl6), a rate‐limiting enzyme catalyzing the elongation of saturated and monounsaturated long‐chain fatty acid, in the regulation of phenotypic switching of VSMC. Methods and Results Neointima formation following wire injury was markedly inhibited in Elovl6‐null (Elovl6−/−) mice, and cultured VSMCs with siRNA‐mediated knockdown of Elovl6 was barely responsive to PDGF‐BB. Elovl6 inhibition induced cell cycle suppressors p53 and p21 and reduced the mammalian targets of rapamycin (mTOR) phosphorylation and VSMC marker expression. These changes are ascribed to increased palmitate levels and reduced oleate levels, changes that lead to reactive oxygen species (ROS) production and resulting AMP‐activated protein kinase (AMPK) activation. Notably, Elovl6 inhibition robustly induced the pluripotency gene Krüppel‐like factor 4 (KLF4) expression in VSMC, and KLF4 knockdown significantly attenuated AMPK‐induced phenotypic switching of VSMC, indicating that KLF4 is a bona fide target of AMPK. Conclusions We demonstrate for the first time that dysregulation of Elovl6‐driven long‐chain fatty acid metabolism induces phenotypic switching of VSMC via ROS production and AMPK/KLF4 signaling that leads to growth arrest and downregulation of VSMC marker expression. The modulation of Elovl6‐mediated cellular processes may provide an intriguing approach for tackling atherosclerosis and postangioplasty restenosis.