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Alterations in plasma triglycerides and ceramides: links with cardiac function in humans with type 2 diabetes

2020; Elsevier BV; Volume: 61; Issue: 7 Linguagem: Inglês

10.1194/jlr.ra120000669

ISSN

1539-7262

Autores

Linda R. Peterson, Xuntian Jiang, Ling Chen, Anne C. Goldberg, Marsha Farmer, Daniel S. Ory, Jean E. Schaffer,

Tópico(s)

Diabetes, Cardiovascular Risks, and Lipoproteins

Resumo

Cardiac dysfunction in T2D is associated with excessive FA uptake, oxidation, and generation of toxic lipid species by the heart. It is not known whether decreasing lipid delivery to the heart can effect improvement in cardiac function in humans with T2D. Thus, our objective was to test the hypothesis that lowering lipid delivery to the heart would result in evidence of decreased "lipotoxicity," improved cardiac function, and salutary effects on plasma biomarkers of cardiovascular risk. Thus, we performed a double-blind randomized placebo-controlled parallel design study of the effects of 12 weeks of fenofibrate-induced lipid lowering on cardiac function, inflammation, and oxidation biomarkers, and on the ratio of two plasma ceramides, Cer d18:1 (4E) (1OH, 3OH)/24:0 and Cer d18:1 (4E) (1OH, 3OH)/16:0 (i.e., "C24:0/C16:0"), which is associated with decreased risk of cardiac dysfunction and heart failure. Fenofibrate lowered plasma TG and cholesterol but did not improve heart systolic or diastolic function. Fenofibrate treatment lowered the plasma C24:0/C16:0 ceramide ratio and minimally altered oxidative stress markers but did not alter measures of inflammation. Overall, plasma TG lowering correlated with improvement of cardiac relaxation (diastolic function) as measured by tissue Doppler-derived parameter e′. Moreover, lowering the plasma C24:0/C16:0 ceramide ratio was correlated with worse diastolic function. These findings indicate that fenofibrate treatment per se is not sufficient to effect changes in cardiac function; however, decreases in plasma TG may be linked to improved diastolic function. In contrast, decreases in plasma C24:0/C16:0 are linked with worsening cardiac function. Cardiac dysfunction in T2D is associated with excessive FA uptake, oxidation, and generation of toxic lipid species by the heart. It is not known whether decreasing lipid delivery to the heart can effect improvement in cardiac function in humans with T2D. Thus, our objective was to test the hypothesis that lowering lipid delivery to the heart would result in evidence of decreased "lipotoxicity," improved cardiac function, and salutary effects on plasma biomarkers of cardiovascular risk. Thus, we performed a double-blind randomized placebo-controlled parallel design study of the effects of 12 weeks of fenofibrate-induced lipid lowering on cardiac function, inflammation, and oxidation biomarkers, and on the ratio of two plasma ceramides, Cer d18:1 (4E) (1OH, 3OH)/24:0 and Cer d18:1 (4E) (1OH, 3OH)/16:0 (i.e., "C24:0/C16:0"), which is associated with decreased risk of cardiac dysfunction and heart failure. Fenofibrate lowered plasma TG and cholesterol but did not improve heart systolic or diastolic function. Fenofibrate treatment lowered the plasma C24:0/C16:0 ceramide ratio and minimally altered oxidative stress markers but did not alter measures of inflammation. Overall, plasma TG lowering correlated with improvement of cardiac relaxation (diastolic function) as measured by tissue Doppler-derived parameter e′. Moreover, lowering the plasma C24:0/C16:0 ceramide ratio was correlated with worse diastolic function. These findings indicate that fenofibrate treatment per se is not sufficient to effect changes in cardiac function; however, decreases in plasma TG may be linked to improved diastolic function. In contrast, decreases in plasma C24:0/C16:0 are linked with worsening cardiac function. coronary heart disease fractional shortening hemoglobin A1c 1H-magnetic resonance spectroscopy high sensitivity C-reactive protein interleukin 6 left ventricular T2D is associated with an increased risk of heart failure in the absence of obstructive coronary heart disease (CHD), valvular abnormalities, alcoholism, or hypertension. Asymptomatic left ventricular (LV) diastolic dysfunction can be detected in up to 75% of people with T2D even in the absence of comorbidities (1Boyer J.K. Thanigaraj S. Schechtman K.B. Perez J.E. Prevalence of ventricular diastolic dysfunction in asymptomatic, normotensive patients with diabetes mellitus.Am. J. Cardiol. 2004; 93: 870-875Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 2Diamant M. Lamb H.J. Groeneveld Y. Endert E.L. Smith J.W. Bax J.J. Romijn J.A. de Roos A. Radder J.K. 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Studies of rodent models of T2D provide evidence that lipotoxicity is caused by increased plasma FFA and TG delivery to and oxidation/storage by the heart, which leads to oxidative stress and significantly contributes to risk of cardiac dysfunction (8Taegtmeyer H. Golfman L. Sharma S. Razeghi P. van Arsdall M. Linking gene expression to function: metabolic flexibility in the normal and diseased heart.Ann. N. Y. Acad. Sci. 2004; 1015: 202-213Crossref PubMed Scopus (168) Google Scholar, 9Young M.E. McNulty P. Taegtmeyer H. Adaptation and maladaptation of the heart in diabetes: Part II: potential mechanisms.Circulation. 2002; 105: 1861-1870Crossref PubMed Scopus (381) Google Scholar, 10Belke D.D. Larsen T.S. Gibbs E.M. Severson D.L. Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice.Am. J. Physiol. Endocrinol. Metab. 2000; 279: E1104-E1113Crossref PubMed Google Scholar, 11Zhou Y.T. Grayburn P. Karim A. Shimabukuro M. Higa M. Baetens D. Orci L. 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Saeed I.M. Recklein C. Coggan A.R. Demoss A.J. Schechtman K.B. Dence C.S. Gropler R.J. Potentiation of abnormalities in myocardial metabolism with the development of diabetes in women with obesity and insulin resistance.J. Nucl. Cardiol. 2011; 18: 421-429, quiz 432Crossref PubMed Scopus (26) Google Scholar, 27McGavock J.M. Lingvay I. Zib I. Tillery T. Salas N. Unger R. Levine B.D. Raskin P. Victor R.G. Szczepaniak L.S. Cardiac steatosis in diabetes mellitus: a 1H-magnetic resonance spectroscopy study.Circulation. 2007; 116: 1170-1175Crossref PubMed Scopus (434) Google Scholar, 28Sharma S. Adrogue J.V. Golfman L. Uray I. Lemm J. Youker K. Noon G.P. Frazier O.H. Taegtmeyer H. Intramyocardial lipid accumulation in the failing human heart resembles the lipotoxic rat heart.FASEB J. 2004; 18: 1692-1700Crossref PubMed Scopus (522) Google Scholar, 29Alavaikko M. Elfving R. Hirvonen J. Jarvi J. Triglycerides, cholesterol, and phospholipids in normal heart papillary muscle and in patients suffering from diabetes, cholelithiasis, hypertension, and coronary atheroma.J. Clin. Pathol. 1973; 26: 285-293Crossref PubMed Scopus (11) Google Scholar). This metabolic pattern is associated with decreased efficiency, impaired energetics, and diastolic dysfunction (2Diamant M. Lamb H.J. Groeneveld Y. Endert E.L. Smith J.W. Bax J.J. Romijn J.A. de Roos A. Radder J.K. Diastolic dysfunction is associated with altered myocardial metabolism in asymptomatic normotensive patients with well-controlled type 2 diabetes mellitus.J. Am. Coll. Cardiol. 2003; 42: 328-335Crossref PubMed Scopus (300) Google Scholar, 26McGill J.B. Peterson L.R. Herrero P. Saeed I.M. Recklein C. Coggan A.R. Demoss A.J. Schechtman K.B. Dence C.S. Gropler R.J. Potentiation of abnormalities in myocardial metabolism with the development of diabetes in women with obesity and insulin resistance.J. Nucl. Cardiol. 2011; 18: 421-429, quiz 432Crossref PubMed Scopus (26) Google Scholar). Nonetheless, it has been difficult to causally link myocardial lipid overload to myocardial dysfunction in humans with diabetes. In the ACCORD lipid trial, the trend for lower rates of fatal or nonfatal congestive heart failure in the fenofibrate-treated versus placebo group was not significant (30ACCORD Study Group Ginsberg H.N. Elam M.B. Lovato L.C. Crouse J.R. Leiter L.A. Linz P. Friedewald W.T. Buse J.B. Gerstein H.C. et al.Effects of combination lipid therapy in type 2 diabetes mellitus.N. Engl. J. Med. 2010; 362: 1563-1574Crossref PubMed Scopus (2005) Google Scholar), and cardiac function was not quantified as an outcome. More recently, the REDUCE-IT trial revealed lower hazard ratios for a composite cardiovascular end point in both diabetic and nondiabetic subjects, but myocardial function and heart failure were not reported as separate outcomes (31Bhatt D.L. Steg P.G. Miller M. Brinton E.A. Jacobson T.A. Ketchum S.B. Doyle R.T. Juliano R.A. Jiao L. Granowitz C. REDUCE-IT Investigators et al.Cardiovascular risk reduction with icosapent ethyl for hypertriglyceridemia.N. Engl. J. Med. 2019; 380: 11-22Crossref PubMed Scopus (927) Google Scholar). The purpose of this study was to test whether lowering lipid delivery to the heart improves cardiac function in human subjects with T2D. We employed a randomized, double-blinded trial design in which subjects were treated with fenofibrate or placebo. In addition to echocardiographic endpoints, we evaluated changes blood-based markers of ceramide metabolism, inflammation, and oxidative stress. We were particularly interested in the plasma ratio of Cer d18:1 (4E) (1OH, 3OH)/24:0 and Cer d18:1 (4E) (1OH, 3OH)/16:0 (i.e., C24:0/C16:0 for brevity). This biomarker is increased in patients with end-stage heart failure and improves following treatment with LV assist devices (20Ji R. Akashi H. Drosatos K. Liao X. Jiang H. Kennel P.J. Brunjes D.L. Castillero E. Zhang X. Deng L.Y. et al.Increased de novo ceramide synthesis and accumulation in failing myocardium.JCI Insight. 2017; 2: 1-19Crossref Scopus (17) Google Scholar). Furthermore, this ratio is inversely associated with incident heart failure in community-based populations (32Peterson L.R. Xanthakis V. Duncan M.S. Gross S. Friedrich N. Völzke H. Felix S.B. Jiang H. Sidhu R. Nauck M. et al.Ceramide remodeling and risk of cardiovascular events and mortality.J. Am. Heart Assoc. 2018; 7: e007931Crossref PubMed Scopus (52) Google Scholar). We chose treatment with fenofibrate for this interventional trial because it has been shown to effectively lower plasma TG, even in subjects treated with statin medications for hypercholesterolemia (33May H.T. Anderson J.L. Pearson R.R. Jensen J.R. Horne B.D. Lavasani F. Yannicelli H.D. Muhlestein J.B. Comparison of effects of simvastatin alone versus fenofibrate alone versus simvastatin plus fenofibrate on lipoprotein subparticle profiles in diabetic patients with mixed dyslipidemia (from the Diabetes and Combined Lipid Therapy Regimen study).Am. J. Cardiol. 2008; 101: 486-489Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 34Muhlestein J.B. May H.T. Jensen J.R. Horne B.D. Lanman R.B. Lavasani F. Wolfert R.L. Pearson R.R. Yannicelli H.D. Anderson J.L. The reduction of inflammatory biomarkers by statin, fibrate, and combination therapy among diabetic patients with mixed dyslipidemia: the DIACOR (Diabetes and Combined Lipid Therapy Regimen) study.J. Am. Coll. Cardiol. 2006; 48: 396-401Crossref PubMed Scopus (131) Google Scholar). We assessed the effect of adding fenofibrate to the existing medical regimens of subjects with T2D who were not already taking fibrates. There was protocol approval from the Institutional Review Board of Washington University School of Medicine. Informed consent was obtained from the human subjects or their representatives, and the study abides by the Declaration of Helsinki principles. All subjects underwent a history and physical exam, stress echocardiography, and phlebotomy for eligibility screening. Subjects were recruited from the St. Louis metropolitan area. In order to minimize possible confounding effects on cardiac function, subjects were excluded if they were 65 years old, smoked, performed >3.5 h/weekk rigorous cardiovascular exercise, had a blood pressure ≥140/90 mm Hg, atrial fibrillation, evidence of CHD on screening stress echocardiography, heart failure, hemoglobin A1c (HbA1c) >10%, recreational drug or severe alcohol use, history of liver disease, renal dysfunction (creatinine >1.5 mg/dl), use of anticoagulants, presence of paradoxical septal motion or more than mild valvular heart disease, or evidence for pericardial or infiltrative myocardial diseases. Women who were pregnant or lactating were also excluded. Subjects were instructed to continue on their usual medications, including lipid-lowering therapy throughout the study. Subjects were randomized (using randomization.com to create block randomization) in a blinded fashion to treatment with fenofibrate (160 mg/day) or identical-appearing placebo for 12 weeks. Subjects were instructed to continue their usual level of physical activity and an ATPIII diet consistent with American Diabetes Association recommendations. At the end of the intervention, 1H-magnetic resonance spectroscopy (1H-MRS) imaging of liver was repeated to assess the effects of intervention on systemic lipid exposure, and fasting blood and echocardiographic studies were repeated. Study physicians provided medical monitoring throughout the intervention to ensure safety of the participants, with interim monthly visits for history and pill counts to assess compliance. Study subjects recorded blood glucose concentrations daily, which were reviewed at monthly visits, and kept an acute event log, since TGs are acute phase reactants. Plasma insulin was measured using a sandwich electrochemiluminescence immunoassay and a Roche Cobas c601 analyzer, Basel, Switzerland). Plasma glucose levels were measured using enzymatic colorimetric assays on the Roche c501 analyzer (Roche Diagnostics, North America). After a 12 h fast, HDL, TG, and total cholesterol levels were measured from plasma using enzymatic colorimetric assays run on the Roche c501 analyzer. HbA1c was measured in whole blood samples using a turbidimetric inhibition immunoassay run on the Roche c501 analyzer. Glycated serum protein was measured using kits from Diazyme, and albumin was measured using kits from Roche Diagnostics, both on a Roche cobas c501. Serum FFAs were quantified using a WAKO colorimetric kit, and systemic inflammatory markers [high sensitivity C-reactive protein (hsCRP), TNF-α, and interleukin 6 (IL-6) were quantified using reagent kits from Roche Diagnostics]. Markers of oxidative stress (hexanoyl-lysine, propanoyl-lysine) were quantified in plasma and urine samples by LC-MS/MS (35Kato Y. Osawa T. Detection of lipid-lysine amide-type adduct as a marker of PUFA oxidation and its applications.Arch. Biochem. Biophys. 2010; 501: 182-187Crossref PubMed Scopus (28) Google Scholar, 36Zhang H. Il'yasova D. Sztaray J. Young S.P. Wang F. Millington D.S. Quantification of the oxidative damage biomarker 2,3-dinor-8-isoprostaglandin-F(2alpha) in human urine using liquid chromatography-tandem mass spectrometry.Anal. Biochem. 2010; 399: 302-304Crossref PubMed Scopus (24) Google Scholar, 37Kato Y. Yoshida A. Naito M. Kawai Y. Tsuji K. Kitamura M. Kitamoto N. Osawa T. Identification and quantification of N(epsilon)-(hexanoyl)lysine in human urine by liquid chromatography/tandem mass spectrometry.Free Radic. Biol. Med. 2004; 37: 1864-1874Crossref PubMed Scopus (30) Google Scholar). Plasma Cer d18:1 (4E) (1OH, 3OH)/24:0 and Cer d18:1 (4E) (1OH, 3OH)/16:0 were analyzed using an ABI 4000 QTRAP LC-MS/MS tandem quadrupole mass spectrometer with a linear ion trap interfaced to a Shimadzu high-performance LC system. Because the assay measured endogenous analytes in plasma, we compared the concentration-response of the analyte in a surrogate matrix (e.g., 5% BSA) to that of plasma samples. To determine recovery, the plasma or surrogate matrix was spiked with differing amounts of d4-C22:0 and d4-C24:0, extracted with chloroform/methanol and analyzed as above. Differences in matrix effects and extraction recoveries were compensated by internal standards. Details of the metabolomic analytical methods used were previously reported by our group (38Jiang H. Hsu F.F. Farmer M.S. Peterson L.R. Schaffer J.E. Ory D.S. Jiang X. Development and validation of LC-MS/MS method for determination of very long acyl chain (C22:0 and C24:0) ceramides in human plasma.Anal. Bioanal. Chem. 2013; 405: 7357-7365Crossref PubMed Scopus (14) Google Scholar). Subjects underwent baseline anthropomorphic and metabolic phenotyping with dual-energy X-ray absorptiometry analysis for determination of body composition (iDXA; GE Healthcare, Madison, WI). 1H-MRS for quantification of hepatic TG used 1.5 T Avanto or a 3 T Trio scanner, a PRESS sequence without water suppression and breath-hold techniques (39Schonfeld G. Patterson B.W. Yablonskiy D.A. Tanoli T.S. Averna M. Elias N. Yue P. Ackerman J. Fatty liver in familial hypobetalipoproteinemia: triglyceride assembly into VLDL particles is affected by the extent of hepatic steatosis.J. Lipid Res. 2003; 44: 470-478Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Liver fat percentage was calculated as area under the lipid peak (methyl and methylene resonances) divided by the sum of area under water and lipid peaks, with correction for T2 relaxation. Cardiac structure and function were assessed using 2D-, Doppler, tissue Doppler, and strain echocardiography according to guidelines from the American Society of Echocardiography (40Lang R.M. Bierig M. Devereux R.B. Flachskampf F.A. Foster E. Pellikka P.A. Picard M.H. Roman M.J. Seward J. Shanewise J.S. et al.Recommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology.J. Am. Soc. Echocardiogr. 2005; 18: 1440-1463Abstract Full Text Full Text PDF PubMed Scopus (8748) Google Scholar, 41Nagueh S.F. Appleton C.P. Gillebert T.C. Marino P.N. Oh J.K. Smiseth O.A. Waggoner A.D. Flachskampf F.A. Pellikka P.A. Evangelisa A. Recommendations for the evaluation of left ventricular diastolic function by echocardiography.Eur. J. Echocardiogr. 2009; 10: 165-193Crossref PubMed Scopus (1611) Google Scholar). FS, a measure of systolic function, and tissue Doppler-derived e′, a measure of diastolic function, were primary outcome variables. Although our study was not powered to evaluate global longitudinal strain measures as endpoints, data using these approaches were analyzed in an exploratory fashion, as these newer techniques may be more sensitive to detect dysfunction (42Galderisi M. Cosyns B. Edvardsen T. Cardim N. Delgado V. Di Salvo G. Donal E. Sade L.E. Ernande L. Garbi M. 2016–2018 EACVI Scientific Documents Committee et al.Standardization of adult transthoracic echocardiography reporting in agreement with recent chamber quantification, diastolic function, and heart valve disease recommendations: an expert consensus document of the European Association of Cardiovascular Imaging.Eur. Heart J. Cardiovasc. Imaging. 2017; 18: 1301-1310Crossref PubMed Scopus (164) Google Scholar). It should be noted that global longitudinal strain could be analyzed in only 27 subjects in the placebo group and 29 subjects in the fenofibrate group due to acoustic window limitations in some subjects. SAS version 9.4 (SAS Inc, Cary, NC) was used for data analyses. The primary endpoint was change in systolic function as quantified by FS and diastolic function as quantified by e′. The secondary endpoint was change in plasma C24:0/C16:0 ceramide ratio. Other cardiac, inflammatory, and oxidative stress endpoints were tertiary endpoints. Two-sample t-tests were used for comparing continuous variables between groups and Chi-square tests were used for categorical variables. Satterthwaite analysis was performed if variables had unequal variances. For comparison of the proportion of subjects taking certain medications, two-sample Z test for proportions was used. One sample t-tests were used to compare means of the deltas of the endpoints. ANCOVA was used for comparison of the change in the endpoints between the groups after adjustment for predetermined baseline variables including age, sex, race, body fat percent, BMI, diastolic blood pressure, HbA1c, systolic blood pressure, fasting glucose, fasting TG, ethnicity, and hsCRP. Pearson correlation coefficients were determined between the change in TGs and the cardiac endpoints as well as among the Δ TGs, Δ liver fat, and Δ C24:0/C16:0 ceramide ratio. Shapiro-Wilk tests for normality of distribution were performed and appropriate data transformations were used when needed. Outlier values were determined using the rule that defines an outlier as a value larger than three times of interquartile range from the 75% percentile or smaller than three times of interquartile range from the 25% percentile, and outliers were removed from analysis as noted in the legends. Data are reported as mean ± SD. All statistical analyses were two-sided at significance level 0.05. The CONSORT diagram (Fig. 1) outlines the recruitment, enrollment, screen failures, drop-outs, and those who finished the study. In short, of the 70 subjects randomized to fenofibrate (n = 34) or placebo (n = 36), 65 completed the study (31 in the fenofibrate group). Reasons for noncompletion included rash and pruritis (n = 2), elevated liver function tests (n = 1), unrelated health issue (n = 1), and noncompliance (n = 1). The characteristics of both groups are shown in Table 1. The two groups were very similar with no significant differences in age, sex, racial composition, anthropomorphic and metabolic characteristics, oxidative stress markers, or hemodynamic measurements. Only one inflammatory marker, hsCRP, was different, higher at baseline in the fenofibrate group. The placebo group had a slightly lower relative wall thickness than the fenofibrate group at baseline, but LV mass and volume and systolic (FS, ejection fraction, global longitudinal strain, tissue Doppler s′ average) and diastolic (e′ average, E/e′ septal) functional measurements were not significantly different (Table 1). The proportion of subjects in both groups who were taking statins, diabetes medications (including insulin), calcium channel blockers, diuretics, angiotensin enzyme-converting inhibitors/angiotensin receptor blockers, or β-blockers was not different (Table 2). Thus, recruitment and randomization led to entry of highly similar subjects in treatment and placebo groups. There were no diff

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