Faculty Bibliography

Cholesterol reduction has markedly reduced major cardiovascular disease (CVD) events and shown regression of atherosclerosis in some studies. However, CVD has for decades also been associated with increased levels of circulating triglyceride (TG)-rich lipoproteins. Whether this is due to a direct toxic effect of these lipoproteins on arteries or whether this is merely an association is unresolved. More recent genetic analyses have linked genes that modulate TG metabolism with CVD. Moreover, analyses of subgroups of hypertriglyceridemic (HTG) subjects in clinical trials using fibric acid drugs have been interpreted as evidence that TG reduction reduces CVD events. This review will focus on how HTG might cause CVD, whether TG reduction makes a difference, what pathophysiological defects cause HTG, and what options are available for treatment.

Kruppel-like factors (KLF) have important roles in metabolism. We previously found that KLF5 is a positive transcriptional regulator of peroxisome proliferator-activated receptor alpha (Ppara), a central regulator of cardiac fatty acid oxidation (FAO). Mice with cardiomyocyte-specific Klf5 ablation (alphaMHCKlf5 ) had reduced cardiac Ppara expression and FAO. At age 6-12 months these mice develop distinct cardiac dysfunction. The role of PPARalpha activation in I/R injury is unclear as both beneficial and detrimental effects have been reported. We aimed to study if Ppara expression changes during I/R are driven by KLF5 and explore its protective or detrimental role. Wild type mice were subjected to in vivo I/R or sham surgery. I/R resulted in an initial increase in Ppara, and its target gene pyruvate dehydrogenase kinase 4 (Pdk4) mRNA after 2h reperfusion, followed by decreased expression after 24h reperfusion. The Ppara expression is associated with parallel changes in cardiac Klf5 mRNA expression. Concurrent, there was a decrease of cardiac FAO-related genes carnitine palmitoyl-transferase 1beta (Cpt1b), very long chain acyl-CoA dehydrogenase (Vlcad), and acyl-CoA oxidase (Aox) in mice with I/R. To define the cell type causing the temporal changes in Klf5 and Ppara after I/R we isolated primary cardiomyocytes and fibroblasts. Our data suggest a similar effect in primary isolated cardiomyocytes only. Klf5 mRNA expression is increased after 2 hour hypoxia and normalized after 4 hour re-oxygenation in cardiomyocytes, whereas there are no changes after hypoxia/normoxia in fibroblasts. To assess the importance of cardiomyocyte KLF5 in I/R we used alphaMHC-Klf5 mice. Interestingly, despite reduced FAO, 7 month old alphaMHC-Klf5 mice subjected to I/R had a marked increase in mortality; 4 of 7 alphaMHCKlf5 mice died within the first 24h of reperfusion while no mortality was observed in age-matched wild type mice that underwent I/R. In conclusion, I/R is associated with an increase in Klf5 and Ppara in the first hours of reperfusion followed by a decrease in Klf5 and Ppara, likely accounted for by cardiomyocytes. Increased mortality for alphaMHC-Klf5 mice with I/R injury suggests that the initial increase may be an adaptive response that is critical for survival

The heart utilizes large amounts of fatty acids as energy providing substrates. The physiological balance of lipid uptake and oxidation prevents accumulation of excess lipids. Several processes that affect cardiac function, including ischemia, obesity, diabetes mellitus, sepsis, and most forms of heart failure lead to altered fatty acid oxidation and often also to the accumulation of lipids. There is now mounting evidence associating certain species of these lipids with cardiac lipotoxicity and subsequent myocardial dysfunction. Experimental and clinical data are discussed and paths to reduction of toxic lipids as a means to improve cardiac function are suggested.

Pulmonary surfactant is synthesized by type II pneumocytes and is mainly composed of phospholipids and minor amounts of cholesterol and specific proteins. Surfactant allows the lungs to inflate by decreasing alveolar surface tension and the work needed for inspiration in each breathing cycle. A delicate balance between synthesis, secretion, recycling and degradation of the lipids is required to maintain surfactant function and lung performance. Single nucleotide polymorphisms in the LDL receptor associated protein 1 (LRP-1) are associated with decreased lung function in patients with chronic obstructive pulmonary disease (COPD). But the function of LRP1 in the lung is not known. Amongst other roles, LRP-1 participates in liver lipoprotein metabolism, in adipocyte cell differentiation and in macrophage inflammatory cascades. We deleted LRP-1 in type II pneumocyte-derived A549 cells using stable transfection. LRP1 shRNA-treated cells and scrambled shRNA-treated cells were cultured in collagen coated transwell inserts at the air-liquid interphase, exposing the cellular basal side to the culture media and the apical side to the air. LRP-1 shRNA decreased surfactant phospholipid, cholesterol and cholesteryl esters on the apical surface of the cells. LRP-1 shRNA cells showed reduced gene expression of the rate limiting enzymes for fatty acid synthesis FAS, ACAT1 and SCD1, and of phosphatidylcholine synthesis enzymes choline transferase and choline kinase. Surprisingly, triglycerides and cholesteryl esters accumulated in intracellular lipid droplets in LRP-1 shRNA cells, while phospholipids and unesterified cholesterol were not changed when compared to scrambled shRNA cells. LRP-1 shRNA cells showed >3 fold overexpression of the fatty acid transporter CD36 and reduced gene expression of acyl-CoA oxidase. After an overnight incubation with fresh media in the basal side, LRP-1 shRNA cells showed higher media depletion of phospholipid, cholesterol esters and unesterifed cholesterol. Uptake of LDL-derived phosphatidylcholine was not altered by LRP-1 deletion, but the gene expression of phospholipid and cholesterol exporters ABCA1 and ABCG1 was reduced. Insulin signaling was affected by LRP-1 deletion. LRP-1 shRNA cells showed decreased protein expression of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and increased phospho-serine IRS-1/total IRS-1 ratio. After IGF-1 treatment, LRP-1 shRNA cells failed to Tyr-phosphorylate the IGF-1 receptor b, and phosphorylation of the downstream target Akt was lower than in scrambled shRNA cells These data suggest that LRP-1 in type II pneumocytes is involved in lipid export, intracellular surfactant lipid metabolism and insulin signaling

RATIONALE: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator-activated receptor (PPAR)alpha and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown. OBJECTIVE: To determine the role of Kruppel-like factor 5 (KLF5) in transcriptional regulation of Ppara. METHODS AND RESULTS: We discovered that KLF5 activates Ppara gene expression via direct promoter binding. This is blocked in hearts of septic mice by c-Jun, which binds an overlapping site on the Ppara promoter and reduces transcription. We generated cardiac myocyte-specific Klf5 knockout mice that showed reduced expression of cardiac Ppara and its downstream fatty acid metabolism-related targets. These changes were associated with reduced cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation and cardiac dysfunction. Diabetic mice showed parallel changes in cardiac Klf5 and Ppara expression levels. CONCLUSIONS: Cardiac myocyte KLF5 is a transcriptional regulator of Ppara and cardiac energetics.

3,5,3'-triiodo-L-thyronine (T3) and 3,5-diiodo-L-thyronine (T2), when administered to a model of familial hypercholesterolemia, i.e., low density lipoprotein receptor (LDLr)-knockout (Ldlr-/-) mice fed with a Western type diet (WTD), dramatically reduce circulating total and very low-density lipoprotein/LDL cholesterol with decreased liver apolipoprotein B (ApoB) production. The aim of the study was to highlight putative molecular mechanisms to manage cholesterol levels in the absence of LDLr. A comprehensive comparative profiling of changes in expression of soluble proteins in livers from Ldlr-/- mice treated with either T3 or T2 was performed. From a total proteome of 450 liver proteins, 25 identified proteins were affected by both T2 and T3, 18 only by T3 and 9 only by T2. Using in silico analyses, an overlap was observed with 11/14 pathways common to both iodothyronines, with T2 and T3 preferentially altering sub-networks centered around hepatocyte nuclear factor 4 alpha (HNF4alpha) and peroxisome proliferator-activated receptor alpha (PPARalpha), respectively. Both T2 and T3 administration significantly reduced nuclear HNF4alpha protein content, while T2, but not T3, decreased the expression levels of the HNFalpha transcriptional coactivator PGC-1alpha. Lower PPARalpha levels were found only following T3 treatment while both T3 and T2 lowered liver X receptor alpha (LXRalpha) nuclear content. Overall, this study, although it was not meant to investigate the use of T2 and T3 as a therapeutic agent, provides novel insights into the regulation of hepatic metabolic pathways involved in T3- and T2-driven cholesterol reduction in Ldlr-/- mice.

Elevated cardiac triacylglycerol (TAG) content is traditionally equated with cardiolipotoxicity and suggested to be a culprit in cardiac dysfunction. However, previous work demonstrated that myosin heavy-chain-mediated cardiac-specific overexpression of diacylglycerol transferase 1 (MHC-DGAT1), the primary enzyme for TAG synthesis, preserved cardiac function in two lipotoxic mouse models despite maintaining high TAG content. Therefore, we examined whether increased cardiomyocyte TAG levels due to DGAT1 overexpression led to changes in cardiac TAG turnover rates under normoxia and ischemia-reperfusion conditions. MHC-DGAT1 mice had elevated TAG content and synthesis rates, which did not alter cardiac function, substrate oxidation, or myocardial energetics. MHC-DGAT1 hearts had ischemia-induced lipolysis; however, when a physiologic mixture of long-chain fatty acids was provided, enhanced TAG turnover rates were associated with improved functional recovery from low-flow ischemia. Conversely, exogenous supply of palmitate during reperfusion suppressed elevated TAG turnover rates and impaired recovery from ischemia in MHC-DGAT1 hearts. Collectively, this study shows that elevated TAG content, accompanied by enhanced turnover, does not adversely affect cardiac function and, in fact, provides cardioprotection from ischemic stress. In addition, the results highlight the importance of exogenous supply of fatty acids when assessing cardiac lipid metabolism and its relationship with cardiac function.

Although diabetes is mainly diagnosed based on elevated glucose levels, dyslipidemia is also observed in these patients. Chronic kidney disease (CKD), a frequent occurrence in patients with diabetes, is associated with major abnormalities in circulating lipoproteins and renal lipid metabolism. At baseline, most renal epithelial cells rely on fatty acids as their energy source. CKD, including that which occurs in diabetes, is characterized by tubule epithelial lipid accumulation. Whether this is due to increased uptake or greater local fatty acid synthesis is unknown. We have recently shown that CKD also leads to decreased fatty acid oxidation, which might be an additional mechanism leading to lipid accumulation. Defective fatty acid utilization causes energy depletion resulting in increased apoptosis and dedifferentiation, which in turn contributes to fibrosis and CKD progression. Enhanced fatty acid oxidation in the kidney induced by fenofibrate, a peroxisomal proliferator-activated receptor (PPAR)-alpha agonist, showed benefit in mouse models of CKD. Fenofibrate treatment also reduced albuminuria in patients with diabetes in multiple clinical trials. Taken together, these findings suggest that further understanding of lipid metabolism in diabetic kidney disease may lead to novel therapeutic approaches.

In this Roundtable, our intent is to discuss those rare genetic disorders that impair the function of lipoprotein lipase. These cause severe hypertriglyceridemia that appears in early childhood with Mendelian inheritance and usually with full penetrance in a recessive pattern. Dr Ira Goldberg from New York University School of Medicine and Dr Stephen Young from the University of California, Los Angeles have agreed to answer my questions about this topic. Both have done fundamental work in recent years that has markedly altered our views on lipoprotein lipase function. I am going to start by asking them to give us a brief history of this enzyme system as a clinical entity.