Kruppel-like Factor 15 Is a Critical Regulator of Cardiac Lipid Metabolism

Background: Metabolic homeostasis is central to normal cardiac function. The molecular mechanisms underlying metabolic plasticity in the heart remain poorly understood. Results: Kruppel-like factor 15 (KLF15) is a direct and independent regulator of myocardial lipid flux. Conclusion: KLF15 is a core component of the transcriptional circuitry that governs cardiac metabolism. Significance: This work is the first to implicate the KLF transcription factor family in cardiac metabolism. The mammalian heart, the body\u27s largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism

HIF drives lipid deposition and cancer in ccRCC via repression of fatty acid metabolism

Clear cell renal cell carcinoma (ccRCC) is histologically defined by its lipid and glycogen-rich cytoplasmic deposits. Alterations in the VHL tumor suppressor stabilizing the hypoxiainducible factors (HIFs) are the most prevalent molecular features of clear cell tumors. The significance of lipid deposition remains undefined. We describe the mechanism of lipid deposition in ccRCC by identifying the rate-limiting component of mitochondrial fatty acid transport, carnitine palmitoyltransferase 1A (CPT1A), as a direct HIF target gene. CPT1A is repressed by HIF1 and HIF2, reducing fatty acid transport into the mitochondria, and forcing fatty acids to lipid droplets for storage. Droplet formation occurs independent of lipid source, but only when CPT1A is repressed. Functionally, repression of CPT1A is critical for tumor formation, as elevated CPT1A expression limits tumor growth. In human tumors, CPT1A expression and activity are decreased versus normal kidney; and poor patient outcome associates with lower expression of CPT1A in tumors in TCGA. Together, our studies identify HIF control of fatty acid metabolism as essential for ccRCC tumorigenesis

Validated Method for the Quantification of Free and Total Carnitine, Butyrobetaine, and Acylcarnitines in Biological Samples

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)­HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes