CPT1c Is Localized in Endoplasmic Reticulum of Neurons and Has Carnitine Palmitoyltransferase Activity

Abstract

CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.

Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes

“…In agreement with the literature (16), we found that CPT-1a and -1c were the most abundant CPT-1 isoforms expressed in brain cells. However, the expression of CPT-1c in cultured astrocytes is not consistent with earlier studies showing a neuronal localization in the adult mouse brain (44,45). This discrepancy could be related to the age of the animals used to generate astrocytes (P1) and/or the culture conditions.…”

C75 is converted to C75-CoA in the hypothalamus, where it inhibits carnitine palmitoyltransferase 1 and decreases food intake and body weight

“…is located in the mitochondrial membrane and CPT1C is expressed in the endoplasmic reticulum of neurons where although it has CPT1 activity, it does not participate in mitochondrial fatty-acid oxidation [12]. Interestingly, our group has generated a mutant form of CPT1A insensitive to malonyl-CoA (CPT1A M593S), and a mutant form of carnitine acetyltransferase (CrAT) that swaps its preference from short to LCFA-CoA (CrAT D356A/M564G).…”

Alternative Exon Usage in the Single CPT1 Gene of Drosophila Generates Functional Diversity in the Kinetic Properties of the Enzyme

“…Thus, for example, the H483A mutant of rat CPT1A shows a 50% decrease in the affinity for malonyl-CoA (18) and the A478D mutation affected the affinity for palmitoyl-CoA (18). Interestingly, Ala 478 is not conserved in the most recently described mammalian isoform, CPT I-C, which has a very low catalytic constant when carnitine and long chain acyl-CoA are used as substrates (14,24). In all of the insect CPT1 genes studied, the residues equivalent to His 483 and Ala 478 in rat CPT1A are both encoded within the alternatively spliced exons (6A/6B) and, importantly, are conserved in both variants generated from each of the insect CPT1 genes analyzed (Fig.…”