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. 2014 Apr;55(4):635-44.
doi: 10.1194/jlr.M043448. Epub 2014 Jan 6.

Obesity and lipid stress inhibit carnitine acetyltransferase activity

Sarah E Seiler  1 , Ola J Martin, Robert C Noland, Dorothy H Slentz, Karen L DeBalsi, Olga R Ilkayeva, Jie An, Christopher B Newgard, Timothy R Koves, Deborah M Muoio
Affiliations

Obesity and lipid stress inhibit carnitine acetyltransferase activity

Sarah E Seiler et al. J Lipid Res. 2014 Apr.

Abstract

Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes.

Keywords: acetyl-coenzyme A; acylcarnitines; diabetes; lipid metabolism; mitochondria; muscle.

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Figures

Fig. 1.
Fig. 1.
CrAT specific activity is diminished by obesity and diabetes. Gastrocnemius muscles were harvested from obese ZDF rats and lean control animals, and from adult Wistar rats fed a 10% LF or 45% HF diet for 20 weeks. (A) Tandem mass spectrometry-based measurement of the acetylcarnitine:acetyl-CoA ratio. (B) CrAT mRNA expression normalized to 18S. (C) Representative western blots and (D) protein abundance of CrAT normalized to the MEMCode stain. (E) Total CrAT enzyme activity and (F) CrAT specific activity corrected for CrAT protein abundance. Data are expressed as fold change relative to the lean control group and represent means ± SE of 5–8 animals per group. * P < 0.05 lean versus obese.
Fig. 2.
Fig. 2.
CrAT prefers SCACoAs. CrAT enzyme activity was measured with 0.45 mM acyl-CoA substrates of various chain lengths and 5.0 mM l-carnitine using (A) purified CrAT from pigeon breast muscle, (B) lysates from primary HSkMCs expressing recombinant rat CrAT, and (C) isolated mitochondria from mouse gastrocnemius muscle (MG). (D) CrAT activity as a function of increasing carnitine concentration measured in the presence of 0.45 mM acetyl-CoA. Results are expressed as percent of maximal CrAT activity. Data represent means ± SE from 3 to 5 separate experiments.
Fig. 3.
Fig. 3.
LCACoAs inhibit CrAT activity. CrAT activity as a function of increasing palmitoyl-CoA concentration was measured using (A) purified protein from pigeon breast, (B) isolated mitochondria from primary HSkMCs expressing recombinant rat CrAT, and (C, D) isolated mitochondria from mouse gastrocnemius muscle. Assays in A, B, and E were performed with 0.45 mM acetyl-CoA and 5 mM l-carnitine. (E) Palmitoyl-CoA inhibition of CrAT activity as a function of l-carnitine concentration using isolated mitochondria from mouse gastrocnemius muscle. Data represent means ± SE from 3 to 5 separate experiments (A, B) or 5–8 animals per group (C–E).
Fig. 4.
Fig. 4.
Fatty acid exposure and CPT1 overexpression decrease SCAC production in HSkMCs. Acylcarnitine levels were measured in lysates prepared from primary HSkMCs treated with rAd encoding β-gal (filled bars) or rat CPT1b (open bars). Cells were treated with adenoviruses on differentiation day 3 and harvested on day 7 following 24 h exposure to 1% BSA alone or complexed with 100 or 500 µM 1:1 oleate:palmitate, along with 1 mM l-carnitine. Specific acylcarnitine species shown include (A) palmitoylcarnitine (C16), (B) 3-hydroxypalmitoylcarnitine (C16:OH), (C) lauroylcarnitine (C12), (D) butyrylcarnitine (C4), (E) propionylcarnitine (C3), (F) acetylcarnitine (C2), and (G) free carnitine. (H) Cellular levels of free carnitine and acetylcarnitine were strongly correlated. * P < 0.05 fatty acid treatment versus BSA control; # P < 0.05 rAd-CPT1b versus β-gal control virus.
Fig. 5.
Fig. 5.
Proposed model of lipid-induced inhibition of CrAT activity. Fatty acid-derived LCACoAs cross the mitochondrial double membrane via the carnitine/acylcarnitine transport system. After traversing the inner membrane through the action of carnitine acylcarnitine translocase (CT), the acylcarnitine products of CPT1 are converted back to LCACoAs by CPT2, thereby replenishing the matrix pool of free carnitine. CrAT coverts SCACoA intermediates of fat, glucose, and amino acid metabolism to SCAC metabolites that can efflux from the mitochondria when nutrient supply and catabolism exceed flux through the tricarboxylic acid cycle (TCAC) and the electron transport system (ETS). CrAT lowers acetyl-CoA and regenerates free CoA, which together disinhibits PDH and promotes glucose oxidation. Excessive formation and cellular efflux of LCAC depletes the matrix pool of free carnitine, thereby limiting CrAT activity. Additionally, LCACoAs act as direct allosteric inhibitors of CrAT. These mechanisms might serve to counterregulate the activities of CrAT and PDH to spare glucose and promote β-oxidation when lipids are plentiful.
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