Carnitine insufficiency caused by aging and overnutrition compromises mitochondrial performance and metabolic control

Abstract

In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine diminution was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete beta-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome.

FIGURE 1.

Dimunition of free carnitine levels in insulin-resistant skeletal muscle. A heat map representation (a) of the pairwise correlation matrix was generated using metabolic profiles of gastrocnemius muscles from lean and obese Zucker diabetic fatty (ZDF) rats as described in a previous study (5). Each square represents the Pearson correlation coefficient between the metabolite of the column with that of the row, according to the color scale. Metabolite order was determined by unsupervised clustering of the correlations. Several organic acids (OA) and long chain acylcarnitines (LCAC) clustered together (clusters 2 and 4, respectively). Free carnitine (C0) correlated positively with several short chain acylcarnitines and amino acids (cluster 1) and negatively with several LCAC and long chain CoAs (LC-CoA (cluster 3)). The strong negative relationship between free carnitine and total LCAC is shown in panel b. Tandem MS/MS was used to measure free carnitine content of mixed gastrocnemius muscle specimens obtained from lean and ZDF rats (c), animals fed a standard chow (SC) or high fat (HF) diet for 3 months (d), young (5 month), and aged (20 month) rats fed an SC diet (e), and rats artificially selected for running capacity that exhibit disparate susceptibility to HF diet-induced insulin resistance (f and g). Tissues were harvested 4 h after food withdrawal (a–e), or after an overnight fast (f and g). Data represent means ± S.E. from 5–8 animals per group. Results were analyzed by one-way (c–e) or two-way (f) analysis of variance using Tukey's post hoc test to determine differences between groups. *, p < 0.05 versus control group; #, p < 0.05 diabetes prone versus diabetes resistant rats.

FIGURE 2.

Whole body carnitine status is compromised by lifelong high fat feeding and restored by carnitine supplementation. Tandem MS/MS was used to measure free carnitine levels in mixed gastrocnemius (a), liver (b), kidney (c), plasma (d), and urine (e) harvested 4 h after food withdrawal from young (Y, 5 month) or old (O, 15 month) animals fed a standard (SC) or high fat (HF) diet. Quantitative reverse transcription-PCR was used to measure the expression of hepatic genes involved in cellular (OCTN2) and mitochondrial (CACT and OCTN1) carnitine transport, as well as genes encoding the first (Trimethyllysine hydroxylase-ϵ (Tmlhe)), third (aldehyde dehydrogenase-9 family, member A1 (Aldh9a1)), and fourth/final (γ-butyrobetaine hydroxylase-1 (Bbox1)) reactions involved in carnitine biosynthesis; young (black), old (white), and old-HF (gray) (f). Data represent means ± S.E. from 5–8 animals per group. A one-way analysis of variance with Tukey's post hoc test was used to examine the effects of aging, whereas a Student's t test was used to determine differences due to HF diet and carnitine supplementation. *, p < 0.05 due to aging; #, p < 0.05 due to HFD; ^$, p < 0.05 due to carnitine supplementation.

FIGURE 3.

Carnitine supplementation improved whole body glucose tolerance. Male Wistar rats were fed either a standard chow (SC) or high fat (HF) diet for 12 months, with or without oral carnitine therapy during the final 2 months (HF/Carn). Intraperitoneal glucose tolerance tests (a) were performed 5 weeks after initiation of carnitine therapy. Plasma insulin values obtained at basal and 30 min post glucose injection (b) were used to calculate the homeostatic assessment model index of insulin resistance (c). Data represent means ± S.E. from 5–8 animals per group. Results were analyzed by Student's t test for between group differences, and a paired t test was applied to detect the within group effect of carnitine. #, p < 0.05 difference versus SC controls; *, p < 0.05 due to carnitine supplementation.

FIGURE 4.

Carnitine supplementation reverses diet-induced mitochondrial dysregulation. Mitochondria were isolated from mixed gastrocnemius muscles of young (2 month) or old (15 month) male Wistar rats fed either standard chow (SC) or high fat (HF) diet for 12 months, without or with oral carnitine supplementation administered during the final 2 months (HF/Carn). Oxidation of [1-¹⁴C]oleate (100 μm) to ¹⁴CO₂ (a) or ¹⁴C-labeled acid soluble metabolites (ASM; panel b) was measured ± pyruvate (5 mm) as an index of substrate switching. PDH activity (c) and pyruvate oxidation (d) were assessed as the liberation of ¹⁴CO₂ from [1-¹⁴C]pyruvate (5 mm) ± l-carnitine (5 mm) or [2-¹⁴C]pyruvate (5 mm) ± oleate (100 μm), respectively. Data represent means ± S.E. from 5–8 animals per group. A Student's t test was used to evaluate between group differences, and paired t tests were applied to detect within-group responses to pyruvate, carnitine, and oleate. *, p < 0.05 pyruvate-induced inhibition of oleate oxidation; ^$, p < 0.05 difference in substrate switching between SC versus HF diet groups; #, p < 0.05 difference between SC and HF diet groups; ^‡, p < 0.05 effect of l-carnitine on PDH activity; ^, p < 0.05 oleate-induced inhibition of pyruvate oxidation.

FIGURE 5.

Effects of carnitine supplementation on acylcarnitine profiles. Tandem MS/MS was used to measure acylcarnitine levels in mixed gastrocnemius (MG), plasma, and urine harvested from male Wistar rats at 15 months of age fed either standard chow (SC) or high fat (HF) diet for 12 months, without or with oral carnitine supplementation administered during the final 2 months (HF/Carn). Data represent means ± S.E. from 5–8 animals per group. Results were analyzed by Student's t test. #, p < 0.05 compared with SC diet; ^$, p < 0.05 compared with HF group without carnitine.

FIGURE 6.

Tissue distribution of CrAT. CrAT mRNA (a) and protein (b) expression were determined in mixed gastrocnemius (MG), heart, kidney, liver, and white adipose tissue (WAT). Recombinant rat CrAT (rCrAT) was expressed in human skeletal myotubes and served as a positive control. CrAT mRNA levels were unaffected by experimental treatments (c). Relationships between free carnitine and acetylcarnitine (d) or total long chain acylcarnitines (LCAC) (e) were evaluated using metabolites measured in soleus, red and white quadriceps, and extensor digitorum longus muscles.

FIGURE 7.

Acylcarnitine production and export by primary human skeletal myotubes. Primary human skeletal myotubes were pulsed with a 1:1 mixture of oleate:palmitate (1 mm) for 24 h in the absence of carnitine, followed by 24-h exposure to chase medium containing 100–5000 μm carnitine, but lacking fatty acid. Mass spectrometry was used to assess free carnitine content within skeletal myotubes (a), total acylcarnitine content (medium plus cell lysates) (b), and acetylcarnitine exported into the medium. Intracellular free carnitine content correlated positively with the amount of acetylcarnitine exported (c). The effect of 24-h lipid exposure (500 μm oleate:palmitate plus 500 μm carnitine) on free carnitine and LCAC content of cells was measured in the absence or presence of 100 μm etomoxir (Etx) (d). Data represent the mean ± S.E. from at least two separate experiments.

FIGURE 8.

Overexpression of CrAT in primary human skeletal myotubes promotes glucose uptake and oxidation. Skeletal myocytes were pretreated with recombinant adenoviruses encoding β-galactoside (β-gal) or Myc-tagged rat CrAT. Protein expression of rCrAT in primary human myotubes was detected using an antibody that recognizes rodent but not human CrAT (a). CrAT localization was evaluated using mitochondrial (Mito) and cytosolic (Cyto) fractions and whole cell lysates (WC) prepared from C2C12 myoblasts exposed to rAdCMV-CrAT, followed by SDS-PAGE and Western blot analysis using the aforementioned antibody (b). Metabolic experiments were performed 72 h after virus treatment. Acetylcarnitine levels in cell lysates and medium (c) was assessed 24 h after addition of carnitine. Myotube oxidation of [U-¹⁴C]glucose (d) and cellular uptake of [2-³H]deoxyglucose (e) were assessed during a 2-h exposure to radiolabel in the presence or absence of 100 μm oleate. Data represent the mean ± S.E. from 3–4 separate experiments. Results were analyzed by two-way analysis of variance. *, p < 0.05, effect of oleate compared with basal. #, p < 0.05, effect of CrAT compared with β-galactosidase.

FIGURE 9.

Proposed role of carnitine and CrAT in regulating mitochondrial energetics. TheLCAC product of CPT1 traverses the inner mitochondrial membrane (imm) via CACT and is then delivered to CPT2, which regenerates acyl-CoA on the matrix side of the membrane. The enzymes of β-oxidation degrade long chain acyl-CoAs to shorter species through a recurrent, multistep process that yields one two-carbon molecule of acetyl-CoA in each successive cycle. When acyl-CoA production exceeds consumption, these intermediates can be converted back to their acylcarnitine counterparts and exported from the mitochondria into the general circulation. CrAT, a mitochondrial matrix enzyme that prefers short-chain acyl esters, regulates mitochondrial metabolism by lowering the acetyl-CoA/free CoA ratio and regenerating free CoA, which is used by PDH and the tricarboxylic acid (TCA) cycle enzyme, α-ketoglutarate dehydrogenase. Futile production and export of LCAC might compromise the intramitochondrial pool of carnitine, thus limiting CrAT activity.