Aging is associated with the development of chronic diseases such as insulin resistance and type 2 diabetes. A reduction in mitochondrial fat oxidation is postulated to be a key factor contributing to the progression of these diseases. Our aim was to investigate the contribution of impaired mitochondrial fat oxidation toward age-related disease. Mice deficient for malonyl CoA decarboxylase (MCD−/−), a mouse model of reduced fat oxidation, were allowed to age while life span and a number of physiological parameters (glucose tolerance, insulin tolerance, indirect calorimetry) were assessed. Decreased fat oxidation in MCD−/− mice resulted in the accumulation of lipid intermediates in peripheral tissues, but this was not associated with a worsening of age-associated insulin resistance and, conversely, improved longevity. This improvement was associated with reduced oxidative stress and reduced acetylation of the antioxidant enzyme superoxide dismutase 2 in muscle but not the liver of MCD−/− mice. These findings were recapitulated in aged mice treated with an MCD inhibitor (CBM-3001106), and these mice also demonstrated improvements in glucose and insulin tolerance. Therefore, our results demonstrate that in addition to decreasing fat oxidation, MCD inhibition also has novel effects on protein acetylation. These combined effects protect against age-related metabolic dysfunction, demonstrating that MCD inhibitors may have utility in the battle against chronic disease in the elderly.
It is well established that aging is associated with the development of a number of chronic diseases, some of which include insulin resistance, type 2 diabetes, and cardiovascular disease (1,2). The development of chronic diseases with aging is due in part to a reduction in mitochondrial function, which manifests as a decline in fatty acid oxidative capacity, and is believed to result in the accumulation of intracellular lipid intermediates that interfere with normal organ function (3,4).
Malonyl CoA decarboxylase (MCD) is an important enzyme involved in the regulation of fatty acid oxidation, since it controls cellular levels of malonyl CoA, a potent endogenous inhibitor of carnitine palmitoyl transferase 1, the rate-limiting enzyme of mitochondrial fatty acid uptake (5). We have shown that acute inhibition of MCD decreases fatty acid oxidation rates in the heart and improves functional recovery during reperfusion after ischemia (6). We observed similar results in a mouse model of MCD deficiency (MCD−/−) (7) and have shown that these mice are also protected against skeletal muscle and cardiac insulin resistance induced by high-fat feeding (8,9).
Although MCD inhibition protects against acute disease, owing to the suggestion that impaired mitochondrial fatty acid oxidative capacity can contribute to disease progression in the elderly (3,4), it became important to determine the potential consequences of lifelong MCD deficiency on whole-body physiology, which was examined in aged MCD−/− mice and their wild-type (WT) littermates.
Research Design and Methods
All animals received care according to the guidelines of the Canadian Council on Animal Care and the University of Alberta Health Sciences Animal Welfare Committee. MCD−/− mice and their WT littermates were placed on a standard chow/low-fat diet (4% kcal from lard) and separated into two cohorts. In the first cohort, WT and MCD−/− mice were monitored until death to determine average life span. In the second cohort, male WT and MCD−/− mice at 70–75 weeks of age were subjected to a number of experimental procedures, described below. In a separate study, 16-month-old C57BL/6J mice were treated with either vehicle control or an MCD inhibitor (CBM-3001106 dissolved in a 1:1:1:7 mixture of ethanol:dimethylsulfoxide:cremophore:water, 10 mg/kg via daily oral gavage for 4 weeks) and subjected to a number of experimental procedures, described below. Once all in vivo physiological parameters were assessed, animals were euthanized via an injection of sodium pentobarbital (12 mg i.p.) in the random fed state 4 h into their dark cycle. Tissues were excised and immediately frozen in liquid N2 for biochemical analyses.
Palmitate Oxidation Rates
A separate cohort of WT and MCD−/− mice was treated with either vehicle control or CBM-3001106 (10 mg/kg) via oral gavage once daily for 2 days (injections at 5:00 p.m.). The following morning, mice were euthanized and hearts extracted for isolated working heart perfusion with 5 mmol/L glucose and 0.4 mmol/L [9,10-3H]palmitate bound to 3% BSA, and palmitate oxidation rates were assessed as previously described (10).
Glucose/Pyruvate/Insulin Tolerance Tests
Glucose, pyruvate, and insulin tolerance tests were performed 6 h after food withdrawal in mice using glucose, pyruvate, and insulin doses of 2 g/kg, 2 g/kg, and 0.7 units/kg, respectively. Plasma insulin concentrations were determined via use of a commercially available kit (Alpco Diagnostics) as previously described (11).
Whole-Body In Vivo Metabolic Assessment and Exercise Tolerance
In vivo metabolic assessment via indirect calorimetry was performed using the Oxymax Comprehensive Laboratory Animal Monitoring System (CLAMS) (Columbus Instruments). Animals were initially acclimatized in the system for a 24-h period; the subsequent 24-h period was used for data collection. Exercise capacity was performed by running animals on a calibrated, motor-driven treadmill (Columbus Instruments) as previously described (12).
Enzyme Activity Assays
Determination of Lipid Intermediates
Tissue triacylglycerols (TAGs) were extracted with a 2:1 chloroform:methanol solution and quantified with a commercially available enzymatic assay kit (Roche), whereas diacylglycerols (DAGs) were extracted in 0.8 mL of 1 mmol/L NaCl and quantified via thin layer chromatography using the DAG kinase assay (8,12). Tissue was extracted for ceramide with 1 mL of a 1:1:1 chloroform:methanol:1 N HCl in the presence of 0.3 mL saline solution, while long-chain acyl CoA was extracted in 200 μL of cold 100 mmol/L KH2PO4 for high-performance liquid chromatography (HPLC) analysis as previously described (8,12). For determination of malonyl CoA content, tissue was extracted in 300 μL of 6% (volume for volume) perchloric acid and 2 mmol/L dithiothreitol for HPLC analysis as previously described (8,12).
Tissue Homogenate Preparation and Immunoblot Analysis
Frozen gastrocnemius or liver tissue (25–30 mg) was homogenized in buffer containing 50 mmol/L Tris HCl (pH 8 at 4°C), 1 mmol/L EDTA, 10% glycerol (weight for volume), 0.02% Brij-35 (weight for volume), 1 mmol/L dithiothreitol, and protease and phosphatase inhibitors (Sigma) and subjected to Western blotting protocols as previously described (11). Primary antibodies were prepared in a 1/1,000 dilution in 5% BSA. Immunoprecipitation with anti–acetyl-lysine antibodies followed by Western blotting methods was also used to detect overall protein acetylation and superoxide dismutase 2 (SOD)2 acetylation as previously described (14).
Protein Carbonylation and Lipid Peroxidation
Protein carbonylation was assessed in gastrocnemius muscle and liver extracts following instructions from a kit from Millipore (S7150). Overall carbonylation was normalized against β-actin protein expression. Lipid peroxidation was assessed in gastrocnemius muscle extracts following instructions from a lipid hydroperoxide assay kit from Millipore (437639).
All values are presented as mean ± SE. The significance of differences was determined by the use of a Kaplan-Meier survival analysis, an unpaired two-tailed Student t test, or a two-way ANOVA followed by a Bonferroni post hoc analysis where appropriate.
Increased Malonyl CoA Content in Aged MCD−/− Mice Is Associated With a Substrate Switch in Metabolism
As predicted based on the increase in malonyl CoA content in muscle and liver (Fig. 1A and B), use of a CLAMS revealed that whole-body fatty acid oxidation rates are decreased in aged MCD−/− mice, evidenced by the reduction in whole-body oxygen consumption rates and elevation in respiratory exchange ratio (Fig. 1C and D). Further support for a reduction in fatty acid oxidation rates in aged MCD−/− mice was also seen during aerobic isolated working heart perfusion experiments, where a significant decrease in palmitate oxidation rates was observed (Fig. 1E). Consistent with a fuel substrate switch favoring carbohydrate oxidation, gastrocnemius muscle PDH activity, the rate-limiting enzyme of glucose oxidation, was elevated in aged MCD−/− mice (Fig. 1F) and was in a more active dephosphorylated state (Supplementary Fig. 1A). The decreased oxygen consumption rates in aged MCD−/− mice could not be explained by changes in gastrocnemius mitochondrial citrate synthase activity (Supplementary Fig. 1B) or protein expression of PGC1α (Supplementary Fig. 1C), a key transcriptional regulator of mitochondrial biogenesis. Furthermore, the decrease in oxygen consumption and fatty acid oxidation in aged MCD−/− mice did not impair their exercise tolerance (Supplementary Fig. 1D and E), affect overall body weight (Fig. 1G), or increase their adiposity, as epididymal and perirenal fat mass were comparable with that observed in their aged WT littermates (Fig. 1H and I ). Although malonyl CoA levels in the brain are important regulators of appetite and peripheral activity (15), food intake and ambulatory activity were not different between aged MCD−/− mice and their aged WT littermates (Supplementary Fig. 2A and B).
Muscle and Hepatic Lipid Intermediate Accumulation in Aged MCD−/− Mice Does Not Impair Glycemic Control
Aged MCD−/− mice demonstrated no accumulation of TAG or ceramide levels in gastrocnemius muscle but did accumulate certain species of long-chain acyl CoA and showed a trend toward an accumulation of DAG (Fig. 1J–M). Although liver weights and plasma levels of free fatty acids and TAGs were not altered in aged MCD−/− mice (Supplementary Fig. 3), hepatic TAG levels were elevated, with no change in long-chain acyl CoA esters or ceramide content (Fig. 2A, B, and D). In contrast, hepatic DAG content was significantly decreased in aged MCD−/− mice (Fig. 2C). Despite these alterations in muscle and hepatic lipid content, random fed and fasted glucose and insulin levels remained similar (Fig. 2E and F), while both glucose and pyruvate tolerance were not impaired in aged MCD−/− mice (Fig. 2G and H). Conversely, the age-related impairment in insulin tolerance was not as severe in MCD−/− mice (Fig. 2I). Furthermore, Akt and GSK3β phosphorylation, key elements in the insulin signaling cascade, were similar in the liver and muscle of aged MCD−/− mice (Fig. 2J and K). Although we did not directly assess glycolysis rates, protein expression of a number of glycolytic enzymes was similar in gastrocnemius muscles and livers from aged MCD−/− mice and their WT littermates (Supplementary Fig. 4A and B). Additional support indicating that MCD deficiency does not affect glycolysis was seen in young MCD−/− mice and their WT littermates, where we observed similar glycolysis rates during aerobic isolated working heart perfusions (Supplementary Fig. 4C), reproducing our previous findings (7).
Improved Life Span in MCD−/− Mice Is Associated With Decreased Oxidative Stress
Reduced fatty acid oxidation rates and lipid accumulation in MCD−/− mice were not associated with reduced longevity; on the contrary, we observed an increased life span in MCD−/− mice (28% increase) versus their WT littermates (Fig. 3A). In addition, we observed an increased expression of MCD protein and subsequent reduction in malonyl CoA content in the gastrocnemius muscle of aged WT mice versus their young counterparts (Fig. 3B and C). Expression of sirtuin 3 (SIRT3), a critical factor influencing mitochondrial function, was not altered in gastrocnemius muscle and livers of aged MCD−/− mice (Fig. 3D). Interestingly, we observed a significant decrease in oxidative stress in gastrocnemius muscle from aged MCD−/− mice (Fig. 3E and F), which was associated with a significant reduction in the acetylation of the antioxidant enzyme SOD2 (Fig. 3G). These results appeared to be specific to skeletal muscle, as SOD2 acetylation and markers of oxidative stress were not different in livers from aged MCD−/− mice versus their WT littermates (Supplementary Figs. 5A and B and 6).
MCD Inhibition Decreases Muscle Acetylation and Improves Age-Related Impairments in Glycemic Control
Recent studies have suggested that in addition to controlling fatty acid oxidation rates via malonyl CoA, MCD may also function as an acetylase (16). In support of this, similar to SOD2 acetylation, overall protein acetylation was also reduced in gastrocnemius muscle from aged MCD−/− mice (Supplementary Fig. 7). Moreover, we treated C2C12 myotubes with an MCD inhibitor (10 μmol/L; CBM-3001106) that we previously showed to decrease fatty acid oxidation rates (17) and observed a decrease in overall protein acetylation (Fig. 4A). Next, we treated aged C57BL/6J mice with CBM-3001106 for 4 weeks (10 mg/kg via daily oral gavage—a dose that did not affect food intake or animal activity [Supplementary Fig. 2C and D]), which recapitulated the acetylation effects observed in gastrocnemius muscles from aged MCD−/− mice, though SOD2 acetylation only showed a trend toward a reduction (Fig. 4B and C). Also similar to results in aged MCD−/− mice, no differences were observed for total liver protein acetylation or liver SOD2 acetylation in CBM-3001106–treated aged mice (Supplementary Fig. 5C and D). In contrast to findings in aged MCD−/− mice, MCD inhibition via CBM-3001106 treatment for 3 weeks did not reduce oxygen consumption in aged C57BL/6J mice (Fig. 4E), though we did observe a mild increase in respiratory exchange ratio in the latter half of the dark cycle (Fig. 4F). In addition, gastrocnemius muscle long-chain acyl CoA and ceramide content were unaltered in aged mice treated with CBM-3001106 (Fig. 4G and H). Interestingly, pharmacological MCD inhibition with CBM-3001106 for 4 weeks yielded a significant improvement in both glucose and insulin tolerance in aged mice (Fig. 4I and J). These findings were independent of changes in body weight (Fig. 4K) and adiposity, as nuclear MRI demonstrated similar body fat content in aged mice treated with CBM-3001106 (Fig. 4L). Illustrating that our results treating aged mice with CBM-3001106 were likely due to an inhibition of MCD, isolated working heart perfusion studies from MCD−/− mice and their WT littermates treated for 2 days with CBM-3001106 demonstrated that CBM-3001106 only reduced palmitate oxidation rates in the WT mouse heart, but not in hearts from MCD−/− mice (Fig. 4M). Furthermore, treatment of MCD−/− mice with CBM-30011006 had no effect on protein acetylation in gastrocnemius muscle (Supplementary Fig. 8).
Recent studies postulate that decreased mitochondrial fatty acid oxidation is responsible for the accumulation of intracellular lipid intermediates that contribute to the development of numerous chronic diseases in the elderly (3,18). Therefore, MCD−/− mice, which have decreased fatty acid oxidation rates, should be at an increased risk for developing insulin resistance, heart failure, and hepatic steatosis. All of these risks in combination would be expected to shorten the overall life span of these animals.
In contrast, MCD−/− mice displayed a significant increase in life span of ∼30%, which was unexpected based on their lower overall fatty acid oxidative capacity. However, it is important to note that our n number for these experiments is not optimal for a true life span study, despite our findings being statistically significant (19). Nevertheless, the critical observation that must be highlighted from our study is that we did not observe any premature metabolic disease development or early death in MCD−/− mice, despite the accumulation of lipid intermediates in key metabolic tissues. Indeed, we observed a strong trend toward an increase in muscle DAG content, which numerous studies have implicated as a key lipid intermediate in mediating skeletal muscle insulin resistance (20). Despite this trend, glucose tolerance was not worse in aged MCD−/− mice, and their age-related impairment in insulin tolerance was not as severe in comparison with their WT littermates. This may be due to the fact that muscle ceramide content was not altered in aged MCD−/− mice, and we (12) and others (21) have shown that increased ceramide content is a key factor precipitating skeletal muscle insulin resistance. With regard to insulin signaling, the aging-associated reductions in skeletal muscle and hepatic Akt and GSK3β phosphorylation were comparable in aged MCD−/− mice versus their aged WT littermates, but these findings would be strengthened if measured in the fasted and insulin-stimulated state (our assessment was carried out in tissues from animals sacrificed 4 h into their dark cycle).
Our findings in aged MCD−/− mice are not meant to insinuate that the accumulation of tissue lipid intermediates does not cause insulin resistance, as there is a mounting body of evidence supporting that ceramide and DAG are critical to the pathogenesis of insulin resistance (22–24). However, it is likely that some other factor or mechanism outweighs the harmful effects of increased lipid intermediates and somehow protects aged MCD−/− mice from developing metabolic disease. Indeed, we have previously shown that young MCD−/− mice are actually protected from obesity-induced skeletal muscle insulin resistance, which may arise from a reduction in the incomplete oxidation of fatty acids observed during obesity (9). Thus, it is possible that incomplete fatty acid oxidation contributes to the insulin resistance in elderly patients and may explain why the age-related impairment in insulin tolerance is not as severe in aged MCD−/− mice. On the other hand, elegant experiments from Bouzakri et al. (25) demonstrate a critical role for MCD in regulating substrate selection in human muscle, whereby inhibition of MCD decreases palmitate oxidation, which is associated with a corresponding increase in insulin-stimulated GLUT4 translocation, glucose uptake, and glucose oxidation. Accordingly, our respiratory exchange ratio data and gastrocnemius PDH activity in aged MCD−/− mice are consistent with the observations of Bouzakri et al., as aged MCD−/− mice demonstrate a switch in substrate preference for carbohydrates.
Another significant concern with reduction in fatty acid oxidation in the elderly population is that it may increase their risk of developing hepatic steatosis and nonalcoholic fatty liver disease (20). Although hepatic TAG content was greater in aged MCD−/− mice, they had a subsequent reduction in hepatic DAG content. This finding is of particular interest, as DAG accumulation has been proposed to be a key player in the development of hepatic insulin resistance observed in elderly patients and those with type 2 diabetes (20). These differences we observed with regard to DAG content in the muscle and liver illustrate and support the notion that the most powerful effect of lowering DAG content to improve insulin sensitivity appears to take place in the liver (26). To our surprise, despite this marked lowering in hepatic DAG content, the reduction in oxidative stress in aged MCD−/− mice was only observed in skeletal muscle and not the liver. However, these findings do not rule out the decreased hepatic DAG content contributing to the overall metabolic health benefits seen in aged MCD−/− mice.
Since our findings in MCD−/− mice could potentially be model specific and due to compensatory adaptations to global MCD deletion throughout development, we examined the effect of CBM-3001106, an MCD inhibitor, in aged mice. Our findings in this study are the first ever published in vivo findings with this MCD inhibitor, and similar to genetic MCD deficiency, we observed protection against age-related insulin resistance with pharmacological MCD inhibition. On the contrary, we also observed protection against glucose intolerance after pharmacological MCD inhibition. Reasons for this discrepancy between our genetic and pharmacological data are unclear at the moment, but could be due to the fact that pharmacological MCD inhibition produces robust effects on both inhibition of fatty acid oxidation and stimulation of glucose oxidation (6), whereas genetic MCD deficiency produces a metabolic phenotype that only becomes apparent under metabolic stress (7,8). Nevertheless, in spite of these minor discrepancies, a key critical take-home message from our findings is that an inhibition of fatty acid oxidation, via either genetic or pharmacological MCD inhibition, does not worsen age-related metabolic disease as would be predicted based on current dogma (3,20,24).
Of interest, our results support the recent suggestion that MCD may be a bifunctional enzyme that also acts as an acetylase regulating protein acetylation (16). However, our findings do not confirm whether MCD acts as an acetylase itself or simply controls protein acetylation via controlling acetyl CoA content (Supplementary Fig. 9). It will be important for future studies to investigate this further and determine whether our MCD inhibitor, CBM-3001106, which inhibits MCD’s decarboxylase activity, also reduces protein acetylation via inhibiting an independent acetylase activity of MCD. We believe that the reduced protein acetylation after treatment with CBM-3001106 is likely a result of a reduction in acetyl CoA supply for mitochondrial acetylases (Supplementary Fig. 9). Indeed, MCD localizes to both the mitochondria and peroxisomes (5), and one target we observed to have lower acetylation after genetic or pharmacological MCD inhibition, SOD2, primarily localizes to the mitochondria in mammals (27). Our results are limited though by encompassing measurements of acetylation in whole cellular extracts versus mitochondrial extracts, which would be the important compartment to measure regarding SOD2 acetylation. In spite of this limitation, because ∼95% of acetyl CoA, which is the substrate for protein acetylation, is localized to the mitochondria (28), we do believe our total protein and SOD2 acetylation results likely reflect changes within the mitochondrial compartment. Furthermore, the deacetylases that regulate SOD2 acetylation, such as SIRT3, are also regulated via NAD+ levels (29). Although muscle SIRT3 protein expression was similar in aged MCD−/− mice, our future studies will need to measure mitochondrial NAD+ content specifically, which may potentially mediate the observed changes in SOD2 acetylation by increasing SIRT3 activity.
To our surprise, the observed changes in gastrocnemius protein acetylation were not observed in livers from aged MCD−/− mice or aged mice treated with CBM-3001106. We are not certain why such changes would only be observed in the muscle and not the liver after MCD deficiency and/or inhibition, but this could be related to our finding that MCD protein expression is only increased in muscle during aging—not in the liver (Supplementary Fig. 10). In addition, it should be noted, though, that our acetylation results were completed via immunoprecipitation with anti–acetyl-lysine antibodies followed by immunoblotting techniques with a SOD2 antibody to detect SOD2 acetylation. Thus, more sophisticated proteome techniques that examine protein-specific changes to the acetylome are necessary before we can truly say that MCD inhibition also affects protein acetylation, in addition to reducing fatty acid oxidation.
In conclusion, our data challenge the hypothesis that decreased fat oxidation accelerates the progression of age-related diseases. Instead, we demonstrate that lifelong whole-body deficiency of MCD is associated with normal insulin sensitivity, decreased whole-body fatty acid oxidation, increased whole-body carbohydrate utilization, and normal liver function. Furthermore, pharmacological MCD inhibition produced marked improvements in glucose homeostasis in aged mice, though it will be important in future studies to determine whether MCD inhibition improves glucose homeostasis in aged obese animals, as the world’s elderly population is frequently also overweight and/or obese. Preliminary studies in our laboratory suggest that pharmacological MCD inhibition also protects against obesity-induced insulin resistance in young mice (data not shown). Whether such findings will translate to aged obese mice remains to be determined, and with the combined elements of both aging and obesity leading to increases in organ lipid accumulation, MCD inhibition may not be favorable in this scenario. Nevertheless, our previous studies have shown that MCD inhibition and/or deficiency in young mice is associated with protection against ischemia/reperfusion injury (6,7,10) and high-fat diet–induced insulin resistance (9), while even reducing appetite and body weight gain (15). Taken together with our observations that MCD deficiency and/or inhibition appears to protect against age-related insulin resistance, we illustrate an important paradigm shift, whereby chronic inhibition of fatty acid oxidation does not exacerbate age-related disease but may instead have overall metabolic health benefits in the elderly.
Acknowledgments. The authors thank the dedicated staff of the University of Alberta Cardiovascular Research Centre (CVRC) HPLC Core Facility for the measurement of lipid intermediates and the University of Alberta CVRC Animal Physiology Core Facility for the in vivo metabolic assessment via the CLAMS apparatus.
Funding. This study was supported by a grant from the Heart and Stroke Foundation of Alberta and the Canadian Institutes of Health Research (MOP-10865) to G.D.L. G.D.L. is an Alberta Heritage Foundation for Medical Research Medical Scientist.
Duality of Interest. J.R.B.D. and G.D.L. are shareholders and officers of Metabolic Modulators Research Ltd., a company with commercial interests in the development of MCD inhibitors. No other potential conflicts of interest relevant to this article were reported.
Author Contributions. J.R.U. designed the study, researched data, and wrote the manuscript. N.F. researched data, contributed to the discussion, and reviewed and edited the manuscript. W.K., L.Z., J.M., V.K.S., A.F., K.G., D.G.L., and C.S.W. researched data and reviewed and edited the manuscript. J.S.J. and J.R.B.D. contributed to the discussion and reviewed and edited the manuscript. G.D.L. designed the research and wrote the manuscript. G.D.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.