Brennan K. Smith

FAT/CD36 is located on the outer mitochondrial membrane, upstream of long-chain acyl-CoA synthetase, and regulates palmitate oxidation.

FAT/CD36 (fatty acid translocase/Cluster of Differentiation 36), a plasma membrane fatty-acid transport protein, has been found on mitochondrial membranes; however, it remains unclear where FAT/CD36 resides on this organelle or its functional role within mitochondria. In the present study, we demonstrate, using several different approaches, that in skeletal muscle FAT/CD36 resides on the OMM (outer mitochondrial membrane). To determine the functional role of mitochondrial FAT/CD36 in this tissue, we determined oxygen consumption rates in permeabilized muscle fibres in WT (wild-type) and FAT/CD36-KO (knockout) mice using a variety of substrates. Despite comparable muscle mitochondrial content, as assessed by unaltered mtDNA (mitochondrial DNA), citrate synthase, β-hydroxyacyl-CoA dehydrogenase, cytochrome c oxidase complex IV and respiratory capacities [maximal OXPHOS (oxidative phosphorylation) respiration] in WT and KO mice, palmitate-supported respiration was 34% lower in KO animals. In contrast, palmitoyl-CoA-supported respiration was unchanged. These results indicate that FAT/CD36 is key for palmitate-supported respiration. Therefore we propose a working model of mitochondrial fatty-acid transport, in which FAT/CD36 is positioned on the OMM, upstream of long-chain acyl-CoA synthetase, thereby contributing to the regulation of mitochondrial fatty-acid transport. We further support this model by providing evidence that FAT/CD36 is not located in mitochondrial contact sites, and therefore does not directly interact with carnitine palmitoyltransferase-I as original proposed.

In Vivo, Fatty Acid Translocase (CD36) Critically Regulates Skeletal Muscle Fuel Selection, Exercise Performance, and Training-induced Adaptation of Fatty Acid Oxidation

Background: CD36-mediated lipid transport may regulate muscle fuel selection and adaptation. Results: CD36 ablation impaired fatty acid oxidation and prevented its exercise training-induced up-regulation. Without altering mitochondrial content, CD36 overexpression mimicked exercise training effects on fatty acid oxidation. Conclusion: CD36 contributes to regulating fatty acid oxidation and adaptation in a mitochondrion-independent manner. Significance: This work identified another mechanism regulating muscle fatty acid oxidation. For ∼40 years it has been widely accepted that (i) the exercise-induced increase in muscle fatty acid oxidation (FAO) is dependent on the increased delivery of circulating fatty acids, and (ii) exercise training-induced FAO up-regulation is largely attributable to muscle mitochondrial biogenesis. These long standing concepts were developed prior to the recent recognition that fatty acid entry into muscle occurs via a regulatable sarcolemmal CD36-mediated mechanism. We examined the role of CD36 in muscle fuel selection under basal conditions, during a metabolic challenge (exercise), and after exercise training. We also investigated whether CD36 overexpression, independent of mitochondrial changes, mimicked exercise training-induced FAO up-regulation. Under basal conditions CD36-KO versus WT mice displayed reduced fatty acid transport (−21%) and oxidation (−25%), intramuscular lipids (less than or equal to −31%), and hepatic glycogen (−20%); but muscle glycogen, VO2max, and mitochondrial content and enzymes did not differ. In acutely exercised (78% VO2max) CD36-KO mice, fatty acid transport (−41%), oxidation (−37%), and exercise duration (−44%) were reduced, whereas muscle and hepatic glycogen depletions were accelerated by 27–55%, revealing 2-fold greater carbohydrate use. Exercise training increased mtDNA and β-hydroxyacyl-CoA dehydrogenase similarly in WT and CD36-KO muscles, but FAO was increased only in WT muscle (+90%). Comparable CD36 increases, induced by exercise training (+44%) or by CD36 overexpression (+41%), increased FAO similarly (84–90%), either when mitochondrial biogenesis and FAO enzymes were up-regulated (exercise training) or when these were unaltered (CD36 overexpression). Thus, sarcolemmal CD36 has a key role in muscle fuel selection, exercise performance, and training-induced muscle FAO adaptation, challenging long held views of mechanisms involved in acute and adaptive regulation of muscle FAO.

AMPK phosphorylation of ACC2 is required for skeletal muscle fatty acid oxidation and insulin sensitivity in mice

Aims/hypothesisObesity is characterised by lipid accumulation in skeletal muscle, which increases the risk of developing insulin resistance and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status and is activated in skeletal muscle by exercise, hormones (leptin, adiponectin, IL-6) and pharmacological agents (5-amino-4-imidazolecarboxamide ribonucleoside [AICAR] and metformin). Phosphorylation of acetyl-CoA carboxylase 2 (ACC2) at S221 (S212 in mice) by AMPK reduces ACC activity and malonyl-CoA content but the importance of the AMPK–ACC2–malonyl-CoA pathway in controlling fatty acid metabolism and insulin sensitivity is not understood; therefore, we characterised Acc2 S212A knock-in (ACC2 KI) mice.MethodsWhole-body and skeletal muscle fatty acid oxidation and insulin sensitivity were assessed in ACC2 KI mice and wild-type littermates.ResultsACC2 KI mice were resistant to increases in skeletal muscle fatty acid oxidation elicited by AICAR. These mice had normal adiposity and liver lipids but elevated contents of triacylglycerol and ceramide in skeletal muscle, which were associated with hyperinsulinaemia, glucose intolerance and skeletal muscle insulin resistance.Conclusions/interpretationThese findings indicate that the phosphorylation of ACC2 S212 is required for the maintenance of skeletal muscle lipid and glucose homeostasis.

Lack of Adipocyte AMPK Exacerbates Insulin Resistance and Hepatic Steatosis through Brown and Beige Adipose Tissue Function

Brown (BAT) and white (WAT) adipose tissues play distinct roles in maintaining whole-body energy homeostasis, and their dysfunction can contribute to non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes. The AMP-activated protein kinase (AMPK) is a cellular energy sensor, but its role in regulating BAT and WAT metabolism is unclear. We generated an inducible model for deletion of the two AMPK β subunits in adipocytes (iβ1β2AKO) and found that iβ1β2AKO mice were cold intolerant and resistant to β-adrenergic activation of BAT and beiging of WAT. BAT from iβ1β2AKO mice had impairments in mitochondrial structure, function, and markers of mitophagy. In response to a high-fat diet, iβ1β2AKO mice more rapidly developed liver steatosis as well as glucose and insulin intolerance. Thus, AMPK in adipocytes is vital for maintaining mitochondrial integrity, responding to pharmacological agents and thermal stress, and protecting against nutrient-overload-induced NAFLD and insulin resistance.

Treatment of nonalcoholic fatty liver disease: role of AMPK

Nonalcoholic fatty liver disease (NAFLD) is a growing worldwide epidemic and an important risk factor for the development of insulin resistance, type 2 diabetes, nonalcoholic steatohepatitis (NASH), and hepatic cellular carcinoma (HCC). Despite the prevalence of NAFLD, lifestyle interventions involving exercise and weight loss are the only accepted treatments for this disease. Over the last decade, numerous experimental compounds have been shown to improve NAFLD in preclinical animal models, and many of these therapeutics have been shown to increase the activity of the cellular energy sensor AMP-activated protein kinase (AMPK). Because AMPK activity is reduced by inflammation, obesity, and diabetes, increasing AMPK activity has been viewed as a viable therapeutic strategy to improve NAFLD. In this review, we propose three primary mechanisms by which AMPK activation may improve NAFLD. In addition, we examine the mechanisms by which AMPK is activated. Finally, we identify 27 studies that have used AMPK activators to reduce NAFLD. Future considerations for studies examining the relationship between AMPK and NAFLD are highlighted.

Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity.

Metformin is the mainstay therapy for type 2 diabetes (T2D) and many patients also take salicylate-based drugs [i.e., aspirin (ASA)] for cardioprotection. Metformin and salicylate both increase AMP-activated protein kinase (AMPK) activity but by distinct mechanisms, with metformin altering cellular adenylate charge (increasing AMP) and salicylate interacting directly at the AMPK β1 drug-binding site. AMPK activation by both drugs results in phosphorylation of ACC (acetyl-CoA carboxylase; P-ACC) and inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme controlling fatty acid synthesis (lipogenesis). We find doses of metformin and salicylate used clinically synergistically activate AMPK in vitro and in vivo, resulting in reduced liver lipogenesis, lower liver lipid levels and improved insulin sensitivity in mice. Synergism occurs in cell-free assays and is specific for the AMPK β1 subunit. These effects are also observed in primary human hepatocytes and patients with dysglycaemia exhibit additional improvements in a marker of insulin resistance (proinsulin) when treated with ASA and metformin compared with either drug alone. These data indicate that metformin-salicylate combination therapy may be efficacious for the treatment of non-alcoholic fatty liver disease (NAFLD) and T2D.

The Na+/glucose co-transporter inhibitor canagliflozin activates AMP-activated protein kinase by inhibiting mitochondrial function and increasing cellular AMP levels

Canagliflozin, dapagliflozin, and empagliflozin, all recently approved for treatment of type 2 diabetes, were derived from the natural product phlorizin. They reduce hyperglycemia by inhibiting glucose reuptake by sodium/glucose cotransporter (SGLT) 2 in the kidney, without affecting intestinal glucose uptake by SGLT1. We now report that canagliflozin also activates AMPK, an effect also seen with phloretin (the aglycone breakdown product of phlorizin), but not to any significant extent with dapagliflozin, empagliflozin, or phlorizin. AMPK activation occurred at canagliflozin concentrations measured in human plasma in clinical trials and was caused by inhibition of Complex I of the respiratory chain, leading to increases in cellular AMP or ADP. Although canagliflozin also inhibited cellular glucose uptake independently of SGLT2, this did not account for AMPK activation. Canagliflozin also inhibited lipid synthesis, an effect that was absent in AMPK knockout cells and that required phosphorylation of acetyl-CoA carboxylase (ACC) 1 and/or ACC2 at the AMPK sites. Oral administration of canagliflozin activated AMPK in mouse liver, although not in muscle, adipose tissue, or spleen. Because phosphorylation of ACC by AMPK is known to lower liver lipid content, these data suggest a potential additional benefit of canagliflozin therapy compared with other SGLT2 inhibitors.

Trans-fatty acids and cancer: a mini-review.

The association between trans-fatty acids (TFA) and cancer risk is poorly understood and remains controversial. It is recognised that unique biological effects are associated with specific isoforms within families of fatty acids such as those belonging to the n-3 fatty acids. Furthermore, the interactions between diet and genetic polymorphisms are increasingly recognised for their potential risk-modifying effects on human health and disease. Therefore, the aim of the present review is to evaluate whether specific TFA isomers and genetic polymorphisms differentially modify cancer risk in prostate, colon and breast cancers in animal and human models. Potential mechanisms of action by which TFA may affect cancer development are also reviewed. Overall, across a number of experimental models and human studies, there is insufficient and inconsistent evidence linking specific TFA isomers to cancers of the prostate, colon and breast. A number of methodological limitations and experimental considerations were identified which may explain the inconsistencies observed across these studies. Therefore, further research is warranted to accurately assess the relationship between TFA and cancer risk.

A decreased n-6/n-3 ratio in the fat-1 mouse is associated with improved glucose tolerance

A reduction in skeletal muscle fatty acid oxidation (FAO), manifested as a reduction in mitochondrial content and (or) FAO within mitochondria, may contribute to the development of insulin resistance. n-3 polyunsaturated fatty acids (PUFA) have been observed to increase the capacity for FAO and improve insulin sensitivity. We used the fat-1 mouse model, a transgenic animal capable of synthesizing n-3 PUFA from n-6 PUFA, to examine this relationship. Fat-1 mice exhibited a approximately 20-fold decrease in the n-6/n-3 ratio in skeletal muscle, and plasma glucose and the area under the glucose curve were significantly (p < 0.05) lower in fat-1 mice during a glucose challenge test. The improvement in whole-body glucose tolerance in the fat-1 mouse was associated with a approximately 21% (p < 0.05) decrease in whole-muscle citrate synthase (CS) activity (in red muscle only), without alterations in CS activity of isolated mitochondria (either red or white muscle; p > 0.05). These data suggest that the fat-1 mouse has decreased skeletal muscle mitochondrial content. However, the intrinsic ability of mitochondria to oxidize fatty acids was not altered in the fat-1 mouse, as rates of palmitate oxidation in isolated mitochondria from both red and white muscle were unchanged. Overall, this study demonstrates that a decrease in the n-6/n-3 ratio can enhance glucose tolerance in healthy animals, independent of changes in mitochondrial content.

Liver-specific ATP-citrate lyase inhibition by bempedoic acid decreases LDL-C and attenuates atherosclerosis

Despite widespread use of statins to reduce low-density lipoprotein cholesterol (LDL-C) and associated atherosclerotic cardiovascular risk, many patients do not achieve sufficient LDL-C lowering due to muscle-related side effects, indicating novel treatment strategies are required. Bempedoic acid (ETC-1002) is a small molecule intended to lower LDL-C in hypercholesterolemic patients, and has been previously shown to modulate both ATP-citrate lyase (ACL) and AMP-activated protein kinase (AMPK) activity in rodents. However, its mechanism for LDL-C lowering, efficacy in models of atherosclerosis and relevance in humans are unknown. Here we show that ETC-1002 is a prodrug that requires activation by very long-chain acyl-CoA synthetase-1 (ACSVL1) to modulate both targets, and that inhibition of ACL leads to LDL receptor upregulation, decreased LDL-C and attenuation of atherosclerosis, independently of AMPK. Furthermore, we demonstrate that the absence of ACSVL1 in skeletal muscle provides a mechanistic basis for ETC-1002 to potentially avoid the myotoxicity associated with statin therapy.