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Fatty acids are vital for the survival of eukaryotes, but when present in excess can have deleterious consequences. The AMP-activated protein kinase (AMPK) is an important regulator of multiple branches of metabolism. Studies in purified enzyme preparations and cultured cells have shown that AMPK is allosterically activated by small molecules as well as fatty acyl-CoAs through a mechanism involving Ser108 within the regulatory AMPK β1 isoform. However, the in vivo physiological significance of this residue has not been evaluated. In the current study, we generated mice with a targeted germline knock-in (KI) mutation of AMPKβ1 Ser108 to Ala (S108A-KI), which renders the site phospho-deficient. S108A-KI mice had reduced AMPK activity (50 to 75%) in the liver but not in the skeletal muscle. On a chow diet, S108A-KI mice had impairments in exogenous lipid-induced fatty acid oxidation. Studies in mice fed a high-fat diet found that S108A-KI mice had a tendency for greater glucose intolerance and elevated liver triglycerides. Consistent with increased liver triglycerides, livers of S108A-KI mice had reductions in mitochondrial content and respiration that were accompanied by enlarged mitochondria, suggestive of impairments in mitophagy. Subsequent studies in primary hepatocytes found that S108A-KI mice had reductions in palmitate- stimulated Cpt1a and Ppargc1a mRNA, ULK1 phosphorylation and autophagic/mitophagic flux. These data demonstrate an important physiological role of AMPKβ1 Ser108 phosphorylation in promoting fatty acid oxidation, mitochondrial biogenesis and autophagy under conditions of high lipid availability. As both ketogenic diets and intermittent fasting increase circulating free fatty acid levels, AMPK activity, mitochondrial biogenesis, and mitophagy, these data suggest a potential unifying mechanism which may be important in mediating these effects.

Perioperative hyperglycemia is common and is associated with significant morbidity. Although patient characteristics and surgery influence perioperative glucose metabolism, anesthetics have a significant impact. We hypothesized that mice that were obese and insulin-resistant would experience greater hyperglycemia in response to sevoflurane anesthesia compared with lean controls. We further hypothesized that sevoflurane-induced hyperglycemia would be attenuated by salsalate pre-treatment. Lean and obese male C57BL/6J mice were anesthetized with sevoflurane for 60 min with or without pre-treatment of 62.5 mg·kg−1 salsalate. Blood glucose, plasma insulin, and glucose uptake into different tissues were measured. Under sevoflurane anesthesia, obese mice had higher blood glucose compared to lean mice. Increases in blood glucose were attenuated with acute salsalate pre-treatment at 60 min under anesthesia in obese mice (mean ± standard error of the mean [SEM], delta blood glucose; vehicle 5.79 ± 1.09 vs salsalate 1.91 ± 1.32 mM; P = 0.04) but did not reach statistical significance in lean mice (delta blood glucose, vehicle 4.39 ± 0.55 vs salsalate 2.79 ± 0.71 mM; P = 0.10). This effect was independent of changes in insulin but associated with an approx. 1.7-fold increase in glucose uptake into brown adipose tissue (vehicle 45.28 ± 4.57 vs salsalate 76.89 ± 12.23 µmol·g−1 tissue·hr−1; P < 0.001). These data show that salsalate can reduce sevoflurane-induced hyperglycemia in mice. This indicates that salsalate may represent a new class of therapeutics that, in addition to its anti-inflammatory and analgesic properties, may be useful to reduce perioperative hyperglycemia.

Long-chain fatty acids (LCFAs) play important roles in cellular energy metabolism, acting as both an important energy source and signalling molecules1. LCFA-CoA esters promote their own oxidation by acting as allosteric inhibitors of acetyl-CoA carboxylase, which reduces the production of malonyl-CoA and relieves inhibition of carnitine palmitoyl-transferase 1, thereby promoting LCFA-CoA transport into the mitochondria for β-oxidation2,3,4,5,6. Here we report a new level of regulation wherein LCFA-CoA esters per se allosterically activate AMP-activated protein kinase (AMPK) β1–containing isoforms to increase fatty acid oxidation through phosphorylation of acetyl-CoA carboxylase. Activation of AMPK by LCFA-CoA esters requires the allosteric drug and metabolite site formed between the α-subunit kinase domain and the β-subunit. β1 subunit mutations that inhibit AMPK activation by the small-molecule activator A769662, which binds to the allosteric drug and metabolite site, also inhibit activation by LCFA-CoAs. Thus, LCFA-CoA metabolites act as direct endogenous AMPK β1–selective activators and promote LCFA oxidation.

Objective Salsalate is a prodrug of salicylate that lowers blood glucose in people with type 2 diabetes. AMP-activated protein kinase (AMPK) is an αβγ heterotrimer which inhibits macrophage inflammation and the synthesis of fatty acids and cholesterol in the liver through phosphorylation of acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), respectively. Salicylate binds to and activates AMPKβ1-containing heterotrimers that are highly expressed in both macrophages and liver, but the potential importance of AMPK and ability of salsalate to reduce atherosclerosis have not been evaluated. Methods ApoE−/− and LDLr−/− mice with or without (−/−) germline or bone marrow AMPKβ1, respectively, were treated with salsalate, and atherosclerotic plaque size was evaluated in serial sections of the aortic root. Studies examining the effects of salicylate on markers of inflammation, fatty acid and cholesterol synthesis and proliferation were conducted in bone marrow–derived macrophages (BMDMs) from wild-type mice or mice lacking AMPKβ1 or the key AMPK-inhibitory phosphorylation sites on ACC (ACC knock-in (KI)-ACC KI) or HMGCR (HMGCR-KI). Results Salsalate reduced atherosclerotic plaques in the aortic roots of ApoE−/− mice, but not ApoE−/− AMPKβ1−/− mice. Similarly, salsalate reduced atherosclerosis in LDLr−/− mice receiving wild-type but not AMPKβ1−/− bone marrow. Reductions in atherosclerosis by salsalate were associated with reduced macrophage proliferation, reduced plaque lipid content and reduced serum cholesterol. In BMDMs, this suppression of proliferation by salicylate required phosphorylation of HMGCR and the suppression of cholesterol synthesis. Conclusions These data indicate that salsalate suppresses macrophage proliferation and atherosclerosis through an AMPKβ1-dependent pathway, which may involve HMGCR phosphorylation and cholesterol synthesis. Since rapidly-proliferating macrophages are a hallmark of atherosclerosis, these data indicate further evaluation of salsalate as a potential therapeutic agent for treating atherosclerotic cardiovascular disease., Highlights • Salsalate (a dimer of salicylate) activates AMPK in macrophages and reduces atherosclerosis. • Salicylate-induced reductions in atherosclerosis are associated with reduced macrophage proliferation and serum cholesterol. • AMPK phosphorylation of HMG-CoA reductase is required for suppressing cholesterol synthesis and macrophage proliferation.

Obesity results from a caloric imbalance between energy intake, absorption and expenditure. In both rodents and humans, diet-induced thermogenesis contributes to energy expenditure and involves the activation of brown adipose tissue (BAT). We hypothesize that environmental toxicants commonly used as food additives or pesticides might reduce BAT thermogenesis through suppression of uncoupling protein 1 (UCP1) and this may contribute to the development of obesity. Using a step-wise screening approach, we discover that the organophosphate insecticide chlorpyrifos suppresses UCP1 and mitochondrial respiration in BAT at concentrations as low as 1 pM. In mice housed at thermoneutrality and fed a high-fat diet, chlorpyrifos impairs BAT mitochondrial function and diet-induced thermogenesis, promoting greater obesity, non-alcoholic fatty liver disease (NAFLD) and insulin resistance. This is associated with reductions in cAMP; activation of p38MAPK and AMPK; protein kinases critical for maintaining UCP1 and mitophagy, respectively in BAT. These data indicate that the commonly used pesticide chlorpyrifos, suppresses diet-induced thermogenesis and the activation of BAT, suggesting its use may contribute to the obesity epidemic., Chlorpyrifos is a widely-used pesticide and a common residue on vegetables and fruits. Here the authors show that at non-neurotoxic doses, chlorpyrifos reduces energy expenditure, by inhibiting diet induced thermogenesis, and promotes obesity and insulin resistance.

Background Globally, medical cannabis legalization has increased in recent years and medical cannabis is commonly used to treat chronic pain. However, there are few randomized control trials studying medical cannabis indicating expert guidance on how to dose and administer medical cannabis safely and effectively is needed. Methods Using a multistage modified Delphi process, twenty global experts across nine countries developed consensus-based recommendations on how to dose and administer medical cannabis in patients with chronic pain. Results There was consensus that medical cannabis may be considered for patients experiencing neuropathic, inflammatory, nociplastic, and mixed pain. Three treatment protocols were developed. A routine protocol where the clinician initiates the patient on a CBD-predominant variety at a dose of 5 mg CBD twice daily and titrates the CBD-predominant dose by 10 mg every 2 to 3 days until the patient reaches their goals, or up to 40 mg/day. At a CBD-predominant dose of 40 mg/day, clinicians may consider adding THC at 2.5 mg and titrate by 2.5 mg every 2 to 7 days until a maximum daily dose of 40 mg/day of THC. A conservative protocol where the clinician initiates the patient on a CBD-predominant variety at a dose of 5 mg once daily and titrates the CBD-predominant dose by 10 mg every 2 to 3 days until the patient reaches their goals, or up to 40 mg/day. At a CBD-predominant dose of 40 mg/day, clinicians may consider adding THC at 1 mg/day and titrate by 1 mg every 7 days until a maximum daily dose of 40 mg/day of THC. A rapid protocol where the clinician initiates the patient on a balanced THC:CBD variety at 2.5–5 mg of each cannabinoid once or twice daily and titrates by 2.5–5 mg of each cannabinoid every 2 to 3 days until the patient reaches his/her goals or to a maximum THC dose of 40 mg/day. Conclusions In summary, using a modified Delphi process, expert consensus-based recommendations were developed on how to dose and administer medical cannabis for the treatment of patients with chronic pain.

Aims Opioid misuse and overuse have contributed to a widespread overdose crisis and many patients and physicians are considering medical cannabis to support opioid tapering and chronic pain control. Using a five‐step modified Delphi process, we aimed to develop consensus‐based recommendations on: 1) when and how to safely initiate and titrate cannabinoids in the presence of opioids, 2) when and how to safely taper opioids in the presence of cannabinoids and 3) how to monitor patients and evaluate outcomes when treating with opioids and cannabinoids. Results In patients with chronic pain taking opioids not reaching treatment goals, there was consensus that cannabinoids may be considered for patients experiencing or displaying opioid‐related complications, despite psychological or physical interventions. There was consensus observed to initiate with a cannabidiol (CBD)‐predominant oral extract in the daytime and consider adding tetrahydrocannabinol (THC). When adding THC, start with 0.5‐3 mg, and increase by 1‐2 mg once or twice weekly up to 30‐40 mg/day. Initiate opioid tapering when the patient reports a minor/major improvement in function, seeks less as‐needed medication to control pain and/or the cannabis dose has been optimised. The opioid tapering schedule may be 5%–10% of the morphine equivalent dose (MED) every 1 to 4 weeks. Clinical success could be defined by an improvement in function/quality of life, a ≥30% reduction in pain intensity, a ≥25% reduction in opioid dose, a reduction in opioid dose to

Introduction Proper insulin injection technique has demonstrated positive clinical outcomes in patients with diabetes. A Canadian-based practice reflective was undertaken to evaluate the current state of understanding of injection technique practices by patients administering insulin, and the importance physicians place on proper injection technique. Methods Twenty-four sites across Canada completed a practice profile survey and enrolled adult non-pregnant patients with either type 1 or type 2 diabetes injecting insulin using an insulin pen. Seven areas of proper injection technique to be evaluated were identified by the study steering committee: size of injection site, use of a skin lift, needle reuse, length of the needle, duration of the needle in the skin, injection into lipohypertrophic tissue, and applied injection force. During a scheduled visit, each patient filled out the Injection Technique Survey and the physician documented the answers via an electronic database. Results Almost all physicians surveyed agreed (96%) that proper insulin injection technique is important or very important and 80% indicated they were either completely confident or fairly confident in discussing overall insulin injection technique. All patients surveyed were making at least one insulin injection technique error within the following categories: applied injection force (76%), area size of injection site (64%), duration of pen needle in skin (61%), pen needle reuse (39%), performs a skin lift with a 4 or 5 mm needle (38%), uses a longer pen needle than required (34%), and injection of insulin into lipohypertrophic tissue (37%). Conclusion Patients commonly make insulin injection errors. Patient and physician education on optimal insulin injection technique continues to be an unmet medical need for the treatment of patients with diabetes. Prospective trials examining the impact of new technology, diabetes educational teams, and e-learning as educational interventions are potential avenues to explore in future studies to support improved insulin injection technique. Electronic Supplementary Material The online version of this article (10.1007/s13300-020-00913-y) contains supplementary material, which is available to authorized users.

Dysregulation of hepatic lipid and cholesterol metabolism is a significant contributor to cardiometabolic health, resulting in excessive liver lipid accumulation and ultimately non-alcoholic steatohepatitis (NASH). Therapeutic activators of the AMP-Activated Protein Kinase (AMPK) have been proposed as a treatment for metabolic diseases; we show that the AMPK β1-biased activator PF-06409577 is capable of lowering hepatic and systemic lipid and cholesterol levels in both rodent and monkey preclinical models. PF-06409577 is able to inhibit de novo lipid and cholesterol synthesis pathways, and causes a reduction in hepatic lipids and mRNA expression of markers of hepatic fibrosis. These effects require AMPK activity in the hepatocytes. Treatment of hyperlipidemic rats or cynomolgus monkeys with PF-06409577 for 6 weeks resulted in a reduction in circulating cholesterol. Together these data suggest that activation of AMPK β1 complexes with PF-06409577 is capable of impacting multiple facets of liver disease and represents a promising strategy for the treatment of NAFLD and NASH in humans., Highlights • PF-06409577 is a potent activator of AMPK β1 containing complexes. • PF-06409577 improves liver function and systemic dyslipidemia in rodents through hepatic AMPK activation. • PF-06409577-mediated reductions in circulating cholesterol was observed in monkeys and diabetic rats. NAFLD and NASH remain poorly treated and are diseases which are growing rapidly in societal cost. Therapeutic mechanisms that impact multiple aspects of the dysregulated metabolic regulation of NAFLD and NASH are needed. Pharmacological AMPK activation has long held promise as a treatment for NAFLD because of its impact on hepatic lipid and cholesterol synthesis, as well as its proposed anti-inflammatory and anti-lipolytic actions. Recent development of clinically viable small molecule AMPK activators, including PF-06409577, has enabled their more thorough characterization in preclinical disease models.

Obesity is a major risk factor for type 2 diabetes (T2D), non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease that arises from a caloric surplus of as little as 10–30 kcal per day. And while increased consumption of energy dense foods and reduced physical activity are commonly thought to be the major contributors to this caloric imbalance, diet-induced thermogenesis is a quantitatively important component of the energy balance equation. In adult humans, recent studies have indicated that diet-induced thermogenesis requires the activation of brown adipose tissue (BAT), however, the determinants regulating this process and why they may differ between individuals are not fully understood. We hypothesized that environmental toxicants commonly used as food additives or pesticides might reduce diet-induced thermogenesis through suppression of uncoupling protein 1, the defining protein of human BAT thermogenesis. Through a screening approach of pesticides/toxicants chosen from the Toxcast chem Library, we discovered that the organophosphate insecticide chlorpyrifos potently suppressed the expression of uncoupling protein 1 (UCP1) and mitochondrial respiration in brown adipocytes at concentrations as low as 1 pM. Chloropyrifos-induced suppression of brown adipocyte thermogenesis was also observed in mice fed a diet high in fat and housed at thermoneutrality where it promoted greater obesity, non-alcoholic fatty liver disease and insulin resistance. Reductions in thermogenesis by chlorpyrifos were associated with impaired activation of the β3-adrenergic receptor and protein kinases critical for regulating UCP1 and mitophagy. These data indicate that the commonly used pesticide chlorpyrifos, at doses found within the food supply, suppresses the activation of brown adipose tissue, suggesting that its use may contribute to the obesity epidemic.

Background: Recent approvals for novel agents such as the small molecule Janus kinase inhibitors (JAKi), combined with the advent of biosimilars has widened the gamut of available therapeutic options in the treatment of rheumatoid arthritis (RA). This combined with the introduction of mandatory non- medical switches to biosimilars in some jurisdictions by both public and private payors has led to a significant increase in the volume of therapeutic changes for patients. Pharmacists are well positioned to ensure effective and safe transitions, however there is a significant unmet need for objective and subjective clinical guidance around therapy as well disease state monitoring in RA that facilitates best practices throughout the patient journey. Objective: In this paper we aim to create a consensus derived monitoring algorithm for pharmacists to facilitate best practices throughout therapeutic transitions from originator biologic to other originator biologics, biosimilars, and Janus kinase inhibitors in RA. Methods: The Nominal Group Technique (NGT) was used to understand if consensus could be found among the participants. Clinically relevant questions were developed to capture solutions to the identified unmet need. The faculty considered the questions as individuals, and privately generated answers/ideas. After discussion and consideration, the participants ranked the ideas and established a consensus. Results: Based on the outcome of the consensus discussions, an algorithm was created to help guide pharmacists through therapeutic transitions in RA. The tool covers important topics such as pre-transition considerations, avoiding the nocebo effect for biosimilars, specific considerations for each drug or class, monitoring efficacy, and when to refer. Conclusions: New classes of anti-rheumatic drugs including JAKi, along with the introduction of biosimilars are presenting more opportunity for therapeutic changes and monitoring in patients with RA. We hope our evidence-based consensus derived guidance tool will assist frontline pharmacists in supporting their patients to a successful therapeutic transition in RA.

BACKGROUND: Physical inactivity impairs insulin sensitivity, which is exacerbated with aging. We examined the impact of 2 wk of acute inactivity and recovery on glycemic control, and integrated rates of muscle protein synthesis in older men and women. METHODS: Twenty-two overweight, prediabetic older adults (12 men, 10 women, 69 ± 4 y) undertook 7 d of habitual activity (baseline; BL), step reduction (SR

TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle.

Objective The sodium-glucose transporter 2 (SGLT2) inhibitors Canagliflozin and Dapagliflozin are recently approved medications for type 2 diabetes. Recent studies indicate that SGLT2 inhibitors may inhibit the growth of some cancer cells but the mechanism(s) remain unclear. Methods Cellular proliferation and clonogenic survival were used to assess the sensitivity of prostate and lung cancer cell growth to the SGLT2 inhibitors. Oxygen consumption, extracellular acidification rate, cellular ATP, glucose uptake, lipogenesis, and phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase, and the p70S6 kinase were assessed. Overexpression of a protein that maintains complex-I supported mitochondrial respiration (NDI1) was used to establish the importance of this pathway for mediating the anti-proliferative effects of Canagliflozin. Results Clinically achievable concentrations of Canagliflozin, but not Dapagliflozin, inhibit cellular proliferation and clonogenic survival of prostate and lung cancer cells alone and in combination with ionizing radiation and the chemotherapy Docetaxel. Canagliflozin reduced glucose uptake, mitochondrial complex-I supported respiration, ATP, and lipogenesis while increasing the activating phosphorylation of AMPK. The overexpression of NDI1 blocked the anti-proliferative effects of Canagliflozin indicating reductions in mitochondrial respiration are critical for anti-proliferative actions. Conclusion These data indicate that like the biguanide metformin, Canagliflozin not only lowers blood glucose but also inhibits complex-I supported respiration and cellular proliferation in prostate and lung cancer cells. These observations support the initiation of studies evaluating the clinical efficacy of Canagliflozin on limiting tumorigenesis in pre-clinical animal models as well epidemiological studies on cancer incidence relative to other glucose lowering therapies in clinical populations., Highlights • Canagliflozin inhibits the proliferation and clonogenic survival of cancer cells. • Canagliflozin enhances the anti-clonogenic effects of radiation and Docetaxel. • Canagliflozin reduces glucose uptake and complex-I supported respiration. • Canagliflozin decreases intracellular ATP and inhibits lipogenesis. • Bypassing complex-1 supported respiration reversed the effects of Canagliflozin.

Salsalate is a prodrug of salicylate that lowers blood glucose in patients with type 2 diabetes (T2D) and reduces nonalcoholic fatty liver disease (NAFLD) in animal models; however, the mechanism mediating these effects is unclear. Salicylate directly activates AMPK via the β1 subunit, but whether salsalate requires AMPK-β1 to improve T2D and NAFLD has not been examined. Therefore, wild-type (WT) and AMPK-β1–knockout (AMPK-β1KO) mice were treated with a salsalate dose resulting in clinically relevant serum salicylate concentrations (∼1 mmol/L). Salsalate treatment increased VO2, lowered fasting glucose, improved glucose tolerance, and led to an ∼55% reduction in liver lipid content. These effects were observed in both WT and AMPK-β1KO mice. To explain these AMPK-independent effects, we found that salicylate increases oligomycin-insensitive respiration (state 4o) and directly increases mitochondrial proton conductance at clinical concentrations. This uncoupling effect is tightly correlated with the suppression of de novo lipogenesis. Salicylate is also able to stimulate brown adipose tissue respiration independent of uncoupling protein 1. These data indicate that the primary mechanism by which salsalate improves glucose homeostasis and NAFLD is via salicylate-driven mitochondrial uncoupling.

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.

Objective Skeletal muscle AMP-activated protein kinase (AMPK) is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. R419 is a mitochondrial complex-I inhibitor that has recently been shown to acutely activate AMPK in myotubes. Our main objective was to examine whether R419 treatment improves insulin sensitivity and exercise capacity in obese insulin resistant mice and whether skeletal muscle AMPK was important for mediating potential effects. Methods Glucose homeostasis, insulin sensitivity, exercise capacity, and electron transport chain content/activity were examined in wildtype (WT) and AMPK β1β2 muscle-specific null (AMPK-MKO) mice fed a high-fat diet (HFD) with or without R419 supplementation. Results There was no change in weight gain, adiposity, glucose tolerance or insulin sensitivity between HFD-fed WT and AMPK-MKO mice. In both HFD-fed WT and AMPK-MKO mice, R419 enhanced insulin tolerance, insulin-stimulated glucose disposal, skeletal muscle 2-deoxyglucose uptake, Akt phosphorylation and glucose transporter 4 (GLUT4) content independently of alterations in body mass. In WT, but not AMPK-MKO mice, R419 improved treadmill running capacity. Treatment with R419 increased muscle electron transport chain content and activity in WT mice; effects which were blunted in AMPK-MKO mice. Conclusions Treatment of obese mice with R419 improved skeletal muscle insulin sensitivity through a mechanism that is independent of skeletal muscle AMPK. R419 also increases exercise capacity and improves mitochondrial function in obese WT mice; effects that are diminished in the absence of skeletal muscle AMPK. These findings suggest that R419 may be a promising therapy for improving whole-body glucose homeostasis and exercise capacity., Highlights • The beneficial effects of a novel complex I inhibitor, R419, were examined in skeletal muscle AMPK deficient obese mice. • R419 improves skeletal muscle insulin sensitivity in obese mice. • R419 improves exercise capacity in obese mice via an AMPK dependent pathway. • These findings suggest that R419 may have therapeutic importance in improving exercise capacity and skeletal muscle insulin sensitivity.

Mitochondrial ADP transport may represent a convergence point unifying two prominent working models for the development of insulin resistance, as reactive lipids (specifically palmitoyl-CoA [P-CoA]) can inhibit ADP transport and subsequently increase mitochondrial reactive oxygen species emissions. In the current study, we aimed to determine if exercise training in humans diminished P-CoA attenuation of mitochondrial ADP respiratory sensitivity. Six weeks of exercise training increased whole-body glucose homeostasis and skeletal muscle Akt signaling and reduced markers of oxidative stress without reducing maximal mitochondrial H2O2 emissions. To ascertain if enhanced mitochondrial ADP transport contributed to the improvement in the in vivo oxidative state, we determined mitochondrial ADP sensitivity in the presence and absence of P-CoA. In the absence of P-CoA, exercise training reduced mitochondrial ADP sensitivity. In contrast, exercise training increased mitochondrial ADP sensitivity with P-CoA present. We further show that P-CoA noncompetitively inhibits mitochondrial ADP transport and the ability of ADP to attenuate mitochondrial H2O2 emission. Altogether, the current data provide a potential mechanism for how P-CoA contributes to insulin resistance and highlight the ability of exercise training to diminish P-CoA attenuation in mitochondrial ADP transport.

Mitochondrial dysfunction and reactive oxygen species (ROS) have been implicated in the aetiology of skeletal muscle insulin resistance, although there is considerable controversy regarding these concepts. Mitochondrial function has been traditionally assessed in the presence of saturating ADP, but ATP turnover and the resultant ADP is thought to limit respiration in vivo. Therefore, we investigated the potential link between submaximal ADP-stimulated respiration rates, ROS generation and skeletal muscle insulin sensitivity in a model of type 2 diabetes mellitus, the ZDF rat. Utilizing permeabilized muscle fibres we observed that submaximal ADP-stimulated respiration rates (250-2000 μm ADP) were lower in ZDF rats than in lean controls, which coincided with decreased adenine nucleotide translocase 2 (ANT2) protein content. This decrease in submaximal ADP-stimulated respiration occurred in the absence of a decrease in electron transport chain function. Treating ZDF rats with resveratrol improved skeletal muscle insulin resistance and this was associated with elevated submaximal ADP-stimulated respiration rates as well as an increase in ANT2 protein content. These results coincided with a greater ability of ADP to attenuate mitochondrial ROS emission and an improvement in cellular redox balance. Together, these data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for mitochondrial ROS emission.

In skeletal muscle, mitochondria exist as two subcellular populations known as subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS mitochondria preferentially respond to exercise training, suggesting divergent transcriptional control of the mitochondrial genomes. The transcriptional co-activator peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and mitochondrial transcription factor A (Tfam) have been implicated in the direct regulation of the mitochondrial genome in mice, although SS and IMF differences may exist, and the potential signalling events regulating the mitochondrial content of these proteins have not been elucidated. Therefore, we examined the potential for PGC-1α and Tfam to translocate to SS and IMF mitochondria in human subjects, and performed experiments in rodents to identify signalling mechanisms regulating these translocation events. Acute exercise in humans and rats increased PGC-1α content in SS but not IMF mitochondria. Acute exposure to 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside in rats recapitulated the exercise effect of increased PGC-1α protein within SS mitochondria only, suggesting that AMP-activated protein kinase (AMPK) signalling is involved. In addition, rendering AMPK inactive (AMPK kinase dead mice) prevented exercise-induced PGC-1α translocation to SS mitochondria, further suggesting that AMPK plays an integral role in these translocation events. In contrast to the conserved PGC-1α translocation to SS mitochondria across species (humans, rats and mice), acute exercise only increased mitochondrial Tfam in rats. Nevertheless, in rat resting muscle PGC-1α and Tfam co-immunoprecipate with α-tubulin, suggesting a common cytosolic localization. These data suggest that exercise causes translocation of PGC-1α preferentially to SS mitochondria in an AMPK-dependent manner.

Intestinal dysbiosis contributes to obesity and insulin resistance, but intervening with antibiotics, prebiotics, or probiotics can be limited by specificity or sustained changes in microbial composition. Postbiotics include bacterial components such as lipopolysaccharides, which have been shown to promote insulin resistance during metabolic endotoxemia. We found that bacterial cell wall-derived muramyl dipeptide (MDP) is an insulin-sensitizing postbiotic that requires NOD2. Injecting MDP lowered adipose inflammation and reduced glucose intolerance in obese mice without causing weight loss or altering the composition of the microbiome. MDP reduced hepatic insulin resistance during obesity and low-level endotoxemia. NOD1-activating muropeptides worsened glucose tolerance. IRF4 distinguished opposing glycemic responses to different types of peptidoglycan and was required for MDP/NOD2-induced insulin sensitization and lower metabolic tissue inflammation during obesity and endotoxemia. IRF4 was dispensable for exacerbated glucose intolerance via NOD1. Mifamurtide, an MDP-based drug with orphan drug status, was an insulin sensitizer at clinically relevant doses in obese mice.

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.

Carnitine palmitoyltransferase I has been viewed historically as the sole regulator of fatty acid oxidation. However, we have identified fatty acid translocase/CD36 as an additional control point. Specifically, fatty acid translocase/CD36 seems to have a novel dual mechanism of action with regard to fatty acid oxidation during exercise, influencing transport of lipids across the sarcolemmal membrane and into the mitochondria.

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. 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.

The association betweentrans-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 then-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.

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.

The maintenance of blood glucose involves the coordination of multiple organ systems. Two new studies indicate that metformin and resveratrol activate metabolic sensors in the duodenum and initiate a neural loop that reduces liver glucose production in rat models of type 2 diabetes.

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.

Mitochondrial dysfunction and reactive oxygen species (ROS) have been implicated in the aetiology of skeletal muscle insulin resistance, although there is considerable controversy regarding these concepts. Mitochondrial function has been traditionally assessed in the presence of saturating ADP, but ATP turnover and the resultant ADP is thought to limit respiration in vivo. Therefore, we investigated the potential link between submaximal ADP-stimulated respiration rates, ROS generation and skeletal muscle insulin sensitivity in a model of type 2 diabetes mellitus, the ZDF rat. Utilizing permeabilized muscle fibres we observed that submaximal ADP-stimulated respiration rates (250–2000 μm ADP) were lower in ZDF rats than in lean controls, which coincided with decreased adenine nucleotide translocase 2 (ANT2) protein content. This decrease in submaximal ADP-stimulated respiration occurred in the absence of a decrease in electron transport chain function. Treating ZDF rats with resveratrol improved skeletal muscle insulin resistance and this was associated with elevated submaximal ADP-stimulated respiration rates as well as an increase in ANT2 protein content. These results coincided with a greater ability of ADP to attenuate mitochondrial ROS emission and an improvement in cellular redox balance. Together, these data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for mitochondrial ROS emission.

Published values regarding the sensitivity (IC50) of CPT-I (carnitine palmitoyltransferase I) to M-CoA (malonyl-CoA) inhibition in isolated mitochondria are inconsistent with predicted in vivo rates of fatty acid oxidation. Therefore we have re-examined M-CoA inhibition kinetics under various P-CoA (palmitoyl-CoA) concentrations in both isolated mitochondria and PMFs (permeabilized muscle fibres). PMFs have an 18-fold higher IC50 (0.61 compared with 0.034 μM) in the presence of 25 μM P-CoA and a 13-fold higher IC50 (6.3 compared with 0.49 μM) in the presence of 150 μM P-CoA compared with isolated mitochondria. M-CoA inhibition kinetics determined in PMFs predicts that CPT-I activity is inhibited by 33% in resting muscle compared with >95% in isolated mitochondria. Additionally, the ability of M-CoA to inhibit CPT-I appears to be dependent on P-CoA concentration, as the relative inhibitory capacity of M-CoA is decreased with increasing P-CoA concentrations. Altogether, the use of PMFs appears to provide an M-CoA IC50 that better reflects the predicted in vivo rates of fatty acid oxidation. These findings also demonstrate that the ratio of [P-CoA]/[M-CoA] is critical for regulating CPT-I activity and may partially rectify the in vivo disconnect between M-CoA content and CPT-I flux within the context of exercise and Type 2 diabetes.