New insights into activation and function of the AMPK.

The classical role of AMP-activated protein kinase (AMPK) is as a cellular energy sensor activated by falling energy status, signalled by increases in AMP to ATP and ADP to ATP ratios. Once activated, AMPK acts to restore energy homeostasis by promoting ATP-producing catabolic pathways while inhibiting energy-consuming processes. In this Review, we provide an update on this canonical (AMP/ADP-dependent) activation mechanism, but focus mainly on recently described non-canonical pathways, including those by which AMPK senses the availability of glucose, glycogen or fatty acids and by which it senses damage to lysosomes and nuclear DNA. We also discuss new findings on the regulation of carbohydrate and lipid metabolism, mitochondrial and lysosomal homeostasis, and DNA repair. Finally, we discuss the role of AMPK in cancer, obesity, diabetes, nonalcoholic steatohepatitis (NASH) and other disorders where therapeutic targeting may exert beneficial effects.

GDF15 promotes weight loss by enhancing energy expenditure in muscle.

Caloric restriction that promotes weight loss is an effective strategy for treating non-alcoholic fatty liver disease and improving insulin sensitivity in people with type 2 diabetes1. Despite its effectiveness, in most individuals, weight loss is usually not maintained partly due to physiological adaptations that suppress energy expenditure, a process known as adaptive thermogenesis, the mechanistic underpinnings of which are unclear2,3. Treatment of rodents fed a high-fat diet with recombinant growth differentiating factor 15 (GDF15) reduces obesity and improves glycaemic control through glial-cell-derived neurotrophic factor family receptor α-like (GFRAL)-dependent suppression of food intake4-7. Here we find that, in addition to suppressing appetite, GDF15 counteracts compensatory reductions in energy expenditure, eliciting greater weight loss and reductions in non-alcoholic fatty liver disease (NAFLD) compared to caloric restriction alone. This effect of GDF15 to maintain energy expenditure during calorie restriction requires a GFRAL-ß-adrenergic-dependent signalling axis that increases fatty acid oxidation and calcium futile cycling in the skeletal muscle of mice. These data indicate that therapeutic targeting of the GDF15-GFRAL pathway may be useful for maintaining energy expenditure in skeletal muscle during caloric restriction.

Restoration of the ER stress response protein TDAG51 in hepatocytes mitigates NAFLD in mice.

Endoplasmic reticulum stress is associated with insulin resistance and the development of nonalcoholic fatty liver disease. Deficiency of the endoplasmic reticulum stress response T-cell death-associated gene 51 (TDAG51) (TDAG51-/-) in mice promotes the development of high-fat diet (HFD)-induced obesity, fatty liver, and hepatic insulin resistance. However, whether this effect is due specifically to hepatic TDAG51 deficiency is unknown. Here, we report that hepatic TDAG51 protein levels are consistently reduced in multiple mouse models of liver steatosis and injury as well as in liver biopsies from patients with liver disease compared to normal controls. Delivery of a liver-specific adeno-associated virus (AAV) increased hepatic expression of a TDAG51-GFP fusion protein in WT, TDAG51-/-, and leptin-deficient (ob/ob) mice. Restoration of hepatic TDAG51 protein was sufficient to increase insulin sensitivity while reducing body weight and fatty liver in HFD fed TDAG51-/- mice and in ob/ob mice. TDAG51-/- mice expressing ectopic TDAG51 display improved Akt (Ser473) phosphorylation, post-insulin stimulation. HFD-fed TDAG51-/- mice treated with AAV-TDAG51-GFP displayed reduced lipogenic gene expression, increased beta-oxidation and lowered hepatic and serum triglycerides, findings consistent with reduced liver weight. Further, AAV-TDAG51-GFP-treated TDAG51-/- mice exhibited reduced hepatic precursor and cleaved sterol regulatory-element binding proteins (SREBP-1 and SREBP-2). In vitro studies confirmed the lipid-lowering effect of TDAG51 overexpression in oleic acid-treated Huh7 cells. These studies suggest that maintaining hepatic TDAG51 protein levels represents a viable therapeutic approach for the treatment of obesity and insulin resistance associated with nonalcoholic fatty liver disease.

The phosphorylation of AMPKß1 is critical for increasing autophagy and maintaining mitochondrial homeostasis in response to fatty acids.

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 ß1 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 AMPKß1 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 AMPKß1 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.

Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy.

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5' AMP-activated protein kinase (AMPKα1/α2/ß2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite-binding site.

AMP-activated protein kinase (AMPK) is a central energy sensor that coordinates the response to energy challenges to maintain cellular ATP levels. AMPK is a potential therapeutic target for treating metabolic disorders, and several direct synthetic activators of AMPK have been developed that show promise in preclinical models of type 2 diabetes. These compounds have been shown to regulate AMPK through binding to a novel allosteric drug and metabolite (ADaM)-binding site on AMPK, and it is possible that other molecules might similarly bind this site. Here, we performed a high-throughput screen with natural plant compounds to identify such direct allosteric activators of AMPK. We identified a natural plant dihydrophenathrene, Lusianthridin, which allosterically activates and protects AMPK from dephosphorylation by binding to the ADaM site. Similar to other ADaM site activators, Lusianthridin showed preferential activation of AMPKß1-containing complexes in intact cells and was unable to activate an AMPKß1 S108A mutant. Lusianthridin dose-dependently increased phosphorylation of acetyl-CoA carboxylase in mouse primary hepatocytes, which led to a corresponding decrease in de novo lipogenesis. This ability of Lusianthridin to inhibit lipogenesis was impaired in hepatocytes from ß1 S108A knock-in mice and mice bearing a mutation at the AMPK phosphorylation site of acetyl-CoA carboxylase 1/2. Finally, we show that activation of AMPK by natural compounds extends to several analogs of Lusianthridin and the related chemical series, phenanthrenes. The emergence of natural plant compounds that regulate AMPK through the ADaM site raises the distinct possibility that other natural compounds share a common mechanism of regulation.

A novel direct adenosine monophosphate kinase activator ameliorates disease progression in preclinical models of Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) mainly results from mutations in the PKD1 gene, which encodes polycystin 1. It is the most common inherited kidney disease and is characterized by a progressive bilateral increase in cyst number and size, often leading to kidney failure. The cellular energy sensor and regulator adenosine monophosphate stimulated protein kinase (AMPK) has been implicated as a promising new therapeutic target. To address this hypothesis, we determined the effects of a potent and selective clinical stage direct allosteric AMPK activator, PXL770, in canine and patient-derived 3D cyst models and an orthologous mouse model of ADPKD. PXL770 induced AMPK activation and dose-dependently reduced cyst growth in principal-like Madin-Darby Canine Kidney cells stimulated with forskolin and kidney epithelial cells derived from patients with ADPKD stimulated with desmopressin. In an inducible, kidney epithelium-specific Pkd1 knockout mouse model, PXL770 produced kidney AMPK pathway engagement, prevented the onset of kidney failure (reducing blood urea by 47%), decreased cystic index by 26% and lowered the kidney weight to body weight ratio by 35% compared to untreated control Pkd1 knockout mice. These effects were accompanied by a reduction of markers of cell proliferation (-48%), macrophage infiltration (-53%) and tissue fibrosis (-37%). Thus, our results show the potential of direct allosteric AMPK activation in the treatment of ADPKD and support the further development of PXL770 for this indication.

Inhibition of basal and glucagon-induced hepatic glucose production by 991 and other pharmacological AMPK activators.

Pharmacological AMPK activation represents an attractive approach for the treatment of type 2 diabetes (T2D). AMPK activation increases skeletal muscle glucose uptake, but there is controversy as to whether AMPK activation also inhibits hepatic glucose production (HGP) and pharmacological AMPK activators can have off-target effects that contribute to their anti-diabetic properties. The main aim was to investigate the effects of 991 and other direct AMPK activators on HGP and determine whether the observed effects were AMPK-dependent. In incubated hepatocytes, 991 substantially decreased gluconeogenesis from lactate, pyruvate and glycerol, but not from other substrates. Hepatocytes from AMPKß1-/- mice had substantially reduced liver AMPK activity, yet the inhibition of glucose production by 991 persisted. Also, the glucose-lowering effect of 991 was still seen in AMPKß1-/- mice subjected to an intraperitoneal pyruvate tolerance test. The AMPK-independent mechanism by which 991 treatment decreased gluconeogenesis could be explained by inhibition of mitochondrial pyruvate uptake and inhibition of mitochondrial sn-glycerol-3-phosphate dehydrogenase-2. However, 991 and new-generation direct small-molecule AMPK activators antagonized glucagon-induced gluconeogenesis in an AMPK-dependent manner. Our studies support the notion that direct pharmacological activation of hepatic AMPK as well as inhibition of pyruvate uptake could be an option for the treatment of T2D-linked hyperglycemia.

Prostate-Specific Membrane Antigen (PSMA) Expression Predicts Need for Early Treatment in Prostate Cancer Patients Managed with Active Surveillance.

Metabolic dysregulation is an early event in carcinogenesis. Here, we examined the expression of enzymes involved in de novo lipogenesis (ATP-citrate lyase: ACLY), glucose uptake (Glucose Transporter 1: GLUT1), and folate-glutamate metabolism (Prostate-Specific Membrane Antigen: PSMA) as potential biomarkers of risk for early prostate cancer progression. Patients who were managed initially on active surveillance with a Gleason score of 6 or a low-volume Gleason score of 7 (3 + 4) were accrued from a prostate cancer diagnostic assessment program. Patients were asked to donate their baseline diagnostic biopsy tissues and permit access to their clinical data. PSMA, GLUT1, and ACLY expression were examined with immunohistochemistry (IHC) in baseline biopsies, quantitated by Histologic Score for expression in benign and malignant glands, and compared with patient time remaining on active surveillance (time-on-AS). All three markers showed trends for elevated expression in malignant compared to benign glands, which was statistically significant for ACLY. On univariate analysis, increased PSMA and GLUT1 expression in malignant glands was associated with shorter time-on-AS (HR: 5.06, [CI 95%: 1.83-13.94] and HR: 2.44, [CI 95%: 1.10-5.44], respectively). Malignant ACLY and benign gland PSMA and GLUT1 expression showed non-significant trends for such association. On multivariate analysis, overexpression of PSMA in malignant glands was an independent predictor of early PC progression (p = 0.006). This work suggests that the expression of metabolic enzymes determined by IHC on baseline diagnostic prostate biopsies may have value as biomarkers of risk for rapid PC progression. PSMA may be an independent predictor of risk for progression and should be investigated further in systematic studies.

Increased gut serotonin production in response to bisphenol A structural analogs may contribute to their obesogenic effects.

Obesogens are synthetic, environmental chemicals that can disrupt endocrine control of metabolism and contribute to the risk of obesity and metabolic disease. Bisphenol A (BPA) is one of the most studied obesogens. There is considerable evidence that BPA exposure is associated with weight gain, increased adiposity, poor blood glucose control, and nonalcoholic fatty liver disease in animal models and human populations. Increased usage of structural analogs of BPA has occurred in response to legislation banning their use in some commercial products. However, BPA analogs may also cause some of the same metabolic impairments because of common mechanisms of action. One key effector that is altered by BPA and its analogs is serotonin, however, it is unknown if BPA-induced changes in peripheral serotonin pathways underlie metabolic perturbations seen with BPA exposure. Upon ingestion, BPA and its analogs act as endocrine-disrupting chemicals in the gastrointestinal tract to influence serotonin production by the gut, where over 95% of serotonin is produced. The purpose of this review is to evaluate how BPA and its analogs alter gut serotonin regulation and then discuss how disruption of serotonergic networks influences host metabolism. We also provide evidence that BPA and its analogs enhance serotonin production in gut enterochromaffin cells. Taken together, we propose that BPA and many BPA analogs represent endocrine-disrupting chemicals that can influence host metabolism through the endogenous production of gut-derived factors, such as serotonin.

AMPK mediates energetic stress-induced liver GDF15.

Growth differentiating factor-15 (GDF15) is an emerging target for the treatment of obesity and metabolic disease partly due to its ability to suppress food intake. GDF15 expression and secretion are thought to be regulated by a cellular integrated stress response, which involves endoplasmic reticulum (ER) stress. AMPK is another cellular stress sensor, but the relationship between AMPK, ER stress, and GDF15 has not been assessed in vivo. Wildtype (WT), AMPK ß1 deficient (AMPKß1-/- ), and CHOP-/- mice were treated with three distinct AMPK activators; AICAR, which is converted to ZMP mimicking the effects of AMP on the AMPKγ isoform, R419, which indirectly activates AMPK through inhibition of mitochondrial respiration, or A769662, a direct AMPK activator which binds the AMPKß1 isoform ADaM site causing allosteric activation. Following treatments, liver Gdf15, markers of ER-stress, AMPK activity, adenine nucleotides, circulating GDF15, and food intake were assessed. AICAR and R419 caused ER and energetic stress, increased GDF15 expression and secretion, and suppressed food intake. Direct activation of AMPK ß1 containing complexes by A769662 increased hepatic Gdf15 expression, circulating GDF15, and suppressed food intake, independent of ER stress. The effects of AICAR, R419, and A769662 on GDF15 were attenuated in AMPKß1-/- mice. AICAR and A769662 increased GDF15 to a similar extent in WT and CHOP-/- mice. Herein, we provide evidence that AMPK plays a role in mediating the induction of GDF15 under conditions of energetic stress in mouse liver in vivo.

AMPK activation by SC4 inhibits noradrenaline-induced lipolysis and insulin-stimulated lipogenesis in white adipose tissue.

The effects of small-molecule AMP-activated protein kinase (AMPK) activators in rat epididymal adipocytes were compared. SC4 was the most effective and submaximal doses of SC4 and 5-amino-4-imidazolecarboxamide (AICA) riboside were combined to study the effects of AMPK activation in white adipose tissue (WAT). Incubation of rat adipocytes with SC4 + AICA riboside inhibited noradrenaline-induced lipolysis and decreased hormone-sensitive lipase (HSL) Ser563 phosphorylation, without affecting HSL Ser565 phosphorylation. Preincubation of fat pads from wild-type (WT) mice with SC4 + AICA riboside inhibited insulin-stimulated lipogenesis from glucose or acetate and these effects were lost in AMPKα1 knockout (KO) mice, indicating AMPKα1 dependency. Moreover, in fat pads from acetyl-CoA carboxylase (ACC)1/2 S79A/S212A double knockin versus WT mice, the effect of SC4 + AICA riboside to inhibit insulin-stimulated lipogenesis from acetate was lost, pinpointing ACC as the main AMPK target. Treatment with SC4 + AICA riboside decreased insulin-stimulated glucose uptake, an effect that was still observed in fat pads from AMPKα1 KO versus WT mice, suggesting the effect was partly AMPKα1-independent. SC4 + AICA riboside treatment had no effect on the insulin-induced increase in palmitate esterification nor on sn-glycerol-3-phosphate-O-acyltransferase activity. Therefore in WAT, AMPK activation inhibits noradrenaline-induced lipolysis and suppresses insulin-stimulated lipogenesis primarily by inactivating ACC and by inhibiting glucose uptake.

The gut microbiome regulates host glucose homeostasis via peripheral serotonin.

The gut microbiome is an established regulator of aspects of host metabolism, such as glucose handling. Despite the known impacts of the gut microbiota on host glucose homeostasis, the underlying mechanisms are unknown. The gut microbiome is also a potent mediator of gut-derived serotonin synthesis, and this peripheral source of serotonin is itself a regulator of glucose homeostasis. Here, we determined whether the gut microbiome influences glucose homeostasis through effects on gut-derived serotonin. Using both pharmacological inhibition and genetic deletion of gut-derived serotonin synthesis, we find that the improvements in host glucose handling caused by antibiotic-induced changes in microbiota composition are dependent on the synthesis of peripheral serotonin.

Impact of pesticide exposure on adipose tissue development and function.

Obesity is a leading cause of morbidity, mortality and health care expenditure whose incidence is rapidly rising across the globe. Although the cause of the obesity epidemic is typically viewed as a product of an increased availability of high calorie foods and/or a reduction in physical activity, there is mounting evidence that exposure to synthetic chemicals in our environment may play an important role. Pesticides, are a class of chemicals whose widespread use has coincided with the global rise of obesity over the past two decades. Importantly, given their lipophilic nature many pesticides have been shown to accumulate with adipose tissue depots, suggesting they may be disrupting the function of white adipose tissue (WAT), brown adipose tissue (BAT) and beige adipose tissue to promote obesity and metabolic diseases such as type 2 diabetes. In this review, we discuss epidemiological evidence linking pesticide exposure with body mass index (BMI) and the incidence of diabetes. We then review preclinical studies in rodent models which have directly evaluated the effects of different classes of insecticides and herbicides on obesity and metabolic dysfunction. Lastly, we review studies conducted in adipose tissue cells lines and the purported mechanisms by which pesticides may induce alterations in adipose tissue function. The review of the literature reveals major gaps in our knowledge regarding human exposure to pesticides and our understanding of whether physiologically relevant concentrations promote obesity and elicit alterations in key signaling pathways vital for maintaining adipose tissue metabolism.

Effects of PKB/Akt inhibitors on insulin-stimulated lipogenesis and phosphorylation state of lipogenic enzymes in white adipose tissue.

We investigated acute effects of two allosteric protein kinase B (PKB) inhibitors, MK-2206 and Akti-1/2, on insulin-stimulated lipogenesis in rat epididymal adipocytes incubated with fructose as carbohydrate substrate. In parallel, the phosphorylation state of lipogenic enzymes in adipocytes and incubated epididymal fat pads was monitored by immunoblotting. Preincubation of rat epididymal adipocytes with PKB inhibitors dose-dependently inhibited the following: insulin-stimulated lipogenesis, increased PKB Ser473 phosphorylation, increased PKB activity and decreased acetyl-CoA carboxylase (ACC) Ser79 phosphorylation. In contrast, the effect of insulin to decrease the phosphorylation of pyruvate dehydrogenase (PDH) at Ser293 and Ser300 was not abolished by PKB inhibition. Insulin treatment also induced ATP-citrate lyase (ACL) Ser454 phosphorylation, but this effect was less sensitive to PKB inhibitors than ACC dephosphorylation by insulin. In incubated rat epididymal fat pads, Akti-1/2 treatment reversed insulin-induced ACC dephosphorylation, while ACL phosphorylation by insulin was maintained. ACL and ACC purified from white adipose tissue were poor substrates for PKBα in vitro. However, effects of wortmannin and torin, along with Akti-1/2 and MK-2206, on recognized PKB target phosphorylation by insulin were similar to their effects on insulin-induced ACL phosphorylation, suggesting that PKB could be the physiological kinase for ACL phosphorylation by insulin. In incubated epididymal fat pads from wild-type versus ACC1/2 S79A/S212A knockin mice, effects of insulin to increase lipogenesis from radioactive fructose or from radioactive acetate were reduced but not abolished. Together, the results support a key role for PKB in mediating insulin-stimulated lipogenesis by decreasing ACC phosphorylation, but not by decreasing PDH phosphorylation.

The SGLT2 inhibitor canagliflozin suppresses lipid synthesis and interleukin-1 beta in ApoE deficient mice.

Sodium-glucose cotransporter 2 inhibitors such as canagliflozin lower blood glucose and reduce cardiovascular events in people with type 2 diabetes through mechanisms that are not fully understood. Canagliflozin has been shown to increase the activity of the AMP-activated protein kinase (AMPK), a metabolic energy sensor important for increasing fatty acid oxidation and energy expenditure and suppressing lipogenesis and inflammation, but whether AMPK activation is important for mediating some of the beneficial metabolic effects of canagliflozin has not been determined. We, therefore, evaluated the effects of canagliflozin in female ApoE-/- and ApoE-/-AMPK ß1-/- mice fed a western diet. Canagliflozin increased fatty acid oxidation and energy expenditure and lowered adiposity, blood glucose and the respiratory exchange ratio independently of AMPK ß1. Canagliflozin also suppressed liver lipid synthesis and the expression of ATP-citrate lyase, acetyl-CoA carboxylase and sterol response element-binding protein 1c independently of AMPK ß1. Canagliflozin lowered circulating IL-1ß and studies in bone marrow-derived macrophages indicated that in contrast with the metabolic adaptations, this effect required AMPK ß1. Canagliflozin had no effect on the size of atherosclerotic plaques in either ApoE-/- and ApoE-/-AMPK ß1-/- mice. Future studies investigating whether reductions in liver lipid synthesis and macrophage IL-1ß are important for the cardioprotective effects of canagliflozin warrant further investigation.

Sevoflurane-induced hyperglycemia is attenuated by salsalate in obese insulin-resistant mice.

PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

RéSUMé: OBJECTIF: L'hyperglycémie périopératoire est fréquente et est associée à une morbidité significative. Bien que les caractéristiques propres au patient et à la chirurgie influencent le métabolisme périopératoire du glucose, les anesthésiques ont un impact significatif. Nous avons émis l'hypothèse que l'hyperglycémie en réponse à une anesthésie à base de sévoflurane serait plus prononcée chez des souris obèses et insulino-résistantes que chez des souris témoins maigres. Nous avons en outre émis l'hypothèse que l'hyperglycémie induite par le sévoflurane serait atténuée par un prétraitement au salsalate. MéTHODE: Des souris mâles C57BL/6J maigres et obèses ont été anesthésiées avec du sévoflurane pendant 60 min avec ou sans prétraitement de 62,5 mg·kg−1 de salsalate. La glycémie, l'insuline plasmatique et l'absorption glycémique ont été mesurées dans différents tissus. RéSULTATS: Sous une anesthésie au sévoflurane, les souris obèses ont affiché une glycémie plus élevée que les souris maigres. Des augmentations de glucose sanguin ont été atténuées lors d'un prétraitement aigu à base de salsalate à 60 min sous anesthésie chez les souris obèses (moyenne ± erreur-type sur la moyenne [ETM], delta glycémique; véhicule 5,79 ± 1,09 vs salsalate 1,91 ± 1,32 mM, P = 0,04), mais elles n'étaient pas statistiquement significative chez les souris maigres (delta glycémique, véhicule 4,39 ± 0,55 vs salsalate 2,79 ± 0,71 mM; P = 0,10). Cet effet était indépendant des changements de l'insuline mais associé à une augmentation d'environ 1,7 fois de l'absorption glycémique dans les tissus adipeux bruns (véhicule 45,28 ± 4,57 vs salsalate 76,89 ± 12,23 µmol·g−1 tissu·h−1; P < 0,001). CONCLUSION: Ces données montrent que le salsalate peut réduire l'hyperglycémie induite par le sévoflurane chez la souris. Ceci indique que le salsalate pourrait constituer une nouvelle classe d'agents thérapeutiques qui, en plus de leurs propriétés anti-inflammatoires et analgésiques, pourraient être utiles pour réduire l'hyperglycémie périopératoire.

The citrus flavonoid nobiletin confers protection from metabolic dysregulation in high-fat-fed mice independent of AMPK.

Obesity, dyslipidemia, and insulin resistance, the increasingly common metabolic syndrome, are risk factors for CVD and type 2 diabetes that warrant novel therapeutic interventions. The flavonoid nobiletin displays potent lipid-lowering and insulin-sensitizing properties in mice with metabolic dysfunction. However, the mechanisms by which nobiletin mediates metabolic protection are not clearly established. The central role of AMP-activated protein kinase (AMPK) as an energy sensor suggests that AMPK is a target of nobiletin. We tested the hypothesis that metabolic protection by nobiletin required phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in mouse hepatocytes, in mice deficient in hepatic AMPK (Ampkß1-/-), in mice incapable of inhibitory phosphorylation of ACC (AccDKI), and in mice with adipocyte-specific AMPK deficiency (iß1ß2AKO). We fed mice a high-fat/high-cholesterol diet with or without nobiletin. Nobiletin increased phosphorylation of AMPK and ACC in primary mouse hepatocytes, which was associated with increased FA oxidation and attenuated FA synthesis. Despite loss of ACC phosphorylation in Ampkß1-/- hepatocytes, nobiletin suppressed FA synthesis and enhanced FA oxidation. Acute injection of nobiletin into mice did not increase phosphorylation of either AMPK or ACC in liver. In mice fed a high-fat diet, nobiletin robustly prevented obesity, hepatic steatosis, dyslipidemia, and insulin resistance, and it improved energy expenditure in Ampkß1-/-, AccDKI, and iß1ß2AKO mice to the same extent as in WT controls. Thus, the beneficial metabolic effects of nobiletin in vivo are conferred independently of hepatic or adipocyte AMPK activation. These studies further underscore the therapeutic potential of nobiletin and begin to clarify possible mechanisms.

AMPK-ACC signaling modulates platelet phospholipids and potentiates thrombus formation.

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation.

Two isoprenylated flavonoids from Dorstenia psilurus activate AMPK, stimulate glucose uptake, inhibit glucose production and lower glycemia.

Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze-thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKß1-/- mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.