Adenosine Monophosphate Kinase Activation Research Articles

Vitamin D3 mitigates myopathy and metabolic dysfunction in rats with metabolic syndrome: the potential role of dipeptidyl peptidase-4.

Metabolic syndrome is associated with vitamin D3 deficiency. This work aims to examine the efficacy of vitamin D3 in inhibiting MetS-induced myopathy and to determine whether the beneficial effects of vitamin D3 are mediated by the inhibition of dipeptidyl peptidase-4 (DPP-4). An in silico study investigated the potential effectiveness of vitamin D3 on the inhibition of the DPP-4 enzyme. An in vitro assay of the DPP-4 inhibitory effect of vitamin D3 was performed. In vivo and over 12 weeks, both diet (with 3% salt) and drinking water (with 10% fructose) were utilized to induce MetS. In the seventh week, rats received either vitamin D3, vildagliptin, a combination of both, or vehicles. Serum lipids, adipokines, glycemic indices, and glucagon-like peptide-1 (GLP-1), muscular glucose transporter type-4 (GLUT-4) content, DPP-4, adenosine monophosphate kinase (AMPK) activities, and Sudan Black B-stained lipids were assessed. Muscular reactive oxygen species (ROS), caspase-3, and desmin immunostaining were used to determine myopathy. MetS-induced metabolic dysfunction was ameliorated by vitamin D3, which also reduced intramuscular glycogen and lipid accumulation. This is demonstrated by the attenuation of MetS-induced myopathy by vitamin D3, decreased oxidative stress, increased desmin immuno-expression, and caspase-3 activity. Our in silico data demonstrated that vitamin D3 is capable of inhibiting DPP-4, which is further supported by biochemical findings. Vitamin D3 increased serum GLP-1, muscular AMPK activity, and GLUT-4 content, whereas the levels of muscular ROS were decreased in MetS. Vildagliptin and its combination with vitamin D3 yielded comparable results. It is suggested that the DPP-4 inhibitory potential of vitamin D3 is responsible for the amelioration of MetS-induced metabolic changes and myopathy.

Maternal AMPK pathway activation with uterine artery blood flow and fetal growth maintenance during hypoxia.

High-altitude (HA) hypoxia lowers uterine artery (UtA) blood flow during pregnancy and birth weight. Adenosine monophosphate kinase (AMPK) activation has selective, uteroplacental vasodilator effects that lessen hypoxia-associated birth weight reductions. In this study, we determined the relationship between AMPK-pathway gene expression and metabolites in the maternal circulation during HA pregnancy as well as with the maintenance of UtA blood flow and birth weight at HA. Residents at HA (2,793 m) versus low altitude (LA; 1,640 m) had smaller UtA diameters at weeks 20 and 34, lower UtA blood flow at week 20, and lower birth weight babies. At week 34, women residing at HA versus women residing at LA had decreased expression of upstream and downstream AMPK-pathway genes. Expression of the α1-AMPK catalytic subunit, PRKAA1, correlated positively with UtA diameter and blood flow at weeks 20 (HA) and 34 (LA). Downstream AMPK-pathway gene expression positively correlated with week 20 fetal biometry at both altitudes and with UtA diameter and birth weight at LA. Reduced gene expression of AMPK activators and downstream targets in women residing at HA versus women residing at LA, together with positive correlations between PRKAA1 gene expression, UtA diameter, and blood flow suggest that greater sensitivity to AMPK activation at midgestation at HA may help offset later depressant effects of hypoxia on fetal growth.NEW & NOTEWORTHY Fetal growth restriction (FGR) is increased and uterine artery (UtA) blood flow is lower at high altitudes (HA) but not all HA pregnancies have FGR. Here we show that greater UtA diameter and blood flow at week 20 are positively correlated with higher expression of the gene encoding the α1-catalytic subunit of AMP protein kinase, PRKAA1, suggesting that increased AMPK activation may help to prevent the detrimental effects of chronic hypoxia on fetal growth.

Role of SELENBP1 and SELENOF in prostate cancer bioenergetics

The contribution of selenium and selenoproteins in prostate cancer etiology remains elusive, potentially due to insufficient information regarding the biochemical pathways in which they are involved. There are twenty-five human selenocysteine-containing proteins or selenoproteins as well as a smaller class of selenium-containing proteins that do not include selenocysteine, and their cancer-associated aberrations, both genetic and functional, have evoked special interest, although their contribution to the metabolic reprogramming of prostate cancers remains has not been extensively studied. While benign prostate tissue exhibits a glycolytic phenotype, neoplastic events restore the truncated tricarboxylic acid cycle and enhance oxidative phosphorylation. Two selenium-containing proteins, selenium binding protein 1 and selenoprotein F, affect prostate cancer phenotypes by modulating tumor cell metabolic profiles with significant effects on mitochondrial biology, including oxidative phosphorylation and ATP synthesis. One of the pathways affected by both proteins is the activation of adenosine monophosphate kinase and its downstream signaling with concomitant induction of glycolysis. This review focuses on highlighting the role of these two proteins in modulating the bioenergetic profile of prostate cancer and in maintaining the metabolic plasticity of these cells rendering growth advantage and possible therapeutic resistance.

Metformin: A Possible Option in Cancer Chemotherapy.

Metformin has been used for a long time as an antidiabetic medication for type 2 diabetes. It is used either as a monotherapy or in combination with other antidiabetic medications. The drug came into prominence in diabetes and other conditions with cardiovascular risk after the landmark study of 1995 by the United Kingdom Prospective Diabetes Study which emphasized its importance. However, the drug has been used in experimental trials in various aspects of medicine and pharmacology such as in reproductive medicine, cancer chemotherapy, metabolic diseases, and neurodegenerative diseases. It has been in use in the treatment of polycystic ovarian disease and obesity and is being considered in type 1 diabetes. This study seeks to evaluate the relevance of metformin in cancer management. Different mechanisms have been proposed for its antitumor action which involves the following: (a) the activation of adenosine monophosphate kinase, (b) modulation of adenosine A1 receptor (ADORA), (c) reduction in insulin/insulin growth factors, and (d) the role of metformin in the inhibition of endogenous reactive oxygen species (ROS); and its resultant damage to deoxyribonucleic acid (DNA) molecule is another paramount antitumor mechanism.

Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system.

BackgroundHepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver.ObjectiveIdentifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system.AnimalsLiver organoids obtained from 6 cats.MethodsEight different drugs were tested, and the 2 most promising were further studied using a quantitative TAG assay, lipid droplet staining, and qPCR.ResultsBoth T863 (a diacylglycerol O‐acyltransferase 1 [DGAT1] inhibitor) and 5‐aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside (AICAR; an adenosine monophosphate kinase activator) decreased TAG accumulation by 55% (P < .0001) and 46% (P = .0003), respectively. Gene expression of perilipin 2 (PLIN2) increased upon the addition of FFAs to the medium and decreased upon treatment with AICAR but not significantly after treatment with T863.Conclusions and Clinical ImportanceTwo potential drugs useful in the treatment of hepatic lipidosis in cats were identified. The drug T863 inhibits DGAT1, indicating that DGAT1 is the primary enzyme responsible for TAG synthesis from external fatty acids in cat organoids. The drug AICAR may act as a lipid‐lowering compound via decreasing PLIN2 mRNA. Liver organoids can be used as an in vitro tool for drug testing in a species‐specific system and provide the basis for further clinical testing of drugs to treat steatosis.

Exercise improves outcomes of surgery on fatty liver and can be substituted through AMPK activation - an experimental study

Background: Steatosis is a serious risk factor for liver surgery and transplantation, mostly because of its impact on ischemic tolerance (IT) and regenerative capacity (RC) of the liver. Exercise is most effective in counteracting steatosis, but how it affects IT and RC is unclear. Compound mimetics of physical activity are of major interest for patients who are unable to comply with exercise. Methods: A standardized mouse model of diet-induced steatosis was used. Obese mice were subjected for four weeks to regular exercise or treatment with AICAR, an activator of the key energy sensor induced through exercise, AMPK (adenosine monophosphate kinase). Mice were then exposed to hepatic ischemia or hepatectomy. Results: Exercise activated AMPK, normalized metabolic parameters, and improved IT and RC of fatty liver. Similar, albeit smaller, improvements were noted for lean liver, indicating steatosis-independent effects of exercising. AICAR treatment had an impact on fatty liver akin to exercise. The power of AMPK activation in improving surgical outcomes was demonstrated in an established model of the small-for-size-syndrome, with exercising raising survival from 20 to 80%. Conclusion: Any safe intervention reducing hepatic fat accumulation and improving IT and RC is most welcome in liver surgery. Exercising is powerful but its application is limited. Our findings suggest that pharmacological AMPK activation is a promising strategy to sustain the surgical benefits of exercise in the absence of compliance issues.

Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts.

Hypertonicity increases water permeability, independently of vasopressin, in the inner medullary collecting duct (IMCD) by increasing aquaporin-2 (AQP2) membrane accumulation. We investigated whether protein kinase C (PKC) and adenosine monophosphate kinase (AMPK) are involved in hypertonicity-regulated water permeability. Increasing perfusate osmolality from 150 to 290 mosmol/kgH2O and bath osmolality from 290 to 430 mosmol/kgH2O significantly stimulated osmotic water permeability. The PKC inhibitors chelerythrine (10 µM) and rottlerin (50 µM) significantly reversed the increase in osmotic water permeability stimulated by hypertonicity in perfused rat terminal IMCDs. Chelerythrine significantly increased phosphorylation of AQP2 at S261 but not at S256. Previous studies show that AMPK is stimulated by osmotic stress. We tested AMPK phosphorylation under hypertonic conditions. Hypertonicity significantly increased AMPK phosphorylation in inner medullary tissues. Blockade of AMPK with Compound C decreased hypertonicity-stimulated water permeability but did not alter phosphorylation of AQP2 at S256 and S261. AICAR, an AMPK stimulator, caused a transient increase in osmotic water permeability and increased phosphorylation of AQP2 at S256. When inner medullary tissue was treated with the PKC activator phorbol dibutyrate (PDBu), the AMPK activator metformin, or both, AQP2 phosphorylation at S261 was decreased with PDBu or metformin alone, but there was no additive effect on phosphorylation with PDBu and metformin together. In conclusion, hypertonicity regulates water reabsorption by activating PKC. Hypertonicity-stimulated water reabsorption by PKC may be related to the decrease in endocytosis of AQP2. AMPK activation promotes water reabsorption, but the mechanism remains to be determined. PKC and AMPK do not appear to act synergistically to regulate water reabsorption.

Dapagliflozin Attenuates Na+/H+ Exchanger-1 in Cardiofibroblasts via AMPK Activation.

We assessed whether the SGLT-2 inhibitor dapagliflozin (Dapa) attenuates the upregulation of the cardiac Na+/H+ exchanger (NHE-1) in vitro in mouse cardiofibroblasts stimulated with lipopolysaccharides (LPS) and whether this effect is dependent on adenosine monophosphate kinase (AMPK) activation. Mouse cardiofibroblasts were exposed for 16h to Dapa (0.4μM), AMPK activator (A769662 (10μM)), AMPK inhibitor (compound C (CC) (10μM)), an SGLT-1 and SGLT-2 inhibitor (phlorizin (PZ) (100μM)), Dapa+CC, or Dapa+PZ, and then stimulated with LPS (10ng/ml) for 3h. NHE-1 mRNA levels were assessed by rt-PCR and total AMPK, phosphorylated-AMPK (P-AMPK), NHE-1, and heat shock protein-70 (Hsp70) protein levels in the whole cell lysate by immunoblotting. In addition, NHE-1 protein levels attached to Hsp70 were assessed by immunoprecipitation. Exposure to LPS significantly reduced P-AMPK levels in the cardiofibroblasts. A769662 and Dapa equally increased P-AMPK. The effect was blocked by CC. Phlorizin had no effect on P-AMPK. LPS exposure significantly increased NHE-1 mRNA levels. Both Dapa and A769662 equally attenuated this increase. The effect of Dapa was blocked with CC. Interestingly, none of the compounds significantly affected NHE-1 and Hsp70 protein levels in the whole cell lysate. However, LPS significantly increased the concentration of NHE-1 attached to Hsp70. Both Dapa and A69662 attenuated this association and CC blocked the effect of Dapa. Again, phlorizin had no effect and did not alter the effect of Dapa. Dapa increases P-AMPK in cardiofibroblasts exposed to LPS. Dapa attenuated the increase in NHE-1 mRNA and the association between NHE-1 and Hsp70. This effect was dependent on AMPK.

CRISPR correction of the PRKAG2 gene mutation in the patient's induced pluripotent stem cell-derived cardiomyocytes eliminates electrophysiological and structural abnormalities

Mutations in the PRKAG2 gene encoding the γ-subunit of adenosine monophosphate kinase (AMPK) cause hypertrophic cardiomyopathy (HCM) and familial Wolff-Parkinson-White (WPW) syndrome. Patients carrying the R302Q mutation in PRKAG2 present with sinus bradycardia, escape rhythms, ventricular preexcitation, supraventricular tachycardia, and atrioventricular block. This mutation affects AMPK activity and increases glycogen storage in cardiomyocytes. The link between glycogen storage, WPW syndrome, HCM, and arrhythmias remains unknown. The purpose of this study was to investigate the pathological changes caused by the PRKAG2 mutation. We tested the hypothesis that patient's induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) display clinical aspects of the disease. Using clustered regularly interspaced short palindromic repeats (CRISPR) technology, we corrected the mutation and then generated isogenic iPSC-CMs. Action potentials were recorded from spontaneously firing and paced cardiomyocytes using the patch clamp technique. Using a microelectrode array setup, we recorded electrograms from iPSC-CMs clusters. Transmission electron microscopy was used to detect ultrastructural abnormalities in the mutated iPSC-CMs. PRKAG2-mutated iPSC-CMs exhibited abnormal firing patterns, delayed afterdepolarizations, triggered arrhythmias, and augmented beat rate variability. Importantly, CRISPRcorrection eliminated the electrophysiological abnormalities, the augmented glycogen, storage, and cardiomyocyte hypertrophy. PRKAG2-mutated iPSC-CMs displayed functional and structural abnormalities, which were abolished by correcting the mutation in the patient's iPSCs using CRISPR technology.

Quercetin, a Lead Compound against Type 2 Diabetes Ameliorates Glucose Uptake via AMPK Pathway in Skeletal Muscle Cell Line.

Herein we investigated the molecular mechanism of action of the citrus flavonoid, quercetin in skeletal muscle cells (L6 myotubes). Taking advantage of protein kinase inhibitors, we proved that the effect of quercetin on 2-NBDG uptake in L6 myotubes was not through insulin signaling pathway, but through adenosine monophosphate kinase (AMPK) pathway and its downstream target p38 MAPK. An increase in the cellular AMP to ATP ratio on pretreatment may account for AMPK activation which was coupled with a transient change in mitochondrial membrane potential. In addition, quercetin triggered a rise in intracellular calcium suggesting that calcium-calmodulin mediated protein kinase (CaMKK) may also be involved. Quercetin shared a similar mechanism with the well-known drug metformin, highlighting it as a promising compound for the management of type 2 diabetes. The AMPK signaling pathway could contribute to correction of insulin resistance through bypassing the insulin-regulated system for GLUT4 translocation.

Glucose deprivation regulates the progranulin-sortilin axis in PC12 cells.

Progranulin (PGRN) is a growth factor implicated in several neurodegenerative diseases, such as frontotemporal lobar degeneration. Despite its important role in the central nervous system (CNS), the mechanisms controlling PGRN expression in the CNS are largely unknown. Recent evidence, however, suggested that several stressors, such as hypoxia, acidosis, or oxidative stress, induce PGRN expression. The present study was mainly aimed at determining whether and, if so, how glucose deprivation affects PGRN expression in PC12 cells. Initially, it was found that glucose deprivation gradually induced PGRN gene expression in PC12 cells. To elucidate the underlying molecular mechanisms, several intracellular signalings that were modified in response to glucose deprivation were examined. Both adenosine monophosphate kinase (AMPK) activation and changes in osmotic pressure, which are modified by extracellular glucose concentration, had no effect on PGRN gene expression; on the other hand, p38 activation in response to glucose deprivation played an important role in inducing PGRN gene expression. It was also found that expression of sortilin, a PGRN receptor implicated in PGRN endocytosis, was dramatically reduced by glucose deprivation. In contrast to glucose‐dependent regulation of PGRN gene expression, AMPK activation played a central role in reducing sortilin expression. Overall, the present study suggests that the PGRN–sortilin axis is modulated by glucose deprivation via two distinct mechanisms. As PGRN is neuroprotective, this system may represent a new neuroprotective mechanism activated by glucose deprivation in the CNS.

Erratum to: Adenosine Monophosphate-Activated Protein Kinase Abates Hyperglycaemia-Induced Neuronal Injury in Experimental Models of Diabetic Neuropathy: Effects on Mitochondrial Biogenesis, Autophagy and Neuroinflammation.

Impaired adenosine monophosphate kinase (AMPK) signalling under hyperglycaemic conditions is known to cause mitochondrial dysfunction in diabetic sensory neurons. Facilitation of AMPK signalling is previously reported to ameliorate inflammation and induce autophagic response in various complications related to diabetes. The present study assesses the role of AMPK activation on mitochondrial biogenesis, autophagy and neuroinflammation in experimental diabetic neuropathy (DN) using an AMPK activator (A769662). A769662 (15 and 30 mg/kg, i.p) was administered to Sprague–Dawley rats (250–270 g) for 2 weeks after 6 weeks of streptozotocin (STZ) injection (55 mg/kg, i.p.). Behavioural parameters (mechanical/thermal hyperalgesia) and functional characteristics (motor/sensory nerve conduction velocities (MNCV and SNCV) and sciatic nerve blood flow (NBF)) were assessed. For in vitro studies, Neuro2a (N2A) cells were incubated with 25 mM glucose to simulate high glucose condition and then studied for mitochondrial dysfunction and protein expression changes. STZ administration resulted in significant hyperglycaemia (>250 mg/dl) in rats. A769662 treatment significantly improved mechanical/thermal hyperalgesia threshold and enhanced MNCV, SNCV and NBF in diabetic animals. A769662 exposure normalised the mitochondrial superoxide production, membrane depolarisation and markedly increased neurite outgrowth of N2A cells. Further, AMPK activation also abolished the NF-κB-mediated neuroinflammation. A769662 treatment increased Thr-172 phosphorylation of AMPK results in stimulated PGC-1α-directed mitochondrial biogenesis and autophagy induction. Our study supports that compromised AMPK signalling in hyperglycaemic conditions causes defective mitochondrial biogenesis ultimately leading to neuronal dysfunction and associated deficits in DN and activation of AMPK can be developed as an attractive therapeutic strategy for the management of DN.

A high-fat jelly diet restores bioenergetic balance and extends lifespan in the presence of motor dysfunction and lumbar spinal cord motor neuron loss in TDP-43A315T mutant C57BL6/J mice.

ABSTRACTTransgenic transactivation response DNA-binding protein 43 (TDP-43) mice expressing the A315T mutation under control of the murine prion promoter progressively develop motor function deficits and are considered a new model for the study of amyotrophic lateral sclerosis (ALS); however, premature sudden death resulting from intestinal obstruction halts disease phenotype progression in 100% of C57BL6/J congenic TDP-43A315T mice. Similar to our recent results in SOD1G93A mice, TDP-43A315T mice fed a standard pellet diet showed increased 5′ adenosine monophosphate-activated protein kinase (AMPK) activation at postnatal day (P)80, indicating elevated energetic stress during disease progression. We therefore investigated the effects of a high-fat jelly diet on bioenergetic status and lifespan in TDP-43A315T mice. In contrast to standard pellet-fed mice, mice fed high-fat jelly showed no difference in AMPK activation up to P120 and decreased phosphorylation of acetly-CoA carboxylase (ACC) at early-stage time points. Exposure to a high-fat jelly diet prevented sudden death and extended survival, allowing development of a motor neuron disease phenotype with significantly decreased body weight from P80 onward that was characterised by deficits in Rotarod abilities and stride length measurements. Development of this phenotype was associated with a significant motor neuron loss as assessed by Nissl staining in the lumbar spinal cord. Our work suggests that a high-fat jelly diet improves the pre-clinical utility of the TDP-43A315T model by extending lifespan and allowing the motor neuron disease phenotype to progress, and indicates the potential benefit of this diet in TDP-43-associated ALS.

Early glycogen synthase kinase-3β and protein phosphatase 2A independent tau dephosphorylation during global brain ischaemia and reperfusion following cardiac arrest and the role of the adenosine monophosphate kinase pathway.

Abnormal tau phosphorylation (p‐tau) has been shown after hypoxic damage to the brain associated with traumatic brain injury and stroke. As the level of p‐tau is controlled by Glycogen Synthase Kinase (GSK)‐3β, Protein Phosphatase 2A (PP2A) and Adenosine Monophosphate Kinase (AMPK), different activity levels of these enzymes could be involved in tau phosphorylation following ischaemia. This study assessed the effects of global brain ischaemia/reperfusion on the immediate status of p‐tau in a rat model of cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We reported an early dephosphorylation of tau at its AMPK sensitive residues, Ser396 and Ser262after 2 min of ischaemia, which did not recover during the first two hours of reperfusion, while the tau phosphorylation at GSK‐3β sensitive but AMPK insensitive residues, Ser202/Thr205 (AT8), as well as the total amount of tau remained unchanged. Our data showed no alteration in the activities of GSK‐3β and PP2A during similar episodes of ischaemia of up to 8 min and reperfusion of up to 2 h, and 4 weeks recovery. Dephosphorylation of AMPK followed the same pattern as tau dephosphorylation during ischaemia/reperfusion. Catalase, another AMPK downstream substrate also showed a similar pattern of decline to p‐AMPK, in ischaemic/reperfusion groups. This suggests the involvement of AMPK in changing the p‐tau levels, indicating that tau dephosphorylation following ischaemia is not dependent on GSK‐3β or PP2A activity, but is associated with AMPK dephosphorylation. We propose that a reduction in AMPK activity is a possible early mechanism responsible for tau dephosphorylation.

Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts.

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations.

Adenosine Monophosphate-Activated Protein Kinase Abates Hyperglycaemia-Induced Neuronal Injury in Experimental Models of Diabetic Neuropathy: Effects on Mitochondrial Biogenesis, Autophagy and Neuroinflammation.

Impaired adenosine monophosphate kinase (AMPK) signalling under hyperglycaemic conditions is known to cause mitochondrial dysfunction in diabetic sensory neurons. Facilitation of AMPK signalling is previously reported to ameliorate inflammation and induce autophagic response in various complications related to diabetes. The present study assesses the role of AMPK activation on mitochondrial biogenesis, autophagy and neuroinflammation in experimental diabetic neuropathy (DN) using an AMPK activator (A769662). A769662 (15 and 30mg/kg, i.p) was administered to Sprague-Dawley rats (250-270g) for 2weeks after 6weeks of streptozotocin (STZ) injection (55mg/kg, i.p.). Behavioural parameters (mechanical/thermal hyperalgesia) and functional characteristics (motor/sensory nerve conduction velocities (MNCV and SNCV) and sciatic nerve blood flow (NBF)) were assessed. For in vitro studies, Neuro2a (N2A) cells were incubated with 25mM glucose to simulate high glucose condition and then studied for mitochondrial dysfunction and protein expression changes. STZ administration resulted in significant hyperglycaemia (>250mg/dl) in rats. A769662 treatment significantly improved mechanical/thermal hyperalgesia threshold and enhanced MNCV, SNCV and NBF in diabetic animals. A769662 exposure normalised the mitochondrial superoxide production, membrane depolarisation and markedly increased neurite outgrowth of N2A cells. Further, AMPK activation also abolished the NF-κB-mediated neuroinflammation. A769662 treatment increased Thr-172 phosphorylation of AMPK results in stimulated PGC-1α-directed mitochondrial biogenesis and autophagy induction. Our study supports that compromised AMPK signalling in hyperglycaemic conditions causes defective mitochondrial biogenesis ultimately leading to neuronal dysfunction and associated deficits in DN and activation of AMPK can be developed as an attractive therapeutic strategy for the management of DN.

Glycolytic potential and activity of adenosine monophosphate kinase (AMPK), glycogen phosphorylase (GP) and glycogen debranching enzyme (GDE) in steer carcasses with normal (< 5.8) or high (> 5.9) 24 h pH determined in M. longissimus dorsi

Muscle glycogen concentration (MGC) and lactate (LA), activity of glycogen debranching enzyme (GDE), glycogen phosphorylase (GP) and adenosine monophosphate kinase (AMPK) were determined at 0.5h (T0) and 24h (T24) post-mortem in Longissimus dorsi samples from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24h postmortem. MGC, LA and glycolytic potential were higher (P<0.05) in normal pH carcasses. GDE activity was similar (P>0.05) in both pH categories. GP activity increased between T0 and T24 only in normal pH carcasses. AMPK activity was four times higher in normal pH v/s high pH carcasses, without changing its activity over time. Results reinforce the idea that differences in postmortem glycogenolytic/glycolytic flow in L. dorsi of steers showing normal v/s high muscle pH at 24h, could be explained not only by the higher initial MGC in normal pH carcasses, but also by a high and sustained activity of AMPK and an increased GP activity at 24h postmortem.

Stilbene Analogs of Resveratrol Improve Insulin Resistance through Activation of AMPK

Resveratrol (RSV), 3,5,4'-trihydroxy-trans-stilbene, is known to have many beneficial physiological activities. We have synthesized several stilbene analogues and have reported that the hydroxyl group in the 4' position of RSV exhibited strong radical scavenging action. Using stilbene analogs, we investigated the structure of RSV to explain its protective effect against obesity and type 2 diabetes. All six analogs used in this study inhibited the differentiation of 3T3-L1 adipocytes. 3-Hydroxy-trans stilbene (3(OH)ST), and 3,4'-dihydroxy-trans stilbene (3,4'(OH)2ST) increased glucose uptake and induced adenosine monophosphate kinase (AMPK) phosphorylation in C2C12 myotubes independently of insulin. An in vivo study using mice fed high-fat diets indicated that 3(OH)ST was more effective than RSV in improving insulin resistance. In conclusion, RSV and its derivatives, particularly 3(OH)ST, inhibited adipocyte differentiation and enhanced glucose uptake in the myotubes, resulting in a reduction of obesity and an improvement in glucose tolerance in vivo.

Effect of metformin on the urinary metabolites of diet-induced-obese mice studied by ultra performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-TOF/MS)

Obesity is becoming a health concern worldwide and metformin, a first line anti-diabetic drug, was associated with weight loss under different backgrounds. However, most researches focused on the anti-diabetic mechanism and less attention has been paid on the mechanism of weight loss of metformin. Therefore, we established a metabonomic method to evaluate metformin action in preventing obesity in a high fat diet-induced-obesity (DIO) mice model. 36 male C57BL/6 mice (8-week old) were randomly divided into control group (n=12, normal chow), model group (n=12, high fat chow) and metformin group (n=12, high fat chow and dosed with metformin) over 16 weeks. A urinary metabonomic study using UPLC-TOF/MS was performed in combination with multivariate statistical analysis. In addition, indices of body weight and food intake as well as fasting blood glucose, fed blood glucose, oral glucose tolerance test (OGTT) and plasma insulin were collected. Significant weight loss in metformin-treated mice was achieved and 21 potential biomarkers were identified. Decreased glucose, myristic acid, stearidonic acid, lysoPC (16:0), lysoPC (18:0), L-glutamic acid, L-methionine, L-threonine, L-phenylalanine, L-histidine, L-carnitine, L-malic acid and pantothenic acid in urine indicated that metformin may have exerted effects on energy metabolism. Further, based on the biomarkers, we cautiously propose that tricarboxylic acid cycle (TCA) may have been compromised by metformin and might contribute to the activation of adenosine monophosphate kinase (AMPK), then AMPK activation led to more β-oxidation of certain fatty acids and augmented lipolysis and thus induced weight loss. Related cellular and molecular studies are being considered to further investigate the underlying mechanism.

Abstract 44: Restoration of Angiogenic Balance and Improved Endothelial Cell Function via Ampk Activation is Hypoxia and Ischemia Dependent in Trophoblast Cells and Pregnant Rats

Previous studies indicate restoration of angiogenic balance lowers blood pressure and improves vascular endothelial cell function in several experimental models of preeclampsia. Stimulation of the adenosine monophosphate kinase (AMPK) pathway is known to increase VEGF expression. We hypothesized stimulation of AMPK with aminoimidazole carboxamide ribonucleotide (AICAR) would increase plasma VEGF, improve endothelial cell (EC) function and attenuate reduced uterine perfusion pressure (RUPP)-induced hypertension. RUPP was induced on day 14 of gestation (term=21), AMPK activation was stimulated in vivo by AICAR administered i.p. (50mg/kg) twice daily. Arterial pressure (AP) and tissues were collected on day 19. Human derived trophoblast cells were cultured in physiologic normoxic (8% O 2 ) and hypoxic (1.5% O 2 ) conditions. Angiogenic balance and EC function were assessed with an endothelial tube formation assay using human umbilical vascular endothelial cells (HUVEC) cultured for 8h with 5% serum from each treatment group. AICAR increased ( P &lt;0.05) placental phosphorylation of AMPKα in RUPP+AICAR (0.33±0.06) compared to normal pregnant (NP), NP+A and RUPP rats (0.19±0.03 vs. 0.16±0.04 vs. 0.18±0.03). AICAR increased ( P &lt;0.05) free plasma VEGF in the RUPP+AICAR (884±128 pg/ml) compared to NP+AICAR, NP and RUPP rats (708±179 vs. 843±52 vs. 420±75 pg/ml). EC function as assessed by tube formation was decreased by treatment with serum from RUPP vs . NP (25±2 vs. 51±7; tubes/frame; P &lt;0.05) rats and this deficit was abrogated with serum from RUPP+AICAR rats (86±9 tubes/frame; P &lt;0.05). RUPP hypertension was mitigated by AICAR infusion (NP 95±3; RUPP 123±2; NP+AICAR 104±3; RUPP+AICAR 101±5mmHg; P &lt;0.05). Hypoxia increased ( P &lt;0.05) VEGF secretion 30% by BeWo cells and this effect was augmented a further 60% ( P &lt;0.05) by treatment with the AICAR mimetic metformin. AICAR increased levels of placental AMPK activation, increased free VEGF concentrations in plasma, restored endothelial cell function and reduced blood pressure RUPP rats but had no effect on these measures in NP control rats. These findings show AMPK activation and stimulation of VEGF with AICAR is ischemia/hypoxia dependent and may be useful for management of hypertension in pregnancy.