Subunit Of AMPK Research Articles

PHARMACODYNAMIC OF METFORMIN IN TYPE 2 DIABETES MELLITUS PATIENTS: REVIEW OF THE PRKAA2 AND SLC22A3 GENES

Type 2 diabetes mellitus (type 2 DM) is a disease including on metabolic disorder that characterized by an increase in glucose levels above normal. This glucose increase is caused by reduced insulin function which can be in the form of abnormalities in insulin secretion, impaired insulin action in peripheral tissues, or both. Metformin is one of the recommended therapies for type 2 DM. AMPK is the main pathway of metformin's mechanism. AMPKa2 encoded by PRKAA2 gene is an AMPK subunit that plays an important role in AMPK activation. OCT3 encoded by SLC22A3 plays a role in the metformin transport mechanism. OCT3 is needed in the metformin pharmacokinetic like absorption and elimination, this process determines metformin bioavailability, clearance, and its pharmacological effects. Mutations in PRKAA2 and SLC22A3 cause variations in the pharmacodynamic effects and pharmacokinetic of metformin in individuals directly. These changes will ultimately affect the effectiveness of metformin in type 2 DM patients.

Autophagy gene expression in skeletal muscle of older individuals is associated with physical performance, muscle volume and mitochondrial function in the study of muscle, mobility and aging (SOMMA).

Autophagy is essential for proteostasis, energetic balance, and cell defense and is a key pathway in aging. Identifying associations between autophagy gene expression patterns in skeletal muscle and physical performance outcomes would further our knowledge of mechanisms related with proteostasis and healthy aging. Muscle biopsies were obtained from participants in the Study of Muscle, Mobility, and Aging (SOMMA). For 575 participants, RNA was sequenced and expression of 281 genes related to autophagy regulation, mitophagy, and mTOR/upstream pathways was determined. Associations between gene expression and outcomes including mitochondrial respiration in muscle fiber bundles (MAX OXPHOS), physical performance (VO2 peak, 400 m walking speed, and leg power), and thigh muscle volume, were determined using negative binomial regression models. For autophagy, key transcriptional regulators including TFE3 and NFKB-related genes (RELA, RELB, and NFKB1) were negatively associated with outcomes. On the contrary, regulators of oxidative metabolism that also promote overall autophagy, mitophagy, and pexophagy (PPARGC1A, PPARA, and EPAS1) were positively associated with multiple outcomes. In line with this, several mitophagy, fusion, and fission-related genes (NIPSNAP2, DNM1L, and OPA1) were also positively associated with outcomes. For mTOR pathway and related genes, expression of WDR59 and WDR24, both subunits of GATOR2 complex (an indirect inhibitor of mTORC1), and PRKAG3, which is a regulatory subunit of AMPK, were negatively correlated with multiple outcomes. Our study identifies autophagy and selective autophagy such as mitophagy gene expression patterns in human skeletal muscle related to physical performance, muscle volume, and mitochondrial function in older persons which may lead to target identification to preserve mobility and independence.

AMPKα2 promotes tumor immune escape by inducing CD8+ T-cell exhaustion and CD4+ Treg cell formation in liver hepatocellular carcinoma

BackgroundAdenosine monophosphate-activated protein kinase (AMPK) is associated with the development of liver hepatocellular carcinoma (LIHC). AMPKα2, an α2 subunit of AMPK, is encoded by PRKAA2, and functions as the catalytic core of AMPK. However, the role of AMPKα2 in the LIHC tumor immune environment is unclear.MethodsRNA-seq data were obtained from the Cancer Genome Atlas and Genotype-Tissue Expression databases. Using the single-cell RNA-sequencing dataset for LIHC obtained from the China National Genebank Database, the communication between malignant cells and T cells in response to different PRKAA2 expression patterns was evaluated. In addition, the association between PRKAA2 expression and T-cell evolution during tumor progression was explored using Pseudotime analysis, and the role of PRKAA2 in metabolic reprogramming was explored using the R “scMetabolis” package. Functional experiments were performed in LIHC HepG2 cells.ResultsAMPK subunits were expressed in tissue-specific and substrate-specific patterns. PRKAA2 was highly expressed in LIHC tissues and was associated with poor patient prognosis. Tumors with high PRKAA2 expression displayed an immune cold phenotype. High PRKAA2 expression significantly promoted LIHC immune escape. This result is supported by the following evidence: 1) the inhibition of major histocompatibility complex class I (MHC-I) expression through the regulation of interferon-gamma activity in malignant cells; 2) the promotion of CD8+ T-cell exhaustion and the formation of CD4+ Treg cells in T cells; 3) altered interactions between malignant cells and T cells in the tumor immune environment; and 4) induction of metabolic reprogramming in malignant cells.ConclusionsOur study indicate that PRKAA2 may contribute to LIHC progression by promoting metabolic reprogramming and tumor immune escape through theoretical analysis, which offers a theoretical foundation for developing PRKAA2-based strategies for personalized LIHC treatment.

ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA.

Tuberculosis has the highest mortality rate worldwide for a chronic infectious disease caused by a single pathogen. RNA-binding proteins (RBPs) are involved in autophagy - a key defense mechanism against Mycobacterium tuberculosis (M. tuberculosis) infection - by modulating RNA stability and forming intricate regulatory networks. However, the functions of host RBPs during M. tuberculosis infection remain relatively unexplored. Zinc finger NFX1-type containing 1 (ZNFX1), a conserved RBP critically involved in immune deficiency diseases and mycobacterial infections, is significantly upregulated in M. tuberculosis-infected macrophages. Here, we aimed to explore the immunoregulatory functions of ZNFX1 during M. tuberculosis infection. We observed that Znfx1 knockout markedly compromised the multifaceted immune responses mediated by macrophages. This compromise resulted in reduced phagocytosis, suppressed macrophage activation, increased M. tuberculosis burden, progressive lung tissue injury, and chronic inflammation in M. tuberculosis-infected mice. Mechanistic investigations revealed that the absence of ZNFX1 inhibited autophagy, consequently mediating immune suppression. ZNFX1 critically maintained AMPK-regulated autophagic flux by stabilizing protein kinase AMP-activated catalytic subunit alpha 2 mRNA, which encodes a key catalytic α subunit of AMPK, through its zinc finger region. This process contributed to M. tuberculosis growth suppression. These findings reveal a function of ZNFX1 in establishing anti-M. tuberculosis immune responses, enhancing our understanding of the roles of RBPs in tuberculosis immunity and providing a promising approach to bolster antituberculosis immunotherapy.

Loss of tubulin tyrosine ligase, a novel feature of sporadic and familial AD, is an enhancer of tau phosphorylation

AbstractBackgroundWe have recently shown that loss of tubulin tyrosine ligase (TTL) and subsequent disruption of a‐tubulin retyrosination is a feature of AD and a promoter of synaptic loss by inhibiting microtubule entries into dendritic spines. TTL+/‐ mice have cognitive deficits, altered LTP, synapse loss and increased levels of detyrosinated and D2 tubulin, two tubulin modifications that accumulate on non‐dynamic microtubules. We also found that low levels of phospho‐tau correlated with higher levels of detyrosinated and D2 tubulin in pyramidal neurons residing in the anterior hippocampal formation of human AD brains, raising the question of whether improper microtubule longevity drives early stages of tau hyperphosphorylation in AD. Here, we demonstrate that loss of TTL enhances both AMPK activation and tau phosphorylation in cultured rat hippocampal and human cortical neurons, and present preliminary evidence on the mechanisms by which this regulation may occur and contribute to TTL‐loss dependent synaptic dysfunction.MethodHuman cortical neurons derived from iPSC lines in which the London familial APP mutation V717I was knocked into one allele of the IMR90 control using CRISPR/Cas9, were used as a cellular model of familial AD. Lentiviral infection with shRNA against TTL was used to silence TTL expression. Microtubule dynamics were measured by confocal time‐lapse microscopy in neurons expressing EB3‐EGFP, a protein that tracks the growing plus ends of microtubules. Immunoblotting was performed to measure both unmodified and modified levels of TLL, tubulins, AMPK subunits, total tau, p262 and AT8 phospho‐tau.ResultIn the APP neurons, we found that a reduction in TTL levels correlated with an increase in p262/AT8 phospho‐tau variants and phospho‐AMPK, the active state of one of the kinases that phosphorylates serine 262, a key residue in the binding of tau to microtubules. Knockdown of TTL was sufficient to reduce microtubule dynamics and increase both active AMPK levels and phospho‐tau.ConclusionOur results indicate that loss of TTL alone reduces dynamic microtubules, triggers tau kinase activation, and results in hyperphosphorylated tau. Together with our previously published data, these findings suggest that reduced levels of TTL act as one of the early drivers of tau hyperphosphorylation in AD.

HSP90 inhibition in the mouse spinal cord enhances opioid signaling by suppressing an AMPK-mediated negative feedback loop.

Opioids and other agonists of the μ-opioid receptor are effective at managing acute pain, but their chronic use can lead to tolerance that limits their efficacy. We previously reported that inhibiting the chaperone protein HSP90 in the spinal cords of mice promotes the antinociceptive effects of opioids in a manner that involved increased activation of the kinase ERK. Here, we found that the underlying mechanism involves the relief of a negative feedback loop mediated by the kinase AMPK. Intrathecal treatment of male and female mice with the HSP90 inhibitor 17-AAG decreased the abundance of the β1 subunit of AMPK in the spinal cord. The antinociceptive effects of 17-AAG with morphine were suppressed by intrathecal administration of AMPK activators and enhanced by an AMPK inhibitor. Opioid treatment increased the abundance of phosphorylated AMPK in the dorsal horn of the spinal cord, where it colocalized with a neuronal marker and the neuropeptide CGRP. Knocking down AMPK in CGRP-positive neurons enhanced the antinociceptive effects of morphine and demonstrated that AMPK mediated the signal transduction between HSP90 inhibition and ERK activation. These data suggest that AMPK mediates an opioid-induced negative feedback loop in CGRP neurons of the spinal cord and that this loop can be disabled by HSP90 inhibition to enhance the efficacy of opioids.

Effect of heat and hypoxia stress on mitochondrion and energy metabolism in the gill of hard clam

Aquatic animals suffer from heat and hypoxia stress more frequently due to global climate change and other anthropogenic activities. Heat and hypoxia stress can significantly affect mitochondrial function and energy metabolism. Here, the response and adaptation characteristics of mitochondria and energy metabolism in the gill of the hard clam Mercenaria mercenaria under heat (35 °C), hypoxia (0.2 mg/L), and heat plus hypoxia stress (35 °C, 0.2 mg/L) after 48 h exposure were investigated. Mitochondrial membrane potentials were depolarized under environmental stress. Mitochondrial fusion, fission and mitophagy played a key role in maintain mitochondrion function. The AMPK subunits showed different expression under environmental stress. Acceleration of enzyme activities (phosphofructokinase, pyruvate kinase and lactic dehydrogenase) and accumulation of anaerobic metabolites in glycolysis and TCA cycle implied that the anaerobic metabolism might play a key role in providing energy. Accumulation of amino acids might help to increase tolerance under heat and heat combined hypoxia stress. In addition, urea cycle played a key role in amino acid metabolism to prevent ammonia/nitrogen toxicity. This study improved our understanding of the mitochondrial and energy metabolism responses of marine bivalves exposed to environmental stress.

Effects of dietary berberine on growth performance, lipid metabolism, antioxidant capacity and lipometabolism-related genes expression of AMPK signaling pathway in juvenile black carp (Mylopharyngodon piceus) fed high-fat diets.

This study aimed to investigate the effects of high-fat diet (HFD) supplemented with berberine on growth, lipid metabolism, antioxidant capacity and lipometabolism-related genes expression of AMPK signaling pathway in juvenile black carp (Mylopharyngodon piceus). Five hundred and forty healthy fish (4.04 ± 0.01g) were randomly distributed into six groups, and fed six experimental diets: normal-fat diet (NFD, 5% fat), HFD (15% fat), and four HFDs supplemented with graded levels of berberine, respectively. The results showed that, compared with fish fed NFD, HFD had no effects on the growth of fish except for reducing survival rate, whereas HFD caused extensive lipid accumulation, oxidative stress injury and hepatic abnormalities. However, compared with the HFD group, fish fed HFD containing an appropriate berberine (98.26 or 196.21mg/kg) improved the growth performance, increased hepatic lipid metabolism and antioxidant enzymes activities, and up-regulated the mRNA expression levels of ampk subunits and lipolysis genes such as pparα, cpt-1, acox, atgl and hsl (P < 0.05). Meanwhile, HFD supplemented with an appropriate berberine reduced crude lipid contents in liver and whole-body, decreased serum lipid contents, and ALT and AST activities, and down-regulated the mRNA expression levels of lipogenesis genes such as srebp-1, acc1, gpat, fas and pparγ, and lipid transporter genes such as fatp, fabp and fat/cd36 (P < 0.05). Thus, HFD supplemented with an appropriate berberine could improve growth of black carp, promote lipid metabolism and enhance antioxidant capacity. The lipid-lowering mechanism of berberine might be mediated by activating AMPK pathway, up-regulating lipolysis genes expression, and down-regulating lipogenesis and transport genes expression.

Arginine Reduces Glycation in γ2 Subunit of AMPK and Pathologies in Alzheimer's Disease Model Mice.

We studied the effect of glycated γ2 subunit of AMPK on its activity in N2a cells. In 3 × Tg mice, we administrated L-arginine once every two days for 45 days and evaluated the glycation level of γ2 subunit and function of AMPK and alternation of pathologies. The glycation level of γ2 subunit was significantly elevated in 3 × Tg mice as compared with control mice, meanwhile, the level of pT172-AMPK was obviously lower in 3 × Tg mice than that in control mice. Moreover, we found that arginine protects the γ2 subunit of AMPK from glycation, preserves AMPK function, and improves pathologies and cognitive deficits in 3 × Tg mice. Arginine treatment decreases glycated γ2 subunit of AMPK and increases p-AMPK levels in 3 × Tg mice, suggesting that reduced glycation of the γ2 subunit could ameliorate AMPK function and become a new target for AD therapy in the future.

Molecular cloning, expression and appetite regulation function of adiponectin in Siberian sturgeon (Acipenser baerii)

Adiponectin (AdipoQ) as an adipocytokine has the potential to regulate feeding behavior, but the information about adipoq in fish is limited. In this study, Siberian sturgeon adiponectin (Ssadipoq) gene was cloned encoding 264 amino acids. The amino acid identity of SsAdipoQ was low compared with that of mammals, birds, amphibians and teleost fishes. The expression of Ssadipoq in the hypothalamus was significantly decreased at 1 h and 3 h post feeding, and increased after 15-day fasting. The mature domain of AdipoQ (fAd) was inserted into expression vector pET32a and successfully expressed in Escherichia coli BL21 (DE3) after stimulated by isopropyl-β-d-thiogalactoside. Food intake at 1 h and 3 h post treatment with SsfAd protein decreased significantly (P < 0.05). The mRNA expression of pyy and cck in the valvula intestine was promoted and hypothalamic npy, agrp and pomc mRNA expression were inhibited after treatment with SsfAd protein. Furthermore, hypothalamic ampk subunits expression was associated with peripheral SsfAd treatment. In summary, present study indicate that SsfAd plays an important role in the regulation of food intake and appetite signals in Siberian sturgeon, which provides a basis for further study application of prokaryotic AdipoQ in feeding behavior regulation.

Increased Connexin 43 expression in AMPK deficient cardiomyocytes in response to Doxorubicin treatment

ContextDoxorubicin (DOX) is an anthracycline anticancer drug that can cause dose‐dependent cardiomyopathy and chronic heart failure. DOX cardiotoxicity has been attributed to excess production of ROS and genotoxicity that leads to cell death. AMPK is a kinase composed of α, β and γ subunits. AMPK has been proposed to modulate DOX‐induced cardiotoxicity, but the downstream mediators or effectors remain unclear. Connexin 43 (Cx43) is a gap junction protein abundantly expressed in the cardiomyocyte and is essential for heart function. Studies have suggested a role for Cx43 in DOX cardiotoxicity, but it remains unknown whether AMPK interacts with Cx43 to regulate cardiac response to DOX chemotherapy.ObjectiveTo determine the role of AMPK in the regulation of the Cx43 in response to DOX‐induced cardiotoxicity.MethodsH9c2 rat cardiomyocytes were seeded on 6‐well plates and cultured in DMEM with 6.5% fetal bovine serum. To investigate the role of AMPK, the cells were transfected with siRNAs targeting different AMPK isoforms using Lipofectamine RNAiMAX and Opti‐MEM I reduced serum medium. After 48 hours of transfection, half of the wells received 0.5 µM DOX treatment, and the other half received equal amounts of saline. Protein samples were collected 16 hours later using the Cell lysis buffer. Western blot analysis was performed to confirm the knockdown of AMPK isoforms and to determine the protein expression levels of Cx43.ResultsOur findings showed that siRNA transfections effectively reduced the protein levels of AMPK isoform a1 and a2, respectively, as indicated by the Western blot analysis. DOX treatment increased the expression levels of the Cx43 in cardiomyocytes. Also, knockdown (KD) of AMPKα1 isoform resulted in a similar increase in Cx43 expression levels as did DOX, but AMPKα2 KD did not significantly alter Cx43 levels, suggesting an AMPKa1 specific effect on Cx43 expression. Interestingly, AMPKα1 KD, together with DOX treatment, did not produce a synergistic effect to increase Cx43 expression levels further, suggesting the possibility that DOX might have elevated the Cx43 expression through its effect on AMPK.ConclusionOur results confirmed the previous studies showing that treatment of the cardiomyocytes with DOX increased the expression of Cx43. However, our investigations produced a novel finding that knocking down AMPKα1 also increased the Cx43 but did not enhance the effect of DOX on Cx43 expression, suggesting a role for AMPK in the pathway that regulates the Cx43 to mediate the DOX‐induced cardiotoxicity. Our investigations also showed an isoform‐specific effect of AMPK on Cx43 expression since only α1 but not α2 KD increased Cx43 levels. Whether the increase in Cx43 is due to increased gene expression or inhibited protein degradation remains to be determined. Further investigation is also warranted to elucidate the roles of other AMPK subunits or isoforms in regulating the Connexin 43 expression in the context of doxorubicin cardiotoxicity.

Can aerobic exercise really be a 'warm-up' for brown adipose tissue?

Can aerobic exercise really be a 'warm-up' for brown adipose tissue?

Metformin-induced AMPK activation suppresses larval growth and molting probably by disrupting 20E synthesis and glycometabolism in fall webworm, Hyphantria cunea Drury

Metformin, considered to be a potent AMPK activator, is widely used for clinical therapy of cancer and diabetes due to its distinct function in regulating cell energy balance and body metabolism. However, the effect of metformin-induced AMPK activation on the growth and development of insects remains largely unknown. In the present study, we focused on the role of metformin in regulating the growth and development of Hyphantria cunea, a notorious defoliator in the forestry. Firstly, we obtained the complete coding sequences of HcAMPKα2, HcAMPKβ1, HcAMPKγ2 from H. cunea, which encoded a protein of 512, 281, and 680 amino acids respectively. Furthermore, the phylogenetic analysis revealed that these three subunits were highly homologous with the AMPK subunits from other lepidopteran species. According to the bioassay, we found metformin remarkably restrained the growth and development of H. cunea larvae, and caused molting delayed and body weight reduced. In addition, expressions of HcAMPKα2, HcAMPKβ1, and HcAMPKγ2 were upregulated 3.30-, 5.93- and 5.92-folds at 24 h after treatment, confirming that metformin activated AMPK signaling at the transcriptional level in H. cunea larvae. Conversely, the expressions of two vital Halloween genes (HcCYP306A1 and HcCYP314A1) in the 20E synthesis pathway were remarkably suppressed by metformin. Thus, we presumed that metformin delayed larval molting probably by impeding 20E synthesis in the H. cunea larvae. Finally, we found that metformin accelerated glycogen breakdown, elevated in vivo trehalose level, promoted chitin synthesis, and upregulated transcriptions of the genes in chitin synthesis pathway. Taken together, the findings provide a new insight into the molecular mechanisms by which AMPK regulates carbohydrate metabolism and chitin synthesis in insects.

Effect of AMPK Subunit Alpha 2 Genetic Polymorphisms on Susceptibility to Postherpetic Pain in Chinese Southwest Han Population

Effect of AMPK Subunit Alpha 2 Genetic Polymorphisms on Susceptibility to Postherpetic Pain in Chinese Southwest Han Population

Subacute cadmium exposure promotes M1 macrophage polarization through oxidative stress-evoked inflammatory response and induces porcine adrenal fibrosis

Cadmium (Cd) is a widely distributed environmental pollutant with immunotoxicity and endocrine toxicity. M1/M2 macrophages participate in the immune response and exert an essential influence on fibrosis. Nevertheless, whether Cd can induce porcineadrenal fibrosis by affecting the polarization of M1/M2 macrophages and its potential regulatory mechanism have not been explored. We added 20 mg/kg CdCl2 to the pig diet for 40 days to investigate the fibrogenic effect of subacute Cd exposure on the adrenal gland. The results indicated that the ACTH and CORT in serum were decreased by 15.26 % and 21.99 %, respectively. The contents of adrenal mineral elements Cd, Cr, Mn were increased up to 34, 1.93, 1.42 folds and Co, Zn, Sn were reduced by 21.57 %, 20.52 %, 15.75 %. Concurrently, the pro-oxidative indicators (LPO, MDA and H2O2) were increased by 1.85, 2.20, 2.77 folds and 3.60, 11.15, 4.11 folds upregulated mRNA levels of TLR4, NF-κB, NLRP3 were observed. Subsequently, the expression of M1 macrophages polarization markers (IL-6, iNOS, TNF-α, CCL2 and CXCL9) were raised by 2.03, 2.30, 2.35, 1.58, 1.56 folds, while M2 macrophages (IL-4, CCL24, Arg1, IL-10, MRC1) showed a 62.34 %, 31.88 %, 50.26 %, 74.00 %, 69.34 % downregulation. The expression levels of AMPK subunits and genes related to glycolysis, oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) were also markedly increased. Additionally, the expression level of TGF-β1, Smad2/3 and downstream pro-fibrotic markers was obviously upregulated. Taken together, we conclude that Cd activates the oxidative stress-mediated TLR4/NF-κB/NLRP3 inflammatory signal transduction, leading to porcine adrenal fibrosis by promoting macrophage polarization toward M1.

SNP discovery of PRKAB1 gene and their associations with growth traits in goats

AMPK plays an important role in regulating the metabolism of carbohydrate, lipid and protein in an organism, and is considered to be a key regulator of cellular energy homeostasis. In recent years, attention has been drawn to AMPK subunit polymorphisms and their association with economical traits of domestic animals and fowls. PRKAB1 encodes the β1 regulatory subunit of AMPK, and it has been reported that PRKAB1 may be applied in breeding programs of meat-type chicken. To date, the polymorphism of goat PRKAB1 gene and its associations remain unknown. In this paper, the polymorphism of PRKAB1 gene was detected in 316 goats of three breeds. A total of four novel single nucleotide polymorphisms (SNPs) of PRKAB1 gene were revealed by sequence analysis. Among them, three were in the coding region (285 C > A, 297 C > A, 309 C > T), and they were all synonymous. One was in the intron (229 A > G). The associations between polymorphic loci and the growth traits of Xuhuai and Haimen goats were analyzed, and significant associations were found in body length index and trunk index (p < 0.05) for Xuhuai breed, while no significant associations in Haimen breed. Our results provide useful information for the improvement and breeding of Chinese native goats.

Impairment of AMPK-α2 augments detrusor contractions in bladder ischemia.

PurposeIschemia disrupts cellular energy homeostasis. Adenosine monophosphate-activated protein kinase alpha-2 (AMPK-α2) is a subunit of AMPK that senses cellular energy deprivation and signals metabolic stress. Our goal was to examine the expression levels and functional role of AMPK-α2 in bladder ischemia.Materials and MethodsIliac artery atherosclerosis and bladder ischemia were engendered in apolipoprotein E knockout rats by partial arterial endothelial denudation using a balloon catheter. After eight weeks, total and phosphorylated AMPK-α2 expression was analyzed by western blotting. Structural integrity of AMPK-α2 protein was assessed by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Functional role of AMPK-α2 was examined by treating animals with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D ribofuranoside (AICAR). Tissue contractility was measured in the organ bath and bladder nerve density was examined by immunostaining.ResultsTotal AMPK-α2 expression increased in bladder ischemia, while phosphorylated AMPK-α2 was significantly downregulated. LC-MS/MS suggested post-translational modification of AMPK-α2 functional domains including phosphorylation sites, suggesting accumulation of catalytically inactive AMPK-α2 in bladder ischemia. Treatment of rats with AICAR diminished the force of overactive detrusor contractions and increased bladder capacity but did not have a significant effect on the frequency of bladder contractions. AICAR diminished contractile reactivity of ischemic tissues in the organ bath and prevented loss of nerve fibers in bladder ischemia.ConclusionsIschemia induces post-translational modification of AMPK-α2 protein. Impairment of AMPK-α2 may contribute to overactive detrusor contractions and loss of nerve fibers in bladder ischemia. AMPK activators may have therapeutic potential against detrusor overactivity and neurodegeneration in bladder conditions involving ischemia.

Increased metabolic rate associated with immune stimulation of heat-killed Vibrio anguillarum at different temperatures in zebrafish (Danio rerio).

The action of the immune response in zebrafish (Danio rerio) has been a target of many studies. However, the energetic demands involved in the immune response are poorly understood in ectothermic poikilotherms, such as fish. This research aims to characterize the energetic response of zebrafish to an immune challenge of heat-killed Vibrio anguillarum at 22°C and 27.5°C. Zebrafish were either not injected, injected intraperitoneally with 10μl of saline and Freund's incomplete adjuvant (sham), or heat-killed Vibrio anguillarum & Freund's incomplete adjuvant (1.21×1010 cfu/ml). Respirometry was then performed on these zebrafish for a period of 27h. Following this, spleen was collected for quantitative PCR analysis of the catalytic subunit of AMPK (ampka1 & ampka2), the nuclear factor kappa-light-chain-enhancer of activated B cells (nf-kb), and several cytokines (tnfa, il-1b, il-8, il-10). While there was no increase in oxygen consumption with any treatment at 22°C, there was a marked 30% increase in oxygen consumption in zebrafish injected with heat-killed Vibrio at 27.5°C. Furthermore, temperature had a strong effect on the timing of the immune response. At 22°C, there was a 2-3-fold increase in the cytokines measured associated with heat-killed Vibrio injection, whereas there were no differences found at 27.5°C. Furthermore, while there was an increase in ampka2 at 22°C, there was a sharp decrease in ampka2 at 27.5°C, although the changes in ampka2 transcript abundance could not be solely attributed to heat-killed Vibrio, as there were similar changes associated with the sham group. The results of this study demonstrate some of the first evidence that zebrafish increase routine metabolic rate associated with immune stimulation.

Synergistic activation of AMPK prevents from polyglutamine-induced toxicity in Caenorhabditis elegans

Expression of abnormally long polyglutamine (polyQ) tracks is the source of a range of dominant neurodegenerative diseases, such as Huntington disease. Currently, there is no treatment for this devastating disease, although some chemicals, e.g., metformin, have been proposed as therapeutic solutions. In this work, we show that metformin, together with salicylate, can synergistically reduce the number of aggregates produced after polyQ expression in Caenorhabditis elegans. Moreover, we demonstrate that incubation polyQ-stressed worms with low doses of both chemicals restores neuronal functionality. Both substances are pleitotropic and may activate a range of different targets. However, we demonstrate in this report that the beneficial effect induced by the combination of these drugs depends entirely on the catalytic action of AMPK, since loss of function mutants of aak-2/AMPKα2 do not respond to the treatment. To further investigate the mechanism of the synergetic activity of metformin/salicylate, we used CRISPR to generate mutant alleles of the scaffolding subunit of AMPK, aakb-1/AMPKβ1. In addition, we used an RNAi strategy to silence the expression of the second AMPKβ subunit in worms, namely aakb-2/AMPKβ2. In this work, we demonstrated that both regulatory subunits of AMPK are modulators of protein homeostasis. Interestingly, only aakb-2/AMPKβ2 is required for the synergistic action of metformin/salicylate to reduce polyQ aggregation. Finally, we showed that autophagy acts downstream of metformin/salicylate-related AMPK activation to promote healthy protein homeostasis in worms.

AMPK Profiling in Rodent and Human Pancreatic Beta-Cells under Nutrient-Rich Metabolic Stress

Chronic exposure of pancreatic β-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in β-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in β-cells. Then, we investigated the expression profile of AMPK pathways in β-cells following metabolic stresses. INS-1E β-cells and human islets were exposed for 3 days to glucose (5.5–25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E β-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 (Prkaa1) and α2 (Prkaa2). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulated insulin secretion, did not change AMPK expression, either in insulinoma cells or in human islets. Expression profile of the six AMPK subunits was marginally modified by the different diabetogenic conditions. However, the expression of some upstream kinases and downstream AMPK targets, including K-ATP channel subunits, exhibited stress-specific signatures. Interestingly, at the protein level, chronic fructose treatment favored fasting-like phenotype in human islets, as witnessed by AMPK activation. Collectively, previously published and present data indicate that, in the β-cell, AMPK activation might be implicated in the pre-diabetic state, potentially as a protective mechanism.