Increased AMP-activated Protein Kinase Phosphorylation Research Articles

8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway.

8-Prenylgenistein (8PG), a genistein derivative, is present in fermented soybeans (Glycine max), including cheonggukjang (CGJ), and exhibits osteoprotective, osteogenic, and antiadipogenic properties. However, the hepatoprotective effects of 8PG and its underlying molecular mechanisms remain largely unexplored. Here, we identified the high binding affinity of 8PG with AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), which acts as a potent AMPK activator that counteracts hepatic steatosis. Notably, 8PG exhibited better pharmacokinetics with greater absorption and higher plasma binding than the positive controls for the target proteins. Moreover, 8PG exerted non-carcinogenic activity in rats and significantly increased AMPK phosphorylation. Compound C, an AMPK inhibitor, did not antagonize 8PG-activated AMPK in HepG2 cells. 8PG significantly attenuated palmitate-induced lipid accumulation and enhanced phosphorylated AMPK and its downstream target, acetyl-CoA carboxylase. Further, 8PG activated nuclear SIRT1 at the protein level, which promoted fatty acid oxidation in palmitate-treated HepG2 cells. Overall, 8PG acts as a potent AMPK activator, further attenuating hepatic steatosis via the SIRT1-mediated pathway and providing new avenues for dietary interventions to treat metabolic dysfunction-associated steatotic liver disease (MASLD).

Empagliflozin mitigates methotrexate-induced nephrotoxicity in male albino rats: insights on the crosstalk of AMPK/Nrf2 signaling pathway

BackgroundThe anti-diabetic drug, empagliflozin (EMPA), has many pleiotropic actions and is challenged recently to possess renoprotective properties. This renoprotective potential is proposed to be mediated via the activation of AMP-activated protein kinase (AMPK)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. This research investigated the renoprotective potential and the mechanistic pathway of EMPA against methotrexate (MTX)-induced nephrotoxicity and evaluated the role of AMPK by utilizing an AMPK inhibitor, dorsomorphin (Dorso).MethodsThirty male Wistar rats, weighing 180–200 g, were divided equally into five groups. Group I represented the control group. Nephrotoxicity was induced in the remaining rats through the administration of a single intraperitoneal injection of MTX (20 mg/kg). Rats were then randomly assigned to: Group 2 (received MTX injection only); Group 3 (received MTX and EMPA 30 mg/kg/day); Group 4 (received MTX and Dorso 0.2 mg/kg/day), Group 5 (received MTX, Dorso, EMPA). After one week, blood samples were collected, the rats were euthanized, and renal tissues were harvested for biochemical and histomorphometric assessments.ResultsMTX produced a significant rise in serum creatinine and tissue MDA levels; an increase in BAX, p53, cytochrome-c expression; a reduction in Bcl2 level; and disruption of renal microarchitecture. In contrast, EMPA therapy in group 3, resulted in a significant improvement of all these parameters, correlated with significant increase in AMPK phosphorylation and Nrf2 expression. Importantly, the co-administration of Dorso, in group 5, prevented EMPA’s beneficial effects.ConclusionEMPA has a potential protective effect against MTX-induced toxicity through the activation of the AMPK/Nrf2 signaling pathway.

Inhibitory effect of aqueous extract of Scrophularia ningpoensis on β-cell pyroptosis in diabetic mice

Scrophularia ningpoensis Hemsl. (SN) is an herbal medicine used as a functional food in China. SN has traditionally been used to treat diabetes mellitus (DM), but its mechanism of action is not well understood. Our previous study indicated that the aqueous extract of SN can reduce hepatic insulin resistance and correct the irregular shape of pancreatic islets in diabetic mice. This study aims to investigate whether the aqueous extract of SN can regulate pyroptosis in pancreatic β-cells and explore its underlying mechanisms. Db/db mice were orally given an aqueous extract of SN for eight weeks. Subsequently, their pancreatic tissues were collected for histopathological analysis and immunoblotting. Rat insulinoma (INS-1) cells were cultured either alone or with the aqueous extract of SN and evaluated for cell viability, pyroptotic body formation, AMP-activated protein kinase (AMPK) activity, the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome and gasdermin D (GSDMD) activation, and reactive oxygen species (ROS) production under high glucose (HG) exposure conditions. The aqueous extract of SN reduced insulitis and pancreatic β-cell death in db/db mice. Increased AMPK phosphorylation and decreased NLRP3 inflammasome and GSDMD activation were observed in pancreatic tissues after administration of the aqueous extract of SN. In vitro, the aqueous extract of SN increased cell viability and AMPK phosphorylation in a concentration-dependent manner. Additionally, the aqueous extract of SN corrected excessive ROS generation, NLRP3 inflammasome and GSDMD activation, and pyroptotic body formation. Inhibition of AMPK negated these beneficial effects of the aqueous extract of SN on INS-1 cells. The study concluded that the aqueous extract of SN alleviates β-cell pyroptosis by inhibiting NLRP3/GSDMD activation in an AMPK-dependent manner.

AMPK boosts ADAM10 shedding activity in human aortic endothelial cells by promoting Rab14-dependent ADAM10 cell surface translocation

A disintegrin and metalloprotease 10 (ADAM10) regulates the expression of cell surface receptors such as tumor necrosis factor receptor 1, toll-like receptor 4, and the receptor for advanced glycation end products (RAGE) by cleaving their extracellular regions. To function as a sheddase, ADAM10 should translocate from the intracellular compartments to the cell surface, but the translocation mechanism remains unclear. In this study, we explored the possible role of adenosine monophosphate-activated protein kinase (AMPK) in the induction of ADAM10 shedding activity. In cultured human aortic endothelial cells (HAECs), 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator, boosted ADAM10 cell surface translocation and ectodomain shedding of RAGE. ADAM10 inhibition with GI 254023X and ADAM10 siRNA silencing both prevented AICAR-induced RAGE ectodomain shedding. AICAR increased AMPK phosphorylation as well. Both Compound C-mediated AMPK inhibition and AMPKα1-siRNA-mediated AMPK depletion suppressed AICAR-induced ADAM10 cell surface translocation and RAGE ectodomain shedding. On the other hand, siRNA knockdown of Rab14, a small GTPase that facilitates the intracellular trafficking of transmembrane proteins, prevented AICAR-induced ADAM10 cell surface translocation and RAGE ectodomain shedding. In conclusion, AMPK activation is an obvious inducer of ADAM10 shedding activity. Our findings suggest that AMPK boosts ADAM10 shedding activity in HAECs by promoting Rab14-dependent ADAM10 cell surface translocation.

Yogurt starter strains ameliorate intestinal barrier dysfunction via activating AMPK in Caco-2 cells

ABSTRACT Lactic acid bacteria (LAB) are commonly used probiotics that improve human health in various aspects. We previously reported that yogurt starter strains, Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, potentially enhance the intestinal epithelial barrier function by inducing the expression of antimicrobial peptides in the small intestine. However, their effects on physical barrier functions remain unknown. In this study, we found that both strains ameliorated the decreased trans-epithelial resistance and the increased permeability of fluorescein isothiocyanate-dextran induced by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. We also demonstrated that LAB prevented a decrease in the expression and disassembly of tight junctions (TJs) induced by TNF-α and IFN-γ. To assess the repair activity of TJs, a calcium switch assay was performed. Both strains were found to promote the reassembly of TJs, and their activity was canceled by the inhibitor of AMP-activated protein kinase (AMPK). Moreover, these strains showed increased AMPK phosphorylation. These observations suggest that the strains ameliorated physical barrier dysfunction via the activation of AMPK. The activities preventing barrier destruction induced by TNF-α and IFN-γ were strain-dependent. Several strains containing L. bulgaricus 2038 and S. thermophilus 1131 significantly suppressed the barrier impairment, and L. bulgaricus 2038 showed the strongest activity among them. Our findings suggest that the intake of L. bulgaricus 2038 and S. thermophilus 1131 is a potential strategy for the prevention and repair of leaky gut.

The Inhibition of Zinc Excitotoxicity and AMPK Phosphorylation by a Novel Zinc Chelator, 2G11, Ameliorates Neuronal Death Induced by Global Cerebral Ischemia.

AMP-activated protein kinase (AMPK) is necessary for maintaining a positive energy balance and essential cellular processes such as glycolysis, gene transcription, glucose uptake, and several other biological functions. However, brain injury-induced energy and metabolic stressors, such as cerebral ischemia, increase AMPK phosphorylation. Phosphorylated AMPK contributes to excitotoxicity, oxidative, and metabolic problems. Furthermore, brain disease-induced release of zinc from synaptic vesicles contributes to neuronal damage via mechanisms including ROS production, apoptotic cell death, and DNA damage. For this reason, we hypothesized that regulating zinc accumulation and AMPK phosphorylation is critical for protection against global cerebral ischemia (GCI). Through virtual screening based on the structure of AMPK subunit alpha 2, we identified a novel compound, 2G11. In this study, we verified that 2G11 administration has neuroprotective effects via the blocking of zinc translocation and AMPK phosphorylation after GCI. As a result, we demonstrated that 2G11 protected hippocampal neurons against GCI and OGD/R-derived cellular damage. In conclusion, we propose that AMPK inhibition and zinc chelation by 2G11 may be a promising tool for preventing GCI-induced hippocampal neuronal death.

Antagonism of the Muscarinic Acetylcholine Type 1 Receptor Enhances Mitochondrial Membrane Potential and Expression of Respiratory Chain Components via AMPK in Human Neuroblastoma SH-SY5Y Cells and Primary Neurons

Impairments in mitochondrial physiology play a role in the progression of multiple neurodegenerative conditions, including peripheral neuropathy in diabetes. Blockade of muscarinic acetylcholine type 1 receptor (M1R) with specific/selective antagonists prevented mitochondrial dysfunction and reversed nerve degeneration in in vitro and in vivo models of peripheral neuropathy. Specifically, in type 1 and type 2 models of diabetes, inhibition of M1R using pirenzepine or muscarinic toxin 7 (MT7) induced AMP-activated protein kinase (AMPK) activity in dorsal root ganglia (DRG) and prevented sensory abnormalities and distal nerve fiber loss. The human neuroblastoma SH-SY5Y cell line has been extensively used as an in vitro model system to study mechanisms of neurodegeneration in DRG neurons and other neuronal sub-types. Here, we tested the hypothesis that pirenzepine or MT7 enhance AMPK activity and via this pathway augment mitochondrial function in SH-SY5Y cells. M1R expression was confirmed by utilizing a fluorescent dye, ATTO590-labeled MT7, that exhibits great specificity for this receptor. M1R antagonist treatment in SH-SY5Y culture increased AMPK phosphorylation and mitochondrial protein expression (OXPHOS). Mitochondrial membrane potential (MMP) was augmented in pirenzepine and MT7 treated cultured SH-SY5Y cells and DRG neurons. Compound C or AMPK-specific siRNA suppressed pirenzepine or MT7-induced elevation of OXPHOS expression and MMP. Moreover, muscarinic antagonists induced hyperpolarization by activating the M-current and, thus, suppressed neuronal excitability. These results reveal that negative regulation of this M1R-dependent pathway could represent a potential therapeutic target to elevate AMPK activity, enhance mitochondrial function, suppress neuropathic pain, and enhance nerve repair in peripheral neuropathy.

L-Arginine increases AMPK phosphorylation and the oxidation of energy substrates in hepatocytes, skeletal muscle cells, and adipocytes.

Previous work has shown that dietary L-arginine (Arg) supplementation reduced white fat mass in obese rats. The present study was conducted with cell models to define direct effects of Arg on energy-substrate oxidation in hepatocytes, skeletal muscle cells, and adipocytes. BNL CL.2 mouse hepatocytes, C2C12 mouse myotubes, and 3T3-L1 mouse adipocytes were treated with different extracellular concentrations of Arg (0, 15, 50, 100 and 400µM) or 400µM Arg + 0.5mM NG-nitro-L-arginine methyl ester (L-NAME; an NOS inhibitor) for 48h. Increasing Arg concentrations in culture medium dose-dependently enhanced (P < 0.05) the oxidation of glucose and oleic acid to CO2 in all three cell types, lactate release from C2C12 cells, and the incorporation of oleic acid into esterified lipids in BNL CL.2 and 3T3-L1 cells. Arg at 400µM also stimulated (P < 0.05) the phosphorylation of AMP-activated protein kinase (AMPK) in all three cell types and increased (P < 0.05) NO production in C2C12 and BNL CL.2 cells. The inhibition of NOS by L-NAME moderately reduced (P < 0.05) glucose and oleic acid oxidation, lactate release, and the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in BNL CL.2 cells, but had no effect (P > 0.05) on these variables inC2C12 or 3T3-L1 cells. Collectively, these results indicate that Arg increased AMPK activity and energy-substrate oxidation in BNL CL.2, C2C12, and 3T3-L1 cells through both NO-dependent and NO-independent mechanisms.

Methanolic Extract of Boswellia serrata Gum Protects the Nigral Dopaminergic Neurons from Rotenone-Induced Neurotoxicity

Boswellia serrata gum is a natural product that showed beneficial effects on neurodegenerative diseases in recent studies. In this study, we investigated the effects of Boswellia serrata resin on rotenone-induced dopaminergic neurotoxicity. Firstly, we attempted to see if the resin can induce AMP-activated protein kinase (AMPK) signaling pathway which has been known to have broad neuroprotective effects. Boswellia increased AMPK phosphorylation and reduced phosphorylation of mammalian target of rapamycin (p-mTOR) and α-synuclein (p-α-synuclein) in the striatum while increased the expression level of Beclin1, a marker for autophagy and brain-derived neurotrophic factor. Next, we examined the neuroprotective effects of the Boswellia extract in the rotenone-injected mice. The results showed that Boswellia evidently attenuated the loss of the nigrostriatal dopaminergic neurons and microglial activation caused by rotenone. Moreover, Boswellia ameliorated rotenone-induced decrease in the striatal dopamine and impairment in motor function. Accumulation of α-synuclein meditated by rotenone was significantly ameliorated by Boswellia. Also, we showed that β-boswellic acid, the active constituents of Boswellia serrata gum, induced AMPK phosphorylation and attenuated α-synuclein phosphorylation in SHSY5 cells. These results suggest that Boswellia protected the dopaminergic neurons from rotenone neurotoxicity via activation of the AMPK pathway which might be associated with attenuation of α-synuclein aggregation and neuroinflammation. Further investigations are warranted to identify specific molecules in Boswellia which are responsible for the neuroprotection.

Abstract 6160: Tie2-mediated AMPK activation by ferritin-based protein C nanoparticles inhibits advanced prostate cancer development through induction of vasodilation

Abstract Prostate cancer (PCa) is one of the most common malignancies and the second-leading cause of cancer-related mortality in men in Western countries. Androgen deprivation therapy (ADT) is an initial systemic therapy for advanced PCa, but almost all cancer eventually becomes castration resistant. Recently, we introduced ferritin-based protein C nanoparticles (PCNs), known as TFG and TFMG, which enhance normalization of the tumor vasculature. However, the exact underlying mechanism how PCNs induces AMP-activated protein kinase (AMPK) activation and anti-tumor activity in castration-resistant prostate cancer (CRPC) remains unknown. Here, we investigated the role of PCNs for CRPC development in endothelial cells. We found that TFMG inhibits prostate cancer castration resistance by activating AMPK through the Tie2-mediated signaling pathway. TFMG treatment increased AMPK phosphorylation at Thr172 and acetyl-CoA carboxylase (ACC) at Ser79 in EA.hy926 cells. TFMG treatment also significantly increased the phosphorylation of endothelial nitric oxide synthase (eNOS) with inducing nitric oxide (NO) production in EA.hy926 cells. In addition, TFMG treatment induced the vasodilatory effect in isolated rat mesenteric resistance arteries (MRAs), which was blocked by NO synthase inhibitor L-NAME. Interestingly, these effects were all abolished by pretreatment with Compound C (an AMPK inhibitor), rebastinib (selective Tie2 Inhibitor), AMPK knockdown using siRNA, or dominant negative AMPKα recombinant adenovirus. Finally, we found that TFMG functionally abrogates CRPC development in mouse model. In summary, TFMG exerted vasodilatation through activating Tie2/AMPK/eNOS signaling in endothelial cells, which leads to the overcoming vasoconstriction induced ADT. This study provides the potential value of TFMG in vasodilation of blood vessels leading suppression of CRPC development. Citation Format: You Mie Lee, Ji-Hak Jeong, Hyunha Jang, Jihye You. Tie2-mediated AMPK activation by ferritin-based protein C nanoparticles inhibits advanced prostate cancer development through induction of vasodilation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6160.

Attenuation of the Counter-Regulatory Glucose Response in CVLM C1 Neurons: A Possible Explanation for Anorexia of Aging

This study aimed to determine the effect of age on CVLM C1 neuron glucoregulatory proteins in the feeding pathway. Male Sprague Dawley rats aged 3 months and 24 months old were divided into two subgroups: the treatment group with 2-deoxy-d-glucose (2DG) and the control group. Rat brains were dissected to obtain the CVLM region of the brainstem. Western blot was used to determine protein expression of tyrosine hydroxylase (TH), phosphorylated TH at Serine40 (pSer40TH), AMP-activated protein kinase (AMPK), phosphorylated AMPK (phospho AMPK), and neuropeptide Y Y5 receptors (NPY5R) in CVLM samples. Immunofluorescence was used to determine TH-, AMPK-, and NPY5R-like immunoreactivities among other brain coronal sections. Results obtained denote a decrease in basal TH phosphorylation levels and AMPK proteins and an increase in TH proteins among aged CVLM neurons. Increases in the basal immunoreactivity of TH+, AMPK+, NPY5R+, TH+/AMPK+, and TH+/NPY5R+ were also observed among old rats. Young treatment-group rats saw a decrease in TH phosphorylation and AMPK proteins following 2DG administration, while an increase in AMPK phosphorylation and a decrease in TH proteins were found among the old-treatment-group rats. These findings suggest the participation of CVLM C1 neurons in counter-regulatory responses among young and old rats. Altering protein changes in aged CVLM C1 neurons may attenuate responses to glucoprivation, thus explaining the decline in food intake among the elderly.

Activation of transient receptor potential vanilloid 4 protects articular cartilage against inflammatory responses via CaMKK/AMPK/NF-\u03baB signaling pathway

Transient receptor potential vanilloid 4 (TRPV4) plays an important role in chondrocytes via Ca2+ signaling. However, its role in the progression of osteoarthritis is unclear. This study aimed to evaluate the effects of TRPV4 activation on articular cartilage and chondrocytes stimulated with interleukin (IL)-1β. Bovine and human articular chondrocytes were stimulated with various agents, including IL-1β, GSK1016790A (GSK101; a TRPV4 agonist), Compound C (an AMP-activated protein kinase (AMPK) inhibitor), and STO-609 (a calmodulin-dependent protein kinase kinase (CaMKK) inhibitor), and were processed for Western blot analysis and real-time PCR. The dimethylmethylene blue (DMMB) assay and Safranin O staining were also performed. GSK101 reversed the IL-1β-induced increase in expression of matrix metalloproteinase (MMP)-13 and decrease in expression of aggrecan. GSK101 also decreased proteoglycan release in the DMMB assay and retained Safranin O staining of articular cartilage tissue. Furthermore, GSK101 increased AMPK phosphorylation and decreased IL-1β-induced nuclear factor kappa B (NF-κB) phosphorylation. Compound C and STO-609 reversed the suppressive effects of GSK101 on NF-κB activation and MMP-13 expression. In conclusion, TRPV4 activation had chondroprotective effects on articular cartilage stimulated with IL-1β by activating CaMKK/AMPK and suppressing the NF-κB pathway. TRPV4 activators may offer a promising therapeutic option for preventing the progression of osteoarthritis.

Extracts of Vine Tea Improve Diet-Induced Non-Alcoholic Steatohepatitis Through AMPK-LXRα Signaling.

Chinese vine tea can improve glucose and lipid metabolic disorders. However, its protective effects in non-alcoholic steatohepatitis (NASH) and its underlying molecular mechanisms remain unclear. Liver X receptor α (LXRα) inhibition and adenosine monophosphate-(AMP)-activated protein kinase (AMPK) activation can enhance control of NASH. AMPK activators have also been shown to inactivate LXRα. Here, the anti-NASH effects of vine tea extract (VTE) dosed at 1 g.100 g−1 diet were investigated using NASH mice challenged with a methionine and choline-deficient l-amino acid diet (MCDD) and a high-fat diet (HFD). Pharmacological mechanisms of VTE were explored using TUNEL staining, AMPK inhibition, Western blot, reporter assays, qRT-PCR analyses, and immunofluorescence. VTE treatment improved fatty liver in HFD-induced mice, while it alleviated the progression of NASH including protecting against liver lipid accumulation, steatosis, endoplasmic reticulum stress, apoptosis, inflammation, and functional injury in MCDD-fed mice. VTE reduced the action of hepatic lipogenic genes, F4/80, pro-inflammatory cytokines, CHOP, and cleaved Caspase-3 expression, while promoting expression of fatty acid oxidation genes CPT1α, ß. VTE also enhanced AMPK and blocked LXRα signaling in mouse livers. In vitro results indicated that VTE increased AMPK phosphorylation and reduced LXRα activity in HepG2 cells. Conversely, the antagonistic effect of VTE on LXRα was decreased through AMPK inhibition. Our data suggests that VTE may improve diet-induced NASH, which involves the pharmacological modulation of the AMPK-LXRα signaling pathway.

Dapagliflozin attenuates arrhythmic vulnerabilities by regulating connexin43 expression via the AMPK pathway in post-infarcted rat hearts

We have demonstrated that dapagliflozin, a sodium-glucose cotransporter (SGLT) 2 inhibitor, attenuates reactive oxygen species (ROS) production. Connexin43 playing a role in ventricular arrhythmia is sensitive to redox status. No data are available on the effects of dapagliflozin on arrhythmogenesis. This study was to determine whether dapagliflozin attenuated arrhythmias through modulating AMP-activated protein kinase (AMPK)/free radicals-induced connexin43 after myocardial infarction. After coronary ligation, normoglycemic male Wistar rats were randomized to either vehicle or dapagliflozin (0.1 mg/kg per day) for 4 weeks. Myocardial ROS levels were significantly increased (p < 0.05) and connexin43 levels were substantially decreased after myocardial infarction (p < 0.05). Dapagliflozin administration was associated with increased SGLT1, attenuated ROS and increased connexin43 levels in myocardium (all p < 0.05). During programmed electrical stimulation, arrhythmic severity was significantly improved in the dapagliflozin-treated infarcted rats than those in the vehicle-treated infarcted rats (p < 0.05). Dapagliflozin significantly increased AMPK phosphorylation compared to vehicle after infarction (p < 0.05). Inhibition of AMPK signaling by SBI-0206965 prevented increased SGLT1 and blocked the effects of dapagliflozin on attenuated ROS levels and increased connexin43 phosphorylation (all p < 0.05). SGLT1 inhibited by KGA-2727 showed attenuated ROS levels and increased connexin43 phosphorylation (both p < 0.05) although AMPK phosphorylation was not changed, implying SGLT1 activation was mediated by AMPK in dapagliflozin-treated hearts. Dapagliflozin-treated hearts had significantly increased connexin43 phosphorylation (p < 0.05), which was significantly decreased after adding 3-morpholinosydnonimine (p < 0.05). These data indicate that clinically-relevant dapagliflozin concentrations decreased free radicals content and increased connexin43 levels through AMPK-dependent and SGLT1-independent mechanisms, which attenuated ventricular arrhythmias in the normoglycemic infarcted rats.

Metformin protects against insulin resistance induced by high uric acid in cardiomyocytes via AMPK signalling pathways in vitro and in vivo.

High uric acid (HUA) is associated with insulin resistance (IR) in cardiomyocytes. We investigated whether metformin protects against HUA‐induced IR in cardiomyocytes. We exposed primary cardiomyocytes to HUA, and cellular glucose uptake was quantified by measuring the uptake of 2‐NBDG, a fluorescent glucose analog. Western blot was used to examine the levels of signalling protein. Membrane of glucose transporter type 4 (GLUT4) was analysed by immunofluorescence. We monitored the impact of metformin on HUA‐induced IR and in myocardial tissue of an acute hyperuricaemia mouse model established by potassium oxonate treatment. Treatment with metformin protected against HUA‐reduced glucose uptake induced by insulin in cardiomyocytes. HUA directly inhibited the phosphorylation of Akt and the translocation of GLUT4 induced by insulin, which was blocked by metformin. Metformin promoted phosphorylation of AMP‐activated protein kinase (AMPK) and restored the insulin‐stimulated glucose uptake in HUA‐induced IR cardiomyocytes. As a result of these effects, in a mouse model of acute hyperuricaemia, metformin improved insulin tolerance and glucose tolerance, accompanied by increased AMPK phosphorylation, Akt phosphorylation and translocation of GLUT4 in myocardial tissues. As expected, AICAR, another AMPK activator, had similar effects to metformin, demonstrating the important role of AMPK activation in protecting against IR induced by HUA in cardiomyocytes. Metformin protects against IR induced by HUA in cardiomyocytes and improves insulin tolerance and glucose tolerance in an acute hyperuricaemic mouse model, along with the activation of AMPK. Consequently, metformin may be an important potential new treatment strategy for hyperuricaemia‐related cardiovascular disease.

An AMPK activator as a therapeutic option for congenital nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) patients produce large amounts of dilute urine. NDI can be congenital, resulting from mutations in the type-2 vasopressin receptor (V2R), or acquired, resulting from medications such as lithium. There are no effective treatment options for NDI. Activation of PKA is disrupted in both congenital and acquired NDI, resulting in decreased aquaporin-2 phosphorylation and water reabsorption. We show that adenosine monophosphate–activated protein kinase (AMPK) also phosphorylates aquaporin-2. We identified an activator of AMPK, NDI-5033, and we tested its ability to increase urine concentration in animal models of NDI. NDI-5033 increased AMPK phosphorylation by 2.5-fold, confirming activation. It increased urine osmolality in tolvaptan-treated NDI rats by 30%–50% and in V2R-KO mice by 50%. Metformin, another AMPK activator, can cause hypoglycemia, which makes it a risky option for treating NDI patients, especially children. Rats with NDI receiving NDI-5033 showed no hypoglycemia in a calorie-restricted, exercise protocol. Congenital NDI therapy needs to be effective long-term. We administered NDI-5033 for 3 weeks and saw no reduction in efficacy. We conclude that NDI-5033 can improve urine concentration in animals with NDI and holds promise as a potential therapy for patients with congenital NDI due to V2R mutations.

Testosterone activates glucose metabolism through AMPK and androgen signaling in cardiomyocyte hypertrophy

BackgroundTestosterone regulates nutrient and energy balance to maintain protein synthesis and metabolism in cardiomyocytes, but supraphysiological concentrations induce cardiac hypertrophy. Previously, we determined that testosterone increased glucose uptake—via AMP-activated protein kinase (AMPK)—after acute treatment in cardiomyocytes. However, whether elevated glucose uptake is involved in long-term changes of glucose metabolism or is required during cardiomyocyte growth remained unknown. In this study, we hypothesized that glucose uptake and glycolysis increase in testosterone-treated cardiomyocytes through AMPK and androgen receptor (AR).MethodsCultured cardiomyocytes were stimulated with 100 nM testosterone for 24 h, and hypertrophy was verified by increased cell size and mRNA levels of β-myosin heavy chain (β-mhc). Glucose uptake was assessed by 2-NBDG. Glycolysis and glycolytic capacity were determined by measuring extracellular acidification rate (ECAR).ResultsTestosterone induced cardiomyocyte hypertrophy that was accompanied by increased glucose uptake, glycolysis enhancement and upregulated mRNA expression of hexokinase 2. In addition, testosterone increased AMPK phosphorylation (Thr172), while inhibition of both AMPK and AR blocked glycolysis and cardiomyocyte hypertrophy induced by testosterone. Moreover, testosterone supplementation in adult male rats by 5 weeks induced cardiac hypertrophy and upregulated β-mhc, Hk2 and Pfk2 mRNA levels.ConclusionThese results indicate that testosterone stimulates glucose metabolism by activation of AMPK and AR signaling which are critical to induce cardiomyocyte hypertrophy.

Inhibitory Effects of Thymol Isolated from Curcuma longa L. on Adipogenesis in HepG2 Cells

Non-alcoholic fatty liver disease (NAFLD) is a disease associated with metabolic syndromes such as diabetes and obesity, regardless of alcohol consumption, and refers to the accumulation of triacylglycerols in the liver. Thymol (THY) is a vegetable essential oil that is naturally contained in the Zingiberaceae and Lamiaceae families. THY was isolated from Curcuma longa L. The rhizomes of Curcuma longa L. were dried, sliced and extracted with 50% ethanol and then isolated through repeated column chromatography. This study was conducted to investigate the inhibitory effect of THY, even in non-alcoholic fatty liver disease, in relation to the inhibiting hyperlipidemia effect of THY, which was demonstrated in previous studies. Hepatocytes were treated with oleate (OA) containing THY to observe lipid accumulation by Oil Red O staining (ORO). We also tested the effect of THY on triacylglycerols (TG) and total cholesterol (TC) in HepG2 cells. Western blot and real-time RT-PCR using sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), CCAAT-enhancer-binding protein (C/EBP), proliferator-activated receptor γ (PPARγ), and adenosine monophosphate (AMP)-activated protein kinase (AMPK) expressions were carried out. Consequently, inhibition of lipogenesis by THY (100 μM or 200 μM) in NAFLD treated with OA in HepG2 cells was confirmed. The results of TG and TC experiments confirmed a decrease in the degree of fat accumulation in the liver. Furthermore, inhibition of the SREBP-1c, FAS, ACC, C/EBP and PPARγ expressions that mediated fat accumulation and increased AMPK phosphorylation was observed. Taken together, THY is proposed as a potential natural constituent for the treatment of NAFLD.

Synphilin-1 Interacts with AMPK and Increases AMPK Phosphorylation.

A role for the cytoplasmic protein synphilin-1 in regulating energy balance has been demonstrated recently. Expression of synphilin-1 increases ATP levels in cultured cells. However, the mechanism by which synphilin-1 alters cellular energy status is unknown. Here, we used cell models and biochemical approaches to investigate the cellular functions of synphilin-1 on the AMP-activated protein kinase (AMPK) signaling pathway, which may affect energy balance. Overexpression of synphilin-1 increased AMPK phosphorylation (activation). Moreover, synphilin-1 interacted with AMPK by co-immunoprecipitation and GST (glutathione S-transferase) pull-down assays. Knockdown of synphilin-1 reduced AMPK phosphorylation. Overexpression of synphilin-1 also altered AMPK downstream signaling, i.e., a decrease in acetyl CoA carboxylase (ACC) phosphorylation, and an increase in p70S6K phosphorylation. Treatment of compound C (an AMPK inhibitor) reduced synphilin-1 binding with AMPK. In addition, compound C diminished synphilin-1-induced AMPK phosphorylation, and the increase in cellular ATP (adenosine triphosphate) levels. Our results demonstrated that synphilin-1 couples with AMPK, and they exert mutual effects on each other to regulate cellular energy status. These findings not only identify novel cellular actions of synphilin-1, but also provide new insights into the roles of synphilin-1 in regulating energy currency, ATP.

Shikonin Attenuates Hepatic Steatosis by Enhancing Beta Oxidation and Energy Expenditure via AMPK Activation.

Shikonin, a natural plant pigment, is known to have anti-obesity activity and to improve insulin sensitivity. This study aimed to examine the effect of shikonin on hepatic steatosis, focusing on the AMP-activated protein kinase (AMPK) and energy expenditure in Hepa 1-6 cells and in high-fat fed mice. Shikonin increased AMPK phosphorylation in a dose- and time-dependent manner, and inhibition of AMPK with compound C inhibited this activation. In an oleic acid-induced steatosis model in hepatocytes, shikonin suppressed oleic acid-induced lipid accumulation, increased AMPK phosphorylation, suppressed the expression of lipogenic genes, and stimulated fatty acid oxidation-related genes. Shikonin administration for four weeks decreased body weight gain and the accumulation of lipid droplets in the liver of high-fat fed mice. Furthermore, shikonin promoted energy expenditure by activating fatty acid oxidation. In addition, shikonin increased the expression of PPARγ coactivator-1α (PGC-1α), carnitine palmitoyltransferase-1 (CPT1) and other mitochondrial function-related genes. These results suggest that shikonin attenuated a high fat diet-induced nonalcoholic fatty liver disease by stimulating fatty acid oxidation and energy expenditure via AMPK activation.