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Glucose metabolism disturbances may result in diabetes-associated cognitive decline (DACI). Methionine restriction (MR) diet has emerged as a potential dietary strategy for managing glucose homeostasis. However, the effects and underlying mechanisms of MR on DACI have not been fully elucidated. Here, we found that a 13-week MR (0.17 % methionine, w/w) intervention starting at 8 weeks of age improved peripheral insulin sensitivity in male db/db mice, a model for type 2 diabetes. Notably, MR significantly improved working as well as long-term memory in db/db mice, accompanied by increased PSD-95 level and reduced neuroinflammatory factors, malondialdehyde (MDA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG). We speculate that this effect may be mediated by MR activating hepatic fibroblast growth factor 21 (FGF21) and the brain FGFR1/AMPK/GLUT4 signaling pathway to enhance brain glucose metabolism. To further delineate the mechanism, we used intracerebroventricular injection of adeno-associated virus to specifically knock down FGFR1 in the brain to verify the role of FGFR1 in MR-mediated DACI. It was found that the positive effects of MR on DACI were offset, reflected in decreased cognitive function, impaired synaptic plasticity, upregulated neuroinflammation, and balanced enzymes regulating reactive oxygen species (Sod1, Sod2, Nox4). Of note, the FGFR1/AMPK/GLUT4 signaling pathway and brain glucose metabolism were inhibited. In summary, our study demonstrated that MR increased peripheral insulin sensitivity, activated brain FGFR1/AMPK/GLUT4 signaling through FGF21, maintained normal glucose metabolism and redox balance in the brain, and thereby alleviated DACI. These results provide new insights into the effects of MR diet on cognitive dysfunction caused by impaired brain energy metabolism.

Promotion of brown adipose tissue phenotype, able to transform energy into heat, is a promising target against obesity development. We investigated the effects of a standardized extract from Cynara cardunculus L. (CCLE) leaves, considered a waste food resource, on adipogenesis inhibition in 3T3-L1 cells, through the thermogenic process involvement. CCLE inhibited adipogenesis (C/EBPβ, PPAR-γ, FABP4, SREBP-1, and FASN) and increased thermogenesis via upregulation of AMPK phosphorylation. Furthermore, CCLE induced FOXO1 pathway as adipogenesis repressive mechanism affecting PPAR-γ activity and induced the expression of cell cycle inhibitor p21. However, pretreatment with the AMPK inhibitor compound C abolished CCLE effects on FOXO1, p21, C/EBPβ, and PPAR-γ. These data confirmed that the antiadipogenic effect of the extract is related to AMPK activation. Therefore, this work provides new perspectives for the use of this extract in obesity prevention and management. Additionally, reuse/recycling of waste food resources provides added value from a circular economy perspective.

Acute kidney injury (AKI) disrupts energy metabolism. Targeting metabolism through AMP-activated protein kinase (AMPK) may alleviate AKI. ATX-304, a pan-AMPK activator, was evaluated in C57Bl/6 mice and tubular epithelial cell (TEC) cultures. Mice received ATX-304 (1 mg/g) or control chow for 7 days before cisplatin-induced AKI (CI-AKI). Primary cultures of tubular epithelial cells (TECs) were pre-treated with ATX-304 (20 µM, 4 h) prior to exposure to cisplatin (20 µM, 23 h). ATX-304 increased acetyl-CoA carboxylase phosphorylation, indicating AMPK activation. It protected against CI-AKI measured by serum creatinine (control 0.05 + 0.03 mM vs ATX-304 0.02 + 0.01 mM, P = 0.03), western blot for neutrophil gelatinase-associated lipocalin (NGAL) (control 3.3 + 1.8-fold vs ATX-304 1.2 + 0.55-fold, P = 0.002), and histological injury (control 3.5 + 0.59 vs ATX-304 2.7 + 0.74, P = 0.03). In TECs, pre-treatment with ATX-304 protected against cisplatin-mediated injury, as measured by lactate dehydrogenase release, MTS cell viability, and cleaved caspase 3 expression. ATX-304 protection against cisplatin was lost in AMPK-null murine embryonic fibroblasts. Metabolomic analysis in TECs revealed that ATX-304 (20 µM, 4 h) altered 66/126 metabolites, including fatty acids, tricarboxylic acid cycle metabolites, and amino acids. Metabolic studies of live cells using the XFe96 Seahorse analyzer revealed that ATX-304 increased the basal TEC oxygen consumption rate by 38%, whereas maximal respiration was unchanged. Thus, ATX-304 protects against cisplatin-mediated kidney injury via AMPK-dependent metabolic reprogramming, revealing a promising therapeutic strategy for AKI.

Moderate exercise decreases the risk for atrial fibrillation (AF), an effect which is probably mediated via exercise-stimulated release of exerkines. β-Aminoisobutyric acid (BAIBA), a novel exerkine, has been reported to provide protective benefits against many cardiovascular diseases, yet its role in AF remains elusive. Herein, using a mouse model of obesity-related AF through high-fat diet (HFD) feeding, we found that 12-week drinking administration of BAIBA (170 mg/kg/day) decreased AF susceptibility in obese mice. Atrial remodeling assessment showed that BAIBA attenuated obesity-induced atrial hypertrophy and interstitial fibrosis, thereby ablating the substrate for AF. Of note, to our knowledge, this is the first report of the direct association of BAIBA and hypertrophy. BAIBA has been reported to be a key regulator of glucose and lipid metabolism, and we found that BAIBA alleviated insulin resistance in obese mice. Transcriptional analysis of metabolism-related genes showed that BAIBA increased the transcription of fatty acids metabolism-related genes in the atria of lean mice but not in that of obese mice. Mechanistic investigation showed that BAIBA stimulated AMP-activated protein kinase (AMPK) signaling in the atria of obese mice and palmitic acid (PA)-treated neonatal rat cardiomyocytes (NRCM), whereas inhibition of AMPK via Compound C attenuated BAIBA-conferred cardioprotection against hypertrophy and insulin resistance in PA-treated NRCM. Collectively, BAIBA attenuates AF susceptibility in obese mice via activated AMPK signaling and resultant improvement of insulin sensitivity, thereby providing perspectives on the potential therapeutic role of BAIBA in AF treatment.

The present study aimed to evaluate the anti-obesity effect of the ethanolic extract of Korean Curcuma longa (CLE) on 3T3-L1 adipocytes and high-fat diet (HFD)-induced obesity in mouse. CLE treatment suppressed lipid accumulation and restored the differentiation-induced changes in adipogenesis- and lipolysis-related proteins by recovering phosphorylation of AMP-activated protein kinase (AMPK). Moreover, in CLE-administered HFD-induced mouse model, body weight and lipid accumulation were reduced, and the changes in factors related to adipogenesis and lipolysis were regulated in the adipose and liver tissues, confirming that these effects were due to a restoration in the phosphorylation of AMPK. In summary, these results provide important insights into the anti-obesity role of CLE by demonstrating its anti-obesity effects on adipocytes and obese mouse models, thereby suggesting the potential application of CLE as a preventive agent for obesity.

Acetaminophen (APAP) overdose has long been considered a major cause of drug-induced liver injury. Ferroptosis is a type of programmed cell death mediated by iron-dependent lipid peroxidation. Endoplasmic reticulum (ER) stress is a systemic response triggered by the accumulation of unfolded or misfolded proteins in the ER. Ferroptosis and ER stress have been proven to contribute to the progression of APAP-induced acute liver injury (ALI). It was reported that salidroside protects against APAP-induced ALI, but the potential mechanism remain unknown. In this study, male C57BL/6 J mice were intraperitoneally (i.p.) injected APAP (500 mg/kg) to induce an ALI model. Salidroside was i.p. injected at a dose of 100 mg/kg 2 h prior to APAP administration. Mice were sacrificed 12 h after APAP injection and the liver and serum of the mice were obtained for histological and biochemistry analysis. AML12 cells were used in in vitro assays. The results indicated that salidroside mitigated glutathione degradation via inhibiting cation transport regulator homolog 1 (CHAC1) to attenuate ferroptosis, and simultaneously suppressing PERK-eIF2α-ATF4 axis-mediated ER stress, thus alleviating APAP-induced ALI. However, PERK activator CCT020312 and overexpression of ATF4 inhibited the protective function of salidroside on CHAC1-mediated ferroptosis. Besides this, activation of the AMPK/SIRT1 signaling pathway by salidroside was demonstrated to have a protective effect against APAP-induced ALI. Interestingly, selective inhibition of SIRT1 ameliorated the protective effects of salidroside on ER stress and ferroptosis. Overall, salidroside plays a significant part in the mitigation of APAP-induced ALI by activating the AMPK/SIRT1 signaling to inhibit ER stress-mediated ferroptosis in the ATF4-CHAC1 axis.

Objective: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. Methods: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. Results: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. Conclusions: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

ABSTRACT: Arginine, one of the conditionally essential AA, has been reported to affect fat synthesis and metabolism in nonruminant animals by influencing adenosine monophosphate activated protein kinase (AMPK) in some organs. In dairy cows, the effect of Arg on milk fat production is not clear, and any potential mechanism that underlies the effect is unknown. We tested the hypothesis that Arg infusion would improve the production of milk fat, and explored possible mechanism that might underlie any effect. We used 6 healthy lactating cows at 20 ± 2 d in milk, in fourth parity, with a body weight of 508 ± 14 kg, body condition score of 3.0 ± 0, and a milk yield of 30.6 ± 1.8 kg/d (mean ± standard deviation). The cows were blocked by days in milk and milk yield and each cow received 3 treatments in a replicated 3 × 3 Latin square design, with each of the experimental periods lasting 7 d with a 14-d washout between each period. The treatments, delivered in random order, were (1) infusion of saline (control); (2) infusion of 0.216 mol/d of l-Arg in saline (Arg); (3) infusion of 0.868 mol/d of l-Ala in saline (the Arg and Ala treatments were iso-nitrogenous) through a jugular vein. On the last day of each experimental period, blood was sampled to measure insulin, nitric oxide, glucose, and nonesterified fatty acid, and the liver and mammary gland were biopsied to measure the expression of genes. Milk yield was recorded, and milk fat percentage was measured daily during each of the experimental periods. The yield and composition of fatty acid (FA) in milk was measured daily on the last 3 d during each of the experimental periods. The data were analyzed using a mixed model with treatment as a fixed factor, and cow, period, and block as random factors. The daily milk yield and milk fat yield when the cows were infused with Arg were 2.2 kg and 76 g, respectively, higher than that in control, and 1.8 kg and 111 g, respectively, higher than that in Ala. When the cows were infused with Arg they had higher concentration and yield of de novo synthesized FA, than when they received the control or Ala infusions, although milk fat percentage, daily feed intake, and the digestibility of nutrients were not affected by treatment. The serum concentration of nitric oxide and insulin were higher during Arg than during control or Ala, with no difference between control and Ala. In the liver, the expression of the genes coding for AMPK (PRKAA1, PRKAB1, and PRKAG1) and genes related to the oxidation of FA were higher during Arg than during control or Ala, whereas in the mammary gland the expression PRKAB1 was lowest, and the expression of genes involved in the synthesis of milk fat were highest, during Arg infusion. The results suggest the intravenous infusion of Arg enhanced the production of milk fat by promoting the de novo synthesis of FA and increasing milk yield.

Aim: Insulin resistance (IR) is a pivotal metabolic disorder associated with type 2 diabetes and metabolic syndrome. This study investigated the potential of hypoxanthine (Hx), a purine metabolite and uric acid precursor, in ameliorating IR and regulating hepatic glucose and lipid metabolism., Methods: We utilized both in vitro IR-HepG2 cells and in vivo diet-induced IR mice to investigate the impact of Hx. The HepG2 cells were treated with Hx to evaluate its effects on glucose production and lipid deposition. Activity-based protein profiling (ABPP) was applied to identify Hx-target proteins and the underlying pathways. In vivo studies involved administration of Hx to IR mice, followed by assessments of IR-associated indices, with explores on the potential regulating mechanisms on hepatic glucose and lipid metabolism., Key Findings: Hx intervention significantly reduced glucose production and lipid deposition in a dose-dependent manner without affecting cell viability in IR-HepG2 cells. ABPP identified key Hx-target proteins engaged in fatty acid and pyruvate metabolism. In vivo, Hx treatment reduced IR severities, as evidenced by decreased HOMA-IR, fasting blood glucose, and serum lipid profiles. Histological assessments confirmed reduced liver lipid deposition. Mechanistic insights revealed that Hx suppresses hepatic gluconeogenesis and fatty acid synthesis, and promotes fatty acid oxidation via the AMPK/mTOR/PPARα pathway., Significance: This study delineates a novel role of Hx in regulating hepatic metabolism, offering a potential therapeutic strategy for IR and associated metabolic disorders. The findings provide a foundation for further investigation into the role of purine metabolites in metabolic regulation and their clinical implications., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Autophagy is a crucial intracellular degradation process that provides energy and supports nutrient deprivation adaptation. However, the mechanisms by which these cells detect lipid scarcity and regulate autophagy are poorly understood. In this study, we demonstrate that protein kinase A (PKA)-dependent lipolysis delays autophagy initiation during short-term nutrient deprivation by inhibiting AMP-activated protein kinase (AMPK). Using coherent anti-Stokes Raman spectroscopy, we visualized free fatty acids (FFAs) in vivo and observed that lipolysis-derived FFAs were used before the onset of autophagy. Our data suggest that autophagy is triggered when the supply of FFAs is insufficient to meet energy demands. Furthermore, PKA activation promotes lipolysis and suppresses AMPK-driven autophagy during early fasting. Disruption of this regulatory axis impairs motility and reduces the lifespan of Caenorhabditis elegans during fasting. These findings establish PKA as a critical regulator of catabolic pathways, prioritizing lipolysis over autophagy by modulating AMPK activity to prevent premature autophagic degradation during transient nutrient deprivation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

AMP-activated protein kinase (AMPK) is a typical sensor of intracellular energy metabolism. Our previous study revealed the role of activated AMPK in the suppression of osteogenic differentiation and traumatic heterotopic ossification, but the underlying mechanism remains poorly understood. The E3 ubiquitin ligase Smurf1 is a crucial regulator of osteogenic differentiation and bone formation. We report here that Smurf1 is primarily SUMOylated at a C-terminal lysine residue (K324), which enhances its activity, facilitating ALK2 proteolysis and subsequent bone morphogenetic protein (BMP) signaling pathway inhibition. Furthermore, SUMOylation of the SUMO E3 ligase PIAS3 and Smurf1 SUMOylation was suppressed during the osteogenic differentiation and traumatic heterotopic ossification. More importantly, we found that AMPK activation enhances the SUMOylation of Smurf1, which is mediated by PIAS3 and increases the association between PIAS3 and AMPK. Overall, our study revealed that Smurf1 can be SUMOylated by PIAS3, Furthermore, Smurf1 SUMOylation mediates osteogenic differentiation and traumatic heterotopic ossification through suppression of the BMP signaling pathway. This study revealed that promotion of Smurf1 SUMOylation by AMPK activation may be implicated in traumatic heterotopic ossification treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phragmites rhizome has been documented to have antioxidative stress and anti-inflammation properties in several pathogenic models. We aimed to investigate the therapeutic effects and molecular mechanism of the aqueous extract of Phragmites rhizome (AP) in in vitro and in vivo hepatic steatosis models. We found that treatment with AP dose-dependently reduced lipid accumulation and endoplasmic reticulum (ER) stress in palmitate-treated human primary hepatocytes and attenuated hepatic steatosis and ER stress in high-fat diet (HFD)-fed mice. AMPK phosphorylation and autophagy markers in hepatocytes were augmented by AP. siRNA targeting AMPK or 3-methyladenine (3-MA) abolished the impacts of AP on palmitate-treated hepatocytes. These results suggest that AP treatment suppresses hepatic ER stress through AMPK/autophagy signaling, leading to attenuation of hepatic steatosis. The present study may provide a novel therapeutic approach for treating nonalcoholic fatty liver disease (NAFLD) and ER stress-mediated metabolic diseases using natural substances.

Background & Aims: Metabolic imbalance and inflammation are common features of chronic liver diseases. Molecular factors controlling these mechanisms represent potential therapeutic targets. CD73 is the major enzyme that dephosphorylates extracellular adenosine monophosphate (AMP) to form the anti-inflammatory adenosine. CD73 is expressed on pericentral hepatocytes, which are important for long-term liver homeostasis. We aimed to determine if CD73 has nonredundant hepatoprotective functions. Methods: Liver-specific CD73 knockout (CD73-LKO) mice were generated by targeting the Nt5e gene in hepatocytes. The CD73-LKO mice and hepatocytes were characterized using multiple approaches. Results: Deletion of hepatocyte Nt5e resulted in an approximately 70% reduction in total liver CD73 protein (P < .0001). Male and female CD73-LKO mice developed normally during the first 21 weeks without significant liver phenotypes. Between 21 and 42 weeks, the CD73-LKO mice developed spontaneous-onset liver disease, with significant severity in male mice. Middle-aged male CD73-LKO mice showed hepatocyte swelling and ballooning (P < .05), inflammation (P < .01), and variable steatosis. Female CD73-LKO mice had lower serum albumin levels (P < .05) and increased inflammatory genes (P < .01), but did not show the spectrum of histopathologic changes in male mice, potentially owing to compensatory induction of adenosine receptors. Serum analysis and proteomic profiling of hepatocytes from male CD73-LKO mice showed significant metabolic imbalance, with increased blood urea nitrogen (P < .0001) and impairments in major metabolic pathways, including oxidative phosphorylation and AMP-activated protein kinase (AMPK) signaling. There was significant hypophosphorylation of AMPK substrates in CD73-LKO livers (P < .0001), while in isolated hepatocytes treated with AMP, soluble CD73 induced AMPK activation (P < .001). Conclusions: Hepatocyte CD73 supports long-term metabolic liver homeostasis through AMPK in a sex-dependent manner. These findings have implications for human liver diseases marked by CD73 dysregulation.

Background: Myocardial ischemia-reperfusion(I/R)injury constitute the fundamental pathophysiology of acute myocardial infarction (AMI). Ischemic heart releases macrophage migration inhibitory factor (MIF), which activates MIF- AMPK signaling pathway. Depression is a significant risk factor for AMI. In a state of depression, peripheral expression of cannabinoid receptor 2 (CNR2) genes was downregulated., Aims: We investigated the mechanism by which depression exacerbates myocardial I/R injury through the CNR2 and MIF-AMPK signaling pathways., Methods: We established mouse models of depression and myocardial I/R. Left ventricular function was assessed using cardiac ultrasound and TTC staining. The protein levels of myocardial CNR2, MIF, AMPK, and ACC were determined by Western blot, while the expression level of CNR2 was measured using RT-qPCR. Additionally, MIF content in peripheral blood was quantified using ELISA., Results: After I/R, the expression level of CNR2 was found to be lower in the depression group, leading to a deterioration in left heart function. Depressed mice exhibited lower secretion of MIF, accompanied by a decrease in the activation of the MIF-AMPK signaling pathway. However, injection of CNR2 agonist JWH133 prior to ischemia increased the activation of the MIF-AMPK signaling pathway, while CNR2 inhibitor AM630 decreased the activation., Limitations: Further research is needed to investigate the specific neuroendocrine mechanism affecting myocardial CNR2 expression in depression. And these experimental conclusions require further verification at the cellular level., Conclusions: The activation of CNR2 in myocardium following I/R is impeded by depression, thereby exacerbating myocardial I/R injury through attenuation of the MIF-AMPK signaling pathway activation., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

AMP-activated protein kinase (AMPK) is essential for maintaining energy balance and has a crucial role in various inflammatory pathways. In this study, AMPK levels positively correlated with many inflammatory indexes in rheumatoid arthritis (RA) patients, especially in the affected synovium. In RA sera, a positive correlation between phosphorylated (p-)AMPK-α1 levels and DAS28 (disease activity score 28) activity (r = 0.270, p < 0.0001) was found. Similarly, a positive correlation was observed between AMPK-α1 and tumor necrosis factor α (TNF-α) levels (r = 0.460, p = 0.0002). Differentially expressed genes between osteoarthritis (OA) and RA synovium from NCBI GEO profiles and our RNA sequencing data suggested activation of metabolic pathways specific to RA-fibroblast-like synoviocytes (FLSs). AMPK-α1 was highly expressed in the synovium of RA but not OA patients. An AMPK activator, metformin, inhibited FLS proliferation at higher but not lower concentrations, whereas the inhibitor dorsomorphin promoted the proliferation of RA-FLSs. Interestingly, both metformin and dorsomorphin inhibited the migration of RA-FLSs. After metformin treatment, expression of interleukin 6 (IL-6), TNF-α, and IL-1β were significantly downregulated in RA-FLSs; however, increased expression of p-AMPK-α1, protein kinase A (PKA)-α1, and HAPLN1 (hyaluronan and proteoglycan link protein 1) was observed. Increased levels of HAPLN1 in RA-FLSs by an AMPK activator could potentially be beneficial in protecting the joints. Hence, our results demonstrate the potential of an AMPK activator as a therapeutic for RA.

The purpose of the current study was to observe the anti-diabetic and anti-obesity effects of blue honeysuckle (BH; Lonicera caerulea L.) extracts on obese diabetic mice. BH extracts dose-dependently and significantly inhibited abnormal endocrine pancreas histopathological changes. Moreover, favorable anti-diabetic effects and non-alcoholic fatty liver disease, nephropathy, and hyperlipidemia refinement activities in BH extracts were shown to be dose dependent. Particularly, BH extract 200 mg/kg showed favorable modulatory activities on PPAR γ and AMPK in hepatic and adipose tissues as comparable to those of positive control, conjugated linoleic acid (CLA) administration. Results from the current study suggest that BH extracts could be a potential functional ingredient for anti-obesity and anti-diabetic effects through the inhibition of lipogenesis and hepatic glucose production by regulating lipid metabolism-related mRNA expressions.

The prevalence of nonalcoholic steatohepatitis (NASH) caused by estrogen deficiency increased sharply in recent decades and has become a major threat to liver health in postmenopausal women. There is no effective strategy to control the incidence and development of NASH. Dehydroepiandrosterone (DHEA) is the most abundant circulating steroid with immune and metabolic regulatory properties, and its level markedly declines with increasing age in humans. Importantly, DHEA can convert into active sex hormones depending on the local needs of target tissues with little diffusion, which serves to avoid systemic side-effects from other tissues’ exposure to estrogen. Here, we found that DHEA prevented the incidence and development of NASH, which is characterized by the reduction of hepatic steatosis, fibrosis, and inflammation in female mice fed with high-fat/high-cholesterol diets and effectively attenuated lipid accumulation, inflammatory response, and oxidative stress in palmitic acid-challenged hepatocytes. Mechanistically, in vitro and in vivo studies showed that the anti-NASH function of DHEA depended on its biotransformation into estrogen rather than androgen, and which up-regulates the expression of G protein-coupled estrogen receptor (GPR30), a non-classical estrogen receptor. The activation of GPR30-mediated AMP-activated protein kinase signaling is a necessary prerequisite for the alleviative effects of DHEA on NASH. Collectively, our data show the mechanisms of DHEA treatment and its effects on NASH that were previously overlooked; the data also show that GPR30 can be used as a target for treating lipid metabolism disorders and related diseases, such as NASH. Furthermore, these findings have the potential to help researchers develop new strategies for preventing NASH in postmenopausal women.

Aims: Differentiation-inducing factor-1 (DIF-1) is a polyketide produced by Dictyostelium discoideum that inhibits growth and migration, while promoting the differentiation of Dictyostelium stalk cells through unknown mechanisms. DIF-1 localizes in stalk mitochondria. In addition to its effect on Dictyostelium, DIF-1 also inhibits growth and migration, and induces mitochondrial fission followed by mitophagy in mammalian cells, at least in part by activating AMP-activated protein kinase (AMPK). In a previous study, we found that DIF-1 binds to mitochondrial malate dehydrogenase (MDH2) and inhibits its activity in HeLa cells. In the present study, we investigated whether MDH2 serves as a pharmacological target of DIF-1 in mammalian cells., Main Methods: To examine the enzymatic activity of MDH, mitochondrial morphology, and molecular mechanisms of DIF-1 action, we conducted an MDH reverse reaction assay, immunofluorescence staining, western blotting, and RNA interference using mammalian cells such as human umbilical vein endothelial cells, human cervical cancer cells, mouse endothelial cells, and mouse breast cancer cells., Key Findings: DIF-1 inhibited mitochondrial but not cytoplasmic MDH activity. Similar to DIF-1, LW6, an authentic MDH2 inhibitor, induced phosphorylation of AMPK, resulting in the phosphorylation of acetyl-CoA carboxylase (ACC) and the dephosphorylation of p70 S6 kinase with approximately the same potency. DIF-1 and LW6 induced mitochondrial fission. Furthermore, MDH2 knockdown using siRNA reproduced the DIF-1 action on the AMPK signaling and mitochondrial morphology. Conversely, an AMPK inhibitor prevented DIF-1-induced mitochondrial fission., Significance: We propose that MDH2 is a mammalian target of DIF-1 for the activation of AMPK and induction of mitochondrial fission., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning. Methods: Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and β subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a β3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function. Results: Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and β2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and β2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice. Conclusions: These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for β3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.

Objective: Skeletal muscle is an attractive target for blood glucose-lowering pharmacological interventions. Oral dosing of small molecule direct pan-activators of AMPK that bind to the allosteric drug and metabolite (ADaM) site, lowers blood glucose through effects in skeletal muscle. The molecular mechanisms responsible for this effect are not described in detail. This study aimed to illuminate the mechanisms by which ADaM-site activators of AMPK increase glucose uptake in skeletal muscle. Further, we investigated the consequence of co-stimulating muscles with two types of AMPK activators i.e., ADaM-site binding small molecules and the prodrug AICAR. Methods: The effect of the ADaM-site binding small molecules (PF739 and 991), AICAR or co-stimulation with PF739 or 991 and AICAR on muscle glucose uptake was investigated ex vivo in m. extensor digitorum longus (EDL) excised from muscle-specific AMPKα1α2 as well as whole-body AMPKγ3-deficient mouse models. In vitro complex-specific AMPK activity was measured by immunoprecipitation and molecular signaling was assessed by western blotting in muscle lysate. To investigate the transferability of these studies, we treated diet-induced obese mice in vivo with PF739 and measured complex-specific AMPK activation in skeletal muscle. Results: Incubation of skeletal muscle with PF739 or 991 increased skeletal muscle glucose uptake in a dose-dependent manner. Co-incubating PF739 or 991 with a maximal dose of AICAR increased glucose uptake to a greater extent than any of the treatments alone. Neither PF739 nor 991 increased AMPKα2β2γ3 activity to the same extent as AICAR, while co-incubation led to potentiated effects on AMPKα2β2γ3 activation. In muscle from AMPKγ3 KO mice, AICAR-stimulated glucose uptake was ablated. In contrast, the effect of PF739 or 991 on glucose uptake was not different between WT and AMPKγ3 KO muscles. In vivo PF739 treatment lowered blood glucose levels and increased muscle AMPKγ1-complex activity 2-fold, while AMPKα2β2γ3 activity was not affected. Conclusions: ADaM-site binding AMPK activators increase glucose uptake independently of AMPKγ3. Co-incubation with PF739 or 991 and AICAR potentiates the effects on muscle glucose uptake and AMPK activation. In vivo, PF739 lowers blood glucose and selectively activates muscle AMPKγ1-complexes. Collectively, this suggests that pharmacological activation of AMPKγ1-containing complexes in skeletal muscle can increase glucose uptake and can lead to blood glucose lowering.

The aim of this study was to explore the possible mechanisms of Ficus carica leaf (FCL) extract in suppressing hepatic gluconeogenesis in diabetic mice. Diabetic mice (streptozotocin-induced) received 1 g/kg of FCL extract twice a day for 6 weeks. Fasting blood glucose levels were measured and a 2-h oral glucose tolerance test was conducted. AMP-activated protein kinase (AMPK), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) expression was examined. HepG2 hepatocytes were treated with FCL extract and an AMPK inhibitor (compound C) or agonist (AICAR), and PEPCK, G6pase, PGC-1α, AMPK, forkhead transcription factor O1 (FOXO1), and hepatic nuclear factor 4α (HNF4α) expression was determined. The results showed that FCL extract inhibited the expression of PEPCK and G6Pase in the liver of diabetic mice and HepG2 hepatocytes. FCL extract activated AMPK and decreased PGC-1α, HNF4α, and FOXO1 expression. The AMPK inhibitor attenuated those effects through inhibiting gluconeogenesis, while the AMPK agonist partially enhanced gluconeogenesis. In conclusion, FCL extract inhibits hepatic gluconeogenesis via activation of AMPK and down-regulation of gluconeogenic enzymes.

Non-alcoholic fatty liver disease (NAFLD) has been a leading cause of chronic metabolic disease, seriously posing healthy burdens to the public, whereas interventions available for it are limited to date. Patchouli oil had been reported to attenuate hepatic steatosis in our previous study. β-patchoulene (β-PAE) is a representative component separated from patchouli oil with multiple activities, but its effect against NAFLD is still unknown. To investigate the effect and potential mechanism of β-PAE on NAFLD, we used high fat diet (HFD) in vivo and free fatty acid (FFA) in vitro to induce hepatic steatosis in rats and L02 cells, respectively. Histological examination was evaluated via Hematoxylin-eosin and oil red O staining. The parameters for hepatic steatosis were estimated via biochemical kits, western blotting and quantitative real-time PCR. Compound C, the inhibitor of AMPK, was applied further to examine the precise mechanism of β-PAE on NAFLD. Our results indicated that β-PAE significantly attenuated HFD-induced weight gain, hepatic injury, lipid deposition in serum and hepatic tissue as well as FFA induced-lipid accumulation. Besides, β-PAE markedly improved the expression of AMP-activated protein kinase (AMPK) and its downstream factors which correlate with hepatic lipid synthesis and oxidation in vivo and in vitro. Nevertheless, Compound C abrogated the benefits derived from β-PAE in L02 cells. In conclusion, these results suggest that β-PAE exerts AMPK agonist-like effect to regulate hepatic lipid synthesis and oxidation, eventually prevent NAFLD progression.

The present study was conducted to identify anti-fatigue peptides in porcine liver hydrolysate (LH) and to evaluate their effects. Peptides in LH were fractionated into hydrophilic and hydrophobic fractions and further into peptides with amino groups (Pep-NH2) and pyroglutamyl peptide fractions (pE). Low dose of hydrophobic Pep-NH2 fraction reversed the decrease in locomotor activity after forced walking in a mouse model upon intraperitoneal (i.p.) administration. The anti-fatigue effects of i.p. injection of Asp-Val, Asp-Leu, and Asp-Phe with α and β peptide bonds and D- and L- asparatyl residue, which appeared in blood after oral administration of LH, were examined. In particular, Asp-Leu (Lβ), Asp-Phe (Dα), and Asp-Phe (Lα) exerted anti-fatigue effects at a low concentration (0.03 mg/kg). We found that Asp-Phe (Lα), but not other dipeptides, activated adenosine monophosphate-activated protein kinase (AMPK) in the liver, indicating that Asp-Phe (Lα) could induce recovery from fatigue via AMPK activation.

Objective: Glycogen is a major energy reserve in liver and skeletal muscle. The master metabolic regulator AMP-activated protein kinase (AMPK) associates with glycogen via its regulatory β subunit carbohydrate-binding module (CBM). However, the physiological role of AMPK-glycogen binding in energy homeostasis has not been investigated in vivo. This study aimed to determine the physiological consequences of disrupting AMPK-glycogen interactions. Methods: Glycogen binding was disrupted in mice via whole-body knock-in (KI) mutation of either the AMPK β1 (W100A) or β2 (W98A) isoform CBM. Systematic whole-body, tissue and molecular phenotyping was performed in KI and respective wild-type (WT) mice. Results: While β1 W100A KI did not affect whole-body metabolism or exercise capacity, β2 W98A KI mice displayed increased adiposity and impairments in whole-body glucose handling and maximal exercise capacity relative to WT. These KI mutations resulted in reduced total AMPK protein and kinase activity in liver and skeletal muscle of β1 W100A and β2 W98A, respectively, versus WT mice. β1 W100A mice also displayed loss of fasting-induced liver AMPK total and α-specific kinase activation relative to WT. Destabilisation of AMPK was associated with increased fat deposition in β1 W100A liver and β2 W98A skeletal muscle versus WT. Conclusions: These results demonstrate that glycogen binding plays critical roles in stabilising AMPK and maintaining cellular, tissue and whole-body energy homeostasis.

Rice bran is a by-product of rice milling, which contains variety of nutrients, and has been reported to possess various activities. Here, we used high fat (HF) diet induced mice to investigate the effects of rice bran phenolic extracts (RBPE) on lipid metabolism. Sixteen-week intake of RBPE (200 mg/kg/d and 400 mg/kg/d) significantly improved the serum lipid profiles of HF diet-fed mice and ameliorated HF diet-induced liver steatosis. Further study showed that RBPE might regulate lipid metabolism through increasing the levels of serum adiponectin, and thereby promoting the activation of 5′ adenosine monophosphate-activated protein kinase α (AMPKα) in liver. Taken together, RBPE might regulate lipid metabolism through adiponectin-mediated AMPK activation and the subsequent reduction of lipid biosynthesis together with the increase of fatty acid oxidation in liver. These results provide us new evidence of the health benefits of rice bran.

The excessive generation of reactive oxygen species (ROS) plays crucial roles in the development of acute liver injury. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is responsible for the robust production of ROS under inflammatory circumstance, but the pathological roles of NOX and the pharmacological significance of NOX inhibitor in acute liver injury remains unclear. In the present study, the potential roles of NOX in acute liver injury were investigated in a mouse model with lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced acute liver injury. The results indicated that LPS/D-Gal exposure time-dependently increased the level of ROS in liver tissue. Pretreatment with the NOX inhibitor apocynin suppressed LPS/D-Gal induced upregulation of ROS, 8-hydroxy-2-deoxyguanosine (8-OH-dG), protein carbonyl content and thiobarbituric acid reactive substances (TBARS). Pretreatment with apocynin also suppressed LPS/D-Gal-induced elevation of aminotransferase, alleviated histological abnormalities, inhibited the production of pro-inflammatory cytokine tumor necrosis factor α (TNF-α), blocked the activation of caspase cascade, reduced the count of TUNEL-positive cells and prevented LPS/D-Gal-induced mortality. Interestingly, post insult treatment with apocynin also suppressed LPS/D-Gal-induced oxidative stress, hepatocyte apoptosis, liver damage but improved the survival rate. Mechanistically, posttreatment with apocynin prohibited LPS/D-Gal-induced activation of the late stage pro-apoptotic AMP-activated protein kinase (AMPK)/c-Jun N-terminal kinase (JNK) pathway. Post-insult treatment with the antioxidant N-acetylcysteine also resulted in suppressed activation of AMPK/JNK, mitigated apoptosis and alleviated liver injury. These data suggest that NOX-derived ROS might be a crucial late stage detrimental factor in LPS/D-Gal-induced acute liver injury via promoting the activation of the pro-apoptotic AMPK/JNK pathway, and the NOX inhibitor might have important value in the pharmacological intervention of inflammation-base liver damage.

To appraise the effects and involved mechanism of Dendropanax morbifera leaves extract (DLE) on diabetes and retinal degeneration, in this study, male C57BLKS/J-m+/db and five groups of db/db mice were treated with 0.5% CMC or metformin or three doses of DLE for 6 weeks. Administration of DLE stimulated AMP-activated protein kinase in the liver, skeletal muscle and adipose tissue, leading to the improvement of gene and protein expression related with insulin resistance and glucose homeostasis in db/db mice. Morphological changes and insulin depletion of pancreatic islets were also attenuated by treatment of DLE. Moreover, DLE treatment prevented the deterioration of retina by regulating not only the gene expression related with apoptosis and neovascularization, but also the level of activator protein-1 in retina. Therefore, DLE improves diabetic phenotype and retinal degeneration, suggesting that DLE could be a prospective agent for preventing diabetes and related eye complication.

The role of serine in metabolic processes has recently attracted much attention as it could be used for the biosynthesis of purine, sphingolipids, and glutathione. The present study was conducted to investigate the effects of serine on LPS-induced intestinal inflammation and barrier dysfunction. The results showed that pretreatment with serine prevented the LPS-induced inflammatory response and oxidative stress both in vivo and in vitro. Moreover, serine maintained intestinal barrier function and permeability. In addition, we found that serine improved glutathione synthesis and AMP-activated protein kinase (AMPK) activity. However, following the small interfering RNA (siRNA) inhibition of p53 expression or inhibition of glutathione synthesis in IPEC-1 cells, the protective effects of serine on LPS-induced damage were diminished. Our results indicated that serine may prevent LPS-induced damage in the intestine by improving glutathione synthesis and AMPK activity in a p53-dependent manner.

A high intake of products containing fructose is known to mediate insulin resistance. In the liver, AMPD2, an isoform of AMPD, has important glucose metabolic homeostasis functions including maintenance of AMP-activated protein kinase (AMPK). We speculated that AMPD2 induces impaired glucose tolerance in individuals who consume a high-fructose diet. We gave either a normal-chow (NCD) or high-fructose (HFrD) diet for 40 days to 8-week-old male wild-type (WT) and Ampd2−/− homozygote (A2−/−) C57BL/6 mice. A glucose tolerance test (GTT) and pyruvate tolerance test (PTT) were used to evaluate glucose metabolism. In addition, gluconeogenesis and glycolysis enzymes, and AMPK phosphorylation in the liver were investigated. With consumption of the HFrD, A2−/− mice showed enhanced glucose tolerance in GTT and PTT results as compared to the WT mice, which were independent of changes in body weight. Also, the levels of phosphoenolpyruvate carboxy kinase and glucose-6-phosphatase (hepatic gluconeogenic enzymes) were significantly reduced in A2−/− as compared to WT mice. The hepatic glycolytic enzymes glucokinase, phosphofructokinase, and pyruvate kinase were also examined, though there were no significant differences between genotypes in regard to both mRNA expression and protein expression under HFrD. Surprisingly, hepatic AMPK phosphorylation resulted in no changes in the A2−/− as compared to WT mice under these conditions. Our results indicated that Ampd2–deficient mice are protected from high fructose diet-induced glycemic dysregulation, mainly because of gluconeogenesis inhibition, and indicate a novel therapeutic target for type 2 diabetes mellitus.

AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism, is a potential target for type 2 diabetes. Although extensive in vitro studies have revealed the complex regulation of AMPK, much remains unknown about the regulation in vivo. We therefore developed transgenic mice expressing a highly sensitive fluorescence resonance energy transfer (FRET)-based biosensor for AMPK, called AMPKAR-EV. AMPKAR-EV allowed us to readily examine the role of LKB1, a canonical stimulator of AMPK, in drug-induced activation and inactivation of AMPK in vitro. In transgenic mice expressing AMPKAR-EV, the AMP analog AICAR activated AMPK in muscle. In contrast, the antidiabetic drug metformin activated AMPK in liver, highlighting the organ-specific action of AMPK stimulators. Moreover, we found that AMPK was activated primarily in fast-twitch muscle fibers after tetanic contraction and exercise. These observations suggest that the AMPKAR-EV mouse will pave a way to understanding the heterogeneous responses of AMPK among cell types in vivo.

This study investigated the effect and the underlying mechanism of ginseng berry (GB) in a mouse model of type 2 diabetes, supplied with 0.05% or 0.1% dietary GB for 12 weeks. GB significantly improved hyperglycemia and insulin resistance, as demonstrated by the blood glucose and insulin levels, HOMA-IR, and GTT. Moreover, the expression of gluconeogenic genes such as PEPCK and G6Pase and the hepatic metabolites involved in the pathway of gluconeogenesis, such as glucose-6-phosphate and dihydroxyacetone phosphate, were significantly reduced by GB. Hepatic steatosis was also significantly ameliorated; TG content and expression of lipogenic enzymes and transcriptional factors such as FAS, ACC, and SREBP1 were reduced by GB. Simultaneously, AMPK phosphorylation was increased both in fatty liver and in lipid-accumulated HepG2 cells by GB. In conclusion, GB improved hyperglycemia by downregulating hepatic glucose production and hepatic steatosis, and AMPK appeared to be an important regulator of these effects.

This study revealed the activity of a Ligularia fischeri extract (LFE) and its constituents to protect against obesity-induced NAFLD using in vitro and in vivo models. In HepG2 cells, LFE and its chemical constituent 3,4-dicaffeoylquinic acid inhibited lipid accumulation by altering lipid metabolism towards fatty acid oxidation through activation of the AMP-activated protein kinase (AMPK) pathway. In high-fat diet-induced obese C57BL/6J mice, oral administration of LFE attenuated the progression of obesity and improved signs of insulin resistance. LFE treatment attenuated development of NAFLD by decreasing fat droplet accumulation and the triacylglycerol content. In vivo activity of LFE was displayed through regulation of hepatic lipid metabolism and activation of hepatic AMPK. Furthermore, LFE was protected against NAFLD by lowering the level of lipid peroxidation through activation of the antioxidant defence system via NRF2 activation. These results indicate that LFE is a potent agent to improve NAFLD.

AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFβ signaling.

Introduction: Focal segmental glomerulosclerosis (FSGS), the most common primary glomerular disease leading to end-stage kidney disease (ESKD), is characterized by podocyte injury and depletion, whereas minimal change disease (MCD) has better outcomes despite podocyte injury. Identifying mechanisms capable of preventing podocytopenia during injury could transform FSGS to an "MCD-like" state. Preclinical data have reported conversion of an MCD-like injury to one with podocytopenia and FSGS by inhibition of AMP-kinase (AMPK) in podocytes. Conversely, in FSGS, AMPK-activation using metformin (MF) mitigated podocytopenia and azotemia. Observational studies also support beneficial effects of MF on proteinuria and chronic kidney disease (CKD) outcomes in diabetes. A randomized controlled trial (RCT) to test MF in podocyte injury with FSGS has not yet been conducted., Methods: We report the rationale and design of phase 2, double-blind, placebo-controlled RCT evaluating the efficacy and safety of MF as adjunctive therapy in FSGS. By randomizing 30 patients with biopsy-confirmed FSGS to MF or placebo (along with standard immunosuppression), we will study mechanistic biomarkers that correlate with podocyte injury or depletion and evaluate outcomes after 6 months. We specifically integrate novel urine, blood, and tissue markers as surrogates for FSGS progression along with unbiased profiling strategies., Results and Conclusion: Our phase 2 trial will provide insight into the potential efficacy and safety of MF as adjunctive therapy in FSGS-a crucial step to developing a larger phase 3 study. The mechanistic assays here will guide the design of other FSGS trials and contribute to understanding AMPK activation as a potential therapeutic target in FSGS. By repurposing an inexpensive agent, our results will have implications for FSGS treatment in resource-poor settings., (© 2024 International Society of Nephrology. Published by Elsevier Inc.)

The Western meat-rich diet is both high in protein and fat. Although the hazardous effect of a high fat diet (HFD) upon liver structure and function is well recognized, whether the co-presence of high protein intake contributes to, or protects against, HF-induced hepatic injury remains unclear. Increased intake of branched chain amino acids (BCAA, essential amino acids compromising 20% of total protein intake) reduces body weight. However, elevated circulating BCAA is associated with non-alcoholic fatty liver disease and injury. The mechanisms responsible for this quandary remain unknown; the role of BCAA in HF-induced liver injury is unclear. Utilizing HFD or HFD + BCAA models, we demonstrated BCAA supplementation attenuated HFD-induced weight gain, decreased fat mass, activated mammalian target of rapamycin (mTOR), inhibited hepatic lipogenic enzymes, and reduced hepatic triglyceride content. However, BCAA caused significant hepatic damage in HFD mice, evidenced by exacerbated hepatic oxidative stress, increased hepatic apoptosis, and elevated circulation hepatic enzymes. Compared to solely HFD-fed animals, plasma levels of free fatty acids (FFA) in the HFD + BCAA group are significantly further increased, due largely to AMPKα2-mediated adipocyte lipolysis. Lipolysis inhibition normalized plasma FFA levels, and improved insulin sensitivity. Surprisingly, blocking lipolysis failed to abolish BCAA-induced liver injury. Mechanistically, hepatic mTOR activation by BCAA inhibited lipid-induced hepatic autophagy, increased hepatic apoptosis, blocked hepatic FFA/triglyceride conversion, and increased hepatocyte susceptibility to FFA-mediated lipotoxicity. These data demonstrated that BCAA reduces HFD-induced body weight, at the expense of abnormal lipolysis and hyperlipidemia, causing hepatic lipotoxicity. Furthermore, BCAA directly exacerbate hepatic lipotoxicity by reducing lipogenesis and inhibiting autophagy in the hepatocyte.

Hepatic steatosis and insulin resistance are highly prevalent and play a vital role in the development of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). The present study examined the protective effects of naturally occurring flaxseed lignan secoisolariciresinol diglucoside (SDG) on these metabolic disorders resulting from high fat diet (HFD). SDG (0.05%, w/w) supplementation for 16 weeks attenuated body weight gain, hyperlipidaemia and hepatic steatosis in HFD (60% kcal from fat)-challenged C57BL/6J mice. Moreover, SDG lowered fasting serum glucose and insulin, and improved homeostasis model assessment of insulin resistance (HOMA-IR) index, glucose tolerance, and insulin response in HFD-fed mice. Furthermore, SDG prevented HFD-induced inhibition of protein kinase B, insulin receptor substrate-1 and AMP-activated protein kinase activation in the liver of HFD-fed mice. These findings indicate that SDG supplementation alleviates hepatic steatosis and insulin resistance, at least in part, by enhancing insulin signalling and AMPK activation.

Background: In many cancer cells, L-type amino acid transporter 1 (LAT1) transports neutral amino acids with bulky side chain, which activate mammalian target of rapamycin (mTOR) to cause cell proliferation. An anti-diabetic drug, metformin, has been shown to activate AMP-activated protein kinase (AMPK), which leads to inhibition of mTOR. LAT1 inhibition in combination with metformin could result in more prominent suppression of mTOR activity. Purpose: Anti-proliferative effect of a newly developed LAT1 specific inhibitor JPH203 in combination with metformin is evaluated in 2 head and neck cancer cell lines, Ca9-22 and HEp-2 cells and in nude mice inoculated with Ca9-22 cells. Results and Discussion: By MTT assay, 0.5 mM metformin inhibited proliferation of Ca9-22 cells to 70% of control. In the presence of 100 μM JPH203, proliferation of Ca9-22 cells was inhibited to 60% of control. By combining these 2 drugs, proliferation of Ca9-22 was significantly inhibited to 40% of control. However, this regimen was not very effective against HEp-2 cells. This combination also suppressed in vivo growth of Ca9-22 cells in a xenotransplant model. A combination of anti-LAT1 drug with metformin may be an effective anti-proliferative therapy for certain subsets of cancers.

Obesity and nonalcoholic fatty liver disease (NAFLD) are characterized by excessive accumulation of triacylglycerol and dysregulation of lipid metabolism. This study investigated the effects of Youngia denticulata extract (YDE) on obesity, hepatic steatosis, inflammation, and antioxidant defences in high-fat diet (HFD)-induced obesity in mice and the molecular mechanisms activated by YDE in HepG2 hepatoma cells. YDE reduced lipid accumulation in HepG2 cells by regulating the expression of lipid metabolism-related enzymes and activating AMP-activated protein kinase (AMPK) signalling. Oral administration of YDE (25 or 100 mg/kg day) to HFD-fed mice lowered the total body weight, adipose and liver tissue weight, insulin resistance, fat deposition, and serum lipids and leptin levels, and augmented the antioxidant enzymes in serum. YDE also altered the expression of lipid metabolism-related enzymes and activated AMPK signalling in liver and white adipose tissue. These results indicate that YDE may be a potent anti-obesity agent.

The triterpenes 3β,25-dihydroxy-7β-methoxycucurbita-5,23(E)-diene (DHM) and 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al (THC) were previously isolated from Momordica charantia (bitter melon) and identified as hypoglycaemic principles. This study further investigated their hypoglycaemic mechanisms. FL83B cells were treated with tumour necrosis factor-α to result in insulin resistance, a feature of type 2 diabetes. DHM and THC increased the tyrosine phosphorylation of insulin receptor substrate isoform 1 and the phosphorylation of Akt only in the presence of insulin in insulin-resistant cells, suggesting that they are insulin sensitizers. However, they enhanced the phosphorylation of AS160 (Akt substrate of 160 kDa), the migration of glucose transporter-4 and the glucose uptake of insulin-resistant cells in the absence of insulin, suggesting that they can substitute for insulin to promote glucose clearance. The insulin substitution function was blocked by an AMP-activated protein kinase (AMPK) inhibitor, whereas the insulin-sensitizing function may involve the inhibition of protein-tyrosine phosphatase-1B (PTP-1B). The IC50 of DHM and THC to PTP-1B is 92.84 µM and 25.42 µM, respectively. In summary, DHM and THC have insulin-sensitizing and insulin-substitution functions, which are likely correlated with their effects on inhibiting PTP-1B and activating AMPK, respectively.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pumpkin has been suggested to have an antidiabetic effect, but the bioactives involved and molecular mechanisms mediating its hypoglycaemic activity are not clear. This study characterized some of the hypoglycaemic constituents in the crude extract of the Cucurbita moschata stem and investigated the mechanisms of their action. The C. moschata stem extract showed a hypoglycaemic effect in vivo in streptozotocin-induced diabetic mice. Several hypoglycaemic fractions of the extract were identified via cell-based screening, and two of them were confirmed to have hypoglycaemic activities in vivo. Ten compounds were afforded from the 2 fractions. Compounds 9 [(22E,24R)-24-methyl-6β-methoxy-5α-cholesta-7,22-diene-3β,5-diol] and 10 [3β-hydroxy-(22E,24R)-ergosta-5,8,22-trien-7-one] showed an insulin-like activity in normal cells that may be mediated by AMP-activated protein kinase. Compounds 4 (ferulic acid), 8 (syringaresinol) and 9 exhibited an insulin sensitizing and/or insulin substitution function in insulin-resistant cells. Thus, the C. moschata stem contains compounds with potential for managing type 1 or type 2 diabetes.

Recent studies on animals have suggested that vinegar consumption may confer an antiobesity effect through the activation of the AMP-activated protein kinase (AMPK) signaling pathway. However, mechanisms of action in humans remain largely unknown. A randomized, double-blind, placebo-controlled trial was performed to examine whether a pomegranate vinegar (PV) beverage alleviates adiposity in overweight subjects, with emphasis on AMPK activation. Seventy-eight overweight women (BMI ≥ 25) were randomly assigned to receive either PV (1.5 g acetic acid and 700 μg ellagic acid/200 mL/day) or a placebo for 8 weeks. The PV reduced visceral adipose tissue, as measured by computed tomography (P = 0.037), and enhanced AMPK phosphorylation (P = 0.013) compared with the placebo group. The PV tended to suppress downstream gene expression, such as that of sterol regulatory element binding protein-1c and acetyl coenzyme carboxylase, in adipose tissue. Together, these data suggest that PV is an excellent AMPK activator and may exert beneficial effects on adiposity.

Ginseng saponins exert various important pharmacological effects with regard to the control of many diseases, including cancer. In this study, the anticancer effect of ginsenosides on human cancer cells was investigated and compared. Among the tested compounds, ginsenoside-Rh2 displays the highest inhibitory effect on cell viability in HepG2 cells. Ginsenoside-Rh2, a ginseng saponin isolated from the root of Panax ginseng, has been suggested to have potential as an anticancer agent, but the underlying mechanisms remain elusive. In the present study, we have shown that cancer cells have differential sensitivity to ginsenoside-Rh2-induced apoptosis, raising questions regarding the specific mechanisms responsible for the discrepant sensitivity to ginsenoside-Rh2. In this study, we demonstrate that AMP-activated protein kinase (AMPK) is a survival factor under ginsenoside-Rh2 treatment in cancer cells. Cancer cells with acute responsiveness of AMPK display a relative resistance to ginsenoside-Rh2, but cotreatment with AMPK inhibitor resulted in a marked increase of ginsenoside-Rh2-induced apoptosis. We also observed that p38 MAPK (mitogen-activated protein kinase) acts as another survival factor under ginsenoside-Rh2 treatment, but there was no signaling crosstalk between AMPK and p38 MAPK, suggesting that combination with inhibitor of AMPK or p38 MAPK can augment the anticancer potential of ginsenoside Rh2.

Acetaminophen (APAP) overdose has long been considered a major cause of drug-induced liver injury. Ferroptosis is a type of programmed cell death mediated by iron-dependent lipid peroxidation. Endoplasmic reticulum (ER) stress is a systemic response triggered by the accumulation of unfolded or misfolded proteins in the ER. Ferroptosis and ER stress have been proven to contribute to the progression of APAP-induced acute liver injury (ALI). It was reported that salidroside protects against APAP-induced ALI, but the potential mechanism remain unknown. In this study, male C57BL/6 J mice were intraperitoneally (i.p.) injected APAP (500 mg/kg) to induce an ALI model. Salidroside was i.p. injected at a dose of 100 mg/kg 2 h prior to APAP administration. Mice were sacrificed 12 h after APAP injection and the liver and serum of the mice were obtained for histological and biochemistry analysis. AML12 cells were used in in vitro assays. The results indicated that salidroside mitigated glutathione degradation via inhibiting cation transport regulator homolog 1 (CHAC1) to attenuate ferroptosis, and simultaneously suppressing PERK-eIF2α-ATF4 axis-mediated ER stress, thus alleviating APAP-induced ALI. However, PERK activator CCT020312 and overexpression of ATF4 inhibited the protective function of salidroside on CHAC1-mediated ferroptosis. Besides this, activation of the AMPK/SIRT1 signaling pathway by salidroside was demonstrated to have a protective effect against APAP-induced ALI. Interestingly, selective inhibition of SIRT1 ameliorated the protective effects of salidroside on ER stress and ferroptosis. Overall, salidroside plays a significant part in the mitigation of APAP-induced ALI by activating the AMPK/SIRT1 signaling to inhibit ER stress-mediated ferroptosis in the ATF4-CHAC1 axis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

ETC-1002 is an investigational drug currently in Phase 2 development for treatment of dyslipidemia and other cardiometabolic risk factors. In dyslipidemic subjects, ETC-1002 not only reduces plasma LDL cholesterol but also significantly attenuates levels of hsCRP, a clinical biomarker of inflammation. Anti-inflammatory properties of ETC-1002 were further investigated in primary human monocyte-derived macrophages and in in vivo models of inflammation. In cells treated with ETC-1002, increased levels of AMP-activated protein kinase (AMPK) phosphorylation coincided with reduced activity of MAP kinases and decreased production of proinflammatory cytokines and chemokines. AMPK phosphorylation and inhibitory effects of ETC-1002 on soluble mediators of inflammation were significantly abrogated by siRNA-mediated silencing of macrophage liver kinase B1 (LKB1), indicating that ETC-1002 activates AMPK and exerts its anti-inflammatory effects via an LKB1-dependent mechanism. In vivo, ETC-1002 suppressed thioglycollate-induced homing of leukocytes into mouse peritoneal cavity. Similarly, in a mouse model of diet-induced obesity, ETC-1002 restored adipose AMPK activity, reduced JNK phosphorylation, and diminished expression of macrophage-specific marker 4F/80. These data were consistent with decreased epididymal fat-pad mass and interleukin (IL)-6 release by inflamed adipose tissue. Thus, ETC-1002 may provide further clinical benefits for patients with cardiometabolic risk factors by reducing systemic inflammation linked to insulin resistance and vascular complications of metabolic syndrome.

Diabetic neuropathy is a neurological complication of diabetes that causes significant morbidity and, because of the obesity-driven rise in incidence of type 2 diabetes, is becoming a major international health problem. Mitochondrial phenotype is abnormal in sensory neurons in diabetes and may contribute to the etiology of diabetic neuropathy where a distal dying-back neurodegenerative process is a key component contributing to fiber loss. This review summarizes the major features of mitochondrial dysfunction in neurons and Schwann cells in human diabetic patients and in experimental animal models (primarily exhibiting type 1 diabetes). This article attempts to relate these findings to the development of critical neuropathological hallmarks of the disease. Recent work reveals that hyperglycemia in diabetes triggers nutrient excess in neurons that, in turn, mediates a phenotypic change in mitochondrial biology through alteration of the AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling axis. This vital energy sensing metabolic pathway modulates mitochondrial function, biogenesis and regeneration. The bioenergetic phenotype of mitochondria in diabetic neurons is aberrant due to deleterious alterations in expression and activity of respiratory chain components as a direct consequence of abnormal AMPK/PGC-1α signaling. Utilization of innovative respirometry equipment to analyze mitochondrial function of cultured adult sensory neurons from diabetic rodents shows that the outcome for cellular bioenergetics is a reduced adaptability to fluctuations in ATP demand. The diabetes-induced maladaptive process is hypothesized to result in exhaustion of the ATP supply in the distal nerve compartment and induction of nerve fiber dissolution. The role of mitochondrial dysfunction in the etiology of diabetic neuropathy is compared with other types of neuropathy with a distal dying-back pathology such as Friedreich ataxia, Charcot–Marie–Tooth disease type 2 and human immunodeficiency virus-associated distal-symmetric neuropathy.

In this study, we demonstrate that activation of AMP-activated protein kinase (AMPK) with glabridin alleviates adiposity and hyperlipidemia in obesity. In several obese rodent models, glabridin decreased body weight and adiposity with a concomitant reduction in fat cell size. Further, glabridin ameliorated fatty liver and plasma levels of triglyceride and cholesterol. In accordance with these findings, glabridin suppressed the expression of lipogenic genes such as sterol regulatory element binding transcription factor (SREBP)-1c, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD)-1 in white adipose tissues and liver, whereas it elevated the expression of fatty acid oxidation genes such as carnitine palmitoyl transferase (CPT)1, acyl-CoA oxidase (ACO), and peroxisome proliferator-activated receptor (PPAR)α in muscle. Moreover, glabridin enhanced phosphorylation of AMPK in muscle and liver and promoted fatty acid oxidation by modulating mitochondrial activity. Together, these data suggest that glabridin is a novel AMPK activator that would exert therapeutic effects in obesity-related metabolic disorders.

In the present study, the anti-diabetic properties and mechanisms underlying the actions of Korean red pepper using C2C12 myotube were evaluated. An ethanol (70%) extract of Korean red pepper (Ekrp) markedly increased 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose uptake in a concentration-dependent manner. To examine the mechanism by which Ekrp increased glucose uptake in C2C12 myotube, the phosphorylation levels of AMP-activated protein kinase (AMPK) and Acetyl-CoA carboxylase (ACC), a marker of AMPK activity were investigated using western blot analysis. Ekrp significantly increased AMPK and ACC phosphorylations in a dose dependent manner. Moreover, we also examined whether the transcriptional activity of peroxisome proliferator-activated receptor gamma (PPAR-γ) was involved in Ekrp-stimulated glucose uptake. Ekrp increased the transactivation of PPAR-γ in a concentration-dependent manner. The total phenolic compound content of Ekrp was measured (508.97 ± 24.93 mg/100 g), and several major compounds contents such as capsaicin and dihydrocapsaicin were also measured. Finally, we investigated the anti-obesity properties of Ekrp by using 3T3-L1 adipocyte differentiation systems. Ekrp did not inhibit adipocyte differentiation. These results suggest that Korean red pepper exhibits anti-diabetic effects via activation of PPAR-γ and AMPK, and that Korean red pepper may be useful for preventing diabetes.

Skeletal muscle triglyceride accumulation is associated with insulin resistance in obesity. Recently, it has been suggested that α lipoic acid (ALA) improves insulin sensitivity by lowering triglyceride accumulation in nonadipose tissues via activation of skeletal muscle AMP-activated protein kinase (AMPK). We examined whether chronic ALA supplementation prevents muscular lipid accumulation that is associated with high-fat diets via activation of AMPK. In addition, we tested if ALA supplementation was able to improve insulin sensitivity in rats fed low- and high-fat diets (LFD, HFD). Supplementing male Wistar rats with 0.5% ALA for 8 weeks significantly reduced body weight, both on LFD and HFD (−24% LFD+ALA vs. LFD, P < 0.01, and −29% HFD+ALA vs. HFD, P < 0.001). Oil red O lipid staining revealed a 3-fold higher lipid content in skeletal muscle after HFD compared with LFD and ALA-supplemented groups (P < 0.05). ALA improved whole body glucose tolerance (∼20% lower total area under the curve (AUC) in ALA supplemented groups vs. controls, P < 0.05). These effects were not mediated by increased muscular AMPK activation or ALA-induced improvement of muscular insulin sensitivity. To conclude, the prevention of HFD-induced muscular lipid accumulation and the improved whole body glucose tolerance are likely secondary effects due to the anorexic nature of ALA.

This study was designed to investigate the effects of prolonged activation of AMP-activated protein kinase (AMPK) on lipid partitioning and the potential molecular mechanisms involved in these processes in white adipose tissue (WAT). Rat epididymal adipocytes were incubated with 5′-aminoimidasole-4-carboxamide-1-β-d-ribofuranoside (AICAR;0.5 mM) for 15 h. Also, epididymal adipocytes were isolated 15 h after AICAR was injected (i.p. 0.7 g/kg body weight) in rats. Adipocytes were utilized for various metabolic assays and for determination of gene expression and protein content. Time-dependent in vivo plasma NEFA concentrations were determined. AICAR treatment significantly increased AMPK activation, inhibited lipogenesis, and increased FA oxidation. This was accompanied by upregulation of peroxisome proliferator-activated receptor (PPAR)α, PPARδ, and PPARγ-coactivator-1α (PGC-1α) mRNA levels. Lipolysis was first suppressed, but then increased, both in vitro and in vivo, with prolonged AICAR treatment. Exposure to AICAR increased adipose triglyceride lipase (ATGL) content and FA release, despite inhibition of basal and epinephrine-stimulated hormone-sensitive lipase (HSL) activity. Here, we provide evidence that prolonged AICAR-induced AMPK activation can remodel adipocyte metabolism by upregulating pathways that favor energy dissipation versus lipid storage in WAT. Additionally, we show novel time-dependent effects of AICAR-induced AMPK activation on lipolysis, which involves antagonistic modulation of HSL and ATGL.