AMP-activated Kinase Research Articles

Metformin (The Miracle Drug) Kinetics in Different Diseases such as Cancer

: Metformin, a miracle drug that was introduced a century ago, could be considered for various aspects of diseases such as diabetes (type 1 and 2), cancer prevention or chemotherapy, metabolic and neurodegenerative disease. It is well known that the frequency of cancer is higher in patients with type 2 diabetes mellitus. This review aims to provide updated information regarding clinical pharmacokinetics and the mechanism of action of Metformin in different diseases such as cancer. Diabetes type 1 is another chronic autoimmune disease detected usually in early childhood due to immune-mediated devastation of insulin-producing pancreatic beta-cells. Because of the lack of effective therapeutic approaches, its prevalence is increasing. Regarding cancer, an estimated 19.3 million new cancer cases and almost 10.0 million cancer deaths were reported in 2020 worldwide. By 50-60% bioavailability, the main route of metformin excretion is through urine. Its mechanism of action is based on 1) initiation of adenosine monophosphate-activated kinase, 2) block proinflammatory paths in perivascular adipose tissue, 3) decrease in monocyte-to-macrophage differentiation in vascular tissues, and 4) improvement in endothelial function. Metformin induces adenosine monophosphate-activated protein kinase signaling and suppresses gluconeogenesis. Antitumor properties of Metformin include a decrease in reactive oxygen species generation and inducing autophagy. In addition to glucose-lowering effects, Metformin has moderate anti-inflammatory and antioxidative effects. It could improve lipid profile and reduce overweight individuals' body mass and arterial blood pressure. In type 1 diabetes, Metformin reduces the requirement for daily insulin and improves glycemia. Its long-term use decreases cardiovascular events. In addition to inhibiting the synthesis of lipids via a reduction in oxidative stress, Metformin inhibits inflammation and increases energy metabolism. Finally, by reducing micro- and macro-vascular consequences, mortality-related diabetes and cancer decline by metformin administration. Therefore, in addition to diabetes, Metformin could reduce the proliferation of cancer cells and the possibility of malignancies in different types of cancer.

Effect of acupuncture on AMPK/ULK1 signaling pathway of hippocampus in chronic stress-induced depression rats

To observe the effect of manual acupuncture stimulation on changes of behavior and hippocampal signaling pathways of AMP-activated kinase (AMPK)/UNC-51-like kinase 1 (ULK1) in rats with chronic unpredictable mild stress (CUMS)-induced depression, so as to explore its molecular mechanism underlying improvement of depression. Thirty-two male SD rats were randomly divided into normal control, model, acupuncture and medication (fluoxetine) groups, with 8 rats in each group. The depression model was established by using CUMS (fasting, water depredation, restraint, swimming in cold water, crowding, stroboscope stimulation, and tail clipping, each of which was used once a week) for 6 weeks. Manual acupuncture stimulation was applied to "Baihui"(GV20) and "Yintang"(EX-HN3) for 10 min, once daily, 6 times a week for 6 weeks before every-day modeling. Rats of the medication group received gavage of fluoxetine (10 mg/kg) once a day for 6 weeks before every-day modeling. The percentage of sucrose preference, and the percentages of open arm times and open arm time in the elevated plus maze experiments were detected. The expression levels of AMPK, phosphorylated (p)-AMPK, ULK1, p-ULK1, mammalian target of rapamycin (mTOR) and p-mTOR proteins in the hippocampus tissue were detected by Western blot, and the expression of autophagy effecting protein (Beclin-1) and the selective autophagy receptors (p62) mRNA in the hippocampal tissue was detected using real-time fluorescence quantitative PCR, respectively. Compared with the normal group, the percentage of sucrose preference, percentage of times of entering the open arm and percentage of open arm time, the expression levels of p-AMPK /AMPK ratio, p-ULK1 /ULK1 ratio of proteins, and Beclin-1 mRNA were significantly decreased (P<0.01, P<0.05), and the expression levels of p-mTOR /mTOR ratio and p62 mRNA were significantly increased (P<0.01, P<0.05) in the model group. In comparison with the model group, the percentage of sucrose preference, percentage of open arm times and open arm time, the expression levels of p-AMPK /AMPK ratio, p-ULK1/ULK1 ratio of proteins, and Beclin-1 mRNA were significantly increased in both acupuncture and medication groups (P<0.01, P<0.05), while the expression levels of p-mTOR /mTOR ratio of proteins, and p62 mRNA were markedly decreased only in the medication group (P<0.05). Acupuncture treatment can alleviate depression-like behavior in CUMS rats, which may be related to its functions in up-regulating AMPK/ULK1 signaling pathway to induce cellular autophagy in the hippocampus.

AMPK activation induces RALDH+ tolerogenic dendritic cells by rewiring glucose and lipid metabolism.

Dendritic cell (DC) activation and function are underpinned by profound changes in cellular metabolism. Several studies indicate that the ability of DCs to promote tolerance is dependent on catabolic metabolism. Yet the contribution of AMP-activated kinase (AMPK), a central energy sensor promoting catabolism, to DC tolerogenicity remains unknown. Here, we show that AMPK activation renders human monocyte-derived DCs tolerogenic as evidenced by an enhanced ability to drive differentiation of regulatory T cells, a process dependent on increased RALDH activity. This is accompanied by several metabolic changes, including increased breakdown of glycerophospholipids, enhanced mitochondrial fission-dependent fatty acid oxidation, and upregulated glucose catabolism. This metabolic rewiring is functionally important as we found interference with these metabolic processes to reduce to various degrees AMPK-induced RALDH activity as well as the tolerogenic capacity of moDCs. Altogether, our findings reveal a key role for AMPK signaling in shaping DC tolerogenicity and suggest AMPK as a target to direct DC-driven tolerogenic responses in therapeutic settings.

Bayesian mapping of protein kinases to vasopressin-regulated phosphorylation sites in renal collecting duct.

Vasopressin controls water permeability in the renal collecting duct by regulating the water channel protein, aquaporin-2 (AQP2). Phosphoproteomic studies have identified multiple proteins that undergo phosphorylation changes in response to vasopressin. The kinases responsible for the phosphorylation of most of these sites have not been identified. Here, we use large-scale Bayesian data integration to predict the responsible kinases for 51 phosphoproteomically identified vasopressin-regulated phosphorylation sites in the renal collecting duct. To do this, we applied Bayes' rule to rank the 515 known mammalian protein kinases for each site. Bayes' rule was applied recursively to integrate each of the seven independent datasets, each time using the posterior probability vector of a given step as the prior probability vector of the next step. In total, 30 of the 33 phosphorylation sites that increase with vasopressin were predicted to be phosphorylated by protein kinase A (PKA) catalytic subunit-α, consistent with prior studies implicating PKA in vasopressin signaling. Eighteen of the vasopressin-regulated phosphorylation sites were decreased in response to vasopressin and all but three of these sites were predicted to be targets of extracellular signal-regulated kinases, ERK1 and ERK2. This result implies that ERK1 and ERK2 are inhibited in response to vasopressin V2 receptor occupation, secondary to PKA activation. The six phosphorylation sites not predicted to be phosphorylated by PKA or ERK1/2 are potential targets of other protein kinases previously implicated in aquaporin-2 regulation, including cyclin-dependent kinase 18 (CDK18), calmodulin-dependent kinase 2δ (CAMK2D), AMP-activated kinase catalytic subunit-α-1 (PRKAA1) and CDC42 binding protein kinase β (CDC42BPB).NEW & NOTEWORTHY Vasopressin regulates water transport in the renal collecting duct in part through phosphorylation or dephosphorylation of proteins that regulate aquaporin-2. Prior studies have identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. This study uses Bayesian data integration techniques to combine information from multiple prior proteomics and transcriptomics studies to predict the protein kinases that phosphorylate the 51 sites. Most of the regulated sites were predicted to be phosphorylated by protein kinase A or ERK1/ERK2.

Development of a Dual Reporter System to Simultaneously Visualize Ca2+ Signals and AMPK Activity.

In this study, we introduce a new separation of phases-based activity reporter of kinase (SPARK) for AMP-activated kinase (AMPK), named AMPK-SPARK, which reports the AMPK activation by forming bright fluorescent clusters. Furthermore, we introduce a dual reporter system, named GCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based Ca2+ biosensor, GCaMP6f, into our initial design, enabling simultaneous monitoring of Ca2+ levels and AMPK activity. This system offers the essential quality of information by single-channel fluorescence microscopy without the need for coexpression of different biosensors and elaborate filter layouts to overcome spectral limitations. We used AMPK-SPARK to map endogenous AMPK activity in different cell types and visualized the dynamics of AMPK activation in response to various stimuli. Using GCaMP-AMPK-SPARK, we revealed cell-to-cell heterogeneities in AMPK activation by Ca2+ mobilization. We anticipate that this dual reporter strategy can be employed to study the intricate interplays between different signaling networks and kinase activities.

PPARγ drives mitochondrial stress signaling and the loss of atrial cardiomyocytes in newborn mice exposed to hyperoxia

Diastolic dysfunction is increasingly common in preterm infants exposed to supplemental oxygen (hyperoxia). Previous studies in neonatal mice showed hyperoxia suppresses fatty acid synthesis genes required for proliferation and survival of atrial cardiomyocytes. The loss of atrial cardiomyocytes creates a hypoplastic left atrium that inappropriately fills the left ventricle during diastole. Here, we show that hyperoxia stimulates adenosine monophosphate-activated kinase (AMPK) and peroxisome proliferator activated receptor-gamma (PPARγ) signaling in atrial cardiomyocytes. While both pathways can regulate lipid homeostasis, PPARγ was the primary pathway by which hyperoxia inhibits fatty acid gene expression and inhibits proliferation of mouse atrial HL-1 cells. It also enhanced the toxicity of hyperoxia by increasing expression of activating transcription factor (ATF) 5 and other mitochondrial stress response genes. Silencing PPARγ signaling restored proliferation and survival of HL-1 cells as well as atrial cardiomyocytes in neonatal mice exposed to hyperoxia. Our findings reveal PPARγ enhances the toxicity of hyperoxia on atrial cardiomyocytes, thus suggesting inhibitors of PPARγ signaling may prevent diastolic dysfunction in preterm infants.

Antidiabetic potential of fenugreek (Trigonella foenum-graecum): A magic herb for diabetes mellitus.

Fenugreek (Trigonella foenum-graecum) is a widely grown dietary herb in Asia, and its seeds are traditionally used for several diseases, including diabetes. The seeds and leaves possess a variety of compounds that play an important role in regulating their hypoglycemic effect. However, so far, no extensive systematic review exists on its antidiabetic effect, highlighting the molecular mechanisms and isolated compounds. The purpose of this review is to summarize the preclinical and clinical antidiabetic properties of fenugreek and its isolated compounds by focusing on underlying mechanisms. PubMed, Google Scholar, Science Direct, and Scopus databases were searched to retrieve articles until June, 2024. Preclinical studies demonstrated that the antidiabetic effect of fenugreek was mostly associated with enhanced glucose transporter type-4 (GLUT4) translocation and hexokinase activity, decreased glucose-6-phosphatase and fructose-1,6-bisphosphatase activities, inhibited α-amylase and maltase activities, protected β cells, and increased insulin release. Furthermore, few studies have reported its role as a glucagon-like peptide-1 (GLP-1) modulator, 5'-AMP-activated kinase (AMPK) activator, and dipeptidyl peptidase-IV (DPP-IV) inhibitor. Further clinical trials showed that fenugreek seeds improved blood glucose levels, insulin resistance, insulin sensitivity, and lipid profiles. This study highlights significant evidence of the antidiabetic effect of fenugreek and its isolated compounds; therefore, it could be a potential therapy for diabetes.

P054 REDUCTION OF BLOOD PRESSURE AND CARDIOVASCULAR RISK WITH IMPROVEMENT OF GLUCOSE AND LIPID METABOLISM BY A NUTRACEUTICAL BASED IN MULBERRY LEAF AND FENUGREEK IN SUBJECTS WITH PREDIABETES

Background and Objective: In Spain there are &gt;5000000 prediabetic subjects, of which &gt;500000 develop type 2 diabetes annually. Alpha-glucosidase inhibitors such as acarbose have been shown to prevent type 2 diabetes and reduce blood pressure, development of hypertension and cardiovascular risk. White mulberry (Morus alba) leaves contain 1-deoxynojirimycin, a powerful competitive alpha-glucosidase inhibitor. A nutraceutical containing Reducose (standardized white mulberry leaf extract), fenugreek (Trigonella foenum-graecum, an AMP-kinase activator), inuline (soluble fiber), Lactiplantibacillus plantarum (probiotic), niacin (vitamin B3), chromium and zinc (oligoelements) has been developed for use in patients with prediabetes. We endeavored to study its tolerability and efficacy on the metabolic profile, blood pressure and cardiovascular risk of the patients, with the change in the HOMA2-IR index of insulin resistance (as a proxy for the risk of developing type 2 diabetes) as the main outcome. Methods: In this ongoing open study, adult patients with prediabetes have been recruited to receive the mentioned nutraceutical daily for 6 months with an intermediate visit after 3 months. BMI, blood pressure, fasting glucose, insulin, lipid profile and HOMA2-IR were determined at each visit. Results: Of 94 recruited patients, 37 have completed the second visit. The HOMA-2 IR dropped by 21.5% (p=0.0016), the cardiovascular risk estimated by SCORE2 by 14.6% (p=0.0009), fasting glycemia by 7.6 mg/dL (p=0.0005), HbA1c by 0.22% (p&lt;0.0001), non-HDL cholesterol by 12.4 mg/dL (p=0.0037), systolic blood pressure by 6.3 mmHg (p=0.0001), diastolic blood pressure by 5.2 mmHg (p=0.0236); the body weight and BMI did not change significantly, and there were no withdrawals due to adverse effects or serious tolerance issues. Conclusions: This nutraceutical was well tolerated and improved the metabolic (both glycemic and lipidic) profile, the blood pressure and the cardiovascular risk of patients with prediabetes. At present there are no licensed drugs for prediabetes in Spain, but this nutraceutical may be an option for these patients in combination with lifestyle changes.

Compartmentalization in cardiomyocytes modulates creatine kinase and adenylate kinase activities.

Intracellular molecules are transported by motor proteins or move by diffusion resulting from random molecular motion. Cardiomyocytes are packed with structures that are crucial for function, but also confine the diffusional spaces, providing cells with a means to control diffusion. They form compartments in which local concentrations are different from the overall, average concentrations. For example, calcium and cyclic AMP are highly compartmentalized, allowing these versatile second messengers to send different signals depending on their location. In energetic compartmentalization, the ratios of AMP and ADP to ATP are different from the average ratios. This is important for the performance of ATPases fuelling cardiac excitation-contraction coupling and mechanical work. A recent study suggested that compartmentalization modulates the activity of creatine kinase and adenylate kinase in situ. This could have implications for energetic signaling through, for example, AMP-activated kinase. It highlights the importance of taking compartmentalization into account in our interpretation of cellular physiology and developing methods to assess local concentrations of AMP and ADP to enhance our understanding of compartmentalization in different cell types.

Neuroprotective and cognitive enhancing effects of herbecetin against thioacetamide induced hepatic encephalopathy in rats via upregulation of AMPK and SIRT1 signaling pathways

Acute liver injury, there is a risky neurological condition known as hepatic encephalopathy (HE). Herbacetin is a glycosylated flavonoid with many pharmacological characteristics. The purpose of this study was to assess the ability of herbacetin to protect against the cognitive deficits associated with thioacetamide (TAA) rat model and delineate the underlying behavioral and pharmacological mechanisms. Rats were pretreated with herbacetin (20 and 40 mg/kg) for 30days. On 30th day, the rats were injected with TAA (i.p. 350 mg/kg) in a single dose. In addition to a histpathological studies, ultra-structural architecture of the brain, liver functions, oxidative stress biomarkers, and behavioral tests were evaluated. Compared to the TAA-intoxicated group, herbacetin improved the locomotor and cognitive deficits, serum hepatotoxicity indices and ammonia levels. Herbacetin reduced brain levels of malodialdeyde, glutamine synthetase (GS), tumor necrosis factor- alpha (TNF-α), interleukin 1 B (IL-1β), annexin v, and increased brain GSH, Sirtuin 1 (SIRT1), and AMP-activated kinase (AMPK) expression levels. Also, herbacetin improve the histopathological changes and ultra- structure of brain tissue via attenuating the number of inflammatory and apoptotic cells. Herbacetin treatment significantly reduced the toxicity caused by TAA. These findings suggest that herbacetin might be taken into account as a possible neuroprotective and cognitive enhancing agent due to its ability to reduce oxidative stress, inflammation and apoptosis associated with TAA.

Lack of Sestrins Is Associated with Increased Glycolytic Flux

Introduction: Sestrin1, 2, and 3 are a family of stress-response proteins implicated in many diseases. How Sestrins affect cell survival through AMP-activated kinase (AMPK) and protein synthesis through mechanistic target of rapamycin in complex 1 (mTORC1) is most studied; however, how Sestrins affect metabolism is poorly understood. Hypothesis: Independent of AMPK/mTORC1 activation, Sestrins negatively regulate glycolytic flux by inhibiting glycolytic enzyme activity through binding inhibition. Results: Wildtype (WT) HEK293T cells were transfected with plasmids expressing individual FLAG-tagged Sestrins and then deprived of glucose for 180 min or deprived of glucose for 150 min followed by glucose addback for 30 min. FLAG-tagged Sestrins were immunoprecipitated and found to directly interact with Hexokinase2 and Phosphofructokinase such that binding inversely correlates with glucose concentration. Additionally, WT and Sestrin-knockout (TKO) HEK293T cells were subjected to the glycolytic and mitochondrial stress tests and analyzed for metabolic flux via Seahorse analysis. TKO cells demonstrated higher glycolytic flux, but lower mitochondrial flux, compared to WT cells. Cells were then treated with inhibitors against AMPK and mTORC1 and subjected to the glycolytic stress test. Surprisingly, glycolytic flux in TKO cells was unaffected by the inhibitors while WT cells reduced glycolytic flux suggesting Sestrins can regulate glycolytic flux independent of AMPK/mTORC1 activation. Thereafter, WT and TKO cells were glucose-deprived for 120 min or glucose-deprived for 110 min followed by glucose addback for 10 min and then characterized by steady-state metabolomics. As expected, TKO cells possessed elevations in glycolytic metabolites with glucose addback compared to WT cells. Similarly, WT and TKO cells were deprived of glucose for 110 min followed by stable isotope tracing with [13C6]glucose for 10- and 30-minutes and metabolites were profiled by mass spectrometry. TKO cells possessed higher abundances of glycolytic metabolites as well as elevated labeled extracellular lactate. Conclusions: These data support a model wherein Sestrins directly act as negative regulators for glycolytic enzyme activity, thus regulating glycolytic flux. This work is funded by F32DK126312 (P.A.R.), P30DK048520 Pilot and Feasibility Award (P.A.R.), T32DK120521 (A.E.L.), P30CA046934 Cancer Center Support Grant, R01AR080051 (A.B.), R01DK015658 (S.R.K.), P30DK048520 (P.S.M.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of Prostate Cancer Cell Survival and Proliferation by Carnosic Acid Is Associated with Inhibition of Akt and Activation of AMPK Signaling.

Prostate cancer, accounting for 375,304 deaths in 2020, is the second most prevalent cancer in men worldwide. While many treatments exist for prostate cancer, novel therapeutic agents with higher efficacy are needed to target aggressive and hormone-resistant forms of prostate cancer, while sparing healthy cells. Plant-derived chemotherapy drugs such as docetaxel and paclitaxel have been established to treat cancers including prostate cancer. Carnosic acid (CA), a phenolic diterpene found in the herb rosemary (Rosmarinus officinalis) has been shown to have anticancer properties but its effects in prostate cancer and its mechanisms of action have not been examined. CA dose-dependently inhibited PC-3 and LNCaP prostate cancer cell survival and proliferation (IC50: 64, 21 µM, respectively). Furthermore, CA decreased phosphorylation/activation of Akt, mTOR, and p70 S6K. A notable increase in phosphorylation/activation of AMP-activated kinase (AMPK), acetyl-CoA carboxylase (ACC) and its upstream regulator sestrin-2 was seen with CA treatment. Our data indicate that CA inhibits AKT-mTORC1-p70S6K and activates Sestrin-2-AMPK signaling leading to a decrease in survival and proliferation. The use of inhibitors and small RNA interference (siRNA) approaches should be employed, in future studies, to elucidate the mechanisms involved in carnosic acid's inhibitory effects of prostate cancer.

25-Hydroxycholesterol regulates lysosome AMP kinase activation and metabolic reprogramming to educate immunosuppressive macrophages

Macrophages are critical to turn noninflamed "cold tumors" into inflamed "hot tumors". Emerging evidence indicates abnormal cholesterol metabolites in the tumor microenvironment (TME) with unclear function. Here, we uncovered the inducible expression of cholesterol-25-hydroxylase (Ch25h) by interleukin-4 (IL-4) and interleukin-13 (IL-13) via the transcription factor STAT6, causing 25-hydroxycholesterol (25HC) accumulation. scRNA-seq analysis confirmed that CH25Hhi subsets were enriched in immunosuppressive macrophage subsets and correlated to lower survival rates in pan-cancers. Targeting CH25H abrogated macrophage immunosuppressive function to enhance infiltrating Tcell numbers and activation, which synergized with anti-PD-1 to improve anti-tumor efficacy. Mechanically, lysosome-accumulated 25HC competed with cholesterol for GPR155 binding to inhibit the kinase mTORC1, leading to AMPKα activation and metabolic reprogramming. AMPKα also phosphorylated STAT6 Ser564 to enhance STAT6 activation and ARG1 production. Together, we propose CH25H as an immunometabolic checkpoint, which manipulates macrophage fate to reshape CD8+ Tcell surveillance and anti-tumor response.

Canagliflozin inhibits PASMCs proliferation via regulating SGLT1/AMPK signaling and attenuates artery remodeling in MCT-induced pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) was a devastating disease characterized by artery remodeling, ultimately resulting in right heart failure. The aim of this study was to investigate the effects of canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor (SGLT2i) with mild SGLT1 inhibitory effects, on rats with PAH, as well as its direct impact on pulmonary arterial smooth muscle cells (PASMCs). PAH rats were induced by injection of monocrotaline (MCT) (40 mg/kg), followed by four weeks of treatment with CANA (30 mg/kg/day) or saline alone. Pulmonary artery and right ventricular (RV) remodeling and dysfunction in PAH were alleviated with CANA, as assessed by echocardiography. Hemodynamic parameters and structural of pulmonary arteriole, including vascular wall thickness and wall area, were reduced by CANA. RV hypertrophy index, cardiomyocyte hypertrophy, and fibrosis were decreased with CANA treatment. PASMCs proliferation was inhibited by CANA under stimulation by platelet-derived growth factor (PDGF)-BB or hypoxia. Activation of AMP kinase (AMPK) was induced by CANA treatment in cultured PASMCs in a time- and concentration-dependent manner. These effects of CANA were attenuated when treatment with compound C, an AMPK inhibitor. Abundant expression of SGLT1 was observed in PASMCs and pulmonary arteries, while SGLT2 expression was undetectable. SGLT1 increased in response to PDGF-BB or hypoxia stimulation, while PASMCs proliferation was inhibited and beneficial effects of CANA were counteracted by knockdown of SGLT1. Our research demonstrated for the first time that CANA inhibited the proliferation of PASMCs by regulating SGLT1/AMPK signaling and thus exerted an anti-proliferative effect on MCT-induced PAH.

AMPK inhibition sensitizes acute leukemia cells to BH3 mimetic-induced cell death.

BH3 mimetics, including the BCL2/BCLXL/BCLw inhibitor navitoclax and MCL1 inhibitors S64315 and tapotoclax, have undergone clinical testing for a variety of neoplasms. Because of toxicities, including thrombocytopenia after BCLXL inhibition as well as hematopoietic, hepatic and possible cardiac toxicities after MCL1 inhibition, there is substantial interest in finding agents that can safely sensitize neoplastic cells to these BH3 mimetics. Building on the observation that BH3 mimetic monotherapy induces AMP kinase (AMPK) activation in multiple acute leukemia cell lines, we report that the AMPK inhibitors (AMPKis) dorsomorphin and BAY-3827 sensitize these cells to navitoclax or MCL1 inhibitors. Cell fractionation and phosphoproteomic analyses suggest that sensitization by dorsomorphin involves dephosphorylation of the proapoptotic BCL2 family member BAD at Ser75 and Ser99, leading BAD to translocate to mitochondria and inhibit BCLXL. Consistent with these results, BAD knockout or mutation to BAD S75E/S99E abolishes the sensitizing effects of dorsomorphin. Conversely, dorsomorphin synergizes with navitoclax or the MCL1 inhibitor S63845 to induce cell death in primary acute leukemia samples ex vivo and increases the antitumor effects of navitoclax or S63845 in several xenograft models in vivo with little or no increase in toxicity in normal tissues. These results suggest that AMPK inhibition can sensitize acute leukemia to multiple BH3 mimetics, potentially allowing administration of lower doses while inducing similar antineoplastic effects.

BCAA metabolism in pancreatic cancer affects lipid balance by regulating fatty acid import into mitochondria

BackgroundPancreatic ductal adenocarcinoma (PDAC) has been associated with the host dysmetabolism of branched-chain amino acids (BCAAs), however, the implications for the role of BCAA metabolism in PDAC development or progression are not clear. The mitochondrial catabolism of valine, leucine, and isoleucine is a multistep process leading to the production of short-chain R-CoA species. They can be subsequently exported from mitochondria as short-chain carnitines (SC-CARs), utilized in anabolic pathways, or released from the cells.MethodsWe examined the specificities of BCAA catabolism and cellular adaptation strategies to BCAA starvation in PDAC cells in vitro. We used metabolomics and lipidomics to quantify major metabolic changes in response to BCAA withdrawal. Using confocal microscopy and flow cytometry we quantified the fluorescence of BODIPY probe and the level of lipid droplets (LDs). We used BODIPY-conjugated palmitate to evaluate transport of fatty acids (FAs) into mitochondria. Also, we have developed a protocol for quantification of SC-CARs, BCAA-derived metabolites.ResultsUsing metabolic profiling, we found that BCAA starvation leads to massive triglyceride (TG) synthesis and LD accumulation. This was associated with the suppression of activated FA transport into the mitochondrial matrix. The suppression of FA import into mitochondria was rescued with the inhibitor of the acetyl-CoA carboxylase (ACC) and the activator of AMP kinase (AMPK), which both regulate carnitine palmitoyltransferase 1A (CPT1) activation status.ConclusionsOur data suggest that BCAA catabolism is required for the import of long chain carnitines (LC-CARs) into mitochondria, whereas the disruption of this link results in the redirection of activated FAs into TG synthesis and its deposition into LDs. We propose that this mechanism protects cells against mitochondrial overload with LC-CARs and it might be part of the universal reaction to amino acid perturbations during cancer growth, regulating FA handling and storage.

Magnolia officinalis Rehder & E. Wilson ameliorates white adipogenesis by upregulating AMPK and SIRT1 in vitro and in vivo

Although there is an established link between Magnolia Cortex (MO) and lipid metabolism in previous research, its exploration within the context of obesity has been limited. Therefore, the present study investigated the therapeutic effects of MO on obesity and its mechanism of action in vitro and in vivo. Our chromatography analysis revealed that Honokiol and Magnolol are contained in MO extract. In vitro experiments showed that lipid droplets, adipogenic, and lipogenic genes were notably diminished by increasing sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK) protein expression in MO-treated 3T3-L1 adipocytes. In vivo experiments exhibited that MO administration significantly recovered the adipogenesis, lipogenesis, and fatty acid oxidation genes by increasing the SIRT1 and AMPK expression in white adipose tissue. Furthermore, hepatic steatosis by HFD feeding was ameliorated in MO-administered obese mice. We conclude that MO could be important manager for treating obesity through AMPK and SIRT1 regulation.

Rationale and Design of a Phase 2, Double-blind, Placebo-Controlled, Randomized Trial Evaluating AMP Kinase-Activation by Metformin in Focal Segmental Glomerulosclerosis

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. 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. 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.

Lactobacillus Casei-fermented Amomum Xanthioides Mitigates non-alcoholic fatty liver disease in a high-fat diet mice model

Non-alcoholic fatty liver disease (NAFLD) is a substantial global health issue owing to its high prevalence and the lack of effective therapies. Fermentation of medicinal herbs has always been considered a feasible strategy for enhancing efficacy in treating various ailments. This study aimed to investigate the potential benefits of the Lactobacillus casei-fermented Amomum xanthioides (LAX) on NAFLD in a high-fat diet model. HFD-fed C57BL6/j mice were administered with 200 mg/kg of LAX or unfermented Amomum xanthioides (AX) or 100 mg/kg of metformin for 6 weeks from the 4th week. The 10-week HFD-induced alterations of hepatic lipid accumulation and hepatic inflammation were significantly attenuated by LAX dominantly (more than AX or metformin), which evidenced by pathohistological findings, lipid contents, inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)− 6 and IL-1β, oxidative parameters such as reactive oxygen species (ROS) and malondialdehyde (MDA), and molecular changes reversely between lipogenic proteins such as glycerol-3-phosphate acyltransferase (GPAM) and sterol regulatory element-binding protein (SREBP)− 1, and lipolytic proteins including peroxisome proliferator-activated receptor (PPAR-α) and AMP-activated kinase (AMPK)-α in the liver tissues. In addition, the abnormal serum lipid parameters (triglyceride, total cholesterol and LDL-cholesterol) notably ameliorated by LAX. In conclusion, these findings support the potential of LAX as a promising plant-derived remedy for NAFLD.

Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake via Direct Inhibition of Mitochondrial Malate Dehydrogenase in Mouse 3T3-L1 Cells.

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 μM dose-dependently suppressed growth, whereas LW6 at 20 μM, but not at 2-10 μM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 μM significantly promoted glucose uptake, with the strongest effect at 20 μM DIF-1, whereas LW6 at 2-20 μM significantly promoted glucose uptake, with the strongest effect at 10 μM LW6. Western blot analyses showed that LW6 (10 μM) and DIF-1 (20 μM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.