Metabolic and Innate Immune Cues Merge into a Specific Inflammatory Response via the UPR.

Innate immune responses are intricately linked with intracellular metabolism of myeloid cells. Toll-like receptor (TLR) stimulation shifts intracellular metabolism toward glycolysis, while anti-inflammatory signals depend on enhanced mitochondrial respiration. How exogenous metabolic signals affect the immune response is unknown. We demonstrate that TLR-dependent responses of dendritic cells (DCs) are exacerbated by a high-fatty-acid (FA) metabolic environment. FAs suppress the TLR-induced hexokinase activity and perturb tricarboxylic acid cycle metabolism. These metabolic changes enhance mitochondrial reactive oxygen species (mtROS) production and, in turn, the unfolded protein response (UPR), leading to a distinct transcriptomic signature with IL-23 as hallmark. Interestingly, chemical or genetic suppression of glycolysis was sufficient to induce this specific immune response. Conversely, reducing mtROS production or DC-specific deficiency in XBP1 attenuated IL-23 expression and skin inflammation in an IL-23-dependent model of psoriasis. Thus, fine-tuning of innate immunity depends on optimization of metabolic demands and minimization of mtROS-induced UPR.

Myeloid Cell-Specific Deletion of AMPKα1 Worsens Ocular Bacterial Infection by Skewing Macrophage Phenotypes.

AMP-activated protein kinase (AMPK) plays a crucial role in governing essential cellular functions such as growth, proliferation, and survival. Previously, we observed increased vulnerability to bacterial (Staphylococcus aureus) endophthalmitis in global AMPKα1 knockout mice. In this study, we investigated the specific involvement of AMPKα1 in myeloid cells using LysMCre;AMPKα1fl mice. Our findings revealed that whereas endophthalmitis resolved in wild-type C57BL/6 mice, the severity of the disease progressively worsened in AMPKα1-deficient mice over time. Moreover, the intraocular bacterial load and inflammatory mediators (e.g., IL-1ß, TNF-α, IL-6, and CXCL2) were markedly elevated in the LysMCre;AMPKα1fl mice. Mechanistically, the deletion of AMPKα1 in myeloid cells skewed macrophage polarization toward the inflammatory M1 phenotype and impaired the phagocytic clearance of S. aureus by macrophages. Notably, transferring AMPK-competent bone marrow from wild-type mice to AMPKα1 knockout mice preserved retinal function and mitigated the severity of endophthalmitis. Overall, our study underscores the role of myeloid-specific AMPKα1 in promoting the resolution of inflammation in the eye during bacterial infection. Hence, therapeutic strategies aimed at restoring or enhancing AMPKα1 activity could improve visual outcomes in endophthalmitis and other ocular infections.

CDK4 Phosphorylates AMPKα2 to Inhibit Its Activity and Repress Fatty Acid Oxidation.

The roles of CDK4 in the cell cycle have been extensively studied, but less is known about the mechanisms underlying the metabolic regulation by CDK4. Here, we report that CDK4 promotes anaerobic glycolysis and represses fatty acid oxidation in mouse embryonic fibroblasts (MEFs) by targeting the AMP-activated protein kinase (AMPK). We also show that fatty acid oxidation (FAO) is specifically induced by AMPK complexes containing the α2 subunit. Moreover, we report that CDK4 represses FAO through direct phosphorylation and inhibition of AMPKα2. The expression of non-phosphorylatable AMPKα2 mutants, or the use of a CDK4 inhibitor, increased FAO rates in MEFs and myotubes. In addition, Cdk4-/- mice have increased oxidative metabolism and exercise capacity. Inhibition of CDK4 mimicked these alterations in normal mice, but not when skeletal muscle was AMPK deficient. This novel mechanism explains how CDK4 promotes anabolism by blocking catabolic processes (FAO) that are activated by AMPK.

Myeloid AMPK signaling restricts fibrosis but is not required for metformin improvements during CDAHFD-induced NASH in mice.

Metabolic programming underpins inflammation and liver macrophage activation in the setting of chronic liver disease. Here, we sought to identify the role of an important metabolic regulator, AMP-activated protein kinase (AMPK), specifically within myeloid cells during the progression of non-alcoholic steatohepatitis (NASH) and whether treatment with metformin, a firstline therapy for diabetes and activator of AMPK could stem disease progression. Male and female Prkaa1fl/fl/Prkaa2fl/fl (Flox) control and Flox-LysM-Cre+ (MacKO) mice were fed a low-fat control or a choline-deficient, amino acid defined 45% Kcal high-fat diet (CDAHFD) for 8 weeks, where metformin was introduced in the drinking water (50 or 250 mg/kg/day) for the last 4 weeks. Hepatic steatosis and fibrosis were dramatically increased in response to CDAHFD-feeding compared to low-fat control. While myeloid AMPK signaling had no effect on markers of hepatic steatosis or circulating markers, fibrosis as measured by total liver collagen was significantly elevated in livers from MacKO mice, independent of sex. Although treatment with 50 mg/kg/day metformin had no effect on any parameter, intervention with 250 mg/kg/day metformin completely ameliorated hepatic steatosis and fibrosis in both male and female mice. While the protective effect of metformin was associated with lower final body weight, and decreased expression of lipogenic and Col1a1 transcripts, it was independent of myeloid AMPK signaling. These results suggest that endogenous AMPK signaling in myeloid cells, both liver-resident and infiltrating, acts to restrict fibrogenesis during CDAHFD-induced NASH progression but is not the mechanism by which metformin improves markers of NASH.

Exercise training adaptations in liver glycogen and glycerolipids require hepatic AMP-activated protein kinase in mice.

Regular exercise elicits adaptations in glucose and lipid metabolism that allow the body to meet energy demands of subsequent exercise bouts more effectively and mitigate metabolic diseases including fatty liver. Energy discharged during the acute exercise bouts that comprise exercise training may be a catalyst for liver adaptations. During acute exercise, liver glycogenolysis and gluconeogenesis are accelerated to supply glucose to working muscle. Lower liver energy state imposed by gluconeogenesis and related pathways activates AMP-activated protein kinase (AMPK), which conserves ATP partly by promoting lipid oxidation. This study tested the hypothesis that AMPK is necessary for liver glucose and lipid adaptations to training. Liver-specific AMPKα1α2 knockout (AMPKα1α2fl/fl+AlbCre) mice and littermate controls (AMPKα1α2fl/fl) completed sedentary and exercise training protocols. Liver nutrient fluxes were quantified at rest or during acute exercise following training. Liver metabolites and molecular regulators of metabolism were assessed. Training increased liver glycogen in AMPKα1α2fl/fl mice, but not in AMPKα1α2fl/fl+AlbCre mice. The inability to increase glycogen led to lower glycogenolysis, glucose production, and circulating glucose during acute exercise in trained AMPKα1α2fl/fl+AlbCre mice. Deletion of AMPKα1α2 attenuated training-induced declines in liver diacylglycerides. In particular, training lowered the concentration of unsaturated and elongated fatty acids comprising diacylglycerides in AMPKα1α2fl/fl mice, but not in AMPKα1α2fl/fl+AlbCre mice. Training increased liver triacylglycerides and the desaturation and elongation of fatty acids in triacylglycerides of AMPKα1α2fl/fl+AlbCre mice. These lipid responses were independent of differences in tricarboxylic acid cycle fluxes. In conclusion, AMPK is required for liver training adaptations that are critical to glucose and lipid metabolism.NEW & NOTEWORTHY This study shows that the energy sensor and transducer, AMP-activated protein kinase (AMPK), is necessary for an exercise training-induced: 1) increase in liver glycogen that is necessary for accelerated glycogenolysis during exercise, 2) decrease in liver glycerolipids independent of tricarboxylic acid (TCA) cycle flux, and 3) decline in the desaturation and elongation of fatty acids comprising liver diacylglycerides. The mechanisms defined in these studies have implications for use of regular exercise or AMPK-activators in patients with fatty liver.

The energy sensor AMPK regulates T cell metabolic adaptation and effector responses in vivo.

Naive T cells undergo metabolic reprogramming to support the increased energetic and biosynthetic demands of effector T cell function. However, how nutrient availability influences T cell metabolism and function remains poorly understood. Here we report plasticity in effector T cell metabolism in response to changing nutrient availability. Activated T cells were found to possess a glucose-sensitive metabolic checkpoint controlled by the energy sensor AMP-activated protein kinase (AMPK) that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability. T cells lacking AMPKα1 displayed reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation in vitro or pathogenic challenge in vivo. Finally, we demonstrated that AMPKα1 is essential for T helper 1 (Th1) and Th17 cell development and primary T cell responses to viral and bacterial infections in vivo. Our data highlight AMPK-dependent regulation of metabolic homeostasis as a key regulator of T cell-mediated adaptive immunity.

Paradoxical activation of AMPK by glucose drives selective EP300 activity in colorectal cancer.

Coordination of gene expression with nutrient availability supports proliferation and homeostasis and is shaped by protein acetylation. Yet how physiological/pathological signals link acetylation to specific gene expression programs and whether such responses are cell-type-specific is unclear. AMP-activated protein kinase (AMPK) is a key energy sensor, activated by glucose limitation to resolve nutrient supply-demand imbalances, critical for diabetes and cancer. Unexpectedly, we show here that, in gastrointestinal cancer cells, glucose activates AMPK to selectively induce EP300, but not CREB-binding protein (CBP). Consequently, EP300 is redirected away from nuclear receptors that promote differentiation towards ß-catenin, a driver of proliferation and colorectal tumorigenesis. Importantly, blocking glycogen synthesis permits reactive oxygen species (ROS) accumulation and AMPK activation in response to glucose in previously nonresponsive cells. Notably, glycogen content and activity of the ROS/AMPK/EP300/ß-catenin axis are opposite in healthy versus tumor sections. Glycogen content reduction from healthy to tumor tissue may explain AMPK switching from tumor suppressor to activator during tumor evolution.

Role of liver AMPK and GCN2 kinases in the control of postprandial protein metabolism in response to mid-term high or low protein intake in mice.

PURPOSE: Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake. METHODS: To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5%), NP (14%) and HP (54%). Two hours after a 1 g test meal, protein synthesis rate was measured after a 13C valine flooding dose. The gene expression of key enzymes involved in proteolysis and GNC2 signaling pathway were quantified. RESULTS: The HP diet but not the LP diet was associated with a decrease in fractional synthesis rate by 29% in the liver compared to NP diet. The expression of mRNA encoding ubiquitin and Cathepsin D was not sensitive to the protein content. The deletion of AMPK or GCN2 in the liver did not affect nor protein synthesis rates and neither proteolysis markers in the liver or in the muscle, whatever the protein intake. In the postprandial state, protein level alters protein synthesis in the liver but not in the muscle. CONCLUSIONS: Taken together, these results suggest that liver AMPK and GCN2 are not involved in this adaptation to high- and low-protein diet observed in the postprandial period.

At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism.

STUDY QUESTION: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)? SUMMARY ANSWER: The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response. WHAT IS KNOWN ALREADY: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin. STUDY DESIGN, SIZE, DURATION: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2). PARTICIPANTS/MATERIALS, SETTING, METHODS: α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9). MAIN RESULTS AND THE ROLE OF CHANCE: Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3ß-hydroxysteroid dehydrogenase (3ßHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3ßHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9). LARGE SCALE DATA: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048. LIMITATIONS, REASONS FOR CAUTION: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY ¼ funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. TRIAL REGISTRATION NUMBER: N/A.

TIM-4 glycoprotein-mediated degradation of dying tumor cells by autophagy leads to reduced antigen presentation and increased immune tolerance.

Phagocytosis of apoptotic cells by myeloid cells has been implicated in the maintenance of immune homeostasis. In this study, we found that T cell immunoglobulin- and mucin domain-containing molecule-4 (TIM-4) repressed tumor-specific immunity triggered by chemotherapy-induced tumor cell death. TIM-4 was found to be highly expressed on tumor-associated myeloid cells such as macrophages (TAMs) and dendritic cells (TADCs) and danger-associated molecular patterns (DAMPs) released from chemotherapy-damaged tumor cells induced TIM-4 on tumor-associated myeloid cells recruited from bone marrow-derived precursors. TIM-4 directly interacted with AMPKα1 and activated autophagy-mediated degradation of ingested tumors, leading to reduced antigen presentation and impaired CTL responses. Consistently, blockade of the TIM-4-AMPKα1-autophagy pathway augmented the antitumor effect of chemotherapeutics by enhancing tumor-specific CTL responses. Our finding provides insight into the immune tolerance mediated by phagocytosis of dying cells, and targeting of the TIM-4-AMPKα1 interaction constitutes a unique strategy for augmenting antitumor immunity and improving cancer chemotherapy.

AMPK activation by SC4 inhibits noradrenaline-induced lipolysis and insulin-stimulated lipogenesis in white adipose tissue.

The effects of small-molecule AMP-activated protein kinase (AMPK) activators in rat epididymal adipocytes were compared. SC4 was the most effective and submaximal doses of SC4 and 5-amino-4-imidazolecarboxamide (AICA) riboside were combined to study the effects of AMPK activation in white adipose tissue (WAT). Incubation of rat adipocytes with SC4 + AICA riboside inhibited noradrenaline-induced lipolysis and decreased hormone-sensitive lipase (HSL) Ser563 phosphorylation, without affecting HSL Ser565 phosphorylation. Preincubation of fat pads from wild-type (WT) mice with SC4 + AICA riboside inhibited insulin-stimulated lipogenesis from glucose or acetate and these effects were lost in AMPKα1 knockout (KO) mice, indicating AMPKα1 dependency. Moreover, in fat pads from acetyl-CoA carboxylase (ACC)1/2 S79A/S212A double knockin versus WT mice, the effect of SC4 + AICA riboside to inhibit insulin-stimulated lipogenesis from acetate was lost, pinpointing ACC as the main AMPK target. Treatment with SC4 + AICA riboside decreased insulin-stimulated glucose uptake, an effect that was still observed in fat pads from AMPKα1 KO versus WT mice, suggesting the effect was partly AMPKα1-independent. SC4 + AICA riboside treatment had no effect on the insulin-induced increase in palmitate esterification nor on sn-glycerol-3-phosphate-O-acyltransferase activity. Therefore in WAT, AMPK activation inhibits noradrenaline-induced lipolysis and suppresses insulin-stimulated lipogenesis primarily by inactivating ACC and by inhibiting glucose uptake.

The Marine-Derived Macrolactone Mandelalide A Is an Indirect Activator of AMPK.

The mandelalides are complex macrolactone natural products with distinct macrocycle motifs and a bioactivity profile that is heavily influenced by compound glycosylation. Mandelalides A and B are direct inhibitors of mitochondrial ATP synthase (complex V) and therefore more toxic to mammalian cells with an oxidative metabolic phenotype. To provide further insight into the pharmacology of the mandelalides, we studied the AMP-activated protein kinase (AMPK) energy stress pathway and report that mandelalide A is an indirect activator of AMPK. Wild-type mouse embryonic fibroblasts (MEFs) and representative human non-small cell lung cancer (NSCLC) cells showed statistically significant increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in response to mandelalide A. Mandelalide L, which also harbors an A-type macrocycle, induced similar increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in U87-MG glioblastoma cells. In contrast, MEFs co-treated with an AMPK inhibitor (dorsomorphin), AMPKα-null MEFs, or NSCLC cells lacking liver kinase B1 (LKB1) lacked this activity. Mandelalide A was significantly more cytotoxic to AMPKα-null MEFs than wild-type cells, suggesting that AMPK activation serves as a protective response to mandelalide-induced depletion of cellular ATP. However, LKB1 status alone was not predictive of the antiproliferative effects of mandelalide A against NSCLC cells. When EGFR status was considered, erlotinib and mandelalide A showed strong cytotoxic synergy in combination against erlotinib-resistant 11-18 NSCLC cells but not against erlotinib-sensitive PC-9 cells. Finally, prolonged exposures rendered mandelalide A, a potent and efficacious cytotoxin, against a panel of human glioblastoma cell types regardless of the underlying metabolic phenotype of the cell. These results add biological relevance to the mandelalide series and provide the basis for their further pre-clinical evaluation as ATP synthase inhibitors and secondary activators of AMPK.

Glucose availability but not changes in pancreatic hormones sensitizes hepatic AMPK activity during nutritional transition in rodents.

The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic regulator that mediates adaptation to nutritional variations to maintain a proper energy balance in cells. We show here that suckling-weaning and fasting-refeeding transitions in rodents are associated with changes in AMPK activation and the cellular energy state in the liver. These nutritional transitions were characterized by a metabolic switch from lipid to glucose utilization, orchestrated by modifications in glucose levels and the glucagon/insulin ratio in the bloodstream. We therefore investigated the respective roles of glucose and pancreatic hormones on AMPK activation in mouse primary hepatocytes. We found that glucose starvation transiently activates AMPK, whereas changes in glucagon and insulin levels had no impact on AMPK. Challenge of hepatocytes with metformin-induced metabolic stress strengthened both AMPK activation and cellular energy depletion under limited-glucose conditions, whereas neither glucagon nor insulin altered AMPK activation. Although both insulin and glucagon induced AMPKα phosphorylation at its Ser485/491 residue, they did not affect its activity. Finally, the decrease in cellular ATP levels in response to an energy stress was additionally exacerbated under fasting conditions and by AMPK deficiency in hepatocytes, revealing metabolic inflexibility and emphasizing the importance of AMPK for maintaining hepatic energy charge. Our results suggest that nutritional changes (i.e. glucose availability), rather than the related hormonal changes (i.e. the glucagon/insulin ratio), sensitize AMPK activation to the energetic stress induced by the dietary transition during fasting. This effect is critical for preserving the cellular energy state in the liver.

Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation.

The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3-3H]glucose relative to [2-3H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2-13C2]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate.

AMPKα1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis.

Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPKα1-/- MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPKα1-/- MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1-/- phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1-/- MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.

AICAR and compound C negatively modulate HCC-induced primary human hepatic stellate cell activation in vitro.

Tumor stroma and microenvironment have been shown to affect hepatocellular carcinoma (HCC) growth, with activated hepatic stellate cells (HSC) as a major contributor in this process. Recent evidence suggests that the energy sensor adenosine monophosphate-activated kinase (AMPK) may mediate a series of essential processes during carcinogenesis and HCC progression. Here, we investigated the effect of different HCC cell lines with known TP53 or CTNBB1 mutations on primary human HSC activation, proliferation, and AMPK activation. We show that conditioned media obtained from multiple HCC cell lines differently modulate human hepatic stellate cell (hHSC) proliferation and hHSC AMPK activity in a paracrine manner. Pharmacological treatment of hHSC with AICAR and Compound C inhibited the HCC-induced proliferation/activation of hHSC through AMPK-dependent and AMPK-independent mechanisms, which was further confirmed using mouse embryonic fibroblasts (MEFs) deficient of both catalytic AMPKα isoforms (AMPKα1/α2-/-) and wild type (wt) MEF. Both compounds induced S-phase cell-cycle arrest and, in addition, AICAR inhibited the mTORC1 pathway by inhibiting phosphorylation of 4E-BP1 and S6 in hHSC and wt MEF. Data mining of the Cancer Genome Atlas (TCGA) and the Liver Cancer (LICA-FR) showed that AMPKα1 (PRKAA1) and AMPKα2 (PRKAA2) expression differed depending on the mutation (TP53 or CTNNB1), tumor grading, and G1-G6 classification, reflecting the heterogeneity in human HCC. Overall, we provide evidence that AMPK modulating pharmacological agents negatively modulate HCC-induced hHSC activation and may therefore provide a novel approach to target the mutual, tumor-promoting interactions between hHSC and HCC.NEW & NOTEWORTHY HCC is marked by genetic heterogeneity and activated hepatic stellate cells (HSC) are considered key players during HCC development. The paracrine effect of different HCC cell lines on the activation of primary hHSC was accompanied by differential AMPK activation depending on the HCC line used. Pharmacological treatment inhibited the HCC-induced hHSC activation through AMPK-dependent and AMPK-independent mechanisms. This heterogenic effect on HCC-induced AMPK activation was confirmed by data mining TCGA and LICA-FR databases.

Myeloid deletion and therapeutic activation of AMPK do not alter atherosclerosis in male or female mice.

The dysregulation of myeloid-derived cell metabolism can drive atherosclerosis. AMP-activated protein kinase (AMPK) controls various aspects of macrophage dynamics and lipid homeostasis, which are important during atherogenesis. Using LysM-Cre to drive the deletion of both the α1 and α2 catalytic subunits (MacKO), we aimed to clarify the role of myeloid-specific AMPK signaling in male and female mice made acutely atherosclerotic by injection of AAV vector encoding a gain-of-function mutant PCSK9 (PCSK9-AAV) and WD feeding. After 6 weeks of WD feeding, mice received a daily injection of either the AMPK activator A-769662 or a vehicle control for an additional 6 weeks. Following this (12 weeks total), we assessed myeloid cell populations and differences between genotype or sex were not observed. Similarly, aortic sinus plaque size, lipid staining, and necrotic area did not differ in male and female MacKO mice compared with their littermate floxed controls. Moreover, therapeutic intervention with A-769662 showed no treatment effect. There were also no observable differences in the amount of circulating total cholesterol or triglyceride, and only minor differences in the levels of inflammatory cytokines between groups. Finally, CD68+ area and markers of autophagy showed no effect of either lacking AMPK signaling or AMPK activation. Our data suggest that while defined roles for each catalytic AMPK subunit have been identified, complete deletion of myeloid AMPK signaling does not significantly impact atherosclerosis. Additionally, these findings suggest that intervention with the first-generation AMPK activator A-769662 is not able to stem the progression of atherosclerosis.

Inhibition of mitochondrial complex 1 by the S6K1 inhibitor PF-4708671 partly contributes to its glucose metabolic effects in muscle and liver cells.

mTOR complex 1 (mTORC1) and p70 S6 kinase (S6K1) are both involved in the development of obesity-linked insulin resistance. Recently, we showed that the S6K1 inhibitor PF-4708671 (PF) increases insulin sensitivity. However, we also reported that PF can increase glucose metabolism even in the absence of insulin in muscle and hepatic cells. Here we further explored the potential mechanisms by which PF increases glucose metabolism in muscle and liver cells independent of insulin. Time course experiments revealed that PF induces AMP-activated protein kinase (AMPK) activation before inhibiting S6K1. However, PF-induced glucose uptake was not prevented in primary muscle cells from AMPK α1/2 double KO (dKO) mice. Moreover, PF-mediated suppression of hepatic glucose production was maintained in hepatocytes derived from AMPK α1/2-dKO mice. Remarkably, PF could still reduce glucose production and activate AMPK in hepatocytes from S6K1/2 dKO mice. Mechanistically, bioenergetics experiments revealed that PF reduces mitochondrial complex I activity in both muscle and hepatic cells. The stimulatory effect of PF on glucose uptake was partially reduced by expression of the Saccharomyces cerevisiae NADH:ubiquinone oxidoreductase in L6 cells. These results indicate that PF-mediated S6K1 inhibition is not required for its effect on insulin-independent glucose metabolism and AMPK activation. We conclude that, although PF rapidly activates AMPK, its ability to acutely increase glucose uptake and suppress glucose production does not require AMPK activation. Unexpectedly, PF rapidly inhibits mitochondrial complex I activity, a mechanism that partially underlies PF's effect on glucose metabolism.