The different types of muscle fibres respond in a specific way to hypertrophy or atrophy. The mechanisms underlying these heterogeneous adaptations remain poorly understood. Using single-nucleus RNA sequencing, we propose that fast glycolytic fibres show genetic limitations to hypertrophy induced by mechanical overload. We show that a prior fibre transition, achieved by reducing SIX1 protein expression (hypomorphism), enhances and accelerates overload-induced hypertrophy, bypassing the genetic limitations of fast glycolytic fibres. In contrast and unexpectedly, Six1 knockout in myofibers abolished overload-induced hypertrophy and instead caused atrophy of IIb/IIx fibers, despite the induction of a strong slow oxidative phenotype. In particular, Six1 deletion leads to metabolic defects caused by inhibition of glycolysis, AMPK and mitochondrial biogenesis. Our findings highlight the critical role of SIX1/AMPK/glycolysis-dependent aerobic metabolism in muscle growth and suggest that fibre type transitions, coupled with preserved metabolic function, may optimise hypertrophic responses. ### Competing Interest Statement The authors have declared no competing interest.

Patients with metastatic high-grade serous ovarian carcinoma are often unresponsive to immunotherapies; here we identify salt-inducible kinases (SIKs) as key drivers of immunosuppression. Human T cells in the presence of patient ascites express high levels of SIK and the upstream kinase LKB1, whereas SIK inhibition reprograms human T cells and strongly activates antitumor responses. In syngeneic mice with resistant high-grade serous ovarian carcinoma, genetic ablation and pharmaceutical inhibition of SIK consistently demonstrated therapeutic efficacy and survival advantages, and combination of PD-1 blockade with SIK inhibition further extended survival. We identified a major role of T cell-intrinsic SIK2 and -3 signaling in driving immunosuppression in part by TXNIP induction and LYST suppression. Multi-omics analyses on SIK inhibitor therapy revealed reduced disease progression, increased T cell infiltration with enhanced cytotoxicity and effector cytokine IFN-γ, and a shift from immunosuppressive to immunostimulatory cellular niche. We propose SIK inhibitors as a new immunotherapy.

Alveolar bone supports and anchors teeth. The parathyroid hormone-related protein (PTHrP) pathway plays a key role in alveolar bone biology. Salt-inducible kinases (SIKs) are important downstream regulators of PTH/PTHrP signaling in the appendicular skeleton, where SIK inhibition increases bone formation and trabecular bone mass. However, the function of these kinases in alveolar bone remains unknown. Here, we report a critical role for SIK2/SIK3 in alveolar bone development, homeostasis, and socket healing after tooth extraction. Inducible SIK2/SIK3 (Ubq-creERt;Sik2f/f;Sik3f/f) deletion led to dramatic alveolar bone defects without changes in tooth eruption. Ablating these kinases impairs alveolar bone formation due to disrupted osteoblast maturation, a finding associated with ectopic periostin expression by fibrous cells in regions of absent alveolar bone at steady state and following molar extraction. Notably, this phenotype is the opposite of the increased trabecular bone mass observed in long bones following SIK2/SIK3 deletion. Distinct phenotypic consequences of SIK2/SIK3 deletion in appendicular versus craniofacial bones prompted us to identify a specific transcriptomic signature in alveolar versus long bone osteoblasts. Thus, SIK2/SIK3 deletion illuminates a key role for these kinases in alveolar bone biology and highlights the emerging concept that different osteoblast subsets utilize unique genetic programs.

Inhibition of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is under increasing investigation for its therapeutic potential in many diseases. Existing AMPK inhibitors are however limited, with poor selectivity and substantial off-target effects. Here, we provide mechanistic insights and describe the cellular selectivity of the recently identified AMPK inhibitor BAY-3827. A 2.5-Å cocrystal structure of the AMPK kinase domain with BAY-3827 revealed distinct features including a disulfide bridge between the αD helix Cys106 and the activation loop residue Cys174. This bridge appears to stabilize the activation loop such that Asn162 repositions the Asp-Phe-Gly (DFG) motif Phe158 toward the C-terminal lobe, displacing His137 and disrupting the regulatory spine, promoting an inactive kinase state. In hepatocytes, BAY-3827 blocked AMPK activator (MK-8722)-mediated phosphorylation of ACC1 and corresponding inhibition of lipogenesis. Transcriptome analysis revealed that BAY-3827 down-regulated ~30% of MK-8722-stimulated AMPK-dependent genes. We establish the molecular and cellular basis of BAY-3827's selectivity and utility for delineating AMPK functions while highlighting its limitations.

AMPK acts downstream of the tumor suppressor LKB1, yet its role in cancer has been controversial. AMPK is activated by biguanides, such as metformin and phenformin, and metformin use in diabetics has been associated with reduced cancer risk. However, whether this is mediated by cell-autonomous AMPK activation within tumor progenitor cells has been unclear. We report that T-cell-specific loss of AMPK-α1 caused accelerated growth of T cell acute lymphoblastic leukemia/lymphoma (T-ALL) induced by PTEN loss in thymic T cell progenitors. Oral administration of phenformin, but not metformin, delayed onset and growth of lymphomas, but only when T cells expressed AMPK-α1. This differential effect of biguanides correlated with detection of phenformin, but not metformin, in thymus. Phenformin also enhanced apoptosis in T-ALL cells both in vivo and in vitro. Thus, AMPK-α1 can be a cell-autonomous tumor suppressor in the context of T-ALL, and phenformin may have potential for the prevention of some cancers.

Metabolic reprogramming controls protective and pathogenic T helper 17 (TH17) cell responses. When naïve T cells are differentiated into TH17 cells in vitro, the presence of the cytokine activin A promotes their maturation into a nonpathogenic state. Here, we found that nonpathogenic TH17 cells induced by activin A displayed reduced aerobic glycolysis and increased oxidative phosphorylation (OXPHOS). In response to activin A, signaling through the adenosine A2A receptor (A2AR) and AMP-activated protein kinase (AMPK) enhanced OXPHOS and reprogrammed pathogenic TH17 cells toward nonpathogenic states that did not induce central nervous system autoimmunity in a mouse model of multiple sclerosis. In pathogenic TH17 cells, the transcriptional coactivator p300/CBP-associated factor (PCAF) increased acetylation at histone 3 Lys9 (H3K9ac) of genes involved in aerobic glycolysis and TH17 pathogenic programs. In contrast, in nonpathogenic activin A-treated TH17 cells, AMPK signaling suppressed PCAF-mediated H3K9ac modification of genes involved in aerobic metabolism and enhanced H3K9ac modification of genes involved in OXPHOS and nonpathogenic TH17 programs. Together, our findings uncover A2AR-AMPK signaling as a central metabolic checkpoint that suppresses TH17 cell pathogenicity.

Inhibition of AMP-activated protein kinase (AMPK) is under increasing investigation for its therapeutic potential in many diseases, including certain cancers. However, existing AMPK-inhibitors available as tool compounds are largely limited to compound C/dorsomorphin and SBI-0206965, both of which suffer from poor selectivity and off-target effects. Here we describe the structure-based molecular insights and cellular actions of a recently identified potent AMPK inhibitor, BAY-3827. Kinase selectivity profiling and sequence analyses of kinases that are highly or weakly inhibited by BAY-3827 uncovered key conserved residues involved in its inhibitory mechanism. A 2.5 Å co-crystal structure of the AMPK kinase domain (KD)-BAY-3827 complex and comparison with known KD-inhibitor structures, revealed an overlapping site in the ATP-binding pocket and an αC helix-out conformation. A distinct feature of the BAY-3827-bound state is the formation of a disulfide bridge between the αD helix Cys106 and the activation loop residue Cys174. This bridge appears to stabilize the activation loop such that Asn162 repositions the DFG motif Phe158 toward the C-terminal kinase lobe, displacing His137 and disrupting the regulatory spine, thereby promoting an inactive state. In hepatocytes, 2.5-5 μM BAY-3827, but not the structurally resembling inactive BAY-974, fully blocked AMPK activator (MK-8722)-mediated phosphorylation of ACC1 and corresponding inhibition of lipogenesis. Unbiased transcriptome analysis in MK-8722-treated wild-type and AMPK-null hepatocytes revealed that 5 μM BAY-3827 downregulated >30% of MK-8722-stimulated AMPK-dependent genes. Based on its greater selectivity and potency substantiated by comprehensive structural and cellular investigations, BAY-3827 is a powerful tool to delineate AMPK functions. ### Competing Interest Statement The authors have declared no competing interest.

Metformin currently serves as the basis of oral treatment for patients with type 2 diabetes. While metformin's effect on glycemia is well established, its mode of action remains unclear. In clinical studies, long-term metformin treatment improved glucose disposal and reduced hepatic gluconeogenesis. The effects on gluconeogenesis have been confirmed in experimental animals and cell preparations, but mostly at higher doses than those used in humans. Proposed hepatic mechanisms can be grouped into those with and without AMP-activated protein kinase (AMPK) activation; the latter include inhibition of mitochondrial complex I and mitochondrial glycerophosphate dehydrogenase. Experimental studies on the effects of metformin on skeletal muscles suggest that AMPK activation and anti-inflammatory activities are possible mechanisms for increasing glucose disposal. Inhibition of renal gluconeogenesis may contribute to the extraintestinal glycemia-lowering effects of metformin. Following the observation that short-term intravenous metformin lacks glycemia-lowering effects in humans, intestinal mechanisms have been investigated. Suggested mechanisms include inhibition of intestinal glucose absorption owing to increased glycolysis driven by complex I inhibition in the mitochondria of enterocytes, stimulation of glucose transport into the colon, and stimulation of glucagon-like peptide-1 (GLP-1) secretion. Intestinal GLP-1 activates the gut-brain-liver axis, which impairs hepatic gluconeogenesis through vagal stimulation. Metformin can enhance intestinal GLP-1 secretion by L-cells directly through AMPK activation via complex I inhibition or indirectly by increasing the availability of glucose, bile acids, and/or metabolites produced by intestinal bacteria. Thus, metformin improves muscle glucose disposal, reduces gluconeogenesis, and has several intestinal effects that impact glycemia. Inhibition of mitochondrial complex I in different organs appears to be an important mechanism of metformin's glucose-lowering effect. SIGNIFICANCE STATEMENT: Most previous studies on the mechanism of metformin's glycemia-reducing effect focused on inhibition of hepatic gluconeogenesis. However, clinical studies show that increased glucose transport into skeletal muscle is at least as important. Furthermore, recent studies suggest that intestinal effects, including inhibition of glucose absorption, stimulation of the gut-liver and gut-brain-liver axes, and changes in the intestinal microbiota, contribute to metformin's glycemia-lowering effect. Thus, metformin's glycemia-reducing effect is multifactorial, affecting glucose metabolism in the gut, liver, and skeletal muscle.

Metabolic reprogramming controls protective and pathogenic T helper 17 (T H 17) cell responses. When naïve T cells are differentiated into T H 17 cells in vitro, the presence of the cytokine activin A promotes their maturation into a nonpathogenic state. Here, we found that nonpathogenic T H 17 cells induced by activin A displayed reduced aerobic glycolysis and increased oxidative phosphorylation (OXPHOS). In response to activin A, signaling through the adenosine A 2A receptor (A 2A R) and AMP-activated protein kinase (AMPK) enhanced OXPHOS and reprogrammed pathogenic T H 17 cells toward nonpathogenic states that did not induce central nervous system autoimmunity in a mouse model of multiple sclerosis. In pathogenic T H 17 cells, the transcriptional coactivator p300/CBP-associated factor (PCAF) increased acetylation at histone 3 Lys 9 (H3K9ac) of genes involved in aerobic glycolysis and T H 17 pathogenic programs. In contrast, in nonpathogenic activin A–treated T H 17 cells, AMPK signaling suppressed PCAF-mediated H3K9ac modification of genes involved in aerobic metabolism and enhanced H3K9ac modification of genes involved in OXPHOS and nonpathogenic T H 17 programs. Together, our findings uncover A 2A R-AMPK signaling as a central metabolic checkpoint that suppresses T H 17 cell pathogenicity.

OBJECTIVES:Small-molecule activators targeting the allosteric drug and metabolite (ADaM) site of AMPK enhance insulin-independent glucose uptake in skeletal muscle and lower glucose in preclinical models of hyperglycemia. The regulatory AMPKγ subunit plays a central role in energy sensing. While the skeletal muscle-selective γ3 isoform is essential for AMP/ZMP-induced glucose uptake, it is dispensable for ADaM site-binding activators. We hypothesized that the predominant γ1 isoform is required for ADaM site activator-stimulated glucose uptake in skeletal muscle. METHODS:Single-nucleus RNA sequencing (snRNA-seq) was performed on mouse and human skeletal muscle mapping AMPK subunit isoform distribution across resident cell types. To determine γ isoform-specific requirements for activator-stimulated glucose uptake, skeletal muscle-specific inducible AMPKγ1/γ3 double knockout (imγ1-/-/γ3-/-) and single knockout (imγ1-/- and imγ3-/-) mice were generated. Ex vivo glucose uptake was measured following treatment with AICAR (AMP-mimetic) or MK-8722 (ADaM site activator), and in vivo MK-8722-induced blood glucose lowering was assessed. RESULTS:snRNA-seq revealed distinct AMPK isoform distribution: γ1 was ubiquitously expressed, whereas γ3 was enriched in glycolytic myofibers in both mouse and human skeletal muscle. Ex vivo, glucose uptake stimulated by either AICAR or MK-8722 was severely blunted in imγ1-/-/γ3-/- muscle, and MK-8722-induced blood glucose lowering was significantly blunted in vivo. AICAR but not MK-8722-stimulated muscle glucose uptake was abolished in imγ3-/-, whereas both activators fully retained effects on glucose uptake and glucose lowering in imγ1-/- mice. CONCLUSIONS:While γ1 predominates in stabilizing the AMPKα2β2γ1 complex, it is dispensable for AMPK activator-stimulated glucose uptake in skeletal muscle, whether mediated via the nucleotide-binding or ADaM site.

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 AMPKa1 knockout mice. In this study, we investigated the specific involvement of AMPKa1 in myeloid cells using LysMCre;AMPKa1flmice. Our findings revealed that whereas endophthalmitis resolved in wild-type C57BL/6 mice, the severity of the disease progressively worsened in AMPKa1-deficient mice over time. Moreover, the intraocular bacterial load and inflammatory mediators (e.g., IL-1b, TNF-a, IL-6, and CXCL2) were markedly elevated in the LysMCre;AMPKa1flmice. Mechanistically, the deletion of AMPKa1 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 AMPKa1 knockout mice preserved retinal function and mitigated the severity of endophthalmitis. Overall, our study underscores the role of myeloid-specific AMPKa1 in promoting the resolution of inflammation in the eye during bacterial infection. Hence, therapeutic strategies aimed at restoring or enhancing AMPKa1 activity could improve visual outcomes in endophthalmitis and other ocular infections. The Journal of Immunology, 2024, 213: 1656-1665.

Phloroglucinol (PHG), an analgesic and spasmolytic drug, shows promise in preventing high-fat-diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) and insulin resistance. In Wistar rats, 10 weeks of PHG treatment did not prevent HFD-induced weight gain but significantly mitigated fasting hyperglycemia, impaired insulin responses, and liver steatosis. This protective effect was not linked to hepatic lipogenesis or AMP-activated protein kinase (AMPK) activation. Instead, PHG improved mitochondrial function by reducing oxidative stress, enhancing ATP production, and increasing anti-oxidant enzyme activity. PHG also relaxed gastric smooth muscles via potassium channel activation and nitric oxide (NO) signaling, potentially delaying gastric emptying. A pilot intervention in pre-diabetic men confirmed PHG’s efficacy in improving postprandial glycemic control and altering lipid metabolism. These findings suggest PHG as a potential therapeutic for NAFLD and insulin resistance, acting through mechanisms involving mitochondrial protection, anti-oxidant activity, and gastric motility modulation. Further clinical evaluation is warranted to explore PHG’s full therapeutic potential.

Salt-inducible kinases (SIKs), a family of serine/threonine kinases, were found to be critical determinants of female fertility. SIK2 silencing results in increased ovulatory response to gonadotropins. In contrast, SIK3 knockout results in infertility, gonadotropin insensitivity, and ovaries devoid of antral and preovulatory follicles. This study hypothesizes that SIK2 and SIK3 differentially regulate follicle growth and fertility via contrasting actions in the granulosa cells (GCs), the somatic cells of the follicle. Therefore, SIK2 or SIK3 GC-specific knockdown (SIK2GCKD and SIK3GCKD, respectively) mice were generated by crossing SIK floxed mice with Cyp19a1pII-Cre mice. Fertility studies revealed that pup accumulation over 6 months and the average litter size of SIK2GCKD mice were similar to controls, although in SIK3GCKD mice were significantly lower compared to controls. Compared to controls, gonadotropin stimulation of prepubertal SIK2GCKD mice resulted in significantly higher serum estradiol levels, whereas SIK3GCKD mice produced significantly less estradiol. Cyp11a1, Cyp19a1, and StAR were significantly increased in the GCs of gonadotropin-stimulated SIK2GCKD mice. However, Cyp11a1 and StAR remained significantly lower than controls in SIK3GCKD mice. Interestingly, Cyp19a1 stimulation in SIK3GCKD was not statistically different compared to controls. Superovulation resulted in SIK2GCKD mice ovulating significantly more oocytes, whereas SIK3GCKD mice ovulated significantly fewer oocytes than controls. Remarkably, SIK3GCKD superovulated ovaries contained significantly more preantral follicles than controls. SIK3GCKD ovaries contained significantly more apoptotic cells and fewer proliferating cells than controls. These data point to the differential regulation of GC function and follicle development by SIK2 and SIK3 and supports the therapeutic potential of targeting these kinases for treating infertility or developing new contraceptives.

ABSTRACT 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 (LAKO) and wild type (WT) mice 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 WT mice, but not in LAKO mice. The inability to increase glycogen led to lower glycogenolysis, glucose production, and circulating glucose during acute exercise in trained LAKO 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 WT mice, but not in LAKO mice. Training increased liver triacylglycerides and the desaturation and elongation of fatty acids in triacylglycerides of LAKO 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, is necessary for an exercise training-induced: i) increase in liver glycogen that is necessary for accelerated glycogenolysis during exercise, ii) decrease in liver glycerolipids independent of TCA cycle flux, and iii) 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 cellular mechanisms underlying axonal morphogenesis are essential to the formation of functional neuronal networks. We previously identified the autism-linked kinase NUAK1 as a central regulator of axon branching through the control of mitochondria trafficking. However, (1) the relationship between mitochondrial position, function and axon branching and (2) the downstream effectors whereby NUAK1 regulates axon branching remain unknown. Here, we report that mitochondria recruitment to synaptic boutons supports collateral branches stabilization rather than formation in mouse cortical neurons. NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration, and upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Finally, we found that NUAK1 regulates axon branching through the mitochondria-targeted microprotein BRAWNIN. Our results demonstrate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondrial distribution and metabolic activity.

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 nonalcoholic steatohepatitis (NASH) and whether treatment with metformin, a firstline therapy for diabetes and activator of AMPK could stem disease progression. Male and female Prkaa1 fl / fl /Prkaa2 fl / fl (Flox) control and Flox- LysM -Cre & thorn; (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.