The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity.

Current therapies for treating heterogeneous cancers such as head and neck squamous cell carcinoma (HNSCC) are non-selective and are administered independent of response biomarkers. Therapy resistance subsequently emerges, resulting in increased cellular proliferation that is associated with loss of differentiation. Whether a cancer cell differentiation potential can dictate therapy responsiveness is still currently unknown. A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity was employed in a HNSCC mouse model, primary patients' data, and human cell lines to assess the potential of functional differentiation in predicting therapy response. Interestingly, a subset of HNSCC with effective GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-Myc, and STAT3 signaling. Furthermore, we identified the GRHL3-differentiation target gene Filaggrin (FLG) as a response biomarker and more importantly, stratified HNSCC subsets as treatment resistant based on their FLG mutational profile. The loss of FLG in sensitive HNSCC resulted in a dramatic resistance to targeted therapies while the GRHL3-FLG signature predicted a favorable patient prognosis. This study provides evidence for a functional GRHL3-FLG tumor-specific differentiation axis that regulates targeted therapy response in HNSCC and establishes a rationale for clinical investigation of differentiation-paired targeted therapy in heterogeneous cancers.

AMPK phosphorylation of ACC2 is required for skeletal muscle fatty acid oxidation and insulin sensitivity in mice.

AIMS/HYPOTHESIS: Obesity is characterised by lipid accumulation in skeletal muscle, which increases the risk of developing insulin resistance and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status and is activated in skeletal muscle by exercise, hormones (leptin, adiponectin, IL-6) and pharmacological agents (5-amino-4-imidazolecarboxamide ribonucleoside [AICAR] and metformin). Phosphorylation of acetyl-CoA carboxylase 2 (ACC2) at S221 (S212 in mice) by AMPK reduces ACC activity and malonyl-CoA content but the importance of the AMPK-ACC2-malonyl-CoA pathway in controlling fatty acid metabolism and insulin sensitivity is not understood; therefore, we characterised Acc2 S212A knock-in (ACC2 KI) mice. METHODS: Whole-body and skeletal muscle fatty acid oxidation and insulin sensitivity were assessed in ACC2 KI mice and wild-type littermates. RESULTS: ACC2 KI mice were resistant to increases in skeletal muscle fatty acid oxidation elicited by AICAR. These mice had normal adiposity and liver lipids but elevated contents of triacylglycerol and ceramide in skeletal muscle, which were associated with hyperinsulinaemia, glucose intolerance and skeletal muscle insulin resistance. CONCLUSIONS/INTERPRETATION: These findings indicate that the phosphorylation of ACC2 S212 is required for the maintenance of skeletal muscle lipid and glucose homeostasis.

AMP-activated protein kinase (AMPK) beta1beta2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose uptake during exercise.

AMP-activated protein kinase (AMPK) ß1 or ß2 subunits are required for assembling of AMPK heterotrimers and are important for regulating enzyme activity and cellular localization. In skeletal muscle, α2ß2γ3-containing heterotrimers predominate. However, compensatory up-regulation and redundancy of AMPK subunits in whole-body AMPK α2, ß2, and γ3 null mice has made it difficult to determine the physiological importance of AMPK in regulating muscle metabolism, because these models have normal mitochondrial content, contraction-stimulated glucose uptake, and insulin sensitivity. In the current study, we generated mice lacking both AMPK ß1 and ß2 isoforms in skeletal muscle (ß1ß2M-KO). ß1ß2M-KO mice are physically inactive and have a drastically impaired capacity for treadmill running that is associated with reductions in skeletal muscle mitochondrial content but not a fiber-type switch. Interestingly, young ß1ß2M-KO mice fed a control chow diet are not obese or insulin resistant but do have impaired contraction-stimulated glucose uptake. These data demonstrate an obligatory role for skeletal muscle AMPK in maintaining mitochondrial capacity and contraction-stimulated glucose uptake, findings that were not apparent in mice with single mutations or deletions in muscle α, ß, or γ subunits.

The C-terminal protein interaction domain of the chromatin reader Yaf9 is critical for pathogenesis of Candida albicans.

Fungal infections cause a large health burden but are treated by only a handful of antifungal drug classes. Chromatin factors have emerged as possible targets for new antifungals. These targets include the reader proteins, which interact with posttranslationally modified histones to influence DNA transcription and repair. The YEATS domain is one such reader recognizing both crotonylated and acetylated histones. Here, we performed a detailed structure/function analysis of the Candida albicans YEATS domain reader Yaf9, a subunit of the NuA4 histone acetyltransferase and the SWR1 chromatin remodeling complex. We have previously demonstrated that the homozygous deletion mutant yaf9Δ/Δ displays growth defects and is avirulent in mice. Here we show that a YEATS domain mutant expected to inactivate Yaf9's chromatin binding does not display strong phenotypes in vitro, nor during infection of immune cells or in a mouse systemic infection model, with only a minor virulence reduction in vivo. In contrast to the YEATS domain mutation, deletion of the C-terminal domain of Yaf9, a protein-protein interaction module necessary for its interactions with SWR1 and NuA4, phenocopies the null mutant. This shows that the C-terminal domain is essential for Yaf9 roles in vitro and in vivo, including C. albicans virulence. Our study informs on the strategies for therapeutic targeting of Yaf9, showing that approaches taken for the mammalian YEATS domains by disrupting their chromatin binding might not be effective in C. albicans, and provides a foundation for studying YEATS proteins in human fungal pathogens.IMPORTANCEThe scarcity of available antifungal drugs and rising resistance demand the development of therapies with new modes of action. In this context, chromatin regulation may be a target for novel antifungal therapeutics. To realize this potential, we must better understand the roles of chromatin regulators in fungal pathogens. Toward this goal, here, we studied the YEATS domain chromatin reader Yaf9 in Candida albicans. Yaf9 uses the YEATS domain for chromatin binding and a C-terminal domain to interact with chromatin remodeling complexes. By constructing mutants in these domains and characterizing their phenotypes, our data indicate that the Yaf9 YEATS domain might not be a suitable therapeutic drug target. Instead, the Yaf9 C-terminal domain is critical for C. albicans virulence. Collectively, our study informs how a class of chromatin regulators performs their cellular and pathogenesis roles in C. albicans and reveals strategies to inhibit them.

AMPK couples plasma renin to cellular metabolism by phosphorylation of ACC1.

Salt reabsorption is the major energy-requiring process in the kidney, and AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism. Mice with targeted deletion of the ß1-subunit of AMPK (AMPK-ß1(-/-) mice) had significantly increased urinary Na(+) excretion on a normal salt diet. This was associated with reduced expression of the ß-subunit of the epithelial Na(+) channel (ENaC) and increased subapical tubular expression of kidney-specific Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2) in the medullary thick ascending limb of Henle. AMPK-ß1(-/-) mice fed a salt-deficient diet were able to conserve Na(+), but renin secretion increased 180% compared with control mice. Cyclooxygenase-2 mRNA also increased in the kidney cortex, indicating greater signaling through the macula densa tubular salt-sensing pathway. To determine whether the increase in renin secretion was due to a change in regulation of fatty acid metabolism by AMPK, mice with a mutation of the inhibitory AMPK phosphosite in acetyl-CoA carboxylase 1 [ACC1-knockin (KI)(S79A) mice] were examined. ACC1-KI(S79A) mice on a normal salt diet had no increase in salt loss or renin secretion, and expression of NKCC2, Na(+)-Cl(-) cotransporter, and ENaC-ß were similar to those in control mice. When mice were placed on a salt-deficient diet, however, renin secretion and cortical expression of cyclooxygenase-2 mRNA increased significantly in ACC1-KI(S79A) mice compared with control mice. In summary, our data suggest that renin synthesis and secretion are regulated by AMPK and coupled to metabolism by phosphorylation of ACC1.

Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells.

INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. METHODS: MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann-Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05). RESULTS: Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity. CONCLUSIONS: This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.

Dual functions of ASCIZ in the DNA base damage response and pulmonary organogenesis.

Zn²(+)-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn²(+)-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.

Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling.

Elevated levels of tumor necrosis factor (TNFalpha) are implicated in the development of insulin resistance, but the mechanisms mediating these chronic effects are not completely understood. We demonstrate that TNFalpha signaling through TNF receptor (TNFR) 1 suppresses AMPK activity via transcriptional upregulation of protein phosphatase 2C (PP2C). This in turn reduces ACC phosphorylation, suppressing fatty-acid oxidation, increasing intramuscular diacylglycerol accumulation, and causing insulin resistance in skeletal muscle, effects observed both in vitro and in vivo. Importantly even at pathologically elevated levels of TNFalpha observed in obesity, the suppressive effects of TNFalpha on AMPK signaling are reversed in mice null for both TNFR1 and 2 or following treatment with a TNFalpha neutralizing antibody. Our data demonstrate that AMPK is an important TNFalpha signaling target and is a contributing factor to the suppression of fatty-acid oxidation and the development of lipid-induced insulin resistance in obesity.

AMPK beta1 deletion reduces appetite, preventing obesity and hepatic insulin resistance.

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that regulates appetite and fuel metabolism. We have generated AMPK beta1(-/-) mice on a C57Bl/6 background that are viable, fertile, survived greater than 2 years, and display no visible brain developmental defects. These mice have a 90% reduction in hepatic AMPK activity due to loss of the catalytic alpha subunits, with modest reductions of activity detected in the hypothalamus and white adipose tissue and no change in skeletal muscle or heart. On a low fat or an obesity-inducing high fat diet, beta1(-/-) mice had reduced food intake, reduced adiposity, and reduced total body mass. Metabolic rate, physical activity, adipose tissue lipolysis, and lipogenesis were similar to wild type littermates. The reduced appetite and body mass of beta1(-/-) mice were associated with protection from high fat diet-induced hyperinsulinemia, hepatic steatosis, and insulin resistance. We demonstrate that the loss of beta1 reduces food intake and protects against the deleterious effects of an obesity-inducing diet.

Whole body deletion of AMP-activated protein kinase {beta}2 reduces muscle AMPK activity and exercise capacity.

AMP-activated protein kinase (AMPK) ß subunits (ß1 and ß2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK ß2 (ß2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that ß2 KO mice are viable and breed normally. ß2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the ß1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in ß2 KO mice. During treadmill running ß2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of ß2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet ß2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK ß2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.

Adipose triglyceride lipase regulation of skeletal muscle lipid metabolism and insulin responsiveness.

Adipose triglyceride lipase (ATGL) is important for triglyceride (TG) metabolism in adipose tissue, and ATGL-null mice show increased adiposity. Given the apparent importance of ATGL in TG metabolism and the association of lipid deposition with insulin resistance, we examined the role of ATGL in regulating skeletal muscle lipid metabolism and insulin-stimulated glucose disposal. ATGL expression in myotubes was reduced by small interfering RNA and increased with a retrovirus encoding GFP-HA-ATGL. ATGL was also overexpressed in rats by in vivo electrotransfer. ATGL was down-regulated in skeletal muscle of obese, insulin-resistant mice and negatively correlated with intramyocellular TG levels. ATGL small interfering RNA in myotubes reduced TG hydrolase activity and increased TG content, whereas ATGL overexpression induced the reciprocal response, indicating that ATGL is an essential TG lipase in skeletal muscle. ATGL overexpression in myotubes increased the oxidation of fatty acid liberated from TG and diglyceride and ceramide contents. These responses in cells were largely recapitulated in rats overexpressing ATGL. When ATGL protein expression and TG hydrolase activity in obese, insulin-resistant rats were restored to levels observed in lean rats, TG content was reduced; however, the insulin resistance induced by the high-fat diet persisted. In conclusion, ATGL TG hydrolysis in skeletal muscle is a critical determinant of lipid metabolism and storage. Although ATGL content and TG hydrolase activity are decreased in obese, insulin-resistant phenotypes, overexpression does not rescue the condition, indicating reduced ATGL is unlikely to be a primary cause of obesity-associated insulin resistance.

Inhibition of Adenosine Monophosphate-Activated Protein Kinase-3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Signaling Leads to Hypercholesterolemia and Promotes Hepatic Steatosis and Insulin Resistance.

Adenosine monophosphate-activated protein kinase (AMPK) regulates multiple signaling pathways involved in glucose and lipid metabolism in response to changes in hormonal and nutrient status. Cell culture studies have shown that AMPK phosphorylation and inhibition of the rate-limiting enzyme in the mevalonate pathway 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase (HMGCR) at serine-871 (Ser871; human HMGCR Ser872) suppresses cholesterol synthesis. In order to evaluate the role of AMPK-HMGCR signaling in vivo, we generated mice with a Ser871-alanine (Ala) knock-in mutation (HMGCR KI). Cholesterol synthesis was significantly suppressed in wild-type (WT) but not in HMGCR KI hepatocytes in response to AMPK activators. Liver cholesterol synthesis and cholesterol levels were significantly up-regulated in HMGCR KI mice. When fed a high-carbohydrate diet, HMGCR KI mice had enhanced triglyceride synthesis and liver steatosis, resulting in impaired glucose homeostasis. Conclusion: AMPK-HMGCR signaling alone is sufficient to regulate both cholesterol and triglyceride synthesis under conditions of a high-carbohydrate diet. Our findings highlight the tight coupling between the mevalonate and fatty acid synthesis pathways as well as revealing a role of AMPK in suppressing the deleterious effects of a high-carbohydrate diet.

AMPK beta subunit targets metabolic stress sensing to glycogen.

AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients, as well as hormones, including adiponectin and leptin. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK. Mammalian AMPK is an alphabetagamma heterotrimer with multiple isoforms of each subunit comprising alpha1, alpha2, beta1, beta2, gamma1, gamma2, and gamma3, which have varying tissue and subcellular expression. Mutations in the AMPK gamma subunit cause glycogen storage disease in humans, but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK beta subunit contains a functional glycogen binding domain (beta-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished beta-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK beta1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.

Mutations in the gal83 glycogen-binding domain activate the snf1/gal83 kinase pathway by a glycogen-independent mechanism.

The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK beta1 subunit, and the sequence is conserved in the Snf1 kinase beta subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK beta1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

Structural basis for glycogen recognition by AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK beta subunit acts as a scaffold for the alpha catalytic and gamma regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK beta glycogen binding domain in complex with beta-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. beta-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two beta-cyclodextrin glucose units and a leucine residue piercing the beta-cyclodextrin ring. Mutation of key beta-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate's helical surface.

Exemplary multiplex bisulfite amplicon data used to demonstrate the utility of Methpat.

BACKGROUND: DNA methylation is a complex epigenetic marker that can be analyzed using a wide variety of methods. Interpretation and visualization of DNA methylation data can mask complexity in terms of methylation status at each CpG site, cellular heterogeneity of samples and allelic DNA methylation patterns within a given DNA strand. Bisulfite sequencing is considered the gold standard, but visualization of massively parallel sequencing results remains a significant challenge. FINDINGS: We created a program called Methpat that facilitates visualization and interpretation of bisulfite sequencing data generated by massively parallel sequencing. To demonstrate this, we performed multiplex PCR that targeted 48 regions of interest across 86 human samples. The regions selected included known gene promoters associated with cancer, repetitive elements, known imprinted regions and mitochondrial genomic sequences. We interrogated a range of samples including human cell lines, primary tumours and primary tissue samples. Methpat generates two forms of output: a tab-delimited text file for each sample that summarizes DNA methylation patterns and their read counts for each amplicon, and a HTML file that summarizes this data visually. Methpat can be used with publicly available whole genome bisulfite sequencing and reduced representation bisulfite sequencing datasets with sufficient read depths. CONCLUSIONS: Using Methpat, complex DNA methylation data derived from massively parallel sequencing can be summarized and visualized for biological interpretation. By accounting for allelic DNA methylation states and their abundance in a sample, Methpat can unmask the complexity of DNA methylation and yield further biological insight in existing datasets.

Skeletal muscle ACC2 S212 phosphorylation is not required for the control of fatty acid oxidation during exercise.

During submaximal exercise fatty acids are a predominant energy source for muscle contractions. An important regulator of fatty acid oxidation is acetyl-CoA carboxylase (ACC), which exists as two isoforms (ACC1 and ACC2) with ACC2 predominating in skeletal muscle. Both ACC isoforms regulate malonyl-CoA production, an allosteric inhibitor of carnitine palmitoyltransferase 1 (CPT-1); the primary enzyme controlling fatty acyl-CoA flux into mitochondria for oxidation. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is activated during exercise or by pharmacological agents such as metformin and AICAR. In resting muscle the activation of AMPK with AICAR leads to increased phosphorylation of ACC (S79 on ACC1 and S221 on ACC2), which reduces ACC activity and malonyl-CoA; effects associated with increased fatty acid oxidation. However, whether this pathway is vital for regulating skeletal muscle fatty acid oxidation during conditions of increased metabolic flux such as exercise/muscle contractions remains unknown. To examine this we characterized mice lacking AMPK phosphorylation sites on ACC2 (S212 in mice/S221 in humans-ACC2-knock-in [ACC2-KI]) or both ACC1 (S79) and ACC2 (S212) (ACC double knock-in [ACCD-KI]) during submaximal treadmill exercise and/or ex vivo muscle contractions. We find that surprisingly, ACC2-KI mice had normal exercise capacity and whole-body fatty acid oxidation during treadmill running despite elevated muscle ACC2 activity and malonyl-CoA. Similar results were observed in ACCD-KI mice. Fatty acid oxidation was also maintained in muscles from ACC2-KI mice contracted ex vivo. These findings indicate that pathways independent of ACC phosphorylation are important for regulating skeletal muscle fatty acid oxidation during exercise/muscle contractions.

Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

AMP-activated protein kinase is not down-regulated in human skeletal muscle of obese females.

Obesity in humans is associated with lipid accumulation in skeletal muscle, insulin and leptin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) is an important regulator of fatty acid (FA) metabolism in skeletal muscle. To address the hypothesis that lipid accumulation in skeletal muscle of obese subjects may be due to down-regulation of AMPK, we measured mRNA and protein levels of AMPK isoforms, AMPKalpha1 and -alpha2 activity, AMPK kinase activity, acetyl-coenzyme A carboxylase (ACCbeta) expression and phosphorylation, and FA metabolism in biopsies of rectus abdominus muscle from lean and obese women. We also examined the effect of 5-aminoimidazole-4-carboxamide riboside (AICAR) on AMPK activity and the effects of AICAR and leptin on FA metabolism. Skeletal muscle of obese subjects had increased total FA uptake and triglyceride esterification, and leptin failed to stimulate FA oxidation. However, AMPK mRNA and protein expression, AMPKalpha1 and -alpha2 activities, AMPK kinase activity, ACCbeta phosphorylation, and FA oxidation were similar in lean and obese subjects. Moreover, AICAR increased AMPKalpha2 activity, ACCbeta phosphorylation, and palmitate oxidation to a similar degree in muscle from lean and obese subjects. We conclude that the abnormal lipid metabolism and leptin resistance of skeletal muscle of obese subjects is not due to down-regulation of AMPK. In addition, the similar stimulation by AICAR of AMPK in skeletal muscle of lean and obese subjects suggests that direct pharmacological activation of AMPK may be a therapeutic approach for stimulating FA oxidation in the treatment of human obesity.