OBJECTIVE-Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms. RESEARCH DESIGN AND METHODS AND RESULTS-Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-␤-D-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription.CONCLUSIONS-These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes. Diabetes 57:860-867, 2008

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that plays a pivotal role in regulating cellular and whole-body metabolism. Activation of AMPK reverses many of the metabolic defects associated with obesity and type 2 diabetes, and therefore AMPK is considered a promising target for drugs to treat these diseases. Recently, the thienopyridone A769662 has been reported to directly activate AMPK by an unexpected mechanism. Here we show that A769662 activates AMPK by a mechanism involving the beta subunit carbohydrate-binding module and residues from the gamma subunit but not the AMP-binding sites. Furthermore, A769662 exclusively activates AMPK heterotrimers containing the beta1 subunit. Our findings highlight the regulatory role played by the beta subunit in modulating AMPK activity and the possibility of developing isoform specific therapeutic activators of this important metabolic regulator.

The AMP-activated protein kinase (AMPK) is an ␣␤␥ heterotrimer that regulates appetite and fuel metabolism. We have generated AMPK ␤1 ؊/؊ 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 ␣ 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, ␤1 ؊/؊ 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 ␤1 ؊/؊ mice were associated with protection from high fat diet-induced hyperinsulinemia, hepatic steatosis, and insulin resistance. We demonstrate that the loss of ␤1 reduces food intake and protects against the deleterious effects of an obesity-inducing diet.The AMP-activated protein kinase (AMPK) 6 is an evolutionarily conserved serine/threonine protein kinase that functions as a metabolic regulatory enzyme at both the cellular and whole body level (1). AMPK is activated in response to physiological processes that raise intracellular levels of AMP, such as exercise and hypoxia. It restores cellular energy balance by switching off ATP-consuming anabolic pathways and switching on ATPgenerating catabolic pathways by direct phosphorylation of downstream targets. Modulation of AMPK activity by hormones adds an additional layer of control, allowing cellular energy supply and demand to be balanced with the energy requirements of the whole organism (2).AMPK functions as an ␣␤␥ heterotrimer. Different isoforms for each of the subunits exist (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3) as well as some splice variants, allowing more than 12 heterotrimeric combinations to be generated that may mediate unique tissue-specific functions (3, 4). The 63-kDa AMPK ␣ subunits, designated ␣1 and ␣2, contain a serine/threonine protein kinase catalytic domain that is activated by phosphorylation of Thr-172 in the activation loop (5, 6). We, and others have shown that the C terminus of the ␤ subunits are essential for AMPK heterotrimer assembly by anchoring the ␣ and ␥ subunits (7,8). The ␤1 and ␤2 subunits show 82% identity from residue 73 to 270, but only 43% identity for the N-terminal residues 1-72 (9). The ␤1 subunit is N-terminally myristoylated and is phosphorylated on multiple serines (10); however, the physiological importance of these phosphorylation sites is poorly understood. Northern blot analysis of human tissues revealed that AMPK ␤1 expression is highest in the liver and brain and low in kidney and skeletal muscle, whereas ␤2 is most highly expressed in skeletal muscle with lower expression in kidney, liver, and lung (11).Hepatic AMPK is thought to play important roles in regulating lipid metabolism, glucose homeostasis, and insulin sensitivity (1)....

AMP-activated protein kinase (AMPK) ␤ subunits (␤1 and ␤2) provide scaffolds for binding ␣ and ␥ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK ␤2 (␤2 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 ␤2 KO mice are viable and breed normally. ␤2 KO mice had a reduction in skeletal muscle AMPK ␣1 and ␣2 expression despite up-regulation of the ␤1 isoform. Heart AMPK ␣2 expression was also reduced but this did not affect resting AMPK ␣1 or ␣2 activities. AMPK ␣1 and ␣2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in ␤2 KO mice. During treadmill running ␤2 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 ␤2 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 ␤2 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 ␤2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.The AMP-activated protein kinase (AMPK) 5 is an evolutionary conserved serine/threonine protein kinase that functions as a metabolic regulatory enzyme at both the intracellular and whole body level (1, 2). As a metabolic stress-sensing enzyme, AMPK is activated through phosphorylation of Thr 172 in the ␣-catalytic subunit by upstream kinases, liver kinase B1 (LKB1) and calcium/calmodulin-dependent kinase kinase in response to physiological processes that consume ATP (exercise) or inhibit ATP production (ischemia or hypoxia) (3). Following activation, AMPK acutely regulates lipid, protein, and carbohydrate metabolism through phosphorylation induced changes that alter enzyme activities by switching off ATP consuming anabolic pathways and switching on ATP generating catabolic pathways (4). In addition to these acute effects, AMPK regulates transcription factors to influence gene expression (4). Modulation of AMPK activity by hormones and cytokines adds a complex layer of regulation allowing energy supply and demand within a cell to be integrated with the energy requirements of the whole organism (5).AMPK functions as an ␣␤␥ heterotrimer where the C terminus of the ␤ isoforms (␤1 and ␤2) contains the subunit-binding sequence that is essential for binding the ␥ and ␣ subunits (6, 7). In addition to their structural role in maintaining the AMPK heterotrimer, AMPK ␤ subunits contain an evolutionary ...

AMP-activated protein kinase (AMPK) is a ␣␤␥ heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the ␣ subunit and by AMP allosteric control previously thought to be mediated by both ␣ and ␥ subunits. Here we present evidence that adjacent ␥ 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 ␥ 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 ␥ homolog, snf4 contains a His151Gly substitution, and when this is introduced into ␥ 1 , AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in ␥ 1 corresponds to the site of mutation in human ␥ 2 and pig ␥ 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 ␣ and ␥ subunits and that AMP functions to derepress AMPK activity.Keywords: AMPK; AMP; CBS sequences; ␥ subunit; allosteric and intrasteric control AMPK is a multisubstrate enzyme that is activated in response to both hormones and intracellular metabolic stress signals including exercise, hypoxia, and nutrient deprivation. AMPK regulates many metabolic processes including glucose transport, glycolysis, and lipid metabolism, coupling energy metabolism to physiological functions including protein synthesis and gene transcription (Kemp et al. 2003). The extent of AMPKs regulatory functions are well illustrated in lipid metabolism where it inhibits multiple steps; fatty acid synthesis by phosphorylation of acetyl-CoA carboxylase-␣, cholesterol synthesis by phosphorylation of HMG-CoA reductase, triglyceride synthesis by phosphorylation of GPAT (glycerol-phosphate acyl transferase), and hormone-sensitive lipase (Hardie and Hawley 2001). In many cell types AMPK regulates ␤-oxidation of fatty acids by phosphorylation of acetyl CoA carboxylase-␤. Furthermore, AMPK exerts powerful transcriptional control of genes involved in lipid and carbohydrate metabolism (Zhou et al.

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable ␣␤␥ heterotrimer comprising a catalytic ␣ and two non-catalytic subunits, ␤ and ␥. The ␤ subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the ␤ subunit, ␤1-(186 -270), is sufficient to form an active AMP-dependent heterotrimer ␣1␤1- AMPK 1 is a multi-substrate enzyme that is activated in response to both hormones and intracellular metabolic stress generated by exercise, hypoxia, and nutrient deprivation. There are multiple isoforms of each AMPK subunit, with ␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3 forming heterotrimers that differ in tissue and subcellular localization (reviewed in Ref. 1). The ␣ subunit contains an N-terminal catalytic core (1-312) and a C-terminal sequence (313-548) responsible for autoregulation and binding the ␤␥ subunits (2). Maximum activity requires all three subunits (3). The catalytic AMPK ␣1-(1-312) fragment is constitutively active whereas the ␣1-(1-392) fragment is autoinhibited, and neither bind ␤␥ subunits (2). The three ␥ subunits each contain four CBS sequence repeats that were named after the corresponding sequences in cystathionine ␤-synthase (CBS) along with variable N-terminal extensions (4). AMP binds to the ␥ subunit and is responsible for the allosteric regulation of AMPK (5). There are two binding sites for AMP formed by the CBS1/2 and CBS3/4 sequence pairs (5), and because pairs of the CBS sequences form a discrete functional structure, they have now been termed Bateman modules (6). The ␤ subunit N-terminal myristoyl group is responsible for targeting AMPK to the membrane (7), and an internal glycogen-binding domain (68 -163) targets AMPK to glycogen (8, 9).The AMPK ␣ subunit is a homolog of yeast Snf1p kinase (10), which also binds ␤␥ subunit homologs (11, 12). There are three yeast ␤ subunit homologs, Gal83p, Sip1p, and Sip2p, and a single ␥ subunit homolog, Snf4p (13). In contrast to mammalian AMPK, which requires all three subunits for optimal activity (3), Snf1p/Snf4p forms a stable active complex that can be readily isolated from bakers' yeast without a ␤ homolog (Gal83p, Sip1p, or Sip2p) (11,14). Deletion of either Snf1p or Snf4p blocks growth on sucrose, as does deletion of all three ␤ subunit homologs (15). Studies of the subunit interactions in yeast using the two-hybrid approach have identified two regions within the yeast ␤ homologs termed KIS (kinase interacting sequence) and ASC (association with Snf1p complex), respectively. The C-terminal 85-residue ASC sequence of Gal83p was shown to bind Snf4p by two-hybrid analysis (13). The internal KIS sequence of Gal83p, Sip1p, or Sip2p interacted with the non-catalytic C terminus of Snf1p (13). In contrast, studies on mammalian AMPK have shown that the corresponding int...

Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.

IntroductionEpithelial-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.MethodsMYB, 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).ResultsParental 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.ConclusionsThis 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.