The AMP-activated protein kinase (AMPK) is a critical regulator of energy balance at both the cellular and whole-body levels. Two upstream kinases have been reported to activate AMPK in cell-free assays, i.e., the tumor suppressor LKB1 and calmodulin-dependent protein kinase kinase. However, evidence that this is physiologically relevant currently only exists for LKB1. We now report that there is a significant basal activity and phosphorylation of AMPK in LKB1-deficient cells that can be stimulated by Ca2+ ionophores, and studies using the CaMKK inhibitor STO-609 and isoform-specific siRNAs show that CaMKKbeta is required for this effect. CaMKKbeta also activates AMPK much more rapidly than CaMKKalpha in cell-free assays. K(+)-induced depolarization in rat cerebrocortical slices, which increases intracellular Ca2+ without disturbing cellular adenine nucleotide levels, activates AMPK, and this is blocked by STO-609. Our results suggest a potential Ca(2+)-dependent neuroprotective pathway involving phosphorylation and activation of AMPK by CaMKKbeta.

The AMP-activated protein kinase is a sensor of cellular energy status that is found in all eukaryotic cells. It is activated by rising AMP and falling ATP by a complex mechanism that results in an ultrasensitive response. The functions of the di¡erent domains on the three subunits of the K KL LQ Q heterotrimer are slowly being unravelled, and a recent development has been the identi¢cation of a glycogen-binding domain on the L L subunit. Along with ¢ndings that high cellular glycogen represses kinase activation, this suggests that the system may be a sensor of glycogen content as well as of AMP and ATP. New insights have been obtained into the sequence and structural features by which the kinase recognises its downstream target proteins, and these are discussed. Once activated by depletion of cellular energy reserves, the kinase switches on ATP-producing catabolic pathways and switches o¡ ATP-consuming processes, both via direct phosphorylation of regulatory proteins and via indirect e¡ects on gene expression. A survey of the range of downstream targets for this important signalling pathway is presented.

Decreased insulin sensitivity is associated with decreased concentrations of polyunsaturated fatty acids in skeletal-muscle phospholipids, raising the possibility that changes in the fatty-acid composition of muscles modulate the action of insulin.

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that is activated by low cellular energy status and affects a switch away from energy-requiring processes and toward catabolism. While it is primarily regulated by AMP and ATP, high muscle glycogen has also been shown to repress its activation. Mutations in the gamma2 and gamma3 subunit isoforms lead to arrhythmias associated with abnormal glycogen storage in human heart and elevated glycogen in pig muscle, respectively. A putative glycogen binding domain (GBD) has now been identified in the beta subunits. Coexpression of truncated beta subunits lacking the GBD with alpha and gamma subunits yielded complexes that were active and normally regulated. However, coexpression of alpha and gamma with full-length beta caused accumulation of AMPK in large cytoplasmic inclusions that could be counterstained with anti-glycogen or anti-glycogen synthase antibodies. These inclusions were not affected by mutations that increased or abolished the kinase activity and were not observed by using truncated beta subunits lacking the GBD. Our results suggest that the GBD binds glycogen and can lead to abnormal glycogen-containing inclusions when the kinase is overexpressed. These may be related to the abnormal glycogen storage bodies seen in heart disease patients with gamma2 mutations.

The cellular basis of insulin resistance is still unknown, however, relationships have been demonstrated between insulin action in muscle and the fatty acid profile of the major membrane structural lipid (phospholipid). The present study aimed to further investigate the hypothesis that insulin action and adiposity are associated with changes in the structural lipid composition of the cell. In 52 adult male Pima Indians, insulin action (euglycemic clamp), percentage body fat (pFAT; underwater weighing), and muscle phospholipid fatty acid composition (percutaneous biopsy of vastus lateralis) were determined. Insulin action (highdose clamp; MZ) correlated with composite measures of membrane unsaturation (% C20-22 polyunsaturated fatty acids [r = 0.463, P < 0.001], unsaturation index [r = -0.369, P < 0.01]), a number of individual fatty acids and with A5 desaturase activity (r = 0.451, P < 0.001). pFAT (range 14-53% ) correlated with a number of individual fatty acids and A5 desaturase activity (r = -0.610, P < 0.0001). Indices of elongase activity (r = -0.467, P < 0.001), and A9 desaturase activity (r = 0.332, P < 0.05) were also related to pFAT but not insulin action. The results demonstrate that A5 desaturase activity is independently related to both insulin resistance and obesity. While determining the mechanisms underlying this relationship is important for future investigations, strategies aimed at restoring "normal" enzyme activities, and membrane unsaturation, may have therapeutic importance in the "syndromes of insulin resistance." (J. Clin. Invest. 1995. 96:2802-2808

No abstract

Polyunsaturated fatty acids (PUFA) suppress hepatic lipogenic gene transcription through a peroxisome proliferator activated receptor ␣ (PPAR␣)-and cyclooxygenase-independent mechanism. Recently, the sterol response element-binding protein 1 (SREBP1) was implicated in the nutrient control of lipogenic gene expression. In this report, we have assessed the role SREBP1 plays in the PUFA control of three hepatic genes, fatty acid synthase, L-pyruvate kinase (LPK), and the S14 protein (S14). PUFA suppressed both the hepatic mRNA SREBP1 through a PPAR␣-independent mechanism as well as SREBP1c nuclear content (nSREBP1c, 65 kDa). Co-transfection of primary hepatocytes revealed a differential sensitivity of the fatty acid synthase, S14, and LPK promoters to nSREBP1c overexpression. Of the three promoters examined, LPK was the least sensitive to overexpressed nSREBP1c. Promoter deletion and gel shift analyses of the S14 promoter localized a functional SREBP1c cis-regulatory element to an E-box-like sequence ( ؊139 TCGCCTGAT ؊131 ) within the S14 PUFA response region. Although overexpression of nSREBP1c significantly reduced PUFA inhibition of S14CAT, overexpression of other factors that induced S14CAT activity, such as steroid receptor co-activator 1 or retinoid X receptor ␣, had no effect on S14CAT PUFA sensitivity. These results suggest that PUFA regulates hepatic nSREBP1c, a factor that functionally interacts with the S14 PUFA response region. PUFA regulation of nSREBP1c may account for the PUFA-mediated suppression of hepatic S14 gene transcription.

Many of the sugary-i mutants of maize (Zea mays L.) have the highly branched water-soluble polysaccharide, phytoglycogen, in quantities equal to or greater than starch as an endosperm storage product in mature seeds. We find that all sugary mutants investigated are deficient in debranching enzyme la-(1, 6)-glucosidasel activity in endosperm tissue 23 days postpollination and suggest that this deficiency is the primary biochemical lesion leading to phytoglycogen accumulation in sugary endosperms. This would indicate that the amylopectin component of starch depends on an equilibrium between the activities of branching enzymes introducing a-1,6 branch points into the linear a-1,4 glucans and debranching enzymes. The debranching enzyme activities from nonsugary endosperms can be separated into three peaks on a hydroxyapatite column. The sugary endosperm extracts lack one ofthese peaks ofactivity while the other two fractions have much reduced activity. The embryos of developing seeds (23 days after pollination) from both sugary and nonsugary genotypes have equivalent debranching activity. The debranching enzyme activity of developing endosperms is proportional to the number of copies (O to 3) of the nonmutant (Su) allele present suggesting that the Su allele may be the structural gene for this debranching enzyme, although this is not definitive. This identification of debranching enzyme activity as being the biochemical lesion in sugary endosperms is consistent with several previous observations on the mutant.The sugary-l (sul) mutant of maize, Zea mays L., which is the usual sweet corn of commerce, has been known and utilized since preColumbian times (21), and specific efforts to improve particular varieties of sweet corn date back to the middle of the nineteenth century (18). In spite of this lengthy association with man, the specific biochemical lesion induced by mutations at the su locus has not been known. Sumner and Somers (20) reported that the principal polysaccharide storage product in su endosperms was not starch, but a highly branched, water-soluble polysaccharide of high mol wt which Sumner and Somers referred to as phytoglycogen. Erlander ( 11) MATERIALS AND METHODS Biological Materials. Self-pollinated seeds ofthe maize inbred W64A or the hybrid W64A x 182E were used as the nonsugary controls. The first group of sugary mutants examined contained the reference allele at the su locus, su-R, and an intermediate allele su-Bn2. These alleles had been placed in the W64A genotype by repeated backcrosses. A second group of sugary alleles contained su-8115, su-8116, su-8117, and su-8135. These were presumed to have been independently induced by ethylmethanesulfonate (EMS) treatment since they were isolated in the progeny of inbred W22 plants exposed to the mutagen. For simplicity, we refer to su endosperms throughout the report. It should be understood that this means homozygous sugary (sul su/su) endosperms.