Mechanisms underlying the metabolic actions of galegine that contribute to weight loss in mice.

BACKGROUND AND PURPOSE: Galegine and guanidine, originally isolated from Galega officinalis, led to the development of the biguanides. The weight-reducing effects of galegine have not previously been studied and the present investigation was undertaken to determine its mechanism(s) of action. EXPERIMENTAL APPROACH: Body weight and food intake were examined in mice. Glucose uptake and acetyl-CoA carboxylase activity were studied in 3T3-L1 adipocytes and L6 myotubes and AMP activated protein kinase (AMPK) activity was examined in cell lines. The gene expression of some enzymes involved in fat metabolism was examined in 3T3-L1 adipocytes. KEY RESULTS: Galegine administered in the diet reduced body weight in mice. Pair-feeding indicated that at least part of this effect was independent of reduced food intake. In 3T3-L1 adipocytes and L6 myotubes, galegine (50 microM-3 mM) stimulated glucose uptake. Galegine (1-300 microM) also reduced isoprenaline-mediated lipolysis in 3T3-L1 adipocytes and inhibited acetyl-CoA carboxylase activity in 3T3-L1 adipocytes and L6 myotubes. Galegine (500 microM) down-regulated genes concerned with fatty acid synthesis, including fatty acid synthase and its upstream regulator SREBP. Galegine (10 microM and above) produced a concentration-dependent activation of AMP activated protein kinase (AMPK) in H4IIE rat hepatoma, HEK293 human kidney cells, 3T3-L1 adipocytes and L6 myotubes. CONCLUSIONS AND IMPLICATIONS: Activation of AMPK can explain many of the effects of galegine, including enhanced glucose uptake and inhibition of acetyl-CoA carboxylase. Inhibition of acetyl-CoA carboxylase both inhibits fatty acid synthesis and stimulates fatty acid oxidation, and this may to contribute to the in vivo effect of galegine on body weight.

AMP-activated protein kinase: the energy charge hypothesis revisited.

The AMP-activated protein kinase cascade is a sensor of cellular energy charge, and its existence provides strong support for the energy charge hypothesis first proposed by Daniel Atkinson in the 1960s. The system is activated in an ultrasensitive manner by cellular stresses that deplete ATP (and consequently elevate AMP), either by inhibiting ATP production (e.g., hypoxia), or by accelerating ATP consumption (e.g., exercise in muscle). Once activated, it switches on catabolic pathways, both acutely by phosphorylation of metabolic enzymes and chronically by effects on gene expression, and switches off many ATP-consuming processes. Recent work suggests that activation of AMPK is responsible for many of the effects of physical exercise, both the rapid metabolic effects and the adaptations that occur during training. Dominant mutations in regulatory subunit isoforms (gamma2 and gamma3) of AMPK, which appear to increase the basal activity in the absence of AMP, lead to hypertrophy of cardiac and skeletal muscle respectively.

AMP-activated protein kinase: an ultrasensitive system for monitoring cellular energy charge.

The AMP-activated protein kinase cascade is activated by elevation of AMP and depression of ATP when cellular energy charge is compromised, leading to inhibition of anabolic pathways and activation of catabolic pathways. Here we show that the system responds in intact cells in an ultrasensitive manner over a critical range of nucleotide concentrations, in that only a 6-fold increase in activating nucleotide is required in order for the maximal activity of the kinase to progress from 10% to 90%, equivalent to a co-operative system with a Hill coefficient (h) of 2.5. Modelling suggests that this sensitivity arises from two features of the system: (i) AMP acts at multiple steps in the cascade (multistep sensitivity); and (ii) the upstream kinase is initially saturated with the downstream kinase (zero-order ultrasensitivity).

AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform.

Mammalian AMP-activated protein kinase (AMPK) is the downstream component of a cascade that is activated by cellular stresses associated with ATP depletion. AMPK exists as heterotrimeric alphabetagamma complexes, where the catalytic subunit has two isoforms (alpha1 and alpha2) with different tissue distributions. The budding yeast homologue is the SNF1 kinase complex, which is essential for derepression of glucose-repressed genes, and seems to act by the direct phosphorylation of transcription factors in the nucleus. AMPK complexes containing the alpha2 rather than the alpha1 isoform have a greater dependence on AMP (approx. 5-fold stimulation compared with approx. 2-fold) both in direct allosteric activation and in reactivation by the upstream kinase. We have also examined their subcellular localization by using Western blotting of nuclear preparations, and by using two detection methods in the confocal microscope, i.e. indirect immunofluorescence of endogenous proteins and transfection of DNA species encoding green fluorescent protein-alpha-subunit fusions. By all three methods a significant proportion of alpha2, but not alpha1, is localized in the nucleus. Like SNF1, AMPK-alpha2 complexes could therefore be involved in the direct regulation of gene expression. The observed differences in the regulation of alpha1 and alpha2 complexes by AMP might result in differential responses to ATP depletion in distinct cellular and subcellular locations.

Glucose repression/derepression in budding yeast: SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP:ATP ratio.

BACKGROUND: Genetic studies of Saccharomyces cerevisiae have shown that Snf1p and Snf4p, which together form the SNF1 complex, are essential for gene derepression on removal of glucose from the medium. However the metabolic signal(s) involved, and the exact role of SNF1, have remained enigmatic. Recently, the AMP-activated protein kinase (AMPK) was shown to be the mammalian homologue of SNF1. AMPK is activated by the elevation of the cellular AMP:ATP ratio, which occurs during cellular stress in mammalian cells. The mechanism of activation involves phosphorylation of AMPK by an upstream protein kinase (AMPKK). We have investigated whether a similar mechanism might explain the role of SNF1 in yeast in the response to the stress of glucose starvation. RESULTS: The protein kinase activity of SNF1 was dramatically and rapidly activated by phosphorylation on removal of glucose from the medium. SNF1 was not activated directly by AMP, but could be inactivated by protein phosphatases and reactivated by mammalian AMPKK. We also demonstrated that an endogenous SNF1-reactivating factor, most likely an upstream protein kinase, is present in yeast extracts. Under a variety of different growth conditions, there was a correlation between cellular adenine nucleotide levels and the activation state of SNF1. CONCLUSIONS: Apart from the lack of direct allosteric activation of SNF1 by AMP, the regulation of the mammalian AMPK and yeast SNF1 protein kinase cascades is highly conserved. Adenine nucleotides are now good candidates for metabolic signals which indicate the lack of glucose in the medium, triggering activation of SNF1 and derepression of glucose-repressed genes.

Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase.

We have developed a sensitive assay for the AMP-activated protein kinase kinase, the upstream component in the AMP-activated protein kinase cascade. Phosphorylation and activation of the downstream kinase by the upstream kinase absolutely requires AMP and is antagonized by high (millimolar) concentrations of ATP. We have purified the upstream kinase >1000-fold from rat liver; a variety of evidence indicates that the catalytic subunit may be a polypeptide of 58 kDa. The physical properties of the downstream and upstream kinases, e.g. catalytic subunit masses (63 versus 58 kDa) and native molecular masses (190 versus 195 kDa), are very similar. However, unlike the downstream kinase, the upstream kinase is not inactivated by protein phosphatases. The upstream kinase phosphorylates the downstream kinase at a single major site on the alpha subunit, i.e. threonine 172, which lies in the "activation segment" between the DFG and APE motifs. This site aligns with activating phosphorylation sites on many other protein kinases, including Thr177 on calmodulin-dependent protein kinase I. As well as suggesting a mechanism of activation of AMP-activated protein kinase, this finding is consistent with our recent report that the AMP-activated protein kinase kinase can slowly phosphorylate and activate calmodulin-dependent protein kinase I, at least in vitro (Hawley, S. A., Selbert, M. A., Goldstein, E. G., Edelman, A. M., Carling, D., and Hardie, D. G. (1995) J. Biol. Chem. 270, 27186-27191).

5'-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms.

AMP-activated protein kinase (AMPK) and Ca2+/calmodulin (CaM)-dependent protein kinase I (CaMKI) are protein kinases that are regulated both by allosteric activation (AMP and Ca2+/CaM, respectively) and by phosphorylation by upstream protein kinases (AMPK kinase (AMPKK) and CaMKI kinase (CaMKIK), respectively). We now report that AMPKK can activate CaMKI and that, conversely, CaMKIK can activate AMPK. CaMKIK is 68-fold more effective at activating CaMKI than AMPK, while AMPKK is 17-fold more effective at activating AMPK than CaMKI. Our results suggest that CaMKIK and AMPKK are distinct enzymes dedicated to their respective kinase targets but with some overlap in their substrate specificities. The availability of alternative substrates for AMPKK and CaMKIK allowed the unequivocal demonstration that AMP and Ca2+/calmodulin promote the activation of AMPK and Ca2+/calmodulin promote the activation of AMPK and CaMKI, respectively, via three independent mechanisms: 1) direct activation of AMPK and CaMKI, 2) activation of AMPKK and CaMKIK, and 3) by binding to AMPK and CaMKI, inducing exposure of their phosphorylation sites. Since AMP and Ca2+/calmodulin each has a triple effect in its respective system, in vivo, the two systems would be expected to be exquisitely sensitive to changes in concentration of their respective activating ligands.

5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?

The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways, the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidazole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK, i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose, heat shock), AICAR does not perturb the cellular contents of ATP, ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation, causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase), and almost total cessation of two of the known target pathways, i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase, previously demonstrated in cell-free assays, also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade.

The AMP-activated protein kinase gene is highly expressed in rat skeletal muscle. Alternative splicing and tissue distribution of the mRNA.

The AMP-activated protein kinase (AMPK) phosphorylates, and thereby inactivates, a number of enzymes involved in the regulation of lipid metabolism. We have studied the expression of the AMPK gene in a variety of rat tissues. The gene is transcribed into a message of approximately 9.5 kb as detected by Northern blotting. Highest expression of the AMPK message was found in skeletal muscle, which contained 20 amol/micrograms total RNA as determined by competitive reverse-transcription/polymerase chain reaction (RT-PCR). In liver, kidney, brain, mammary glands, heart and lung, AMPK mRNA levels ranged over 1-4 amol/micrograms total RNA. Adipose tissue contained less than 1 amol/microgram total RNA. A second AMPK mRNA form was detected by RT-PCR that was 142 bases shorter than the functional transcript. This transcript was apparently generated by alternative splicing of a single exon within the 5'-coding region. The shorter of the two messages, which is not translated into AMPK protein, contributed between 35-60% of AMPK mRNA in most tissues, but only 15-20% in skeletal muscle and heart. As a result, functional AMPK mRNA was sevenfold higher in skeletal muscle than in liver, although AMPK activity was much lower. By Western blotting, relatively large amounts of AMPK protein were detected in skeletal muscle compared to liver. AMPK isolated from skeletal muscle was not activated by treatment with AMPK kinase under conditions where liver AMPK was fully activated. A single 63-kDa polypeptide was immunoprecipitated from rat skeletal muscle using anti-peptide IgG against AMPK. In contrast, two additional polypeptides with apparent molecular masses of 38 kDa and 36 kDa co-precipitated with the 63-kDa AMPK protein from rat liver. These results indicate that the muscle enzyme has a different subunit organization compared to the liver enzyme, which may account for its low catalytic activity. Together, our results indicate a physiological role for AMPK in muscle, in addition to its previously described role in lipid metabolism.

Purification of the AMP-activated protein kinase on ATP-gamma-sepharose and analysis of its subunit structure.

The AMP-activated protein kinase has been purified by affinity chromatography on ATP-gamma-Sepharose. A proportion of the activity can be eluted using AMP, while the remainder is eluted using ATP. The AMP eluate contains three polypeptides of 63, 38 and 35 kDa (p63, p38 and p35) in a molar ratio (by Coomassie blue binding) close to 1:1:1. p63 was previously identified as the AMP-binding catalytic subunit [Carling, D., Clarke, P. R., Zammit, V. A. & Hardie, D. G. (1989) Eur. J. Biochem. 186, 129-136]. All three polypeptides exactly comigrate both on native gel electrophoresis and on gel filtration, suggesting that p38 and p35 are additional subunits. Estimation of Stokes radius (5.4-5.8 nm) by gel filtration, and sedimentation coefficient (7.9-8.4 S) by glycerol gradient centrifugation, suggest that the kinase has an asymmetric structure with a native molecular mass for the complex of 190 +/- 10 kDa. Thus the native enzyme appears to be a heterotrimer with a p63/p38/p35 (1:1:1) structure. Despite the fact that the ATP eluate has a higher specific activity than the AMP eluate (3.5 +/- 0.2 vs 2.3 +/- 0.2 mumol.min-1.mg-1), it appears to be less pure, containing p63, p38 and p35 plus other polypeptides. Experiments examining the effects of protein phosphatase-2A and kinase kinase, and analysis by Western blotting with anti-p63 antibody, suggests that the AMP eluate is entirely in the low-activity dephosphorylated form, while the ATP eluate is a mixture of that form and the high-activity phosphorylated form. As well as establishing the subunit structure of the AMP-activated protein kinase, these results suggest that the kinase can bind to ATP-gamma-Sepharose through either the allosteric (AMP/ATP) site or the catalytic (ATP) site, and that phosphorylation by the kinase kinase increases the affinity for the latter site.

Activation of rat liver AMP-activated protein kinase by kinase kinase in a purified, reconstituted system. Effects of AMP and AMP analogues.

AMP-activated protein kinase, purified from rat liver as far as the diethylaminoethyl-Sepharose step, is inactivated by treatment with protein phosphatase 2C, and reactivated by an endogenous 'kinase kinase'. Further purification of AMP-activated protein kinase on Blue Sepharose removes the kinase kinase, but the system can be reconstituted by adding back the flow-through from the Blue-Sepharose column. The kinase kinase can be further purified by subjecting the flow-through from the Blue-Sepharose column to chromatography on a Mono-Q column. A single peak of kinase kinase activity is obtained. Using this fraction, and the most highly purified preparation of AMP-activated protein kinase, phosphorylation of the 63-kDa polypeptide, previously identified as the catalytic subunit of AMP-activated protein kinase, can be demonstrated. As previously shown in the partially purified system, phosphorylation of the 63-kDa polypeptide is markedly stimulated by AMP. The kinase and kinase kinase reactions exhibit similar dependence on AMP concentration. The structurally related AMP analogue, 8-aza-9-deazaadenosine-5'-monophosphate, mimics the effect of AMP on both allosteric activation and phosphorylation of the kinase, while adenosine (5')tetraphospho(5')adenosine antagonizes both effects. These results suggest that both the allosteric effect of AMP, and the promotion of phosphorylation and activation by the kinase kinase, are due to binding of AMP to a single site on the kinase.

Molecular analysis of cis-regulatory sequences at the alpha-amylase locus in Drosophila melanogaster.

The Amylase locus in Drosophila melanogaster contains duplicate, divergently transcribed structural genes for alpha-amylase, AmyA and AmyB. A sensitive and reliable transient expression assay was developed for testing amylase activities produced by exogenous Amy genes in somatically transformed larvae of an amylase-null strain of flies. Alleles tested, AmyA and AmyB, came from recombinant clone lambda Dm65, which contains genomic DNA from a Canton-S strain. The transient assay was used in a deletion analysis aimed at locating cis-regulatory sequences within the 5' region of AmyB. Results suggest that upstream regulatory sequences for correct spatial expression of AmyA and AmyB in third-instar larvae are located within 446 and 430 bp of their respective starts for transcription. A sequence required for high levels of AmyB expression was located within its 5' upstream region between the base pairs at -332 and -219. AmyA does not appear to have a comparable regulatory element in its 5'-flanking sequence. Barely detectable expression of AmyB was observed when it was flanked by only 92 bp of upstream sequence. A model is proposed for incomplete coordinate control of the duplicate Amy genes.

Amylase gene expression in intraspecific and interspecific somatic transformants of Drosophila.

The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.

Molecular genetics of a three-gene cluster in the Amy region of Drosophila.

Analysis of amylase RNA levels in the anterior and posterior midgut regions of flies from the Amy1,6 mapA and c Amy2,3 mapC strains of D. melanogaster, reared on yeast and on yeast supplemented with glucose, indicates that the trans-acting map gene controls the abundance of amylase RNA tissue-specifically, i.e., in the adult posterior midgut. This is consistent with the view that its role in controlling Amy expression is that of a transcription factor. Dietary glucose represses Amy expression in the anterior and posterior midgut regions of adults, reducing the abundance of amylase RNA, which suggests that it also controls Amy transcriptional activity. However, the mechanism for glucose repression appears to act systemically in the midgut, in a manner that is independent of the effects of map on Amy expression. A new glucose repressible TU was identified that is located just proximal to the Amy locus in region 54A of polytene chromosome 2R. It is transcribed in the direction opposite to that of the proximal Amy gene and encodes an RNA about 1500 bases long. Its RNA is expressed in both larvae and adults of the above strains of D. melanogaster, but the nature of the product it encodes is unknown. We speculate that all three genes in the cluster at 54A, namely the two Amy gene copies and the new glucose repressible TU, are coordinately controlled by the same mechanism that regulates Amy gene expression in response to dietary glucose. Somatic transformation experiments suggest that 5' cis-regulatory mechanisms required for the correct spatial expression of the proximal and distal Amylase genes from a Canton-S strain of D. melanogaster, Amy-p1 and Amy-d3, are located within 450 bp and 463 bp of their respective translation start sites. These regions also contain sequences responsive to dietary glucose repression, which is mediated at the DNA level of exogenous Amy genes in somatically transformed larvae reared on a yeast + glucose diet. A positive activator is located in the upstream region of Amy-d3 between the nucleotide pairs at -365 and -252 from the translation start site, but a comparable activator does not appear to exist in the upstream region of Amy-p1. Deletion analysis of the 5' sequence flanking the coding region of Amy-d3 indicates 125 nucleotide pairs of flanking DNA is sufficient for its functional activity. A model is proposed for coordinate control, in part, of the duplicated Amy genes.

Structural organization of the alpha-amylase gene locus in Drosophila melanogaster and Drosophila miranda.

Chromosomal sites belonging to the alpha-amylase gene family have been identified in D. melanogaster and D. miranda and in the sibling species of miranda, pseudoobscura, and persimilis. Two sites occur in chromosome 2 of melanogaster; one contains the Amy gene locus (54A) and the other an amylase "pseudogene" (53CD). Two sites of homology exist at 73A and 78C and perhaps another at 81BC in chromosome 3 of pseudoobscura and persimilis and in the homologous regions of the X2 chromosome in miranda. The active Amy locus is apparently at 73A. The structural organization of cloned sequences from this multigene family in melanogaster and miranda is under analysis, with emphasis on the functional Amy gene region. Electrophoretic variants of amylase have served as invaluable tools in these studies. For melanogaster, their use as genetic markers enabled us to positively identify our lambda Dm65 clone of the Amy locus and to show that it contains two functional copies of the structural gene for alpha-amylase. Amylase isozymes are now being used in P element-mediated transformation experiments aimed at defining regulatory elements for the temporal and spatial control of amylase expression during development and in response to dietary glucose. In miranda, electrophoretic variants of amylase were useful in assigning the Amy locus to chromosome X2, and they continue to serve as essential markers in our study of the evolution of dosage compensation for amylase expression in males of this species. Restriction maps of the Amy locus in 7 strains of D. melanogaster indicate that despite the worldwide origins of the chromosome samples, all contain a duplication of the amylase structural gene at this locus regardless of whether they produce two alpha-amylase isozymes, a single variant, or none. We have aligned these maps with the genetic and cytological maps of chromosome 2R in melanogaster and assigned alleles for different amylase isozymes to either the proximal or distal Amy gene copy in a number of strains. Restriction site polymorphism is relatively limited at the Amy locus, but some strain-specific rearrangements exist. The locus of two strains with reduced amylase activity, Amy1 (CA 1) and Amy "null", contain anomalies--an insertion in the former and an inversion in the latter. Causal relationships are being sought between the level of amylase expression in these strains and the position of their respective anomalies.(ABSTRACT TRUNCATED AT 400 WORDS)

An electrophoretic study of reversible protein denaturation: chymotrypsinogen at high pressures.

When reversible denaturation of chymotrypsinogen is produced at elevated hydrostatic pressures, conformational relaxation can occur quite slowly, allowing electrophoretic separation of the principal states from the equilibrium mixture. In this work we report experimental concentration distribution patterns obtained at pH 2.03 at a temperature of 20.5 degrees and find them to be reasonably consistent with the behavior that is expected from a simple two-state isomerism. However, the results do not at all rule out the existence of low levels of intermediate states.