Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hormone-sensitive lipase and cholesterol ester hydrolase of chicken adipose tissue were markedly activated by adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase (on the average, 235 to 275%; occasionally as much as 1000%). Diglyceride and monoglyceride hydrolases were also activated, but to a lesser extent (60 to 87%). The activation of all four hydrolases was inhibited by protein kinase inhibitor and reversed by the addition of exogenous protein kinase. Following activation by cAMP-dependent protein kinase, all four hydrolases were deactivated in a Mg2+-dependent reaction and then reactivated to or near initial levels on incubation with cAMP and Mg2+-ATP. The reversible deactivation is assumed to reflect activity of one or more protein phosphatases. The maximum activation obtainable for the four hydrolases decreased when the tissue had been previously exposed to glucagon, indicating that the glucagon-induced activation was probably similar to or identical with the activation demonstrated in cell-free preparations. The pH optima for the four hydrolase activities were similar (7.13 to 7.38). Although the absolute activities and relative degrees of kinase activation differed according to the particular emulsified substrates used, the results do not rule out the possibility that all four hydrolase activities are referable to a single hormone-sensitive hydrolase. Hormone-sensitive acyl hydrolases were separated from lipoprotein lipase by heparin-Sepharose affinity chromatography. Lipoprotein lipase was active against triolein, diolein, and monoolein, but not cholesterol oleate. Incubation of lipoprotein lipase with exogenous protein kinase, cAMP, and Mg2+ATP had no effect on any of the three hydrolase activities. Lipoprotein lipase was further purified to homogeneity and used to prepare antiserum in rabbits. The immunoglobin G fraction from these antisera completely inhibited lipoprotein lipase eluted from heparin-Sepharose columns. However, the hormone-sensitive hydrolase activities (not retained on heparin-Sepharose affinity chromatography) were not inhibited by anti-lipoprotein lipase immunoglobin G, and anti-lopoprotein lipase immunoglobin G did not affect the activation process in crude fractions. Thus, hormone-sensitive lipase and lipoprotein lipase, functionally distinct enzymes, have been physically resolved and immunochemically distinguished. Apparently lipoprotein lipase activity is not regulated, at least directly, by cAMP-dependent protein kinase.
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PMID:Triglyceride, diglyceride, monoglyceride, and cholesterol ester hydrolases in chicken adipose tissue activated by adenosine 3':5'-Monophosphate-dependent protein kinase. Chromatographic resolution and immunochemical differentiation from lipoprotein lipase. 0 45

Hormone-sensitive lipase activity (HSL), which is found in the supernatant of centrifuged homogenates of lipolytically quiet isolated rat adipocytes, was greatly reduced in or absent from the supernatant of lipolytically stimulated cells. The lipase was purified 100- to 250-fold from the supernatant of lipolytically quiet cells to 10-20% purity by a single passage over phenyl-Sepharose resin with high (greater than 70%) activity yields. Western blotting of adipocyte homogenate fractions with polyclonal antiserum raised against HSL showed that the enzyme shifted quantitatively from the supernatant of control cells to the floating "fat cake" of lipolytically stimulated cells. A similar shift to the fat cake was observed when cells were disrupted by hypotonic lysis and centrifugation rather than by homogenization. We propose that upon lipolytic activation of adipocytes and phosphorylation of HSL by cAMP-dependent protein kinase, the critical event is not an increase in catalytic activity (i.e., turnover number) but a translocation of the lipase to its substrate at the surface of the lipid storage droplet.
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PMID:Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet. 152 59

Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.
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PMID:Domain-structure analysis of recombinant rat hormone-sensitive lipase. 891 75

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The enzyme is also expressed in steroidogenic tissues, mammary gland, muscle tissues and macrophages. A novel HSL mRNA termed hHSL-S, 228 bp shorter than the full-length HSL mRNA, was detected in human adipocytes. hHSL-S mRNA results from the in-frame skipping of exon 6, which encodes the serine residue of the catalytic triad. The corresponding 80 kDa protein was identified in human adipocytes after immunoprecipitation. The truncated protein expressed in COS cells showed neither lipase nor esterase activity but was phosphorylated by cAMP-dependent protein kinase. hHSL-S mRNA was found in all human tissues expressing HSL, except brown adipose tissue from newborns. It represented approx. 20% of total HSL transcripts in human subcutaneous adipocytes. No alternative splicing was detected in other mammals. Human and mouse three-exon HSL minigenes transfected into primate and rodent cell lines reproduced the splicing pattern of the endogenous HSL genes. Analysis of hybrid human/mouse minigenes transfected into human cell lines showed that cis-acting elements responsible for the skipping of human exon 6 were restricted to a 247 bp region including exon 6 and the first 19 nt of intron 6. Moreover, divergence in exonic splicing elements between mouse and human was shown to be critical for the species-specific alternative splicing.
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PMID:Species-specific alternative splicing generates a catalytically inactive form of human hormone-sensitive lipase. 935 44

Hormone-sensitive lipase catalyzes the rate-limiting step in the release of fatty acids from triacylglycerol-rich lipid storage droplets of adipocytes, which contain the body's major energy reserves. Hormonal stimulation of cAMP formation and the activation of cAMP-dependent protein kinase leads to the phosphorylation of hormone-sensitive lipase and a large increase in lipolysis in adipocytes. By contrast, phosphorylation of hormone-sensitive lipase by the kinase in vitro results in a comparatively minor increase in catalytic activity. In this study, we investigate the basis for this discrepancy by using immunofluorescence microscopy to locate hormone-sensitive lipase in lipolytically stimulated and unstimulated 3T3-L1 adipocytes. In unstimulated cells, hormone-sensitive lipase is diffusely distributed throughout the cytosol. Upon stimulation of cells with the beta-adrenergic receptor agonist, isoproterenol, hormone-sensitive lipase translocates from the cytosol to the surfaces of intracellular lipid droplets concomitant with the onset of lipolysis, as measured by the release of glycerol to the culture medium. Both hormone-sensitive lipase translocation and lipolysis are reversed by the incubation of cells with the beta-adrenergic receptor antagonist, propranolol. The treatment of cells with cycloheximide fails to inhibit lipase translocation or lipolysis, indicating that the synthesis of nascent proteins is not required. Cytochalasin D and nocodazole used singly and in combination also failed to have a major effect, thus suggesting that the polymerization of microfilaments and microtubules and the formation of intermediate filament networks is unnecessary. Hormone-sensitive lipase translocation and lipolysis were inhibited by N-ethylmaleimide and a combination of deoxyglucose and sodium azide. We propose that the major consequence of the phosphorylation of hormone-sensitive lipase following the lipolytic stimulation of adipocytes is the translocation of the lipase from the cytosol to the surfaces of lipid storage droplets.
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PMID:The lipolytic stimulation of 3T3-L1 adipocytes promotes the translocation of hormone-sensitive lipase to the surfaces of lipid storage droplets. 1063 41

A possible mechanism(s) behind exercise training-enhanced lipolysis was investigated in rat adipocytes. Exercise training (9 weeks; running) enhanced the activity of cAMP-dependent protein kinase (PKA) and the protein expressions of PKA subunits (catalytic, RII alpha, and RII beta) in P(40) fraction (sedimenting at 40,000g), but not in I(40) fraction (infranatant of 40,000g) of adipocyte homogenate. The expression of PKA-anchoring protein 150 (AKAP150) in P(40) fraction was greater in exercise-trained (TR) than in control (C) rats. Hormone-sensitive lipase (HSL) activities in both fractions were also greater in TR. On the other hand, stimulated lipolysis was accompanied by increased activities of HSL in P(40) but not in I(40) fraction. The decreases in stimulated lipolysis due to St-Ht31 were greater in TR rats. Thus, the mechanisms behind exercise training-enhanced adipocyte lipolysis could involve the increased activities of PKA and HSL with enhanced expressions of AKAP150 and some subunits of PKA, all of which may be compartmentalized within adipocytes.
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PMID:Possible mechanisms by which adipocyte lipolysis is enhanced in exercise-trained rats. 1215 Sep 37

Hormone-sensitive lipase (HSL) is presumed to be essential for lipolysis, which is defined as the mobilization of free fatty acids from adipocytes. In the present study, we investigated the effects of various lipolytic hormones on the lipolysis in adipocytes derived from mouse embryonic fibroblasts (MEF adipocytes) prepared from HSL-deficient mice (HSL-/-). HSL-/- MEF differentiated into mature adipocytes in a manner indistinguishable from that of wild-type mice. Both isoproterenol (ISO) and tumor necrosis factor (TNF)-alpha stimulated the rate of lipolysis in HSL-/- MEF adipocytes, although to a lesser extent than in wild-type cells, and these lipolytic activities were inhibited by H-89, a cAMP-dependent protein kinase inhibitor, and troglitazone, respectively. Thus, the responses of the residual lipolytic activity to lipolytic hormones and TNF-alpha were well conserved in the absence of HSL. Extracts from HSL-/- MEF adipocytes hydrolyzed triacylglycerol (TG) but not cholesterol ester, indicating that the residual lipolytic activity was mediated by another TG-specific lipase. The TG lipase activity, which was decreased in cytosolic fraction in response to ISO, was increased in fat cake fraction. Therefore, translocation of the TG lipase may explain, at least partially, the ISO-stimulated lipolysis in HSL-/- adipocytes. In conclusion, lipolysis is mediated not only by HSL but also by the non-HSL TG lipase, whose responses to lipolytic hormones are similar to those of HSL. We propose that both lipases are regulated by common mechanism of lipolysis.
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PMID:Lipolysis in the absence of hormone-sensitive lipase: evidence for a common mechanism regulating distinct lipases. 1245 88

Hormone-sensitive lipase (HSL) is important for the degradation of triacylglycerol in adipose and muscle tissue, but the tissue-specific regulation of this enzyme is not fully understood. We investigated the effects of adrenergic stimulation and AMPK activation in vitro and in circumstances where AMPK activity and catecholamines are physiologically elevated in humans in vivo (during physical exercise) on HSL activity and phosphorylation at Ser(563) and Ser(660), the PKA regulatory sites, and Ser(565), the AMPK regulatory site. In human experiments, skeletal muscle, subcutaneous adipose and venous blood samples were obtained before, at 15 and 90 min during, and 120 min after exercise. Skeletal muscle HSL activity was increased by approximately 80% at 15 min compared with rest and returned to resting rates at the cessation of and 120 min after exercise. Consistent with changes in plasma epinephrine, skeletal muscle HSL Ser(563) and Ser(660) phosphorylation were increased by 27% at 15 min (P < 0.05), remained elevated at 90 min, and returned to preexercise values postexercise. Skeletal muscle HSL Ser(565) phosphorylation and AMPK signaling were increased at 90 min during, and after, exercise. Phosphorylation of adipose tissue HSL paralleled changes in skeletal muscle in vivo, except HSL Ser(660) was elevated 80% in adipose compared with 35% in skeletal muscle during exercise. Studies in L6 myotubes and 3T3-L1 adipocytes revealed important tissue differences in the regulation of HSL. AMPK inhibited epinephrine-induced HSL activity in L6 myotubes and was associated with reduced HSL Ser(660) but not Ser(563) phosphorylation. HSL activity was reduced in L6 myotubes expressing constitutively active AMPK, confirming the inhibitory effects of AMPK on HSL activity. Conversely, in 3T3-L1 adipocytes, AMPK activation after epinephrine stimulation did not prevent HSL activity or glycerol release, which coincided with maintenance of HSL Ser(660) phosphorylation. Taken together, these data indicate that HSL activity is maintained in the face of AMPK activation as a result of elevated HSL Ser(660) phosphorylation in adipose tissue but not skeletal muscle.
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PMID:Regulation of HSL serine phosphorylation in skeletal muscle and adipose tissue. 1618 6