Anti-lipolytic Action of AMP-activated Protein Kinase in Rodent Adipocytes

Abstract

Despite its importance in terms of energy homeostasis, the role of AMP-activated protein kinase in adipose tissue remains controversial. Initial studies have described an anti-lipolytic role for AMP-activated protein kinase, whereas more recent studies have suggested the converse. Thus we have addressed the role of AMP-activated protein kinase in adipose tissue by modulating AMP-activated protein kinase activity in primary rodent adipocytes using pharmacological activators or by adenoviral expression of dominant negative or constitutively active forms of the kinase. We then studied the effects of AMP-activated protein kinase activity modulation on lipolytic mechanisms. Finally, we analyzed the consequences of a genetic deletion of AMP-activated protein kinase in mouse adipocytes. AMP-activated protein kinase activity in adipocytes is represented mainly by the α1 isoform and is induced by all of the stimuli that increase cAMP in adipocytes, including fasting. When AMP-activated protein kinase activity is increased by 5-aminoimidazole-4-carboxamide-riboside, phenformin, or by the expression of a constitutively active form, isoproterenol-induced lipolysis is strongly reduced. Conversely, when AMP-activated protein kinase activity is decreased either by a dominant negative form or in AMP-activated protein kinase α1 knock-out mice, lipolysis is increased. We present data suggesting that AMP-activated protein kinase acts on hormone-sensitive lipase by blocking its translocation to the lipid droplet. We conclude that, in mature adipocytes, AMP-activated protein kinase activation has a clear anti-lipolytic effect. Despite its importance in terms of energy homeostasis, the role of AMP-activated protein kinase in adipose tissue remains controversial. Initial studies have described an anti-lipolytic role for AMP-activated protein kinase, whereas more recent studies have suggested the converse. Thus we have addressed the role of AMP-activated protein kinase in adipose tissue by modulating AMP-activated protein kinase activity in primary rodent adipocytes using pharmacological activators or by adenoviral expression of dominant negative or constitutively active forms of the kinase. We then studied the effects of AMP-activated protein kinase activity modulation on lipolytic mechanisms. Finally, we analyzed the consequences of a genetic deletion of AMP-activated protein kinase in mouse adipocytes. AMP-activated protein kinase activity in adipocytes is represented mainly by the α1 isoform and is induced by all of the stimuli that increase cAMP in adipocytes, including fasting. When AMP-activated protein kinase activity is increased by 5-aminoimidazole-4-carboxamide-riboside, phenformin, or by the expression of a constitutively active form, isoproterenol-induced lipolysis is strongly reduced. Conversely, when AMP-activated protein kinase activity is decreased either by a dominant negative form or in AMP-activated protein kinase α1 knock-out mice, lipolysis is increased. We present data suggesting that AMP-activated protein kinase acts on hormone-sensitive lipase by blocking its translocation to the lipid droplet. We conclude that, in mature adipocytes, AMP-activated protein kinase activation has a clear anti-lipolytic effect. AMP-activated protein kinase (AMPK) 1The abbreviations used are: AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-riboside; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; DN, dominant negative; CA, constitutively active; Ad, adenovirus; CREB, cAMP-responsive element-binding protein; HSL, hormone-sensitive lipase; KO, knockout; pfu, plaque-forming unit; WT, wild type. is a widely expressed serine/threonine kinase that is considered to act as an intracellular sensor of energy. AMPK is a heterotrimeric complex consisting of a catalytic (α) and two regulatory (β and γ) subunits (1Hardie D.G. Endocrinology. 2003; 144: 5179-5183Crossref PubMed Scopus (834) Google Scholar). For each subunit, several isoforms have been identified, although the exact function of the different isoforms remains unclear. Numerous studies have shown that AMPK is activated by stresses that increase the AMP/ATP ratio into the cell such as hypoxia, exercise, and long term starvation (1Hardie D.G. Endocrinology. 2003; 144: 5179-5183Crossref PubMed Scopus (834) Google Scholar, 2Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar). AMPK is activated by phosphorylation of the threonine residue 172 within the activation loop of the α catalytic subunit by an AMPK kinase. AMPK kinase has been recently identified as LKB1, a tumor suppressor mutated in humans with Peutz-Jeger syndrome. This disorder is associated with increased risk of colon, stomach, and lung carcinomas (3Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1340) Google Scholar, 4Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. De-Pinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1445) Google Scholar, 5Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar). The activation of AMPK switches on catabolic pathways that produce ATP and switches off anabolic pathways that consume ATP. The activation of AMPK leads to the phosphorylation of a number of proteins that results in increased glucose uptake and metabolism as well as fatty acid oxidation and simultaneously in an inhibition of hepatic lipogenesis, cholesterol synthesis, and glucose production (1Hardie D.G. Endocrinology. 2003; 144: 5179-5183Crossref PubMed Scopus (834) Google Scholar). AMPK is also responsible for increased fatty acid oxidation in response to the adipocyte-derived hormones, leptin and adiponectin (6Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar, 7Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3460) Google Scholar). Because AMPK activation would have beneficial metabolic consequences for diabetic patients, AMPK has emerged as a potential target for the treatment of obesity and type 2 diabetes (8Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-10PubMed Google Scholar, 9Ruderman N. Prentki M. Nat. Rev. Drug Discov. 2004; 3: 340-351Crossref PubMed Scopus (377) Google Scholar). It has been demonstrated that two classes of anti-diabetic drugs, metformin and thiazolidinediones, could act at least in part through an activation of AMPK in liver and muscle (10Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4432) Google Scholar, 11Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). Whereas the function of AMPK in liver and muscle has been well illustrated, its role in adipose tissue remains poorly documented and controversial. Adipose tissue is an important actor for the preservation of energetic homeostasis. The breakdown of triglycerides through lipolysis during fasting is a major function in adipocytes. Previously, Sullivan et al. (12Sullivan J.E. Brocklehurst K.J. Marley A.E. Carey F. Carling D. Beri R.K. FEBS Lett. 1994; 353: 33-36Crossref PubMed Scopus (416) Google Scholar) reported that prior activation of AMPK by 5-aminoimidazole-4-carboxamide-riboside (AICAR) in isolated rat adipocytes inhibits isoproterenol-induced lipolysis. However, recently, a study performed in the 3T3-L1 adipocyte cell line has reached the opposite conclusion. Based on the fact that β-adrenergic agents activate AMPK activity in adipocytes and that lipolysis is lower in the presence of a dominant negative form of the kinase, it was proposed in this latter study (13Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) that AMPK stimulates the lipolytic pathway. To clarify the role of AMPK, we have modulated the activity of the kinase in primary rodent adipocytes and in 3T3-L1 adipocytes using pharmacological activators or with adenoviruses expressing either dominant negative or constitutively active forms of the kinase and have studied the effects of these manipulations on lipolytic mechanisms. We have also analyzed the consequences of genetic deletion of AMPK activity in mouse adipocytes. Animals—Animal studies were conducted according to the French guidelines for the care and use of experimental animals. Male Sprague-Dawley rats (200-300-g body weight) and male CBA mice (25-35 g) were obtained from Charles River Laboratory (L'Arbresle, France). All of the experiments were performed on adipocytes isolated from epididymal fat pads. The AMPK α1 knock-out (KO) mice were as previously described (14Jorgensen S.B. Viollet B. Andreelli F. Frosig C. Birk J.B. Schjerling P. Vaulont S. Richter E.A. Wojtaszewski J.F. J. Biol. Chem. 2004; 279: 1070-1079Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). Epididymal and inguinal fat pads of 5-month-old wild type (WT) and AMPK α1 KO mice were used in this study. Animals were housed in cages at a constant temperature (22 °C) with light from 7-19 h and free access to water and laboratory chow (UAR, France). For the fasting/refeeding experiments, rats were fasted for 12 or 24 h and then refed for 1 or 3 h with a high carbohydrate diet (72% carbohydrate, 1% fat, and 27% protein (% energy)). Adipocyte Isolation and Determination of Fat Cell Size and Number—Mature adipocytes were isolated by collagenase treatment of epididymal fat pads according to Rodbell (15Rodbell M. J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar). The diameter of adipocytes was measured using a light microscope fitted with a camera and Perfect-Image software (Clara Vision). A frequency distribution plot of cell diameters was used to determine the mean fat cell diameter and mean ± S.D. Both mean fat cell volume and surface area were then determined using standard equations. Mean fat cell weight was calculated from cell volume, assuming that the density of lipid is to that of of in the cell fat cell number was calculated by the lipid of the cell by the mean fat cell 3T3-L1 from J. were in Dulbecco's modified Eagle's with serum and adipocyte was induced by and for 2 were in with serum and for an and then in with 3T3-L1 adipocytes were used for experiments and as described for mature adipocytes. of and AMPK adipocytes were in with in a for were then with to and then for 1 h in in a in the presence or of the different AMPK adipocytes were in 1 1 1 and with 1% The were by at for at and the was to and at the of proteins was in A. were used to the AMPK activity using in the presence of of as described previously M. Carling D. C. Ferre P. Foufelle F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of AMPK were from of adipocyte proteins by with either or to protein for 2 h at rat α1 and catalytic subunits were by D. G. Hardie of AMPK α1 proteins and were from adipocyte using an to protein France). were by and in A. AMPK activity in the complex was determined by phosphorylation of the as described previously M. Carling D. C. Ferre P. Foufelle F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of with a constitutively active form and a dominant negative form of AMPK were as previously described A. D. M. P. Ferre P. Foufelle F. Carling D. Biol. PubMed Scopus Google Scholar). of the expression the major with was used as were in by density and at were in 2 of with of to for h at was to with with 1% BSA, and The of was by the presence of protein in adipocytes, is from an by the was were for 24 h with the and for h with the prior to the lipolysis were in by and to a The was for 1 h at temperature in and a with primary at the were with and with the to were using The and proteins a A. D. M. P. Ferre P. Foufelle F. Carling D. Biol. PubMed Scopus Google Scholar) and were using an lipase and were by D. G. Hardie and C. of and were obtained from and were by Cell and was obtained from was using to fat were with AMPK or with AMPK activators as described were then at for 1 h in of with or 1 in an of in to lipolysis 3T3-L1 adipocytes were in for 3 then were for 1 h with or by a treatment with or 1 of the of rat adipocytes or 3T3-L1 adipocytes was with of The was and then at for at A volume of of was and with of and for France). of ATP fat were with AMPK or with AMPK activators as described The was then and were in acid the cell was with and the ATP was measured using a Isolation of were isolated according to et W. J. PubMed Google Scholar). were in 2 of 1 and were by at for at The was with an volume of and with 2 of 2 of and 2 of 1 and 1 at for different were from the lipid to the of the and The lipid were then with and at for at was measured according to with as expressed as the mean ± The of in the was calculated by with the of AMPK activity 1 and and lipolysis in was calculated by the of adipocyte cell and lipolysis in the adipose tissue of AMPK α1 KO α1 and AMPK were measured from of inguinal fat pads of and AMPK α1 KO mice at when with AMPK α1 activity measured in adipocytes of adipocytes were isolated from the inguinal and epididymal fat pads of and AMPK α1 KO mice and for each fat The number and the of adipocytes were determined as described The results the frequency distribution of adipocyte cell in and KO adipocytes from epididymal fat pads were for 1 h in the or the presence of AICAR, and then 1 was within 1 h to activate lipolysis. the mean ± from two each the adipose of mice were experimental has been performed in at when with with at when with lipolytic in AMPK the α1 AMPK of the two catalytic subunits or that to stimuli (6Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar, Y. T. N. Kim Y.B. A. B. Mu J. Foufelle F. Ferre P. Birnbaum M.J. Kahn B.B. Nature. 2004; PubMed Scopus Google we determined the of the catalytic subunit for AMPK activity in adipose rat adipocytes were for 1 h in the or presence of AICAR, a of of adipocyte the of the α1 catalytic subunit To this AMPK were from using α1 or subunits or a of α1 and to AMPK activity was by as well as in the activity of AMPK is and for AMPK activity measured and the activity of AMPK is it is also by and AMPK in of isolated adipocytes with a β-adrenergic or a of the is by a increase of AMPK activity as previously described (13Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, FEBS Lett. PubMed Scopus Google Scholar). of the and have also a on AMPK Thus AMPK activity is of increased cAMP Because fasting a β-adrenergic of adipocytes, we have AMPK activity in the fat of fasted and refed AMPK activity is in fasted with refed rats This is associated with an increase of the phosphorylation of of ACC, a target of AMPK, whereas is modified in these of AMPK in AMPK activity is increased during a in lipolysis is strongly we the AMPK and lipolytic isolated rat adipocytes, the lipolytic is increased in the presence of of adipocytes with or phenformin, a of activate AMPK as shown by the increase of the phosphorylation of AMPK and strongly the isoproterenol-induced lipolysis in a To that the effects of and on lipolysis to an of the protein kinase A we the presence of or the ATP in adipocytes. shown previously by S.A. Hardie D.G. J. Biochem. PubMed Scopus Google Scholar, Hardie D.G. W.W. Am. J. Physiol. PubMed Google ATP in adipocytes ± 3 of in adipocytes ± in ATP in adipocytes ± of However, with the phosphorylation on of in the presence of This that activation of lipolysis is in the presence of these and activators of Thus to that effects on the lipolytic were by AMPK, we in adipocytes. The to a catalytic α1 subunit in the phosphorylation has been mutated to an acid in to phosphorylation A. D. M. P. Ferre P. Foufelle F. Carling D. Biol. PubMed Scopus Google Scholar). of adipocyte h of the number of adipocytes is in ± or in ± adipocytes. ATP into adipocytes is modified in the presence of the ± of in ± in of the in mature adipocytes leads to an increased AMPK activity to that measured in adipocytes for 1 h with and the phosphorylation of on to the This increase of AMPK activity inhibits the isoproterenol-induced lipolysis This the results obtained using and and that the anti-lipolytic of these agents was at least in part by of AMPK a of AMPK results shown that, when AMPK is it inhibits the lipolytic pathway. this is the of AMPK activity in adipocytes increase the isoproterenol-induced lipolytic To this we in adipocytes A. D. M. P. Ferre P. Foufelle F. Carling D. Biol. PubMed Scopus Google Scholar). The of cell 24 h of ± adipocytes in ± adipocytes in and ATP ± of ± of The dominant negative form of the α1 subunit is it the activation of AMPK by and as well as the phosphorylation of on in response to and Finally, the inhibition of AMPK has a and on stimulates the isoproterenol-induced lipolysis that, in lipolytic the role of AMPK is to the lipolytic of by AMPK the is the lipolysis in adipocytes. The activity of is by several including phosphorylation by a number of protein and translocation from the to the surface of the lipid Biochem. J. 2004; PubMed Scopus Google Scholar). It has been previously reported that the phosphorylation of on has on activity the phosphorylation of protein kinase A on the D.G. Carling D. Hardie D.G. R.J. J. Biochem. PubMed Scopus Google Thus we have analyzed the effects of AMPK on phosphorylation and translocation to the lipid droplet. of adipocytes with or expression of the the phosphorylation of on A of adipocytes in the presence of the translocation of from the 1 to the lipid activation of AMPK by treatment with or strongly the of to the 2 3 and and and To that the in translocation treatment is by AMPK, we the form to AMPK the translocation of to the lipid and inhibits this of AMPK activity the of on translocation AMPK in 3T3-L1 and of to the opposite results of have been obtained in 3T3-L1 adipocytes (13Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (240) Google we have to of experiments in this cell of 3T3-L1 for 1 h with or a increase in AMPK activity ± of ATP in ± of ATP in ± of ATP in previously in mature rat adipocytes prior activation of AMPK by or the isoproterenol-induced lipolysis Finally, the treatment of 3T3-L1 with the translocation of induced by to the lipid these results that, as previously demonstrated in mature adipocytes, the activation of AMPK inhibits the lipolytic in 3T3-L1 adipocytes. Adipocyte in AMPK α1 the activity of the AMPK complex is in adipose tissue the importance of AMPK in adipose tissue metabolism in AMPK α1 KO in a number of KO by a isoform is we measured α1 and AMPK activity in adipocytes of and AMPK α1 KO shown previously in rats AMPK α1 activity was the activity measured in adipocytes of mice adipose tissue of AMPK α1 KO mice, AMPK α1 activity was and increase in AMPK was The body weight of the AMPK α1 KO mice was different from that of mice, although it has a to lower However, the weight of different adipose tissue is strongly in the KO We then measured the of adipocytes in and that the mean diameter was decreased in the KO mice The lipolytic was increased in the presence of in adipocytes of mice, an that is when the with AMPK α1 KO mice, lipolysis as well as isoproterenol-induced lipolysis was increased in adipocytes when with and effect. This in the potential anti-lipolytic role of AMPK during of the AMPK α1 KO AMPK KO weight ± ± fat pads ± ± at when with fat pads ± ± at when with at when with at when with animals. in a AMPK a when it was that its activation could have beneficial effects in the metabolic through an increase in muscle glucose decreased hepatic glucose and increased fatty acid oxidation in muscle and Adipose tissue is a major of energy and a in the of through fatty acid and the role of AMPK in adipocytes is for a of effects of AMPK the importance of this and the results in the we to the exact function of AMPK on lipolysis using in and in we that AMPK α1 is the subunit expressed in adipocytes. this has a is although it that AMPK this isoform to S.A. A. A. Carling D. Hardie D.G. Biochem. J. PubMed Scopus Google Scholar). we demonstrated as (13Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, FEBS Lett. PubMed Scopus Google Scholar) that cAMP and agents activate AMPK in adipocytes, although the exact remains We have a study (12Sullivan J.E. Brocklehurst K.J. Marley A.E. Carey F. Carling D. Beri R.K. FEBS Lett. 1994; 353: 33-36Crossref PubMed Scopus (416) Google Scholar) that strongly inhibits the lipolytic induced by in primary adipocytes. we have shown that phenformin, has a to has a effect. The use of expression of dominant and negative forms of the kinase in adipocytes has to the role of AMPK activation on lipolysis. results at with of et al. (13Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) have reached opposite in 3T3-L1 (13Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). is on the fact that adenoviral expression of a dominant negative form of the subunit of the kinase inhibits isoproterenol-induced lipolysis. However, it that in experiments using this form, AMPK activity was measured and it is to conclude the of the modulation of AMPK activity in these the we demonstrated that treatment with and AMPK activity and strongly the isoproterenol-induced lipolysis We have addressed the of AMPK modulation in adipocytes. AMPK activity is increased by fasting a with lipolysis in mice the α subunit isoform we have that the of adipocytes is that and lipolysis in these is that of and that has on all results with an of AMPK on the lipolytic This is in with in studies an anti-lipolytic of R. P. 2001; PubMed Scopus Google Scholar). could AMPK the lipolytic Because AMPK activation is associated in adipocytes with an increase in glucose uptake PubMed Scopus Google it could and fatty acid However, the of glucose is Initial studies have shown that the phosphorylation of on the phosphorylation on by protein kinase A D.G. Carling D. Hardie D.G. R.J. J. Biochem. PubMed Scopus Google Scholar). However, it was then demonstrated that and were the of protein kinase activation L. C. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). lipolysis is to activation also to its translocation to the lipid droplet. A of to the translocation of to the lipid in 3T3-L1 on an function of an phosphorylation on C. C. C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). we have shown in rat adipocytes and in 3T3-L1 that translocation of to the lipid a major for an active is strongly by AMPK a with increased phosphorylation of on of AMPK activity by the form the of with AMPK activators the of AMPK in this The with the results of et al. C. C. C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) that the of phosphorylation at translocation is clear could to the fact that a to in such a that it the translocation C. C. C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). AMPK could and the function of protein in translocation such as This The study that activation of AMPK fatty acid in rodent adipocytes. It is to on the of such an it could as suggested by and FEBS Lett. PubMed Scopus Google a the of lipolysis and the intracellular of fatty that to the these studies to the activation of AMPK in adipose tissue by the of fatty could beneficial in such as obesity and type 2 We S. for care and B. for of the We D. Hardie for the of and and Carling for the We and for the of