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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

This study investigated the role of adenosine monophosphate–activated protein kinase (AMPK) in the regulation of lipolysis in visceral (VC) and subcutaneous (SC) rat adipocytes and the molecular mechanisms involved in this process. VC (epididymal and retroperitoneal) and SC (inguinal) adipocytes were isolated from male Wistar rats (160–180 g). Adipocytes were incubated either in the absence or in the presence of the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, 0–500 µmol/l). AMPK and acetyl-CoA carboxylase (ACC) phosphorylation, basal and epinephrine-stimulated (100 nmol/l) glycerol release, and hormone-sensitive lipase (HSL) phosphorylation and activity were determined. AICAR-induced (500 µmol/l) AMPK activation inhibited basal glycerol release by ∼42, 41, and 44% in epididymal, retroperitoneal, and inguinal adipocytes, respectively. Epinephrine-stimulated glycerol release was almost completely prevented by AICAR treatment in adipocytes from all fat depots. The AMPK inhibitor compound C (20 µmol/l) prevented AICAR-induced phosphorylation of AMPK and significantly increased basal (∼1.3-, 1.4-, and 1.7-fold) and epinephrine-stimulated (∼1.3-, 1.2-, 1.4-fold) glycerol release in epididymal, retroperitoneal, and inguinal adipocytes, respectively. AICAR increased phosphorylation of HSLSer565 and inhibited epinephrine-induced phosphorylation of HSLSer563 and HSLSer660. This was also accompanied by a 73% reduction in epinephrine-stimulated HSL activity. Compound C prevented the phosphorylation of HSLSer565 induced by AICAR and partially prevented the inhibitory effect of this drug on basal and epinephrine-stimulated lipolysis in adipocytes in VC and SC fat depots. In summary, despite different fat depots eliciting distinct rates of lipolysis, acute AICAR-induced AMPK activation suppressed HSL phosphorylation/activation and exerted similar antilipolytic effects on both VC and SC adipocytes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Storage of fat and supply of energy are the major physiological functions of white adipose tissue (WAT). Under conditions such as fasting and exercise, visceral (VC) and subcutaneous (SC) adipose tissues undergo lipolysis and release nonesterified fatty acids (NEFAs) into the blood stream to be used by peripheral tissues for energy production (1). Catecholamine-stimulated and insulin-mediated suppression of cyclic adenosine monophosphate–dependent protein kinase A (PKA) activation are recognized as the two primary signaling cascades in the regulation of lipolysis (1). Although both VC and SC adipocytes release NEFAs into the circulation, human (2,3) and rat (4) studies have reported higher lipolytic rates from VC than SC adipocytes in response to catecholamines. In addition, isolated VC adipocytes exhibit a blunted response to the antilipolytic effect of insulin when compared to SC adipocytes (3). Importantly, dysregulation of NEFA metabolism is a well-documented abnormality in upper body/VC obesity, and differences in fat depot function have been implicated in various metabolic dysfunctional alterations of obesity (5). Therefore, a better understanding of the molecular mechanisms that govern the pathways of lipolysis and lipid storage in VC and SC WAT is of particular therapeutic importance for obesity and its related metabolic disorders.

The underlying mechanisms of site-related differences in the regulation of lipolysis in fat cells is poorly understood; however, contributing factors have been attributed to variations in β-adrenergic receptor expression and sensitivity to agonists (6), α2:β-adrenergic receptor ratio (7), hormone-sensitive lipase (HSL) mRNA levels, and HSL activity (4,6). In the lipolytic signaling cascade, PKA-mediated phosphorylation and activation of HSL is a major determinant of fatty acid mobilization in adipose tissue. In fact, HSL is suggested to be the major rate-limiting enzyme in the hydrolysis of diacylglycerol as studies report a marked reduction in catecholamine-stimulated glycerol and NEFA release and an accumulation of diacylglycerol in HSL-deficient mice (8,9). PKA stimulates activation of HSL through the phosphorylation of three serine residues, 563, 659, and 660, which is essential for stimulating lipolysis particularly with respect to the latter two serine residues (10). In addition to HSL, a recently discovered lipase called adipose triglyceride lipase (ATGL) exhibits high substrate specificity for triacylglycerol and very low activity toward diacylglycerol (11). Unlike HSL, ATGL is closely associated with the lipid droplet under basal conditions and is not a substrate of PKA-mediated phosphorylation (11). Moreover, it has been reported that adipocytes incubated with a selective HSL inhibitor completely abolished catecholamine-stimulated lipolysis and reduced basal lipolysis by 50% (12). Therefore, HSL and ATGL have been considered to be the major regulators of lipolysis under catecholamine-stimulated and basal lipolysis, respectively.

In vitro studies have reported that HSL can also be phosphorylated on Ser-565 by adenosine monophosphate–activated protein kinase (AMPK) (13), which does not affect the activity of HSL per se, but may prevent subsequent phosphorylation of other HSL serine residues by PKA that seem to be crucial for activation of this lipase (13,14). Based on these observations, it was originally proposed that AMPK exerted an antilipolytic role in WAT (13,14). In accordance, a few studies have reported a decrease in lipolysis by increasing AMPK activity in adipocytes using adenovirus-mediated overexpression of a constitutively active form of the kinase (15) or using the pharmacological AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) (15,16). Also, in vivo human (17) and rat (18) studies have demonstrated that acute intravenous AICAR administration reduces whole-body lipolysis. Conversely, others report that AMPK exerts a prolipolytic effect and that activation of this enzyme is required for maximal activation of the lipolytic pathway in adipocytes (19,20). In addition, PKA-stimulated translocation of cytosolic HSL to the lipid droplet, which is essential for lipolysis to occur (21,22), was found to be abolished in cultured cells expressing a mutation to the AMPK-targeted HSLSer565 phosphorylation site (23), also indicating that AMPK was necessary for lipolysis to occur. More recently, evidence has been provided that activation of AMPK in adipocytes by pharmacological agents that induce lipolysis is secondary to an increase in the intracellular adenosine monophosphate:adenosine triphosphate ratio rather than a direct effect of cyclic adenosine monophosphate or PKA activation on the enzyme (24). Therefore, AMPK activation as a consequence of lipolysis has been proposed to limit NEFA release and operate as a mechanism to restrain energy depletion in WAT. Currently, there is no clear consensus regarding the role of AMPK activation in adipocyte lipolysis. In addition, no studies have investigated whether or not lipolysis is regulated differently in VC and SC WAT by AMPK. Therefore, this study was designed to determine the effect of acute AICAR-induced AMPK activation on both VC and SC WAT lipolysis under basal and epinephrine-stimulated conditions. We also studied the phosphorylation status of HSL on serine residues 563, 565, and 660 and the activity of this enzyme. Here, we provide evidence that despite different fat depots eliciting distinct rates of lipolysis, acute AICAR-induced AMPK activation exerted similar antilipolytic effects on both VC and SC adipocytes. Alternatively, inhibition of AMPK with compound C increased epinephrine-stimulated HSLSer563/660 phosphorylation and partially prevented the inhibition of basal and epinephrine-stimulated lipolysis by AICAR in VC and SC adipocytes.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Reagents. AICAR was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada); “compound C” (6-[4-(2-piperidin-1-yl-etoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]pyrimidine), an AMPK inhibitor, was kindly provided by MERCK Research Laboratories (Rahway, NJ). Epinephrine, fatty acid-free bovine serum albumin, free glycerol determination kit, phosphatidylcholine, phosphatidylinositol, 1-oleoyl-2-acetyl-sn-glycerol, and MTT (3[-4,5-dimethylthiazol-2-yl]2,5idiphenyl tetrazolium bromide) assay kit were purchased from Sigma-Aldrich (St Louis, MO). [1-14C]diolein was from American Radiolabeled Chemicals (St. Louis, MO). Specific antibodies against phospho-AMPK, phospho-HSL serines 563, 565, and 660, and secondary antibody horseradish peroxidase–conjugated antirabbit were from Cell Signaling Technology (Beverley, MA). Phospho-acetyl-CoA carboxylase (ACC) antibody was from Upstate (Charlottesville, VA). Glyceraldehyde 3-phosphate dehydrogenase was from Abcam (Cambridge, MA). All other chemicals were of the highest grade available.

Animals, adipocyte isolation, and cytotoxicity. Male albino rats (Wistar strain), weighing 160–180 g were maintained on a 12/12 h light/dark cycle at 22 °C and fed (ad libitum) standard laboratory chow. We certify that all applicable institutional and governmental regulations concerning the ethical use of animals were followed during this research. The York University Animal Care Ethics Committee approved the experimental procedures. Fat cells from VC (epididymal and retroperitoneal) and SC (inguinal) WAT depots were isolated as described previously (25). Cytotoxicity was tested using the MTT (3[-4,5-dimethylthiazol-2-yl]2,5idiphenyl tetrazolium bromide) assay (26). Cell viability was also assessed by counting the number of intact adipocytes before and after the exposure to all compounds used in this study. Results of the MTT assay showed no significant differences between control and the cells exposed to all treatments (Figure 1a). In addition, there was no significant difference in the number of intact cells before or after treatment of VC and SC adipocytes with all compounds (data not shown).

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Figure 1. Cytotoxicity and AMPK and ACC phosphorylation in VC and SC adipocytes treated with various drugs. Cytotoxicity (a) in epididymal (Epi), retroperitoneal (Retro), and inguinal (Ing) adipocytes was assessed in control cells (Con, 0.5 M HCl vehicle) and in cells exposed to epinephrine (E, 100 nmol/l), AICAR (A, 500 µmol/l), compound C (C, 20 µmol/l), AICAR plus epinephrine (A+E), compound C plus epinephrine (C+E), compound C plus AICAR (C+A), and compound C plus AICAR plus epinephrine (C+A+E). Data were compiled from two independent experiments and expressed as mean ± s.e.m. absorbance at 570 nm. The phosphorylation states of (b) AMPK and (c) ACC were assessed in control (Con, cells not receiving any treatment) and in cells exposed to AICAR (A, 500 µmol/l), compound C (C, 20 µmol/l), and compound C plus AICAR (C+A). Immunoblots are representative of three independent experiments. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) immunoblots were used as a loading control. ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AMPK, adenosine monophosphate–activated protein kinase.

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Determination of glycerol release and HSL activity. After isolation, adipocytes were washed three times and resuspended in Krebs Ringer buffer medium containing 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid and 3.5% bovine serum albumin and allowed to equilibrate for 30 min at 37 °C. Equal cell aliquots were incubated in plastic tubes for 30 min either in the absence or in the presence of various AICAR concentrations (0–500 µmol/l). For AMPK inhibition, cells were preincubated with compound C (20 µmol/l) for 30 min before adding AICAR. After all treatments, aliquots (750 µl) of cell suspension were distributed into plastic vials containing either epinephrine (100 nmol/l final concentration) or vehicle (0.5 M HCl, control) and incubated at 37 °C for 75 min. A cell-free aliquot of the infranatant was collected at time zero and after 75 min of incubation for the determination of glycerol release. Total lipid extraction (27) was performed to normalize glycerol release in each treatment condition. Activity of HSL was measured in cells (∼1.4 × 106) treated with AICAR (500 µmol/l) for 30 min and stimulated with epinephrine (100 nmol/l) for an additional 30 min. Cells were then immediately lysed in two volumes of homogenization buffer (0.25 M sucrose, 1 mmol/l EDTA, 1 mmol/l DTT, 20 µg/ml leupeptin, 2 µg/ml antipain, 1 µg/ml pepstatin, pH 7.0) and centrifuged (11,000 g, 45 min, 4 °C) (28). The infranatant was collected, and 100 µl of the sample was placed into glass tubes containing labeled DO ([1-14C]diolein; 0.5 µCi/ml), unlabeled DO (1-oleoyl-2-acetyl-sn-glycerol; 12.1 mg), and phospholipid mixture (phosphatidylcholine:phosphatidylinositol, weight ratios, 3:1) (28). The reaction was carried out at 37 °C for 10 min and terminated by adding 3.25 ml of extraction buffer (methanol:chloroform:heptane; 10:9:7) and 1.05 ml of a 0.1 M K2CO3, 0.1 M boric acid solution, pH 10.5 (28). After centrifugation (800 g), 1 ml from the upper phase was extracted and counted for radioactivity.

Western blot determination of p-AMPK, p-ACC, and p-HSL. VC and SC adipocytes (∼1 × 107 cells) were incubated in plastic tubes containing either AICAR (0–500 µmol/l), epinephrine (100 nmol/l), compound C (20 µmol/l), AICAR plus epinephrine, compound C plus epinephrine, compound C plus AICAR, or compound C plus AICAR plus epinephrine. Exposure to compound C always preceded AICAR treatment and stimulation of adipocytes with epinephrine. Immediately after all incubations, cells were snap-frozen in liquid nitrogen and lysed in buffer containing 25 mmol/l Tris-HCl and 25 mmol/l NaCl, pH 7.4, 1 mmol/l MgCl2, 2.7 mmol/l KCl, and protease and phosphatase inhibitors (0.5 mmol/l Na3VO4, 1 mmol/l NaF, 1 µmol/l leupeptin, 1 µmol/l pepstatin, 1 µmol/l okadaic acid, and 1 mmol/l phenylmethylsulfonyl fluoride (PMSF)). Protein concentration in each sample was determined using the Bradford method. Before loading onto sodium dodecyl sulfate–polyacrylamide gels, the samples were diluted 1:1 (v/v) with 2 × Laemmli sample buffer (62.5 mmol/l Tris-HCl, pH 6.8, 2% w/v sodium dodecyl sulfate, 50 mmol/l DTT, 0.01% w/v bromophenol blue). Aliquots of cell lysates were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Mississauga, Canada), and probed for phospho-AMPK (Thr-172), phospho-ACC (Ser-79), and phospho-HSL (Ser-563, 565, and 660). All primary antibodies were used in a dilution of 1:1,000 with the exception of phospho-AMPK (1:500). Equal loading of samples was confirmed using glyceraldehyde 3-phosphate dehydrogenase (1:5,000).

Statistical analysis. Statistical significance was determined using either one- or two-way ANOVA with Tukey-Kramer multiple comparison post hoc tests. The level of significance was set to P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Effects of AICAR on AMPK/ACC phosphorylation, basal and epinephrine-stimulated lipolysis, and HSL activity. AICAR (0–500 µmol/l) increased AMPK and ACC phosphorylation in a dose-dependent manner in VC and SC adipocytes (Figure 2a,b). As expected, epinephrine (100 nmol/l) significantly increased glycerol release by ∼8.9-, 7.1-, and 10.5-fold in epididymal, retroperitoneal, inguinal adipocytes, respectively. Furthermore, VC adipocytes elicited a higher lipolytic activity than SC adipocytes either under basal or epinephrine-stimulated conditions (Figure 2c). AICAR (500 µmol/l) inhibited basal glycerol release by ∼42, 41, and 44% in epididymal, retroperitoneal, and inguinal adipocytes, respectively. Moreover, epinephrine-stimulated glycerol release was powerfully reduced to almost basal levels by AICAR (Figure 2c). The epinephrine-induced increase in HSL activity (Figure 2d) was suppressed by ∼73% in epididymal adipocytes exposed to AICAR.

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Figure 2. Effects of AICAR (0–500 µmol/l) on phosphorylation of (a) AMPK and (b) ACC in isolated epididymal (Epi), retroperitoneal (Retro), and inguinal (Ing) adipocytes. Lipolysis (c) was assessed in control cells (Con, 0.5 M HCl vehicle), epinephrine-stimulated (E, 100 nmol/l), AICAR (A, 500 µmol/l), and AICAR plus epinephrine (A+E) treated cells. HSL activity (d) was determined in epididymal adipocytes under the same conditions as for lipolysis. Immunoblots are representatives of three independent experiments. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) immunoblots were used as a loading control. *P < 0.05 vs. Con, E (Ing), AICAR, and A+E; #P < 0.05 vs. Con, AICAR, and A+E; §P < 0.05 vs. Con, E, and A+E; $P < 0.05 vs. Con, AICAR, and A+E; †P < 0.05 vs. Con, E, and AICAR. ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AMPK, adenosine monophosphate–activated protein kinase; HSL, hormone-sensitive lipase.

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Effects of compound C on AICAR-induced AMPK activation and lipolysis. Preincubation in the presence of the AMPK inhibitor compound C (20 µmol/l) prevented AICAR-induced phosphorylation of AMPK and ACC in adipocytes isolated from VC and SC adipocytes (Figure 1b,c). In the absence of AICAR, compound C significantly increased basal (∼1.3-, 1.4-, and 1.7-fold) and epinephrine-stimulated (∼1.3-, 1.2-, and 1.4-fold) glycerol release in epididymal, retroperitoneal, and inguinal isolated adipocytes, respectively (Table 1). As the concentration of AICAR in the medium increased from 0 to 500 µmol/l, basal and epinephrine-stimulated lipolysis were progressively suppressed reaching values corresponding to ∼58, 59, and 56%, and to ∼26, 20, and 32% of controls in epididymal, retroperitoneal, and inguinal adipocytes, respectively. Incubation of adipocytes with compound C before adding the various concentrations of AICAR partially prevented the inhibitory effect of this AMPK agonist on basal and epinephrine-stimulated lipolysis in VC and SC adipocytes (Table 1). In the absence of AICAR, compound C caused the highest increase (1.7-fold) in basal lipolysis in inguinal adipocytes (Table 1). However, the ability of this AMPK inhibitor to counteract AICAR-induced suppression of basal lipolysis in inguinal adipocytes was only observed in the presence of the lowest AICAR concentration (10 µmol/l) (Table 1). Although compound C did not prevent the suppression of basal lipolysis observed at higher AICAR concentrations (100 and 500 µmol/l), it was able to increase epinephrine-stimulated lipolysis in inguinal adipocytes by ∼1.5- and 1.6-fold in the presence of 100 and 500 µmol/l of AICAR, respectively (Table 1).

Table 1.  Effects of AICAR and compound C on visceral and subcutaneous adipocyte lipolysis
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Effects of AICAR and compound C on HSL phosphorylation. HSL phosphorylation at Ser-565 was powerfully increased in a dose-dependent manner by AICAR in epididymal, retroperitoneal, and inguinal adipocytes (Figure 3a). Conversely, compound C prevented the phosphorylation of HSLSer565 induced by AICAR (Figure 3b). Epinephrine treatment significantly increased HSL phosphorylation on the PKA-targeted serine residues 563 and 660 in epididymal, retroperitoneal, and inguinal isolated adipocytes (Figure 3c,d). However, when cells were incubated with AICAR, epinephrine-induced phosphorylation of HSLSer563 and HSLSer660 was inhibited (Figure 3c,d). Preincubation of adipocytes with compound C did not affect basal HSL phosphorylation but potentiated the effect of epinephrine on HSLSer563/660 phosphorylation (Figure 3c,d). In addition, compound C prevented the inhibitory effect of AICAR treatment on epinephrine-stimulated phosphorylation of HSLSer660 (Figure 3d), but not the suppression of HSLSer563 phosphorylation (Figure 3c) by this AMPK agonist.

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Figure 3. Effects of (a) AICAR (0–500 µmol/l) and (b) AICAR (500 µmol/l), compound C (C, 20 µmol/l), and compound C plus AICAR (C+A) on HSLSer565 phosphorylation in isolated epididymal (Epi), retroperitoneal (Retro), and inguinal (Ing) adipocytes. The phosphorylation states of (c) HSLSer563 and (d) HSLSer660 were assessed in cells exposed to epinephrine (E, 100 nmol/l), AICAR (A, 500 µmol/l), compound C (C, 20 µmol/l), AICAR plus epinephrine (A+E), compound C plus epinephrine (C+E), compound C plus AICAR (C+A), and compound C plus AICAR plus epinephrine (C+A+E). Immunoblots are representatives of three independent experiments. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) immunoblots were used as a loading control. ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AMPK, adenosine monophosphate–activated protein kinase; HSL, hormone-sensitive lipase.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Here, we show that acute treatment of VC and SC adipocytes with AICAR stimulated phosphorylation and activation of AMPK in a dose-dependent manner, suppressed basal lipolysis by ∼40%, and almost completely prevented epinephrine-stimulated lipolysis. AICAR also caused ∼73% reduction in epinephrine-stimulated HSL activity in epididymal adipocytes. These results are in line with previous studies indicating that acute AMPK activation exerts an antilipolytic effect in adipocytes (15,16,24) and skeletal muscle cells (29). Phosphorylation of HSLSer565 by AMPK has been proposed to impair PKA-targeted HSLSer563 phosphorylation and lipolysis (13). However, mutagenesis experiments have provided evidence that phosphorylation of Ser-660 but not Ser-563 is critical for translocation of cytosolic HSL to the lipid droplet and therefore maximal activation of adipocyte lipolysis (10,23). Thus, uncertainty remained regarding whether AMPK-induced HSLSer565 phosphorylation could indeed impair PKA-mediated lipolysis. We have found that acute AICAR treatment caused a dose-dependent increase in HSLSer565 phosphorylation. Importantly, this was also accompanied by the inhibition of epinephrine-stimulated phosphorylation of HSL not only on Ser-563, but also on Ser-660 in adipocytes isolated from either VC or SC fat depots. Therefore, even if suppression of HSLSer563 phosphorylation was not sufficient to impair PKA-mediated activation of HSL, suppression of HSLSer660 phosphorylation could inhibit catecholamine-induced lipolysis in the presence of AICAR.

Treatment of adipocytes with the AMPK inhibitor compound C prevented the phosphorylation/activation of AMPK and the phosphorylation of HSLSer565 induced by AICAR. Interestingly, compound C potentiated the effect of epinephrine on HSLSer563/660 phosphorylation and completely prevented the inhibitory effect of AICAR on HSLSer660 phosphorylation, although it did not prevent inhibition of HSLSer563 phosphorylation induced by AICAR. All effects of compound C antagonizing AICAR-induced AMPK activation and HSLSer565 phosphorylation were followed by an increase in glycerol release under both basal and epinephrine-stimulated conditions in VC and SC adipocytes. However, even though compound C increased lipolysis in the absence of AICAR, it only completely prevented the suppression of lipolysis induced by low (10 µmol/l) AICAR concentrations. It is clear from our data that lipolysis was not fully maintained by compound C despite the fact that this AMPK inhibitor fully prevented AICAR-induced AMPK phosphorylation/activation, inhibited HSLSer565 phosphorylation, and increased epinephrine-stimulated HSLSer563/660 phosphorylation in VC and SC adipocytes. Therefore, it is important to consider that AICAR-induced AMPK activation could also be affecting lipolysis by regulating intracellular steps that take place in parallel to or downstream of HSL phosphorylation/activation. In this context, ATGL activity could also be affected by AMPK activation and lead to impairment of lipolysis. Although ATGL can be phosphorylated, the activity of this enzyme does not seem to be regulated by covalent modification (11). Activity of ATGL is greatly enhanced (∼20-fold in mice adipocytes) by its interaction with comparative gene identification-58 (CGI-58) (30). In the basal state, CGI-58 binds to perilipin A, a phosphoprotein that coats the lipid droplet (31). Following catecholamine stimulation, perilipin A is phosphorylated by PKA at several serine residues including Ser-517 (32). This causes CGI-58 to dissociate from perilipin A allowing its interaction with ATGL and activation of triacylglycerol hydrolysis. It is currently unknown whether phosphorylation of perilipin A Ser-517 affects the direct biding of CGI-58 with ATGL. It could be possible that AICAR-induced AMPK activation affects perilipin A function and interferes with the interaction between ATGL and CGI-58, leading to impairment of adipocyte lipolysis. However, further studies are necessary to address these possibilities.

Inhibition of lipolysis by AMPK seems counterintuitive at first, because this kinase is activated to restore intracellular energy levels. Furthermore, fasting and exercise, two conditions in which circulating NEFAs is increased, have been demonstrated to upregulate AMPK activity in WAT (24). Thus, inhibition of lipolysis could be seen as restricting substrate availability for adenosine triphosphate generation. However, it is noteworthy that the release of NEFAs under energy depleting conditions such as exercise and fasting surpasses the ability of peripheral tissues to uptake and oxidize this substrate (33). As a consequence, the NEFAs remaining in the circulation return to WAT to be re-esterified into triacylglycerol (34). Re-esterification of NEFAs requires acylation of fatty acids, which is costly for adipocytes and further reduces intracellular energy availability. Therefore, under physiological conditions, activation of AMPK has been proposed to restrict the release of NEFAs and minimize the cost of re-esterification and energy depletion (24). Our results demonstrating that acute AICAR-induced AMPK activation inhibits lipolysis in VC and SC adipocytes support this hypothesis. Importantly, in our experiments, we activated AMPK by exposing VC and SC adipocytes to AICAR before stimulating with epinephrine, whereas in a physiological setting, activation of AMPK would occur as a consequence of catecholamine-stimulated lipolysis. Therefore, our data indicate that pharmacological activation of AMPK in WAT may be an alternative approach to reducing circulating levels of NEFAs and prevent lipotoxicity in obesity.

In summary, we provide evidence that acute AICAR-induced AMPK activation promotes phosphorylation of HSLSer565 and inhibits HSLSer563/660 phosphorylation in VC and SC adipocytes, leading to suppression of HSL activity and lipolysis. Moreover, pharmacological inhibition of AMPK activation prevents the effects of this kinase on HSL phosphorylation and enhances both basal and epinephrine-stimulated lipolysis. Despite different fat depots eliciting distinct rates of lipolysis, AMPK activation exerts similar antilipolytic effects on both VC and SC adipocytes.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

This research was funded by the Natural Science and Engineering Research Council (NSERC), the Canadian Institute of Health Research (CIHR), and the Canadian Diabetes Association (CDA) through operating grants awarded to R.B.C. R.B.C. is also a recipient of the CIHR New Investigator Award. N.M.A. was supported by a CIHR Canadian Graduate Scholarship—Masters Award. M.P.G. was supported by a CIHR Canadian Graduate Scholarship—Doctoral Award.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES