This study investigated the molecular mechanisms by which a high-fat diet (HFD) dysregulates lipolysis and lipid metabolism in mouse epididymal (visceral, VC) and inguinal (subcutaneous, SC) adipocytes. Eight-weeks of HFD feeding increased adipose triglyceride lipase (ATGL) content and comparative gene identification-58 (CGI-58) expression, whereas hormone-sensitive lipase (HSL) phosphorylation and perilipin content were severely reduced. Adipocytes from HFD mice elicited increased basal but blunted epinephrine-stimulated lipolysis and increased diacylglycerol content in both fat depots. Consistent with impaired adrenergic receptor signaling, HFD also increased adipose-specific phospholipase A(2) expression in both fat depots. Inhibition of E-prostanoid 3 receptor increased basal lipolysis in control adipocytes but failed to acutely alter the effects of HFD on lipolysis in both fat depots. In HFD visceral adipocytes, activation of adenylyl cyclases by forskolin increased HSL phosphorylation and surpassed the lipolytic response of control cells. However, in HFD subcutaneous adipocytes, forskolin induced lipolysis without detectable HSL phosphorylation, suggesting activation of an alternative lipase in response to HFD-induced suppression of HSL in VC and SC adipocytes. HFD also powerfully inhibited basal, epinephrine-, and forskolin-induced AMP kinase (AMPK) activation as well peroxisome proliferator-activated receptor gamma coactivator-1alpha expression, citrate synthase activity, and palmitate oxidation in both fat depots. In summary, novel evidence is provided that defective adrenergic receptor signaling combined with upregulation of ATGL and suppression of HSL and AMPK signaling mediate HFD-induced alterations in lipolysis and lipid utilization in VC and SC adipocytes, which may play an important role in defective lipid mobilization and metabolism seen in diet-induced obesity.

This study was designed to investigate the effects of prolonged activation of AMP-activated protein kinase (AMPK) on lipid partitioning and the potential molecular mechanisms involved in these processes in white adipose tissue (WAT). Rat epididymal adipocytes were incubated with 5′-aminoimidasole-4-carboxamide-1-b-D-ribofuranoside (AICAR;0.5 mM) for 15 h. Also, epididymal adipocytes were isolated 15 h after AICAR was injected (i.p. 0.7 g/kg body weight) in rats. Adipocytes were utilized for various metabolic assays and for determination of gene expression and protein content. Timedependent in vivo plasma NEFA concentrations were determined. AICAR treatment significantly increased AMPK activation, inhibited lipogenesis, and increased FA oxidation. This was accompanied by upregulation of peroxisome proliferator-activated receptor (PPAR)a, PPARd, and PPARgcoactivator-1a (PGC-1a) mRNA levels. Lipolysis was first suppressed, but then increased, both in vitro and in vivo, with prolonged AICAR treatment. Exposure to AICAR increased adipose triglyceride lipase (ATGL) content and FA release, despite inhibition of basal and epinephrine-stimulated hormonesensitive lipase (HSL) activity. Here, we provide evidence that prolonged AICAR-induced AMPK activation can remodel adipocyte metabolism by upregulating pathways that favor energy dissipation versus lipid storage in WAT. Additionally, we show novel time-dependent effects of AICAR-induced AMPK activation on lipolysis, which involves antagonistic modulation of HSL and ATGL.

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 HSL Ser565 and inhibited epinephrine-induced phosphorylation of HSL Ser563 and HSL Ser660 . This was also accompanied by a 73% reduction in epinephrine-stimulated HSL activity. Compound C prevented the phosphorylation of HSL Ser565 induced by AICAR and partially prevented the inhibitory effect of this drug on basal and epinephrinestimulated 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.

This study investigated the acute effects of troglitazone (Tro) on glucose and fatty acid (FA) partitioning in skeletal muscle. Exposure of L6 myotubes to Tro for 1h resulted in ∼1.3‐ and ∼1.6‐fold increases in palmitate oxidation and CPT‐1 activity, respectively. Tro inhibited basal (∼25%) and insulin‐stimulated (∼35%) palmitate uptake but significantly increased basal (∼2.2‐fold) and insulin‐stimulated (∼2.7‐fold) glucose uptake. Inhibition of AMPK prevented the effects of Tro on palmitate oxidation and glucose uptake. Although Tro exerted an insulin sensitizing effect, it reduced basal and insulin‐stimulated rates of glycogen synthesis, incorporation of glucose into lipids, and glucose oxidation to values corresponding to ∼30%, ∼60%, and 30% of the controls, respectively. These effects were accompanied by an increase in basal and insulin‐stimulated phosphorylation of AktThr308, AktSer473, and GSK3α/β. Tro also powerfully suppressed pyruvate decarboxylation, which was followed by a significant increase in basal (∼3.5‐fold) and insulin‐stimulated (∼5.5‐fold) rates of lactate production. In summary, we provide novel evidence that Tro exerts acute insulin sensitizing effects by increasing FA oxidation, reducing FA uptake, suppressing pyruvate dehydrogenase activity, and shifting glucose metabolism towards lactate production in muscle cells. Funding was provided by NSERC, CIHR, and CDA.

AICAR‐induced AMP‐activated protein kinase (AMPK) activation suppresses basal and epinephrine‐stimulated lipolysis in adipocytes. Our goal was to investigate whether the anti‐lipolytic effects of AICAR‐induced AMPK activation are mediated via inhibition of hormone sensitive lipase (HSL). Isolated rat epididymal (Epi), retroperitoneal (Ret), and subcutaneous (Sc) adipocytes were acutely (1h) incubated either in the absence or presence of AICAR (10 – 500μM), under basal and epinephrine (10μM) stimulated conditions. The lipolytic response and the phosphorylation status of HSL on Ser563 and Ser660 were determined. AICAR treatment significantly inhibited basal (77%, 31%, and 80%) and epinephrine stimulated (84%, 68%, and 86%) lipolysis in Epi, Ret, and Sc adipocytes, respectively. HSL phosphorylation on both serine residues was unaffected by AICAR under basal and epinephrine stimulated conditions. Interestingly, pharmacological inhibition of AMPK increased basal and epinephrine‐stimulated lipolysis and also reversed the anti‐lipolytic effect of low (10–100μM) AICAR concentrations. Even though lipolysis was increased by the AMPK inhibitor; HSL phosphorylation was not modified. These data indicate that the anti‐lipolytic effects of AICAR‐induced AMPK activation is independent of HSL phosphorylation, and appears to occur downstream of this enzyme. Funding was provided by NSERC, CIHR and CDA.