Intracellular fatty acids suppress β-adrenergic induction of PKA-targeted gene expression in white adipocytes

Abstract

β-Adrenergic receptor (β-AR) activation elevates cAMP levels in fat cells and triggers both metabolic and transcriptional responses; however, the potential interactions between these pathways are poorly understood. This study investigated whether lipolysis affects β-AR-mediated gene expression in adipocytes. Acute β(3)-adrenergic receptor (β(3)-AR) stimulation with CL 316,243 (CL) increased expression of PKA-targeted genes PCG-1α, UCP1, and NOR-1 in mouse white fat. Limiting lipolysis via inhibition of hormone-sensitive lipase (HSL), a direct target of PKA, sharply potentiated CL induction of PCG-1α, UCP1, and NOR-1. CL also induced greater expression of PKA-targeted genes in white fat of HSL-null mice compared with wild-type littermates, further indicating that HSL activity limits PKA-mediated gene expression. Inhibiting HSL in 3T3-L1 adipocytes also potentiated the induction of PGC-1α, UCP1, and NOR-1 by β-AR activation, as did siRNA knockdown of adipose triglyceride lipase, the rate-limiting enzyme for lipolysis. Conversely, treatments that promote intracellular fatty acid accumulation suppressed induction of PGC-1α and UCP1 through β-AR stimulation. Analysis of β-adrenergic signaling indicated that excessive intracellular fatty acid production inhibits adenylyl cyclase activity and thereby reduces PKA signaling to the nucleus. Lastly, partially limiting lipolysis by inhibition of HSL increased the induction of oxidative gene expression and mitochondrial electron transport chain activity in white adipose tissue and facilitated fat loss in mice treated for 5 days with CL. Overall, our results demonstrate that fatty acids limit the upregulation of β-AR-responsive genes in white adipocytes and suggest that limiting lipolysis may be a novel means of enhancing β-AR signaling.

Fig. 1.

Reducing lipolysis by inhibition of hormone-sensitive lipase (HSL) potentiates PKA-targeted genes during β₃-adrenergic receptor (β₃-AR) activation in white adipose tissue (WAT) of mice. A: mice were pretreated with BAY 59-9435 (BAY) or methylcellulose (MC) for 1 h, followed by CL 316,243 (CL) treatment (10 nmol) or vehicle (H₂O) for 3 h, and epididymal WAT (EWAT) was collected and analyzed for mRNA expression by quantitative PCR (qPCR). Data are from 8 mice/group, and the effect of BAY is indicated. B: serum fatty acid and glycerol levels in mice (n = 2–3) pretreated with BAY or vehicle (MC) after 45 min of CL (10 nmol). The effect of BAY is indicated. ***P < 0.001; **P < 0.01. ns, Nonsignificant. PGC-1α, peroxisome proliferator-activated receptor (PPAR)γ coactivator-1α; UCP1, uncoupling protein 1; %PPIA, percentage of peptidylprolyl cis-trans isomerase A; NOR-1, neuron-derived orphan receptor-1; C/EBPβ, CCAAT/enhancer-binding protein-β; FFA, free fatty acid.

Fig. 2.

HSL knockout (HSL-KO) mice have increased expression of β₃-AR-targeted genes in EWAT. mRNA levels were measured by qPCR and normalized to %PPIA from 3–7 mice/group. The difference between wild-type (WT) and HSL-KO is indicated (*P < 0.05).

Fig. 3.

Inhibition of HSL in 3T3-L1 adipocytes potentiates β-AR induction of PKA-targeted genes. 3T3-L1 adipocytes were treated with BAY or vehicle (DMSO), followed by 10 μM isoproterenol (Iso) or water (H₂O) for 3 h. Gene expression was measured by qPCR and normalized to %PPIA. Data are from 5 separate experiments, and the effect of BAY is indicated (***P < 0.001; *P < 0.05).

Fig. 4.

Knockdown of adipose triglyceride lipase (ATGL) potentiates β-AR induction of PKA-targeted genes. A: Western blot for ATGL, HSL, and GAPDH from 3 separate wells per small interfering (si)RNA. GAPDH serves as a loading control. B: glycerol levels from basal and stimulated (10 μM) control siRNA (siCON) and siATGL adipocytes. The effect of ATGL knockdown (siATGL) is indicated. C: siCON- and siATGL-treated adipocytes were stimulated for 3 h with 10 μM Iso or H₂O. Gene expression was measured by qPCR and normalized to %PPIA. The effect of ATGL knockdown (siATGL) is indicated. Data are from an experiment performed in triplicate and are representative of 2 independent experiments. ***P < 0.001. Ctl, control.

Fig. 5.

Intracellular fatty acids suppress the expression of β-AR-targeted genes. A: 3T3-L1 adipocytes were treated with 5 μM triacsin C or vehicle (DMSO), followed by 10 μM Iso or H₂O. Gene expression was measured by qPCR from 7 separate experiments and normalized to %PPIA. The effect of triacsin C is indicated. B: 3T3-L1 adipocytes were treated as in A, but Iso treatment was for 1 h. Intracellular fatty acids were quantified, and statistical analysis was performed by repeated-measures ANOVA to determine the effect of triacsin C. C: 3T3-L1 adipocytes were incubated in indicated concentrations of BSA and treated with BAY (5 μM) or DMSO followed by Iso (10 μM) for 3 h. mRNA levels were quantified by qPCR, normalized to %PPIA, and expressed as %Iso in the presence of 1% BSA. One-way ANOVA was performed to compare Iso with BAY/Iso at each BSA concentration and to test the effect of Iso at different concentrations of BSA. The fold difference between Iso and BAY/Iso is indicated above the bars. Data are from 3 separate experiments performed in duplicate. **P < 0.01; ***P < 0.001; &P < 0.05; &&&P < 0.001.

Fig. 6.

Fatty acids suppress cAMP/PKA signaling at the level of adenylyl cyclase. A: 3T3-L1 adipocytes were treated with BAY followed by Iso for 10 min. cAMP levels were measured from 3 separate experiments, each performed in duplicate. The effect of BAY is indicated. B: 3T3-L1 adipocytes were treated with triacsin C followed by Iso for 10 min. cAMP levels were measured in 2 separate experiments, each performed in triplicate. The effect of triacsin C is indicated. C: 3T3-L1 adipocytes were treated with vehicle (DMSO) or BAY (5 μM) followed by forskolin (FSK; 4 μM) or 8-Br-cAMP (1 mM). mRNA levels were measured by qPCR and normalized to %PPIA. The BAY effect is indicated. D: 3T3-L1 adipocytes, transfected with a cAMP response element reporter, were treated with vehicle (DMSO), BAY, or triacsin C followed by FSK for 6 h. Reporter activity is expressed as fold of FSK, and statistical analysis was performed on the normalized data (relative light units/β-galactosidase OD) by repeated-measures ANOVA to determine the effect of BAY or triacsin C. ***P < 0.001; *P < 0.05.

Fig. 7.

Partial inhibition of lipolysis enhances markers of white fat browning and mitochondrial activity and promotes fat loss in mice. Mice (n = 8/group) were treated with BAY (30 mg/kg) or MC followed by CL (5D CL; 10 nmol) or saline repeatedly for 5 days. A: mRNA levels in EWAT were measured by qPCR and normalized to %PPIA. The effect of BAY on 5-day CL is shown. B: 2,3,5-triphenyltetrazolium chloride (TTC) reduction as a measurement of mitochondrial electron transport chain activity. The effect of 5D CL treatment is shown. C: body fat composition was measured in mice by MRI and expressed as loss of body fat. The effect of 5D CL is indicated. ***P < 0.001; **P < 0.01.

Fig. 8.

Limiting lipolysis promotes β₃-AR signaling. A: acute stimulation of the β₃-AR activates adenylyl cyclase (AC) and PKA and subsequently ATGL and HSL and transcription factors (TF) that promote the transcription of genes such as PCG-1α, UCP1, and NOR-1. The mobilization of fatty acids causes inflammation and feedback to reduce AC activity. B: during inhibition of HSL in vivo, fatty acid mobilization is reduced, inflammation is suppressed, and the negative feedback on AC is relieved, thereby increasing expression of PKA-targeted genes and promoting fat loss.