Piperonal attenuates visceral adiposity in mice fed a high-fat diet: potential involvement of the adenylate cyclase–protein kinase A dependent pathway

2.1 Animals and diets

Eighteen C57BL/6N mice (5 weeks of age) were purchased from Orient Bio, Gyeonggi-do, Korea. The mice were housed at a pathogen-free facility at 21°C ± 2.0°C, with 50% ± 5% relative humidity and a 12-h light/dark cycle. After a 1-week acclimation period, the mice were randomly distributed into three experimental groups (n = 6 each): normal diet (ND) group, HFD group, and 0.05% piperonal-supplemented HFD (PSD) group. The ND group was fed the purified diet based on the AIN-76 rodent diet composition. The HFD was identical to the ND except for the addition of 200 g fat/kg (170 g of lard plus 30 g of corn oil) and 1% cholesterol. The PSD group was fed the HFD diet supplemented with 0.05% of piperonal (w/w). The experimental diets were provided ad libitum for 10 weeks in the form of pellets. Food intake of the mice was recorded daily, and their body weights were measured weekly during the feeding period. At the end of the experimental period, the animals were anesthetized after 6-h fasting. Blood was collected from the anesthetized mice, and the plasma samples were prepared by centrifuging the blood at 2000 × g for 15 min at 4°C, with subsequent storage at −80°C. The visceral fat pads were harvested, weighed, and stored at −80°C until further use. All animal experiments were performed in accordance with the Korea Food and Drug Administration (FDA) guidelines. All the experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center (Permit no. 2010-0039).

2.2 Biochemical analyses

Plasma concentrations of triglycerides, total cholesterol, free fatty acids, LDL-VLDL cholesterol, HDL-cholesterol, glucose, and activities of alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured using commercial kits (Bio-Clinical System, Gyeonggi-do, South Korea). Plasma levels of adiponectin, tumor necrosis factor-alpha (TNF-α), interlukin-6 (IL-6) and leptin were measured by means of a commercially available mouse ELISA kit (Millipore, MA, USA).

2.3 An Adcy enzymatic assay

The plasma membranes from frozen epididymal adipose tissues of C57BL/6N mice (16 weeks of age) were extracted using HEPES-sucrose buffer (20 mM HEPES, pH 7.4, 4 mM EDTA, 250 mM sucrose, two tablets of a protein inhibitor, 1 mM sodium orthovanadate, and 1% of Triton X-100). The homogenates were centrifuged at 2000 × g for 5 min at 4°C, and the supernatants were centrifuged at 12 000 × g for 1.5 h at 4°C to obtain a membrane preparation. The total protein concentration in membrane fraction extracts was determined by the Bradford assay.

The cAMP assay was performed using the cAMP ELISA kit from Enzo Life Sciences International, Inc. (Plymouth Metting, PA, USA). Fifty microliters of an Adcy reaction mixture (100 mM Tris-acetate, pH 7.4, 20 mM KCl, 10 mM MgCl₂, 20 mM phosphoenolpyruvate, 100 μg/mg pyruvate kinase, increasing concentrations of ATP, 20 μM guanosine triphosphate, 2 mM dithiothreitol, 0.4 mM bovine serum albumin, and 0.1 mM 3-isobutyl-1-methylxanthine) was added into microcentrifuge tubes in duplicate and maintained at 4°C. Enzymatic activities were determined using 125 μg of a membrane protein in the presence of piperonal or forskolin for 30 min at 37°C. The reaction was stopped by heating at 95°C for 5 min. The supernatant was stored after centrifugation at 10 000 × g for 5 min at 4°C. cAMP production rates were recorded in picomoles of cAMP produced per milligram of the membrane protein per minute.

2.4 Isolation of total RNA and semiquantitative RT-PCR

Total RNA was isolated from the epididymal adipose tissue of each mouse with the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was measured using a spectrophotometer, and the RNA was converted to cDNA using a reverse transcriptase (Invitrogen). Supporting Information Table 1 shows the forward (F) and reverse (R) primer sequences. The cDNA was denatured at 94°C for 10 min, followed by 30–35 cycles of PCR (94°C for 30 s, 55°C for 30 s, 72°C for 1 min). Finally, 4 μL of each PCR reaction mixture was mixed with 1 μL of 6 × loading buffer and then loaded onto a 2% agarose gel containing ethidium bromide. The mRNA levels of all genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

2.5 Western blot analysis

The epididymal adipose tissues of each mouse were homogenized in extraction buffer consisting of 100 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM sodium pyrophosphate, 50 mM NaF, 100 mM orthovanadate, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 2 μg/mL aprotinin, 1 μg/mL pepstatin A, and 1 μg/mL leupeptin. The tissue homogenates were centrifuged at 1300 × g for 20 min at 4°C, and the supernatants were collected and stored at −80°C. The protein concentrations of tissue extracts were measured by the Bradford assay (Bio-Rad, Hercules, CA, USA). Lysate samples containing 40 μg of protein were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (Amersham, Buckinghamshire, UK). The samples were incubated overnight with primary antibodies (diluted 1:1000) at 4°C. Western blotting was conducted using antibodies against AMPK, phospho-AMPK (Thr172), HSL, phospho-HSL (Ser563), CREB, and phospho-CREB (Ser133), tuberous sclerosis 2 (TSC2) (Ser1387), mammalian target of rapamycin (mTOR) (Ser 2448), and ribosomal protein S6 kinase (S6K) (Thr389) (Cell Signaling Technology; Beverly, MA, USA). The membranes were incubated with an anti-rabbit IgG HRPsc-2004 secondary antibody (diluted 1:5000; Santa Cruz Biotechnology, Dallas, TX). Finally, immunoreactive signals were detected using the Chemiluminescent Detection System (Amersham) and quantified in the Quantity One software (Bio-Rad).

2.6 Cell culture

Mouse embryo 3T3-L1 fibroblast cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Hyclone, Logan, UT) containing 10% of bovine calf serum (Gibco-BRL, Grand Island, NY) and penicillin–streptomycin (Hyclone, Logan, UT) at 37°C in a humidified incubator at 5% CO₂. For the differentiation assay, preadipocytes were seeded in 12-well plates and cultured until 2 days postconfluence. After the 3T3-L1 cells became confluent, the medium was replaced with DMEM consisting of 10% of fetal bovine serum (FBS; Gibco-BRL), 1% of penicillin–streptomycin, 1 μg/mL insulin, 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), and 0.5 mM isobutylxanthine (Sigma-Aldrich) with or without piperonal (Sigma-Aldrich), SQ22,536 (an Adcy inhibitor, Sigma-Aldrich) and H89 (a PKA Inhibitor, Sigma Aldrich). The cells were then maintained in DMEM containing 10% of FBS with 1 μg/mL insulin for another 2 days, followed by culturing with DMEM containing 10% of FBS for additional 4 days with or without piperonal and SQ22,536.

2.7 Oil red O staining

After 8 days of 3T3-L1 differentiation, the medium was removed from the wells. The cells were washed twice with 1 × PBS and fixed in 10% formaldehyde for 1 h. The fixed cells were stained with a filtered Oil red O solution (0.5% Oil red O in isopropanol) for 30 min at room temperature and washed with distilled water. After washing and complete drying, we added 500 μL of isopropanol to each well, followed by incubation at room temperature for 10 min to release Oil Red O from lipid staining. The extraction solution was transferred to another 96-well plate, which was subjected to OD measurement at a wavelength of 490 nm using a microplate reader (Versa Max). The images of stained cells were captured by means of an Olympus microscope (Tokyo, Japan).

2.8 A PKA enzymatic assay

3T3-L1 cells were lysed in 200 μL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, pH 7.4), and then centrifuged at 2000 × g for 15 min at 4°C. The supernatant was stored at −80°C until use. The samples were added into the appropriate wells, followed by addition of ATP to start the reaction. The reaction was terminated and a specific phospho-substrate antibody was added into the wells. After the addition of an IgG HRP-conjugated secondary antibody, color development was initiated by addition of tetramethylbenzidine (the substrate). The color development was stopped with a stop solution, and intensity of the color was measured on a microplate reader at 450 nm. The kinase assay was performed using the PKA Assay Kit (Kamiya Biomedical Company, Tokyo, Japan).

2.9 Statistical analysis

The results on body weight gain, visceral fat pad weights, and plasma biochemical data are presented as mean ± SEM of six mice per group. RT-PCR and western blot data are shown as mean ± SEM of three independent experiments (n = 2 per experiment) for each group. Data were subjected to one-way analysis of variance (Duncan's multiple range test) in the SPSS software (version 12.0). In all statistical tests, p values less than or equal to 0.05 were assumed to denote statistical significance.

3.1 Piperonal attenuates visceral adiposity and improves plasma lipid levels

Mice fed the PSD for 10 weeks showed significant reductions in final body weight (−10%), body weight gain (−19%), and food efficiency ratio (−15%) as compared with HFD-fed mice, without changes in food intake (Fig. 1B–E). The total visceral (epididymal, perirenal, mesenteric, and retroperitoneal) fat pad weights were significantly lower in the PSD-fed mice than in the HFD-fed mice (Fig. 1F). The PSD-fed mice had lower plasma levels of triglycerides (−21%), total cholesterol (−14%), LDL-VLDL cholesterol (−18%), free fatty acids (−17%) and glucose (−26%) than did HFD-fed mice (Table 1). Piperonal supplementation of the HFD resulted in significantly elevated HDL-cholesterol (+3%), adiponectin (+20%) and decreased leptin (−25%), TNF-α (−22%), and IL-6 (−28%), levels and ALT (−15%) and AST (−27%) activity in the plasma of mice in the plasma of mice (Table 1).

3.2 Piperonal prevents the HFD-induced downregulation of Adcy and PKA genes in WAT

We found that mice fed the PSD exhibited increased Adcy activity in the epididymal adipose tissue of mice compared to the HFD group. (Fig. 2A). The nine isoforms of Adcy (Adcy1 through Adcy9) were all expressed in the visceral adipose tissue of mice. The mRNA expression profile of Adcy isoforms in the WAT of HFD-fed mice differed from that in ND-fed mice, with a significant decrease in the expression of Adcy1, Adcy2, Adcy3, and Adcy8. Piperonal prevented these HFD-induced reductions in Adcy1, Adcy2, Adcy3, and Adcy8 expression, without affecting the expression of the other isoforms (Fig. 2B). Similarly, the downregulation of the PKA gene induced by the HFD in the WAT of mice was completely reversed by piperonal supplementation (Fig. 2C).

3.3 Piperonal prevents the HFD-induced modulation of PKA-related signaling molecules in WAT

The immunoblot results indicated that piperonal supplementation of the HFD significantly prevented the HFD-induced downregulation of phospho-AMPK, phospho-HSL, and phospho-CREB levels: the downstream targets of PKA in the visceral adipose tissues of mice (Fig. 3A). The protein levels levels of tuberous sclerosis 2 (TSC2), which was significantly downregulated by the HFD as compared to the ND, was upregulated back by piperonal supplementation (Fig. 3B). The HFD-induced upregulation of the signaling molecules downstream of TSC2, such as phospho-mTOR, the ribosomal protein S6 kinase (S6K), adipogenic transcription factors (PPAR-γ and CEBPα), and their target genes, FAS and adipocyte fatty acid-binding protein (aP2), was significantly prevented for all the above genes in visceral adipose tissue by piperonal supplementation (Fig. 3C). Piperonal significantly increased the mRNA levels of CPT1, molecules involved in mitochondrial fatty acid oxidation (Fig. 3D), and thermogenic genes, such as PGC-1α, retinoid X receptor (RXR), retinoic acid receptor (RAR), UCP1, PR domain containing 16 (PRDM16), and transcription factor A, mitochondrial (TFAM) (Fig. 3E) in the visceral fat tissues of mice fed the HFD. Piperonal significantly increased and decreased, respectively, the mRNA expression of adiponectin and leptin, in the epididymal adipose tissues of mice fed the HFD (Fig. 3F).

3.4 Piperonal attenuates adipogenic differentiation of 3T3-L1 cells through Adcy

To test our hypothesis that piperonal may act as an allosteric activator of Adcy that is known to promote cAMP production from ATP, the concentration-response activity was measured using the plasma membranes prepared from epididymal adipose tissue of the mice. Piperonal caused a concentration-dependent cAMP accumulation after a 30-min stimulation, yielding a characteristic sigmoidal curve with an EC₅₀ value of 12.1 μM and a maximal response at ∼100 μM (Fig. 4A). Only this maximal effective dose (100 μM) was used in subsequent experiments to determine the effects of piperonal on the kinetic properties of Adcy in the presence of an increasing concentration of the substrate, ATP. EC₅₀ for ATP-dependent cAMP was significantly reduced in the piperonal-treated membrane extracts as compared with untreated extracts. The magnitude of the piperonal-induced decrease in K_m for ATP was similar to that observed in membranes treated with forskolin, a direct activator of Adcy, indicating that the interaction with piperonal affected the function of Adcy in the plasma membranes of WAT in a manner similar to forskolin-induced activation (K_m = 2.1 mM for piperonal and K_m = 1.7 mM for forskolin versus K_m = 3.1 mM for control; Fig. 4B).

To confirm that piperonal exerts its anti-adipogenic effect through the direct activation of Adcy in the plasma membrane, we examined the lipid accumulation and regulation of the Adcy/PKA signaling cascade in 3T3-L1 cells in the presence or absence of a specific Adcy inhibitor, SQ22,536. Piperonal (100 μM) treatment significantly inhibited adipogenic differentiation of 3T3-L1 cells, whereas this positive effect of piperonal was attenuated by the inhibitors of Adcy (100 μM SQ22,536; Fig. 4C) and PKA (100 μM H89; Fig. 4D). 3T3-L1 cells treated with piperonal during differentiation showed significantly elevated Adcy and PKA activities as compared with untreated control cells, and these effects of piperonal were abrogated by the Adcy inhibitor (Fig. 4F and H). Furthermore, mRNA from 3T3-L1 cells treated with piperonal during differentiation showed significantly elevated expressions of Adcy isoforms (1, 2, 3, and 8) and PKA gene as also shown in the WAT of PSD mice (Fig. 4G). Similarly, piperonal-induced downregulation of adipogenic genes—such as adipogenic transcription factors (PPAR-γ and CEBPα) and their target genes (aP2 and FAS)—was almost abrogated by the Adcy inhibitor (Fig. 4H).