AMP-Activated Protein Kinases

Two cell lines, Caco-2 and SAOs, derived from human colorectal adenocarcinoma [42 (link)] and osteosarcoma [43 (link), 44 (link)], respectively, were employed. For the proliferation assays, cells were maintained at low confluence in polystyrene Petri plates (Corning, Milan, Italy) treated to ensure an optimal adhesion. The culture medium used was Dulbecco's modified Eagle medium (DMEM) enriched to 10% foetal bovine serum (FBS) (Life Technologies, Milan, Italy), 1% L-glutamine, 1% not-essential amino acids, and 1% penicillin/streptomycin solution (Life Technologies). Cells were treated as indicated and incubated at a temperature of 37°C in a humidified atmosphere containing 5% CO₂. Caco-2 and SAOs cells were incubated at different times (96–168 h and 48–168 h, resp.) with CEN corresponding to a concentration of 400 and 200 μg/ml (w/v) carotenoid-enriched extract, respectively. At the end of incubation, cell suspension was stained using Trypan blue solution (0.04% v/v), diluted in the culture medium (1 : 1). The cell count was performed using EVE Automatic cell counter (Eve™, NanoEnTek, Seoul, South Korea) and expressed as cell number/ml. CyQuant viability assay was performed to quantify the number of living cells using a nuclear dye that selectively binds to nucleic acids, emitting fluorescence. CyQuant assay was employed to the following treatments: (1) Caco-2 and SAOs cells, treated with CEN at the times and concentration indicated above; (2) SAOs cells added with 0.1 mM AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside, acadesine, N¹-(β-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide; Sigma-Aldrich, Milan, Italy), an AMPK (5′ adenosine monophosphate-activated protein kinase) activator [45 (link)]; and (3) SAOs and Caco-2 cells treated with 20 μM chloroquine during the last 24 h of incubation, or pretreated with 50 nM bafilomycin A1 (Sigma-Aldrich) to assess the activation of a nonprotective autophagy, before treatment with CEN at concentration indicated above for 96 h. Briefly, CyQuant mixture, containing the nuclear dye (CyQuant nuclear stain) and the suppressor of basal fluorescence (background suppressor), was added to the culture medium and incubated for 1 h at 37°C. Fluorescence was measured at the excitation wavelength of 485 nm and 530 nm emission, and the results were expressed as percentage of fluorescence of the untreated control using a microplate reader (Synergy HT BioTek, Milan, Italy).

Acetyl-CoA carboxylase 1 (ACC1), AMP activated protein kinase (AMPK)α1, and AMPKα2 mRNA expression levels were determined in prepared hepatic tissues by real-time RT-PCR. In addition, peroxisome proliferator-activated receptor (PPAR)α, PPARγ, leptin, uncoupling protein (UCP)2, adiponectin, CCAAT-enhancer-binding protein (C/EBP)α, C/EBPβ, fatty acid synthase, and sterol-regulatory-element-binding protein 1c (SREBP1c) mRNA expression levels were determined separately in periovarian adipose tissues, using methods described in previous reports [35 (link),36 (link)]. Briefly, RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA quality and concentrations were measured by the CFX96^TM Real-Time System (Bio-Rad, Hercules, CA, USA). Samples were mixed with recombinant DNase I (DNA-free; Ambion, Austin, TX, USA) to eliminate contaminating DNA. RNA was reverse transcribed by a reagent in the High-Capacity cDNA Reverse Transcription Kit, in accordance with the manufacturer’s instructions. ABI Step One Plus Sequence Detection System (Applied Biosystems, Foster City, CA, USA) were utilized for analysis, and relative expression levels of the vehicle control mice were calculated. The thermal cycling conditions were 10 min at 94 °C, followed by 39 cycles as follows: 15 s at 94 °C, 20 s at 57 °C, and 30 s at 72 °C. The sequences of the PCR oligonucleotide primers were as follows: PPARα, 5′- ATGCCAGTACTGCCGTTTTC-3′ and 5′-GGCCTTGACCTTGTTCATGT-3′; PPARγ, 5′- AGTGGAGACCGCCCAGG-3′ and 5′- GCAGCAGGTTGTCTTGGATGT-3′; Leptin, 5′- CCAAAACCCTCATCAAGACC-3′ and 5′- GTCCAACTGTTGAAGAATGTCCC-3′; UCP2, 5′- CCGCATTGGCCTCTACGACTCT-3′ and 5′- CCCCGAAGGCAGAAGTGAAGTG-3′; Adiponectin, 5′- CCCAAGGGAACTTGTGCAGGTTGGATG-3′ and 5′- GTTGGTATCATGGTAGAGAAGAAAGCC-3′; C/EBPα, 5′- TGGACAAGAACAGCAACGAGTAC-3′ and 5′- CGGTCATTGTCACTGGTCAACT-3′; C/EBPβ, 5′- AAGCTGAGCGACGAGTACAAGA-3′ and 5′- GTCAGCTCCAGCACCTTGTG-3′; SREBP1c, 5′- AGCCTGGCCATCTGTGAGAA-3′ and 5′- CAGACTGGTACGGGCCACAA-3′; FAS, 5′- GCTGCGGAAACTTCAGGAAAT-3′ and 5′- AGAGACGTGTCACTCCTGGACTT-3′; ACC1, 5′- GCCATTGGTATTGGGGCTTAC-3′ and 5′- CCCGACCAAGGACTTTGTTG-3′; AMPKα1, 5′- AAGCCGACCCAATGACATCA-3′ and 5′- CTTCCTTCGTACACGCAAAT-3′; AMPKα2, 5′- GATGATGAGGTGGTGGA-3′ and 5′-GCCGAGGACAAAGTGC-3′; GAPDH, 5′- CATCTTCCAGGAGCGAGACC-3′ and 5′- TCCACCACCCTGTTGCTGTA-3′. The data were normalized to GAPDH mRNA expression level by the comparative threshold cycle method [37 (link)].

Cells were lysed in a lysis buffer, as was described [15 (link)]. Samples were run on a 10% SDS polyacrylamide gel and transferred onto nitrocellulose membranes, as was described [15 (link)]. Blots were incubated with antibodies against AMP-activated protein kinase (AMPK), phosphorylated AMPK (pAMPK), BMAL1, phosphorylated BMAL1 (pBMAL1), S6, phosphorylated S6, FAS (Cell Signaling Technology, Beverly, MA, USA), mTOR, CRY1, and CLOCK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and after several washes, with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA). Anti-mouse antibody (Santa Cruz Biotechnologies) was used to detect actin, the loading control. The immune reaction was detected by enhanced chemiluminescence (Santa Cruz Biotechnologies). Finally, bands were quantified by scanning and densitometry and expressed as arbitrary units.

Liver and isolated hepatocytes were homogenized in RIPA buffer containing a protease inhibitor cocktail (Thermo Scientific, Waltham, MA, USA) and incubated on ice for 10 min. After centrifuging for 15 min at a maximum speed, the protein concentration of the supernatant was determined using the Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty micrograms of liver protein or 5 μg of hepatocyte protein were used for immunoblotting with primary antibodies for fatty acid translocase (FAT/CD36) (AF2519, R&D Systems, Minneapolis, MN, USA), fatty acid binding protein 1 (FABP1) (ab7366, Abcam), fatty acid synthase (FASN) (3180, Cell Signaling Technology, Beverly, MA, USA), acetyl-CoA carboxylase (ACC) (3676, Cell Signaling Technology), insulin receptor (IR) (3025, Cell Signaling Technology), PY99 (p-Tyr, 7020, Santa Cruz Biotechnology, Dallas, TX, USA), insulin receptor substrate-1 (IRS-1) (611394, BD Bioscience, Franklin Lakes, NJ, USA), phosphatidylinositol-3-kinase (PI3-K) (610045, BD Bioscience), sterol regulatory element-binding protein-1c (SREBP-1c) (NB600-582, NB100-2215, Novus Biologicals, Centennial, CO, USA), protein kinase B (Akt) (9272, Cell Signaling Technology), phospho-Akt (Ser473, 9271, Cell Signaling Technology), mammalian target of rapamycin (mTOR) (2983, Cell Signaling Technology), phospho-mTOR (Ser2448, 2971, Cell Signaling Technology), AMP-activated protein kinase α (AMPKα) (2603, Cell Signaling Technology), phospho-AMPKα (Thr172, 2535, Cell Signaling Technology), or β-Actin (A5441, Sigma-Aldrich, St. Louis, MO, USA), at dilutions suggested by the manufacturers. Densitometric analysis was performed by ImageQuant^TM LAS 4000 (GE Healthcare Bioscience, Piscataway, NJ, USA), and quantification values were normalized against β-Actin.

Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Hyclone (Logan, UT, USA), and fetal bovine serum (FBS) and penicillin/streptomycin solution were purchased from Invitrogen (Carlsbad, CA, USA). The EZ-Cytox assay kit, obtained from Daeil Lab Service (Chungcheongkuk-do, South Korea), was used to measure cell viability. The phosphorylation-form or non-phosphorylation-form primary antibodies of ACC (Acetyl-CoA carboxylase), AMPK (AMP-activated protein kinase), AKT (Protein kinase B), CPT-1 (Carnitine palmitoyltransferase-1) were purchased from Cell signaling Technology (Berkeley, CA, USA), and β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), which also supplied secondary antibodies. The oligonucleotide primers for real-time qPCR were produced by Macrogen (Seoul, South Korea).