Anti-insulin receptor subunit β (IRβ) and anti-CD36 antibodies for immunoblotting were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-IRS2 (clone 9.5.2) antibody for immunoblotting was purchased from Merck Millipore (Billerica, MA, USA). Anti-phospho-p70 S6K (Thr389), anti-p70 S6K (49D7), anti-phospho-4E-BP1 (Thr37/46), anti-4E-BP1, and anti-phospho-AMPKα (Thr172) (40H9) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-GAPDH (6C5) antibody was purchased from Abcam (Cambridge, UK). Anti-rabbit and mouse IgG horseradish peroxidase-conjugated secondary antibodies were purchased from GE Healthcare (Little Chalfont, UK).
The experiments were approved by the Animal Usage Committee of the Faculty of Agriculture of the University of Tokyo and performed in accordance with its guidelines (Permission No. P09-375). Male Wistar rats were purchased from Charles River Laboratories International (Kanagawa, Japan). The animals were housed individually in wire cages with free access to food and water. The rats were maintained at a room temperature of 23°C ± 1°C with 50–60% relative humidity under a 12-h light/dark cycle (light from 08:00 to 20:00). In the pre-experimental period, the rats were fed a purified diet containing 15% protein from casein.
2.3. Comparison of the effects of the low-total-amino acid and low-arginine diets
Six-week-old male Wistar rats were randomly divided into three groups with different diets: the amino acid mixture control diet (equivalent of 15% protein in the diet; 15PAA), low-arginine diet in which the concentration of arginine in young rodents was 33% of that in the 15PAA diet (low Arg, n = 8), and the low-total-amino acid diet (equivalent of 5% protein in the diet; 5PAA). This amino acid mixture simulated casein composition . The diet compositions are shown in Tables 1 and 2.Rats were given ad libitum access to tap water and food. Body weight and food intake were measured daily. Fourteen days after initiation of the experimental diets, the rats were anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg) 1 h after removing the diet, and a postprandial blood sample was collected from the carotid artery. This blood samples were obtained at feeding condition. The liver, longissimus muscles, and abdominal fats were removed and weighed. The livers and muscle tissues were soaked in RNAlater (AMBION, Austin, TX, USA) or snap-frozen in liquid nitrogen and stored at −80°C until use. These organ samples were used to analyses tissue TG concentration, western blotting, and RNA expression.
We performed oral glucose tolerance tests (OGTTs) 12 days after initiation of the experimental diets. After 16 h of overnight fasting, glucose (2 g/kg) was orally administered to the rats. Blood was collected from the tail vein into heparinized tubes that were chilled on ice, and preparandial blood samples were obtained at this fasting condition. The blood samples were subjected to centrifugation at 3,000 ×g for 5 min at 4°C and the supernatants were transferred to new tubes. Plasma samples were stored at −80°C until analysis.
2.4. Blood biochemistry
Blood biochemical parameters, including total cholesterol, TG, free fatty acids (FFA), and glucose concentrations, were determined using commercial kits (Cholesterol E-test, Triglyceride E-test, NEFA C-test, and Glucose CII test, respectively; Wako Pure Chemical Industries, Osaka, Japan). The plasma insulin concentration was measured using an insulin measurement kit (Morinaga Institute of Biological Science, Yokohama, Japan) according to the manufacturer’s instructions.
2.5. Lipid extraction and TG measurements
Lipids were extracted from frozen livers and longissimus muscles by a modified Folch method  in a 2:1 (vol/vol) mixture of chloroform/methanol. The extracts were washed with 0.5 volumes of 0.8% KCl and centrifuged at 1,500 ×g for 10 min, and the organic phases were recovered. The TG content in the liver and plasma was also determined using a commercial kit (Wako Pure Chemical Industries) according to the manufacturer’s instructions.
2.6. RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from homogenized livers using NucleoSpin® RNA (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. The total RNA concentration was measured with a NanoDrop® spectrophotometer (ND-1000, NanoDrop, Wilmington, DE, USA). The quality of the RNA was determined by assessing the A260/280 ratio and by agarose gel electrophoresis. The RNA was reverse-transcribed into cDNA using PrimeScript® RT Master Mix (Takara Bio, Shiga, Japan). cDNA was amplified using SYBR® Premix Ex Taq II (Takara Bio) according to the manufacturer’s protocol. We designed the primers for RT-PCR with the design software Primer 3. β-actin was used as an endogenous control. The following PCR primers were used: β-actin (Actb) forward, 5′-GGAGATTACTGCCCTGGCTCCTA-3′, and reverse, 5′-GACTCATCGTACTCCTGCTTGCTG-3′; MTP (Mttp) forward, 5′-AGCAACATGCCTACTTCTTACAC-3′, and reverse, 5′-TCACGGGTTCACTTTCACTG-3′; apolipoprotein A-IV (ApoA4, Apoa4) forward, 5′-ACCCTCTTCCAGGACAAACTTG-3′, and reverse, 5′-CCTTGGTTAGATGTCCACTCAGTTG-3′; and apolipoprotein B (ApoB, Apob) forward, 5′-CCTGTCCATTCAAAACTACCACA-3′, and reverse 5′-CAATGAACGAATCAGAAGGTGA-3′.
2.7. Western blotting
Western blotting analysis was performed as previously described [7, 11]. In brief, frozen livers were homogenized in homogenizing buffer and centrifuged at 100,000 ×g for 1 h at 4°C. The protein content in the supernatant was determined using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes. The membranes were blocked with blocking buffer, and then incubated at 4°C overnight with primary antibodies against IRβ, CD36, and FFA synthase at 1:200, and against PI3 kinase p85, phospho-p70 S6K (Thr389), p70 S6K, phospho-4E-BP1 (Thr37/46), 4E-BP1, phospho-AMPKα (Thr172), AMPKα, phospho-acetyl-CoA carboxylase (Ser79), acetyl CoA carboxylase, and acetyl CoA carboxylase 1 at 1:1,000. Primary mouse anti-IRS2 and anti-GAPDH antibodies were used at dilutions of 1:1,000 and 1:3,000, respectively. We visualized the blots by chemiluminescence after incubating with donkey anti-rabbit IgG or sheep anti-mouse IgG conjugated to horseradish peroxidase (1:2,500). The immunoreactive bands were exposed and the signals were quantified using a cooled charge-coupled device camera system (LAS-3000 Mini; Fujifilm, Kanagawa, Japan).
2.7. Very-low-density lipoprotein (VLDL) excretion test
Male Wistar rats were fed a casein control diet between 10:00 and 18:00 for 7 days prior to the experiment. After habituation, the rats (7.5 weeks of age, 208–229 g) were assigned to the 15PAA (n = 8), 5PAA (n = 8), and low Arg (n = 9) groups. The diet compositions are same as in above section (Table 1 and 2). The experimental diets were provided for 5 h from 9:00 to 14:00. We then administered tyloxapol (200 mg/kg, dissolved in 0.9% NaCl; Triton WR-1339, Sigma-Aldrich, St. Louis, MO, USA) to all rats under isoflurane anesthesia (3–4%, 5 l/min; Dainippon Sumitomo Pharma, Osaka, Japan) via the tail vein. Blood was collected from the tail vein into chilled, heparinized tubes prior to tyloxapol injection, and at 30, 60, 120, and 240 min after injection. The TG concentrations in the plasma were measured with a commercial kit as described in section 2.5 above. Tyloxapol inhibits endogenous lipoprotein lipase and accumulates plasma TG [12, 13]. Therefore, we monitored an increase in plasm TG concentration from0 time. The TG secretion rate was expressed in mg TG /dl/min.
2.8. Respiratory exchange ratio (RER)
Six-week-old male Wistar rats were assigned to the 15PAA (n = 6), 5PAA (n = 6), and low Arg (n = 6) groups. Six days after initiation of the experimental diets, animals were individually placed in a metabolic chamber for 24 h, and VO2 and VCO2 were monitored every 10 min with an OXYMAX system (Columbus Instruments, USA). Rats were allowed free access to water and food during the experiment.
2.9. De novo lipogenesis assay
De novo lipogenesis was measured based on the method described previously with small modifications. Six-week-old male Wistar rats were assigned to the 15PAA (n = 8), 5PAA (n = 8), and low Arg (n = 8) groups (240–280 g) for one night (from 18:00 to 10:00). Rats were allowed free access to food. The following day, rats of each group were further divided into D2O (Sigma Aldrich, USA) or H2O injection group (5 ml/kg body weight, i.p.). Liver samples were collected under isoflurane anesthesia (3–4%, 5 l/min; Dainippon Sumitomo Pharma, Osaka, Japan) 24 hours after injection and stored at -80°C until used for analysis. Lipids were extracted as described in section 2.5 above. The extracted lipids were hydrolyzed and methylated by FFA Methylation Kit (Nacalai Tesque, Japan), and the obtained products were purified by Fatty Acid Methyl Ester Purification Kit (Nacalai Tesque, Japan). Purified FFA esters were analyzed with gas chromatography-mass spectrometry (GCMS-QP2010 Plus, SHIMADZU, Japan) to quantify palmitate isotopomers. Relative amount of fatty acids was normalized by tissue weight and the difference in the total amount of methyl palmitate isotopomers between the D2O-injected group and H2O-injected group was interpreted as the de novo lipogenesis rate.
2.10. Hepatic FFA uptake assay
In vivo FFA uptake assay was conducted according to the previous reports [15, 16].Six-week-old male Wistar rats were assigned to the 15PAA (n = 8), 5PAA (n = 8), and low Arg (n = 8) groups (240–280 g) for one night (from 18:00 to 10:00). The following day, rats of each group were further divided into two groups; one was treated with fluorescent FFA analog (0.1 mg/kg, BODIPY® FL C12, Invitrogen, USA) and the other was treated with only vehicle (0.1% bovine serum albumin in phosphate-buffered saline) by intravenous injection under isoflurane anesthesia (3–4%, 5 l/min; Dainippon Sumitomo Pharma, Osaka, Japan). The liver samples were collected 10 min after injection and stored at -80°C until used for analysis. Lipids were extracted and reconstituted in isopropanol,. Then its fluorescence was measured by the ARVO X3 microplate reader (PerkinElmer, USA). All values were normalized by tissue weight and the value of vehicle-administered group was subtracted as background fluorescence from that of BODIPY-administered group.
2.11 Statistical analysis
All data are presented as the mean ± standard error. The data were statistically analyzed with the Ekuseru-Toukei 2010 software package (Social Survey Research Information, Tokyo, Japan). Statistical significance was calculated using one-way analysis of variance (ANOVA) with the Bonferroni and Tukey–Kramer post-hoc tests to assess differences between groups. P < 0.05 was considered statistically significant.