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.
Comparison of the effects of the low-total-amino acid and low-arginine diets
Six-week-old male Wistar rats were randomly divided into 3 groups with different diets: the amino acid mixture control diet (equivalent to 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 to 5% protein in the diet; 5PAA). The low-total-amino acid mixture simulated the composition of casein . 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 removal of the diet, and a postprandial blood sample was collected from the carotid artery. The liver, longissimus muscles, and abdominal fat 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. The organ samples were used to analyze tissue TG concentrations, and protein and RNA expression.
We performed oral glucose tolerance tests (OGTTs) 12 d after initiation of the experimental diets. After 16 h of overnight fasting, glucose (2 g/kg) was orally administered to the rats. Blood (preprandial) was collected from the tail vein into heparinized tubes that were chilled on ice. 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.
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.
Lipid extraction and TG measurements
Lipids were extracted from frozen livers and longissimus muscles via 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.
RNA extraction and reverse transcription-polymerase chain reaction
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 reverse transcription (RT) polymerase chain reaction (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 or Apoa4) forward, 5′-ACCCTCTTCCAGGACAAACTTG-3′, and reverse, 5′-CCTTGGTTAGATGTCCACTCAGTTG-3′; and apolipoprotein B (ApoB or Apob) forward, 5′-CCTGTCCATTCAAAACTACCACA-3′, and reverse 5′-CAATGAACGAATCAGAAGGTGA-3′.
Western blotting analysis was performed as previously described [9, 16]. 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 (1:200 dilution), 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 (1:1,000 dilution). 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).
Very-low-density lipoprotein excretion test
Male Wistar rats were fed a casein control diet between 10:00 and 18:00 for 7 d 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 shown in Tables 1 and 2. The experimental diets were provided for 5 h from 9:00 to 14:00. One hour after removing the diets, we 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 30, 60, 120, and 240 min after injection to measure plasma TG concentrations. Tyloxapol inhibits endogenous lipoprotein lipase and blocks the clearance of lipid-carrying lipoproteins in the blood [19-21]. Thus, the rise in plasma TG concentration after tyloxapol injection is approximately proportional to the very-low-density lipoprotein (VLDL)-TG level. The TG concentrations in the plasma were measured with a commercial kit as described in “Lipid extraction and TG measurements.” The TG secretion rate was expressed in mg TG/dl/min.
Respiratory exchange ratio
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, Columbus, OH, USA). Rats were allowed free access to water and food during the experiment.
Theoretically, a respiratory exchange rate (RER) of 1.0 represents the dominant consumption of carbohydrates, and a decrease in the RER to approximately 0.7 represents proportionally higher lipid consumption.
De novo lipogenesis assay
De novo lipogenesis was measured based on the method described previously with minor 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 1 night (from 18:00 to 10:00). Rats were allowed free access to food. The following day, the rats of each group were further divided into D2O (Sigma Aldrich, St. Louis, MO, USA) or H2O injection groups (5 ml/kg body weight, intraperitoneally). Liver samples were collected under isoflurane anesthesia (3%–4%, 5 l/min; Dainippon Sumitomo Pharma, Osaka, Japan) 24 h after injection and stored at −80°C until analysis. Lipids were extracted as described in “Lipid extraction and TG measurements” . The extracted lipids were hydrolyzed and methylated with an FFA Methylation Kit (Nacalai Tesque, Kyoto, Japan), and the obtained products were purified with a Fatty Acid Methyl Ester Purification Kit (Nacalai Tesque). Purified FFA esters were analyzed by gas chromatography–mass spectrometry (GCMS-QP2010 Plus, SHIMADZU, Kyoto, Japan) to quantify palmitate isotopomers. The relative amounts of fatty acids were normalized by tissue weight and the difference in the total amount of methyl palmitate isotopomers in the D2O-injected group and H2O-injected group was interpreted as the de novo lipogenesis rate.
Hepatic FFA uptake assay
We performed an in vivo FFA uptake assay according to the method published in previous reports [24, 25]. 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 1 night (from 18:00 to 10:00). The following day, the rats of each group were further divided into 2 groups; one was treated with fluorescent FFA analog (0.1 mg/kg, BODIPY® FL C12, Invitrogen, Carlsbad, CA, 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 analysis. Lipids were extracted  and reconstituted in isopropanol. Then, lipid fluorescence was measured with an ARVO X3 microplate reader (PerkinElmer, Waltham, MA, USA). All values were normalized by tissue weight, and the value of the vehicle-administered group was subtracted as background fluorescence from that of the BODIPY-administered group.
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.