Animals and diets
Four-week-old male C57BL/6 J mice were purchased from CLEA Japan (Tokyo, Japan). Four diets were prepared: normal chow (CLEA Rodent diet CE-2: 12% of calories from fat, 59.1% of calories from carbohydrate, 28.8% of calories from protein), a high fat diet (HFD) (Clea High fat diet 32: 56.7% of calories from fat, 23.1% of calories from carbohydrate, 20% of calories from protein), normal chow containing 0.008% miglitol and HFD containing 0.008% miglitol. A previous study of miglitol in mice used a diet containing 0.08% miglitol. We chose to use a lower dose because it was closer to the dose used in clinical medicine. Mice were divided into 4 groups: a control group (NC), which was fed normal chow, a normal chow plus miglitol (NCM) group, which was fed the normal chow plus miglitol, a high fat (HF) group, which was fed the HFD, and a high fat plus miglitol (HFM) group, which was fed the HFD plus miglitol. The mice were kept in a temperature-controlled room (23°C) on a 12 h light/dark cycle (lights on 07:00 h; off 19:00 h) with free access to food and water. Individual food intake and body weight gain were monitored once a week. At 8 weeks, mice were fasted overnight and anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and blood was obtained by cardiopuncture. Plasma was separated by centrifugation at 4°C and stored at -80°C until assayed. The epididymal and subcutaneous white adipose tissues were dissected and weighed. Interscapular brown adipose tissue and liver were immediately dissected, frozen in liquid nitrogen and stored at -80°C until further analysis. All animal experiments and care procedures were conducted in conformity with the Guidelines of the Animal Care and Use Committee of Kyoto Prefectural University of Medicine.
Blood glucose was determined with a compact glucose analyzer Antsense II (Horiba, Kyoto, Japan). Plasma triglyceride (TG) and total cholesterol (T-Cho) levels were measured with reagents from Wako (Osaka, Japan). Plasma insulin level was measured by an ELISA kit (Morinaga Institute of Biological Science, Kanagawa, Japan). Plasma active glucagon-like peptide 1 (GLP1) levels were measured with an ELISA kit (Shibayagi, Gunma, Japan). All of the assays were performed according to the manufacturer’s instructions. Serum concentration of miglitol was measured by liquid chromatography - tandem mass spectrometry (LC/MS/MS).
Oxygen consumption (VO2) was measured with an O2/CO2 metabolism-measuring system (model MK-5000, Muromachi-Kikai, Tokyo, Japan), which consists of two independent 560-ml chambers (for measuring two animals simultaneously), a suction pump and a computer for data analysis. The mice were placed in the chambers at 23°C and acclimated for more than two hours. Every three minutes, the pump draws air from one of the chambers for one minute at rate of a 650 ml/min to measure O2 concentration. Oxygen consumption (VO2) was calculated as [Oa-Oc]v m-1 t-1, where Oa is the atmospheric oxygen concentration (%) that flows into the chamber, Oc is the oxygen concentration in the chamber (%), v is the flow rate (650 milliliters/min), m is the mass of the mouse in kg and t is the time in hours.
Mice were fasted for 6 hours and anaesthetized (sodium pentobarbital, 30 mg/kg, i.p.). Interscapular temperature surrounding BAT was recorded with a thermal imaging camera (FLIR i3, FLIR Systems, Tokyo, Japan) and analyzed with FLIR QuickReport software.
BAT was fixed in 10% buffered formalin. Sections (5 μm) were stained with hematoxylin and eosin. Slides were examined and photomicrographs taken under the same exposure and magnification. Lipid droplets in cells of BAT were quantified as previously described. One tissue section from each mouse was measured under blinded conditions by one investigator (S.S.) counting the number of nuclei surrounded by four or more lipid vacuoles/cell in two randomly chosen areas (1600 μm2) of each section, and averaging the results.
Western blot analysis
BAT was lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Nacalai Tesque, Kyoto, Japan). Homogenates were centrifuged at 10,000 × g for 10 min at 4°C and supernatants were collected. Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad, Tokyo, Japan). Tissue proteins were resolved on 10% polyacrylamide gels in the presence of sodium dodecyl sulfate, transferred electrophoretically to polyvinylidene difluoride membranes, and blocked by Blocking One (Nacalai Tesque). The primary and secondary antibodies were diluted with Can Get Signal (Toyobo, Osaka, Japan). The membrane was incubated with primary antibodies against proliferator-activated receptor gamma coactivator 1α (PGC1α) (1:10,000) (Abcam, Tokyo, Japan), UCP1 (1:15,000) (Abcam), β3-adrenergic receptor (β3AR) (1:10,000) (Abcam), protein kinase A (PKA) (1:5,000) (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated-protein kinase A (p PKA) (1:5,000) (Santa Cruz Biotechnology, Santa Cruz, CA), hormone-sensitive lipase (HSL) (1:10,000) (Cell Signaling Technology, Tokyo, Japan), carnitine palmitoyltransferase1 (CPT1) (1:5,000) (Lifespan Biosciences, Seattle, WA), p38α mitogen-activated protein kinase (p38αMAPK) (1:5,000) (Cell Signaling Technology), and β-actin (1:5,000) (Cell Signaling Technology). Secondary antibody consisted of a 1:15,000 dilution of HRP-conjugated donkey anti-rabbit IgG (for PGC1α, UCP1, β3AR, PKA, p PKA, HSL, CPT1, p38αMAPK) (GE Healthcare, Tokyo, Japan) or HRP-conjugated sheep anti-mouse IgG (for β-actin) (GE Healthcare). The immunocomplexes were detected using an enhanced HRP-luminol chemiluminescence system (ECL prime) (GE Healthcare) and subjected to autoradiography (New Amersham Hyperfilm) (GE Healthcare). Signals on the immunoblot were quantified using the NIH Image computer program (NIH, Bethesda, MD, version 1.45). To compare the results for protein expression, we assigned a value of 1 to expression in BAT from control mice.
Cyclic AMP (cAMP) assay
The selective β3-adrenergic receptor agonist CL316,243 (Sigma, Tokyo, Japan) (2 mg/kg/body weight) and saline was given by intraperitoneal injection 6 h before the end of the experiment. The amount of cAMP in BAT was measured by a cAMP assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.
Quantitative real-time PCR
Total RNA from BAT and liver were isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany). Template cDNA synthesized from 500 ng total RNA with random hexamer primers was used as the template for each reaction with a SuperScript First-Strand Synthesis System (Invitrogen Life Technology, Osaka, Japan). Quantitative real-time PCR (qRT-PCR) was performed using a SYBR Green master mix (Takara, Shiga, Japan) with 10 μM of each primer in an AB 7300 Real-Time PCR System (Applied Biosystems, Tokyo, Japan). Amplification was performed with the following protocol: 40 cycles (5 sec at 95°C and 31 sec at 60°C) after an initial activation step for 30 sec at 95°C. Primer sequences were shown as follows: β-actin (BAT), forward primer: 5′-GAAATCGTGCGTGACATCAAAG-3′, reverse primer: 5′-TGTAGTTTCATGATGCCACAG-3′; β-actin (liver), forward primer: 5′-GGCTGTATTCCCCTCCATCG-3′, reverse primer: 5′-CCAGTTGGTAACAATGCCATGT-3′; PGC1α, forward primer: 5′-TGAACGCACCTTAAGTGTGGAA-3′, reverse primer: 5′- GGGTTATCTTGGTTGGCTTTATGA-3′; UCP1, forward primer: 5′- AGGCTTCCAGTACCATTAGGT -3′, reverse primer: 5′-CTGAGTGAGGCAAAGCTGATTT-3′; CPT1, forward primer: 5′-CCAATCATCTGGGTGCTGG-3′, reverse primer: 5′-AAGAGACCCCGTAGCCATCA-3′; glucokinase ( GK), forward primer: 5′-CAACTGGACCAAGGGCTTCAA-3′, reverse primer: 5′-TGTGGCCACCGTGTCATTC-3′. β-actin was chosen as an internal standard.
Data are shown as means ± SEM. Single-group data were assessed using Student’s t-test. Repeated measurements of analysis of variance (ANOVA) with Tukey-Kramer post hoc comparisons were performed for multiple comparisons. P values less than 0.05 were considered statistically significant.