All animal experiments were approved by the Animal Care and Use Committee of Keio University. Eight- to 16-week-old male mice (db/db-C57BLKS, WT-C57BLKS, ob/ob-C57BL/6 J, Akita-C57BL/6 J, and WT-C57BL/6 J) were obtained from Charles River (Yokohama, Japan), group-housed, and provided with water and a standard laboratory diet ad libitum. All animals were bred and maintained in the animal facility at Keio University according to institutional guidelines.
Min6 cells were kindly provided by Jun-ichi Miyazaki32 (Osaka University) and were cultured under 5% CO2 at 37 °C in High-Glucose (25 mM) Dulbecco’s modified Eagle’s medium–F12 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 72 μM β-mercaptoethanol53.
Mouse tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 µm. The sections were exposed for 1 h to phosphate-buffered saline (PBS) containing 3% bovine serum albumin, incubated overnight at 4 °C with primary antibodies, washed three times with PBS, and then incubated for 1 h at room temperature with secondary antibodies. Primary antibodies included those to insulin (Abcam, Cambridge, MA), to proinsulin (R&D Systems, Minneapolis, MN), to LAT1 (Transgenic, Kumamoto, Japan), to panCD44 (clone IM7; BD Pharmingen, Franklin Lakes, NJ), to CD44v (Link Genomics, Tokyo, Japan), and to glucagon (Cell Signaling Technology, Danvers, MA). For immunohistochemistry, immune complexes were detected with Histofine Simple Stain (Nichirei, Tokyo, Japan). Images were captured with a microscope (Olympus), and staining was quantified with Image J software (NIH, Bethesda, MD). For immunohistofluorescence staining, immune complexes were detected with Alexa Fluor 594– or Alexa Fluor 488–conjugated secondary antibodies (Molecular Probes, Eugene, OR). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).
For immunocytofluorescence staining, cells were fixed with 4% paraformaldehyde, permeabilized for 10 min with PBS containing 0.1% Triton X-100, exposed to PBS containing 3% bovine serum albumin for 1 h, and then incubated for 2 h at room temperature with primary antibodies. The cells were then washed three times with PBS and incubated for 1 h at room temperature with Alexa Fluor–labeled secondary antibodies. Nuclei were counterstained with Hoechst 33342. Immunofluorescence images were acquired with a BZ9000 inverted fluorescence microscope (Keyence, Osaka, Japan) and digitally processed with Keyence Analysis Software.
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich), the lysates were fractionated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a polyvinylidene difluoride membrane. Immunoblot analysis was performed with antibodies to β-actin (clone C-4; Santa Cruz Biotechnology, Santa Cruz, CA), CD44v (Link Genomics), and to LAT1 (Santa Cruz Biotechnology).
RT and real-time PCR analysis
Total RNA was isolated from Min6 cells with the use of an RNeasy kit (Qiagen, Hilden, German) and was subjected to RT with a Prime Script RT reagent kit (Takara Clontech, Shiga, Japan) followed by real-time PCR analysis with SYBR Green gene expression assays (Qiagen). Primers (sense and antisense, respectively) for mouse mRNAs included those for β-actin (5′-CTGGCTCCTAGCACCATGAAGAT-3′ and 5′-GGTGGACAGTGAGGCCAGGAT-3′), panCD44 (5′-TCCGAATTAGCTGGACACTC-3′ and 5′-CCACACCTTCTCCTACTATTGA-3′), LAT1 (5′-TCTTCCTCATTGCCGTGTCATTC-3′ and 5′-GCTTGTTCTTCCACCAGACACC-3′), INS1 (5′-GAAGCGTGGCATTGTGGAT-3′ and 5′-TGGGCCTTAGTTGCAGTAGTTCT-3′), and INS2 (5′-GCTTCTTCTACACACCCATGTC-3′ and 5′-AGCACTGATCTACAATGCCAC-3′). The abundance of target mRNAs was normalized by that of β-actin mRNA.
CD44 siRNAs were designed to specifically knockdown all splicing variants of the mouse CD44 gene. The siRNA sequences (sense and antisense, respectively) were as follows: CD44 siRNA #1, 5′-UUCCUUCGAUGGACCGGUUtt-3′ and 5′-AACCGGUCCAUCGAAGGAAtt-3′; CD44 siRNA #2, 5′-GUCUCAGGAAAUGGUGCAUtt-3′ and 5′-AUGCACCAUUUCCUGAGACtt-3′; and scrambled control siRNA, 5′-UUCUCCGAACGUGUCACGUtt-3′ and 5′-ACGUGACACGUUCGGAGAAtt-3′. The sequences for a LAT1 siRNA were 5′- GUCAUGUCCUGGAUCAUUCtt-3′ and 5′-GAAUGAUCCAGGACAUGACac-3′.
Min6 cells (3 × 105 per well of six-well plates) were transfected for 48 h with 100 pmol of siRNA with the use of the Lipofectamine RNA iMAX reagent (Invitrogen, Carlsbad, CA).
Expression vectors and cell transfection
Mouse LAT1 cDNA was amplified by PCR from total cDNA derived from the mouse breast cancer cell line 4T1 (American Type Culture Collection, Rockville, MD) and was cloned upstream of enhanced GFP cDNA into the pMXs-IP vector for expression of a LAT1-GFP fusion protein. The modified pMXs-IP vector together with pVSV-G was introduced by transfection into GP2-293 cells with the use of the FuGene reagent (Promega, Madison, WI). The retroviruses released into the culture supernatant were then harvested for infection of Min6 cells. Infected cells were selected with puromycin (2 µg/ml).
BCH20,34,54 was obtained from Sigma-Aldrich. Min6 cells (3 × 105 per well of six-well plates) were cultured for 24 h and then exposed to various concentrations of BCH in culture medium for 24 h before analysis of insulin secretion or insulin content.
Measurement of insulin secretion and insulin content
Min6 cells (3 × 105 per well) were cultured in six-well plates for 48 h, washed with PBS, and then incubated at 37 °C first for 60 min in Krebs-Ringer bicarbonate buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, and 2 mM NaHCO3 at pH 7.4) and then for 60 min in the same solution supplemented with 5 or 25 mM glucose. Insulin released into the incubation buffer or present in cell lysates was then measured with the use of a mouse insulin ELISA kit (Morinaga, Tokyo, Japan) and was normalized by the amount of total protein in each well.
Analysis of metabolites by CE-MS
Cells transfected with control or CD44 siRNAs were plated in 10-cm dishes and incubated with 5 or 25 mM glucose for 1 h, after which metabolites were extracted according to the Human Metabolome Technologies (HMT, Tsuruoka, Japan) metabolite extraction method for adherent cells55. Metabolite concentrations were measured with the use of an Agilent Capillary Electrophoresis System (HMT)56,57 and were normalized by the amount of cellular protein.
Quantitative data are presented as means ± s.d. and were analyzed with the unpaired Student’s t test as performed with Excel 2013 software (Microsoft, Redmond, WA), with the exception that metabolome data were analyzed with Tukey’s HSD and F test. A P value of <0.05 was considered statistically significant.
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.