Ethics statement
This study was performed with the approval of the Institutional Animal Care and Use Committees (IACUC), ethics committee of Kyoto University (approval No. MedKyo 19,301). All sections of this report are based on the ARRIVE Guidelines for reporting animal research [22]. The mice were deeply anesthetized with 40 mg/kg of pentobarbital sodium (Kyoritsu Seiyaku, Tokyo, Japan); according to terminal procedures under anesthesia, blood was withdrawn and tissues were collected. All efforts were made to minimize suffering.
Mulberry leaves
The three races of Mulberry tree (Morus alba L.), “Hayate-sakari,” “Ichinose,” and “Minamisakari” had been stocked in the experimental farm of Kyoto Institute of Technology. These were transplanted to Kyotango Furusato Farm (Kyoto, Japan) by planting branches, and cultured using Japanese standard methods. A mixture of mulberry leaves was harvested from the three races “Hayate-sakari,” “Ichinose,” and “Minamisakari” at a ratio of 5:3:2 in the Kyotango Furusato Farm. Mulberry leaf powder was prepared by drying and milling at 180 °C for 8 s in a hot-air mill (Drymeister, Hosokawa Micron, Osaka, Japan). The average diameter of the dried powder used in this experiment was 20 μm.
Animals and diets
Five-week-old (5-w) male db/db mice and regular chow were purchased from Oriental Bio Service (Kyoto, Kyoto, Japan). The db/db mice were randomly allocated into two diet-groups, control-diet group and ML-diet group. Each diet group was further divided into three groups (each n = 6) by feeding duration from 7-w age to 10, 15, or 20-w age. Mice of all diet groups were provided ad libitum access to regular chow diet and water and were acclimated for at least 7 days under conditions of controlled temperature and a 12 h light/dark cycle. From 7-w of age, mice of the control-diet group were fed regular chow diet consisting of 4.8% fat, 61.4% carbohydrate, 20.8% protein, 8.0% water and 5.0% ash, while the ML-diet group were fed regular chow containing 5% (w/w) dried ML. The diets were stored at 4 °C until use. Mice of each diet group were euthanized at 10, 15 and 20 weeks of age, bloods were collected, and pancreas tissues were removed and frozen in liquid nitrogen. All samples were stored at − 80 °C. All procedures were approved by the Kyoto University.
Blood analysis and glucose tolerance test
All blood samples were collected following an overnight fast. Blood glucose levels and insulin concentrations were measured using the commercial kits, Glucose CII test Wako kit (Mutarotase-GOD method, Wako Pure Chemical Industries, Osaka, Japan) and Morinaga Ultra-Sensitive Mouse/Rat Insulin ELISA kit (MIoBS, Yokohama, Japan), respectively. For the intraperitoneal glucose tolerance test (ipGTT), mice were subjected to fasting for 16 h, and intraperitoneally injected with 1.5 g/kg of D-glucose (Wako). Blood samples were collected before and then sequentially after injection. The ipGTT tests were done on the same day for mice from the same age group of both diet groups.
Immunohistological analysis
The pancreases were fixed overnight in 4% paraformaldehyde (Wako) and embedded in paraffin. Sections were incubated with primary antibodies overnight at 4 °C. DAPI (diamidino-2-phenylindole) dye (Invitrogen, Carlsbad, CA, USA) was used to detect nuclei in immunofluorescent images. Primary antibodies used were as follows: mouse anti-insulin + proinsulin antibody (Abcam, Cambridge, MA, USA, ab8304, 1:1250), rabbit anti-insulin (Santa Cruz Biotechnology, Dallas, TX, USA, sc-9168, 1: 1000), rabbit anti-CHOP (anti-GADD153) (Santa Cruz, sc-575, 1:1000), mouse anti-ATF4 (Proteintech, Rosemont, IL, USA, 10835–1-AP, 1:200), mouse anti-PCNA (Santa Cruz, sc-56, 1:1000), rabbit anti-PDX1 (Abcam, ab134150, 1:500). Alexa Fluor-conjugated chicken antibodies were used as secondary antibodies (Invitrogen).
TUNEL staining
Pancreases were fixed overnight with 4% paraformaldehyde and embedded in paraffin. In order to detect apoptotic cells, a TUNEL assay was performed using the ApopTag fluorescein in situ apoptosis detection kit (Millipore, Massachusetts USA). Sections were triple stained with a TUNEL kit, anti-insulin antibodies, and DAPI. After being stained, the sections were observed under a confocal fluorescence microscope (BZ8100, KEYENCE, Osaka, Japan), and the ratio of TUNEL-positive cells in insulin-positive cells was calculated.
RNA preparation and reverse transcription quantitative PCR (RT-qPCR)
Total RNA was extracted from the pancreases using a RNeasy mini kit (Qiagen, Valencia, CA, USA) and cDNA was prepared via a high-capacity cDNA Reverse Transcription kit (Applied Biosystems, Forster City, CA, USA). Samples were analyzed using the 7300 Real-Time PCR System (Applied Biosystems) using the Power SYBR Green PCR Master Mix (Applied Biosystems). The following primers were used: β-actin: Forward, 5′-CCTGAGCGCAAGTACTCTGTGT-3′; Reverse, 5′-GCTGATCCACATCTGCTGGAA-3′; Atf4: Forward, 5′- GGACAGATTGGATGTTGGAGAAAATG-3′; Reverse, 5′-GGAGATGGCCAATTGGTTCAC-3′; Bip: Forward, 5′-GTTTGCTGAGGAAGACAAAAAGCTC-3′; Reverse, 5′-CACTTCCATAGAGTTTGCTGATAAT-3′; Chop: Forward, 5′-GTCCAGCTGGGAGCTGGAAG-3′; Reverse, 5′-CTGACTGGAATCTGGAGAG-3′; Pdx1: Forward, 5′-ACTTGAGCGTTCCAATACGC-3′, Reverse, 5′-AGAGGGGGAACGACTCTAGG − 3′; proinsulin (pro-ins): Forward, 5′-TCTTCTACACACACCCATGTCCC-3′; Reverse, 5′-GGTGCAGCACTGATCCAC-3′; Xbp1: Forward, 5′-TGGGCATCTCAAACCTGCTT-3′; Reverse, 5′-GCGTCCAGCAGGCAAGA-3′. All experiments were performed in duplicate. Results were normalized to β-actin expression level, and relative mRNA levels of the ML-diet group compared to that of the control-diet group were calculated using the 2^(−ΔΔCt) method.
Statistical analysis
Data were presented as mean ± standard error of the mean (SEM). All data were analyzed using Kaleida Graph software (version 4.5; Synergy software, Reading, PA, USA). The trapezoidal rule was used to determine the net incremental area under the curve (net AUC). The ‘t’ test was used to determine statistically significant differences. Statistical significance was set at P < 0.05.