Rhesus monkeys

The Rhesus monkey MetS model has been reported previously16. All monkeys were housed individually in cages, had free access to water, and were fed ad libitum with pellet monkey chow (Beijing HFK Bio-Technology Co., Ltd, China) that contained 7–10% crude fat, 16–20% crude protein, and 55–65% crude carbohydrate. All animals in this study were maintained in an animal facility at Peking University that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The experimental procedures for monkeys were approved by the Institutional Animal Care and Use Committee of Peking University and were in accordance with the principles of laboratory animal care of the National Academy of Sciences/National Research Council (approval number: IMM-ZhangXQ-1).

Blood sampling and biochemical tests

The blood samples were taken from a hindlimb vein after overnight fasting and anesthesia with ketamine at 10 mg/kg body weight. Blood samples for HbA1c analysis were collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA). For plasma samples, blood was collected in vacutainer tubes containing EDTA, mixed and kept on ice, and centrifuged for 10 min at 1200 × g at 4 °C within an hour. All measurements of plasma lipids and glucose were performed using Roche biochemical test kits. Insulin was measured by Roche Modular Analytics E170 Combinations.

Intravenous glucose tolerance test: After 14–16 h overnight fasting, under ketamine anesthesia, two i.v. cannulae were inserted into veins of both hindlimbs, one for blood sampling and the other for glucose administration. After baseline blood sampling, 50% glucose was administered at a dose of 0.5 ml/kg body weight over a time period of 30 s, which made a total glucose load of 0.25 g/kg body weight. Then blood samples were taken at 1, 3, 5, 10, 15, 20, 30, 45, and 60 min after the challenge. Heparinized saline was infused between each sampling to maintain the patency of the i.v. cannula. All blood samples were immediately put on ice and centrifuged at 4 °C as for plasma isolation. IR was calculated by means of the homeostasis model assessment–IR (HOMA-IR): fasting plasma glucose × fasting insulin/22.5.

Generation of global PRKD2-deficient mice

The mouse PRKD2 DNA clones were isolated from a 129SVJ mouse genomic library (Stratagene). A vector that contains a neomycin selection cassette flanked by FRT sites was used to generate PRKD2 targeting construct. Briefly, two homology arms of PRKD2 gene were cloned into the targeting vector, and a 603 bp fragment containing exon2 of PRKD2 was inserted after the first LoxP site (Supplementary Fig. 5). PRKD2 targeting vector was then linearized with NotI, and subsequently electroporated into R1 embryonic stem (ES) cells. G418-resistant ES clones were screened for homologous recombination by DNA blot analysis, as described below. Two independent homologous recombinant ES clones for each targeting construct were microinjected into blastocysts from C57BL/6J mice to generate male chimeras. Male chimeras were bred with female Black Swiss mice to generate germline transmitted floxed heterozygous mice (PRKD2+/flox-neo), which was subsequently crossed with Pro-Cre mouse50 to generate heterozygous mice (PRKD2+/−). Offspring from intercrosses were genotyped by PCR analysis using mouse tail DNA as previously described51. The experimental procedures for mice were approved by the Institutional Animal Care and Use Committee of Peking University and were in accordance with the principles of laboratory animal care of the National Academy of Sciences/National Research Council (Approval number: IMM-ZhangXQ-6).

DNA analysis for genotyping

Genomic DNA was extracted from G418-resistant ES cell clones and mouse tails, as previously described52. ES cell DNA was digested using Acc65I, and DNA was electrophoresed on a 1% (wt/vol) agarose gel, and subsequently blotted onto a nitrocellulose membrane. A 250 bp fragment was generated by PCR using mouse genomic DNA and was subsequently radiolabeled using [32P] dATP by random priming (Invitrogen). DNA blots were hybridized with the radiolabeled probe and visualized by autoradiography. Offspring from intercrosses were genotyped by PCR analysis using mouse tail DNA and the following primers: PRKD2, WT/flox allele (forward, AGAGCCAGGTAACAGGAACAATAG; reverse, GTGCTAAGGAGGGAGGCTCT), mutant allele (neo-specific primer, AATGGGCTGACCGCTTCCTCGT; reverse, GCAAGCTACTTCCTCCCAAG).

After genotyping, the mice were backcrossed to C57/BL6 for seven generations to reduce contamination from its original mixture with the 129SvJ background. The mice were maintained under specific pathogen-free conditions in an animal facility at Peking University that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The mice had free access to water and food; 4- and 14-week-old male mice were used in the following experiment. All experimental procedures with animals were approved by the Institutional Animal Care and Use Committee of Peking University and were in accord with the principles of laboratory animal care of the National Academy of Sciences/National Research Council.

Library preparation and deep sequencing

Genomic DNA was obtained from blood samples of the monkey animals to prepare libraries for parallel exome capture and deep sequencing53. DNA fragments in exonic regions of rhesus monkey were captured and enriched following the manufacturer’s protocol (Agilent Technologies). Strand-specific RNA-Seq libraries were prepared from skeletal muscle or adipose tissue derived from rhesus monkey as previously reported54,55. Both RNA-Seq and Exome-Seq were performed on a HiSeq 2000 Sequencing System. The deep sequencing data were evaluated following the guidelines of the RhesusBase Quality Score system19. Exome-seq data and RNA-Seq data were then mapped with BWA (v0.7.10–r789) and Tophat (v2.0.8), respectively. Only uniquely mapped reads were subjected to subsequent analyses. All the raw sequencing data are available at SRA under accession numbers SRP070922 and SRP071778.

Identification of candidate genetic variations in rhesus monkeys

In past years, we developed a rhesus monkey MetS model and developed a strategy to identify candidate genetic variations underpinning metabolic diseases through extreme phenotype sampling in 59 rhesus monkeys. Within these monkeys, 20 developed spontaneous MetS, two of which (ID# 950807 and 960109) showed prolonged and extremely high fasting insulin levels.

On the basis of the deep sequencing data, genetic variation calling was performed with the GATK Unified Genotyper (v2.7–0.4). Variations with unusual clustering (≥5 variants within a 25-bp window) or located in tandem repeats were removed, and only variations located in coding regions or splice sites were retained. The effects of these variations were then annotated by snpEff (v3.5a). A three-step pipeline was then used to identify candidate genetic variations: (i) the variations should be specific to the two monkeys with the extreme phenotype, but not in normal controls; (ii) the variations with large effects (such as nonsense variation, frameshift or splice-site variation), as well as missense variations were retained; and (iii) the variations should not be identified in the whole-genome sequencing data on 31 normal monkeys17.

Mouse glucose and ITT

For intraperitoneal glucose tolerance test, WT and PRKD2−/− mice were fasted overnight, and glucose was injected intraperitoneally at 2 g/kg body weight. Blood samples were taken from a tail vein at the indicated time points for glucose and insulin measurement. Glucose levels were measured on a portable glucometer (Roche, ACCU-CHEK® Performa); insulin levels in serum were measured using an ELISA kit (Millipore).

For ITT, WT, and PRKD2−/− mice were fasted for 4 h, and insulin (Humulin-R, LILLY) was injected intraperitoneally at 1 U/kg body weight. Blood samples were taken from tail vein at 0, 15, 30, 60, and 120 min, and levels of blood glucose were measured.

Mouse tissue sample collection

At 4 and 14 weeks of age, the male mice were anesthetized with sodium pentobarbital (100 mg/kg body weight) and sacrificed by cervical dislocation. Blood samples were collected to isolate serum. Samples of skeletal muscle, liver, and pancreas were harvested for further analysis. In brief, the tissues were snap-frozen in liquid nitrogen to isolate protein and mRNA. For histological and immunohistochemically analyses, tissues were fixed in ice-cold 4% paraformaldehyde (PFA) overnight.

Islet and single β-cell isolation

To measure in vitro insulin secretion, the pancreases from 14-week-old mice were exposed by a ventral midline incision and perfused with collagenase P (0.5 mg/ml) through the common bile duct. Then the pancreas was dissected and incubated in collagenase P solution at 37 °C for 24 min. Cooled Hanks buffer was added to the incubation mixture to terminate digestion and separate the pancreatic tissue. Then 100–200 islets were picked out by a pipette under a microscope, and cultured in RPMI1640 (7 mM glucose) at 37 °C overnight in a cell culture incubator. Eighty islets with similar size were pre-incubated for 2 h at 37 °C in fresh RPMI1640, then incubated in a modified Krebs–Ringer bicarbonate buffer (KRBB, pH 7.4) that contained 130 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 2.8 mM glucose, and 10 mM HEPES, supplemented with 0.1% bovine serum albumin for 30 min to synchronize cells. After pre-incubation, the islets were transferred into perfusion buffer containing 2.8 mM glucose, perfused for 3 min, then was changed to perfusion buffer containing 16.7 mM glucose, and perfused for 20 min at 37 °C. The perfusion rate was 500 µl/min and the perfusate was collected to measure insulin.

To obtain single β-cells, isolated mouse islets were pretreated with Hanks buffer without Ca2+/Mg2+ at 37 °C for 4 min, and digested into single cells by trypsin for 4 min at 37 °C. Then the separated β-cells were cultured in RPMI1640 containing 10% fetal bovine serum (FBS) and 7 mM glucose for overnight.

Calcium imaging and patch-clamp analysis in β-cells

Fluo-4 AM was from Thermo Fisher (Cat#: F14217). For Ca2+ imaging experiments, β-cells were incubated with 5 µM Fluo-4 AM in KRBB containing 2.8 mM glucose at 37 °C for 20 min. The cells were perfused with KRBB containing 2.8 mM glucose for 3 min, then switched to KRBB containing 20 mM glucose for 10 min. The depolarization was evoked by high-K+ solution (pH 7.4) containing 70 mM KCl, 67 mM NaCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM HEPES, and 2.8 mM glucose. Dynamic images were captured on a Zeiss LSM710 confocal microscope with a ×40, 1.3 NA oil-immersion objective, with a frequency of 2 Hz. Data were analyzed using ImageJ (NIH, Bethesda, MD).

To investigate L-type Ca2+ currents, patch-clamp was conducted with standard whole-cell recordings using an EPC-10 patch-clamp amplifier (HEKA) at room temperature (~22 °C). Isolated islet cells were stimulated with a train of depolarizing pulses from a holding potential of −70 mV. Cells that exhibited no voltage-dependent Na+ currents were β-cells due to the inactivation of Na+ channel at the −70 mV holding potential56. The extracellular solution containing 138 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl 2 , 1.2 mM MgCl 2 , 5 mM glucose, and 10 mM HEPES (pH 7.4 adjusted with NaOH), while the pipette solution containing 125 mM Cs-glutamate, 0.3 mM Na 2 -GTP, 2 mM Mg-ATP, 1 mM MgCl 2 , and 0.1 mM EGTA (pH 7.2 with CsOH). For depolarization experiments, Ca2+ currents were evoked by a depolarization from −70 mV to 0 mV for 200 ms. For L-type Ca2+ channel blocking experiments, nifedipine with a final concentration of 30 μM was added in the bath solution during depolarization57.

Cell culture

The rat β-cell line (Ins-1) was from the China Infrastructure of Cell Line Resources (Beijing, China). Cells were cultured at 37 °C under 5% CO 2 and 95% O 2 in RPMI1640 with 11.1 mM glucose and supplemented with 10% FBS, 1 mM pyruvate, 50 μM β-mercaptoethanol, 100 U penicillin/ml, and 0.1 mg streptomycin/ml. Lipofectamine RNAiMAX from Invitrogen and PRKD2 siRNA from Dhamarcon (Cat#: L-081927-02-0005) were used to knock down PRKD2 when the cells had reached 60–70% confluence. Insulin in the supernatant was measured after the cells were incubated in KRBB containing 2.8 or 16.7 mM glucose for 30 min. For insulin sensitivity, Ins-1 cells were stimulated with 100 nM insulin for 20 min and proteins were extracted for western blot analysis.

To analyze the effect of PRKD2-knockdown on insulin signaling in peripheral tissue, the mouse liver cell line, Hepa1-6 (China Infrastructure of Cell Line Resources, Beijing, China), was cultured at 37 °C under 5% CO 2 and 95% O 2 in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin (Gibco, Grand Island, NY, USA), and 25 mM glucose. PRKD2 siRNA was used to knock down PRKD2 in the same way as for Ins-1 cells. Insulin signaling activation was determined by western blot of the Akt phosphorylation in proteins collected from Hepa1-6 cells treated with insulin for 15 min.

Western blotting

Total protein from mouse tissue samples and cultured cells were extracted using RIPA lysis buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) supplemented with a protease inhibitor cocktail (Sigma), 10× phosphorylation protease inhibitor (Roche), and 2 mM phenylmethane sulfonyl fluoride. After homogenization with a polytron, the homogenate was incubated on ice for 30 min, and centrifuged at 12,000 g for 15 min at 4 °C. Protein samples (50–100 µg) were separated on 10% SDS-polyacrylamide gel electrophoresis. Blots were incubated at 4 °C overnight with primary antibodies, Rabbbit anti-Akt (1:1000, CST, #4691); Rabbit anti-Phospho-Akt Ser473 (1:1000, CST, #4058L); Mouse anti-PKD2 (1:500, Abcam, ab7281); Mouse anti-PKD2 (1:500, Abcam, ab57114); Rabbit anti-β-actin (1:1000, CST, #8457); Mouse anti-α-Tubulin (1:1000, Sigma, T5168), followed by anti-rabbit or mouse horseradish peroxidase-labeled secondary antibodies (1:1000; Santa Cruz Biotech, sc-2004, sc-2005) for 1 h at room temperature. An electrochemiluminescence detection system revealed the peroxidase label and relative abundance was quantified by densitometry using Quantity One ver. 4.6.7 software (both from Bio-Rad). Uncropped western blots in this study are shown in Supplementary Fig. 9.

Histology and immunohistochemistry of pancreas

Each PFA-fixed pancreas was embedded in paraffin, and 5-µm sections were cut and stained with hematoxylin-eosin (HE) for histological analysis. The HE-stained sections were imaged with a BX51 light microscope and Image-Pro MC 6.0 software (Olympus, Japan) to measure the areas of at least 15 randomly selected islets from each mouse. For Immunohistochemistry, sections were incubated with rabbit anti-insulin (1:400; Santa, sc9168) or mouse anti-glucagon (1:400; Santa, sc57171) antibodies overnight at 4 °C, followed by Alexa Fluor®594 donkey anti-rabbit (1:200; Invitrogen, A21207) or Alexa Fluor®488 goat anti-mouse secondary antibody (1:200; Invitrogen, A11029) for 1 h at room temperature. The slides were imaged with a Zeiss LSM700 confocal microscope. Cells positive for insulin was counted using Image-Pro MC 6.0 software.

Real-time PCR

Total RNA was extracted from PRKD2−/− or WT β-cells (TIANGEN Biotech, Beijing). Reverse transcription was performed by the M-MLV Reverse Transcriptase (Promega). The primers used for CACNA1c amplification were reported in previous study (forward 5′-GGCATCACCAACTTCGACA and reverse 5′-TACACCCAGGGCAACTCATA)58. Primers used for β-actin amplification: forward 5′-CACCATGAAGATCAAGATCATTGCT and reverse 5′-AACGCAGCTCAGTAACAGTCCG.

Nifedipine treatment in rhesus monkeys

To investigate the inhibition function of nifedipine in insulin secretion in monkeys, we anesthetized the monkeys with ketamine at 10 mg/kg body weight after 14–16 h of fasting. Nifedipine solution was prepared by adding 2 mg nifediping to 5 ml ethylalcohol, 25 ml polyethylene glycol, and 20 ml saline solution, for a final concentration of 40 μg/ml59. After baseline blood sampling and blood pressure measurements, Nifedipine was infused at a rate of 5 μg/kg/min for 11 min. Then the infusion rate was changed to 0.5 μg/kg/min for the rest time of the experiment. The IVGTT was started 3 min after changing the infusion rate.

Statistical analyses

Statistical analyses in this study were performed using GraphPad PRISM ver. 5.01 (GraphPad Software, Inc.) and the SPSS 18.0 software package (SPSS Inc.). For the animal studies, sample size was determined based on previous study60. For in vivo studies in mice, PRKD2−/− mice and WT controls were used as different experimental groups. The investigator was blinded to the group allocation during the experiment. Data sets were tested for normality with Kolmogorov–Smirnov tests. Data groups (two groups) with normal distributions were compared using two-sided, unpaired Student’s t-tests. Data groups with multi-factors were compared using two-way ANOVA tests. A p-value <0.05 was considered as statistically significant.

Data availability

The raw deep sequencing data in this study are available at SRA under accession numbers SRP070922 and SRP071778. The rest of the data is available from the authors upon reasonable request.