GP73 is a glucogenic hormone contributing to SARS-CoV-2-induced hyperglycemia

  1. Luming Wan
  2. Qi Gao
  3. Yongqiang Deng
  4. Yuehua Ke
  5. Enhao Ma
  6. Huan Yang
  7. Haotian Lin
  8. Huilong Li
  9. Yilong Yang
  10. Jing Gong
  11. Jingfei Li
  12. Yixin Xu
  13. Jing Liu
  14. Jianmin Li
  15. Jialong Liu
  16. Xuemiao Zhang
  17. Linfei Huang
  18. Jiangyue Feng
  19. Yanhong Zhang
  20. Hanqing Huang
  21. Huapeng Wang
  22. Changjun Wang
  23. Qi Chen
  24. Xingyao Huang
  25. Qing Ye
  26. Dongyu Li
  27. Qiulin Yan
  28. Muyi Liu
  29. Meng Wei
  30. Yunhai Mo
  31. Dongrui Li
  32. Ke Tang
  33. Changqing Lin
  34. Fei Zheng
  35. Lei Xu
  36. Gong Cheng
  37. Peihui Wang
  38. Xiaopan Yang
  39. Feixang Wu
  40. Zhiwei Sun
  41. Chengfeng Qin
  42. Congwen Wei
  43. Hui Zhong

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Abstract

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  1. Version published to 10.1038/s42255-021-00508-2
  2. Version published to 10.21203/rs.3.rs-537089/v1 on Research Square
  3. ScreenIT

    SciScore for 10.1101/2021.04.30.21256060: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsField Sample Permit: The number of replicates carried out for each experiment is described in the figure/table legends.
    IRB: Sample acquisition from COVID-19 patients: The Ethics Committee of Huoshenshan Hospital approved the study (HSSLL036).
    Consent: Given the urgency of the COVID-19 pandemic, the need for informed consent forms was waived by the ethics boards of the hospitals.
    IACUC: All animal experiments were performed at the AMMS Animal Center (Beijing, China) and were approved by the Institutional Animal Care and Use Committee.
    Sex as a biological variabledecompensated chronic renal insufficiency, or severe congestive heart failure), were pregnant or had malignancy (Table 1).
    RandomizationFasting blood glucose and random blood glucose levels were measured weekly.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: All cell lines tested for mycoplasma contamination were incubated in DMEM at 37°C in a humidified atmosphere with 5% CO2.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: Anti-α-tubulin (T6074, 1:5,000 dilution) and anti-Flag (A8592, 1:5,000 dilution) antibodies were purchased from Sigma-Aldrich (Missouri, USA).
    Anti-α-tubulin
    suggested: None
    T6074
    suggested: (Sigma-Aldrich Cat# T6074, RRID:AB_477582)
    anti-Flag
    suggested: (Sigma-Aldrich Cat# A8592, RRID:AB_439702)
    A8592
    suggested: None
    dilution), anti-insulin (ab6995, 1:200 dilution), anti-CREB-phospho S133 (ab32096, 1:1000 dilution), and anti-CREB (ab32515, 1:1000 dilution) antibodies were purchased from Abcam (Illinois, USA).
    anti-insulin
    suggested: (Abcam Cat# ab6995, RRID:AB_305690)
    anti-CREB-phospho
    suggested: None
    anti-CREB
    suggested: (Abcam Cat# ab32515, RRID:AB_2292301)
    ab32515
    suggested: (Abcam Cat# ab32515, RRID:AB_2292301)
    An anti-GP73 antibody (F-12, sc-393372, 1:200 dilution) was purchased from Santa Cruz (Texas, USA)
    sc-393372
    suggested: None
    An anti-His antibody (KM8001, 1:1000 dilution) was purchased from Taihua Lekang Biotechnology (Beijing, China).
    anti-His
    suggested: (LSBio (LifeSpan Cat# LS-C129774-1000, RRID:AB_10832018)
    KM8001
    suggested: None
    dilution), anti-Akt (9272, 1:1000 dilution), anti-phospho-PKA-C-α (Thr197, 5661, 1:1000 dilution), anti-phospho-PKA substrate (RRXpS/T, 9624, 1:1000 dilution), and anti-PKA-C-α (5842, 1:1000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, USA)
    anti-phospho-PKA substrate ( RRXpS/T , 9624
    suggested: None
    anti-PKA-C-α ( 5842
    suggested: None
    Anti-rabbit HRP-IgG (ZB-2301, 1:5000 dilution) and anti-mouse HRP-IgG (ZB-2305, 1:5000 dilution) secondary antibodies were purchased from ZSGB-BIO (Beijing, China).
    Anti-rabbit HRP-IgG
    suggested: None
    ZB-2301
    suggested: (ZSGB-Bio Cat# ZB-2301, RRID:AB_2747412)
    anti-mouse HRP-IgG
    suggested: None
    Sandwich ELISA and Western blot analysis: For endogenous GP73 sandwich ELISA, two custom-made rat monoclonal anti-GP73 antibodies were used as the capture antibody and the detection antibody.
    anti-GP73
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The HepG2 (CRL-10741), Vero E6 (CRL-1568), HK-2 (CRL-2190), 293T (CRL-3216) and L6 (CRL-1658) cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).
    HepG2
    suggested: None
    Vero E6
    suggested: None
    To knock out human GP73 in Huh-7 cells, two small guide RNAs (sgRNAs) targeting GP73 were designed and inserted into the LentiCrispr v2 vector to construct transfer plasmids.
    Huh-7
    suggested: None
    293T cells were transfected with pMD2.
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Experimental Models: Organisms/Strains
    SentencesResources
    GP73 KO mice (T20200316-18[D25]) were generated by Southern Model Biotechnology (Shanghai, China)
    GP73 KO
    suggested: None
    Male C57 BLKS/J db/db and BKS control mice (8 weeks, 36-40 g) were purchased from GemPharma Tech Co. Ltd. (Jiangsu, China).
    C57 BLKS/J
    suggested: None
    STZ (40 mg/kg) in citric acid buffer (0.1 mol/L, pH 4.2) was administered to male C57BL/6N mice via intraperitoneal injection, and the same dose of STZ was injected 24 h later.
    C57BL/6N
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids and cell culture: Mammalian expression vectors encoding Flag-tagged human, mouse and rat GP73 were constructed by inserting the corresponding PCR-amplified fragments into pcDNA3 (Invitrogen, Massachusetts, USA).
    pcDNA3
    suggested: RRID:Addgene_15475)
    293T cells were transfected with pMD2.
    pMD2
    suggested: None
    G, psPAX2 and the corresponding transfer plasmid to produce lentivirus.
    psPAX2
    suggested: RRID:Addgene_12260)
    Recombinant GP73 protein purification: Human, mouse, and rat GP73 cDNAs, each with a six-amino-acid His tag on the N-terminus, were cloned into the pCDNA3.1 vector.
    pCDNA3.1
    suggested: RRID:Addgene_79663)
    Software and Algorithms
    SentencesResources
    triglyceride (TG, 200224) and cholesterol (CHO, 200224) biochemical test kits were purchased from Ruierda Biological Technology (Beijing, China).
    Ruierda Biological
    suggested: None
    The network was represented using Cytoscape ver 3.6.2 (
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    https://cytoscape.org/).
    https://cytoscape.org/
    suggested: (CluePedia Cytoscape plugin, RRID:SCR_015784)
    Protein-protein interactions were retrieved from STRING App (v1.51) (https://string-db.org/).
    STRING
    suggested: (STRING, RRID:SCR_005223)
    For KEGG and GO enrichment analysis, DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/home.jsp) was used.
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    Statistical analysis: The present study used GraphPad Prism 8.0 for statistical calculations and data plotting.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2021.04.30.21256060v1 on medRxiv