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A hormone complex of FABP4 and nucleoside kinases regulates islet function

Abstract

The liberation of energy stores from adipocytes is critical to support survival in times of energy deficit; however, uncontrolled or chronic lipolysis associated with insulin resistance and/or insulin insufficiency disrupts metabolic homeostasis1,2. Coupled to lipolysis is the release of a recently identified hormone, fatty-acid-binding protein 4 (FABP4)3. Although circulating FABP4 levels have been strongly associated with cardiometabolic diseases in both preclinical models and humans4,5,6,7, no mechanism of action has yet been described8,9,10. Here we show that hormonal FABP4 forms a functional hormone complex with adenosine kinase (ADK) and nucleoside diphosphate kinase (NDPK) to regulate extracellular ATP and ADP levels. We identify a substantial effect of this hormone on beta cells and given the central role of beta-cell function in both the control of lipolysis and development of diabetes, postulate that hormonal FABP4 is a key regulator of an adipose–beta-cell endocrine axis. Antibody-mediated targeting of this hormone complex improves metabolic outcomes, enhances beta-cell function and preserves beta-cell integrity to prevent both type 1 and type 2 diabetes. Thus, the FABP4–ADK–NDPK complex, Fabkin, represents a previously unknown hormone and mechanism of action that integrates energy status with the function of metabolic organs, and represents a promising target against metabolic disease.

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Fig. 1: Targeting FABP4 increases beta-cell mass and function.
Fig. 2: Circulating FABP4 forms a hormonal complex with NDPK and ADK to regulate extracellular nucleosides.
Fig. 3: Fabkin inhibits GSIS and is neutralized by a-Ab.
Fig. 4: Fabkin alters beta-cell calcium dynamics and promotes cell death.

Data availability

The datasets generated during the current study are available in the FigShare repository at https://doi.org/10.6084/m9.figshare.16547853Source data are provided with this paper.

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Acknowledgements

We thank members of the Hotamisligil Laboratory and the Sabri Ülker Center, past and present, for their contributions to our understanding of FABP4 and their helpful discussions. We thank staff at the Islet Core and Clinical Islet Laboratory (Alberta Islet Distribution Program, University of Alberta) for providing us with human islets from review-board-approved donors. UCB generated and purified the antibodies used in this study and independently reproduced the interaction data between NDPK, FABP4 and a-Ab. The Hotamisligil laboratory is supported by grants from the National Institutes of Health (NIH DK123458) and the Juvenile Diabetes Research Foundation (JDRF; 2-SRA-2019-660-S-B). A JDRF Postdoctoral Fellowship (3-PDF-2017-400-A-N) funds K.J.P. The Sigrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research and the Otto A. Malm Foundation support J.S. F.E. was supported by grants from the JDRF (JDRF-5-CDA-2014-184-A-N) and the NIH (NIH 5K01DK102488-03).

Author information

Authors and Affiliations

Authors

Contributions

K.J.P. designed and performed the in vitro and in vivo experiments, analysed the data, prepared the figures and wrote and revised the manuscript. F.E., K.E.I., A.L., P.C. and L.T.R. designed and performed the in vivo experiments, analysed data and revised the manuscript. J.S., G.Y.L., K.E., E.O. and E.S.C. designed and performed the in vitro experiments, analysed data and revised the manuscript. O.C. analysed the crystal structure and generated recombinant proteins used in this study, analysed the data and revised the manuscript. P.A. and A.-G.Z. collected and analysed human serum samples and revised the manuscript. G.S.H. conceived, supervised and supported the project, designed experiments, interpreted results and revised the manuscript.

Corresponding author

Correspondence to Gökhan S. Hotamisligil.

Ethics declarations

Competing interests

The Hotamisligil Lab has generated intellectual property (assigned to Harvard University) related to hormonal FABP4 and its therapeutic targeting and receives funding for this project from Lab1636, LLC, an affiliate of Deerfield Management. G.S.H. is on the Scientific Advisory Board of Crescenta Pharmaceuticals and holds equity. Other authors have no conflicts of interest to declare.

Additional information

Peer review information Nature thanks Matthias Hebrok, Andrew Stewart, Thomas Wieland and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 FABP4-/- mice have no difference in alpha cell mass, and FABP4 is not expressed in beta cells.

(a) Dithizone-stained islets in vivo from FABP4−/− mice (Representative image from N = 3). (b) Immunohistochemical staining for glucagon of pancreatic sections from 7-week-old WT or FABP4−/− mice, and (c) quantification of percentage of glucagon positive area per total pancreatic area based on IHC (N = 5/group). (d) Immunofluorescent staining for insulin (green), FABP4 (red) and nuclei (DAPI, blue) in primary isolated mouse islets (N = 15 islets). (e) Western blot for FABP4 and B-Tubulin (B-Tubb) from INS1 cells with and without treatment with FABP4 (N = 3). Baseline matching parameters for 10 week old WT female NOD mice prior to the initiation of dosing showing no difference in (f) body weight, (g) blood glucose, (h) plasma insulin, or (i) plasma FABP4 (N = 46/group). (j) 6 h fasting blood glucose, (k) body weight, and (l) plasma insulin levels among NOD mice that remained non-diabetic for the duration of the treatment period (N = 11 (PBS), 23 (a-Ab), 13 (c-Ab)). (m) Immunofluorescent staining for insulin (green) and glucagon (red) in pancreatic sections from NOD mice treated with a-Ab or c-Ab for 5 wks (N = 4 mice/group). Scale bars are 100um. Data are mean +/− SEM. Two-tailed unpaired t-test (c); One-way ANOVA (f-i); Two-way ANOVA (j-l).

Source data

Extended Data Fig. 2 FABP4 exerts differential activity in vivo and in vitro, and only interacts with a-Ab through the light chain.

(a) GSIS from islets from FABP4−/− mice treated with increasing doses of FABP4 (N = 3). (b) Plasma FABP4 levels following acute injection of 10 µg FABP4 (N = 3 (PBS), 6 (FABP4)). (c) Blood glucose (P = 0.0300) and (d) plasma insulin levels 20 min post-injection (P = 0.0160) with FABP4 or PBS (N = 3 (PBS), 6 (FABP4)).(e) Crystal structure showing FABP4 binding through the light chain of a-Ab. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean +/− SEM. Two-way ANOVA (a,b); Welch’s t-test (c,d).

Source data

Extended Data Fig. 3 Determination of the Binding Epitope for a-Ab on NDPK-A.

(a) Amino acid sequence for human NDPK-A, and indication of the location of each peptide generated for epitope mapping (red). Each peptide is 15 amino acids long with an N-terminal 6xHis Tag for peptide labeling. Each peptide has a 5 amino acid overlap with the preceeding peptide sequentially from the N-terminus of the protein.(b) Table summarizing binding affinity as determined by MicroScale Thermophoresis (MST). Low affinity binding was observed between peptide 2 and peptide 3 with a-Ab. High affinity binding was observed between peptide 8 and a-Ab, comparable to full-length NDPK-A protein. No binding was detected (NBD) between a-Ab and any other peptide examined. The lack of binding between peptide 7 or peptide 9 and a-Ab indicated that the central 5 amino acids of peptide 8, non-overlapping in sequence with either peptide 7 or 9, is likely the primary epitope for a-A binding. (c) Example MST binding curves for full-length NDPK-A, NDPK-A peptide 9 and NDPK-A peptide 8 with a-Ab. No binding is observed between NDPK-A peptide 9 and a-Ab. (d) Crystal structure of human NDPK-A in hexamer conformation (source PDB: 3L7U) with the potential binding region of peptide 8 highlighted in pink and peptide 3 highlighted in blue. The protein structure indicates the potential binding region of peptide 8 is on the surface of the protein, amenable for a-Ab interaction. The tertiary folding of NDPK-A places peptide 3 and peptide 8 in close proximity, suggesting binding of a-Ab may be primarily occurring through residues in peptide 8, and partially facilitated through interaction with residues in peptides 2 and 3. EC50 calculated by Hill Slope.

Extended Data Fig. 4 a-Ab interacts with Fabkin.

(a–e) Representative mesoscale thermophoresis experiments showing 1:1 protein interactions between complex components and a-Ab (N = 6/interaction). EC50 was calculated using Hill Slope. (f) Representtive Western blot and (g) quantification showing relative abundance of complex components interacting with GST-NDPK-A (N = 4 replicates; P < 0.0001). Kinase activity of recombinant ADK to generate (h) ATP and (i) ADP in the presence of complex components (N = 3/group). Kinase activity of recombinant NDPK to generate (j) ADP and (k) ADP in the presence of complex components (N = 3/group). Activity of recombinant (l) ADK and (m) NDPK in the presence of a-Ab or c-Ab alone (N = 3/group). *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean +/− SEM. Two-way ANOVA (g-m).

Source data

Extended Data Fig. 5 Complex components alone do not alter GSIS.

(a) GSIS from WT mouse islets treated with each of the proposed complex components alone (N = 4/condition). (b) GSIS from INS1 cells treated with NDPK-ADK with FABP4, lipid binding mutant (LBM) FABP4, or FABP4 pre-treated with inhibitor BMS-309403 (N = 4/group). *P < 0.05, ***P < 0.001. Data are mean +/− SEM. One-way ANOVA (a,b).

Source data

Extended Data Fig. 6 Treatment with a-Ab preserves beta cell mass and islet number with minimal alterations to the pancreas immune profile.

Cytosolic calcium flux in INS1 cells from (a,b) the extracellular space (N = 2-3 coverslips/treatment; 88 (Control), 100 (NDPK-ADK-FABP4), 47 (MRS2365) cells) and (c,d) the ER in response to thapsigargin as determined by Fura-2 AM staining in control conditions or pretreatment with FABP4-ADK-NDPK with or without MRS2365 (N = 2-3 coverslips/treatment; 50 cells).(e) Cytosolic calcium flux from the ER in INS1 cells under control conditions, or pretreatment with FABP4-ADK-NDPK with or without the adenylyl cyclase inhibitor NKY80 (N = 2-3 coverslips/treatment; 100 (Control, NDPK-ADK-FABP4), 150 (NKY80) cells). (f) Quantification of Western blots from Fig. 4g (N = 4/condition). (g) Gene expression of ER stress markers BIP following 2hr treatment with NDPK-ADK, FABP4-ADK-NDPK, or FABP4-ADK-NDPK with a-Ab in the presence or absence of Tg (N = 3). (h) Cleaved caspase 3/7 activity in INS1 cells treated with increasing concentrations of Tg with or without FABP4-ADK-NDPK (N = 4). (i) Percentage of CD45+ cells (N = 12), (j) regulatory T-cells (N = 6), (k) cytotoxic T-cells (N = 6), (l) T-Helper cells (N = 5), (m) B-cells (N = 6), (n) dendritic cells (N = 6), and (o) granulocytes (N = 6) in whole pancreas from mice treated with PBS, a-Ab or c-Ab for 14 weeks by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean +/− SEM. One-way ANOVA (b,d,i–o); Two-tailed unpaired t-test (f); Two-way ANOVA (g,h).

Source data

Extended Data Table 1 Comparative data on T1D and control groups (BABYDIAB and DIMELLI cohorts)
Extended Data Table 2 Comparative data on T1D and control group (BRI cohort)
Extended Data Table 3 Spearman correlations of circulating FABP4 in the BRI cohort
Extended Data Table 4 Nucleoside abundance in INS1 cell supernatant with high glucose
Extended Data Table 5 Immunephenotyping of peripheral tissues in non-diabetic NOD mice
Extended Data Table 6 List of antibodies used in the study

Supplementary information

Supplementary Figure 1

This file contains the uncropped blots.

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Prentice, K.J., Saksi, J., Robertson, L.T. et al. A hormone complex of FABP4 and nucleoside kinases regulates islet function. Nature 600, 720–726 (2021). https://doi.org/10.1038/s41586-021-04137-3

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