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Toll-like receptors TLR2 and TLR4 block the replication of pancreatic β cells in diet-induced obesity

Nature Immunology volume 20, pages 677–686 (2019)Cite this article

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Abstract

Consumption of a high-energy Western diet triggers mild adaptive β cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of β cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of β cells, but not that of α cells, leading to enlarged β cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of β cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse β cell failure in patients with diabetes.

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Fig. 1: The loss of TLR2 and TLR4 leads to β cell accumulation and hyperinsulinemia in mice on an HFD.
Fig. 2: TLR2 and TLR4 deficiency leads to increased β cell proliferation in an HFD-dependent manner.
Fig. 3: TLR2 and TLR4 deficiency triggers nuclear entry of Ccnd2 in an HFD-dependent manner.
Fig. 4: Redundant effect of TLR2 and TLR4 on β cell proliferation.
Fig. 5: The effect of TLR2 and TLR4 on HFD-induced β cell proliferation is independent of circulating factors.
Fig. 6: The effect of TLR2 and TLR4 on HFD-induced β cell proliferation is islet intrinsic.
Fig. 7: TLR2 and TLR4 inhibit β cell proliferation via the Mek–Erk pathway.

Data availability

Microarray data supporting the findings of this study have been deposited into the Gene Expression Omnibus with accession number GSE101392. All other relevant data are available from the corresponding author upon request.

References

  1. Dor, Y., Brown, J., Martinez, O. I. and Melton, D. A. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429, 41–46 (2004).

    Article  CAS  Google Scholar 

  2. Xu, X. et al. Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas. Cell 132, 197–207 (2008).

    Article  CAS  Google Scholar 

  3. Garofano, A., Czernichow, P. and Breant, B. Impaired beta-cell regeneration in perinatally malnourished rats: a study with STZ. FASEB J. 14, 2611–2617 (2000).

    Article  CAS  Google Scholar 

  4. Teta, M., Long, S. Y., Wartschow, L. M., Rankin, M. M. and Kushner, J. A. Very slow turnover of beta-cells in aged adult mice. Diabetes 54, 2557–2567 (2005).

    Article  CAS  Google Scholar 

  5. Meier, J. J. et al. Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans. Diabetes 57, 1584–1594 (2008).

    Article  CAS  Google Scholar 

  6. Nir, T., Melton, D. A. and Dor, Y. Recovery from diabetes in mice by beta cell regeneration. J. Clin. Invest. 117, 2553–2561 (2007).

    Article  CAS  Google Scholar 

  7. Georgia, S. and Bhushan, A. Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass. J. Clin. Invest. 114, 963–968 (2004).

    Article  CAS  Google Scholar 

  8. Okada, T. et al. Insulin receptors in beta-cells are critical for islet compensatory growth response to insulin resistance. Proc. Natl Acad. Sci. USA 104, 8977–8982 (2007).

    Article  CAS  Google Scholar 

  9. Golson, M. L., Misfeldt, A. A., Kopsombut, U. G., Petersen, C. P. and Gannon, M. High fat diet regulation of beta-cell proliferation and beta-cell mass. Open Endocrinol. J. 4, 66–77 (2010).

    Article  CAS  Google Scholar 

  10. Cox, A. R. et al. Extreme obesity induces massive beta cell expansion in mice through self-renewal and does not alter the beta cell lineage. Diabetologia 59, 1231–1241 (2016).

    Article  CAS  Google Scholar 

  11. Stamateris, R. E., Sharma, R. B., Hollern, D. A. and Alonso, L. C. Adaptive beta-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression. Am. J. Physiol. Endocrinol. Metab. 305, E149–E159 (2013).

    Article  CAS  Google Scholar 

  12. Sachdeva, M. M. and Stoffers, D. A. Minireview: Meeting the demand for insulin: molecular mechanisms of adaptive postnatal beta-cell mass expansion. Mol. Endocrinol. 23, 747–758 (2009).

    Article  CAS  Google Scholar 

  13. Stewart, A. F. et al. Human beta-cell proliferation and intracellular signaling: part 3. Diabetes 64, 1872–1885 (2015).

    Article  CAS  Google Scholar 

  14. Georgia, S. et al. Cyclin D2 is essential for the compensatory beta-cell hyperplastic response to insulin resistance in rodents. Diabetes 59, 987–996 (2010).

    Article  CAS  Google Scholar 

  15. Kushner, J. A. et al. Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth. Mol. Cell Biol. 25, 3752–3762 (2005).

    Article  CAS  Google Scholar 

  16. Alonso, L. C. et al. Glucose infusion in mice: a new model to induce beta-cell replication. Diabetes 56, 1792–1801 (2007).

    Article  CAS  Google Scholar 

  17. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M. and Hoffmann, J. A. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973–983 (1996).

    Article  CAS  Google Scholar 

  18. Medzhitov, R., Preston-Hurlburt, P. and Janeway, C. A. Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394–397 (1997).

    Article  CAS  Google Scholar 

  19. Poltorak, A. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088 (1998).

    Article  CAS  Google Scholar 

  20. Konner, A. C. and Bruning, J. C. Toll-like receptors: linking inflammation to metabolism. Trends Endocrinol. Metab. 22, 16–23 (2011).

    Article  Google Scholar 

  21. Shi, H. et al. TLR4 links innate immunity and fatty acid-induced insulin resistance. J. Clin. Invest. 116, 3015–3025 (2006).

    Article  CAS  Google Scholar 

  22. Donath, M. Y. Targeting inflammation in the treatment of type 2 diabetes: time to start. Nat. Rev. Drug Discov. 13, 465–476 (2014).

    Article  CAS  Google Scholar 

  23. Sun, S., Ji, Y., Kersten, S. and Qi, L. Mechanisms of inflammatory responses in obese adipose tissue. Annu. Rev. Nutr. 32, 261–286 (2012).

    Article  CAS  Google Scholar 

  24. Himes, R. W. and Smith, C. W. Tlr2 is critical for diet-induced metabolic syndrome in a murine model. FASEB J. 24, 731–739 (2010).

    Article  CAS  Google Scholar 

  25. Razolli, D. S. et al. TLR4 expression in bone marrow-derived cells is both necessary and sufficient to produce the insulin resistance phenotype in diet-induced obesity. Endocrinology 156, 103–113 (2015).

    Article  Google Scholar 

  26. Vila, I. K. et al. Immune cell Toll-like receptor 4 mediates the development of obesity- and endotoxemia-associated adipose tissue fibrosis. Cell Rep. 7, 1116–1129 (2014).

    Article  CAS  Google Scholar 

  27. Jia, L. et al. Hepatocyte Toll-like receptor 4 regulates obesity-induced inflammation and insulin resistance. Nat. Commun. 5, 3878 (2014).

    Article  CAS  Google Scholar 

  28. Lee, C. C., Avalos, A. M. and Ploegh, H. L. Accessory molecules for Toll-like receptors and their function. Nat. Rev. Immunol. 12, 168–179 (2012).

    Article  CAS  Google Scholar 

  29. Vikram, A. and Jena, G. S961, an insulin receptor antagonist causes hyperinsulinemia, insulin-resistance and depletion of energy stores in rats. Biochem. Biophys. Res. Commun. 398, 260–265 (2010).

    Article  CAS  Google Scholar 

  30. Ji, Y. et al. Diet-induced alterations in gut microflora contribute to lethal pulmonary damage in TLR2/TLR4-deficient mice. Cell Rep. 8, 137–149 (2014).

    Article  CAS  Google Scholar 

  31. Michael, M. D. et al. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol. Cell 6, 87–97 (2000).

    Article  CAS  Google Scholar 

  32. Rane, S. G. et al. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia. Nat. Genet. 22, 44–52 (1999).

    Article  CAS  Google Scholar 

  33. Dirice, E. et al. Soluble factors secreted by T cells promote beta-cell proliferation. Diabetes 63, 188–202 (2014).

    Article  CAS  Google Scholar 

  34. Xiao, X. et al. M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7. Proc. Natl Acad. Sci. USA 111, E1211–E1220 (2014).

    Article  CAS  Google Scholar 

  35. Jourdan, T. et al. Activation of the Nlrp3 inflammasome in infiltrating macrophages by endocannabinoids mediates beta cell loss in type 2 diabetes. Nat. Med. 19, 1132–1140 (2013).

    Article  CAS  Google Scholar 

  36. El Ouaamari, A. et al. SerpinB1 promotes pancreatic beta cell proliferation. Cell Metab. 23, 194–205 (2016).

    Article  CAS  Google Scholar 

  37. Brissova, M. et al. Islet microenvironment, modulated by vascular endothelial growth factor-A signaling, promotes beta cell regeneration. Cell Metab. 19, 498–511 (2014).

    Article  CAS  Google Scholar 

  38. Terazono, K. et al. A novel gene activated in regenerating islets. J. Biol. Chem. 263, 2111–2114 (1988).

    CAS  PubMed  Google Scholar 

  39. Li, Q. et al. Reg2 expression is required for pancreatic islet compensation in response to aging and high fat diet-induced obesity. Endocrinology 158, 1634–1644 (2017).

    Article  Google Scholar 

  40. Chen, H. et al. PDGF signalling controls age-dependent proliferation in pancreatic beta-cells. Nature 478, 349–355 (2011).

    Article  CAS  Google Scholar 

  41. Carboneau, B. A. et al. Opposing effects of prostaglandin E2 receptors EP3 and EP4 on mouse and human beta-cell survival and proliferation. Mol. Metab. 6, 548–559 (2017).

    Article  CAS  Google Scholar 

  42. Wang, P. et al. Diabetes mellitus – advances and challenges in human beta-cell proliferation. Nat. Rev. Endocrinol. 11, 201–212 (2015).

    Article  CAS  Google Scholar 

  43. Crabtree, J. S. et al. Of mice and MEN1: insulinomas in a conditional mouse knockout. Mol. Cell. Biol. 23, 6075–6085 (2003).

    Article  CAS  Google Scholar 

  44. Imai, J. et al. Regulation of pancreatic beta cell mass by neuronal signals from the liver. Science 322, 1250–1254 (2008).

    Article  CAS  Google Scholar 

  45. Darveau, R. P. et al. Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both toll-like receptors 2 and 4. Infect. Immun. 72, 5041–5051 (2004).

    Article  CAS  Google Scholar 

  46. Ji, Y. et al. Activation of natural killer T cells promotes M2 macrophage polarization in adipose tissue and improves systemic glucose tolerance via interleukin-4 (IL-4)/STAT6 protein signaling axis in obesity. J. Biol. Chem. 287, 13561–13571 (2012).

    Article  CAS  Google Scholar 

  47. Szot, G. L., Koudria, P. and Bluestone, J. A. Transplantation of pancreatic islets into the kidney capsule of diabetic mice. J. Vis. Exp. 9, 404 (2007).

  48. Zmuda, E. J., Powell, C. A. and Hai, T. A method for murine islet isolation and subcapsular kidney transplantation. J. Vis. Exp., 2096 (2011).

  49. Kamran, P. et al. Parabiosis in mice: a detailed protocol. J. Vis. Exp., 50556 (2013).

  50. Ricordi, C. et al. National institutes of health-sponsored clinical islet transplantation consortium phase 3 trial: manufacture of a complex cellular product at eight processing facilities. Diabetes 65, 3418–3428 (2016).

    Article  CAS  Google Scholar 

  51. Adewola, A. F. et al. Microfluidic perifusion and imaging device for multi-parametric islet function assessment. Biomed. Microdevices 12, 409–417 (2010).

    Article  Google Scholar 

  52. Xing, Y. et al. A pumpless microfluidic device driven by surface tension for pancreatic islet analysis. Biomed. Microdevices 18, 80 (2016).

    Article  Google Scholar 

  53. Ji, Y. et al. Short term high fat diet challenge promotes alternative macrophage polarization in adipose tissue via natural killer T cells and interleukin-4. J. Biol. Chem. 287, 24378–24386 (2012).

    Article  CAS  Google Scholar 

  54. Sun, S. et al. IRE1a is an endogenous substrate of endoplasmic-reticulum-associated degradation. Nat. Cell Biol. 17, 1546–1555 (2015).

    Article  CAS  Google Scholar 

  55. Sha, H. et al. The ER-associated degradation adaptor protein Sel1L regulates LPL secretion and lipid metabolism. Cell Metab. 20, 458–470 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L. Hajjar and D. Russell for sharing reagents, Histology and Vision Research Core Facilities at the University of Michigan Medical School and the TEM core facility at the Weill Cornell Medical College for their assistance, and P. Arvan and R. B. Reinert, G. H. Kim and X. Shu, as well as other members of the Qi/Arvan labs and islet club at University of Michigan Diabetes Center, for comments and technical assistance. The Histology Core at the University of Michigan Medical School was supported by the National Cancer Institute of the NIH (award no. P30CA046592). This work was supported by NIH (grant nos. R01DK108921, R03 DK106304), and Juvenile Diabetes Research Foundation (JDRF; grant no. CDA-2016–189; to S.A.S.), VA Merit award 1 (no. I01 BX003744-01 to M.G.), NIH (grant no. R01DK091526), JDRF Microfluidic-Based Functional Facility at the University of Illinois at Chicago, and the Chicago Diabetes Project (to J.O. and Y.W.), NIH (grant no. U01 DK070430) and NIDDK/City of Hope Integrated Islet Distribution Program (to A.N.), NIH (grant nos. R01DK67536, R01DK117639 and R01DK103215 to R.N.K.), and JDRF (grant nos. 47-2012-767 and 1-SRA-2014-251-Q-R), NIH (grant nos. R01DK11174 and R01DK117639; to L.Q.). Y.J. is supported by American Heart Association Scientist Development (grant no. 17SDG33670192) and Michigan Nutrition Obesity Research Center Pilot/Feasibility grant (no. P30DK089503). S.S. was an international student research fellow of the Howard Hughes Medical Institute (no. 59107338) and a Helen Hay Whitney Postdoctoral Fellow. L.Q. is the recipient of the Junior Faculty and Career Development Awards from the American Diabetes Association.

Author information

Author notes

  1. Shengyi Sun

    Present address: Center for Molecular Medicine and Genetics, Department of Microbiology, Immunology and Biochemistry, Wayne State University School of Medicine, Detroit, MI, USA

  2. Laurel B. Darragh

    Present address: Department of Radiation Oncology, School of Medicine, University of Colorado, Aurora, CO, USA

  3. Hana Kim

    Present address: XBiotech USA, Inc., Austin, TX, USA

  4. These authors contributed equally: Y. Ji, S. Sun.

Authors and Affiliations

  1. Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, USA

    Yewei Ji, Neha Shrestha & Ling Qi

  2. Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA

    Yewei Ji, Shengyi Sun, Laurel B. Darragh, Hana Kim, Sander Kersten & Ling Qi

  3. Section of Islet Cell and Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Brigham and Women’s Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA

    Jun Shirakawa & Rohit N. Kulkarni

  4. Department of Surgery, University of Virginia, Charlottesville, VA, USA

    Yuan Xing, Yi He, Jose Oberholzer & Yong Wang

  5. Division of Diabetes, Endocrinology and Metabolism, Vanderbilt University Medical Center, Nashville, TN, USA

    Bethany A. Carboneau & Maureen Gannon

  6. Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY, USA

    Duo An & Minglin Ma

  7. Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA

    Scott A. Soleimanpour & Ling Qi

  8. Department of Surgery, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Chengyang Liu & Ali Naji

  9. Nutrition Metabolism and Genomics group, Wageningen University, Wageningen, the Netherlands

    Sander Kersten

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Contributions

Y.J. and S.S. designed and performed most of the experiments. N.S., L.B.D., H.K., D.A. and M.M. assisted in some of the experiments. J.S., R.N.K., B.A.C. and M.G. measured TLR expression in human and aged mouse islets. S.A.S. provided helpful discussions and suggestions. C.L. and A.N. provided human islets. Y.X., Y.H., Y.W. and J.O. performed functional analysis of the mouse islets. S.K. performed microarray analyses. Y.J. and S.S. wrote the methods and legends. L.Q. designed the experiments, managed the project and wrote the manuscript. All authors edited and approved the manuscript.

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Correspondence to Ling Qi.

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Integrated supplementary information

Supplementary Figure 1

HFD triggers moderate β-cell proliferation and TLR2/TLR4 activation blocks human β-cell proliferation. a, Representative confocal images of Ki67+Ins+ cells in pancreas sections from 4- or 45-week-old B6 mice on either LFD or HFD for 39 weeks, with or without S96λn the last two weeks. Scale bars, 50 µm. n = 3 mice each. b–d, Analysis of TLR effect on human β cell proliferation: human islets were cultured with BrdU-containing medium with 2.8 (low) or 22.8 mM (high) glucose and treated with TLR2/TLR4 agonists in vitro. b, Representative microscopic images of primary human islets of Donor 1. Scale bars, 400 µm (upper) or 200 µm (lower). c, Flow cytometry plot showing percent of BrdU+Ins+ β cells in islets from Donor 1 cultured under low or high glucose with or without LPS and LTA. A total of ~ 5,000 live cells were analyzed for each sample. Quantitation of data from three batches of human islets is shown in d.

Supplementary Figure 2

Accumulation of β-cell, not α cell, in Tlr2−/−Tlr4−/− mice following HFD. a, Body weights of B6 and Tlr2−/−Tlr4−/− mice on HFD for 29 weeks. n = 5, 7 mice (B6, Tlr2−/−Tlr4−/−). b–d, Representative images showing H&E (b), Ins+ β cells (c) and Glucagon+ α cells (d) in pancreatic sections from B6 and Tlr2−/−Tlr4−/− mice on HFD for 14 or 29 weeks. Scale bars, 1 mm and 0.5 mm (d, the insets). n = 3 or 4 mice each genotype and each time point. (e) Quantitation showing number of Glucagon+ α cells per islet from B6 and Tlr2−/−Tlr4−/− mice on LFD or HFD for 14 or 29 weeks. n = 4 mice each group with n = 30, 15, and 31 islets (B6 LFD, 14-week HFD and 29-week HFD) and n = 21, 15, 31 (Tlr2−/−Tlr4−/− LFD, 14-week HFD and 29-week HFD). Values represent mean ± SEM. n.s., not significant, p > 0.05 by two-tailed Student’s t test.

Supplementary Figure 3

Massive β cell accumulation in Tlr2−/−Tlr4−/− mice is HFD-dependent. a, Photos showing pancreas from B6 and Tlr2−/−Tlr4−/− mice on 51-week HFD, highlighting visually visible islets in Tlr2−/−Tlr4−/− mice (arrows). n = 3 mice each. b, Representative images of Ins+ β cells (upper) and Glucagon+ α cells (lower) in pancreatic sections of 20-week-old B6 and Tlr2−/−Tlr4−/− mice on LFD. Scale bars, 0.3 mm. n = 6, 5 mice (B6 and Tlr2−/−Tlr4−/−). c, Quantitation of Ins+ β cell areas normalized to total pancreas area of mice on LFD at the indicated ages. Data from HFD cohorts are included for comparison. From left to right, n = 6, 5, 4, 4, 5, 6 mice. Values represent mean ± SEM. n.s., not significant, p > 0.05 and ***, p < 0.001, using a one-way ANOVA with Newman-Keuls post-test. d, Representative images of H&E and Ins+ β cells in pancreatic sections of 26-weeks-old Tlr2+/+Tlr4+/+ and Tlr2−/−Tlr4−/− littermates on LFD. Scale bars, 2 mm (left) and 0.5 mm (right). n = 3 or 4 mice each.

Supplementary Figure 4

Tlr2/Tlr4 deficiency triggers β cell proliferation in a HFD-dependent manner. a, b, Representative confocal images showing Ki67+Ins+ (a) and Pdx1+Ins+ (b) cells in the pancreas of B6 and Tlr2−/−Tlr4−/− mice on 39-week (w) HFD. Scale bars, 100 µm (a) and 50 µm (b). c, Representative confocal images showing Ki67+Ins+ in the pancreas of 26-weeks-old Tlr2+/+Tlr4+/+ and Tlr2−/−Tlr4−/− littermates on LFD. Scale bars, 50 µm. d, Representative confocal images showing Ki67+ Glucagon+ cells in pancreatic sections from mice on 14-week HFD. Scale bars, 100 µm (upper) and 10 µm (lower). e, f, flow cytometric analysis showing the levels of β cell senescence, p16INK4a (e) and senescence-associated β-galactosidase (f), in Ins+ β cells from B6 and Tlr2−/−Tlr4−/− mice on 39-week HFD. g, Representative confocal images of Ki67+Ins+ β cells in the pancreas of Tlr2−/−Tlr4−/− mice on 32-week HFD followed by 4-week LFD or HFD. Scale bars, 100 µm. Representative data from 3 or 4 mice each with at least 2 repeats.

Supplementary Figure 5

Tlr2/Tlr4 deficiency triggers nuclear entry of Ccnd2 in a HFD-dependent manner. a-b, Representative confocal images of Ccnd2+Glucagon+ α cells of the pancreas from (a) Tlr2+/+Tlr4+/+ and Tlr2−/−Tlr4−/− littermates on 20-week HFD and (b) B6 and Tlr2−/−Tlr4−/− mice on 14-week HFD. Scale bars, 50 µm (upper) and 10 µm (lower). c, Representative confocal images of Ccnd2 localization in β cells of B6 and Tlr2−/−Tlr4−/− mice on LFD for 35 weeks. Scale bars, 50 µm (left) and 10 µm (right). d, Representative confocal images showing Cdk4+ Glucagon+ cells in islets of B6 and Tlr2−/−Tlr4−/− mice on 14-week HFD. Scale bars, 100 µm (left) and 10 µm (right). Representative data from 3 or 4 mice each with 2 repeats.

Supplementary Figure 6

The effect of Tlr2/Tlr4 on islet expansion is islet-intrinsic. a, qPCR analysis showing the expression of Tlr2 and Tlr4 in islets from mice at the indicated ages on LFD. Data are normalized to Hprt (housekeeping gene) and then to that of 2-month (m) timepoint. n = 3, 4, 5, 4 (2, 4, 8 and 12 m). Values represent mean ± SEM. **, p < 0.01; ***, p < 0.001 by one-way ANOVA with Newman-Keuls post test. b-d, Islet transplantation under kidney capsules: (b) Schematic diagram of islet transplantation assay, in which 100 B6 or Tlr2−/−Tlr4−/− primary islets were transplanted under the left and right kidney capsules, respectively, of streptozotocin (STZ)-induced diabetic recipient, either B6 or Tlr2−/−Tlr4−/− mice. The islets from B6 and Tlr2−/−Tlr4−/− mice were transplanted into the same recipient to exclude potential effect from the host. c, Representative photos showing islet grafts under the kidney capsules of B6 (left) and Tlr2−/−Tlr4−/− (right) recipients. n = 4 mice each. d, Representative confocal images showing CD31+ endothelial cells, that is vascularization, in B6 and Tlr2−/−Tlr4−/− islet grafts. The dotted line marks the boundary of the islets (note high auto-fluorescent signals of the surrounding kidney tubules); for Tlr2−/−Tlr4−/− islets, the whole field is full of β cells. Scale bars, 100 µm. n = 4 mice each.

Supplementary Figure 7

Microarray analysis of primary islets reveals a unique signature of a highly proliferative β cell population. a, GSEA analyses of the pathways affected by Tlr2-Tlr4 deficiency in primary islets of mice on 7-month HFD, with top up-regulated pathways shown. b,d, Heat map showing top upregulated genes (greater than 2.0 fold, p < 0.01) in Tlr2−/−Tlr4−/− compared to B6 islets from mice on a HFD for 7 months (b) or 5 weeks (d). n = =4, 4 mice (7-month), n = 2, 4 (5 weeks). p value, by IBMT regularized t test. c, Pie chart analysis of microarray data showing 45 and 101 up-regulated genes in Tlr2−/−Tlr4−/− vs. B6 islets from 5-week and 7-month-HFD mice, respectively.

Supplementary Figure 8

TLR2 and TLR4 agonists reduce nuclear Ccnd2 and Cdk4 level in β cells. a-b, Representative confocal images of Ccnd2+Ins+ (a) and Cdk4+Ins+ (b) in primary islet cells pooled from five 4-month-old B6 mice on a LFD, cultured in 2.8 or 22.8 mM glucose with or without LPS and LTA for 72 h. Merged images are shown in Fig. 7g. Scale bars, 10 µm. c, Immunoblots showing Erk phosphorylation in B6 islets cultured in 2.8 or 22.8 mM glucose with or without LPS and/or LTA for 48 h. Quantitation is shown below the blot. Each lane, pooled islets from 5 mice. d, Immunoblot showing Erk phosphorylation in B6 macrophages cultured in 2.8 or 22.8 mM glucose with LPS+LTA for 24 h with quantitation shown below the blot. e, Immunoblots showing Erk phosphorylation in Ins-1 β cells treated with a MEK inhibitor MEK162 at the indicated concentrations for 1 h. a–e, Representative data from 2 repeats. c–e, The gel was cropped to show relevant bands. f, Quantitation of the percent of ki67+Ins+ β cells from Tlr2−/−Tlr4−/− mice on 8-week HFD treated with MEK162 for the last 2 days, with original representative data shown in Fig. 7h. Each symbol represents an islet. n = 14, 9 (vehicle, MEK162). Values represent mean ± SEM. ***, p < 0.001 by two-tailed Student’s t test.

Supplementary Figure 9

Uncropped gels and blots.

Supplementary Figure 10

Uncropped gels and blots.

Supplementary Figure 11

Uncropped gels and blots.

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Ji, Y., Sun, S., Shrestha, N. et al. Toll-like receptors TLR2 and TLR4 block the replication of pancreatic β cells in diet-induced obesity. Nat Immunol 20, 677–686 (2019). https://doi.org/10.1038/s41590-019-0396-z

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