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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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.
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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.
The authors declare no competing interests.
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Integrated supplementary information
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.
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.
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.
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.
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.
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.
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.
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.
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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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