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β-Klotho promotes glycolysis and glucose-stimulated insulin secretion via GP130

Nature Metabolism volume 4, pages 608–626 (2022)Cite this article

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Abstract

Impaired glucose-stimulated insulin secretion (GSIS) is a hallmark of type-2 diabetes. However, cellular signaling machineries that control GSIS remain incompletely understood. Here, we report that β-klotho (KLB), a single-pass transmembrane protein known as a co-receptor for fibroblast growth factor 21 (FGF21), fine tunes GSIS via modulation of glycolysis in pancreatic β-cells independent of the actions of FGF21. β-cell-specific deletion of Klb but not Fgf21 deletion causes defective GSIS and glucose intolerance in mice and defective GSIS in islets of type-2 diabetic mice is mitigated by adenovirus-mediated restoration of KLB. Mechanistically, KLB interacts with and stabilizes the cytokine receptor subunit GP130 by blockage of ubiquitin-dependent lysosomal degradation, thereby facilitating interleukin-6-evoked STAT3–HIF1α signaling, which in turn transactivates a cluster of glycolytic genes for adenosine triphosphate production and GSIS. The defective glycolysis and GSIS in Klb-deficient islets are rescued by adenovirus-mediated replenishment of STAT3 or HIF1α. Thus, KLB functions as a key cell-surface regulator of GSIS by coupling the GP130 receptor signaling to glucose catabolism in β-cells and represents a promising therapeutic target for diabetes.

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Fig. 1: Depletion of KLB in pancreatic β-cells impairs glucose tolerance and GSIS in mice.
Fig. 2: Glycolytic flux is impaired in islets of KlbRIP mice.
Fig. 3: Klb deficiency downregulates the expression of glycolytic genes in pancreatic β cells.
Fig. 4: Restoration of HIF1α in KlbRIP islets reverses impaired GSIS and glycolysis.
Fig. 5: Knockdown of KLB expression impairs GSIS and glycolysis in cultured human islets.
Fig. 6: KLB preferentially signals through STAT3 to promote pancreatic HIF1α expression and GSIS.
Fig. 7: KLB mediates IL-6-induced STAT3–HIF1α signaling and GSIS.
Fig. 8: KLB interacts with GP130 and inhibits its ubiquitin-dependent lysosomal degradation in β-cells.

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Data availability

The data supporting the findings of this study are available in source data files. Source data are provided with this paper.

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Acknowledgements

This work was supported by the General Research Fund (17121819, A.M.X.) and Area of Excellence (AOE/M/707-18, A.M.X.) from the Research Grant Council of Hong Kong, the National Natural Science Foundation of China (82070860, A.M.X. and 32000816, L.L.G.) and Hong Kong Health and Medical Research Fund (06172956, Q.Z.L.). Y.T.Z. was supported by the Natural Science Foundation of Guangdong Province (2017A030307001).

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Authors and Affiliations

  1. State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong, Hong Kong, China

    Leiluo Geng, Boya Liao, Leigang Jin, Jiasui Yu, Xiaoyu Zhao, Yuntao Zhao, Ling Zhong, Baile Wang, Qizhou Lian & Aimin Xu

  2. Department of Medicine, The University of Hong Kong, Hong Kong, China

    Leiluo Geng, Leigang Jin, Jiasui Yu, Xiaoyu Zhao, Yuntao Zhao, Ling Zhong, Baile Wang, Jie Liu, Qizhou Lian & Aimin Xu

  3. Cord Blood Bank, Guangzhou Institute of Eugenics and Perinatology, Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou, China

    Leiluo Geng, Jie Liu & Qizhou Lian

  4. Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China

    Boya Liao & Aimin Xu

  5. School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China

    Boya Liao, Jiufeng Li & Wei Jia

  6. College of Food Science and Technology, Guangdong Ocean University, Zhanjiang, China

    Yuntao Zhao

  7. Beijing Key Laboratory of Diabetes Research and Care, Beijing Tongren Hospital, Capital Medical University, Beijing, China

    Jin-Kui Yang

  8. Center for Translational Medicine, Shanghai Key Laboratory of Diabetes Mellitus, and Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai Diabetes Institute, Shanghai, China

    Wei Jia

  9. HKUMed Laboratory of Cellular Therapeutics, The University of Hong Kong, Hong Kong, China

    Qizhou Lian

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Contributions

L.L.G. designed the study, carried out the research, analyzed and interpreted the results and wrote the manuscript. B.Y.L. carried out the research and data analysis. L.G.J., X.Y.Z., B.L.W. and J.L. helped with the molecular, cellular and biochemical experiments. J.S.Y. helped data analysis and drawing the schematic diagrams. Y.T.Z. and L.Z. helped with the animal experiments. J.F.L. and W.J. helped with the analysis of glycolytic metabolites. J.K.Y. helped with human islet study. Q.Z.L. advised the study and edited the manuscript. A.M.X. conceived and supervised the study, wrote and edited the manuscript.

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Correspondence to Qizhou Lian or Aimin Xu.

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Nature Metabolism thanks Marc Donath and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary handling editor: Isabella Samuelson, in collaboration with the Nature Metabolism team

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Extended data

Extended Data Fig. 1 FGF21 has no obvious effects on GSIS.

a, Illustration of the experimental design for the effects of recombinant mouse FGF21 (rmFGF21) protein on GSIS in islets isolated from 20-week-old male C57BL/6J lean, diet-induced obese (DIO, fed with a high-fat diet for 12 weeks) and db/db mice. b, Insulin secretion capacities of isolated islets treated with 50 nM rmFGF21 protein for 0-72 h under 2.8 mM or 16.7 mM glucose condition (n = 6 mice). c, Insulin secretion capacities of isolated islets treated with rmFGF21 protein at 0, 50, 100 nM for 48 h under 2.8 mM or 16.7 mM glucose condition (n = 6 mice). d, Islets isolated from lean, DIO and db/db mice were fasted for 12 h and treated with 0, 50, 100 nM rmFGF21 protein for 10 min, followed by examination of p-Erk (Thr202/Tyr204) and t-Erk protein levels by Western blot. Similar results were obtained from three independent experiments. e, Rat INS-1E (n = 6 independent cultures) and mouse MIN6 β-cells (n = 7 independent cultures) were treated with 0, 50, 100 nM rmFGF21 protein for 48 h before exposure to 2.8 mM or 16.7 mM glucose for 30 min. Insulin concentration in the conditioned medium was measured and normalized to cell protein concentrations. f-g, Blood glucose levels (f) and serum insulin levels (g) in male 14-week-old WT (FGF21+/+) and Fgf21 KO (FGF21-/-) mice under feeding or fasting conditions (n = 8 mice). h-i, GTT (h, n = 8 mice) and GSIS (i, n = 6 mice) were performed in male FGF21+/+ and FGF21-/- mice on regular diet for 12 weeks and the values of area under curve (AUC) were quantified. j, Dynamic insulin secretion under basal or high glucose condition was measured in cultured islets isolated from male FGF21+/+ and FGF21-/- mice on regular diet for 12 weeks (n = 6 mice). Data are presented as mean ± SEM.

Source data

Extended Data Fig. 2 Effects of β-cell-selective deletion of Klb in mice.

a, Daily food intake in KlbF/F, KlbRIP and RIP-Cre mice on STC diet for 12 weeks (n = 5 mice for KlbF/F group, n = 6 mice for KlbRIP group, n = 5 for RIP-Cre group). b-c, Body weight and composition in KlbF/F, KlbRIP and RIP-Cre mice on STC diet for 16 weeks (b, n = 6 mice for each group) or HFD for 12 weeks (c, n = 7 mice for KlbF/F group, n = 7 mice for KlbRIP group, n = 8 mice for RIP-Cre group). d-e. Insulin tolerance test (ITT) in KlbF/F, KlbRIP and RIP-Cre mice on STC feeding for 15 weeks (d) or HFD for 11 weeks (e) (n = 6 mice). f, Representative H&E staining of pancreatic sections of STC- and HFD-fed KlbF/F, KlbRIP and RIP-Cre mice. The islets were circled with blue lines. Similar results were obtained from three independent experiments. Scale bar, 50μm. g, Representative immunofluorescence staining of glucagon (red) and insulin (green) in pancreatic sections of STC- and HFD-fed KlbF/F, KlbRIP and RIP-Cre mice. Similar results were obtained from three independent experiments. Scale bar, 50μm. h, The β-cell mass in STC- and HFD-fed KlbF/F, KlbRIP and RIP-Cre mice (n = 7 mice). i, Relative mRNA levels of β-cell marker genes in islets isolated from STC-fed KlbF/F, KlbRIP and RIP-Cre mice (n = 8 mice). Data are presented as mean ± SEM.

Source data

Extended Data Fig. 3 Restoration of KLB expression alleviates defective GSIS in islets isolated from db/db mice.

a, Representative immunoblots and quantification of KLB in islets isolated from C57BL/6J mice fed with STC or HFD for 16 weeks (n = 8 mice). b, Correlation between GSIS capacity and Klb mRNA level in islets isolated from C57BL/6J mice fed with STC or HFD for 16 weeks (n = 16 mice). c, Representative immunoblots and quantification of KLB in islets isolated from 16-week-old C57BKS m + /db and db/db mice (n = 8 mice). d, Correlation between GSIS capacity and the Klb mRNA level in islets isolated from 16-week-old C57BKS m + /db or db/db mice (n = 16 mice). e, Illustration of the experimental design to evaluate the effect of adenovirus-mediated expression of KLB on insulin secretion in diabetic islets. f, Representative microscopic examination of GFP signals in cultured islets infected with various adenoviruses by paraffin sectioning. Similar results were obtained from three independent experiments. Scale bar, 50μm. g, Representative immunoblots of KLB and Tubulin in islets infected with indicated adenoviruses. Similar results were obtained from three independent experiments. h, Dynamic GSIS and quantification of AUC in islets at 48 h after adenovirus infection (n = 6 mice). i-j, Glycolytic flux measured by 3H2O generated from [5-3H]-glucose (i, n = 8 mice) and ATP production (j, n = 6 mice) in isolated islets infected with indicated adenoviruses. Data are presented as mean ± SEM. P values are derived from two-tailed unpaired t-test with Welch’s correction (a, c), two-tailed nonparametric Spearman correlation (b, d), Welch’s ANOVA followed by Dunnett’s T3 multiple comparisons test (h), or ordinary two-way ANOVA followed by Tukey’s multiple-comparisons test (i, j).

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Extended Data Fig. 4 AAV-mediated depletion of KLB in pancreatic β-cells of adult mice impairs glucose uptake and glycolysis.

Islets were isolated from 16-week-old KlbF/F mice injected with AAV–RIP-GFP or AAV–RIP-Cre, or C57BL/6 J mice injected with AAV–RIP-Cre as described in Fig. 1l. a, Glycolytic flux measured by 3H2O generated from [5-3H]-glucose through glycolysis (n = 8 mice for each group). b, Glucose uptake measured by radioisotope-labeled glucose analog 3H-2-deoxyglucose assay (n = 8 mice for KlbF/F-GFP group, n = 8 mice for KlbF/F-Cre group, n = 7 mice for C57-Cre group). c, Enzymatic activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK) (n = 7 mice for each group). Data are presented as mean ± SEM. P values are derived from ordinary two-way ANOVA followed by Tukey’s multiple-comparisons test (a, c), or ordinary one-way ANOVA followed by Tukey’s multiple-comparisons test (b).

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Extended Data Fig. 5 Excessive overexpression of HIF1α is not able to rescue defective GSIS in Klb-null β-cells.

Islets isolated from 16-week-old male KlbRIP or KlbF/F mice were infected with adenovirus encoding Myc-tagged HIF1α (Adv-HIF1α) or GFP (Adv-GFP) at 300 multiplicity of infection (MOI) for 24 h. a, Representative immunoblots with anti-HIF1α, Myc, GFP or Tubulin antibodies in isolated islets infected with different adenoviruses (left) and quantification of HIF1α protein levels normalized to Tubulin (right, n = 6 mice). b-e, Insulin secretion (b, n = 8 mice), glycolytic flux measured by 3H2O generated from [5-3H]-glucose (c, n = 8 mice), mitochondrial TCA flux measured by 14CO2 generated from [2-14C]-pyruvate (d, n = 8 mice for KlbF/F-GFP, n = 8 mice for KlbRIP-GFP group, n = 9 mice for KlbRIP-HIF1α group) and ATP production (e, n = 6 mice) under basal (2.8 mM glucose) or high-glucose (16.7 mM) conditions in isolated islets infected with different adenoviruses. Data are presented as mean ± SEM. P values are derived from Welch’s ANOVA followed by Dunnett’s T3 multiple comparisons test (a), or ordinary two-way ANOVA followed by Tukey’s multiple-comparisons test (b-e).

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Extended Data Fig. 6 STAT3 modulates HIF1α expression and GSIS in β-cells.

a, Phosphorylation and expression levels of several kinases and transcription factors in islets isolated from 16-week-old male KlbF/F and KlbRIP mice as determined by immunoblotting (n = 4 mice for each group). b, Mouse MIN6 β-cells were infected with adenovirus encoding GFP (Adv-GFP) or Flag-tagged constitutively active STAT3 (Adv-STAT3c) at 100 MOI for 48 h, followed by examination of insulin secretion under 2.8 mM or 16.7 mM glucose conditions. The insulin secretion capacities were normalized to the cell protein concentration (n = 7 independent cultures). c, Mouse MIN6 β-cells were treated with the STAT3 inhibitor S3I-201 (200 μM) or PBS for 24 h, followed by immunoblotting analysis of p-STAT3, t-STAT3, HIF1α and Tubulin (left). The expression levels of HIF1α protein were quantified by densitometry analysis (right, n = 6 independent cultures). d, Basal (2.8 mM glucose) and high glucose (16.7 mM)-stimulated insulin secretion capacities were assessed in cells described in panel c (n = 7 independent cultures). e, ChIP assays with a rabbit anti-p-STAT3(Tyr705) polyclonal antibody were performed in islets isolated from 15-week-old male KlbF/F and KlbRIP mice using primers spanning to the SIE1 and SIE2 regions within the mouse Hif1α proximal promoters as described in Fig. 6e. The precipitated chromatin was quantified by real-time PCR and the relative fold change was calculated by normalizing to the genomic 36b4 gene (n = 8 mice for KlbF/F group, n = 9 mice for KlbRIP group). Data are presented as mean ± SEM. P values are derived from ordinary two-way ANOVA followed by Sidak’s multiple comparisons test (b, d, e), or two-tailed unpaired t-test (c).

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Extended Data Fig. 7 Effects of excessive IL6 treatment on glucose control and insulin secretion in mice.

a-d, Schematic diagram of chronic rmIL6 protein administration (a). 15-week-old male KlbF/F and KlbRIP mice were administered with rmIL6 protein (0.2 mg/kg/day) or PBS for 10 days. The blood was collected from tail vein to measure the circulating IL6 concentration every other day (b, n = 4 mice for each group). On the last day, GTT was performed (c, n = 8 mice for each group) and serum insulin levels were measured to assess GSIS (d, n = 6 mice for each group). Data are presented as mean ± SEM. P values are derived from ordinary two-way ANOVA followed by Sidak’s multiple comparisons test (c-d).

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Extended Data Fig. 8 Overexpression of degradation-resistant GP130 reverses impaired GSIS and glycolysis in KlbRIP islets.

a, Islets isolated from male 16-week-old KlbF/F or KlbRIP mice were infected with adenovirus encoding Flag-tagged GP130 with K716R mutation or GFP at 100 multiplicity of infection (MOI) for 24 h. b, Representative immunoblots with anti-GP130, GFP, Flag or Tubulin antibodies in isolated islets infected with different adenoviruses. Similar results were obtained from three independent experiments. c-d, Insulin secretion (c) and glycolytic flux measured by 3H2O generated from [5-3H]-glucose (d) under 2.8 mM or 16.7 mM glucose conditions in isolated islets infected with different adenoviruses (n = 7 mice for each group). e, Immunoblots for p-STAT3(Tyr705) and t-STAT3 in islets infected with different adenoviruses (upper) and densitometric quantification of the relative expression levels of p-/t-STAT3 (lower, n = 4 mice for each group). f, Relative expression levels of Hif1α and glycolytic genes quantified by real-time PCR and normalized to β-actin gene in islets infected with different adenoviruses. The mRNA level in KlbF/F-GFP group was set as 1 (n = 8 mice for each group). Data are presented as mean ± SEM. P values are derived from ordinary two-way ANOVA followed by Tukey’s multiple-comparisons test (c, d) or ordinary one-way ANOVA followed by Tukey’s multiple-comparisons test (e).

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Extended Data Fig. 9 Working model for an FGF21-independent role of KLB in regulating insulin secretion.

KLB interacts with GP130 and prevents ubiquitin-dependent lysosomal degradation of GP130, thereby facilitating IL6-induced STAT3–HIF1α signaling to maintain normal glycolysis and GSIS in pancreatic β-cells.

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Geng, L., Liao, B., Jin, L. et al. β-Klotho promotes glycolysis and glucose-stimulated insulin secretion via GP130. Nat Metab 4, 608–626 (2022). https://doi.org/10.1038/s42255-022-00572-2

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