Cell Biology

Targeting Lysosomal MCOLN1/TRPML1 Ion Channels to Finely Alleviate Diabetes Mellitus via a Ca²⁺- CaMKKβ-AMPK pathway

Jiaxuan Zhu
Zhaonan Pan
Shijia Wang
Yuyuan Wang
Zongxian Ding
Qianhui Wang
Dan Li author has email address

Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, China
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, United States

https://doi.org/10.7554/eLife.112056.1

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Figures and data

Stimulation of the lysosomal TRPML1 channel induces AMPK activation.
(a) ML-SA8 (a TRPML1 specific synthetic agonist) activated AMPK in a dose-dependent manner. HepG2 cells were treated with ML-SA8 (0.3-3 μM) for 2 h. (b) Quantification of results shown in c. from n= 3 independent experiments. (c) AMPK inhibitor blocked ML-SA8-induced AMPK phosphorylation. HepG2 cells were pretreated with CC (10 μM, 1h), followed by cotreatment with ML-SA8 (1 μM, 2 h) and AMPK phosphorylation was measured by Western blotting. (d) Ratio of p-AMPK/total AMPK shown in e. (n= 3). (e) ML-SA8 induced GLUT4 PM translocation. HepG2 cells were treated with ML-SA8 (1 μM, 4 h) and GLUT4 immunoreactivity was measured. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. The graph at the Righter panel shows the fluorescence intensity of GLUT4 with a red line scan across a cell (red line shown in the Lefter images). (f) Western blot analysis of PM GLUT4 expression induced by ML-SA8. Membrane and cytoplasm protein fractions were isolated from cells treated with ML-SA8 (1 µM) or DMSO for 4 h and subjected to western blot analysis. (g) Quantification of PM GLUT4 expression as shown in f. (n= 3 independent experiments). (h) ML-SA8 promoted glucose influx. IR-HepG2 cells were treated with ML-SA8 (0.1-3 μM) for 4 h, followed by 2-NBDG (50 µM, 0.5 h) treatment. Scale bar, 40 μm. (i) Quantification of the relative fluorescent intensity of 2-NBDG in h. from n= 3 independent experiments. (j) The effect of ML-SA8 on glucose consumption in medium. IR-HepG2 cells were treated with or without ML-SA8 (1 μM) for 4 h in DMEM containing 10 mM glucose, no FBS, no phenol red, and glucose consumption in the medium was measured. Metformin (MET, 2 mM) served as a positive control. For all panels, data are presented as mean ± s.e.m.; *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA.

TRPML1 is required for ML-SA8-induced AMPK activation.
(a) Overexpressing TRPML1 further promoted low-dose of ML-SA8- activating AMPK. In the top panel, western blot validation of the GFP-TRPML1 overexpression in HepG2. In the bottom panel, HepG2 cells with or without GFP-TRPML1 overexpression were treated with ML-SA8 (0.03-1 µM) for 2 h and western blotting analysis of p-AMPK/total AMPK protein levels. (b) TRPML1 overexpression promoted cellular glucose uptake (2-NBDG) by low-dose of SA8 (0.03-1 µM, 4 h) in IR-HepG2 cells. Scale bar, 40 μm. (c) Quantification of the relative fluorescent intensity of 2-NBDG shown in b. (n= 3 independent experiments). (d) ML-SI5 (a TRPML1 synthetic inhibitor) blocked ML-SA8-induced AMPK activation. Western blot analysis of AMPK phosphorylation in HepG2 cells pretreated with ML-SI5 (3 μM) for 1 h, followed by cotreated with ML-SA8 (1 μM) or MET (2 mM) for 2 h. (e) Quantification of the results shown in d. from n= 6 independent experiments. (f) The effect of ML-SI5 on ML-SA8-induced cellular glucose uptake. IR-HepG2 were pretreated with ML-SI5 (3 μM) for 1 h, then cotreated with ML-SA8 (1 μM, 4 h), followed by 2-NBDG (50 µM, 0.5 h). Scale bar, 40 μm. (g) Quantification of the intensity of 2-NBDG in f. (n= 3). (h) ML-SI5 blocked ML-SA8-induced glucose consumption. IR-HepG2 cells were pretreated with ML-SI5 (3 μM) for 1 h, then cotreated with ML-SA8 (1 μM) or MET (2 mM) for 4 h, finally glucose levels in the supernatant medium were measured. (i) Western blotting analysis of p-AMPK levels in WT and TRPML1 KO HAP1 cells treated with ML-SA8 (1 μM) or MET (2 mM) for 2 h. (j) Quantitative analysis of p-AMPK/total AMPK levels as shown in i. from n= 4 independent experiments. (k) TRPML1 KO abrogated ML-SA8- but not MET-induced cellular glucose uptake. WT and TRPML1 KO HAP1 cells were treated with PA (0.25 mM, 12 h), followed by ML-SA8 (1 μM) or MET (2 mM) for 4 h. 2-NBDG (50 µM, 0.5 h) was then applied and measured. Scale bar, 40 μm. (l) Quantification of the relative fluorescent intensity of 2-NBDG in k. from n= 4 independent experiments. In all panels, data are presented as mean ± s.e.m.; **p < 0.01, ***p < 0.001.

Ca²⁺- CaMKKβ dependence of ML-SA8 induced-AMPK activation.
(a) BAPTA-AM inhibited ML-SA8-induced AMPK phosphorylation. HepG2 cells were pretreated with BAPTA-AM (fast Ca²⁺ chelator, 20 μM) or EGTA-AM (slow Ca²⁺ chelator, 20 μM) for 1 h, then cotreated with ML-SA8 (1 μM, 2 h), AMPK phosphorylation was then evaluated by Western blotting. (b) Quantification of results shown in a. from n= 3 experiments. (c) The effect of BAPTA-AM on ML-SA8-induced cellular glucose uptake. IR-HepG2 cells were treated with ML-SA8 (1 μM, 4 h) in the presence or absence of BAPTA-AM (20 μM) or EGTA-AM (20 μM), followed by analysis of cellular glucose levels with 2-NBDG. Scale bar, 40 μm. (d) Quantification of results shown in c. from (n= 4). (e) CaMKKβ inhibitor blocked ML-SA8-induced AMPK phosphorylation. HepG2 cells were pretreated with STO609 (10 μM, 1h) and cotreated with ML-SA8 (1 μM, 2 h), and AMPK phosphorylation was assayed by Western blotting. (f) Ratio of p-AMPK/total AMPK shown in e. (n= 3). (g) CC and STO609 abolished ML-SA8-induced cellular glucose uptake. IR-HepG2 cells were treated with ML-SA8 (1 μM) with or without CC (10 μM) or STO609 (10 μM) for 4 h and cellular glucose levels were measured by 2-NBDG. Scale bar, 40 μm. (h) Quantification of the intensity of 2-NBDG shown in g. (n= 3). (i) CaMKKβ KD abolished ML-SA8- induced AMPK activation. CaMKKβ KD efficiency was examined by QPCR (Left panel). HepG2 cells were transfected with CaMKKβ siRNA and then treated with ML-SA8 (1 μM) or MET (2 mM) for 2 h, then p-AMPK levels were measured by Western blot. (j) Quantitation of results shown in i. (k) CaMKKβ KD abrogated ML-SA8- induced cellular glucose uptake. IR-HepG2 cells were treated with ML-SA8 (1 μM) or MET (2 mM) for 4 h. 2-NBDG (50 µM, 0.5 h) was then applied and measured. Scale bar, 40 μm. (l) Quantification of results in k. (n= 3) (m) The effects of CaMKKβ KD on ML-SA8-induced glucose consumption. IR-HepG2 cells were treated with ML-SA8 (1 μM) or MET (2 mM) for 4 h, then glucose levels in the supernatant medium were measured (n=3). (n) A working model to illustrate that small-molecule TRPML1 agonist-ML-SA8 promotes cellular glucose uptake via TRPML1-Ca²⁺-CaMKKβ-AMPK-GLUT4 pathway. ML-SA8 specifically activates TRPML1 channel, which releases Ca²⁺ into the cytoplasm. Elevated cytosolic Ca²⁺ then triggers CaMKKβ kinase activation, which phosphorylates AMPK to initiate its catalytic activity, followed by promoting GLUT4-containing vesicle translocation to the PM. Membrane-inserted GLUT4 facilitates extracellular glucose uptake into cells, ultimately restoring glycemic homeostasis. Arrows denote sequential signaling events, with color-coded molecular entities matching experimental annotations.

Administration of ML-SA8 attenuates metabolic disorder in db/db mice.
(a) ML-SA8 promoted AMPK phosphorylation in the liver tissues of db/db mice with ML-SA8 (4 mg/kg) or vehicle (5% DMSO, 90% PEG300, 5% ddH₂O) for 24 h. Liver tissues were collected and extracted proteins were subjected to detect p-AMPK/total AMPK by Western blotting. (b) Quantification of results shown in a. (n= 4). (c) ML-SA8 promoted GLUT4 expression in the liver tissues of db/db mice with ML-SA8 (4 mg/kg) or vehicle (5% DMSO, 90% PEG300, 5% ddH₂O) for 6 weeks. Liver tissues were collected and extracted proteins were subjected to detect GLUT4 proteins by Western blotting. (d) Quantification of results shown in c. (n= 4). (e) Experimental design. db/db mice were randomly allocated into 4 groups: DB+V group was i.p. injected with vehicle (5% DMSO, 90% PEG300, 5% ddH₂O) daily (n=11); DB+SA8 group was i.p. injected with SA8 (2mg/kg, n=9; 4mg/kg, n=10; 8 mg/kg, n=9). (f) Fasting blood glucose (FBG) levels at 0 and 6 weeks. (g) HbA1c levels. (h) OGTT. Mice daily i.p with SA8 (2, 4, 8 mg/kg) received oral glucose (1g/kg) after 10 h fasting, followed by blood glucose dynamic measurement (0-120 min) with corresponding AUC at the Right panel. (i) ITT. Mice with SA8 (2, 4, 8 mg/kg) treatment were i.p injected insulin (0.75 U/kg) after 4 h fasting, followed by blood glucose measurements. Left panel: Blood glucose levels (0-120 min). Right panel: AUC values. (j) Representative photomicrographs of H&E and PAS staining in liver tissues from mice treated with SA8 (4 mg/kg). Scale bar, 40 μm. (k) Serum ALT levels. (l) Serum AST levels. All data are presented as mean ± s.e.m.; *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA. (m) Pharmacological up-regulation of TRPML1 ameliorates diabetic phenotypes in vivo through Ca²⁺-dependent AMPK activation. ML-SA8 (a specific TRPML1 agonist) activates lysosomal TRPML1 channel, leading to lysosomal Ca²⁺ release from lysosomes. Ca²⁺ triggers Ca²⁺- dependent AMPK phosphorylation and initiates GLUT4 Storage Vesicle (GSV) trafficking to the PM. Subsequently, extracellular glucose is transferred into cells via GLUT4 and blood glucose levels are reduced in vitro and in vivo.

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