Author response:
Public Reviews:
Reviewer #1 (Public review):
(1) This article purports to show that ML-SA8, a synthetic activator of the lysosomal TRPML1 channel, results in AMPK activation and glucose uptake in hepatocytes, and that this action has therapeutic potential for metabolic disease. The final figure shows that glucose levels are improved in db/db mice, although it is not entirely clear whether this is due to an effect on the liver, on other tissues, or on glucose production or uptake. The earlier figures try to make the case that SA8 causes activation and GLUT4 translocation and glucose uptake in liver cells; however, these data are not convincing. GLUT4 is expressed at such low levels in liver that it is likely not physiologically important. The authors use a fluorescent glucose analog to measure glucose uptake, and this molecule has been shown to enter cells largely by fluid phase endocytosis. Overall, this reviewer finds the premise misguided and the data unconvincing.
Thank you for the critical comments and constructive suggestions. We have carefully considered all the concerns raised and provide our point-by-point responses below.
(2) The initial figures show phosphorylation of AMPK on Thr172, but no downstream effects are shown. Usually, to convincingly show that AMPK activity is increased, it would be appropriate to immunoblot phospho-ACC or some other substrate. This is minor.
Suggestion was taken! We will investigate the effect of SA8 on AMPK downstream effectors i.e. ACC activation by Western blotting. Ie p-ACC (Ser79) / total ACC.
(3) Lines 135-148: GLUT4 is not expressed at levels that are significant for physiology in liver cells, and its function in liver is not particularly relevant. The authors cite references 38-40 to support that it may be expressed at low levels in liver, but no knockout studies have been done to show that this expression is physiologically important.
We thank the reviewer for this critical comment. We agree that GLUT4 is not the predominant hepatic glucose transporter, but it is expressed at a relatively low level in the liver compared to other tissues.
Regarding the physiological role of GLUT4, it has been shown to mediate glucose uptake in hepatic stellate and sinusoidal endothelial cells (Tang and Chen 2010, Karim, Liaskou et al. 2014). Furthermore, ischemia‑reperfusion (IR) significantly upregulated the expression of GLUT4 in the liver, rather than GLUT2, and the increased GLUT4 localized to the membrane peripheries of hepatocytes and enhanced glucose uptake, which in turn led to marked glycogen deposition (Kim, Jung et al. 2014, Kurabayashi, Furihata et al. 2022). These studies establish a clear physiological role for GLUT4 in the liver.
Notably, Ranalletta, et. al. (2005) showed that GLUT4-null mice exhibit compensatory alterations in hepatic glucose and lipid metabolism, including increased hepatic glucose uptake and triglycerides conversion (Ranalletta, Jiang et al. 2005), indicating that GLUT4 ablation influences liver metabolism. Nevertheless, these existing evidences including the GLUT4 expression data and the functional changes observed in GLUT4 null mice—supports the relevance of GLUT4 in hepatic glucose metabolism. However, it is necessary to perform the liver-specific GLUT4 KO studies to clarify the role of GLUT4 in the liver. (We will incorporate these points and limitations into the revised Discussion section).
(4) Figure 1e is not convincing. No controls are included to show the specificity of the antibody for immunofluorescent staining. No intracellular GLUT4 is visible in the unstimulated samples.
We understood the reviewer’s concern about the specificity of GLUT4 antibody for immunofluorescent staining. The GLUT4 antibody (Abcam, ab33780) employed in our study has been extensively validated in previous studies for both Western blotting (Xie, Liu et al. 2024, Amanollahi, Holman et al. 2025, Ando, Takeda et al. 2025) and immunofluorescence (see also Johansson, Mannerås-Holm et al. 2013, Xiao, Zhang et al. 2025). in addition, we confirmed its specificity in our system by Western blot (Suppl. Fig.4), which showed a single band at ~45–55 kDa. Collectively, the combination of published validations, our own data supports the specificity of GLUT4 immunofluorescence detection.
About the intracellular GLUT4 signal in Fig 1e. We apologize for the unclear GLUT4 signal in our original Fig. 1e. This was due to an inadvertently short exposure for the control condition. To improve this, we have now acquired new images with uniformly increased exposure time for all groups. As shown in the new Fig. 1e, GLUT4 is now clearly detected in control cells and mainly in cytosol, and ML-SA8 treatment obviously increases its accumulation at the plasma membrane.
(5) In Figure 1f, again, the data are not convincing. The bands seem too sharp for GLUT4, which has 12 membrane-spanning domains as well as an N-linked glycosylation, so that it usually runs as a smear.
We understood the reviewer’s concern. As an N-glycosylated membrane protein, GLUT4 may exhibit broader or diffuse migration patterns on immunoblotting. Nevertheless, the final band pattern is influenced by multiple factors, e.g. antibody specificity, sample preparation, electrophoresis conditions, and detection condition. Of note, multiple independent studies have shown endogenous GLUT4 as a relatively “sharp” immunoreactive band around 55–60 kDa (Gurley, Ilkayeva et al. 2016, Habtemichael, Li et al. 2021, Wu, Yu et al. 2024) (see also Ando et al., 2025; Amanollahi et al., 2025; Xie et al., 2024), which is very consistent with our results. Thus, we are confident that our GLUT4 band is specific and reliable.
(6) Figure 1h. Data are not convincing. 2-NBDG is not a valid approach to measure glucose uptake. 2-NBDG enters cells largely via fluid phase endocytosis, and its accumulation is independent of known GLUT inhibitors such as cytochalasin B (Yazdani et al., MBoC 2022; PMID: 35921166; see also PMID: 42287154). The idea that such a bulky derivative of glucose could enter the transporter channel is not compatible with known structural data.
We thank the reviewer for raising this point. We acknowledge that the uptake mechanism of 2-NBDG is controversial. As the reviewer noted, some studies have reported that it enters cells largely by endocytosis in certain cell types, and this remains a subject of ongoing discussion. Nevertheless, 2-NBDG continues to be widely employed as a glucose uptake tracer in this field, including several recent high-profile studies (Nobs, Kolodziejczyk et al. 2023, Xiong, Helm et al. 2023, Wu, Lv et al. 2025) as following.
(1) Nobs, S.P., et al., Lung dendritic-cell metabolism underlies susceptibility to viral infection in diabetes. Nature, 2023. 624(7992): p. 645-652.
(2) Xiong, L., et al., Nutrition impact on ILC3 maintenance and function centers on a cell-intrinsic CD71-iron axis. Nat Immunol, 2023. 24(10): p. 1671-1684.
(3) Wu, Y., et al., Dalbergia odorifera T.C. Chen leaf extract promotes microglial energy expenditure to phagocytize neutrophils after cerebral ischemia-reperfusion. Phytomedicine, 2025. 149: p. 157508.
In the current study, given the consistency of our results with parallel functional assays, the use of 2‑NBDG is justified in this context.
(7) Supplementary Figure 5 uses 2-NBDG glucose uptake again. This reviewer is not convinced that the data reflect transporter-mediated glucose uptake, as suggested by the authors.
Please see response to #6.
(8) As well, although palmitate treatment of cells can cause an insulin-resistant-like phenotype in some cell types, this is not characterized in the present work.
We understand the reviewer’s concern regarding the characterization of PA-induced insulin resistance model.
First, this PA-induced insulin resistant hepatic model is well-established and validated in several literatures (Lee, Cho et al. 2010, Zhang, Cai et al. 2020, Malik, Inamdar et al. 2024), and it has been used for T2DM natural and synthetic drug screening (Faria, Calixto et al. 2025).
Second, we have characterized this model in our system. As shown in Suppl. Fig. 5a, b, insulin (100 nM, 0.5 h) induced an increase of glucose uptake in HepG2 cells measured by 2-NBDG (Yamada, Nakata et al. 2000). In contrast, in PA-treated HepG2 cells, this effect was almost completely blocked, suggesting that PA-treated HepG2 cells are less sensitive to insulin. Overall, this model has been well validated and is suitable for the purposes of our study.
(9) Finally, as noted, one would not expect hepatocytes to exhibit insulin-responsive glucose transport. Glycogen synthesis is the main insulin-regulated step that might be affected.
As stated in the Responses#9, PA-induced hepatic insulin-resistance has become a widely accepted in vitro model for investigating therapeutic strategies (Lee, Cho et al. 2010, Zhang, Cai et al. 2020, Malik, Inamdar et al. 2024, Faria, Calixto et al. 2025).
(10) The data in Figures 2b,c,f,g,k,l are not convincing. Again, 2-NBDG is used.
Please see response to #6.
(11) For the glucose consumption measurements in other panels of Figure 2, the methods section states that cells were cultured in 10 mM glucose. What volume was used? It is difficult to believe that a monolayer of cells would consume very much of the glucose that is present in the culture medium. Data are shown as a percent of controls, and look reasonable, but it would be helpful to include absolute as well as relative units.
We appreciate the reviewer’s comments and apologize for any confusion about the method.
First, we have revised the methods section to clarify the assay procedure “Following the manufacturer’s protocol, 2.5 μL of sample (medium/standard) was mixed with 250 μL of working solution a 96-well plate. The mixture was then incubated at 37 °C for 10 min and the absorbance was measured…” (line 421-423).
Second, in response to the suggestion to include both absolute and relative units, we will provide the data with absolute value for reviewer’s reference. In the main figures, we have retained the normalized data as this format allows direct comparison of treatment effects across independent experiments.
(12) In Figure 2, in experiments using the TRPML1 KO cells, no panel is shown to demonstrate knockout. The authors cite a previous paper for the construction of these cells, but the control immunoblot should still be shown here.
We thank the reviewer for raising this point. The TRPML1 knockout cell line used in our study was originally generated and provided by Prof. Haoxing Xu’s laboratory, this cell line has been validated in several published literature (Wang, Gao et al. 2015, Zhang, Cheng et al. 2016). We understand the reviewer’s concern, so we will further validate this TRPML1 KO cell line.
(13) In Figure 3, controls are missing in the BAPTA experiment in Figure 3a (only SA8-treated cells were treated with BAPTA and with EGTA). Again, it would be helpful to have p-ACC or some other readout of AMPK activity, and not just AMPK phosphorylation. 2NBDG is again used in this figure.
Suggestion taken! We will add the controls including BAPTA-AM and EGTA only data. p-ACC/total ACC will also be measured. About the 2-NBDG, please see Responses#6.
(14) Line 212-213 the text states "considering our finding that TRPML1-mediated Ca2+ release is essential for AMPK activation." This has not been shown. The work uses chelators and does not necessarily indicate a role for TRPML1. The drug may be specific, as suggested by the authors, but the way this phrase is worded is too strong. As well, AMPK was shown to be phosphorylated, but full activation towards its various substrates has not been shown.
We thank the reviewer for raising this concern. We fully agree that the Ca2+ chelators experiment alone could not specially attribute the effect to TRPML1.
In fact, we have performed experiment to address the TRPML1-dependent mechanism in the original submission. As shown in Fig. 2i-l, In TRPML1 KO HAP1 cells (Qi, Xing et al. 2021), ML-SA8-induced AMPK phosphorylation and cellular glucose uptake were almost completely abolished compared to wild-type (WT) HAP1 cells. Moreover, pharmacological inhibition of TRPML1 with a TRPML1 specific synthetic inhibitor-ML-SI5 completely abolished ML-SA8-triggered AMPK phosphorylation (Fig. 2d, e). In addition, in IR-HepG2 model, ML-SI5 could substantially inhibited ML-SA8-induced cellular glucose uptake (Fig. 2f-h).
Accordingly, we have also revised the statement to” considering our finding that TRPML1-mediated Ca2+ release is necessary for AMPK activation” to make the sentence more rigorous.
(15) Figure 4cd suggests that GLUT4 expression is increased by 2 or 3-fold in the liver of DB+SA8-treated mice, compared to controls. This may be the case, but its abundance is still likely ~1000-fold less in liver compared to skeletal muscle or adipose tissue. This reviewer is still not convinced that this is physiologically relevant. The images in Supplementary Figure 8 suggest a larger increase, but it remains uncertain whether the staining really represents GLUT4.
We understood the reviewer’s concern. About the physiological importance of GLUT4, please see Responses #3. About the specificity of GLUT4 antibody, please see Responses#4.
(16) Data showing that blood glucose and HbA1c are reduced in SA8-treated mice are reasonable, and GTTs and ITTs are shown. Unfortunately, there are no insulin concentrations, and it remains uncertain whether glucose production is reduced or uptake is increased (or if both effects are present).
We will measure the insulin concentrations.
(17) In the discussion, the authors again state that GLUT4 is present in the liver and that it regulates hepatic glucose homeostasis, and they cite reference 63. This review article does not argue that GLUT4 acts in the liver to regulate hepatic glucose homeostasis, but that its actions in muscle and fat have secondary effects on the liver.
Sorry for the oversights. We have supplementary more precise references (Rossetti, Stenbit et al. 1997, Kurabayashi, Furihata et al. 2022, Fan, Jiao et al. 2023, Jiang, Luo et al. 2024).
Reviewer #2 (Public review):
The manuscript contains interesting studies suggesting that pharmacological activation of TRPML1 could be useful to treat T2D by increasing glucose uptake via activation of AMPK. Preclinical studies suggest the inhibitor improved blood glucose in Db/Db mice. Ex vivo studies in cell lines examine both pharmacologic and genetic manipulations, both to activate and to inactivate TRPML1, and the results consistently suggest that TRPML1 activates AMPK and increases glucose uptake.
Strengths:
The manuscript is well written, and the studies are carefully performed.
(18) All mechanistic studies were performed in transformed cell lines; conclusions would be stronger if performed in primary cells. The in vivo studies were only performed in male mice. Performing metabolic studies in both sexes is standard practice now. Whether the findings would extend to females was not tested and remains uncertain. Some controls are missing, such as plasma membrane loading controls for fractionation studies. The GLUT4 staining was performed after fixation and permeabilization, yet control cells appear to be devoid of intracellular (and all) staining, a confusing result that doesn't reflect the expected biology.
Thank you for your support and the constructive suggestions! We will answer these questions in the following point-to-point responses.
Reviewer #3 (Public review):
(19) Zhu et al. present a proof-of-concept for targeting the lysosomal calcium channel MCOLN1/TRPML1endolysosomal ion channels to restore type 2 diabetes mellitus (T2DM). Using synthetic TRPML1 agonists (ML-SA8) and genetic manipulation, the authors demonstrate that TRPML1 stimulation triggers localized lysosomal calcium release. This calcium efflux sequentially activates CaMKKβ and phosphorylates AMPK at Thr172 in various cell models, including palmitic acid-induced insulin-resistant HepG2 cells. This signaling pathway promotes GLUT4 translocation to the plasma membrane and increases intracellular glucose uptake. When administered daily to diabetic db/db mice over six weeks, ML-SA8 lowers fasting and random blood glucose, improves oral glucose and insulin tolerance tests, reduces hepatic steatosis, and lowers serum ALT and AST levels.
Strengths:
Based on the TFEB-independent pathway activated by TRPML1 and the experimental approaches described by Medina's group (PMID: 31822666), the authors use a combination of pharmacological and genetic tools to dissect such an intracellular signaling pathway. Additionally, the animal experiments show consistent phenotypic improvements across independent metabolic parameters. The ability of ML-SA8 to restore glycogen deposition and clear hepatic lipid accumulation in db/db mice without causing weight loss or overt toxicity provides a strong rationale for exploring lysosomal targets in metabolic disease.
Thank you for the support!
(21) The authors focus almost exclusively on hepatic GLUT4 to explain the observed glucose disposal. However, other glucose transporter isoforms such as GLUT2 dominate basal glucose transport. While the authors show increased AMPK phosphorylation in skeletal muscle and adipose tissue, they do not measure GLUT4 translocation or glucose uptake in these primary disposal organs. As a result, attributing systemic glycemic recovery primarily to hepatic GLUT4 translocation overlooks the major physiological roles of peripheral tissues.
We thank the reviewer for this critical comment and we agree with the reviewer. Our initial focus on hepatic GLUT4 was driven by our primary interest in liver metabolism and the fact that we observed a consistent and robust effect of ML‑SA8 on hepatic GLUT4 translocation, AMPK activation and glucose uptake. We acknowledge that the possible contribution of GLUT2 to these effects cannot be excluded. Also, ML‑SA8 may exert similar effects in other tissues, such as skeletal muscle and adipose tissue as we found out that GLUT4 levels were upregulated in these tissues (Suppl.Fig.8b, c). We therefore agree that the systemic glycemic recovery should be attributed to the multi‑tissue effects of ML‑SA8, rather than a liver-restricted phenomenon. Accordingly, we will revise the Discussion section to incorporate this. Note that the precise mechanisms of ML-SAs on GLUT2 and in peripheral tissues warrant further investigation.
(22) In both HepG2 cells and mouse liver tissues, ML-SA8 treatment increases total GLUT4 protein expression in addition to plasma membrane localization. Because total protein pools expand, the enrichment of GLUT4 in plasma membrane fractions cannot be cleanly attributed to acute vesicular translocation alone. The manuscript does not explain the timescale or mechanism behind this rapid total protein upregulation, leaving a mechanistic gap between acute ion channel gating and protein expression.
Great suggestion. we will re-calculated the enrichment of GLUT4 in PM with total GLUT4 protein to clarify.
(23) While the in vitro specificity of ML-SA8 is well-controlled, the systemic animal experiments lack a specific rescue or knockout control. Small-molecule agonists administered intraperitoneally over six weeks can exert off-target effects. Without demonstrating that co-administering the TRPML1 inhibitor ML-SI5 blunts the therapeutic effect in vivo, or showing that ML-SA8 lacks efficacy in TRPML1-null mice, the definitive link between in vivo glycemic recovery and TRPML1 activation remains incomplete.
We understand the reviewer’s concern about the specificity of ML-SAs for TRPML1. In fact, the specificities of ML-SAs and ML-SIs have been rigorously validated in previous studies using TRPML1 knockout (KO) cells (Sahoo, Gu et al. 2017, Yu, Zhang et al. 2020) and further confirmed in the atomic-resolution co-structures (Schmiege, Fine et al. 2017, Schmiege, Fine et al. 2021).
In the current study we provide additional evidence supporting this specificity. We show that ML-SA8-induced AMPK phosphorylation and glucose uptake were abolished in TRPML1 knockout cells (Fig. 2i-l) and blocked by specific inhibitor ML-SI5 (Fig. 2d, e, f, g). In addition, ML‑SA5 has been show to lack efficacy in TRPML1‑null mice in several studies (Yu, Zhang et al. 2020, Zhang, Wang et al. 2024, Xing, Wang et al. 2025), further reinforcing the target specificity of this class of compounds. Hence, multiple lines of evidence support the on-target effect of ML-SA8: (1) in vitro blockade by ML-SI5 and TRPML1 KO; (2) consistent effects across structurally distinct TRPML1 agonists; (3) dose‑dependent responses in vivo; and (4) absence of efficacy in TRPML1‑null mice.
We agree with the reviewer that rescue experiment or in vivo knockout validation would provide the most definitive proof of target specificity. However, breeding TRPML1‑null mice or performing extensive dose-finding studies for ML-SI5 in vivo would require substantial time and resources which probably fall beyond the scope of the current study. We have therefore acknowledge this limitation in the Discussion section and noted that future validation with ML-SI5 co-administration or TRPML1-null mice is warranted. We hope the reviewer finds our response acceptable.
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