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

Reviewer #1 (Public review):

Summary:

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.

Strengths and Weaknesses:

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.

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.

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.

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.

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.

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. 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. 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.

The data in Figures 2b,c,f,g,k,l are not convincing. Again, 2-NBDG is used.

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.

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.

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.

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.

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.

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).

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.

Reviewer #2 (Public review):

Summary:

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.

Weaknesses:

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.

Reviewer #3 (Public review):

Summary:

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.

Weaknesses:

(1) 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.

(2) 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.

(3) 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.