Microtubule-mediated GLUT4 trafficking is disrupted in insulin-resistant skeletal muscle
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This paper provides strong evidence for the important point that microtubule function is required for the proper localization of Glut4 glucose transporters in an insulin responsive compartment. This membrane localization is required in turn for effective translocation of Glut4 to the muscle cell surface in response to the hormone.
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Abstract
Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking.
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Author Response
Reviewer #1 (Public Review):
Weakness of the study include:
- There are no data supporting a role for insulin regulation of microtubule-dependent GLUT4-containg vesicle movement. The data in Fig.2B do not support a differences in the number of "moving" GLUT4 vesicles between basal and insulin-stimulated fibers. The statement on line 103 that they "observed a ~16% but insignificant increase" to be confusing. These data do not support an effect of insulin on the number of moving GLUT4 vesicles that can be detected in an individual experiment. There is also effect of insulin on GLUT4 vesicles in the data reported in Fig.S2D, Fig.S5B, and Fig.S5F. However, the data in Fig. 2C suggest there was a consistent increase in "moving" vesicles in insulin-stimulated conditions in 4 independent experiments (how are these data …
Author Response
Reviewer #1 (Public Review):
Weakness of the study include:
- There are no data supporting a role for insulin regulation of microtubule-dependent GLUT4-containg vesicle movement. The data in Fig.2B do not support a differences in the number of "moving" GLUT4 vesicles between basal and insulin-stimulated fibers. The statement on line 103 that they "observed a ~16% but insignificant increase" to be confusing. These data do not support an effect of insulin on the number of moving GLUT4 vesicles that can be detected in an individual experiment. There is also effect of insulin on GLUT4 vesicles in the data reported in Fig.S2D, Fig.S5B, and Fig.S5F. However, the data in Fig. 2C suggest there was a consistent increase in "moving" vesicles in insulin-stimulated conditions in 4 independent experiments (how are these data normalized?). Because the basis of insulin-regulation of glucose uptake is the control of GLUT4 translocation to the plasma membrane, the authors need to clarify their thinking on why they do not detect insulin robust effects on GLUT4 dynamics in the individual experiments. Is it that they are not measuring the correct parameter? That the assay is not sensitive to the changes?
The small (or no effect) of insulin distracts a bit from the findings that there is microtubule-dependent GLUT4 movement in basal and stimulated muscle fibers, and that disruption of this movement by depolymerization of microtubules or Kif5b knockdown blunts GLUT4 translocation. As noted above, the data strongly support microtubule-dependent GLUT4 dynamics as permissive for insulin-stimulated GLUT4 translocation even if this dynamics might not be a target of insulin action.
In light of the reviewer´s comment and to avoid confusing/distracting readers we have removed figure 2C showing the effect of insulin based on pooled data across all our independent experiments. We discuss several possibilities for the lack of significant insulin effect on GLUT4 movement in individual experiments in the discussion section (lines 342 to 361 in TC version of MS). The discussion has been updated to reflect the points raised by the reviewer. More sensitive techniques than currently available in our lab are required to firmly conclude whether microtubule-based GLUT4 trafficking is directly regulated by insulin.
- The analyses of GLUT4-containing structures are not particularly informative. Co-localization with other markers (beyond syntaxin6) are needed to understand these structures. Defining structures as small, medium or large is incomplete. In particular, it is important to probe the microtubule nucleation site clusters for other membrane markers. Transferrin receptor? IRAP?
While our analysis based on structure-segmentation clearly demonstrate a microtubule-dependent effect on GLUT4 localization, we completely agree that additional work including co-labelling of GLUT4 and various compartment markers is required to fully understand the localization changes observed for GLUT4-containing structures upon microtubule disruption. However, for practical reasons, it is not currently feasible for us to complete these analyses within a reasonable time-frame so we will reserve this for future studies.
- The Kinesore data do not support the authors hypothesis. The data show that Kinesore increases the amount of GLUT4 in the plasma membrane of basal cells and that insulin further increases plasma membrane GLUT4 to the same extent as it does in control cells. How does that provide insight into the role microtubules (or kif5b) in GLUT4 biology? Why does Kinesore increase plasma membrane GLUT4? Is it an effect of Kinesin 1 on GLUT4 vesicles? Kinesore is reported to remodel the microtubule cytoskeleton by a mechanism dependent on Kinesin 1. Is that the reason for the change in GLUT4?
To better understand the effect of kinesore on GLUT4-dependent glucose uptake, we have now incubated EDL and Soleus muscles ± kinesore and ± insulin and measured 2-DG uptake (GLUT4 translocation and glucose transport is considered the rate-limiting step for 2-DG uptake in incubated muscles due to the lack of muscle perfusion in this model) and proximal insulin signaling. In contrast to the enhancing effect on membrane GLUT4 observed following kinesore treatment in basal and insulin stimulated L6 cells, kinesore did not stimulate basal 2-DG uptake in EDL and Soleus. Furthermore, kinesore markedly impaired insulin-stimulated 2-DG uptake (figure 4B). We also tested the effect of 2h kinesore treatment in differentiated primary human myotubes. In this model, kinesore reduced basal glucose uptake and blocked the insulin effect (figure 4C). Together, this suggests that kinesore inhibits GLUT4-dependent glucose uptake in adult muscle and primary human muscle cells, presumably by inhibiting the binding of GLUT4 containing cargo, despite kinesore also having an activating effect on Kinesin-1 motor function. This possibility is discussed in the current version of the manuscript (line 177-180, 203-211). These data are consistent with the KIF5B knockdown data in L6 and support a necessary role of this motor protein in skeletal muscle GLUT4 trafficking.
To better understand, why kinesore led to increased rather than decreased GLUT4 translocation in L6 cells, we also disrupted the microtubule network using nocodazole and colchicine prior to kinesore stimulation. Surprisingly, kinesore stimulation enhanced membrane GLUT4 even in microtubule-disrupted L6 cells, indicating that the effect of kinesore on GLUT4 translocation is microtubule-independent in L6 cells. With three of four data sets supporting a necessary role of Kinesin-1 motor proteins in GLUT4 trafficking, including the adult muscle data, we end up concluding:
…our shRNA data in L6 myoblasts and kinesore data in adult muscle support the requirement of KIF5B-containing Kinesin-1 motor proteins in insulin-stimulated GLUT4-dependent glucose uptake in skeletal muscle.
However, we would also like to include the discrepant effect of Kinesore in L6 myoblasts as this may be useful information to others using this compound and/or studying GLUT4 in cultured cells.
- The analysis of Kif5b is a bit cursory. Depolymerization of microtubules in muscle fibers essentially blocks all GLUT4 movement (only the insulin condition is shown in Fig.2B but I assume basal would be equally inhibited), and fully inhibits insulin-stimulated glucose uptake in muscle fibers. What are the effects of nocodazole in L6 cells (cell used for kif5b studies) and is it similar in magnitude to kif5b knockdown? Those data would identify there are non-Kif5b microtubule-dependent effects.
To address the magnitude of reduced insulin-stimulated GLUT4 translocation in microtubule-disrupted L6 cells, we investigated the effect of nocodazole (13 µM) and colchicine (25 µM) on GLUT4 translocation in L6 cells.
Insulin stimulated GLUT4 translocation was reduced but not blocked by either nocodazole or colchicine. This is in accordance with previous in vitro studies in 3T3 adipocytes and muscle cells (PMID: 11085918, PMID: 11145966, PMID: 24705014). Overall, these data still support that Kif5b is a major microtubule motor protein regulating GLUT4 translocation across cell-types.
- The authors need to show that the fibers isolated from the HFD mice remain insulin-resistant ex vivo by measuring glucose uptake. It is possible that once removed from the mice they "revert" to normal insulin-sensitivity, which might contribute to the differences reported in Fig5.
This is an important point. In figure 5 figure supplement 1E, we show that the fibers isolated from the diet-induced obese mice display impaired insulin-induced p-Akt Thr308 and p-TBC1D4 Thr642 after isolation and in vitro culture. This shows that the insulin resistance is present at the muscular level and is preserved after isolation and in vitro culturing.
- Although it is interesting that the authors have included the insulin-resistance models/experiments, they are not well developed and therefore the conclusions are not particularly strong.
In this study, we induced insulin resistance by two different means (C2 ceramide treatment and diet-induced obesity) and demonstrated at the level of p-Akt and p-TBC1D4 in cultured muscle fibers that we successfully achieved insulin resistance in our models. In particular the high fat diet model is arguably the most common in vivo model of obesity-linked insulin resistance. Thus, we were able to study GLUT4 trafficking on microtubules in normal vs. insulin-resistant muscle fibers and found this to be impaired in insulin-resistant muscle. Although one could always have done more, we believe that our data on adult muscle GLUT4 movement in insulin-resistance are robust, novel and do support our conclusions and title.
- The data do not support the title.
We respectfully disagree. See our reply to comment 6 above.
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eLife assessment
This paper provides strong evidence for the important point that microtubule function is required for the proper localization of Glut4 glucose transporters in an insulin responsive compartment. This membrane localization is required in turn for effective translocation of Glut4 to the muscle cell surface in response to the hormone.
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Reviewer #1 (Public Review):
Muscle is a major insulin-responsive tissue for the disposal of glucose, a process dependent on the translocation of GLUT4 glucose transporter from intracellular compartments to the plasma membrane. Knudsen and co-workers provide an analysis of the impact of microtubule-based movement on GLUT4 biology in muscle cell lines, and rodent and human muscle fibers ex vivo. A role for microtubules in the control of GLUT4 vesicle dynamics in both unstimulated and insulin-stimulated adipocytes (cultured and primary) has been previously reported by a number of groups. Less is known about the requirement for microtubules for GLUT4 translocation in muscle. A strength of this study is that key aspects of the work were performed in muscle fibers rather than muscle cell lines.
Conclusions that are strongly supported by the …
Reviewer #1 (Public Review):
Muscle is a major insulin-responsive tissue for the disposal of glucose, a process dependent on the translocation of GLUT4 glucose transporter from intracellular compartments to the plasma membrane. Knudsen and co-workers provide an analysis of the impact of microtubule-based movement on GLUT4 biology in muscle cell lines, and rodent and human muscle fibers ex vivo. A role for microtubules in the control of GLUT4 vesicle dynamics in both unstimulated and insulin-stimulated adipocytes (cultured and primary) has been previously reported by a number of groups. Less is known about the requirement for microtubules for GLUT4 translocation in muscle. A strength of this study is that key aspects of the work were performed in muscle fibers rather than muscle cell lines.
Conclusions that are strongly supported by the data presented include:
1. Demonstration of constitutive GLUT4 movement along microtubule tracks in both unstimulated and insulin-stimulated muscle fibers. GLUT4 dynamics in unstimulated fibers were captured by fluorescence recover after photobleaching (FRAP) and by quantifying vesicle movements by live cell microscopy, whereas in insulin-stimulated cells GLUT4 dynamics were captured by following the movements of GLUT4-containing vesicles. These data support a model in which intracellular GLUT4 is dynamic in both unstimulated and insulin-stimulated muscle fibers rather than being static in unstimulated conditions and only mobilized upon insulin-stimulation.
2. Similar microscopy analyses of GLUT4-containing vesicles demonstrate that depolymerization of microtubules reduced GLUT4 vesicle movement and impacted insulin-stimulated glucose uptake. Short term depolymerization of microtubules (5 min) did not affect insulin-stimulated glucose uptake, whereas insulin-stimulated glucose uptake was blocked after prolonged depolymerization (2 hrs). The use of a muscle on a chip method to monitor glucose uptake in real time was critical for these experiments.
The changes in glucose uptake were accompanied by changes in the morphologies of intracellular GLUT4-containing structures. The differences between short and long term depolymerization of microtubules support a model in which GLUT4 can be translocated to the plasma membrane by insulin stimulation in the absence of microtubules but an intact microtubule cytoskeleton is required to maintain GLUT4 in a "compartment" that can be recruited by insulin. Stated another way, the microtubule-dependent dynamics of GLUT4-containing vesicles in unstimulated cells is permissive for insulin-stimulated GLUT4 translocation.
3. Knockdown of the microtubule motor protein, Kif5b, blunts insulin-stimulated translocation of GLUT4 to the plasma membrane of cultured muscle cells. These findings agree with previously demonstrated role for Kif5b in adipocytes.
4. In an in vitro model of insulin resistance (incubation of muscle fibers with short chain C2 ceramide) unstimulated and insulin-stimulated GLUT4-containing vesicle movement was blunted and unstimulated and insulin-stimulated microtubule polymerization was reduced.
Weakness of the study include:
1. There are no data supporting a role for insulin regulation of microtubule-dependent GLUT4-containg vesicle movement. The data in Fig.2B do not support a differences in the number of "moving" GLUT4 vesicles between basal and insulin-stimulated fibers. The statement on line 103 that they "observed a ~16% but insignificant increase" to be confusing. These data do not support an effect of insulin on the number of moving GLUT4 vesicles that can be detected in an individual experiment. There is also effect of insulin on GLUT4 vesicles in the data reported in Fig.S2D, Fig.S5B, and Fig.S5F. However, the data in Fig. 2C suggest there was a consistent increase in "moving" vesicles in insulin-stimulated conditions in 4 independent experiments (how are these data normalized?). Because the basis of insulin-regulation of glucose uptake is the control of GLUT4 translocation to the plasma membrane, the authors need to clarify their thinking on why they do not detect insulin robust effects on GLUT4 dynamics in the individual experiments. Is it that they are not measuring the correct parameter? That the assay is not sensitive to the changes?
The small (or no effect) of insulin distracts a bit from the findings that there is microtubule-dependent GLUT4 movement in basal and stimulated muscle fibers, and that disruption of this movement by depolymerization of microtubules or Kif5b knockdown blunts GLUT4 translocation. As noted above, the data strongly support microtubule-dependent GLUT4 dynamics as permissive for insulin-stimulated GLUT4 translocation even if this dynamics might not be a target of insulin action.
2. The analyses of GLUT4-containing structures are not particularly informative. Co-localization with other markers (beyond syntaxin6) are needed to understand these structures. Defining structures as small, medium or large is incomplete. In particular, it is important to probe the microtubule nucleation site clusters for other membrane markers. Transferrin receptor? IRAP?
3. The Kinesore data do not support the authors hypothesis. The data show that Kinesore increases the amount of GLUT4 in the plasma membrane of basal cells and that insulin further increases plasma membrane GLUT4 to the same extent as it does in control cells. How does that provide insight into the role microtubules (or kif5b) in GLUT4 biology? Why does Kinesore increase plasma membrane GLUT4? Is it an effect of Kinesin 1 on GLUT4 vesicles? Kinesore is reported to remodel the microtubule cytoskeleton by a mechanism dependent on Kinesin 1. Is that the reason for the change in GLUT4?
4. The analysis of Kif5b is a bit cursory. Depolymerization of microtubules in muscle fibers essentially blocks all GLUT4 movement (only the insulin condition is shown in Fig.2B but I assume basal would be equally inhibited), and fully inhibits insulin-stimulated glucose uptake in muscle fibers. What are the effects of nocodazole in L6 cells (cell used for kif5b studies) and is it similar in magnitude to kif5b knockdown? Those data would identify there are non-Kif5b microtubule-dependent effects.
5. The authors need to show that the fibers isolated from the HFD mice remain insulin-resistant ex vivo by measuring glucose uptake. It is possible that once removed from the mice they "revert" to normal insulin-sensitivity, which might contribute to the differences reported in Fig5.
6. Although it is interesting that the authors have included the insulin-resistance models/experiments, they are not well developed and therefore the conclusions are not particularly strong.
7. The data do not support the title.
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Reviewer #2 (Public Review):
Overall, this manuscript provides a thorough characterization of the role of microtubules in the movement of GLUT4 in muscle fibers, and demonstrates the need for an intact microtubule network for GLUT4 responsiveness but only after the initial round of response.
The study poses a very interesting question, rooted in studies in the literature studying the effects of Nocodazole (Noco) and C2-ceramide on GLUT4 traffic in cell systems. It is important to validate or refute predictions from those studies and, largely through this group's work, the quest to examine these questions in isolated muscle fibers and intact muscles as feasible is commendable. The authors develop very interesting imaging approaches to this end, and quantify the results in a convincing and elegant fashion. The system to measure 2-DG …Reviewer #2 (Public Review):
Overall, this manuscript provides a thorough characterization of the role of microtubules in the movement of GLUT4 in muscle fibers, and demonstrates the need for an intact microtubule network for GLUT4 responsiveness but only after the initial round of response.
The study poses a very interesting question, rooted in studies in the literature studying the effects of Nocodazole (Noco) and C2-ceramide on GLUT4 traffic in cell systems. It is important to validate or refute predictions from those studies and, largely through this group's work, the quest to examine these questions in isolated muscle fibers and intact muscles as feasible is commendable. The authors develop very interesting imaging approaches to this end, and quantify the results in a convincing and elegant fashion. The system to measure 2-DG uptake and glucose uptake by electrochemical sensing in isolated fibers using the microfluidic pump is very ingenious.
The main conclusion that microtubules are important for GLUT4 proper localization is important and adds mechanistic insight beyond that obtained from work in myoblasts and pre/adipocytes. The observation that microtubules are not engaged in GLUT4 traffic in the first round of insulin action but it is thereafter is also very revealing and should lead to more insights into the first and subsequent rounds of GLUT4 translocation. -
