TBC1D4 Is Necessary for Enhancing Muscle Insulin Sensitivity in Response to AICAR and Contraction

Impaired insulin-stimulated glucose uptake in skeletal muscle is a key defect in the pathogenesis of type 2 diabetes (1). Importantly, the ability of a single bout of exercise to improve insulin-stimulated glucose uptake in skeletal muscle has been demonstrated by several studies in both healthy and insulin-resistant rodents (2–4) and humans (5–8), highlighting the therapeutic potential of exercise. In vivo enhanced insulin-stimulated glucose uptake is likely mediated by a coordinated increase in microvascular perfusion (9) and GLUT4 translocation to the plasma membrane (10) in response to insulin without enhanced proximal insulin signaling (4,8,11–13). We recently reported that prior activation of AMPK in skeletal muscle is necessary to improve muscle and whole-body insulin sensitivity in response to AICAR, contraction, and exercise (14,15). TBC1D4 is regulated by Akt-mediated phosphorylation during insulin stimulation that has been suggested to be important for insulin-stimulated GLUT4 translocation and glucose uptake in skeletal muscle (16,17). TBC1D4 is also regulated by AMPK-mediated phosphorylation, but this seems insufficient in itself to promote glucose uptake (18). A number of studies have reported that elevated insulin-stimulated phosphorylation of TBC1D4 occurs in parallel with improved muscle insulin sensitivity after a single bout of exercise, contraction, and AICAR stimulation (8,13–15,19,20). Importantly, this relationship is abolished in AMPK-deficient muscle (14,15), suggesting that an AMPK-TBC1D4 signaling axis is mediating the insulin-sensitizing effect of these stimuli. However, direct evidence linking AMPK and TBC1D4 to improved muscle insulin sensitivity has not been established.

To address the hypothesis that TBC1D4 is required for enhancing muscle insulin sensitivity after pharmacological- and contraction-induced activation of AMPK, we applied a genetic approach and took advantage of our previously established ex vivo and in situ experimental protocols (14,15).

All animal experiments complied with the European Union Convention for the Protection of Vertebrates Used for Scientific Purposes and were approved by the State Agency for Nature, Environment and Consumer Protection (LANUV, North Rhine-Westphalia, Germany) Ethics Committee and the Danish Animal Experiments Inspectorate. Animals used in this study were whole-body Tbc1d4 knock-out (D4KO) female mice (21) and CRISPR/Cas9-generated muscle-specific Tbc1d4 KO (mD4KO) male and female mice (Supplementary Data) with corresponding wild-type (WT) littermates as controls. Mice (17 ± 6 weeks [mean ± SD]) were maintained on a 12-:12-h light-dark cycle with free access to standard rodent chow and water.

Fed whole-body Tbc1d4 KO female mice (21) and WT littermates were anesthetized by an intraperitoneal injection of tribromoethanol (Avertin) (50 mg/100 g body wt) or pentobarbital (10 mg/100 g body wt) before extensor digitorum longus (EDL) muscles were isolated and suspended in incubation chambers (Multi Wire Myograph System; Danish Myo Technology, Aarhus, Denmark) containing Krebs-Ringer buffer (KRB) supplemented with 0.1% BSA, 8 mmol/L mannitol, and 2 mmol/L pyruvate, as previously described (14). In short, after 10 min of preincubation, EDL muscles were incubated for 50 min in 100% human serum in the absence or presence of 1 mmol/L AICAR (Toronto Research Chemicals, Toronto, Ontario, Canada). Serum was obtained from a young, healthy man after an overnight fast in accordance with a protocol approved by the Ethics Committee of Copenhagen (#H-18045268).

Muscles were allowed to recover for 6 h in KRB supplemented with 0.1% BSA, 5 mmol/L d-glucose, and 5 mmol/L mannitol, after which they were incubated in KRB supplemented with 0.1% BSA, 2 mmol/L pyruvate, and 8 mmol/L mannitol, with or without a submaximal or a maximal insulin concentration (100 and 10,000 μU/mL), for 30 min. 2-Deoxyglucose uptake was measured during the last 10 of the 30-min stimulation period by adding 1 mmol/L [³H]2-deoxyglucose (0.028 MBq/mL) and 7 mmol/L [¹⁴C]mannitol (0.0083 MBq/mL) to the incubation medium. For glucose uptake measurements in response to acute AICAR stimulation, EDL muscles were incubated in 100% human serum for 50 min in the absence or presence of 1 mmol/L of AICAR. Subsequently, muscles were washed for 1 min in KRB supplemented with 0.1% BSA, 8 mmol/L mannitol, and 2 mmol/L pyruvate, after which 2-deoxyglucose uptake was measured during a 10 min incubation period as described above. Also present in the wash and transport buffers was 1 mmol/L AICAR. For all incubations, 2-deoxyglucose uptake was determined as previously described (15).

Fed CRISPR/Cas9-generated muscle-specific Tbc1d4 KO male and female mice as well as WT littermates were anesthetized by an intraperitoneal injection of pentobarbital (10 mg/100 g body wt), after which electrodes were connected to the common peroneal nerve of both legs of the animals. Half of the animals served as sham-operated controls. Immediately after 10 min of in situ contraction (duration: 0.5 s, frequency: 100 Hz, width: 0.1 ms, voltage: 5 V, repeated every 1.5 s) tibialis anterior muscles were dissected and frozen in liquid nitrogen to study the acute response of contraction on intracellular signaling and glycogen levels. After dissection of the tibialis anterior muscles, EDL muscles were dissected and suspended in incubation chambers containing KRB supplemented with 0.1% BSA, 5 mmol/L d-glucose, and 5 mmol/L mannitol to study the submaximal insulin response after contraction, as previously described (15). In short, EDL muscles were incubated for 180 min during which the incubation buffer was replaced every 30 min to maintain a glucose concentration of ∼5 mmol/L. Muscles were subsequently incubated in KRB supplemented with 0.1% BSA, 2 mmol/L pyruvate, and 8 mmol/L mannitol, with or without a submaximal insulin concentration (100 μU/mL), for 30 min. Uptake of 2-deoxyglucose was measured during the last 10 min of the 30-min stimulation period as described above.

Muscles were homogenized, and lysates were collected and frozen (−80°C) for subsequent analyses as previously described (14). The bicinchoninic acid method was used to determine total protein abundance in muscle lysates. Lysates were boiled in Laemmli buffer and subjected to SDS-PAGE and immunoblotting as previously described (14).

Glycogen concentration in tibialis anterior muscle was measured on 500 μg of muscle protein homogenate after acid hydrolysis. In short, muscle homogenates were heated (100°C) for 2 h in 2 mol/L HCl. Glucosyl units were determined in a fraction of the supernatant (22) and related to protein concentration.

Primary antibodies against Akt2, phosphorylated (p)Akt-Ser473, pAkt-Thr308, Erk1/2, pErk1/2 Thr202/Tyr204, pAMPKα-Thr172, pACC-Ser79/212, pTBC1D4-Thr642, pTBC1D1-Thr590, TBC1D1, and hexokinase II were from Cell Signaling Technology. Antibody against pTBC1D1-Ser231 was from Millipore, and antibody against TBC1D4 (AS160) was from Millipore and Abcam. Antibody against AMPKα2 was from Santa Cruz Biotechnology as well as donated by Dr. D. Grahame Hardie (School of Life Sciences, University of Dundee). GLUT4 antibody was from Thermo-Fisher Scientific, and ACC protein was detected using horseradish peroxidase-conjugated streptavidin from Jackson ImmunoResearch. pTBC1D4-Ser711 was detected using antibody donated by Dr. Laurie J. Goodyear (Joslin Diabetes Center and Harvard Medical School) (18).

Data are presented as the means ± SEM unless stated otherwise. Two-way ANOVA with repeated measures and paired/unpaired Student t tests were used to assess statistical significance within and between genotypes, where appropriate. The Student-Newman-Keuls test was used for post hoc testing. Statistical significance was defined as P < 0.05.

Glucose uptake increased similarly in EDL muscle from Tbc1d4 WT and KO mice during AICAR stimulation (Fig. 1A), as also previously reported (21). Acute AICAR stimulation also increased phosphorylation of AMPKα-Thr172 (Fig. 1B) and downstream targets ACC-Ser212 (Fig. 1C) and TBC1D1-Ser231 (Fig. 1D) in EDL muscle from both genotypes. As expected, acute AICAR stimulation increased phosphorylation of TBC1D4-Ser711 in WT EDL muscle only (Fig. 1E). Immediately after AICAR stimulation, total protein abundance of AMPKα2, ACC, and TBC1D4 was not altered; however, we detected a slight but significant increase in TBC1D1 protein in EDL muscle from both genotypes. Collectively, these results suggest that immediately after AICAR stimulation, AMPK activity increased to a similar extent in EDL muscle from WT and whole-body Tbc1d4 KO mice.

In response to in situ contraction, glycogen content decreased to a similar extent in skeletal muscle from WT and muscle-specific Tbc1d4 KO mice (Fig. 2A). In addition, in situ contraction increased phosphorylation of Erk1/2-Thr202/Tyr204 (Fig. 2B), AMPKα-Thr172 (Fig. 2C), ACC-Ser212 (Fig. 2D), and TBC1D1-Ser231 (Fig. 2E) in skeletal muscle to an extent that did not differ between genotypes. This indicates that the in situ contraction protocol induced similar metabolic stress and relevant cellular signaling in both WT and Tbc1d4-deficient skeletal muscle. TBC1D4 protein was not detected in skeletal muscle from muscle-specific Tbc1d4 KO mice, and thus, in situ contraction increased phosphorylation of TBC1D4-Ser711 in WT skeletal muscle only (Fig. 2F and G). Besides a small (∼30%) significant decrease in AMPKα2, total protein abundance of Erk1/2, ACC, TBC1D1, and TBC1D4 were not affected in skeletal muscle from either genotype immediately after in situ contraction (Fig. 2G).

To clarify whether the AMPK-dependent increase in muscle insulin sensitivity after AICAR stimulation is dependent on TBC1D4, we measured submaximal and maximal insulin-stimulated glucose uptake in EDL muscle 6 h after AICAR stimulation. At this time point, the acute effect of AICAR on noninsulin-stimulated glucose uptake (Fig. 1A) had returned to near control levels in muscle from both WT and whole-body Tbc1d4 KO mice (Fig. 3A). Interestingly, prior AICAR stimulation improved submaximal insulin-stimulated glucose uptake in isolated EDL muscle from WT mice only (Fig. 3A). Thus, the incremental increase in submaximal insulin-stimulated glucose uptake was significantly higher (∼70%) in prior AICAR-stimulated EDL muscle from WT mice only (Fig. 3B).

We next investigated the phosphorylation pattern of TBC1D4. We found that activation of AMPK by prior AICAR stimulation increased submaximal insulin-stimulated phosphorylation of TBC1D4-Ser711 and TBC1D4-Thr649 in WT EDL muscle (Fig. 3C and D). This occurred despite that proximal insulin signaling measured at the level of phosphorylated Akt-Thr308 and -Ser473 was not enhanced by prior AICAR stimulation (Fig. 3E and F). As expected, no changes in total muscle protein abundance of Akt2, TBC1D4, HKII, and GLUT4 were found 6 h after AICAR stimulation (Fig. 3G).

Noninsulin-stimulated glucose uptake was slightly elevated 3 h after in situ contraction in EDL muscle from both WT and muscle-specific Tbc1d4 KO mice (Fig. 4A). However, prior in situ contraction enhanced submaximal insulin-stimulated glucose uptake in isolated EDL muscle from WT mice only (Fig. 4A). Thus, a significant incremental increase in submaximal insulin-stimulated glucose uptake (∼150%) was observed in prior contracted EDL muscle from WT mice (Fig. 4B). When examining the phosphorylation pattern of TBC1D4, we found that prior in situ contraction increased submaximal insulin-stimulated phosphorylation of TBC1D4-Ser711 and TBC1D4-Thr649 in WT EDL muscle (Fig. 4C and D). This also occurred without significant enhanced insulin-stimulated phosphorylation of Akt-Thr308 and -Ser473 (Fig. 4E and F). In addition, no changes in total muscle protein abundance of Akt2, TBC1D4, HKII, and GLUT4 were found 3 h after prior in situ contraction (Fig. 4G).

We have previously reported that AMPK activity and downstream signaling are increased in skeletal muscle in the recovery period after AICAR stimulation (14). In accordance, we found that the phosphorylation of AMPKα-Thr172, ACC-Ser212, TBC1D1-Ser231, and TBC1D1-Thr590 was increased in EDL muscle from WT and Tbc1d4 KO mice 6 h after AICAR stimulation (Fig. 5A–D). On average, the increase in phosphorylation of AMPKα-Thr172, ACC-Ser212, and TBC1D1-Ser231 represented ∼38%, ∼80%, and ∼90%, respectively, of the increase observed immediately after AICAR stimulation (Fig. 1). In addition, insulin increased the phosphorylation of TBC1D1-Thr590 in both control and prior AICAR-stimulated muscles independent of genotype (Fig. 5D). We did not detect any changes in total protein abundance of AMPKα2, ACC, and TBC1D1 6 h after AICAR stimulation (Fig. 5E).

In contrast to findings 6 h after AICAR stimulation, we observed that the phosphorylation of AMPKα-Thr172, ACC-Ser212, and TBC1D1-Ser231 had returned to resting levels in EDL muscle 3 h after in situ contraction (Fig. 6A–C) as also previously reported (15). The phosphorylation of TBC1D1-Thr590 was not different between rest and prior contracted muscle but increased in response to insulin in both genotypes (Fig. 6D). We did not detect any changes in total protein abundance of AMPKα2, ACC, and TBC1D1 3 h after in situ contraction (Fig. 6E). Collectively, no differences between genotypes were observed for AMPK signaling in skeletal muscle after in situ contraction and AICAR stimulation.

Skeletal muscle insulin resistance is a key risk factor for developing type 2 diabetes. Importantly, a marked and persistent increase in insulin sensitivity is observed in the previously active muscle after a single bout of exercise (2,3,5,7,11), highlighting the therapeutic potential of exercise. We and others have shown that pharmacological activation of AMPK in isolated muscle is sufficient to increase insulin sensitivity (12,14), and we recently reported that AMPK is also necessary for contraction and exercise to improve muscle insulin sensitivity (15). Moreover, we have provided proof-of-concept that direct pharmacological activation of AMPK in skeletal muscle improves glucose homeostasis in diet-induced obese mice and nonhuman primates (23). Here we provide the first genetic evidence to support that the AMPK downstream target TBC1D4 is necessary for enhancing insulin sensitivity in glycolytic EDL muscle after pharmacological and contraction-mediated activation of AMPK. Because AICAR and in situ contraction fail to increase insulin sensitivity in WT soleus muscle (14,15) and insulin-stimulated glucose uptake is abrogated in soleus muscle from Tbc1d4 KO mice (21), we were unable to determine the role of TBC1D4 in regulating insulin sensitivity in isolated oxidative soleus muscle.

Since the seminal observations in rat skeletal muscle by Arias et al. (19), elevated phosphorylation of TBC1D4 has emerged as an attractive candidate for mediating improvements in muscle insulin sensitivity after a single bout of exercise. Indeed, we reported in several human studies that improved muscle insulin sensitivity after acute exercise coincides with elevated insulin-stimulated phosphorylation of TBC1D4 in the prior exercised muscle (8,9,20). Furthermore, we showed that improved insulin sensitivity after prior contraction and AICAR stimulation is positively associated with elevated phosphorylation of TBC1D4-Thr649 and -Ser711 in WT EDL mouse muscle (14,15). Interestingly, such interplay is lost in AMPK-deficient muscle, supporting the notion of an AMPK-TBC1D4 signaling axis regulating muscle insulin sensitivity.

Evidence suggests that insulin stimulates glucose uptake by Akt-mediated phosphorylation of TBC1D4 (16). Phosphorylation of TBC1D4-Thr649 seems to be the site primarily responsible for enhancing GLUT4 translocation and glucose uptake in skeletal muscle in response to insulin (17). TBC1D4 is also phosphorylated at Ser711 in response to insulin and activation of AMPK, but this seems insufficient in itself to promote glucose uptake (18). However, when a TBC1D4-Ser711Ala mutant is overexpressed in skeletal muscle by gene electrotransfer, the ability of insulin to enhance phosphorylation of TBC1D4-Thr649 is impaired (14), suggesting that Ser711 phosphorylation increases the potential of upstream kinase Akt to phosphorylate TBC1D4. This seemingly increased effect of Akt on TBC1D4 several hours after prior AMPK activation by AICAR and contraction may be mediated by altered cellular localization of TBC1D4 and/or a decreased ability of phosphatases to dephosphorylate TBC1D4 during submaximal insulin concentrations (24,25). Taken together, our observations may suggest that activation of AMPK in some way primes TBC1D4 for a subsequent insulin stimulus, which leads to enhanced insulin-stimulated glucose uptake.

Several heterotrimeric complexes of AMPK exist in skeletal muscle (26). We have shown that the insulin-sensitizing effect of prior AICAR stimulation occurs via the AMPKα2β2γ3 complex (14) and that this complex likely phosphorylates TBC1D4-Ser711 during contraction (15). Based on these findings, we propose that AICAR and contraction enhance muscle insulin sensitivity by activating the AMPKα2β2γ3 complex, which subsequently phosphorylates TBC1D4-Ser711. Thus, in the period after contraction and AICAR stimulation, internalized GLUT4 may relocate to specific intracellular compartments that are highly susceptible to recruitment by a subsequent insulin stimulus leading to enhanced insulin-stimulated glucose uptake as previously hypothesized (27). Alternatively, more TBC1D4 protein may associate with GLUT4 as it moves from the cell surface membrane into the cell. If the recently internalized GLUT4 associates with more protein that senses the insulin signal (i.e., TBC1D4), this could also explain the increase in insulin sensitivity as previously suggested (28).

Similar to our previous study (14), we observed elevated AMPK downstream signaling 6 h after prior AICAR stimulation, which likely derives from a persistent increase in AMPK activity (14). We presume that this is not due to changes in the adenosine nucleotide pools but rather to a lasting effect of 5-amino-4-imidazole carboxamide ribonucleoside 5′-phosphate (ZMP) in skeletal muscle, because accumulation of ZMP after AICAR stimulation does not seem to affect concentrations of AMP, ADP, or ATP in cells and isolated skeletal muscle (29,30). Although the action of AICAR/ZMP in skeletal muscle is likely not confined to AMPK alone, we previously showed in AMPK transgenic animals that the insulin-sensitizing effect of prior AICAR stimulation is indeed mediated via AMPK in skeletal muscle (14).

Because GLUT4 and HKII protein abundance have been shown to be important for insulin-stimulated muscle glucose uptake (31), improved insulin sensitivity or the lack thereof several hours after contraction and AICAR stimulation could potentially be due to changes in muscle protein abundance of GLUT4 and HKII. However, we did not detect changes in total muscle protein abundance of GLUT4 and HKII, suggesting that these proteins are not essential for the insulin-sensitizing effect of contraction and AICAR in EDL muscle from WT mice or the lack thereof in EDL muscle from TBC1D4-deficient mice.

Studies have reported that improved muscle insulin sensitivity after prior AICAR stimulation and contraction occurs without enhanced proximal insulin signaling (12,14). In line, we found that insulin-stimulated phosphorylation of Akt-Thr308 and -Ser473 was not enhanced by prior AICAR stimulation or contraction. These results are similar to findings showing normal proximal insulin signaling in prior exercised and insulin-sensitized muscle (4,8,11,13).

Similar to TBC1D4, it has been shown that TBC1D1 is targeted by both Akt and AMPK and that TBC1D1 may function to regulate glucose uptake in skeletal muscle (32–34). Because the phosphorylation pattern of TBC1D1 was similar in skeletal muscle from WT and Tbc1d4-deficient mice, this indicates that TBC1D1 is not involved in mediating the insulin-sensitizing effect of prior contraction and AICAR stimulation in WT EDL muscle. Nevertheless, we cannot rule out a potential role of TBC1D1 because it may function to regulate muscle insulin sensitivity only in the presence of TBC1D4.

In conclusion, we show for the first time that the ability of AMPK to regulate muscle insulin sensitivity is dependent on TBC1D4 because in situ contraction and AICAR fail to increase insulin sensitivity in mouse EDL muscle lacking TBC1D4. Future studies will have to determine the involved mechanism of interplay between AMPK and TBC1D4, but we hypothesize that phospho-regulation of TBC1D4-Ser711 is important. Furthermore, because insulin-stimulated glucose uptake and phosphorylation of TBC1D4-Ser711 are also potentiated in human skeletal muscle after exercise (8,9), we hypothesize that an AMPK-TBC1D4 signaling axis is also regulating muscle insulin sensitivity in humans.

Acknowledgments. The authors thank Irene Bech Nielsen (Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen) for her skilled technical assistance and also thank Dr. Laurie J. Goodyear (Joslin Diabetes Center and Harvard Medical School, Boston, MA) and Dr. D. Grahame Hardie (School of Life Sciences, University of Dundee, Dundee, Scotland, U.K.) for the donation of phospho-specific TBC1D4-Ser711 and AMPKα2 antibody, respectively.

Funding. This work was supported by grants from the Danish Council for Independent Research – Medical Sciences (FSS8020-00288B) and the Novo Nordisk Foundation (NNF160C0023046), as well as funded in part by a grant from the Deutsche Forschungsgemeinschaft to A.C. (CH1659). This work was also supported by a research grant from the Danish Diabetes Academy, which is funded by the Novo Nordisk Foundation, grant number NNF17SA0031406.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. R.K. conceived and designed the research, performed the experiments, analyzed the data, and drafted the manuscript. A.C., N.O.J., K.K., J.K.L., and C.d.W. performed the experiments. H.A.-H. provided founder mice for the study cohort. J.F.P.W. conceived and designed the research and contributed to drafting the manuscript. All authors interpreted the results, contributed to the discussion, edited and revised the manuscript, and read and approved the final version of the manuscript. J.F.P.W. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in abstract form at the Danish Diabetes Academy Summer School on Diabetes & Metabolism, Ebberup, Denmark, 3–6 September 2018.