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Gene deletion of γ‐actin impairs insulin‐stimulated skeletal muscle glucose uptake in growing mice but not in mature adult mice

Abstract The cortical cytoskeleton, consisting of the cytoplasmic actin isoforms β and/or γ‐actin, has been implicated in insulin‐stimulated GLUT4 translocation and glucose uptake in muscle and adipose cell culture. Furthermore, transgenic inhibition of multiple actin‐regulating proteins in muscle inhibits insulin‐stimulated muscle glucose uptake. The current study tested if γ‐actin was required for insulin‐stimulated glucose uptake in mouse skeletal muscle. Based on our previously reported age‐dependent phenotype in muscle‐specific β‐actin gene deletion (−/−) mice, we included cohorts of growing 8–14 weeks old and mature 18–32 weeks old muscle‐specific γ‐actin−/− mice or wild‐type littermates. In growing mice, insulin significantly increased the glucose uptake in slow‐twitch oxidative soleus and fast‐twitch glycolytic EDL muscles from wild‐type mice, but not γ‐actin−/−. In relative values, the maximal insulin‐stimulated glucose uptake was reduced by ~50% in soleus and by ~70% in EDL muscles from growing γ‐actin−/− mice compared to growing wild‐type mice. In contrast, the insulin‐stimulated glucose uptake responses in mature adult γ‐actin−/− soleus and EDL muscles were indistinguishable from the responses in wild‐type muscles. Mature adult insulin‐stimulated phosphorylations on Akt, p70S6K, and ULK1 were not significantly affected by genotype. Hence, insulin‐stimulated muscle glucose uptake shows an age‐dependent impairment in young growing but not in fully grown γ‐actin−/− mice, bearing phenotypic resemblance to β‐actin−/− mice. Overall, γ‐actin does not appear required for insulin‐stimulated muscle glucose uptake in adulthood. Furthermore, our data emphasize the need to consider the rapid growth of young mice as a potential confounder in transgenic mouse phenotyping studies.


| INTRODUCTION
Whole-body insulin-resistance has been proposed to accelerate a range of non-communicable diseases (Czech, 2020;DeFronzo et al., 2015;González-Muniesa et al., 2017;Scully et al., 2021). Skeletal muscle is quantitatively the largest site of post-prandial insulin-stimulated glucose disposal and a major site of insulin-resistance (DeFronzo & Tripathy, 2009). Mechanistic understanding of how insulin increases glucose uptake into muscle fibers may therefore help prevent/treat muscle insulin-resistance.
We previously used conditional muscle-specific βactin −/− mice to investigate if the cytoplasmic βactin isoform was required for MGU stimulation by different stimuli, including insulin (Madsen et al., 2018). Surprisingly, no genotype-differences in glucose uptake-responsiveness were observed, except for a reduction in maximal insulin-stimulated glucose uptake in 8-13 weeks old mice. This difference was absent in 18-23 weeks old mice, suggesting this to be an indirect and transient growth-dependent phenotype. A similar growth-dependent phenotype has been reported for other skeletal muscle endpoints such as satellite cell-dependent hypertrophy (Murach et al., 2017).
One possibility to explain the normal MGU in mature adult mouse muscles lacking cytoplasmic βactin might be that cytoplasmic γactin is the predominant isoform regulating insulin-stimulated GLUT4 translocation and MGU, consistent with the stronger progressive myopathy phenotype of γactin −/− (Sonnemann et al., 2006) compared to βactin −/− mice (Prins et al., 2011). To test this, we analysed maximal insulin-stimulated glucose uptake and cell signaling in incubated soleus and extensor digitorum longus (EDL) muscles from young growing 9-14 weeks old and mature adult 18-32 weeks old cohorts of γactin −/− mice and corresponding littermates.

| Ethical approval
All experiments and breeding protocol were approved by the Danish Animal Experiments Inspectorate (license: 2017-15-0201-01311) and carried out in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. The experiments conformed to the principles and regulations as described in the editorial by Grundy (Grundy, 2015).
The conditional γactin −/− mice were littermates from the cross-breeding of hemizygous human αskeletal actin (HSA)-Cre and γactin flox/flox mice originally generated by prof. James M Ervasti, University of Minnesota, USA

New findings
1. What is the central question of this study? Is γactin required for insulin-stimulated glucose uptake in mouse skeletal muscle fibers. 2. What is the main finding and its importance? Reduced insulin-stimulated glucose uptake was observed in young, growing but not mature adult mice, suggesting a growth x genotype interaction. Overall, however, γactin is not required for insulin-stimulated glucose uptake in adult mouse muscle. This improves our mechanistic understanding of insulin-regulated metabolism in adult muscle and emphasizes the need to consider the rapid growth of young mice as a potential confounder in transgenic mouse phenotyping studies. (Sonnemann et al., 2006). In brief, the γactin −/− mice develop a progressive myopathy with muscle weakness, centronucleated fibers and more variable fiber sizes being apparent at 3 months mainly in different type II-fiber dominated muscles but not soleus muscle (Sonnemann et al., 2006). Due to poor breeding performance of the mouse strain and no discernible sex-dependent differences in measured endpoints, the male and female mouse data were pooled for statistical analyses. Wild-type and γactin −/− mice were littermates. Mice were maintained at 22-24°C on a 12-h light/dark cycle with ad libitum access to water and rodent chow diet. Mice were categorized as growing when aged 9-14 weeks and mature adults when aged 18-32 weeks.

| Genotyping
Muscle pieces (5-10 mg) were lysed overnight at 55°C in 100 µl of DirectPCR Lysis Reagent (Tail) (250-101-T, Nordic BioSite) with freshly added 0.2 mg/ml proteinase K (Roche Diagnostics). The lysate was centrifuged at 1250 g for 5 min at room temperature, and the supernatant was kept for DNA extraction. The released DNA was diluted ten-fold in dilution buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and 5 μl of this dilution was then amplified in a 25-μl SYBR Green polymerase chain reaction (PCR) containing 1× Quantitect SYBR Green Master Mix (Qiagen) and 200 nM of each primer (gDNA: TCCAGCACGTGGGTCTTAGAGG and CTGGCAGGTAGGCTCAGCAGGT, γactin flox: GGGCGTGACGAGTCATTTTGTG and GAATCGATGCC CCTGAATTCATAAC). The amplification was monitored in real time using the MX3005P real-time PCR machine (Stratagene). All reactions were performed in triplicate, and the mean C T was used for calculations. Flox abundance was calculated as 2^(C T Flox − C T gDNA ) and normalized to the geometric average of all mice.

2-DG uptake
The mice were anaesthetized (pentobarbital 6 mg and 0.2 mg lidocaine/100 g body weight, intraperitoneal injection), followed by excision of soleus and EDL muscles. Immediately after removal of the muscles the mice were killed by cervical dislocation. The excised muscles were allowed to rest for 30 min in incubation chambers (Multi Myograph System, Danish Myo-Technology) containing 30°C Krebs-Ringer-Henseleit (KRH) buffer with 8 mM mannitol and 2 mM pyruvate as previously described . For insulin stimulation, 30 min rested muscles were stimulated with 60 nM insulin for 20 min. 2-Deoxyglucose (2-DG) transport was measured during the last 10 min of incubation using [ 3 H]-2-Deoxy-D-glucose ([ 3 H]-2-DG) and [ 14 C]-mannitol radioactive tracers as previously described . Immediately after ex vivo incubation, the muscles were washed in ice-cold KRH buffer and snap-frozen in liquid nitrogen.

| Data presentation and statistics
Results are presented as means with individual data points. Statistical testing was performed using unpaired two-tailed t-test or two-way analysis of variance (ANOVA) with or without repeated measures as described in the figure legends. If significant (p < 0.05) ANOVA main effects or interactions were found, Tukey's post hoc test was used for multiple comparisons, unless otherwise noted. Non-significant p values between 0.5 and 0.1 are shown on the figures. The statistical analyses were carried using GraphPad Prism Vers. 9.

| RESULTS
To validate muscle specific removal of γactin we first immunoblotted whole-muscle lysate using a γactin antibody. However, we found similar γactin expression in the wild-type and −/− lysate (Figure 1a), likely due to γactin from other cell types dominating γactin pool in whole-muscle lysate. Thus, we next verified the effective excision of the floxed γactin gene in myofibers by PCR similar to our previous studies (Li et al., 2021;Madsen et al., 2018). This yielded a ~50% reduction in floxed gene content in quadriceps muscle and ~30% in soleus (Figure 1b). A reduction of this magnitude is in agreement with the proportion of myofiber nuclei to total nuclei in skeletal muscle (Wang et al., 2014), and supports that efficient muscle-specific excision of the γactin gene had occurred as intended.
In growing wild-type mice 9 to 14 weeks of age insulin increased the muscle glucose uptake by ~125% in soleus and ~75% in EDL (Figure 2a). In contrast, although the insulin-stimulated muscles on average were ~60% and ~20% higher than basal control soleus and EDL muscles from the γactin −/− mice, the effect of insulin on MGU was neither significant in soleus (p = 0.13) nor in EDL (p = 0.67) (Figure 2a). The observed difference (insulin -corresponding basal MGU) were ~50% lower in soleus and ~70% lower in EDL from γactin −/− compared to wild-type ( Figure 2b). As expected insulin increased phospho-Akt at Thr308 and Ser473 in wild-type and γactin −/− mice in both soleus and EDL muscles (Figure 2c and d). However, γactin −/− mice had lower insulin-stimulated phospho-Akt levels at Ser473 in soleus than the wild-type mice (Figure 2d). Total Akt2 protein expression was similar between groups ( Figure 2e). This prompted us to investigate Akt-mediated F I G U R E 1 Verification of the γactin −/− genotype. (a) Immunoblotting against γactin in isolated soleus and EDL muscles from wildtype and muscle specific γactin −/− mice. (b) Quantification of the flox allele in quadriceps and soleus muscles of wild-type and γactin −/− mice. Two-way repeated measures ANOVA was performed followed by a Sidak's post hoc test. ¤¤¤p < 0.001 ANOVA effects. ME = main effect. ***p < 0.001 effect of genotype. For wild-type and γactin −/− n = 10 and 13 animals respectively F I G U R E 2 Impaired insulin-stimulated glucose uptake in soleus and EDL muscles from growing muscle-specific γactin −/− mice. Isolated soleus and EDL muscles excised from growing (8-14 weeks old) wild-type and muscle specific γactin −/− mice were incubated in constantly oxygenated Krebs-Ringer-Henseleit buffer at 30°C for 30 min and then stimulated with insulin (60 nM) or kept in the unstimulated state for 10 min before a final 10 min of [ 3 H]-2-Deoxy-D-glucose (2-DG) tracer accumulation. (a) glucose transport into soleus and EDL muscles. (b) insulin-stimulated muscle glucose uptake calculated as the difference between insulin and basal-stimulated transport. (c) phospho(p)-Akt Ser473, (d) p-Akt Thr308, (e) Akt2, (f) p-p70S6K Thr389, (g) p-ULK1 Ser757, (h) p70S6K, (i) GLUT4 and (j) Rac1 levels quantified from soleus and EDL. (k) representative blots of the quantified proteins in (c-j). Ser = serine, Thr = threonine, ME = main effect, Int. = interaction, geno. = genotype, ins. = insulin. For (a, c, d, f and g) two-way ANOVA was performed followed by a Tukey's post hoc test in case of ANOVA effect. For (e, h, i, and j), a t test was performed. ¤/¤¤/¤¤¤p < 0.1/0.01/0.001 ANOVA effect. */**/***p < 0.05/0.01/0.001 effect of insulin, #p < 0.05 different from corresponding wild-type group. For soleus wild-type basal, γactin −/− basal, wild-type insulin and γactin −/− insulin n = 17, 14, 12, 11 respectively and for EDL in the same order n = 17, 14, 12, 12 mTORC1-regulated phosphorylation sites in the γactin −/− mice. Interestingly, only the wild-type mice responded to insulin at phospho-p70S6K Thr389 and phospho-ULK1 Ser757 (Figure 2f and g) and particularly the insulinstimulated phospho-p70S6K Thr389 were lower in the EDL muscles from γactin −/− mice when compared to the insulin-stimulated wild-type muscles (Figure 2f). Total p70S6K expression was similar across groups (Figure 2h). Total GLUT4 and Rac1 protein expression were not different between the genotypes in any of the muscles (Figure  2i and j). Representative western blots are presented in Figure 2k. In conclusion, insulin-stimulated MGU and cell signaling readout are less responsive in growing γactin −/− mice compared to wild-type.
In mature adult 18-32 week old mice, insulin increased MGU in soleus muscles from both wild-type and γactin −/− mice (Figure 3a). Insulin also increased MGU in wild-type EDL muscles and tended to increase MGU in γactin −/− muscles (p = 0.08). Specifically, insulin increased the MGU by ~300% in wild-type soleus and ~240% in soleus from γactin −/− mice and for EDL the increases were ~120% and ~60% for wild-type and γactin −/− , respectively ( Figure 3a). The calculated effect of insulin on MGU in soleus and EDL is shown in Figure 3b. Signalingwise, insulin increased phospho-Akt Thr308 and Ser473 in both muscles irrespective of genotype, and with no difference in total Akt2 protein content (Figure 3c-e). Insulin also increased phospho-p70S6K Thr389 and phospho-ULK1 Ser757 irrespective of genotype (Figure 3f and g). Interestingly, total p70S6K protein content was lower in EDL muscles from γactin −/− mice than in corresponding wild-type muscles (Figure 3h), while GLUT4 protein content was higher in soleus muscles from γactin −/− mice than in corresponding wild-type muscles (Figure 3i). Rac1 protein content was not significantly affected by the genotype (Figure 3j). Representative western blots are presented in Figure 3k. In conclusion, insulin-stimulated MGU and cell signaling in appear normal in mature-adult γactin −/− mice although total protein content of GLUT4 and p70S6K may be altered by γactin −/− in soleus and EDL, respectively.

| DISCUSSION
In the present study, we investigated whether γactin rather than βactin might be required for insulinstimulated MGU. In young growing γactin −/− mice, the apparent ability of insulin to stimulate MGU in both soleus and EDL muscle was less compared to wild-type since no significant effects of insulin were observed in the γactin −/− muscles. However, in mature adult mice, the effect of insulin was normal in the soleus γactin −/− muscles. Similarly, in EDL muscles there were no genotype differences on insulin-stimulated MGU and the MGU was significantly increased in the wild-type muscles and tended to increase in the γactin −/− muscles. This could indicate that the impaired insulin-stimulated MGU observed in young γactin −/− mice is secondary to the previously reported impaired muscle growth and maturation (Sonnemann et al., 2006) and does not reflect a requirement for γactin in the actual GLUT4 translocation/glucose uptake process.
Numerous pharmacological and transgenic rodent studies have previously suggested that insulin-stimulated glucose uptake in adult skeletal muscle is dependent on the cortical actin-cytoskeleton. Pharmacologically, this is evidenced by the marked reduction of insulinstimulated GLUT4 translocation and glucose uptake by the depolymerizing agent latrunculin B in ex vivo incubated rat epitrochlearis muscle (Brozinick et al., 2004) and of insulin, contraction and passive stretchstimulated glucose uptake in ex vivo incubated mouse soleus and EDL muscle (Sylow et al., 2013a,b;Sylow et al., 2015). In terms of transgenic mouse models, a number of KO models lacking proteins linked to actin regulation have been shown to display reduced insulin-stimulated MGU and/or GLUT4 translocation. The proteins investigated in KO mice include 12/15-Lipoxygenase (20 weeks old mice) (Vahsen et al., 2006), β1 integrin (14 weeks old mice) (Zong et al., 2009), Myo1c (8-10 weeks old mice) (Toyoda et al., 2011), Tropomyosin Tpm3.1 (12-16 weeks old mice) (Kee et al., 2015), dynamin-2 (8-12 weeks old mice) (González-Jamett et al., 2017), F I G U R E 3 Similar insulin-induced glucose uptake in soleus and EDL muscles from adult wild-type and muscle-specific γactin −/− mice. Isolated soleus and EDL muscles excised from adult (18-32 weeks old) wild-type and muscle specific γactin −/− mice were incubated in constantly oxygenated Krebs-Ringer-Henseleit buffer at 30°C for 30 min and then stimulated with insulin (60 nM) or kept in the unstimulated state for 10 min before a final 10 min of [ 3 H]-2-Deoxy-D-glucose (2-DG) tracer accumulation. (a) glucose transport into soleus and EDL muscles. (b) insulin-stimulated muscle glucose uptake calculated as the difference between insulin and basal-stimulated transport. (c) phospho(p)-Akt Ser473, (d) p-Akt Thr308, (e) Akt2, (f) p-70S6K Thr389, (g) p-ULK1 Ser757, (h) p70S6K, (i) GLUT4 and (j) Rac1 levels quantified from soleus and EDL. (k) Representative blots of the quantified proteins in (c-j). Ser = serine, Thr = threonine, ME = main effect, Int. = interaction, geno. = genotype, ins. = insulin. For (a, c, d, f and g) two-way ANOVA was performed followed by a Tukey's post hoc test in case of ANOVA effect. For (e, h, i and j) a t test was performed. ¤/¤¤¤p < 0.1/0.001 ANOVA effect. **/***p < 0.01/0.001 effect of insulin, ##p < 0.01 different from corresponding wild-type group. For soleus wild-type basal, γactin −/− basal, wild-type insulin and γactin −/− insulin n = 12, 18, 12, 19 respectively and for EDL in the same order n = 12, 17, 12, 19 βcatenin (drug-inducible model, 10-20 weeks old mice at time of experiments) (Li et al., 2016), Rac1 (druginducible model, 14-18 weeks at time of experiments (Sylow et al., 2013a,b;Sylow et al., 2015) Rac1 (constitutive, 8-10 weeks old mice) (Ueda et al., 2010) and PAK1 (16-24 weeks old mice) (Wang et al., 2011), although the PAK1 KO mouse phenotype does not seem robust (no difference observed in 10-24 weeks old mice) (Møller et al., 2020). In light of our age-dependent phenotype, we specifically consider it possible that a genotype x growth interaction may have produced some of the observed insulin-resistant phenotypes in the studies using young mice. Also, the reduced insulin-stimulated glucose uptake could also reflect the involvement of these proteins in other processes than actin remodelling. For instance, although a role of Rac1-stimulated actin remodelling is possible, Rac1 appears to regulate GLUT4 translocation and MGU during in vivo exercise via NOX2 mediated production of reactive oxygen species (Henríquez-Olguin et al., 2019). Nonetheless, these studies as a whole indicate that the cortical actin cytoskeleton is involved in insulin-stimulated GLUT4 translocation and glucose uptake in adult muscle similar to cultured cells.
The most obvious explanation of why neither βactin (Madsen et al., 2018) nor γactin are required for insulinstimulated glucose uptake in mature adult muscle would be functional redundancy between the cytoplasmic β and γ isoforms. Cytoplasmic β and γactin are 99% homologous and can produce heteromeric F-actin in vitro (Bergeron et al., 2010;Müller et al., 2013). Muscle-specific β and γactin −/− mice also develop similar phenotypes with gradually developing myopathies characterized by accelerated myofiber turnover and weakness (Prins et al., 2011;Sonnemann et al., 2006), which might also indicate overlapping functions. However, there is also evidence that βand γactin are part of distinct actin structures in different cell types (Perrin & Ervasti, 2010;Tondeleir et al., 2009;Vedula & Kashina, 2018), making the degree to which βand γactin work interchangeably in vivo a continuous point of debate. A study in muscle-specific β/γactin double −/− mice, preferably drug-inducible in adult animals to minimize compensation during development, could be performed to directly test this. In contrast, cytoplasmic actins and muscle αactins, although also highly homologous (~93%), are generally thought to be non-redundant in their functions (Perrin & Ervasti, 2010;Tondeleir et al., 2009;Vedula & Kashina, 2018), making skeletal muscle αactin less likely to compensate for the absence of γactin. That said, ~2000x overexpression of γactin in mouse muscle has also been shown to incorporate readily in skeletal muscle αactin filaments, although this cannot rescue the reduced life-span of skeletal muscle αactin KO mice (Murach et al., 2017). Taken together, we speculate that β and γactin in particular may be functionally redundant in the regulation of insulin-stimulated MGU.
What might explain the smaller effect of insulin on MGU in young γactin −/− mice compared to wild-type? Although the phosphorylation level of Akt Thr308 was similar between wild-type and γactin −/− mice, the genedeleted mice had lower insulin-stimulated Akt Ser473 in soleus. This might reflect inhibition of insulin-signaling secondary to inflammation and potentially other mechanisms which converge onto inhibition of IRS1 function (DeFronzo et al., 2015). A decreased structural integrity of γactin −/− muscle fibers might have exacerbated these mechanisms in young growing mice. However, increased muscle inflammation was not observed in γactin −/− mice although the specific age and muscle investigated was not reported (Sonnemann et al., 2006). Also, whether the relative subtle reduction in insulin-signaling contributes to muscle insulin-resistance is unclear due to the large spare capacity of Akt-signaling to GLUT4 translocation (Hoehn et al., 2008). A related possibility might be increased production of reactive oxygen species (ROS). Hence, a ROS-dependent loss of transverse microtubules has been described in β and γactin −/− muscles, partly depedendent on NOX2 (Nelson et al., 2020). Both NOX2 and other ROS sources such as mitochondria have been implicated in the development of insulin-resistance, with considerable cross-talk known to occur between the two (Henríquez-Olguín et al., 2019). Microtubules are well-described to serve as tracks for long-range GLUT4 trafficking (Klip et al., 2019;Sylow et al., 2021). NOX2-dependent ROS production is also known to increase with mechanical stress in cardiomyocytes and skeletal muscle (Pal et al., 2013;Prosser et al., 2011Prosser et al., , 2013 and contractile activity in vitro and in vivo (Henríquez-Olguin et al., 2019;Pal et al., 2013;Sakellariou et al., 2013). Based on this, increased ROS production due to mechanical hyperactivation of NOX2 or from other sources in growing γactin −/− muscle could contribute to the development of muscle insulin resistance.
Unfortunately, we were unable to directly demonstrate a reduction in γactin in the muscles from the −/− mice using immunoblotting in the present study.
This could indicate that the γactin gene excission was ineffective in our −/− mice. However, since (a) the used model was previously validated as being a muscle-fiber specific protein knockout mouse (Sonnemann et al., 2006) and (b) we observed the expected level of reduction in floxed gene content, we consider it more likely that γactin from non-muscle cell types dominated the γactin signal in whole-muscle lysate. Another study found that knocking out NOX4 specifically in endothelial cells caused a ~80% reduction in whole-muscle NOX4 mRNA (Specht et al., 2021), clearly showing how other cell types than muscle fibers can dominate the whole-muscle lysate signal. Thus, we believe that our used mice were γactin −/− although we acknowledge our inability to directly show this.
In summary, γactin −/− transiently impaired insulinstimulated MGU in young, growing mice but not mature adult mice. The impaired glucose uptake phenotype in young γactin −/− mice is thus likely an indirect effect provoked by rapid growth of the more fragile muscle fibers in γactin −/− mice. Since neither βactin nor γactin gene deletion alone reduced insulin-stimulated glucose uptake, we consider it likely that these actin isoforms are functionally redundant in adult skeletal muscle.