To investigate the mechanism underlying how diabetes mellitus promotes skeletal muscle decline, we utilized a mouse model of diabetes induced by streptozotocin (STZ). Intraperitoneal injection of STZ in mice resulted in marked hyperglycemia (~25 mM) accompanied by hypoinsulinemia within 14 days (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.124952DS1) as well as a loss of body mass (Supplemental Figure 1C) and skeletal muscle mass (Figure 1A) apparent at 21 days. Given that skeletal muscle mass normalized by body mass was decreased in the treated mice, the loss of muscle mass appears to be specific rather than the result of a generalized increase in catabolism.

Figure 1 Skeletal muscle atrophy associated with diabetes is prevented in mice with skeletal muscle deficiency of KLF15. (A–D) Ratio of gastrocnemius or extensor digitorum longus (EDL) muscle mass to body mass (A; n = 12), quantitative reverse transcription PCR (RT-PCR) analysis of Klf15 mRNA in gastrocnemius (B; n = 6), immunoblot analysis of KLF15 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, loading control) in soleus muscle (nuclear fraction from 3 mice was loaded in 1 lane) (C; n = 2), and quantitative RT-PCR analysis of atrophy-related gene expression in gastrocnemius (D; n = 6) for control mice and diabetes-model mice at 21 days after the onset of STZ administration. (E–H) Ratio of muscle mass to body mass (E; n = 12), histological determination of muscle fiber area in EDL (F and G), and atrophy-related gene expression in gastrocnemius (H; n = 6) for WT or M-KLF15KO mice at 21 days after the onset of STZ administration or vehicle (Cont.) injection. In G, the areas of 500 fibers were measured in each condition. All quantitative data are means ± SEM for the indicated numbers of mice. *P < 0.05, **P < 0.01; NS, not significant. Unpaired t test (A, B, and D) or 2-way ANOVA with Bonferroni’s post hoc test (E, G, and H).

Krüppel-like factor 15 (KLF15), a member of the KLF family of transcription factors, regulates carbohydrate, lipid, and protein metabolism (14–18). The expression of KLF15 is upregulated in the liver of diabetic mice and is thought to contribute to their hyperglycemia (15), suggestive of a pathological role for this protein in diabetes. Furthermore, the mRNA abundance of KLF15 is increased by glucocorticoids, and overexpression of KLF15 in muscle cells upregulates genes related to muscle atrophy (19), suggesting that KLF15 is implicated in muscle atrophy induced by glucocorticoids. These findings prompted us to investigate the role of KLF15 in muscle atrophy associated with diabetes.

Different from skeletal muscle atrophy induced by glucocorticoids, the amount of Klf15 mRNA in skeletal muscle of mice with STZ treatment was unaltered (Figure 1B). The abundance of KLF15 protein, however, was increased in skeletal muscle of our diabetic model mice at 21 days after the onset of STZ administration (Figure 1C). The expression of genes related to muscle atrophy — Atrogin1, Murf1, Foxo3a, Prodh, and Tdo2 — was also increased by STZ treatment (Figure 1D).

To examine the effect of KLF15 loss on muscle atrophy, we generated mice lacking KLF15 specifically in skeletal muscle (M-KLF15KO mice) by crossing mice harboring a floxed allele of Klf15 (Supplemental Figure 2) with those expressing Cre recombinase under the control of the myosin light chain 1f gene (Mlc1f) promoter (20). Body mass, skeletal muscle mass, and the cross-sectional area of lower limb muscle (Supplemental Figure 3) as well as blood glucose and plasma insulin levels (Supplemental Figure 4, A and B) were unaltered in the mutant mice. Treatment of M-KLF15KO mice with STZ also resulted in hyperglycemia with an extent and time course similar to that in control mice (Supplemental Figure 4C). In contrast, STZ-induced muscle atrophy — as reflected by changes in muscle mass (Figure 1E), the cross-sectional area of lower limb muscle (Supplemental Figure 5A), and muscle fiber area evaluated histologically (Figure 1, F and G) — was prevented in M-KLF15KO mice. Furthermore, whereas STZ treatment increased and decreased the proportions of small and large muscle fibers, respectively, in wild-type (WT) mice, no such effect was apparent in M-KLF15KO mice (Supplemental Figure 5B). In addition, the STZ-induced increase in the expression of muscle atrophy–related genes was abolished in the mutant mice (Figure 1H). The abundance of proteins encoded by muscle atrophy–related genes Atrogin1 and Foxo3a was also increased in the skeletal muscle of STZ-treated mice, and the STZ-induced increase was inhibited in M-KLF15KO mice (Supplemental Figure 6A). Furthermore, muscle function assessed by a passive wire-hang test as well as by the tolerance for maximum speed and the time for exhaustion on a treadmill exercise load test was decreased in STZ diabetic mice and the STZ-induced decline in muscle function was prevented in M-KLF15KO mice (Supplemental Figure 6, B and C). Together, these results thus indicated that KLF15 is responsible for muscle atrophy as well as decline in muscle function in this model of diabetes.

Both hyperglycemia and hypoinsulinemia accompany the STZ-induced diabetes. We have found that exposure of mouse C2C12 myotubes to glucose increased the amount of KLF15 protein in a concentration- and time-dependent manner (Figure 2A and Supplemental Figure 7A), without affecting that of Klf15 mRNA (Figure 2B), as was seen in skeletal muscle of mice treated with STZ. Furthermore, exposure of the cells to glucose increased the expression of muscle atrophy–related genes Atrogin1 and Murf1 (Figure 2C). In contrast, treatment of the myotubes with insulin had no effect on the amount of Klf15 mRNA or the encoded protein (Supplemental Figure 7, B and C), suggesting that hyperglycemia is directly responsible for the upregulation of KLF15 protein in skeletal muscle of diabetic mice.

Figure 2 Glucose decreases the ubiquitination of, and increases the protein abundance of, KLF15. (A and B) Immunoblot analysis of KLF15 protein (A) and quantitative RT-PCR analysis of Klf15 mRNA (B; n = 4) in C2C12 myotubes exposed to the indicated concentrations of glucose for 24 hours. In A, a representative blot and quantitative data (n = 4) are shown in the left and right panels, respectively. (C) Quantitative RT-PCR analysis of muscle atrophy–related gene expression for myotubes treated as in A. n = 6. (D) Immunoblot analysis of KLF15 in myotubes exposed to 5 or 25 mM glucose in the absence or presence of 15 μM MG132 for 6 hours. A representative blot and quantitative data (n = 2) are shown in the left and right panels, respectively. (E) C2C12 myoblasts expressing HA-ubiquitin (Ub) and FLAG-KLF15 were incubated with 5 or 25 mM glucose for 24 hours and then subjected to immunoprecipitation (IP) with antibodies against FLAG. The resulting precipitates were analyzed by immunoblot with antibodies against HA to detect polyubiquitinated [-(Ub) n ] KLF15, and the original cell lysates were analyzed by immunoblot with antibodies against FLAG. Representative data from 3 independent experiments are shown. All quantitative data are means ± SEM for the indicated numbers of independent experiments. *P < 0.05; NS, not significant. Two-way ANOVA with Bonferroni’s post hoc test (A and B) or unpaired t test (C).

Treatment of C2C12 myotubes with the proteasome inhibitor MG132 also increased the amount of KLF15 protein in the presence of a low glucose concentration (5 mM), and a high concentration of glucose (25 mM) did not further increase KLF15 abundance in the presence of MG132 (Figure 2D). We also found that high glucose inhibited the polyubiquitination of FLAG epitope–tagged KLF15 in C2C12 myotubes also expressing hemagglutinin epitope–tagged (HA-tagged) ubiquitin (Figure 2E), suggesting that glucose upregulates KLF15 protein by inhibiting its ubiquitin-dependent degradation and thereby increases the expression of genes related to muscle atrophy.

To identify the E3 ubiquitin ligase responsible for the glucose-dependent change in polyubiquitination of KLF15, we investigated the expression of genes encoding E3 ubiquitin ligases in skeletal muscle of mice with STZ-induced diabetes. Microarray analysis showed that the expression of 27 genes encoding E3 ubiquitin ligases was downregulated in skeletal muscle of the diabetic mice (Supplemental Table 1). Analysis of a public database of gene expression revealed that 8 of these 27 genes are highly expressed in skeletal muscle (Supplemental Table 1). Among the proteins encoded by these 8 genes, WW domain–containing E3 ubiquitin protein ligase 1 (WWP1) and neural precursor cell–expressed developmentally downregulated 4 (NEDD4) were previously shown to interact with other members of the KLF family (21, 22). Coimmunoprecipitation analysis revealed that WWP1, but not NEDD4, interacted with KLF15 in transfected COS-7 cells (Supplemental Figure 8A). Furthermore, WWP1 was found to be downregulated at both mRNA and protein levels in skeletal muscle of STZ diabetic mice (Supplemental Figure 8, B and C) as well as in C2C12 myotubes treated with glucose (Figure 3, A and B). Insulin did not affect the amount of Wwp1 mRNA in C2C12 myotubes (Supplemental Figure 8D). The decrease in WWP1 protein (~75% reduction) (Supplemental Figure 8C) in skeletal muscle in response to STZ treatment was greater than that of Wwp1 mRNA (~20% reduction) (Supplemental Figure 8B). Greater reduction in the level of WWP1 protein than that of Wwp1 mRNA was also observed in C2C12 myotubes treated with glucose (Figure 3, A and B). Treatment of MG132 with C2C12 myotubes prevented glucose-induced decrease in the WWP1 protein (Supplemental Figure 8E) without the increase in Wwp1 mRNA (Supplemental Figure 8F), suggesting that glucose stimulates the degradation of WWP1 protein through the proteasome pathway.

Figure 3 WWP1 regulates the polyubiquitination and abundance of KLF15. (A and B) Quantitative RT-PCR analysis of Wwp1 mRNA (A; n = 4) and immunoblot analysis of WWP1 protein (B) in C2C12 myotubes exposed to the indicated concentrations of glucose for 24 hours. In B, a representative blot and quantitative data (n = 4) are shown in the left and right panels, respectively. (C) COS-7 cells transfected with vectors for HA-Ub, KLF15, and either WT or C890A mutant forms of WWP1 were subjected to immunoprecipitation with antibodies against KLF15. The resulting precipitates and the original cell lysates were analyzed by immunoblot as indicated. (D) Immunofluorescence analysis of KLF15 and WWP1 in C2C12 myoblasts transfected with vectors for these proteins and exposed to 15 μM MG132 for 6 hours as indicated. Nuclei were stained with DAPI. Scale bar: 10 μm. In C and D, representative data from at least 3 independent experiments are shown. All quantitative data are means ± SEM for the indicated numbers of independent experiments. *P < 0.05, **P < 0.01. Two-way ANOVA with Bonferroni’s post hoc test (A and B).

Forced expression of WWP1 in COS-7 cells reduced the abundance (Supplemental Figure 9A) and increased the polyubiquitination (Figure 3C) of KLF15, whereas expression of a catalytically inactive mutant (C890A) of WWP1 had no effect on KLF15 polyubiquitination (Figure 3C). Immunofluorescence analysis of transfected C2C12 myoblasts revealed that KLF15 immunoreactivity was localized predominantly to the nucleus, with smaller amounts also being detected in the cytosol (Figure 3D). Treatment of the cells with MG132 increased the amount of KLF15 immunoreactivity in the cytosol. In contrast, WWP1 was localized exclusively to the cytosol, and its forced expression resulted in the complete or partial loss of KLF15 in the cytosol and nucleus, respectively, with these effects being prevented in the presence of MG132 (Figure 3D). These results thus suggested that WWP1 interacts with KLF15 in the cytosol, which results in the downregulation of KLF15 in both the cytosol and the nucleus.

We next transfected a vector expressing both enhanced green fluorescent protein (EGFP) and WWP1 or a control vector only expressing EGFP into C2C12 myotubes, and evaluated the effect of glucose on the size of transfected myotubes (detected by EGFP fluorescence). Treatment with glucose decreased the diameters of the myotubes transfected only with EGFP, whereas the effect of glucose was inhibited in myotubes transfected with EGFP plus WWP1 (Supplemental Figure 9, B and C). These results suggest that glucose induces atrophy of the myotubes in a WWP1-dependent manner.

Depletion of WWP1 in C2C12 myotubes by transfection with small interfering RNA (siRNA) (Figure 4A) increased the amount of KLF15 protein (Figure 4B) as well as that of Atrogin1 mRNA (Figure 4C), indicating that the downregulation of WWP1 leads to the upregulation of KLF15 protein. To deplete WWP1 in skeletal muscle of mice, we constructed adeno-associated virus (AAV) vectors encoding both a short hairpin RNA (shRNA) specific for Wwp1 mRNA (Supplemental Figure 9D) and EGFP to visualize virus-infected muscle fibers. Whereas injection of skeletal muscle (tibialis anterior) with the AAV vector led to only an approximately 40% reduction of Wwp1 mRNA in whole-muscle extracts in both WT and M-KLF15KO mice (Figure 4D), fluorescence microscopic analysis revealed that only a part of the muscle fibers was infected with the virus (positive for GFP) (Figure 4E), suggesting that the reduction of the amount of Wwp1 mRNA in the infected muscle fibers was greater than the extent evaluated with the whole-muscle extracts. The cross-sectional area of virus-infected muscle fibers (detected by EGFP fluorescence) was significantly smaller in WT mice injected with the AAV encoding WWP1 shRNA than in those injected with a control AAV (Figure 4, E and F). In contrast, such depletion of WWP1 did not significantly decrease the cross-sectional area of skeletal muscle fibers in M-KLF15KO mice. Infection with the AAV encoding WWP1 shRNA also increased the expression of Atrogin1 and Murf1 in whole-muscle extracts of WT mice but not of M-KLF15KO mice (Figure 4G). Collectively, these results thus indicated that downregulation of WWP1 promotes skeletal muscle atrophy in live animals in a KLF15-dependent manner.

Figure 4 WWP1 regulates skeletal muscle atrophy in a KLF15-dependent manner. (A) Quantitative RT-PCR analysis of Wwp1 mRNA in C2C12 myotubes transfected with control or WWP1 siRNA. n = 6. (B and C) Immunoblot analysis of KLF15 (B) and quantitative RT-PCR analysis of Atrogin1 and Murf1 mRNAs (C; n = 6) in C2C12 myotubes transfected as in A. In B, a representative blot and quantitative data (n = 6) are shown in the left and right panels, respectively. (D) Quantitative RT-PCR analysis of Wwp1 mRNA in tibialis anterior muscle of WT or M-KLF15KO mice injected with AAVs encoding EGFP with or without (Cont.) WWP1 shRNA. n = 4. (E) Fluorescence microscopic detection of EGFP-positive muscle fibers of mice infected as in D. Scale bar: 100 μm. (F) Cross-sectional area of EGFP-positive muscle fibers determined from images as in E. The areas of 100 fibers were measured in each condition. (G) Quantitative RT-PCR analysis of Atrogin1 and Murf1 mRNAs in tibialis anterior muscle of mice infected as in D. n = 4. All quantitative data are means ± SEM for the indicated numbers of independent experiments (A–C) or mice (D, F, and G). *P < 0.05, **P < 0.01; NS, not significant. Unpaired t test (A–C) or 2-way ANOVA with Bonferroni’s post hoc test (D, F, and G).

We lastly examined whether hyperglycemia is responsible for declines of skeletal muscle mass in a chronic model of diabetes. Akita mice, which develop diabetes as a result of pancreatic β cell dysfunction (23), manifested hyperglycemia as early as 5 weeks of age (Figure 5A). Although skeletal muscle mass at 6 weeks of age was similar in Akita and WT mice, that at 10 weeks was smaller in the former animals (Supplemental Figure 10), indicating that this chronic model of diabetes develops age-dependent skeletal muscle atrophy. Inhibitors of sodium-glucose cotransporter 2 (SGLT2) such as empagliflozin lower blood glucose levels in an insulin-independent manner (24) and thus are a useful tool for the evaluation of the pathological significance of blood glucose. Administration of empagliflozin thus lowered the blood glucose concentration of Akita mice to a level similar to that of WT mice within 1 week, without affecting the plasma insulin level (Figure 5, A and B). Empagliflozin prevented the reduction in skeletal muscle mass and muscle fiber area apparent in control Akita mice (Figure 5, C–E). The amount of Wwp1 mRNA in skeletal muscle of Akita mice was smaller than that in WT mice but was increased by empagliflozin treatment (Figure 6A). The amount of KLF15 protein was increased in skeletal muscle of Akita mice, but this increase was attenuated by the administration of empagliflozin (Figure 6B). The amount of Klf15 mRNA in skeletal muscle was similar in Akita and WT mice and was not affected by empagliflozin (Figure 6C). The expression of muscle atrophy–related genes was also increased in skeletal muscle of Akita mice in a manner sensitive to empagliflozin treatment (Figure 6C). Together, these results suggested that a mechanism similar to that operative in STZ diabetic mice contributes to the age-dependent muscle atrophy in this chronic model of diabetes.

Figure 5 SGLT2 inhibitor ameliorates age-dependent muscle atrophy in Akita mice. (A and B) Blood glucose (A; n = 8) and plasma insulin (B; n = 4) levels of WT or Akita mice fed a normal diet (ND) or a diet containing empagliflozin (Empa) beginning at 5 weeks of age. (C–E) Skeletal muscle/body mass ratios (C; n = 8) as well as histological determination of muscle fiber area in tibialis anterior muscle (D and E) of mice as in A at 10 weeks of age. In E, the areas of 200 fibers were measured in each condition. All quantitative data are means ± SEM for the indicated numbers of mice. *P < 0.05, **P < 0.01 for Akita Empa versus Akita ND (A) or for the indicated comparisons. NS, not significant. Two-way ANOVA with Bonferroni’s post hoc test (A–C, and E).