Undercarboxylated Osteocalcin Improves Insulin‐Stimulated Glucose Uptake in Muscles of Corticosterone‐Treated Mice

Short‐term administration of glucocorticoids (GCs) impairs muscle insulin sensitivity at least in part via the reduction of undercarboxylated osteocalcin (ucOC). However, whether ucOC treatment reverses the GC‐induced muscle insulin resistance remains unclear. To test the hypothesis that ucOC directly ameliorates impaired insulin‐stimulated glucose uptake (ISGU) induced by short‐term GC administration in mice muscle and to identify the molecular mechanisms, mice were implanted with placebo or corticosterone (CS) slow‐release pellets. Two days post‐surgery, insulin‐tolerance tests (ITTs) were performed. On day 3, serum was collected and extensor digitorum longus (EDL) and soleus muscles were isolated and treated ex vivo with vehicle, ucOC (30 ng/mL), insulin (60 µU/mL), or both. Circulating hormone levels, muscle glucose uptake, and muscle signaling proteins were assessed. CS administration reduced both serum osteocalcin and ucOC levels, whole‐body insulin sensitivity, and muscle ISGU in EDL. Ex vivo ucOC treatment restored ISGU in CS‐affected muscle, without increasing non‐insulin‐stimulated glucose uptake. In CS‐affected EDL muscle, ucOC enhanced insulin action on phosphorylated (p‐)protein kinase B (Akt)Ser473and the p‐extracellular signal‐regulated kinase isoform 2 (ERK2)Thr202/Tyr204/total (t)ERK2 ratio, which correlated with ISGU. In CS‐affected soleus muscle, ucOC enhanced insulin action on p‐mammalian target of rapamycin (mTOR)Ser2481, the p‐mTORSer2481/tmTOR ratio, p‐Akt substrate of 160kD (AS160)Thr642, and p‐protein kinase C (PKC) (pan)Thr410, which correlated with ISGU. Furthermore, p‐PKC (pan)Thr410 correlated with p‐AktSer473 and p‐AS160Thr642. ucOC exerts direct insulin‐sensitizing effects on CS‐affected mouse muscle, likely through an enhancement in activity of key proteins involved in both insulin and ucOC signaling pathways. Furthermore, these effects are muscle type‐dependent. © 2019 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc.


Introduction
G lucocorticoid (GC) treatment is commonly used in the clinical setting to treat inflammatory or immunological pathologies. (1) It is well-documented that frequent and longterm GC treatment induces adverse side-effects including metabolic disorders such as obesity, glucose intolerance, and type 2 diabetes (T2DM). (1,2) However, recent evidence suggests that even short-term administration of GCs results in the development of skeletal muscle insulin resistance: the initiating and primary defect of T2DM. (3,4) Thus, the investigation of the etiology and therapeutic intervention for short-term GC administration-induced muscle insulin resistance is of great importance for the prevention and control of T2DM during GC treatment.
The adverse metabolic effects of short-term GC treatment on muscle may be partly based on the functional perturbation of bone cells, leading to the reduction of circulating ucOC-a hormone secreted by osteoblasts. Emerging evidence shows that ucOC favors muscle insulin sensitivity, at least in mice. (5)(6)(7) ucOC administration not only enhances muscle insulin sensitivity in healthy animals, but also partially restores insulin sensitivity in insulin-resistant animals that commonly exhibit low ucOC levels. (8)(9)(10) We recently reported that acute GC-induced insulin resistance in humans (20 mg of prednisolone) strongly correlated with suppressed serum ucOC and ucOC-associated signaling pathways in muscle. (3) Furthermore, it has also been reported that the restoration of circulating ucOC during CS administration, via heterotopic expression, rescues whole-body insulin resistance in mice. (10) However, whether ucOC treatment can directly ameliorate GC-induced muscle insulin resistance and the underlying mechanisms still remains unexplored.
Skeletal muscle is a heterogeneous tissue containing different fiber types with various molecular, mechanical, and metabolic traits. (11,12) Glycolytic muscles, such as extensor digitorum longus (EDL) muscle, predominantly contain type II fibers (98% type II fibers in mouse EDL muscle (13) ) and rely largely on glycolytic metabolism to support rapid muscular contraction. In contrast, oxidative muscles, such as soleus muscle, are enriched with type I fibers (55% type I fibers in mouse soleus muscle (13) ) and rely primarily on oxidative metabolism pathways to support lowintensity movements and body posture maintenance. Our recent findings suggest that ucOC likely has effects on insulin-stimulated glucose uptake (ISGU) of healthy mouse muscle in a muscle typespecific manner. For example, ucOC enhances ISGU in soleus muscle only at rest, whereas in EDL muscle ucOC only enhances ISGU following ex vivo muscle contraction. (5,14) Therefore, the possible rescuing effect of ucOC on insulin-resistant muscle, as well as the molecular mechanism, may also be muscle type-dependant.
Previous studies have suggested that the beneficial effect of ucOC on glucose uptake likely occurs through enhanced activation (phosphorylated/total ratio) of mTOR complex 2 (mTORC2)-Akt-AS160 cascade within the insulin signaling pathway, as well as signaling proteins within the ucOC signaling pathway (ERK, adenosine monophosphate kinase [AMPK], and PKC) (3,5,(14)(15)(16)(17) (Supplemental Fig. S1). Furthermore, it was reported that the effect is also likely via an increase in the abundance of these signaling proteins, including the postulated ucOC receptor G proteincoupled receptor, class C, group 6, member A (GPRC6A) (Supplemental Fig. S1). (14,15,18) However, the exact molecular mechanisms of ucOC on muscle insulin resistance, particularly GC-induced insulin resistance, are largely unknown.
We tested the hypotheses that (1) ex vivo ucOC treatment enhances ISGU in CS-affected mouse EDL and/or soleus muscle; and (2) in these muscles, ucOC treatment enhances activation and/or expression of key proteins in the insulin and ucOC signaling pathways.

Animals
Eight-week-old male C57BL/6 J mice (n = 18; Animal Resources Centre, WA, Australia) were housed with a 12-hour light/12hour dark cycle and fed standard laboratory chow (Glen Forrest, WA, Australia) and water ad libitum until 9-to 12-weeks-old (body weight 24.7 ± 1.4 g). The study was approved by the Animal Ethics Committee of Victoria University (Project code: 14/009). The mice for each group in this study were randomly allocated into placebo and CS groups by an animal technician who was independent from the research.

Slow-release pellet implantation
After one week of acclimatization, animals were randomly allocated into CS or placebo groups. Mice were subcutaneously implanted with slow-release pellets containing either 1.5 mg CS (n = 9) or placebo (n = 9; Innovative Research of America, Sarasota, FL, USA), following a previously described protocol. (10) The average delivery rate of CS was about 2.89 mg/kg/d.

Insulin tolerance test (ITT)
Two days after pellet implantation, mice were fasted for 6 hours prior to a baseline blood glucose reading using glucose strips and an Accu Chek Glucometer (Roche, Basel, Switzerland), followed by an intraperitoneal (i.p.) injection of insulin at 0.75 U/kg body weight (Sigma-Aldrich, St. Louis, MO, USA). Blood samples were then collected at 15, 30, 60, 90, and 120 min postinsulin injection via tail prick for blood glucose measurements.

Muscle dissection and serum preparation
Three days after pellet implantation, mice were fasted for 6 hours before deep anesthetization with 60 mg/kg i.p. pentobarbital. EDL and soleus muscle of both legs were excised and evenly divided into halves longitudinally. After muscle isolation, blood samples were collected via heart puncture and fasting blood glucose levels were measured. Then blood samples were left on ice for 30 min, after which they were spun in a centrifuge at 16,000 g at 4°C for 10 min for serum samples. Serum was stored at −80°C until analysis.

2-Deoxyglucose (2DG) uptake measurement and sample homogenization
The methods for muscle 2DG uptake and sample homogenization have previously been described. (14) Briefly, after ex vivo treatment, muscle samples were transferred to chambers containing carbogenated KHB with 2 mM 2-deoxy-d-[1,2-3 H]glucose (PerkinElmer, Waltham, MA, USA) and 16 mM d-[1-14 C] mannitol (PerkinElmer). After 10 min, samples were rapidly rinsed with ice-cold KHB buffer, then snap-frozen via liquid nitrogen. On the day of sample processing, muscle samples were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Beverly, MA, USA) with Inhibitor Cocktail (Cell Signaling Technology) and 100 mM dithiothreitol (Sigma-Aldrich) using TissueLyser II (QIAGEN, Hilden, Germany). Half of the lysate was pipetted into a vial with scintillation cocktail for scintillation counting by Tri-Carb 2910TR Liquid Scintillation Analyzer (PerkinElmer); the other half was used in Western blotting.

Serum hormone measurement
Total osteocalcin was measured using an ELISA kit purchased from Immutopics (San Clemente, CA, USA) according to the manufacturer's instructions. ucOC levels were detected following hydroxyapatite binding as previously described (19) using the same ELISA kit. Serum insulin and CS were measured using ELISA kits purchased from Crystal Chem (Elk Grove Village, IL, USA) based on the kits' instructions.

Statistical analysis
Fisher's LSD test following 2-way ANOVA with repeated measures was used to determine significant differences between placebo and CS groups at each time point in the ITT, and between placebo and CS muscle with the same ex vivo treatment in results of glucose uptake and Western blotting. The significance level calculated from Fisher's LSD test is shown as # (p < 0.05) and ## (p < 0.01).
T tests were used to analyze the differences in serum hormones and fasting blood glucose level between placebo and CS groups, the significance found in the test is shown as * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) ( Fig. 1A-E).
Paired t tests were used to determine significant differences in glucose uptake and quantified Western blot results between muscle split within the same animals, the significance in the test compared with the control group is shown as * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) (  The effect of ex vivo undercarboxylated osteocalcin (ucOC) treatment on proteins in insulin signaling pathway in placebo and CS-affected extensor digitorum longus (EDL) muscle. The phosphorylation, total expression, and phosphor/total ratio of mTOR Ser2481 and mTOR Ser2448 (A-E), Akt Ser473 (F-H), as well as AS160 Thr642 and AS160 Ser588 (I-M) were analyzed in placebo and corticosterone-(CS-) affected EDL muscle. ** and *** represent p < 0.01 and p < 0.001 in paired t tests compared with control-treated muscle. "a" and "b" represent p < 0.05 and p < 0.01 in paired t tests compared with insulin alone-treated muscle. # represents p < 0.05 and ## represents p < 0.01 between placebo and CS muscle with the same ex vivo treatment, using Fisher's LSD following 2-way ANOVA with repeated measures. Fig. 8), and the significance in the test compared with the insulin group is shown as "a" (p < 0.05) and "b" (p < 0.01) (Fig. 1G, I; Fig. 2; Fig. 4; Fig. 6; Fig. 8).
Spearman's correlation was first performed between glucose uptake and increased variables of signaling proteins in CS-affected muscle (insulin versus ucOC + insulin), then Spearman's correlation was carried out between variables of insulin and ucOC signaling proteins that were associated with glucose uptake.
All data are reported as mean ± SEM.

Results
The effect of CS on circulating hormones and the effect of ex vivo ucOC treatment on muscle glucose uptake Compared with mice implanted with placebo pellets, mice implanted with CS pellets had 39.5% higher serum CS levels ( Fig. 1A; p < 0.05). Furthermore, CS-treated mice exhibited 57.3% lower OC levels ( Fig. 1B; p < 0.001), and 35.5% lower ucOC levels ( Fig. 1C; p < 0.05) compared with placebo animals. Serum insulin levels were 2.6-fold higher in CS-treated mice than placebo counterparts ( Fig. 1D; p < 0.05).
The effect of ex vivo ucOC treatment on the insulin signaling pathway in EDL muscle In placebo EDL muscle, insulin alone increased the p-mTOR Ser2448 /tmTOR ratio (p < 0.05; Fig. 2E), p-Akt Ser473 (p < 0.001; Fig. 2F), the p-Akt Ser473 /tAkt ratio (p < 0.001; Fig. 2H), and p-AS160 Thr642 (p < 0.05; Fig. 2I). ucOC alone (without insulin) increased the levels of p-mTOR Ser2481 Journal of Bone and Mineral Research Fig. 4. The effect of ex vivo undercarboxylated osteocalcin (ucOC) treatment on proteins in insulin signaling pathway in placebo and corticosterone-(CS-) affected soleus muscle. The phosphorylation, total expression, and phosphor/total ratio of mTOR Ser2481 and mTOR Ser2448 (A-E), Akt Ser473 (F-H), as well as AS160 Thr642 and AS160 Ser588 (I-M) were analyzed in placebo and CS-affected soleus muscle. * and ** represent p < 0.05 and p < 0.01 in paired t tests compared with control-treated muscle. "a" represents p < 0.05 in paired t tests compared with insulin alone-treated muscle. # represents p < 0.05 between placebo and CS muscle with the same ex vivo treatment, using Fisher's LSD following 2-way ANOVA with repeated measures.

Discussion
Short-term use of GCs in the clinical setting has been suggested to induce muscle insulin resistance via the reduction of ucOC. (3,10) In support, we found that short-term (3-days) CS administration is sufficient to suppress circulating levels of both OC and ucOC. Furthermore, CS treatment led to attenuated insulin-stimulated muscle glucose uptake, especially in EDL muscle, along with hyperinsulinemia, high fasting blood glucose, and the development of whole-body insulin resistance. Previous research indicates that ucOC treatment may be an effective strategy for improving muscle insulin sensitivity; however, its ability to directly rescue GC-induced insulin resistance in skeletal muscle is unclear. We provide evidence that in EDL muscle, and to a lesser extent in soleus muscle, ex  Fig. 7. The correlations between variables of postulated undercarboxylated osteocalcin (ucOC) signaling proteins and glucose uptake, and insulin signaling proteins in corticosterone (CS-) affected extensor digitorum longus (EDL) muscle. The correlations between glucose uptake and p-ERK2 Thr202/Tyr204 (A), the p-ERK2 Thr202/Tyr204 /tERK2 ratio (B), as well as the correlation between the p-ERK2 Thr202/Tyr204 /tERK2 ratio and p-Akt Ser473 (C) were analyzed among insulin and ucOC + insulin groups in CS-affected EDL samples.
vivo ucOC treatment restores impaired ISGU induced by CS administration. In addition to the insulin-sensitizing effect, ucOC alone may also increase muscle glucose uptake. (15,20) Thus, the rescuing effect of ucOC on muscle ISGU could be, at least in part, insulin-independent. However, our results suggest that the ucOC effect on CS-affected muscle was primarily insulin-dependent, as in the absence of insulin stimulation, ucOC did not increase muscle glucose uptake. affected soleus muscle. The phosphorylation, total expression, and/or phosphor/total ratio of GPRC6A (A), ERK2 Thr202/Tyr204 (B-D), AMPKα Thr172 (E-G), p-protein kinase C (PKC) (pan) Thr410 (H), PKCδ/θ Ser643/676 (I), and PKCζ/λ Thr410/403 (J). * and ** represent p < 0.05 and p < 0.01 in paired t tests compared with controltreated muscle. "a" represents p < 0.05 in paired t tests compared with insulin-treated muscle. # represents p < 0.05 and ## represents p < 0.01 between placebo and CS muscle with the same ex vivo treatment, using Fisher's LSD following 2-way ANOVA with repeated measures.

Journal of Bone and Mineral Research
Accumulating evidence suggests that ucOC may exert its beneficial effects on muscle insulin sensitivity through the upregulation of activation and/or expression of mTORC2-Akt-AS160 cascade. (5,14,(21)(22)(23)(24) In support, we report that ucOC increased insulin-stimulated phosphorylation of mTOR Ser2481 in both CS-affected EDL and soleus muscles, and Akt Ser473 in CS-affected EDL muscle. Furthermore, in CSaffected muscles under insulin-treated conditions (both insulin and ucOC + insulin groups), p-Akt Ser473 in both muscles, as well as mTOR Ser2481 in soleus muscle, correlated with ISGU. It seems that the enhancement of insulinstimulated p-Akt Ser473 by ucOC in CS-affected EDL muscle was primarily because of the restoration of decreased Akt levels. Downregulated Akt expression has been previously reported in insulin-resistant skeletal muscle. (25,26) In a case study, in vitro insulin stimulation reduced the Akt protein level in skeletal muscle from a patient with T2DM, (27) which appears consistent with our finding. As such, ucOC-induced restoration of Akt expression may result in increased Akt phosphorylation, leading to improved ISGU in CS-affected EDL muscle.
We also found that ucOC treatment increased insulin action on the phosphor/total ratio of both AS160 Thr642 and AS160 Ser588 in CS-affected EDL muscle, but the overall phosphorylation levels were not changed based on a decrease in AS160 protein abundance. However, in CS-affected soleus muscle, ucOC treatment enhanced insulin action on AS160 Thr642 phosphorylation, which was associated with muscle glucose uptake. Therefore, the increase in AS160 Thr642 phosphorylation may also be involved in the insulin-sensitizing effect of ucOC in CS-affected soleus muscle, but to a lesser extent in CS-affected EDL muscle.
GPRC6A is the putative receptor for ucOC in muscle cells. (20,28) The ucOC effect on muscle cells appears to involve the enhancement of GPRC6A expression. (18) In support, the expression of GPRC6A was increased by ucOC + insulin treatment in placebo EDL muscle compared with insulin alone. However, ucOC did not change GPRC6A expression in muscle from CS-treated animals, suggesting that the alteration of GPRC6A abundance is unlikely to be involved in the rescuing effect of ucOC.
ERK activation has also been reported to mediate the Akt Ser473 phosphorylation induced by ucOC in healthy muscle and C2C12 myotubes, (15,22) although this remains controversial. (6) In the current study, ucOC + insulin treatment enhanced total phosphorylation and phosphor/total ratio of ERK2 Thr202/ Tyr204 in CS-affected EDL muscle compared with insulin alone. However, within the insulin and ucOC + insulin groups, the ERK2 Thr202/Tyr204 /tERK2 ratio was not associated with p-Akt Ser473 , supporting previous findings that ERK activation was not involved in the rescuing effect of ucOC on insulinresistant muscle. (6) The exact mechanism underlying the ucOCinduced increase in Akt phosphorylation in GC-treated muscle requires further investigation.
AMPK plays an important role in muscle energy metabolism. (29) It is possible that ucOC activates AMPK signaling in skeletal muscle, thereby contributing to the beneficial effects of ucOC on glucose uptake. (28) However, we and others have reported an unlikely role of AMPKα Thr172 phosphorylation in the ucOC effects in C2C12 myotubes, ex vivo mouse muscle,

Journal of Bone and Mineral Research
UCOC IMPROVES INSULIN-STIMULATED GLUCOSE UPTAKE 1527 ◼ Fig. 9. The correlations between variables of postulated undercarboxylated osteocalcin (ucOC) signaling proteins and glucose uptake, and insulin signaling proteins in corticosterone-(CS-) affected soleus muscle. The correlations between glucose uptake and p-protein kinase C (p-PKC) (pan) Thr410 (A), as well as the correlations between p-PKC (pan) Thr410 and p-mTOR Ser2481 (B), the p-mTOR Ser2481 /tmTOR ratio (C), p-Akt Ser473 (D), p-AS160 Thr642 (E), the p-AS160 Ser588 /tAS160 ratio (F) were analyzed among insulin and ucOC + insulin groups in CS-affected soleus samples. Fig. 10. Undercarboxylated osteocalcin (ucOC) might enhance insulin-stimulated glucose uptake in glucocorticoid-(GC-) affected mice muscle in a muscle type-specific manner. (A) In GC-affected extensor digitorum longus (EDL) muscle, ucOC treatment enhances insulin-stimulated glucose uptake partly by inducing enhancement in p-Akt Ser473 , mostly via the increase in total Akt expression. This increase of p-Akt Ser473 is independent of ERK activation. (B) In GC-affected soleus muscle, ucOC treatment enhances the Thr410 phosphorylation in certain types of p-protein kinase C (PKC), which leads to enhanced insulin-stimulated activation of Akt (Ser473) and AS160 (Thr642). In addition, ucOC treatment also increases insulin-stimulated p-mTOR Ser2481 via a mechanism that is yet to be identified. Overall, the enhancement in the activation of mTORC2-Akt-AS160 cascade contributes to the insulin-sensitizing effect of ucOC on soleus muscle.
increase increase suggested in current study hypothesized increase and human muscle. (3,14,15,22) In the current study, our findings provide further support that AMPK is unlikely to play a major role in the rescuing effect of ucOC on insulin-resistant muscle.
PKC is an emerging candidate for important upstream regulation in the ucOC cascade. (15,17) However, in our previous work we did not observe significant changes in the phosphorylation of PKCδ/θ in ucOC-treated muscle. (15) However, all three types of PKC (classical, novel, and atypical) have been linked to muscle glucose uptake. (30)(31)(32) In the current study, we report that in CS-affected soleus muscle, insulin-stimulated pan Thr410 phosphorylation of PKC was enhanced by ucOC treatment, whereas p-PKCδ/θ Ser643/676 and p-PKCζ/λ Thr410/403 remain unchanged. Furthermore, in CS-affected soleus muscle, PKC (pan) Thr410 phosphorylation was associated with glucose uptake, p-Akt Ser473 , and p-AS160 Thr642 within insulin and ucOC + insulin groups, but not with p-mTOR Ser2481 . These results suggest that the phosphorylation of PKC may be linked with the ucOC-induced increase in insulin-stimulated activation of Akt and AS160, resulting in insulin sensitization in CSaffected soleus muscle. However, determining which of the PKCs are involved requires further investigation. The current study has two major limitations. First, hyperglycemia and insulin resistance following surgical stress is a welldocumented clinical phenomenon. (33,34) Thus, even though our placebo-treated mice also underwent the surgery with a placebo pellet, the pellet-implantation surgery may have influenced muscle insulin sensitivity 3 days postsurgery. Indeed, we found that placebo soleus muscle did not respond to insulin as well as muscle from animals that had undergone no surgery. (14) Nevertheless, the surgery did not appear to have a large impact on the effect of ucOC, as glucose uptake and AS160 Thr642 phosphorylation responded similarly to our previous work in mouse muscle without any surgery influence. (15) Second, although AS160 plays an important role in insulin-stimulated GLUT4 translocation in muscle, the phosphorylation and expression of TBC1 domain family member 1 (TBC1D1)-a major Akt substrate in mouse EDL muscle (35) -were not assessed in this study. Whether TBC1D1 is involved in the ucOC effect on insulin-resistant muscle, particularly in EDL muscle, needs to be verified in future studies.
Taken together, our findings support the notion that ucOC treatment improves muscle insulin sensitivity in mice that undergo short-term CS administration, without enhancing basal muscle glucose uptake. Furthermore, the mechanisms underlying this ucOC effect involves an enhancement of the activation and abundance of key proteins in both distal insulin and ucOC signaling pathways, in a muscle-specific manner. The potential mechanisms are illustrated in Fig. 10 in detail. In GC-affected EDL muscle (Fig. 10A), ucOC treatment may restore muscle ISGU partly by inducing enhancement in p-Akt Ser473 via the increase in total Akt expression. This increase in p-Akt Ser473 appears to occur independent of ERK activation. In glucocorticoid-affected soleus muscle (Fig. 10B), ucOC treatment enhances the Thr410 phosphorylation in certain types of PKC, which may lead to enhanced insulinstimulated activation of Akt (Ser473) and AS160 (Thr642). In addition, ucOC treatment may also increase insulin-stimulated p-mTOR Ser2481 via a mechanism that is yet to be identified.
Overall, it appears that the enhancement in the activation of mTORC2-Akt-AS160 cascade contributes to the insulin-sensitizing effect of ucOC on soleus muscle. In combination with our previous work, our findings not only implicate ucOC as an effective target for the therapeutic treatment of muscle insulin resistance, particularly with respect to restoring insulin sensitivity during GC therapy, but also provide new mechanisms underlying the insulin-sensitizing effect of ucOC on skeletal muscle. Nevertheless, this therapeutic potential needs to be further explored in future research to test whether in vivo ucOC treatment can improve muscle insulin resistance induced by both short-term and long-term GC administrations.

Disclosures
All authors state that they have no conflicts of interest.