1. Volume 66, Issue 3, Pages 332-344.e4 (May 2017)
Glucose Sensing by Skeletal Myocytes Couples Nutrient Signaling to Systemic Homeostasis 
Zhuo-Xian Meng, Jianke Gong, Zhimin Chen, Jingxia Sun, Yuanyuan Xiao, Lin Wang, Yaqiang Li, Jianfeng Liu, X. Z. Shawn Xu, Jiandie D. Lin 
Molecular Cell 
Volume 66, Issue 3, Pages e4 (May 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 66, 332-344.e4DOI: (10.1016/j.molcel.2017.04.007)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Glucose Activates the Baf60c-Deptor-AKT Axis in Skeletal Myocytes
(A) qPCR analysis of gene expression (top) and immunoblots of total protein lysates (bottom) from quadriceps muscle. Data represent mean ± SEM (n = 3–4). ∗p < 0.05, ∗∗∗p < 0.001, fasted versus fed, one-way ANOVA.
(B) qPCR analysis of gene expression in C2C12 myotubes following treatment for 12 hr. Veh, vehicle; Ins, insulin; AA, amino acids; FA, fatty acids; Glc, glucose; PA, palmitic acid; OA, oleic Acid. Data represent mean ± SD. ∗∗∗p < versus 1 mM glucose, one-way ANOVA.
(C and D) Immunoblots of total cell lysates from C2C12 myotubes (C) and primary human myotubes (D) treated with different concentrations of glucose for 12 hr.
(E) qPCR analysis of gene expression in ex vivo-cultured EDL muscle treated with different concentrations of glucose for 4 hr. Data represent mean ± SEM (n = 3). ∗∗p < 0.01, ∗∗∗p < versus 1 mM glucose; one-way ANOVA.
(F) Immunoblots of total protein lysates from ex vivo-cultured plantaris muscle treated with glucose for 4 hr.
See also Figure S1.
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4. Figure 2
Baf60c-Mediated Glucose Sensing Acts in Concert with Insulin to Facilitate Postprandial Glucose Disposal
(A) Immunoblots of total cell lysates from C2C12 myotubes treated with 1 mM or 10 mM glucose in the absence or presence of 10 μM AKTi for 12 hr.
(B) qPCR analysis of gene expression in siCtr or siBaf C2C12 myotubes treated with different concentrations of glucose for 12 hr. Data represent mean ± SD. ∗∗∗p < siCtr versus siBaf; two-way ANOVA.
(C) Immunoblots of total cell lysates from control and Baf60c knockdown C2C12 myotubes treated with different concentrations of glucose for 12 hr.
(D) Glucose consumption and lactate production in C2C12 myotubes pretreated with different concentrations of glucose for 12 hr, followed by switching to medium containing 5 mM glucose for an additional 12 hr. Data represent mean ± SD. ∗∗∗p < versus 1 mM; one-way ANOVA.
(E) Lactate production in ex vivo-cultured muscles after chase with medium containing 5 mM glucose. Data represent mean ± SEM (n = 4). ∗p < 0.05, ∗∗∗p < 0.001 versus 1 mM; one-way ANOVA.
(F) Lactate production in ex vivo-cultured muscles in the absence or presence of AKTi (10 μM). Data represent mean ± SEM (n = 4). ∗∗∗p < 0.001; two-way ANOVA.
(G) Clustering analysis of glucose-responsive genes in C2C12 myotubes. The red bar indicates a cluster of Baf60c-dependent, glucose-responsive genes.
(H) Immunoblots of total cell lysates from C2C12 myotubes pretreated with 1 mM or 10 mM glucose for 12 hr, followed by treatment with different concentrations of insulin for 30 min.
(I) Blood glucose levels in HFD-fed WT and Tg mice. Data represent mean ± SEM (n = 5–7). ∗∗p < 0.01, ∗∗∗p < 0.001; two-tailed unpaired Student’s t test.
(J) Blood glucose and plasma insulin levels during OGTT. Data represent mean ± SEM (n = 5–7). ∗∗p < 0.01; two-way ANOVA with multiple comparisons. Area under curve (AUC) was evaluated by two-tailed unpaired Student’s t test.
(K) Blood glucose and plasma insulin levels in STZ-treated, HFD-fed WT and Tg mice. Data represent mean ± SEM (n = 5–7). ∗∗p < 0.01; two-tailed unpaired Student’s t test.
(L) Postprandial blood glucose levels in mice as described in (K). Data represent mean ± SEM (n = 5–7). ∗∗p < 0.01; two-way ANOVA with multiple comparisons. AUC was evaluated by two-tailed unpaired Student’s t test.
See also Figure S1.
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5. Figure 3
Glucose Elevates Intramyocellular Calcium Levels through a KATP Channel-Dependent Pathway
(A) qPCR analysis of gene expression in C2C12 myotubes treated with Veh alone or 10 mM of the indicated treatments for 12 hr. Lac, lactate; Gal, galactose; Pyr, pyruvate. Data represent mean ± SD. ∗∗∗p < 0.001; one-way ANOVA.
(B) Immunoblots of total cell lysates from C2C12 myotubes as treated in (A).
(C) qPCR analysis of gene expression in ex vivo-cultured EDL muscles with the indicated treatments for 4 hr. Data represent mean ± SEM (n = 3). ∗∗∗p < 0.001; one-way ANOVA.
(D) Immunoblots of total protein lysates from ex vivo-cultured plantaris muscle as treated in (C).
(E) Top: calcium imaging traces in C2C12 myotubes in response to 10 mM glucose (left), 100 μM DZO pretreatment followed by 10 mM glucose treatment (center), or 10 mM 2-DG (right). Data represent mean ± SD. Bottom: representative fluorescent images of C2C12 myotubes treated with 10 mM glucose.
(F) ATP content in treated C2C12 myotubes. Data represent mean ± SD. ∗∗∗p < 0.001; one-way ANOVA.
(G) Top: calcium imaging traces in primary mouse myotubes in response to 10 mM glucose (left), 100 μM DZO pretreatment followed by 10 mM glucose treatment (center), or 10 mM 2-DG (right). Data represent mean ± SD. Bottom: representative fluorescent images of primary mouse myotubes treated 10 mM with glucose.
(H) qPCR analysis of Baf60c and Deptor expression in C2C12 myotubes treated with 1 or 10 mM glucose in combination with Veh or DZO for 12 hr. Data represent mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001; two-way ANOVA.
(I) Immunoblots of total lysates from C2C12 myotubes treated as in (H).
See also Figures S2 and S3.
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6. Figure 4
Glucose Stimulates HDAC5 Phosphorylation and Nuclear Exclusion in Cultured Myotubes
(A) qPCR analysis of gene expression in C2C12 myotubes treated with 1 mM (open) or 10 mM (filled) glucose in the presence of the indicated concentrations of the CaMKII inhibitor KN-62 for 12 hr. Data represent mean ± SD. ∗∗∗p < 0.001; two-way ANOVA.
(B) Immunoblots of total cell lysates from C2C12 myotubes treated with the indicated concentrations of glucose without or with 40 μM of KN-62 for 12 hr.
(C) qPCR analysis of gene expression in C2C12 myotubes transduced with adenoviruses expressing GFP or constitutive active CaMKII (CA-CaMKII) at low (LD) or high (HD) doses. Data represent mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA.
(D) Immunoblots of total cell lysates from C2C12 myotubes with the indicated treatment for 12 hr.
(E) Time-course response of HDAC5 phosphorylation in C2C12 myotubes treated with 10 mM glucose.
(F) Immunoblots of total protein lysates from quadriceps muscle.
(G) Immunoblots of the cytoplasmic and nuclear fractions from C2C12 myotubes treated with 1 mM or 10 mM glucose for 12 hr.
(H) Immunofluorescence images of endogenous HDAC5 (green) and nuclei (DAPI) in C2C12 myotubes treated as described in (G). Quantitation of myotubes exhibiting cytoplasmic only (C only) or both cytoplasmic and nuclear (C+N) localization of HDAC5 in response to 1 mM (n = 21) or 10 mM (n = 19) glucose is shown on the right. Scale bar, 25 μm.
(I) Immunoblots of total protein lysates from C2C12 myotubes as treated in (B).
(J) Immunoblots of nuclear extracts from C2C12 myotubes as treated in (B).
See also Figure S4.
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7. Figure 5
HDAC5 Mediates the Induction of Glucose-Responsive Genes in Cultured Myotubes
(A and B) ChIP assay of C2C12 myotubes treated with 1 mM (open) or 10 mM (filled) glucose using antibodies against Ace-H3, H3K4m3, or H3K9m2. The locations of the qPCR primers relative to the transcription start site of Baf60c (A) and Deptor (B) are indicated. Data represent mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; two-tailed unpaired Student’s t test.
(C) qPCR analysis of gene expression in transduced C2C12 myotubes. Data represent mean ± SD. ∗∗∗p < 0.001, two-way ANOVA.
(D) Immunofluorescence images of GFP-HDAC5-Mut and nuclei (DAPI) in C2C12 myotubes transduced with an adenovirus expressing GFP-HDAC5-Mut and treated with 1 mM (top) or 10 mM (bottom) glucose. Scale bar, 25 μm.
(E) qPCR analysis of gene expression in C2C12 myotubes transduced with GFP or GFP-HDAC5-mut adenoviruses. Data represent mean ± SD. ∗∗∗p < 0.001, two-way ANOVA.
See also Figure S5.
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8. Figure 6
Skeletal Muscle-Specific Baf60c Deficiency Impairs Glucose Homeostasis
(A) qPCR analysis of gene expression in EDL muscles from control (flox/flox, open) and MKO (blue) mice. Data represent mean ± SEM (n = 5–7). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; two-tailed unpaired Student’s t test.
(B) Immunoblots of total protein lysates from quadriceps muscles from flox/flox and MKO mice fasted for 24 hr (Fasted) or fasted for 24 hr followed by refeeding for 2 hr (Refed).
(C) Lactate production in ex vivo-cultured plantaris (PL), EDL and soleus (Sol) muscles from flox/flox and MKO mice pretreated with 4 or 10 mM glucose for 3 hr, followed by 3 hr chase with medium containing 5 mM glucose. Data represent mean ± SEM (n = 4). ∗∗∗p < 0.001; two-way ANOVA.
(D and E) ITT (D) and OGTT (E) in HFD-fed flox/flox and MKO mice. Data represent mean ± SEM (n = 8–11). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; two-way ANOVA with multiple comparisons. AUC was evaluated by two-tailed unpaired Student’s t test.
(F) Blood glucose levels in HFD-fed flox/flox and MKO mice fasted for 24 hr, followed by refeeding for 8 hr. Data represent mean ± SEM (n = 6–7). ∗∗p < 0.01; two-tailed unpaired Student’s t test.
(G) Plasma insulin and blood glucose levels in STZ-treated, HFD-fed flox/flox and MKO mice. Data represent mean ± SEM (n = 6). ∗∗∗p < 0.01; two-tailed unpaired Student’s t test.
(H) ITT (left) and GTT (right) in mice as described in (G).
(I) Postprandial (fasted/refeeding) blood glucose levels in mice as described in (G).
The data in (H) and (I) represent mean ± SEM (n = 6). ∗p < 0.05, ∗∗∗p < 0.001; two-way ANOVA with multiple comparisons. AUC was evaluated by two-tailed unpaired Student’s t test.
(J) Immunoblots of total protein lysates from quadriceps muscles from STZ-treated HFD-fed flox/flox and MKO mice fasted for 24 hr, followed by refeeding for 2 hr.
See also Figure S6.
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9. Figure 7
Sulfonylurea Engages the Baf60c-Deptor-AKT Pathway in Skeletal Muscle to Lower Blood Glucose
(A and B) Calcium imaging traces (left) and representative fluorescence images (right) in primary mouse myotubes treated with 100 μM tolbutamide (A) or 10 μM glibenclamide (B), as indicated by the red arrows. Data represent mean ± SD.
(C) qPCR analysis of gene expression in ex vivo-cultured EDL muscles treated with Veh or 10 μM glibenclamide (Glib) for 3 hr. Data represent mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01; two-tailed unpaired Student’s t test.
(D and E) Immunoblots of total protein lysates from ex vivo-cultured plantaris muscles, from wild-type mice (D) or from flox/flox and MKO mice (E), treated with Veh or 10 μM Glib for 3 hr.
(F) Plasma insulin and blood glucose levels in STZ-treated, HFD-fed flox/flox and MKO mice in response to Glib treatment (5 mg/kg by gavage). Data represent mean ± SEM (n = 5). ∗∗p < 0.01, flox/flox versus MKO; two-way ANOVA.
See also Figure S7.
Molecular Cell  , e4DOI: ( /j.molcel )
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