Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus

Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus

Expression of DARP in mouse tissues and myotubes.

<p>(A) Quantitative analysis for DARP mRNA expression in various mouse tissues (n = 3 each). DARP was predominantly expressed in skeletal muscle. (B) Quantitative analysis for ANKRD2 or DARP mRNA expression in C2C12 cells during myogenesis (n = 4 each). Expression of DARP was substantially enhanced during myogenesis, and was predominantly expressed in mature myotube. ANKRD2 showed the less degree of up-regulation during myogenesis comparing to that of DARP.</p

DARP regulates AMPK activity in C2C12 cells.

<p>(A) Phosphorylation of AMPK in C2C12 myotubes transfected with either negative (scramble) or DARP siRNA (DARP-KD) was assessed by immunoblotting. Silencing of DARP significantly enhanced the phosphorylation of AMPK. *P<0.05 (n = 3 each). (B) Phosphorylation of AMPK and ACC in C2C12 myotubes in the presence or absence of AICAR was assessed by immunoblotting. Silencing of DARP significantly enhanced the phosphorylation of AMPK and ACC in the absence of AICAR. Treatment with AICAR enhanced phosphorylation of AMPK and ACC in C2C12 myotubes, and the difference of their phosphorylation between scramble and DARP-KD cells disappeared when treated with AICAR. *P<0.05, **P<0.01, #Not significant (n = 3 each). (C) Glucose uptake in C2C12 myotubes was assessed by measuring 2-deoxyglucose uptake using an enzymatic photometric assay. Cells were transfected with either scramble or DARP siRNA (DARP-KD), and the glucose uptake was analyzed in the presence or absence of AICAR. Silencing of DARP significantly increased glucose uptake in C2C12 myotubes comparing to control cells. *P<0.05 (n = 5 each). Treatment with AICAR increased glucose uptake in control cells and abolished the increased glucose uptake induced by DARP-silencing. **P<0.01, #Not significant (n = 5 each). (D) Glucose oxidation in C2C12 myotubes was assessed by ¹⁴CO₂ production from ¹⁴C-D-glucose. Cells were transfected with either scramble or DARP siRNA (DARP KD). DARP-silencing enhanced the glucose oxidation in C2C12 myotubes. **P<0.01 (n = 5 for scramble, n = 4 for DARP KD). (E) Treatment with AICAR abrogated the increase in glucose oxidation induced by DARP-silencing (n = 3 each). (F) C2C12 myoblasts were infected with retroviruses carrying GFP (GFP) or DARP (DARP OE) gene, and then phosphorylation of AMPK was assessed by immunoblotting. Overexpression of DARP significantly reduced the phosphorylation of AMPK. **P<0.01 (n = 3 each).</p

Enhanced AMPK activity is attributable to the better glucose homeostasis in DARP-/- mice.

<p>(A) Glucose tolerance was analyzed in WT or DARP-/- mice treated with AICAR at the age of 24 weeks old (n = 6 for WT mice, n = 7 for DARP-/- mice). Administration of AICAR abolished the better glucose tolerance in DARP-/- mice. (B) Phosphorylation of AMPK and ACC in skeletal muscle of WT or DARP-/- mice treated with AICAR was assessed by immunoblotting. #Not significant (n = 7 each). (C) LKB1 expression in skeletal muscle was assessed by immunoblotting. Protein expression of LKB1 increased in skeletal muscle of DARP-/- mice comparing to that in WT mice. *P<0.05 (n = 4 each). (D) LKB1 expression in C2C12 myotubes transfected with either negative (scramble) or DARP siRNA (DARP-KD) was assessed by immunoblotting. Protein expression of LKB1 increased in DARP-KD myotubes comparing to that in scramble control cells. **P<0.01 (n = 3 each). (E) Quantitative analysis for LKB1 mRNA expression in skeletal muscle of WT or DARP-/- mice (n = 6 for WT and n = 5 for DARP-/-). LKB1 expression in skeletal muscle was not significantly different between WT and DARP-/- mice. (F) Quantitative analysis for LKB1 mRNA expression in C2C12 myotubes transfected with either negative (scramble) or DARP siRNA (DARP-KD). LKB1 expression in myotubes was not significantly different between scramble and DARP-KD cells (n = 4 each).</p

Loss of DARP leads to AMPK activation in skeletal muscle.

<p>(A) Quantitative analysis for GLUT1 and GLUT4 mRNA expression in skeletal muscle of WT or DARP-/- mice. *P<0.05 (n = 6 for WT mice, n = 5 for DARP-/- mice). Expression of GLUT4 in skeletal muscle of DARP-/- mice was similar, while GLUT1 expression in muscle of DARP-/- mice was significantly reduced comparing to those in WT mice. (B) Quantitative analysis for VEGF and Hmox1 mRNA expression in skeletal muscle of WT or DARP-/- mice. #Not significant (n = 5 each). (C) C2C12 myotubes were transfected with either negative (scramble) or DARP siRNA (DARP-KD), and then mRNA expression of DARP, GLUT1 and GLUT4 was quantitatively analyzed. **P<0.01, #Not significant (n = 4 each). (D) Phosphorylation of AMPK in skeletal muscle of WT or DARP-/- mice. *P<0.05 (n = 6 each). DARP-/- mice showed significantly accentuated AMPK phosphorylation in skeletal muscle comparing to WT mice. (E) Phosphorylation of ACC in skeletal muscle of WT or DARP-/- mice. *P<0.05 (n = 6 each). DARP-/- mice showed significantly accentuated ACC phosphorylation in skeletal muscle comparing to WT mice.</p

Nucleotide sequences of primers.

<p>Nucleotide sequences of primers.</p

A Point Mutation R122C in RUNX3 Promotes the Expansion of Isthmus Stem Cells and Inhibits Their Differentiation in the Stomach

10.1016/j.jcmgh.2022.01.010CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY1351317-134