Catestatin induces glycogenesis by stimulating the phosphoinositide 3-kinase-AKT pathway

Abstract

Aim: Defects in hepatic glycogen synthesis contribute to post-prandial hyperglycaemia in type 2 diabetic patients. Chromogranin A (CgA) peptide Catestatin (CST: hCgA_352-372 ) improves glucose tolerance in insulin-resistant mice. Here, we seek to determine whether CST induces hepatic glycogen synthesis.

Methods: We determined liver glycogen, glucose-6-phosphate (G6P), uridine diphosphate glucose (UDPG) and glycogen synthase (GYS2) activities; plasma insulin, glucagon, noradrenaline and adrenaline levels in wild-type (WT) as well as in CST knockout (CST-KO) mice; glycogen synthesis and glycogenolysis in primary hepatocytes. We also analysed phosphorylation signals of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-dependent kinase-1 (PDK-1), GYS2, glycogen synthase kinase-3β (GSK-3β), AKT (a kinase in AKR mouse that produces Thymoma)/PKB (protein kinase B) and mammalian/mechanistic target of rapamycin (mTOR) by immunoblotting.

Results: CST stimulated glycogen accumulation in fed and fasted liver and in primary hepatocytes. CST reduced plasma noradrenaline and adrenaline levels. CST also directly stimulated glycogenesis and inhibited noradrenaline and adrenaline-induced glycogenolysis in hepatocytes. In addition, CST elevated the levels of UDPG and increased GYS2 activity. CST-KO mice had decreased liver glycogen that was restored by treatment with CST, reinforcing the crucial role of CST in hepatic glycogenesis. CST improved insulin signals downstream of IR and IRS-1 by enhancing phospho-AKT signals through the stimulation of PDK-1 and mTORC2 (mTOR Complex 2, rapamycin-insensitive complex) activities.

Conclusions: CST directly promotes the glycogenic pathway by (a) reducing glucose production, (b) increasing glycogen synthesis from UDPG, (c) reducing glycogenolysis and (d) enhancing downstream insulin signalling.

Keywords: catestatin; glucose-6-phosphate; glycogen; glycogen synthase; hyperglycaemia; phospho-AKT.

FIGURE 1

Effects of chronic catestatin (CST) and acute insulin treatments on hepatic glycogen contents in both fed and fasted normal chow diet wild-type (NCD-WT) mice. Mice were treated with CST (2 μg/g body weight/day for 15 days or 1 hr; intraperitoneal) followed by fasting (8 hr) and treatment with saline or insulin (0.4 mU/g body weight for 30 min; intraperitoneal) before harvesting tissues under deep anaesthesia. A, Liver glycogen content in fed (n = 12) and fasted (n = 15) NCD-WT mice. Two-way ANOVA: Interaction: P < .001; Food: P < .001; Treatment: P < .001. B, Plasma insulin levels in fed and fasted WT-NCD mice (n = 14). Student’s t test. C, Liver glycogen content in fed and fasted WT-NCD mice treated with saline (Sal; n = 43), acute CST for 1 hr (Acu; n = 12) or chronic CST for 15 days (Chr; n = 43). Three-way ANOVA: Food: P < .001; Acute vs Chronic: P < .001; Saline vs CST: P < .001; Food × Acute vs Chronic: ns; Food × Saline vs CST: P < .05; Acute vs Chronic × Saline vs CST; P < .001; Food × Acute vs Chronic × Saline vs CST: ns. D, Liver glycogen content after saline (n = 20), insulin for 30 min (n = 9), acute CST plus insulin (n = 9) or chronic CST for 15 days plus insulin for 30 min (n = 11) treatment in fasted NCD-WT mice. One-way ANOVA. E, Glycogen content in fed NCD-WT gastrocnemius muscle after saline or CST (2 μg/g body weight/day for 15 days; intraperitoneal) treatment (n = 10). Student’s t test. F, Morphometric analysis of transmission electron microscopy (TEM) micrographs showing glycogen granules in saline and CST-treated subsarcolemma and myofibril. Two-way ANOVA: Interaction: P < .01; Treatment: P < .001; Zone: P < .001. G, Morphometric assessment of lipid content in the TEM micrographs of steatotic liver of diet-induced obese mice after saline or chronic CST treatments. Student’s t test. *P < .05; ^†P < .01; ^‡P < .001

FIGURE 2

Effects of chronic catestatin (CST) and acute insulin treatments on plasma counterregulatory hormone levels in both fed and fasted normal chow diet wild-type (NCD-WT) mice. Fed or fasted NCD-WT mice were treated with CST (2 μg/g body weight/day for 15 days: intraperitoneal) before harvesting tissues under deep anaesthesia after 24 hrs of last injection. A, Noradrenaline (n = 16), adrenaline (n = 16) and glucagon (n = 6) levels in fed and fasted NCD-WT mice. Three-way ANOVA: Hormones: P < .001; Fed vs Fast: P < .001; Saline vs CST: P < .001; Hormones × Fed vs Fast: P < .05; Hormones × Saline vs CST: ns; Fed vs Fast × Saline vs CST: P < .001; Hormones × Fed vs Fast × Saline vs CST: P < .001. B, Noradrenaline levels in saline or insulin-treated fed or fasted NCD-WT mice (n = 7). Two-way ANOVA: Interaction: P < .01; Treatment: P < .05; Food: P < .01. C, Adrenaline levels in saline or insulin-treated fed or fasted NCD-WT mice (n = 7). Two-way ANOVA: Interaction: P < .01; Treatment: P < .001; Food: P < .001. D, Schematic diagram showing age, diet, CST treatments, glucose and insulin tolerance tests and tissue harvesting. E, Liver glycogen content in fed and fasted diet-induced obese (DIO-WT) mice after saline (n = 27) or CST (n = 16) treatments. Two-way ANOVA: Interaction: P < .01; Treatment: P < .001; Food: P < .001. F, Effects of saline (n = 47), insulin alone (n = 9; 30 min) or in combination with chronic CST (n = 12) on glycogen content in fasted DIO-WT mice. One-way ANOVA: P < .001. ^†P < .01; ^‡P < .001; ns, not significant

FIGURE 3

Effects of intraperitoneal or oral catestatin (CST) treatment on glucose tolerance in diet-induced obese (DIO) mice without causing weight loss. A, B, Oral glucose tolerance test (O-GTT): (A) Dose-dependent effects of oral CST on blood glucose levels during O-GTT (n = 6). Two-way ANOVA: Interaction: P < .05; Time: P < .001; Treatment: P < .001. B, The area under the curve (AUC) of the O-GTT was used to determine EC₅₀ of CST action. One-way ANOVA: P < .001. C, Blood glucose levels during O-GTT after time-course of CST treatment (2 μg/g body weight) (n = 7). Two-way ANOVA: Interaction: P < .001; Time: P < .001; Treatment: P < .001. D, The area under the curve (AUC) of the O-GTT. One-way ANOVA: P < .001. E, Intraperitoneal insulin tolerance test (ip-ITT): Blood glucose levels during ip-ITT after chronic CST treatment (n = 12). Two-way ANOVA: Interaction: P < .001; Time: P < .001; Treatment: P < .001. F, The AUC of the ip-ITT. Student’s t test. *P < .05; ^†P < .01; ^‡P < .001; ns, not significant

FIGURE 4

Effects of chronic catestatin (CST) and acute insulin treatments on hepatic glycogen content and plasma levels of counterregulatory hormones levels in normal chow diet (NCD)-CST-knock-out (KO) mice. NCD-WT and NCD-CST-KO mice were treated with CST (2 μg/g body weight/day for 15 days; intraperitoneal) followed by treatment with insulin (0.4 mU/g body weight for 30 min) before tissue harvesting. Tissues were harvested from fed or fasted (8 hr) mice under deep anaesthesia. A, Hepatic glycogen content in fed and fasted NCD-CST-KO mice compared with NCD-WT mice (n = 12). Two-way ANOVA: Interaction: P < .001; Treatment: P < .001; Food: P < .001. B, Effects of insulin (30 min) on glycogen content in fed and fasted NCD-CST-KO mice (n = 12). Two-way ANOVA: Interaction: ns; Time: Treatment: P < .05; Food: P < .001. C, Liver glycogen content in fasted and fed NCD-CST-KO mice after saline (n = 26) chronic CST treatment (n = 14). Two-way ANOVA: Interaction: ns; Time: Treatment: P < .05; Food: P < .001. D, Effects of insulin (30 min) alone or in combination with chronic CST on liver glycogen content in fasted CST-KO-NCD mice (n = 12). One-way ANOVA: P < .001. E, Plasma insulin levels in fed or fasted NCD-CST-KO mice (n = 14). Student’s t test. F, Noradrenaline (n = 15), adrenaline (n = 15) and glucagon (n = 6) levels in fed or fasted NCD-CST-KO mice after chronic treatments with CST. Three-way ANOVA: Hormones: P < .001; Fed vs Fast: P < .01; Sal vs CST: P < .001; Hormones × Fed vs Fast: ns; Hormones × Sal vs CST: P < .001; Fed vs Fast × Sal vs CST: P < .05; Hormones × Fed vs Fast × Sal vs CST: ns. G, Noradrenaline and adrenaline levels in saline or insulin (30 min)-treated fed and fasted CST-KO-NCD mice (n = 6). Three-way ANOVA: Catecholamines: P < .001; Fed vs Fast: P < .001; Sal vs Insulin: P < .01; Catecholamines × Fed vs Fast: P < .001; Catecholamines × Sal vs Insulin: P < .05; Fed vs Fast × Sal vs CST: ns; Catecholamines × Fed vs Fast × Sal vs CST: ns. *P < .05; ^‡P < .001; ns, not significant

FIGURE 5

Regulation of glycogenesis and glycogenolysis in hepatocytes by catestatin (CST) and regulation of the levels of glucose-6-phosphate (G6P) and uridine diphosphate glucose (UDPG) as well as GYS2 activity in mouse liver by CST. A, Dose-dependent effects of CST on glycogenesis from ¹⁴C-glucose in cultured primary lean hepatocytes. The EC₅₀ was calculated using GraphPad PRISM software. B, Effects of insulin and/or CST on glycogenesis (n = 4–5). One-way ANOVA: P < .001. C, Effects of insulin (Ins), adrenaline, noradrenaline and/or CST on glycogenolysis from preloaded radio-labelled glycogen (n = 6). One-way ANOVA: P < .001. D, Effects of CST on liver G6P levels in fed or fasted normal chow diet (NCD)-wild-type (WT) mice (n = 6). Two-way ANOVA: Interaction: ns; Treatment: P < .001; Food: ns. E, Effects of CST on liver G6P levels in fed or fasted diet-induced obese (DIO)-WT mice (n = 6). Two-way ANOVA: Interaction: ns; Treatment: ns; Food: P < .01. (F) and (G) Effects of CST on UDPG in the liver of fasted NCD-WT or DIO-WT mice (n = 4). Student’s t test. (H) and (I) Effects of CST on GYS2 activities in the liver of fasted NCD-WT and DIO-WT mice (n = 6). Student’s t test. For GYS2 activity, the activation ratios in the presence of low (0.1 mM) and high (10 mM) G6P are shown (n = 6). Tissues were from the same experiments shown in Figures 1 and 2. *P < .05; ^†P < .01; ^‡P < .001, ns, not significant

FIGURE 6

Unchanged tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) and increased PI3-kinase activity by catestatin (CST) in primary hepatocytes. A, Immunoblots show tyrosine phosphorylation (pY) of IR in response to insulin and CST (n = 3). B, Immunoblots show tyrosine phosphorylation (pY) of IRS-1 in response to insulin and CST (n = 3). C, Corresponding density ratio of phospho-/total IR signals. One-way ANOVA: P < .001. D, Corresponding density ratio of phospho-/total IRS-1 signals. One-way ANOVA: P < .01. E, Autoradiograph of thin layer chromatography plate showing the formation of phosphatidyl inositol-3-phosphate (PI3-P) due to increased PI-3-kinase activity in the anti-p85-immunprecipitates after stimulation with insulin and/or CST. *P < .05; ^†P < .01; ns, not significant

FIGURE 7

Stimulation of phosphorylation of AKT at T308 residue by insulin and catestatin (CST) in a PDK-1-dependent manner. A, Immunoblots showing downregulation of PDK-1 protein expression by si-RNA. B, Bar graphs showing knockdown efficiencies from the immunoblot. Student’s t test. C, Immunoblots showing phospho-T308-AKT and total AKT signals after saline (Basal), insulin and CST treatments in the presence and absence of si-RNA against PDK-1. D, Bar graphs showing phospho-/total AKT signals. One-way ANOVA: P < .001. ^‡P < .001; ns, not significant

FIGURE 8

Increased phosphorylation of mammalian/mechanistic target of rapamycin (mTOR) in the mTORC2 complex and AKT-S473 by insulin and catestatin (CST) in primary hepatocytes. A, Immunoblots showing phosphorylation of mTOR at Ser2481 (n = 3). B, Corresponding density ratio of phospho-/total of mTOR signals. One-way ANOVA: P < .001. C, Immunoblots showing phosphorylation of AKT at Ser473 (n = 3). D, Corresponding density ratio of phospho-/total AKT signals. One-way ANOVA: P < .001. *P < .05; ^†P < .01; ^‡P < .001; ns, not significant

FIGURE 9

Catestatin (CST) augments hepatic GYS2 activity (ie, increased dephosphorylation) and decreases GSK-3β activity (ie, increased phosphorylation) in fasted normal chow diet (NCD)-wild-type (WT) and diet-induced obese (DIO)-WT mice. A, Immunoblots showing total and phosphorylated signals for GSK-3 β and GYS2 in NCD-WT and DIO-WT mice. B, Densitometry values of GSK-3β in NCD-WT mice (n = 4). One-way ANOVA: P < .001. C, Densitometry values of GSK-3β in DIO-WT mice (n = 4). One-way ANOVA: P < .001. D, Densitometry values of GYS2 in NCD-WT mice (n = 4). One-way ANOVA: P < .01. E, Densitometry values of GYS2 in DIO-WT mice (n = 4). One-way ANOVA: P < .001. Tissues were from the same experiments as shown in Figures 1 and 2. *P < .05; ^†,‡P < .001; ns, not significant

FIGURE 10

Schematic diagram showing insulin and catestatin-mediated signalling pathways involved in the regulation of hepatic glucose and glycogen metabolism