Discovery of pancreastatin inhibitor PSTi8 for the treatment of insulin resistance and diabetes: studies in rodent models of diabetes mellitus

Abstract

Pancreastatin (PST) is an endogenous peptide which regulates glucose and lipid metabolism in liver and adipose tissues. In type 2 diabetic patients, PST level is high and plays a crucial role in the negative regulation of insulin sensitivity. Novel therapeutic agents are needed to treat the diabetes and insulin resistance (IR) against the PST action. In this regard, we have investigated the PST inhibitor peptide-8 (PSTi8) action against diabetogenic PST. PSTi8 rescued PST-induced IR in HepG2 and 3T3L1 cells. PSTi8 increases the GLUT4 translocation to cell surface to promote glucose uptake in L6-GLUT4myc cells. PSTi8 treatment showed an increase in insulin sensitivity in db/db, high fat and fructose fed streptozotocin (STZ) induced IR mice. PSTi8 improved the glucose homeostasis which is comparable to metformin in diabetic mice, characterized by elevated glucose clearance, enhanced glycogenesis, enhanced glycolysis and reduced gluconeogenesis. PST and PSTi8 both were docked to the GRP78 inhibitor binding site in protein-protein docking, GRP78 expression and its ATPase activity studies. The mechanism of action of PSTi8 may be mediated by activating IRS1/2-phosphatidylinositol-3-kinase-AKT (FoxO1, Srebp-1c) signaling pathway. The discovery of PSTi8 provides a promising therapeutic agent for the treatment of metabolic diseases mainly diabetes.

Figure 1

Pancreastatin inhibitor rescued IR in vitro. (a) PSTi8 attenuated PST induced IR in HepG2 cells. α, control vs insulin (100 nM); β, insulin vs PST (25 nM) + insulin; γ, control vs PSTi8 (150 nM) and δ, insulin +PST vs insulin + PST + PSTi8. (b) PSTi8 controlled the high-glucose induced IR in HepG2 cells. (c) PSTi8 rescued PST elevated IR in 3T3L1 cells. α, control vs insulin (100 nM); β, insulin vs PST (200 nM) + insulin and γ, insulin + PST vs insulin + PST + PSTi8 (800 nM). (d) PSTi8 inhibits the glucose release in the presence of glucagon in HepG2 cells. α, control vs glucagon (6 nM); β, glucagon vs glucagon +PSTi8 (150 nM); γ, glucagon vs glucagon + metformin (100 µM). (e) Effect of insulin, PST and PSTi8 treatment on expression of glycolysis genes (Hk, Pfk1) and gluconeogenesis genes (Pepck and G6pase) in HepG2 cells. α, control vs insulin (100 nM); δ, insulin vs PST (25 nM) + insulin; β, PST + insulin vs PST + insulin + PSTi8 (150 nM). (f) Effect of insulin, PST and PSTi8 treatment on expression of lipogenic genes (Pparα, Cpt1a, Srebp-1c and Scd1) in HepG2 cells. α, control vs insulin (100 nM); β, PST (25 nM) + insulin vs PST + insulin + PSTi8 (150 nM). (g) Effect of insulin, PST and PSTi8 treatment on expression of lipogenic genes (Ucp2, Pparγ, Pgc1α, FoxO1, Akt1 and Pparβ) in 3T3L1 cells. α, control vs insulin (100 nM); δ, insulin vs PST (25 nM) + insulin; β, insulin vs PST (25 nM) + insulin; γ, PST + insulin vs PST + insulin + PSTi8 (150 nM). All genes are normalized to Gapdh and β-actin as reference genes in HepG2 and 3T3L1 cells, respectively. (h) Effect of PSTi8 on glucose uptake in L6 cells. α, control vs metformin (10 µM); β, control vs insulin (100 nM); γ, control vs PSTi8 (100 nM). (i) Effect of PSTi8 (150 nM) and insulin (200 nM) on GLUT4 translocation to surface in L6-GLUT4myc cells. (j) PSTi8 inhibits gluconeogenic gene (Pepck, Pc) expression in glucagon stimulated HepG2 cells. α, control vs glucagon (6 nM); β glucagon vs glu. + metformin (100 µM) and γ glucagon vs glu. + PSTi8 (150 nM). *P < 0.05; **P < 0.01; ***P < 0.001, NS, Non-significant. Error bar indicate mean ± s.e.m.

Figure 2

PSTi8 attenuates IR in HFD-fed, HFrD-fed and db/db diabetic mice. Acute (i.p, 5 mg/kg) and chronic (i.p, 2 mg/kg for 7 days), administration of PSTi8 during i.p GTT (0.5 g/kg) for db/db, (1 gram/kg) for HFD, HFrD mice or ITT (1 IU/kg) for db/db, (0.6 IU/kg) for HFD, HFrD mice or PTT (2 gram/kg) for db/db and HFD, HFrD in fasting (6 h) mice (n = 6). (a,c,e) i.p GTT; (b,d,f) AUC of i.p GTT; (g,i,k) i.p GTT; (h,j,l) AUC of i.p ITT; (m,o,q) i.p PTT; (n,p,r) AUC of i.p PTT. α, db/db, HFD, HFrD vs acute PSTi8 treatment, δ, db/db, HFD, HFrD vs chronic PSTi8 treatment and β, acute PSTi8 treated vs chronic PSTi8 treated db/db, HFD, HFrD. *P < 0.05; **P < 0.01; ***P < 0.001. Error bar indicate mean ± s.e.m.

Figure 3

Efficacy of PSTi8 as compared to metformin. Acute PSTi8 (i.p, 5 mg/kg), chronic PSTi8 (i.p, 2 mg/kg for 7 days), acute metformin (p.o, 300 mg/kg) and chronic metformin (p.o, 300 mg/kg for 7 days) administration (30 min before bolus glucose/insulin) during i.p GTT (1 g/kg) or ITT (0.6 IU/kg) in fasting (6 h) HFrD mice (n = 6). (a) i.pGTT with acute metformin and PSTi8; (b) i.pGTT with chronic metformin and PSTi8; (c) i.p ITT with acute metformin and PSTi8; (d) i.p ITT with chronic metformin and PSTi8; (e) AUC of i.p GTT with acute metformin and PSTi8; (f) AUC of i.p GTT with chronic metformin and PSTi8; (g) AUC of i.p ITT with acute metformin and PSTi8; (h) AUC of i.p ITT with chronic metformin and PSTi8. α, HFrD mice vs drug treatment, β, chronic metformin vs chronic PSTi8 treatment. Glucose reduction experiment after acute administration of PSTi8 (i.p, 5 mg/kg) and metformin (p.o, 300 mg/kg) (i) C57BL/6J mice and (j) db/db mice. (k) Plasma concentration vs time profile of PSTi8 following intraperitoneal administration at dose of 5 mg/kg to db/db mice. Met: metformin *^,#P < 0.05; **^,##P < 0.01; ***^,###P < 0.001. Error bar indicate mean ± s.e.m.

Figure 4

PSTi8 regulate secreation of biomolecule, glycogen storage and hepatic gene expression. Plasma concentrations of (a–c) insulin, (d–f) leptin, (g–i) IL-6, (j–l) MCP1, (p,q) NEFA-C and (m–o) liver glycogen in db/db, HFD, HFrD mice and PSTi8 treated db/db, HFD, HFrD mice. α, db/db, HFD, HFrD mice vs PSTi8 treated db/db, HFD, HFrD mice. (r–t) Effect of PSTi8 treatment on db/db, HFD and HFrD mice for the expression of glucose and lipid metabolic genes, (Pepck, G6pase, Pfk1, Pk, Pparγ, Srebp-1c and Fas) for db/db, (Pepck, G6pase, Pfk1, Pparγ, Srebp-1c, FoxO1 and Fas) for HFD, (Pepck, G6pase, Pfk1, Pk, Pparγ, Pgc1α, Srebp-1c and FoxO1) for HFrD in liver tissue. α, db/db vs db/db + PSTi8, HFD vs HFD + PSTi8, HFrD vs HFrD + PSTi8. *P < 0.05; **P < 0.01; ***P < 0.001. Error bar indicate mean ± s.e.m.

Figure 5

PSTi8 modulate insulin signaling. Western blot analysis of (a–c) phosphorylation of IRS-1 (p-S307IRS-1), (d–f) phosphorylation of Akt (p-T308AKT), (g–i) phosphorylation of Srebp-1c (p-S372SREBP1c) and (j–l) phosphorylation of FoxO1 (p-S256FoxO1) in liver tissues of db/db, HFD, HFrD mice and PSTi8 treated db/db, HFD, HFrD mice. α, control vs db/db, HFD, HFrD and β, db/db vs db/db + PSTi8, HFD vs HFD + PSTi8, HFrD vs HFrD + PSTi8. *P < 0.05; **P < 0.01; ***P < 0.001. Error bar indicate mean ± s.e.m.

Figure 6

PSTi8 competes with PST on GRP78 receptor binding. Molecular docking (a) PST (yellow) is docked in the active site of human GRP78 (blue): the residues of PST in red are showing hydrogen bond with the residues of human GRP78 (green). (b) The superimposed image of PSTi8 (purple) and PST in the active site of human GRP78. (c) PSTi8 is docked in the active site of human GRP78: the residues of PSTi8 in red are showing hydrogen bond with the residues of human GRP78. (d) Competitive binding between different concentrations of PST and Sulphorhodamine labelled PSTi8 (150 nM) in HepG2 cells. (e) Competitive binding between different concentrations of PSTi8 and Sulphorhodamine labelled PST (25 nM) in HepG2 cells. (f) GRP78 ATPase activity in presences of PST (1 µM) with different dose of PSTi8 (0, 1, 2.5, 5 µM). Western blot analysis (g) Effect of PSTi8 (800 nM) on PST (100 nM) inhibited tunicamycin (5 mg/ml) stimulated GRP78 expression in HepG2 cells. α, control vs tunicamycin; β, Tunicamycin vs Tunca. + PST; γ, Tunica. PST vs Tunica. +PST + PSTi8 and δ, control vs PSTi8. *P < 0.05; **P < 0.01; ***P < 0.001, NS, Non-significant. Error bar indicate mean ± s.e.m.

Figure 7

Mechanism of action of PSTi8 towards glucose and energy homeostasis. PSTi8 pre-occupy the GRP78 receptor where PST binds. PST regulation of glucose homeostasis in different tissues which is inhibited by PSTi8 in metabolic syndrome and type-2 diabetes. Schematic diagram showing the inhibitory effect of PSTi8 on PST at PST receptors in the liver. GPCR, G protein-coupled receptor; PKC, protein kinase c; PI-3K, phosphatidylinositol 3-kinase; pFoxO1, phosphorylated FoxO1; Srebp-1c, Sterol regulatory element-binding protein 1c; Akt, protein kinase B; PIP3, Phosphatidylinositol (3,4,5)-trisphosphate; IRS1, Insulin receptor substrate 1; GTP, Guanosine triphosphate; cGMP, Cyclic guanosine monophosphate; NOS, Nitric oxide synthase.