Table 1.
Amino acid sequence and physicochemical properties of esculentin-2CHa and its substituted analogues.
Fig 1.
Dose dependent effects of esculentin-2CHa and its substituted analogues on insulin release from BRIN-BD11 cells (A, B, E, F) and mouse islets (C, D). Effects on lactate dehydrogenase (LDH) release (G) is also shown.
Values are mean ± SEM with n = 8. For A, B, D and F, *P < 0.05, **P < 0.01, ***P < 0.001 compared with 5.6mM glucose. For B and D, ΔP < 0.05, ΔΔP < 0.01 compared with native esculentin-2CHa. For C, *P < 0.05, **P < 0.01, ***P < 0.001 compared with 16.7 mM glucose. For E, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the same concentration of glucose alone. ΔP<0.05 compared with esculentin-2CHa at the same glucose concentration. ++P < 0.01, +++P < 0.001 compared with incubation at 5.6 mM glucose for each peptide. For F, ΔΔΔP < 0.001 compared with respective incubation in the absence of the peptide. +P < 0.05, ++P < 0.01, +++P < 0.001 compared with incubation at 5.6 mM glucose in the presence of esculentin-2CHa alone.
Table 2.
Effects of esculentin-2CHa and its analogues on insulin- and LDH-release, membrane potential and intracellular calcium concentration in BRIN-BD11 cells.
Fig 2.
Effects of esculentin-2CHa on membrane potential (A, B) and intracellular calcium concentration (C, D) in BRIN-BD11 cells expressed as relative fluorescence unit, RFU (A, C) and area under the curve, AUC (B, D).
Cells were incubated with esculentin-2CHa (1 μM) or its substituted analogue and data were collected every 1.52 s over a period of 5 min. Values are mean ± SEM with n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 compared with 5.6 mM glucose.
Fig 3.
Effects of esculentin-2CHa on glucose tolerance (A, B) and plasma insulin response to glucose (C, D) in high fat fed mice expressed as line graph (A, C) and area under the curve, AUC (B, D).
Values are mean ± SEM with n = 8. *P < 0.05 compared with control
Table 3.
Acute effects of esculentin-2CHa and its analogues on glucose tolerance and insulin release in high fat fed mice.
Fig 4.
Effects of [L28K]esculentin-2CHa on body weight (A), cumulative energy intake (B), non-fasting plasma glucose (C) and insulin (D) in lean and high-fat fed mice. Parameters were measured 3 days prior to, and every 72 hours during twice-daily treatment (indicated by the black bar) with saline or [L28K]esculentin-2CHa (75 nmol/kg bodyweight) for 28 days.
Values are mean ± SEM with n = 8 mice. *P < 0.05, *** P < 0.001 compared to high fat fed control. All parameters were significantly lower in lean mice than high fat fed control mice (P < 0.05 –P < 0.001).
Fig 5.
Effects of [L28K]esculentin-2CHa on plasma glucose and insulin concentrations following intraperitoneal (A–D) and oral (E-H) glucose administration (18 mmol/kg body weight) in lean and high-fat fed mice as well as insulin sensitivity (I, J).
Insulin sensitivity tests were performed using 25 U/kg body weight of insulin injected intraperitoneally. All tests were conducted following twice-daily treatment of mice with saline or [L28K]esculentin-2CHa (75 nmol/kg body weight) for 28 days. Values are mean ± SEM with n = 8 mice. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with saline-treated lean mice. ΔP < 0.05, ΔΔP < 0.01 compared with high-fat fed control.
Fig 6.
Effects of [L28K]esculentin-2CHa on (A) pancreatic weight, (B) pancreatic insulin content and (C) insulin-secretory responses of islets isolated from lean and high-fat fed mice treated with saline or [L28K]esculentin-2CHa (75 nmol/kg body weight) for 28 days. Values are means ± SEM for 8 mice.
For Fig 6B, ***P < 0.001 compared with lean control, ΔP < 0.01 compared with high-fat control. For Fig 6C, *P < 0.05,**P < 0.01, ***P < 0.001 compared with the response of islets isolated from the same group of mice at 16.7 mM glucose; ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 compared with the respective response of islets isolated from lean control. For Fig 6D, stimulation index refers to fold-increase in insulin secretion from 1.4 mM glucose to 16.7 mM glucose or from 16.7 mM glucose to that observed in the presence of each secretagogue. *P < 0.05, **P < 0.01 compared with the stimulation index of islets isolated from each group of mice at 16.7mM glucose. ΔP < 0.05, ΔΔP < 0.01 compared the stimulation index of lean control.
Fig 7.
Effects of [L28K]esculentin-2CHa on plasma glucagon content (A), pancreatic glucagon content (B), plasma lipid profile (C), plasma concentrations of AST, ALT and ALP (D) and plasma level of creatinine (E) in lean and high-fat fed mice.
Mice were treated with twice-daily injections of saline or [L28K]esculentin-2CHa (75 nmol/kg body weight) for 28 days prior to measurement of biochemical parameters. Values are mean ± SEM with n = 8. *P < 0.05, **P < 0.01, ***P < 0.01 compared with lean control. ΔP < 0.05 compared with high-fat control.
Fig 8.
Effects of [L28K]esculentin-2CHa on islet morphology (A) and computed islet number (B), islet area (C), beta cell area (D) and alpha cell area (E) in lean and high-fat fed mice.
Mice were treated with twice-daily injections of saline or [L28K]esculentin-2CHa (75 nmol/kg body weight) for 28 days prior to excision of pancreatic tissue for immunohistological analysis. Values are mean ± SEM for 8 observations (~120 islets per group). *P < 0.05, **P < 0.01, ***P < 0.001 compared to lean control. ΔΔΔP < 0.001 compared to high fat control.