A novel neurotensin/xenin fusion peptide enhances β-cell function and exhibits antidiabetic efficacy in high-fat fed mice

Abstract

Neurotensin and xenin possess antidiabetic potential, mediated in part through augmentation of incretin hormone, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), action. In the present study, fragment peptides of neurotensin and xenin, acetyl-neurotensin and xenin-8-Gln, were fused together to create Ac-NT/XN-8-Gln. Following assessment of enzymatic stability, effects of Ac-NT/XN-8-Gln on in vitro β-cell function were studied. Subchronic antidiabetic efficacy of Ac-NT/XN-8-Gln alone, and in combination with the clinically approved GLP-1 receptor agonist exendin-4, was assessed in high-fat fed (HFF) mice. Ac-NT/XN-8-Gln was highly resistant to plasma enzyme degradation and induced dose-dependent insulin-releasing actions (P<0.05 to P<0.01) in BRIN-BD11 β-cells and isolated mouse islets. Ac-NT/XN-8-Gln augmented (P<0.001) the insulinotropic actions of GIP, while possessing independent β-cell proliferative (P<0.001) and anti-apoptotic (P<0.01) actions. Twice daily treatment of HFF mice with Ac-NT/XN-8-Gln for 32 days improved glycaemic control and circulating insulin, with benefits significantly enhanced by combined exendin-4 treatment. This was reflected by reduced body fat mass (P<0.001), improved circulating lipid profile (P<0.01) and reduced HbA1c concentrations (P<0.01) in the combined treatment group. Following an oral glucose challenge, glucose levels were markedly decreased (P<0.05) only in combination treatment group and superior to exendin-4 alone, with similar observations made in response to glucose plus GIP injection. The combined treatment group also presented with improved insulin sensitivity, decreased pancreatic insulin content as well as increased islet and β-cell areas. These data reveal that Ac-NT/XN-8-Gln is a biologically active neurotensin/xenin fusion peptide that displays prominent antidiabetic efficacy when administered together with exendin-4.

Keywords: Neurotensin; Xenin; hybrid; incretin.

Figure 1. Acute effects of acetyl-neurotensin(8-13), xenin-8-Gln, Ac-NT/XN-8-Gln alone and in combination with GIP or GLP-1, on insulin release from BRIN-BD11 cells and isolated mouse islets

BRIN-BD11 cells were incubated (20 min) with a range of concentrations (10⁻¹² to 10⁻⁶ M) of acetyl-neurotensin(8-13), xenin-8-Gln or Ac-NT/XN-8-Gln alone (A,B) or in combination with GIP (C) or GLP-1 (D) in the presence of (A,C,D) 5.6 or (B) 16.7 mM glucose. (E) Murine islets were incubated (60 min) with Ac-NT/XN-8-Gln (10⁻⁸ and 10⁻⁶ M) in the presence 16.7 mM glucose. (A–E) Insulin release was measured using radioimmunoassay. Values represent means ± SEM (n=8 for (A–D) and n=5 for (E)). *P<0.05 and ***P<0.001 compared with respective glucose controls. ^∆P<0.05, ^∆∆P<0.01 and ^∆∆∆P<0.001 compared with respective 10⁻⁷ M GIP or GLP-1 control.

Figure 2. Effect of acetyl-neurotensin(8-13), xenin-8-Gln and Ac-NT/XN-8-Gln on β-cell proliferation and protection against apoptosis

BRIN-BD11 cells were incubated overnight (18 h) with exendin-4, acetyl-neurotensin(8-13), xenin-8-Gln or Ac-NT/XN-8-Gln (each at 10⁻⁸ and 10⁻⁶ M). (A) Proliferation was measured using Ki-67 immunocytochemistry. (B) Caspase-3/7 activation was detected by luminescence. (A) Values represent means ± SEM (n=3–4). **P<0.01 and ***P<0.001 compared with respective media control. ^∆∆P<0.01 and ^∆∆∆P<0.001 compared with respective Ac-NT/XN-8-Gln. (B) Values are mean ± SEM (n=3). **P<0.01 and ***P<0.001 compared with untreated control culture.

Figure 3. Effects of twice-daily administration of Ac-NT/XN-8-Gln and exendin-4 alone, or in combination, on body weight, food intake, glucose, insulin and HbA1c concentrations in HFF mice

(A,B,D,F) Parameters were measured for 6 days before and 32 days during (indicated by black horizontal line in panels B,D,F) twice-daily treatment with saline, exendin-4, Ac-NT/XN-8-Gln or a combination of both (each at 25 nmol/kg) in HFF mice. (C,E) Parameters were assessed on day 32. Values represent mean ± SEM (n=6–8). *P<0.05, **P<0.01 and ***P<0.001 compared with HFF saline control mice.

Figure 4. Effects of twice-daily administration of Ac-NT/XN-8-Gln and exendin-4 alone, or in combination, on glucose tolerance and metabolic response to GIP

Tests were performed following 32 days twice-daily i.p. administration of saline, exendin-4, Ac-NT/XN-8-Gln or a combination of both peptides (each at 25 nmol/kg bw) in 10 h fasted HFF mice. (A–D) Blood glucose (A) and plasma insulin (C) were measured prior to and after oral administration of glucose alone (18 mmol/kg bw). (E–H) Blood glucose (E) and plasma insulin (G) were measured prior to and after i.p. administration of glucose (18 mmol/kg bw) in combination with GIP (25 nmol/kg bw). (B,D,F,H) Corresponding 0–120 min AUC values are also shown. Values represent mean ± SEM (n=6–8). *P<0.05 and **P<0.01 compared with HFF saline control mice. ⁺P<0.05 compared with Ac-NT/XN-8-Gln treated HFF mice. ^∆P<0.05 compared with lean saline control mice. Where appropriate to aid interpretation, lines are used to indicate significance between groups on the bar graphs.

Figure 5. Effects of twice-daily administration of Ac-NT/XN-8-Gln and exendin-4 alone, or in combination, on insulin sensitivity and lipid status in HFF mice

Tests were performed following 32 days twice-daily i.p. administration of saline, exendin-4, Ac-NT/XN-8-Gln or a combination of both peptides (each at 25 nmol/kg bw) in HFF mice. (A,B) Blood glucose was measured prior to and after i.p. administration of insulin (25 U/kg bw), with 0–60 min AAC values also shown. (C–E) Effects of test peptides on circulating (C) triglycerides, (D) total- and (E) LDL-cholesterol concentrations. Values are mean ± SEM (n=6–8). *P<0.05 **P<0.01 and ***P<0.001 compared with HFF saline control mice. ⁺P<0.05 and ⁺⁺P<0.01 compared with Ac-NT/XN-8-Gln treated HFF mice. Where appropriate to aid interpretation, lines are used to indicate significance between groups on the bar graphs.

Figure 6. Effects of twice-daily administration of Ac-NT/XN-8-Gln and exendin-4 alone, or in combination, on pancreatic insulin content and islet histology in HFF mice

Effects of 32 days twice-daily i.p. administration of saline, exendin-4, Ac-NT/XN-8-Gln or a combination of both peptides (each at 25 nmol/kg bw) on (A) pancreatic insulin content as well as (B) islet, (C) β-cell and (D) α-cell areas in HFF mice. Values represent mean ± SEM (n=6–8). ⁺⁺⁺P<0.001 compared with lean control. *P<0.05, **P<0.01 and ***P<0.001 compared with HFF saline control mice. ^ΔΔP<0.01 and ^ΔΔΔP<0.001 compared with Ac-NT/XN-8-Gln treated HFF mice. Where appropriate to aid interpretation, lines are used to indicate significance between groups on the bar graphs.