Combining a dipeptidyl peptidase-4 inhibitor, alogliptin, with pioglitazone improves glycaemic control, lipid profiles and beta-cell function in db/db mice

Abstract

Background and purpose: Alogliptin, a highly selective dipeptidyl peptidase-4 (DPP-4) inhibitor, enhances incretin action and pioglitazone enhances hepatic and peripheral insulin actions. Here, we have evaluated the effects of combining these agents in diabetic mice.

Experimental approach: Effects of short-term treatment with alogliptin alone (0.01%-0.1% in diet), and chronic combination treatment with alogliptin (0.03% in diet) and pioglitazone (0.0075% in diet) were evaluated in db/db mice exhibiting early stages of diabetes.

Key results: Alogliptin inhibited plasma DPP-4 activity up to 84% and increased plasma active glucagon-like peptide-1 by 4.4- to 4.9-fold. Unexpectedly, alogliptin alone lacked clear efficacy for improving glucose levels. However, alogliptin in combination with pioglitazone clearly enhanced the effects of pioglitazone alone. After 3-4 weeks of treatment, combination treatment increased plasma insulin by 3.8-fold, decreased plasma glucagon by 41%, both of which were greater than each drug alone, and increased plasma adiponectin by 2.4-fold. In addition, combination treatment decreased glycosylated haemoglobin by 2.2%, plasma glucose by 52%, plasma triglycerides by 77% and non-esterified fatty acids by 48%, all of which were greater than each drug alone. Combination treatment also increased expression of insulin and pancreatic and duodenal homeobox 1 (PDX1), maintained normal beta-cell/alpha-cell distribution in islets and restored pancreatic insulin content to levels comparable to non-diabetic mice.

Conclusions and implications: These results indicate that combination treatment with alogliptin and pioglitazone at an early stage of diabetes improved metabolic profiles and indices that measure beta-cell function, and maintained islet structure in db/db mice, compared with either alogliptin or pioglitazone monotherapy.

Figure 1

Effects of short-term treatment with alogliptin on plasma DPP-4 activity (A) and active GLP-1 levels (B). Eight week old mice were treated with vehicle or alogliptin at the indicated dosages for 2 days. After treatment, blood samples were collected and plasma DPP-4 activity and plasma active GLP-1 levels were determined. DPP-4 activity is presented as a relative percentage compared with vehicle-treated db/db mice. Plasma DPP-4 activity was significantly (#P ≤ 0.025) reduced at all dosages when compared with vehicle-treated db/db mice by one-tailed Shirley–Williams test. Plasma active GLP-1 was significantly (*P ≤ 0.025) increased at all dosages when compared with vehicle-treated db/db mice by one-tailed Williams' test. Data are presented as means ± SD (n = 8 for db/db mice, n = 5 for db/+ mice). DPP-4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide-1.

Figure 2

Chronic effects of alogliptin-, pioglitazone- and alogliptin plus pioglitazone combination treatment on body weight (A) and food consumption (B). Animals were fed a diet containing alogliptin (Alo; 0.03%), pioglitazone (Pio; 0.0075%) or alogliptin (0.03%) plus pioglitazone (0.0075%) during the study period. Body weight and food consumption were measured at designated intervals throughout the treatment period. Data are presented as means ± SD (n = 8 for db/db mice, n = 4 for db/+ mice). The results from a two-way anova are presented below the figure. NS, not significant.

Figure 3

Chronic effects of alogliptin-, pioglitazone- and alogliptin plus pioglitazone combination treatment on the plasma levels of DPP-4 activity (A), insulin (B), glucagon (C) and adiponectin (D). Animals were fed a diet containing alogliptin (Alo; 0.03%), pioglitazone (Pio; 0.0075%) or alogliptin (0.03%) plus pioglitazone (0.0075%) during the study period. Plasma DPP-4 activity was determined after 21 days, plasma insulin levels were determined after 14 and 21 days, and plasma glucagon and adiponectin levels were determined after 23 days. Alogliptin treatment decreased plasma DPP-4 activity and pioglitazone treatment increased plasma adiponectin levels. Although the combination of alogliptin with pioglitazone was more effective at increasing plasma insulin levels and decreasing glucagon levels, these effects were not statistically additive or synergistic by two-way anova. Data are presented as means ± SD (n = 8 for db/db mice, n = 4 for db/+ mice). The results from a two-way anova are presented below the figure. DPP-4, dipeptidyl peptidase-4; NS, not significant.

Figure 4

Chronic effects of alogliptin-, pioglitazone- and alogliptin plus pioglitazone combination treatment on glycosylated haemoglobin (A), non-fasting plasma glucose (B), fasting plasma glucose and glucose excursion during an OGTT (C) and glucose AUC during an OGTT (D). Animals were fed a diet containing alogliptin (Alo; 0.03%), pioglitazone (Pio; 0.0075%) or alogliptin (0.03%) plus pioglitazone (0.0075%) during the study period. Glycosylated haemoglobin was determined after 21 days, non-fasting plasma glucose levels were determined after 14 and 21 days, and fasting plasma glucose, glucose excursion and glucose AUC during an OGTT were determined after 25 days followed by 17 h fasting. Combination treatment with alogliptin and pioglitazone resulted in additive improvements in glycosylated haemoglobin and glucose AUC during an OGTT, and a synergistic improvement in non-fasting plasma glucose. Although the combination of alogliptin with pioglitazone was more effective at lowering fasting plasma glucose levels, the effect was not statistically additive or synergistic. Data are presented as means ± SD (n = 8 for db/db mice, n = 4 for db/+ mice). The results from a two-way anova are presented below the figure. AUC, area under the curve; NS, not significant; OGTT, oral glucose tolerance test.

Figure 5

Chronic effects of alogliptin-, pioglitazone- and alogliptin plus pioglitazone combination treatment on plasma triglyceride (A), fasting plasma triglyceride (B) and non-fasting plasma NEFA (C). Animals were fed a diet containing alogliptin (Alo; 0.03%), pioglitazone (Pio; 0.0075%) or alogliptin (0.03%) plus pioglitazone (0.0075%) during the study period. Non-fasting plasma triglyceride levels were determined after 14 and 21 days, fasting plasma triglyceride levels were determined after 25 days followed by 17 h fasting, and non-fasting plasma NEFA levels were determined after 21 days. Combination treatment with alogliptin and pioglitazone resulted in additive improvements on these parameters. Data are presented as means ± SD (n = 8 for db/db mice, n = 4 for db/+ mice). The results from a two-way anova are presented below the figure. NEFA, non-esterified fatty acids; NS, not significant.

Figure 6

Chronic effects of alogliptin-, pioglitazone- and alogliptin plus pioglitazone combination treatment on pancreatic insulin content. Animals were fed a diet containing alogliptin (Alo; 0.03%), pioglitazone (Pio; 0.0075%) or alogliptin (0.03%) plus pioglitazone (0.0075%) during the study period. After 25 days of treatment, the animals were fasted for 17 h and then given an OGTT. Upon the completion of the OGTT, the pancreas was isolated for processing and analysis of insulin content. Combination treatment with alogliptin and pioglitazone resulted in a synergistic increase in pancreatic insulin content. Data are presented as means ± SD (n = 8 for db/db mice, n = 4 for db/+ mice). The results from a two-way anova are presented below the figure. OGTT, oral glucose tolerance test.

Figure 7

Chronic effects of alogliptin-, pioglitazone- and alogliptin plus pioglitazone combination treatment on insulin-staining, β-cell/α-cell architecture and PDX1 expression in pancreatic islets. Animals were fed a diet containing alogliptin (0.03%), pioglitazone (0.0075%) or alogliptin (0.03%) plus pioglitazone (0.0075%) for 25 days and were fasted for 17 h. After completing an OGTT, the pancreas was isolated and stained with anti-insulin antibody (A–F), anti-glucagon antibody (G–L) and anti-PDX1 antibody (M–R). Representative images for each group of mice are shown. The combination-treated db/db mice exhibited increased expression of insulin and PDX1, and normal β-cell/α-cell distribution, which were comparable to those observed in the vehicle-treated non-diabetic db/+ mice. Scale bars = 200 µm for insulin and glucagon, and 100 µm for PDX1. PDX1, pancreatic and duodenal homeobox 1.