original article
Effect of sitagliptin therapy on triglyceride-rich lipoprotein kinetics in patients with type 2 diabetes A. J. Tremblay1,2 , B. Lamarche2 , I. Kelly3 , A. Charest2 , M.-C. Lépine2 , A. Droit3 & P. Couture1,2 1 Lipid Research Centre, Centre Hospitalier de l’Université Laval (CHUL) Research Centre, Quebec City, QC, Canada 2 Institute of Nutrition and Functional Foods, Laval University, Quebec City, QC, Canada 3 Proteomic Centre, CHUL Research Centre, Quebec City, QC, Canada
Aim: To investigate the effects of sitagliptin therapy on the kinetics of triglyceride-rich lipoprotein (TRL) apolipoprotein (apo)B-48, VLDL apoB-100, apoE and apoC-III in patients with type 2 diabetes. Methods: Twenty-two subjects with type 2 diabetes were recruited in this double-blind crossover study, during which the subjects received sitagliptin (100 mg/day) or placebo for a 6-week period each. At the end of each phase of treatment, the in vivo kinetics of the different apolipoproteins were assessed using a primed-constant infusion of L-[5,5,5-D3]leucine for 12 h, with the participants in a constantly fed state. Results: Sitagliptin therapy significantly reduced fasting plasma triglyceride (−15.4%, p = 0.03), apoB-48 (−16.3%, p = 0.03) and free fatty acid concentrations (−9.5%, p = 0.04), as well as plasma HbA1c (placebo: 7.0% ± 0.8 vs. sitagliptin: 6.6% ± 0.7, p < 0.0001) and plasma glucose levels (−13.5%, p = 0.001), without any significant effect on insulin levels. Kinetic results showed that treatment with sitagliptin significantly reduced the pool size of TRL apoB-48 by −20.8% (p = 0.03), paralleled by a reduction in the production rate of these particles (−16.0%, p = 0.03). The VLDL apoB-100 pool size was also significantly decreased by sitagliptin therapy (−9.3%, p = 0.03), mainly because of a reduction in the hepatic secretion of these lipoproteins, although this difference did not reach statistical significance (−9.2%, p = 0.06). Conclusions: Treatment with sitagliptin for 6 weeks reduced triglyceride-rich apoB-containing lipoprotein levels by reducing the synthesis of these particles. Keywords: apolipoprotein B-100, apolipoprotein B-48, dipeptidyl peptidase-4, glucagon-like peptide-1, glucose, insulin, kinetic, sitagliptin, type 2 diabetes Date submitted 17 April 2014; date of first decision 23 May 2014; date of final acceptance 21 July 2014
Introduction Type 2 diabetes is a complex and multifactorial disease that is associated with an increased risk of cardiovascular events and is frequently characterized by an atherogenic dyslipidaemia that includes elevated levels of triglycerides (TG), reduced concentrations of HDL cholesterol and increased numbers of small, dense LDL particles [1]. Furthermore, excessive postprandial lipaemia is also now recognized as an inherent feature of diabetic dyslipidaemia and non-diabetic insulin-resistance [2]. There is now convincing evidence that insulin-resistant states are associated with an increased hepatic secretion of apolipoprotein (apo)B-100-containing VLDL and an increased intestinal secretion of apoB-48-containing chylomicrons [3,4]. Elevated levels of both liver- and intestine-derived lipoproteins are potentially atherogenic and associated with an increased risk of cardiovascular disease [2,5]; therefore, there is an interest in identifying specific therapies to improve both postprandial lipaemia and glycaemia in patients with type 2 diabetes. Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are incretin hormones that are Correspondence to: P. Couture, MD, FRCP(C), PhD, Lipid Research Centre, CHUL Research Centre, 2705 Laurier Boulevard, S-102 Québec, QC, G1V 4G2,Canada. E-mail: [email protected]
produced by the gastrointestinal tract after a meal. They play a major role in glucose homeostasis by stimulating insulin secretion, suppressing glucagon secretion, inhibiting gastric emptying and reducing appetite and food intake [6,7]. Both incretin hormones are rapidly degraded and removed from the circulation by the enzyme dipeptidyl peptidase-4 (DPP-4) [8]. In type 2 diabetes, the incretin response is blunted, largely as a result of reduced incretin action [9]; therefore, preventing GLP-1 and GIP inactivation by inhibiting DPP-4 now represents a novel strategy for the treatment of type 2 diabetes. Sitagliptin, a selective DPP-4 inhibitor, reduces both fasting and postprandial plasma glucose concentrations, presumably by inhibiting the inactivation of GLP-1 and GIP and thereby prolonging their duration of action on the pancreatic islets [10]. Although the incretin hormones do not have appreciable effects on fasting lipid levels, they do have significant effects on postprandial lipaemia. Animal studies have suggested that incretin hormones reduce intestinal TG absorption and apolipoprotein production [11] and increase chylomicron catabolism [12]. A recent study from our group showed that sitagliptin therapy for 6 weeks decreased the postprandial plasma levels of TG-rich lipoproteins (TRLs) from both intestinal and hepatic origins in patients with type 2 diabetes [13]. Moreover, a previous study reported that therapy with the DPP-4 inhibitor vildagliptin also
ORIGINAL ARTICLE
Diabetes, Obesity and Metabolism 16: 1223–1229, 2014. © 2014 John Wiley & Sons Ltd
original article reduced postprandial lipaemia in patients with type 2 diabetes, with no significant effect on fasting lipid levels [14]. The objective of this study was to determine the effect of sitagliptin on the kinetics of TG-rich apoB-48- and apoB-100-containing lipoproteins as well as on VLDL apoE and apoC-III in patients with type 2 diabetes. We hypothesized that 6-week therapy with sitagliptin (100 mg/day) would reduce TRL levels by decreasing the production of these atherogenic particles.
Methods Subjects Twenty-two patients (18 men and four postmenopausal women who were not receiving hormone therapy) with type 2 diabetes, as defined by the American Diabetes Association [15], were recruited in the Quebec City area to participate in the study. To be part of the study, participants had to have received stable doses of metformin for at least 3 months before randomization. All eligible subjects had to be withdrawn from lipid-lowering medications or other antidiabetic drugs for at least 6 weeks before the beginning of the study. Subjects with monogenic lipid disorders, type 1 diabetes, insulin treatment, a previous history of cardiovascular disease, a recent history of alcohol or drug abuse, disorders of the haematologic, digestive or central nervous systems, known impairment of renal function, persistent elevations of serum transaminases, uncontrolled diabetes mellitus (HbA1c >8.5%) or a history of cancer or any other conditions that may interfere with optimum participation in the study were ineligible. The study consisted of a 1-week screening period and a 4-week placebo run-in period, followed by two 6-week double-blind, crossover treatment periods with sitagliptin (100 mg/day) and placebo given in random order, with a 4-week wash-out period between the two phases. Fasting blood samples were collected at the end of each treatment. Kinetic studies using primed-constant infusion of deuterated leucine were also performed at the end of each treatment. Participants were instructed to take one capsule before their morning meal. Compliance was assessed by pill counting. The research protocol was approved by the Laval University Medical Center ethical review committee, and written informed consent was obtained from each participant. This trial was registered at clinicaltrials.gov as NCT01334229.
Experimental Protocol for In Vivo Stable Isotope Kinetics To determine the kinetics of TRL apoB-48 and of VLDL apoB-100, apoC-III and apoE, the participants underwent a primed-constant infusion of l-[5,5,5-D3 ] leucine while kept in a constant fed state. Starting at 07:00 h, the subjects received one small cookie every half hour for 15 h, each containing 1/30th of their estimated daily food intake, based on the Harris–Benedict equation [16], with 15% of the calories from protein, 45% from carbohydrates and 40% from fat, as well as 85 mg of cholesterol/1000 kcal. At 10:00 h, with two i.v. lines in place (one for the infusate and one for blood sampling), l-[5,5,5-D3 ] leucine (10 μmol/kg body wt) was injected as a bolus i.v. and then by
1224 Tremblay et al.
DIABETES, OBESITY AND METABOLISM
continuous infusion (10 μmol/kg body weight per h) over a 12-h period. Blood samples (24 ml) were collected at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 11 and 12 h. For sample processing, laboratory measurements, analysis of lipoprotein production and clearance rates, please see supporting information Appendix S1.
Total RNA Extraction, RNA Quantification and Quantitative Real-Time PCR Blood samples that were intended for the gene expression measurements were collected in PAXgene Blood RNA vacutainers (PreAnalytix, Hombechtikon, Switzerland), in both fasting and fed states, and incubated at room temperature for 2 h for RNA stabilization, stored at −20∘ C for at least 24 h and then frozen at −80∘ C until analysis. At this time, RNA was extracted from the blood using the PAXgeneTM Blood RNA System Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total RNA was eluted into 100 μL RNase-free H2 O and stored at −80∘ C. RNA concentrations were determined using
®
a NanoDrop ND-2000C spectrophotometer (Techmo Scientific, Waltham, MA, USA). RNA quantification and quantitative real-time PCR were performed with QuantStudio technology (Life Technologies, Carlsbad, CA, USA).
Statistical Analysis Student’s paired t-tests were used to assess the effects of sitagliptin versus placebo on fasting lipid/lipoprotein profiles, glucose homeostasis, kinetic variables and mRNA expression. Mixed models with proper interaction terms have shown no evidence of a carryover effect of the sitagliptin treatment. Spearman’s correlation coefficients were determined to assess the significance of the associations. Differences were considered significant at p ≤ 0.05. All analyses were performed using JMP Statistical Software (version 10.0, SAS Institute, Cary, NC, USA).
Results Demographic Characteristics and Fasting Biochemical Variables The participants’ mean ± standard deviation (s.d.) age (18 men and 4 postmenopausal women) was 58.2 ± 3.8 years and no significant differences were observed in weight and body mass index between the two phases of treatment. The participant’s fasting lipid/lipoprotein profiles and glycaemic variables after the 6-week treatment with either placebo or sitagliptin (100 mg/day) are shown in Table 1. Sitagliptin led to significant reductions in plasma and VLDL TG concentrations (−12.8%, p = 0.03 and −15.4%, p = 0.03, respectively) and to reductions in apoB-48 and free fatty acid (FFA) levels (−16.3%, p = 0.03 and −9.5%, p = 0.04, respectively). Sitagliptin therapy significantly reduced fasting plasma glucose levels (−13.5%, p = 0.001) but had no impact on fasting plasma insulin levels. Sitagliptin also improved HbA1c levels compared with placebo (placebo: 7.0% vs. sitagliptin: 6.6%; p < 0.0001). As expected, treatment with sitagliptin significantly increased the
Volume 16 No. 12 December 2014
original article
DIABETES, OBESITY AND METABOLISM
levels of GLP-1 (+80.6%; p < 0.0001). Moreover, 𝛽-cell function (+11.1%; p = 0.06) tended to be improved by sitagliptin therapy compared with placebo, whereas the homeostasis model assessment index for insulin resistance tended to be reduced (−20.3%; p = 0.06). There was no change in systolic or diastolic blood pressure after sitagliptin therapy. Finally, the plasma levels of FFAs in the fed state did not change with sitagliptin treatment (placebo: 293.6 ± 123.9 μM vs. sitagliptin: 287.4 ± 112.7 μM; p = 0.6).
Kinetics of TRL apoB-48, VLDL apoB-100, VLDL apoC-III and VLDL apoE Analyses of the deuterated plasma amino acids and the lipid/lipoprotein measurements indicated that plasma leucine enrichments and plasma TG and TRL apoB-48 levels remained constant over the course of the infusion (data not shown). Detailed kinetic information, obtained by conducting a multicompartmental model analysis, is summarized in Table 2. Compared with placebo, treatment with sitagliptin significantly reduced the TRL apoB-48 and VLDL apoB-100 pool sizes in subjects with type 2 diabetes (−20.8%, p = 0.03 and −9.3%,
p = 0.03, respectively). The reduction in TRL apoB-48 levels with sitagliptin therapy was mainly attributable to a significant decrease in the secretion rate (−16.0%; p = 0.03) of this lipoprotein. The reduction in VLDL apoB-100 pool size after sitagliptin therapy was also partly attributable to a reduction in hepatic secretion of these particles (−9.2%; p = 0.06), but this change did not reach statistical significance. Moreover, treatment with sitagliptin did not have any significant effect on VLDL apoC-III and apoE levels compared with placebo; however, the fractional catabolic and production rates of VLDL apoE were significantly reduced by sitagliptin therapy (−10.6%, p = 0.05 and −12.7%, p = 0.04, respectively). Finally, changes in the TRL apoB-48 PR were positively correlated with changes in the VLDL apoE production rate (r = 0.43, p = 0.04) after treatment with sitagliptin (Figure 1).
Peripheral Blood mRNA Levels As shown in Table 3, sitagliptin treatment had no significant effect on the mRNA expression of various key genes that are involved in lipid and lipoprotein metabolism in peripheral blood.
Table 1. Demographic and fasting biochemical characteristics of the participants with type 2 diabetes after a 6-week treatment with sitagliptin 100 mg/day and placebo.
Weight, kg Body mass index, kg/m2 Waist circumference, cm Plasma Cholesterol, mmol/l Triglycerides, mmol/l Apolipoprotein B, g/l Apolipoprotein B-48, mg/dl Free fatty acids, μM VLDL Cholesterol, mmol/l Triglycerides, mmol/l Apolipoprotein B, g/l LDL Cholesterol, mmol/l Triglycerides, mmol/l Apolipoprotein B, g/l HDL Cholesterol, mmol/l Triglycerides, mmol/l Apolipoprotein A-I, g/l Glucose homeostasis HbA1c, % Plasma glucose, mmol/l Plasma insulin, 𝜌mol/l GLP-1 (active 7–36), 𝜌mol/l Homeostasis model assessment of insulin resistance 𝛽-cell function Blood pressure Systolic blood pressure, mm Hg Diastolic blood pressure, mm Hg
Placebo (n = 22) Mean ± s.d.
Sitagliptin (n = 22) Mean ± s.d.
% change*
p
95.1 ± 18.3 32.2 ± 5.4 112.4 ± 14.3
95.1 ± 18.2 32.2 ± 5.4 111.3 ± 15.2
0.0 0.0 −1.0
0.9 0.9 0.05
5.47 ± 1.20 2.50 ± 1.05 1.09 ± 0.24 1.23 ± 0.73 559 ± 187
5.38 ± 1.11 2.18 ± 0.67 1.06 ± 0.24 1.03 ± 0.53 506 ± 175
−1.6 −12.8 −2.8 −16.3 −9.5
0.3 0.03 0.2 0.03 0.04
0.98 ± 0.52 1.88 ± 0.98 0.16 ± 0.07
0.89 ± 0.36 1.59 ± 0.60 0.15 ± 0.05
−9.2 −15.4 −6.3
0.2 0.03 0.3
3.43 ± 1.17 0.30 ± 0.09 0.93 ± 0.23
3.39 ± 1.02 0.28 ± 0.08 0.91 ± 0.23
−1.2 −6.7 −2.2
0.7 0.2 0.4
1.06 ± 0.22 0.33 ± 0.08 1.32 ± 0.19
1.10 ± 0.22 0.32 ± 0.08 1.34 ± 0.21
+3.8 −3.0 +1.5
0.1 0.8 0.6
7.0 ± 0.8 8.9 ± 1.5 179.5 ± 88.1 3.1 ± 5.8 10.3 ± 6.2 105.0 ± 56.8
6.6 ± 0.7 7.7 ± 1.2 161.1 ± 91.7 5.6 ± 6.8 8.2 ± 5.8 116.7 ± 67.5
−5.7 −13.5 −10.3 +80.6 −20.3 +11.1
Effect of sitagliptin therapy on triglyceride-rich lipoprotein kinetics in patients with type 2 diabetes A. J. Tremblay1,2 , B. Lamarche2 , I. Kelly3 , A. Charest2 , M.-C. Lépine2 , A. Droit3 & P. Couture1,2 1 Lipid Research Centre, Centre Hospitalier de l’Université Laval (CHUL) Research Centre, Quebec City, QC, Canada 2 Institute of Nutrition and Functional Foods, Laval University, Quebec City, QC, Canada 3 Proteomic Centre, CHUL Research Centre, Quebec City, QC, Canada
Aim: To investigate the effects of sitagliptin therapy on the kinetics of triglyceride-rich lipoprotein (TRL) apolipoprotein (apo)B-48, VLDL apoB-100, apoE and apoC-III in patients with type 2 diabetes. Methods: Twenty-two subjects with type 2 diabetes were recruited in this double-blind crossover study, during which the subjects received sitagliptin (100 mg/day) or placebo for a 6-week period each. At the end of each phase of treatment, the in vivo kinetics of the different apolipoproteins were assessed using a primed-constant infusion of L-[5,5,5-D3]leucine for 12 h, with the participants in a constantly fed state. Results: Sitagliptin therapy significantly reduced fasting plasma triglyceride (−15.4%, p = 0.03), apoB-48 (−16.3%, p = 0.03) and free fatty acid concentrations (−9.5%, p = 0.04), as well as plasma HbA1c (placebo: 7.0% ± 0.8 vs. sitagliptin: 6.6% ± 0.7, p < 0.0001) and plasma glucose levels (−13.5%, p = 0.001), without any significant effect on insulin levels. Kinetic results showed that treatment with sitagliptin significantly reduced the pool size of TRL apoB-48 by −20.8% (p = 0.03), paralleled by a reduction in the production rate of these particles (−16.0%, p = 0.03). The VLDL apoB-100 pool size was also significantly decreased by sitagliptin therapy (−9.3%, p = 0.03), mainly because of a reduction in the hepatic secretion of these lipoproteins, although this difference did not reach statistical significance (−9.2%, p = 0.06). Conclusions: Treatment with sitagliptin for 6 weeks reduced triglyceride-rich apoB-containing lipoprotein levels by reducing the synthesis of these particles. Keywords: apolipoprotein B-100, apolipoprotein B-48, dipeptidyl peptidase-4, glucagon-like peptide-1, glucose, insulin, kinetic, sitagliptin, type 2 diabetes Date submitted 17 April 2014; date of first decision 23 May 2014; date of final acceptance 21 July 2014
Introduction Type 2 diabetes is a complex and multifactorial disease that is associated with an increased risk of cardiovascular events and is frequently characterized by an atherogenic dyslipidaemia that includes elevated levels of triglycerides (TG), reduced concentrations of HDL cholesterol and increased numbers of small, dense LDL particles [1]. Furthermore, excessive postprandial lipaemia is also now recognized as an inherent feature of diabetic dyslipidaemia and non-diabetic insulin-resistance [2]. There is now convincing evidence that insulin-resistant states are associated with an increased hepatic secretion of apolipoprotein (apo)B-100-containing VLDL and an increased intestinal secretion of apoB-48-containing chylomicrons [3,4]. Elevated levels of both liver- and intestine-derived lipoproteins are potentially atherogenic and associated with an increased risk of cardiovascular disease [2,5]; therefore, there is an interest in identifying specific therapies to improve both postprandial lipaemia and glycaemia in patients with type 2 diabetes. Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are incretin hormones that are Correspondence to: P. Couture, MD, FRCP(C), PhD, Lipid Research Centre, CHUL Research Centre, 2705 Laurier Boulevard, S-102 Québec, QC, G1V 4G2,Canada. E-mail: [email protected]
produced by the gastrointestinal tract after a meal. They play a major role in glucose homeostasis by stimulating insulin secretion, suppressing glucagon secretion, inhibiting gastric emptying and reducing appetite and food intake [6,7]. Both incretin hormones are rapidly degraded and removed from the circulation by the enzyme dipeptidyl peptidase-4 (DPP-4) [8]. In type 2 diabetes, the incretin response is blunted, largely as a result of reduced incretin action [9]; therefore, preventing GLP-1 and GIP inactivation by inhibiting DPP-4 now represents a novel strategy for the treatment of type 2 diabetes. Sitagliptin, a selective DPP-4 inhibitor, reduces both fasting and postprandial plasma glucose concentrations, presumably by inhibiting the inactivation of GLP-1 and GIP and thereby prolonging their duration of action on the pancreatic islets [10]. Although the incretin hormones do not have appreciable effects on fasting lipid levels, they do have significant effects on postprandial lipaemia. Animal studies have suggested that incretin hormones reduce intestinal TG absorption and apolipoprotein production [11] and increase chylomicron catabolism [12]. A recent study from our group showed that sitagliptin therapy for 6 weeks decreased the postprandial plasma levels of TG-rich lipoproteins (TRLs) from both intestinal and hepatic origins in patients with type 2 diabetes [13]. Moreover, a previous study reported that therapy with the DPP-4 inhibitor vildagliptin also
ORIGINAL ARTICLE
Diabetes, Obesity and Metabolism 16: 1223–1229, 2014. © 2014 John Wiley & Sons Ltd
original article reduced postprandial lipaemia in patients with type 2 diabetes, with no significant effect on fasting lipid levels [14]. The objective of this study was to determine the effect of sitagliptin on the kinetics of TG-rich apoB-48- and apoB-100-containing lipoproteins as well as on VLDL apoE and apoC-III in patients with type 2 diabetes. We hypothesized that 6-week therapy with sitagliptin (100 mg/day) would reduce TRL levels by decreasing the production of these atherogenic particles.
Methods Subjects Twenty-two patients (18 men and four postmenopausal women who were not receiving hormone therapy) with type 2 diabetes, as defined by the American Diabetes Association [15], were recruited in the Quebec City area to participate in the study. To be part of the study, participants had to have received stable doses of metformin for at least 3 months before randomization. All eligible subjects had to be withdrawn from lipid-lowering medications or other antidiabetic drugs for at least 6 weeks before the beginning of the study. Subjects with monogenic lipid disorders, type 1 diabetes, insulin treatment, a previous history of cardiovascular disease, a recent history of alcohol or drug abuse, disorders of the haematologic, digestive or central nervous systems, known impairment of renal function, persistent elevations of serum transaminases, uncontrolled diabetes mellitus (HbA1c >8.5%) or a history of cancer or any other conditions that may interfere with optimum participation in the study were ineligible. The study consisted of a 1-week screening period and a 4-week placebo run-in period, followed by two 6-week double-blind, crossover treatment periods with sitagliptin (100 mg/day) and placebo given in random order, with a 4-week wash-out period between the two phases. Fasting blood samples were collected at the end of each treatment. Kinetic studies using primed-constant infusion of deuterated leucine were also performed at the end of each treatment. Participants were instructed to take one capsule before their morning meal. Compliance was assessed by pill counting. The research protocol was approved by the Laval University Medical Center ethical review committee, and written informed consent was obtained from each participant. This trial was registered at clinicaltrials.gov as NCT01334229.
Experimental Protocol for In Vivo Stable Isotope Kinetics To determine the kinetics of TRL apoB-48 and of VLDL apoB-100, apoC-III and apoE, the participants underwent a primed-constant infusion of l-[5,5,5-D3 ] leucine while kept in a constant fed state. Starting at 07:00 h, the subjects received one small cookie every half hour for 15 h, each containing 1/30th of their estimated daily food intake, based on the Harris–Benedict equation [16], with 15% of the calories from protein, 45% from carbohydrates and 40% from fat, as well as 85 mg of cholesterol/1000 kcal. At 10:00 h, with two i.v. lines in place (one for the infusate and one for blood sampling), l-[5,5,5-D3 ] leucine (10 μmol/kg body wt) was injected as a bolus i.v. and then by
1224 Tremblay et al.
DIABETES, OBESITY AND METABOLISM
continuous infusion (10 μmol/kg body weight per h) over a 12-h period. Blood samples (24 ml) were collected at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 11 and 12 h. For sample processing, laboratory measurements, analysis of lipoprotein production and clearance rates, please see supporting information Appendix S1.
Total RNA Extraction, RNA Quantification and Quantitative Real-Time PCR Blood samples that were intended for the gene expression measurements were collected in PAXgene Blood RNA vacutainers (PreAnalytix, Hombechtikon, Switzerland), in both fasting and fed states, and incubated at room temperature for 2 h for RNA stabilization, stored at −20∘ C for at least 24 h and then frozen at −80∘ C until analysis. At this time, RNA was extracted from the blood using the PAXgeneTM Blood RNA System Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total RNA was eluted into 100 μL RNase-free H2 O and stored at −80∘ C. RNA concentrations were determined using
®
a NanoDrop ND-2000C spectrophotometer (Techmo Scientific, Waltham, MA, USA). RNA quantification and quantitative real-time PCR were performed with QuantStudio technology (Life Technologies, Carlsbad, CA, USA).
Statistical Analysis Student’s paired t-tests were used to assess the effects of sitagliptin versus placebo on fasting lipid/lipoprotein profiles, glucose homeostasis, kinetic variables and mRNA expression. Mixed models with proper interaction terms have shown no evidence of a carryover effect of the sitagliptin treatment. Spearman’s correlation coefficients were determined to assess the significance of the associations. Differences were considered significant at p ≤ 0.05. All analyses were performed using JMP Statistical Software (version 10.0, SAS Institute, Cary, NC, USA).
Results Demographic Characteristics and Fasting Biochemical Variables The participants’ mean ± standard deviation (s.d.) age (18 men and 4 postmenopausal women) was 58.2 ± 3.8 years and no significant differences were observed in weight and body mass index between the two phases of treatment. The participant’s fasting lipid/lipoprotein profiles and glycaemic variables after the 6-week treatment with either placebo or sitagliptin (100 mg/day) are shown in Table 1. Sitagliptin led to significant reductions in plasma and VLDL TG concentrations (−12.8%, p = 0.03 and −15.4%, p = 0.03, respectively) and to reductions in apoB-48 and free fatty acid (FFA) levels (−16.3%, p = 0.03 and −9.5%, p = 0.04, respectively). Sitagliptin therapy significantly reduced fasting plasma glucose levels (−13.5%, p = 0.001) but had no impact on fasting plasma insulin levels. Sitagliptin also improved HbA1c levels compared with placebo (placebo: 7.0% vs. sitagliptin: 6.6%; p < 0.0001). As expected, treatment with sitagliptin significantly increased the
Volume 16 No. 12 December 2014
original article
DIABETES, OBESITY AND METABOLISM
levels of GLP-1 (+80.6%; p < 0.0001). Moreover, 𝛽-cell function (+11.1%; p = 0.06) tended to be improved by sitagliptin therapy compared with placebo, whereas the homeostasis model assessment index for insulin resistance tended to be reduced (−20.3%; p = 0.06). There was no change in systolic or diastolic blood pressure after sitagliptin therapy. Finally, the plasma levels of FFAs in the fed state did not change with sitagliptin treatment (placebo: 293.6 ± 123.9 μM vs. sitagliptin: 287.4 ± 112.7 μM; p = 0.6).
Kinetics of TRL apoB-48, VLDL apoB-100, VLDL apoC-III and VLDL apoE Analyses of the deuterated plasma amino acids and the lipid/lipoprotein measurements indicated that plasma leucine enrichments and plasma TG and TRL apoB-48 levels remained constant over the course of the infusion (data not shown). Detailed kinetic information, obtained by conducting a multicompartmental model analysis, is summarized in Table 2. Compared with placebo, treatment with sitagliptin significantly reduced the TRL apoB-48 and VLDL apoB-100 pool sizes in subjects with type 2 diabetes (−20.8%, p = 0.03 and −9.3%,
p = 0.03, respectively). The reduction in TRL apoB-48 levels with sitagliptin therapy was mainly attributable to a significant decrease in the secretion rate (−16.0%; p = 0.03) of this lipoprotein. The reduction in VLDL apoB-100 pool size after sitagliptin therapy was also partly attributable to a reduction in hepatic secretion of these particles (−9.2%; p = 0.06), but this change did not reach statistical significance. Moreover, treatment with sitagliptin did not have any significant effect on VLDL apoC-III and apoE levels compared with placebo; however, the fractional catabolic and production rates of VLDL apoE were significantly reduced by sitagliptin therapy (−10.6%, p = 0.05 and −12.7%, p = 0.04, respectively). Finally, changes in the TRL apoB-48 PR were positively correlated with changes in the VLDL apoE production rate (r = 0.43, p = 0.04) after treatment with sitagliptin (Figure 1).
Peripheral Blood mRNA Levels As shown in Table 3, sitagliptin treatment had no significant effect on the mRNA expression of various key genes that are involved in lipid and lipoprotein metabolism in peripheral blood.
Table 1. Demographic and fasting biochemical characteristics of the participants with type 2 diabetes after a 6-week treatment with sitagliptin 100 mg/day and placebo.
Weight, kg Body mass index, kg/m2 Waist circumference, cm Plasma Cholesterol, mmol/l Triglycerides, mmol/l Apolipoprotein B, g/l Apolipoprotein B-48, mg/dl Free fatty acids, μM VLDL Cholesterol, mmol/l Triglycerides, mmol/l Apolipoprotein B, g/l LDL Cholesterol, mmol/l Triglycerides, mmol/l Apolipoprotein B, g/l HDL Cholesterol, mmol/l Triglycerides, mmol/l Apolipoprotein A-I, g/l Glucose homeostasis HbA1c, % Plasma glucose, mmol/l Plasma insulin, 𝜌mol/l GLP-1 (active 7–36), 𝜌mol/l Homeostasis model assessment of insulin resistance 𝛽-cell function Blood pressure Systolic blood pressure, mm Hg Diastolic blood pressure, mm Hg
Placebo (n = 22) Mean ± s.d.
Sitagliptin (n = 22) Mean ± s.d.
% change*
p
95.1 ± 18.3 32.2 ± 5.4 112.4 ± 14.3
95.1 ± 18.2 32.2 ± 5.4 111.3 ± 15.2
0.0 0.0 −1.0
0.9 0.9 0.05
5.47 ± 1.20 2.50 ± 1.05 1.09 ± 0.24 1.23 ± 0.73 559 ± 187
5.38 ± 1.11 2.18 ± 0.67 1.06 ± 0.24 1.03 ± 0.53 506 ± 175
−1.6 −12.8 −2.8 −16.3 −9.5
0.3 0.03 0.2 0.03 0.04
0.98 ± 0.52 1.88 ± 0.98 0.16 ± 0.07
0.89 ± 0.36 1.59 ± 0.60 0.15 ± 0.05
−9.2 −15.4 −6.3
0.2 0.03 0.3
3.43 ± 1.17 0.30 ± 0.09 0.93 ± 0.23
3.39 ± 1.02 0.28 ± 0.08 0.91 ± 0.23
−1.2 −6.7 −2.2
0.7 0.2 0.4
1.06 ± 0.22 0.33 ± 0.08 1.32 ± 0.19
1.10 ± 0.22 0.32 ± 0.08 1.34 ± 0.21
+3.8 −3.0 +1.5
0.1 0.8 0.6
7.0 ± 0.8 8.9 ± 1.5 179.5 ± 88.1 3.1 ± 5.8 10.3 ± 6.2 105.0 ± 56.8
6.6 ± 0.7 7.7 ± 1.2 161.1 ± 91.7 5.6 ± 6.8 8.2 ± 5.8 116.7 ± 67.5
−5.7 −13.5 −10.3 +80.6 −20.3 +11.1
