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Effect of sitagliptin treatment on metabolism and cardiac function in genetic diabetic mice
European Journal of Pharmacology 723 (2014) 175–180
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Endocrine pharmacology
Effect of sitagliptin treatment on metabolism and cardiac function in genetic diabetic mice Bianca Hemmeryckx a, Melissa Swinnen b, David J Gallacher c, Hua Rong Lu c, H. Roger Lijnen a,n a
Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium Division of Clinical Cardiology, KU Leuven, Leuven, Belgium c Translational Sciences, Safety Pharmacology Research, Janssen Research & Development, Janssen Pharmaceutical NV, Beerse, Belgium b
art ic l e i nf o
a b s t r a c t
Article history: Received 26 September 2013 Received in revised form 8 December 2013 Accepted 14 December 2013 Available online 6 January 2014
To investigate the chronic effect of sitagliptin (7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]5,6,7,8-tetrahydro-(3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine phosphate (1:1) monohydrate, SIT) on metabolism and cardiac function in genetic diabetic Akita mice, 10 weeks old Akita mice were either exposed for 4 months to a high fat and high cholesterol (HF–HC) diet, with or without 10 mg/kg/day SIT, or were fed for 3 months with the same diet with or without 50 mg/kg/day SIT. SIT treatment of Akita mice at either a low or high dose did not affect body or liver weight. A significant increase in subcutaneous and gonadal fat mass was only observed for the 50 mg/kg/day dose of SIT. Furthermore, only the 50 mg/kg/day SIT dose resulted in an improvement of glycemic control, as evidenced by a decrease in fasting blood HbA1c levels and an increase in plasma adiponectin levels. Echocardiographic analysis revealed that Akita mice kept on the HF–HC diet with 10 mg/kg/day of SIT for 4 months showed an increase in ejection fraction and fractional shortening, whereas the higher dose (50 mg/kg/day) had no effect on these parameters, but instead induced left ventricular (LV) hypertrophy as evidenced by an enlarged LV internal diameter, volume and mass. Thus, in the diabetic Akita mouse SIT is cardioprotective at a low dose (10 mg/kg/day), whereas improvement of glycemic control requires a higher dose (50 mg/kg/day) which, however, induces LV hypertrophy. This mouse model may thus be useful to study the safety of antidiabetic drugs. & 2013 Elsevier B.V. All rights reserved.
Keywords: Cardiac function Genetic Akita mice Sitagliptin
1. Introduction Diabetes mellitus (DM) has reached epidemic proportions: globally, in 2010 an estimated 285 million people suffered from this metabolic disease (Shaw et al., 2010); 90% of DM patients is type 2 (T2DM) (Zimmet et al., 2001). The pathology of T2DM is characterized by defects in insulin sensitivity and secretion, resulting in hyperglycemia (Taylor, 1999). Current therapies for T2DM are associated with adverse drug effects such as hypoglycemia (Micheli et al., 2012), weight gain (Quinn et al., 2008), gastrointestinal problems (Stein et al., 2013), liver toxicity (Marcy et al., 2004) and increased risk for cardiovascular diseases (Nesto et al., 2003). To better understand the molecular mechanisms underlying these complications, suitable preclinical rodent models are needed. In a previous study, we have used the type 1 and type 2 diabetic Akita mouse model to establish side effects of the n Correspondence to: Center for Molecular and Vascular Biology, KU Leuven, Campus Gasthuisberg, CDG, Herestraat 49, Box 911, B-3000 Leuven, Belgium. Tel.: þ 32 16 345771; fax: þ32 16 345990. E-mail address: [email protected] (H. Roger Lijnen).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.12.036
thiazolidinedione rosiglitazone (RGZ), including weight gain and increased risk for cardiovascular disease (Hemmeryckx et al., 2013). These mice are heterozygous for a spontaneous mutation (C96Y) in the insulin 2 gene, which prevents proper folding of the insulin protein and results in subsequent pancreatic β-cell endoplasmatic reticulum stress and apoptosis. Akita mice develop hyperglycemia, hypoinsulinemia, polydipsia and polyuria at an age of 3–4 weeks (Hong et al., 2007; Yoshioka et al., 1997). Consequently, insulin production/secretion is very low. In humans, type 1 diabetes is an auto-immune disorder. It is believed that the disease develops due to destruction of the pancreatic β-cells by auto-antibodies evoked by viruses or environmental toxins. As a result, insulin production/secretion is very low or nonexistent. Although the pathophysiology of the disease in humans and the Akita mouse model differs, the outcome is very similar: a very low level of insulin production/secretion. Insulitis however, induced by an auto-immune response in type 1 diabetic patients leading to β-cell death and hypoinsulinemia, does not occur in the Akita mice. In addition, they show symptoms of human type 2 diabetes such as insulin resistance in skeletal muscle (caused by reduced glucose transporter-4 levels), liver and brown adipose tissue that is
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partly due to hyperglycemia (Hong et al., 2007; Yoshioka et al., 1997). However, in T2DM patients insulin-stimulated glucose uptake is reduced and the insulin resistance in skeletal muscle and liver is associated with an increased intracellular lipid accumulation, while in Akita mice the opposite pattern is observed (Hong et al., 2007). The reduced amount of lipids in muscle and liver may be explained by the increased whole body lipid oxidation seen in these mice. A species-associated difference in lipid metabolism therefore exists. To compensate for the altered lipid metabolism, Akita mice display an increase in insulin-stimulated glucose uptake, which allows for glucose to be directed to lipogenesis in white adipose tissues. To further validate this diabetic mouse model in testing the safety of anti-diabetic drugs, sitagliptin (7-[(3R)-3-amino1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-(3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine phosphate (1:1) monohydrate, SIT, Januvias) was administered at two physiological doses (10 and 50 mg/kg/day). SIT is presumed to have a greater impact on type 2 diabetes than RGZ, as its capacity to improve overall glycemic control is higher, and it restores pancreatic islet cell mass (Mu et al., 2009). SIT selectively inhibits the activity of the enzyme dipeptidyl peptidase 4 (DPP-4), leading to increased circulating levels of the incretin hormone glucagon-like peptide 1 (GLP-1) and subsequent promotion of insulin secretion and normalization of blood glucose (Deacon, 2007).
2. Materials and methods 2.1. Animal model Male Akita (Ins2Akita; JAX003548) mice in a C57Bl/6J genetic background were purchased from Jackson Laboratories (Bar Harbor, MN, USA). At the age of 10 weeks, mice kept in individual microisolation cages on a 12 h day/night cycle at 20–22 1C, were started for 4 months on 5 g/day of a high fat-high cholesterol (HF–HC) diet (Ssniff EF Clinton/Cybulsky (II) mod. 19.5% fat and 1.25% cholesterol; E15751-30, Ssniff Spezialitäten GmbH, Soest, Germany), supplemented with (n¼8) or without (n¼11) 10 mg/kg/day SIT (MSD Ltd., Hertfordshire, UK). In a second study, 10 weeks old Akita mice were kept for 3 months on the HF–HC diet ad libitum with (n¼9) or without (n¼10) 50 mg/kg/day SIT. Water was always available ad libitum. Body weight and food intake were measured weekly and daily in the two studies, respectively. Before and at the end of the studies, after a fasting period of 6 h, blood was collected on 3.8% citrate via the retro-orbital sinus to measure fasting blood glucose and glycohemoglobin (HbA1c) levels. Plasma samples were prepared for determination of insulin, adiponectin, glucagon, and GLP-1 levels, and for active DPP4 levels. For comparison, some parameters (insulin and adiponectin) were determined for 10 weeks old wildtype (WT) C57BL/6J male mice (Janvier, Le Genest Saint Isle, France). In addition, transthoracic echocardiographic examinations were performed in anesthetized mice (2% isoflurane; Ecuphar, Oostkamp, Belgium) at the start and end of the treatment, using a MS 400 transducer (18–38 Mhz) (Visualsonics Inc., Toronto, Canada) on a Vevo 2100 equipment. Left ventricle (LV) diameter at end-diastole (LVIDd) and end-systole (LVIDs), muscle thickness in diastole (IVSd) and in systole (IVSs) and LV posterior wall thickness in end-diastole (LVPWd) and end-systole (LVPWs) were measured, and fractional shortening (FS), LV mass, end-diastolic volume (EDV) and endsystolic volume (ESV), ejection fraction (EF), stroke volume (SV) and cardiac output (CO) were calculated (Stypmann et al., 2009). Mice were euthanized by ip injection of 60 mg/kg Nembutal (Abbott Laboratories, North Chicago, IL, USA). Inguinal subcutaneous (SC) and gonadal (GON) fat pads, liver, and heart were removed, weighed and stored for further analysis. The tibia length was also recorded.
All animal experiments were approved by the local ethical committee (KU Leuven P091/2010) and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (1996). 2.2. Assays Glycosylated hemoglobin (HbA1c) in plasma was measured by ion exchange HPLC (Selvin et al., 2005). SIT levels in plasma and extracts of heart tissue were determined by LC–MS/MS (courtesy of Janssen Pharmaceutica N.V., Beerse, Belgium). Blood glucose concentrations were measured using Glucocard strips (Menarini Diagnostics, Firenze, Italy). Plasma levels of insulin (Mercodia, Uppsala, Sweden), adiponectin and glucagon (R&D Systems Europe, Lille, France), GLP-1 (Cusabio, Wuhan, China) and active DPP4 enzyme levels (Enzo Life Sciences, Antwerpen, Belgium) were measured using commercial ELISA0 s. Hematocrit levels in blood samples were determined using a Cell-Dyn 3500R (Abbott Diagnostics, Abbott Park, IL, USA). Plasma volume was estimated as 1 minus the hematocrit value measured at the end of the study multiplied by the estimated blood volume (8% of total body weight) (Ortiz et al., 2002; Reynolds, 1953). 2.3. Statistical analysis Data are shown as means 7 S.E.M. for the number of animals studied. Statistical significance between groups is evaluated by non-parametric Mann–Whitney U-test. Values of P o0.05 are considered statistically significant.
3. Results 3.1. Effect of SIT on body composition and metabolic parameters The body weight of 10 weeks old Akita mice was slightly lower than that of age-matched WT mice (20 g70.17 g versus 21 7 0.28 g, P ¼0.04). Diabetic status was confirmed by higher fasting blood glucose (369714 mg/dL versus 10478.3 mg/dL, Po0.0001) and glycohemoglobin levels (6.870.09% versus 2.670.052%, Po0.0001), as well as hypoinsulinemia (0.07870.010 ng/ml versus 0.2270.088 ng/μl, P40.05) in Akita as compared to WT mice. Upon HF–HC diet feeding, the body weight of the Akita mice increased to 2470.50 g (Po0.0001) after 3 months and to 2470.40 g (P o0.0001) after 4 months. Co-administration of SIT at a dose of 10 mg/kg/day during 4 months or of 50 mg/kg/day during 3 months did not affect food intake, total body weight, liver weight, heart weight or tibia length (Table 1). Whereas the lower dose of SIT did not affect SC or GON fat mass, administration of the higher dose resulted in a significantly increased SC (P¼ 0.04) and GON (P ¼0.01) adipose tissue mass (Table 1). Fasting blood glucose and plasma insulin and glucagon levels were not affected by either dose of SIT, whereas at the higher dose blood HbA1c and plasma GLP-1 levels were decreased and plasma adiponectin concentrations increased as compared to HF–HC diet feeding without addition of SIT (Table 2). 3.2. Effect of SIT on cardiac function Echocardiographic analysis revealed that the hearts of Akita mice upon HF–HC loading showed a significant reduction in FS and EF, due to an increase in diastolic and systolic LVID and LV volume (EDV/ESV), resulting in a higher LV mass. In addition, SV and CO were also enhanced by the diet. These diet-induced changes in cardiac parameters were more pronounced after 4 as compared to 3 months (Table 3).
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Table 1 Effect of a low (10 mg/kg/day during 4 months) or a high dose (50 mg/kg/day during 3 months) sitagliptin (SIT) on body composition of Akita mice fed a high fat-high cholesterol diet 4 months
n Body weight (g) Food intake (g/day) Weight (mg) SC GON Liver Heart Tibia length (mm)
3 months
Akita
Akita þ10 MPK SIT
Akita
Akita þ50 MPK SIT
10 247 0.40 4.9 7 0.042
8 247 1.0 4.9 7 0.038
10 24 70.50 5.8 70.14
9 267 0.47 5.5 7 0.16
234 7 21 308 7 33 21427 172 139 76.2 177 0.074
2467 23 3617 44 2288 7 126 154 77.2 177 0.16
1857 12 288 7 19 21007 113 1157 3.4 177 0.084
2337 16a 403 7 32a 21257 144 1257 6.7 177 0.053
Data are means 7 S.E.M. of n experiments. Abbreviations: subcutaneous (SC) and gonadal (GON). a
Po 0.05 versus untreated Akita mice.
Table 2 Effect of a low (10 mg/kg/day) and a high dose (50 mg/kg/day) sitagliptin (SIT) on metabolic parameters in Akita mice fed a high fat-high cholesterol diet.
n Blood glucose level (mg/dL) Blood HbA1c level (%) Plasma insulin level (ng/mL) Plasma adiponectin level (mg/mL) Plasma glucagon level (pg/ml) Plasma glucagon-like peptide-1 level (ng/ml)
Akita
4 months
3 months
Start
Akita
Akita þ10 MPK SIT
Akita
Akita þ50 MPK SIT
37 369 7 14 6.8 7 0.086 0.0787 0.010 6.6 7 0.34 84 7 9.3 NM
10 228 7 50b 6.2 7 1.1 0.20 7 0.076 3.5 7 0.48b 267 16b 427 741
8 222 7 42b 6.0 71.1b 0.177 0.067 3.2 70.32b ND 454 7 53
10 482 7 45b 7.0 7 0.58 0.137 0.016b 5.4 70.25 68 7 16 4617 37
9 485 7 53b 4.8 7 0.44a,b 0.107 0.027 6.7 7 0.55a 577 17 3747 12a
Data are means 7 S.E.M. of n experiments. Abbreviations: not detectable (ND); not measured (NM). a b
Po 0.05 versus untreated Akita mice. Po 0.05 versus start.
Table 3 Effect of a low (10 mg/kg/day) and a high dose (50 mg/kg/day) sitagliptin (SIT) on cardiac function in Akita mice on a high fat-high cholesterol diet.
n LVIDd (mm) LVPWd (mm) LVIDs (mm) FS (%) LV mass (μl) EDV (μl) ESV (μl) SV (μl) EF (%) CO (μl bpm)
Akita
4 months
3 months
Start
Akita
Akita þ 10 MPK SIT
Akita
Akita þ50 MPK SIT
25 3.2 70.018 0.777 0.0093 2.0 70.012 36 70.35 377 0.35 177 0.29 4.4 70.081 127 0.25 737 0.54 6087 7 132
10 4.0 7 0.056b 0.81 7 0.0028b 3.1 70.046b 247 0.80b 607 1.3b 357 1.5b 177 1.8b 217 1.6b 567 1.4b 10,7107 844b
8 3.2 7 0.074a 0.83 7 0.013a,b 2.2 7 0.059a,b 337 0.66a,b 447 2.1a,b 187 1.2a 5.4 7 0.41a,b 137 0.82a 707 0.89a,b 64687 480a
10 3.5 7 0.086b 0.89 70.022b 2.4 7 0.074b 317 2.2b 52 71.6b 23 71.7b 7.6 7 0.69b 167 1.6 667 3.1b 82187 956b
9 3.9 7 0.081a,b 0.85 7 0.035b 2.5 7 0.13b 347 2.8 607 3.9b 307 2.0a,b 9.17 1.6b 217 1.6a,b 707 3.9 11,0717 937a,b
Data are means 7 S.E.M. of n experiments. Abbreviations: diastolic (d); systolic (s); left ventricular internal diameter (LVID); left ventricular posterior wall thickness (LVPW); fractional shortening (FS); left ventricle (LV); end-diastolic volume (EVD); end-systolic volume (ESV); stroke volume (SV); ejection fraction (EF); and cardiac output (CO). a b
Po 0.05 versus untreated Akita mice. Po 0.05 versus start.
Chronic SIT (10 mg/kg/day) administration to Akita mice did not affect the diastolic and systolic IVS, the systolic LVPW and HR (data not shown). SIT decreased the diastolic and systolic LVID and LV volume, LV mass, SV and CO, while it increased the diastolic LVPW, FS and EF (Table 3). Exposing Akita mice to a high dose of SIT (50 mg/kg/day) for 3 months resulted in an increased LV end
diastolic diameter and LV volume, SV and CO (Table 3), while other cardiac parameters were not affected by the treatment. Estimated plasma volumes in SIT-treated Akita mice were similar to untreated controls for the lower dose (3471.5 mL/kg versus 3973.6 mL/kg), as well as the higher dose (2371.9 mL/kg versus 2271.1 mL/kg). Pharmacokinetic analysis revealed detectable levels of SIT in plasma
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Table 4 Pharmacokinetic analysis of sitagliptin (SIT) concentrations in plasma and heart tissue after 3 h administration, after 21 h discontinuation and at the end of the treatment period. SIT was administered at a dose of 10 mg/kg/day during 4 months or at a dose of 50 mg/kg/day during 3 months. 3h 10 (mg/kg/day) Plasma (ng/mL) Heart (ng/g) 50 mg/kg/day Plasma (ng/mL) Heart (ng/g)
66 711 218 734 479 729 1323 788
21 h
End
11 70.44 31 73.6
107 1.0 NM
5.4 72.0 38 712
367 3.4 NM
Data are means 7 S.E.M. of 3 (3 h, 21 h) or 8 (end) determinations; NM, not measured.
and heart tissue after administration of the low dose during 3h, and about 5–6-fold higher levels at the 5-fold higher dose. Twenty-one hours after discontinuation of the drug, plasma and heart levels had decreased to similar values for both doses, indicating that clearance mechanisms are not saturated at the high dose. After SIT administration for 3 or 4 months, plasma levels (measured at 6 h after end of administration) did not indicate accumulation of SIT over time (Table 4).
4. Discussion The aim of this study was to evaluate whether the genetic diabetic Akita mouse model could reveal beneficial or adverse effects of the anti-diabetic drug SIT. In a previous study, this model detected RGZ-induced weight gain, adiposity and early signs of cardiomyopathy (Hemmeryckx et al., 2013). In the present study, Akita mice were treated with two different doses of the DPP-4 inhibitor SIT, and the effect on body composition, metabolic parameters and cardiac function was analyzed. SIT was previously shown to have a body weight neutrality and anti-hyperglycemic effect (decrease in pre- and postprandial blood glucose and HbA1c levels), to reduce postprandial glucagon levels, while increasing fasting and postprandial plasma insulin and GLP-1 levels, to preserve β-cell mass and to be cardioprotective. The low (10 mg/ kg/day) and high (50 mg/kg/day) doses of SIT are based upon previous experiments in rodents (Ferreira et al., 2010; Goncalves et al., 2012; Mu et al., 2009; Vittone et al., 2012). The rationale for the different time periods (3 versus 4 months) for the low and high dose of SIT stems in the massive induction of plasma levels of the cardiac stress markers endothelin-1 and pro-brain natriuretic peptide by the diet after 4 months of treatment in Akita mice (SIT treated or untreated), masking any effect elicited by SIT treatment. Our main findings are (1) high dose SIT increases fat mass; (2) high dose SIT improves glycemic control; (3) low dose SIT is cardioprotective; and (4) high dose SIT causes LV hypertrophy. 4.1. High dose SIT increases fat mass As observed in other animal models (Lenski et al., 2011; Mu et al., 2009) and humans (Raz et al., 2006; Scott et al., 2007), SIT treatment (either dose) of Akita mice did not affect body weight. Previous studies in rats with doses of 10 mg/kg/day (Reimer et al., 2012) and 30 mg/kg/day (Chen et al., 2011) did not reveal an effect on fat mass; however the effect of SIT treatment on SC or visceral fat mass directly was not investigated. In our study we noticed however that a dose of 50 mg/kg/day SIT induced higher SC and GON fat masses. A high dose SIT may have increased the insulin sensitivity of the adipocytes, as suggested by an increased circulating level of the insulin-sensitizing hormone adiponectin,
leading to an enhanced adipogenic effect of insulin and an increase of the mass of these fat depots. 4.2. High dose SIT improves glycemic control The anti-diabetic action of the DPP-4 inhibitor SIT is reflected by increased concentrations of active GLP-1 and gastric inhibitory peptide (Herman et al., 2011; Mu et al., 2009), lower fasting and postprandial blood glucose and HbA1c levels (Ferreira et al., 2010; Mu et al., 2009) and improved β-cell function, as evidenced by an increased insulin secretion (Ferreira et al., 2010; Mu et al., 2009). In addition, SIT reduces glucagon secretion (Mu et al., 2009) and increases plasma adiponectin levels in diabetic patients (Kubota et al., 2012). In our study, exposure of Akita mice to a low dose of SIT (10 mg/kg/day) for 4 months did not affect fasting blood glucose (indication of current blood glucose level at time of measurement) or HbA1c levels (marker for glycemic control or average blood glucose level over an extended period of time), nor plasma insulin, adiponectin, and GLP-1 levels. In addition, active DPP4 levels were not influenced (data not shown). Since Akita mice are known to display a high level of oxidative stress (Yang and Chen, 2009), and to develop high HbA1c levels, it is conceivable that the 10 mg/kg/day dose was too low to effectively reduce these. In addition, we used a diet containing 19% fat and 1.25% cholesterol to further aggravate the diabetic phenotype and its complications. In a second study with a higher dose (50 mg/kg/ day) of SIT given for 3 months, we indeed observed lower levels of oxidative stress systemically, as evidenced by reduced HbA1c concentrations in the blood, and higher plasma adiponectin levels. In type 2 diabetic patients this negative correlation between adiponectin levels and HbA1c levels exists as well (Stejskal et al., 2003). SIT treatment did, however, not increase fasting plasma insulin levels, probably because of the fact that Akita mice undergo massive apoptosis of pancreatic β-cells due to ER stress, induced by accumulation of mutated pro-insulin (Hong et al., 2007). Furthermore, the diet appears to have masked beneficial effects of SIT on glucose levels. Indeed, blood glucose levels after 3 months exposure to the diet without addition of SIT are much higher than after 4 months. Lenski et al. (2011) previously reported that treatment of diabetic db/db mice for 4 weeks with 16 mg/kg/day SIT added to the R/M–H diet (only 3.3% fat) did not change fasting blood glucose levels, but enhanced the insulin response in a peritoneal glucose tolerance test. One reason could be that before the start of the study, the Akita mice were exposed to standard chow ad libitum. They were fasted for 6 h and blood glucose levels were determined. Subsequently, the mice received every morning a metal cup of 5 g of diet and ate about 50–70% of the diet during the first 3 h. The next morning most (95–100%) of the food was eaten. This means that the animals were in a state of starvation, which resulted in lower blood glucose and HbA1c levels than in the beginning. In addition, we noticed reduced fasting plasma GLP-1 levels in SIT (50 mg/kg/day)-treated Akita versus untreated Akita mice, which may explain the higher blood glucose levels in the treated mice. The reduction in GLP-1 levels may be a defect in nutrient and neuronal stimulation of the intestinal Lcells that are responsible for the basal secretion of GLP-1. However, this remains to be investigated. 4.3. Low dose SIT is cardioprotective At a low dose (10 mg/kg/day), echocardiographic analysis showed that SIT had a beneficial effect on cardiac function, as it improved FS and EF by reducing the diastolic and systolic LV volume and mass. Other preclinical and clinical studies have suggested potential benefits of DPP-4 inhibitors on cardiovascular function (Lenski et al., 2011; Nikolaidis et al., 2004). This may be explained by the protective effect of SIT on GLP-1. It is indeed
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known that elevated GLP-1 levels can improve LV EF and contractile function in patients with acute myocardial infarction (Nikolaidis et al., 2004). Furthermore, SIT prevented myocardial fibrosis in 10 weeks old db/db mice and reduced expression of transforming growth factor β1, 8-hydroxyguanosine (marker of oxidative stress) and advanced glycation end products in myocardium (Lenski et al., 2011). However, as fasting plasma GLP-1 levels were not increased by a low dose of SIT treatment, a role of other endogenous DPP-4 substrates, such as stromal-derived factor-α (Fadini et al., 2010), cannot be excluded.
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Acknowledgments Skillful technical assistance by L. Frederix, A. De Wolf, I. Vorsters, and C. Vranckx is gratefully acknowledged. This study was supported financially by Johnson & Johnson. The Center for Molecular and Vascular Biology, the Division of Cardiovascular Imaging and Dynamics and the Division of Clinical Cardiology are supported by the Programmafinanciering KU Leuven (PF/10/014) and by the Research Foundation-Flanders (FWO; G.0678.10).
References 4.4. High dose SIT causes LV hypertrophy At a higher dose (50 mg/kg/day), SIT did not provide a beneficial effect, as it did not decrease FS nor EF, but instead provoked LV hypertrophy, as evidenced by a higher LVIDd, EDV and a trend towards a higher LV mass, which resulted in an increase in SV and CO. Pharmacokinetic analysis revealed detectable levels of SIT in heart tissue at 3 h after administration, whereas 21 h after discontinuation SIT levels were low, indicating efficient clearance. Chronic SIT exposure did not result in accumulation in plasma. Previous data suggest potential explanations for the induction of LV hypertrophy. Firstly, expansion of plasma volume could lead to LV hypertrophy and increased SV and CO, as an exponent of a dominant compensatory mechanism in chronic stable heart failure (MacIver and Dayer, 2012). In our study, however, plasma volumes were similar for SIT-treated and untreated Akita animals. Secondly, increases in oxidative stress may lead to oxidative myocardial injury and subsequent LV hypertrophy (Takimoto and Kass, 2007). However, high dose SIT treatment of Akita mice for 3 months significantly decreased HbA1c (formed through reactive oxygen species-induced glucose oxidation and subsequent glycation of hemoglobin (Selvaraj et al., 2008)) levels, suggesting a reduction of oxidative stress. Although plasma active DPP4 levels are not influenced by a high dose of SIT (data not shown), fasting plasma GLP-1 levels were significantly lower in SIT-treated versus control animals. As elevated GLP-1 concentrations can improve LV EF and contractile function in patients with acute myocardial infarction (Nikolaidis et al., 2004), decreased levels of GLP-1 may therefore have an adverse effect on cardiac function. Indeed, GLP-1 knockout mice exhibit several cardiac abnormalities including a reduced heart resting rate, an increase in LV end diastolic pressure and wall thickness (Davidson, 2011).
5. Conclusions Treatment of type 1 and type 2 genetic diabetic Akita mice with 10 mg/kg/day SIT predicted some of its beneficial effects such as body weight neutrality and cardioprotective action by improving systolic function. However, metabolically no evidence was found of an improved glycemic control due to a strong impact of the diet. On the other hand, a higher dose of SIT (50 mg/kg/day) showed beneficial as well as adverse side effects. Beneficial effects include body weight neutrality, and improved fasting blood HbA1c levels and plasma adiponectin levels. Adverse side effects include gain of SC and visceral (GON) fat mass and LV hypertrophy, an early sign of heart failure. Thus, the Akita mouse represents a useful preclinical model to reveal beneficial or adverse side effects of anti-diabetic drugs.
Disclosure None.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Endocrine pharmacology
Effect of sitagliptin treatment on metabolism and cardiac function in genetic diabetic mice Bianca Hemmeryckx a, Melissa Swinnen b, David J Gallacher c, Hua Rong Lu c, H. Roger Lijnen a,n a
Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium Division of Clinical Cardiology, KU Leuven, Leuven, Belgium c Translational Sciences, Safety Pharmacology Research, Janssen Research & Development, Janssen Pharmaceutical NV, Beerse, Belgium b
art ic l e i nf o
a b s t r a c t
Article history: Received 26 September 2013 Received in revised form 8 December 2013 Accepted 14 December 2013 Available online 6 January 2014
To investigate the chronic effect of sitagliptin (7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]5,6,7,8-tetrahydro-(3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine phosphate (1:1) monohydrate, SIT) on metabolism and cardiac function in genetic diabetic Akita mice, 10 weeks old Akita mice were either exposed for 4 months to a high fat and high cholesterol (HF–HC) diet, with or without 10 mg/kg/day SIT, or were fed for 3 months with the same diet with or without 50 mg/kg/day SIT. SIT treatment of Akita mice at either a low or high dose did not affect body or liver weight. A significant increase in subcutaneous and gonadal fat mass was only observed for the 50 mg/kg/day dose of SIT. Furthermore, only the 50 mg/kg/day SIT dose resulted in an improvement of glycemic control, as evidenced by a decrease in fasting blood HbA1c levels and an increase in plasma adiponectin levels. Echocardiographic analysis revealed that Akita mice kept on the HF–HC diet with 10 mg/kg/day of SIT for 4 months showed an increase in ejection fraction and fractional shortening, whereas the higher dose (50 mg/kg/day) had no effect on these parameters, but instead induced left ventricular (LV) hypertrophy as evidenced by an enlarged LV internal diameter, volume and mass. Thus, in the diabetic Akita mouse SIT is cardioprotective at a low dose (10 mg/kg/day), whereas improvement of glycemic control requires a higher dose (50 mg/kg/day) which, however, induces LV hypertrophy. This mouse model may thus be useful to study the safety of antidiabetic drugs. & 2013 Elsevier B.V. All rights reserved.
Keywords: Cardiac function Genetic Akita mice Sitagliptin
1. Introduction Diabetes mellitus (DM) has reached epidemic proportions: globally, in 2010 an estimated 285 million people suffered from this metabolic disease (Shaw et al., 2010); 90% of DM patients is type 2 (T2DM) (Zimmet et al., 2001). The pathology of T2DM is characterized by defects in insulin sensitivity and secretion, resulting in hyperglycemia (Taylor, 1999). Current therapies for T2DM are associated with adverse drug effects such as hypoglycemia (Micheli et al., 2012), weight gain (Quinn et al., 2008), gastrointestinal problems (Stein et al., 2013), liver toxicity (Marcy et al., 2004) and increased risk for cardiovascular diseases (Nesto et al., 2003). To better understand the molecular mechanisms underlying these complications, suitable preclinical rodent models are needed. In a previous study, we have used the type 1 and type 2 diabetic Akita mouse model to establish side effects of the n Correspondence to: Center for Molecular and Vascular Biology, KU Leuven, Campus Gasthuisberg, CDG, Herestraat 49, Box 911, B-3000 Leuven, Belgium. Tel.: þ 32 16 345771; fax: þ32 16 345990. E-mail address: [email protected] (H. Roger Lijnen).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.12.036
thiazolidinedione rosiglitazone (RGZ), including weight gain and increased risk for cardiovascular disease (Hemmeryckx et al., 2013). These mice are heterozygous for a spontaneous mutation (C96Y) in the insulin 2 gene, which prevents proper folding of the insulin protein and results in subsequent pancreatic β-cell endoplasmatic reticulum stress and apoptosis. Akita mice develop hyperglycemia, hypoinsulinemia, polydipsia and polyuria at an age of 3–4 weeks (Hong et al., 2007; Yoshioka et al., 1997). Consequently, insulin production/secretion is very low. In humans, type 1 diabetes is an auto-immune disorder. It is believed that the disease develops due to destruction of the pancreatic β-cells by auto-antibodies evoked by viruses or environmental toxins. As a result, insulin production/secretion is very low or nonexistent. Although the pathophysiology of the disease in humans and the Akita mouse model differs, the outcome is very similar: a very low level of insulin production/secretion. Insulitis however, induced by an auto-immune response in type 1 diabetic patients leading to β-cell death and hypoinsulinemia, does not occur in the Akita mice. In addition, they show symptoms of human type 2 diabetes such as insulin resistance in skeletal muscle (caused by reduced glucose transporter-4 levels), liver and brown adipose tissue that is
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partly due to hyperglycemia (Hong et al., 2007; Yoshioka et al., 1997). However, in T2DM patients insulin-stimulated glucose uptake is reduced and the insulin resistance in skeletal muscle and liver is associated with an increased intracellular lipid accumulation, while in Akita mice the opposite pattern is observed (Hong et al., 2007). The reduced amount of lipids in muscle and liver may be explained by the increased whole body lipid oxidation seen in these mice. A species-associated difference in lipid metabolism therefore exists. To compensate for the altered lipid metabolism, Akita mice display an increase in insulin-stimulated glucose uptake, which allows for glucose to be directed to lipogenesis in white adipose tissues. To further validate this diabetic mouse model in testing the safety of anti-diabetic drugs, sitagliptin (7-[(3R)-3-amino1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-(3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine phosphate (1:1) monohydrate, SIT, Januvias) was administered at two physiological doses (10 and 50 mg/kg/day). SIT is presumed to have a greater impact on type 2 diabetes than RGZ, as its capacity to improve overall glycemic control is higher, and it restores pancreatic islet cell mass (Mu et al., 2009). SIT selectively inhibits the activity of the enzyme dipeptidyl peptidase 4 (DPP-4), leading to increased circulating levels of the incretin hormone glucagon-like peptide 1 (GLP-1) and subsequent promotion of insulin secretion and normalization of blood glucose (Deacon, 2007).
2. Materials and methods 2.1. Animal model Male Akita (Ins2Akita; JAX003548) mice in a C57Bl/6J genetic background were purchased from Jackson Laboratories (Bar Harbor, MN, USA). At the age of 10 weeks, mice kept in individual microisolation cages on a 12 h day/night cycle at 20–22 1C, were started for 4 months on 5 g/day of a high fat-high cholesterol (HF–HC) diet (Ssniff EF Clinton/Cybulsky (II) mod. 19.5% fat and 1.25% cholesterol; E15751-30, Ssniff Spezialitäten GmbH, Soest, Germany), supplemented with (n¼8) or without (n¼11) 10 mg/kg/day SIT (MSD Ltd., Hertfordshire, UK). In a second study, 10 weeks old Akita mice were kept for 3 months on the HF–HC diet ad libitum with (n¼9) or without (n¼10) 50 mg/kg/day SIT. Water was always available ad libitum. Body weight and food intake were measured weekly and daily in the two studies, respectively. Before and at the end of the studies, after a fasting period of 6 h, blood was collected on 3.8% citrate via the retro-orbital sinus to measure fasting blood glucose and glycohemoglobin (HbA1c) levels. Plasma samples were prepared for determination of insulin, adiponectin, glucagon, and GLP-1 levels, and for active DPP4 levels. For comparison, some parameters (insulin and adiponectin) were determined for 10 weeks old wildtype (WT) C57BL/6J male mice (Janvier, Le Genest Saint Isle, France). In addition, transthoracic echocardiographic examinations were performed in anesthetized mice (2% isoflurane; Ecuphar, Oostkamp, Belgium) at the start and end of the treatment, using a MS 400 transducer (18–38 Mhz) (Visualsonics Inc., Toronto, Canada) on a Vevo 2100 equipment. Left ventricle (LV) diameter at end-diastole (LVIDd) and end-systole (LVIDs), muscle thickness in diastole (IVSd) and in systole (IVSs) and LV posterior wall thickness in end-diastole (LVPWd) and end-systole (LVPWs) were measured, and fractional shortening (FS), LV mass, end-diastolic volume (EDV) and endsystolic volume (ESV), ejection fraction (EF), stroke volume (SV) and cardiac output (CO) were calculated (Stypmann et al., 2009). Mice were euthanized by ip injection of 60 mg/kg Nembutal (Abbott Laboratories, North Chicago, IL, USA). Inguinal subcutaneous (SC) and gonadal (GON) fat pads, liver, and heart were removed, weighed and stored for further analysis. The tibia length was also recorded.
All animal experiments were approved by the local ethical committee (KU Leuven P091/2010) and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (1996). 2.2. Assays Glycosylated hemoglobin (HbA1c) in plasma was measured by ion exchange HPLC (Selvin et al., 2005). SIT levels in plasma and extracts of heart tissue were determined by LC–MS/MS (courtesy of Janssen Pharmaceutica N.V., Beerse, Belgium). Blood glucose concentrations were measured using Glucocard strips (Menarini Diagnostics, Firenze, Italy). Plasma levels of insulin (Mercodia, Uppsala, Sweden), adiponectin and glucagon (R&D Systems Europe, Lille, France), GLP-1 (Cusabio, Wuhan, China) and active DPP4 enzyme levels (Enzo Life Sciences, Antwerpen, Belgium) were measured using commercial ELISA0 s. Hematocrit levels in blood samples were determined using a Cell-Dyn 3500R (Abbott Diagnostics, Abbott Park, IL, USA). Plasma volume was estimated as 1 minus the hematocrit value measured at the end of the study multiplied by the estimated blood volume (8% of total body weight) (Ortiz et al., 2002; Reynolds, 1953). 2.3. Statistical analysis Data are shown as means 7 S.E.M. for the number of animals studied. Statistical significance between groups is evaluated by non-parametric Mann–Whitney U-test. Values of P o0.05 are considered statistically significant.
3. Results 3.1. Effect of SIT on body composition and metabolic parameters The body weight of 10 weeks old Akita mice was slightly lower than that of age-matched WT mice (20 g70.17 g versus 21 7 0.28 g, P ¼0.04). Diabetic status was confirmed by higher fasting blood glucose (369714 mg/dL versus 10478.3 mg/dL, Po0.0001) and glycohemoglobin levels (6.870.09% versus 2.670.052%, Po0.0001), as well as hypoinsulinemia (0.07870.010 ng/ml versus 0.2270.088 ng/μl, P40.05) in Akita as compared to WT mice. Upon HF–HC diet feeding, the body weight of the Akita mice increased to 2470.50 g (Po0.0001) after 3 months and to 2470.40 g (P o0.0001) after 4 months. Co-administration of SIT at a dose of 10 mg/kg/day during 4 months or of 50 mg/kg/day during 3 months did not affect food intake, total body weight, liver weight, heart weight or tibia length (Table 1). Whereas the lower dose of SIT did not affect SC or GON fat mass, administration of the higher dose resulted in a significantly increased SC (P¼ 0.04) and GON (P ¼0.01) adipose tissue mass (Table 1). Fasting blood glucose and plasma insulin and glucagon levels were not affected by either dose of SIT, whereas at the higher dose blood HbA1c and plasma GLP-1 levels were decreased and plasma adiponectin concentrations increased as compared to HF–HC diet feeding without addition of SIT (Table 2). 3.2. Effect of SIT on cardiac function Echocardiographic analysis revealed that the hearts of Akita mice upon HF–HC loading showed a significant reduction in FS and EF, due to an increase in diastolic and systolic LVID and LV volume (EDV/ESV), resulting in a higher LV mass. In addition, SV and CO were also enhanced by the diet. These diet-induced changes in cardiac parameters were more pronounced after 4 as compared to 3 months (Table 3).
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Table 1 Effect of a low (10 mg/kg/day during 4 months) or a high dose (50 mg/kg/day during 3 months) sitagliptin (SIT) on body composition of Akita mice fed a high fat-high cholesterol diet 4 months
n Body weight (g) Food intake (g/day) Weight (mg) SC GON Liver Heart Tibia length (mm)
3 months
Akita
Akita þ10 MPK SIT
Akita
Akita þ50 MPK SIT
10 247 0.40 4.9 7 0.042
8 247 1.0 4.9 7 0.038
10 24 70.50 5.8 70.14
9 267 0.47 5.5 7 0.16
234 7 21 308 7 33 21427 172 139 76.2 177 0.074
2467 23 3617 44 2288 7 126 154 77.2 177 0.16
1857 12 288 7 19 21007 113 1157 3.4 177 0.084
2337 16a 403 7 32a 21257 144 1257 6.7 177 0.053
Data are means 7 S.E.M. of n experiments. Abbreviations: subcutaneous (SC) and gonadal (GON). a
Po 0.05 versus untreated Akita mice.
Table 2 Effect of a low (10 mg/kg/day) and a high dose (50 mg/kg/day) sitagliptin (SIT) on metabolic parameters in Akita mice fed a high fat-high cholesterol diet.
n Blood glucose level (mg/dL) Blood HbA1c level (%) Plasma insulin level (ng/mL) Plasma adiponectin level (mg/mL) Plasma glucagon level (pg/ml) Plasma glucagon-like peptide-1 level (ng/ml)
Akita
4 months
3 months
Start
Akita
Akita þ10 MPK SIT
Akita
Akita þ50 MPK SIT
37 369 7 14 6.8 7 0.086 0.0787 0.010 6.6 7 0.34 84 7 9.3 NM
10 228 7 50b 6.2 7 1.1 0.20 7 0.076 3.5 7 0.48b 267 16b 427 741
8 222 7 42b 6.0 71.1b 0.177 0.067 3.2 70.32b ND 454 7 53
10 482 7 45b 7.0 7 0.58 0.137 0.016b 5.4 70.25 68 7 16 4617 37
9 485 7 53b 4.8 7 0.44a,b 0.107 0.027 6.7 7 0.55a 577 17 3747 12a
Data are means 7 S.E.M. of n experiments. Abbreviations: not detectable (ND); not measured (NM). a b
Po 0.05 versus untreated Akita mice. Po 0.05 versus start.
Table 3 Effect of a low (10 mg/kg/day) and a high dose (50 mg/kg/day) sitagliptin (SIT) on cardiac function in Akita mice on a high fat-high cholesterol diet.
n LVIDd (mm) LVPWd (mm) LVIDs (mm) FS (%) LV mass (μl) EDV (μl) ESV (μl) SV (μl) EF (%) CO (μl bpm)
Akita
4 months
3 months
Start
Akita
Akita þ 10 MPK SIT
Akita
Akita þ50 MPK SIT
25 3.2 70.018 0.777 0.0093 2.0 70.012 36 70.35 377 0.35 177 0.29 4.4 70.081 127 0.25 737 0.54 6087 7 132
10 4.0 7 0.056b 0.81 7 0.0028b 3.1 70.046b 247 0.80b 607 1.3b 357 1.5b 177 1.8b 217 1.6b 567 1.4b 10,7107 844b
8 3.2 7 0.074a 0.83 7 0.013a,b 2.2 7 0.059a,b 337 0.66a,b 447 2.1a,b 187 1.2a 5.4 7 0.41a,b 137 0.82a 707 0.89a,b 64687 480a
10 3.5 7 0.086b 0.89 70.022b 2.4 7 0.074b 317 2.2b 52 71.6b 23 71.7b 7.6 7 0.69b 167 1.6 667 3.1b 82187 956b
9 3.9 7 0.081a,b 0.85 7 0.035b 2.5 7 0.13b 347 2.8 607 3.9b 307 2.0a,b 9.17 1.6b 217 1.6a,b 707 3.9 11,0717 937a,b
Data are means 7 S.E.M. of n experiments. Abbreviations: diastolic (d); systolic (s); left ventricular internal diameter (LVID); left ventricular posterior wall thickness (LVPW); fractional shortening (FS); left ventricle (LV); end-diastolic volume (EVD); end-systolic volume (ESV); stroke volume (SV); ejection fraction (EF); and cardiac output (CO). a b
Po 0.05 versus untreated Akita mice. Po 0.05 versus start.
Chronic SIT (10 mg/kg/day) administration to Akita mice did not affect the diastolic and systolic IVS, the systolic LVPW and HR (data not shown). SIT decreased the diastolic and systolic LVID and LV volume, LV mass, SV and CO, while it increased the diastolic LVPW, FS and EF (Table 3). Exposing Akita mice to a high dose of SIT (50 mg/kg/day) for 3 months resulted in an increased LV end
diastolic diameter and LV volume, SV and CO (Table 3), while other cardiac parameters were not affected by the treatment. Estimated plasma volumes in SIT-treated Akita mice were similar to untreated controls for the lower dose (3471.5 mL/kg versus 3973.6 mL/kg), as well as the higher dose (2371.9 mL/kg versus 2271.1 mL/kg). Pharmacokinetic analysis revealed detectable levels of SIT in plasma
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Table 4 Pharmacokinetic analysis of sitagliptin (SIT) concentrations in plasma and heart tissue after 3 h administration, after 21 h discontinuation and at the end of the treatment period. SIT was administered at a dose of 10 mg/kg/day during 4 months or at a dose of 50 mg/kg/day during 3 months. 3h 10 (mg/kg/day) Plasma (ng/mL) Heart (ng/g) 50 mg/kg/day Plasma (ng/mL) Heart (ng/g)
66 711 218 734 479 729 1323 788
21 h
End
11 70.44 31 73.6
107 1.0 NM
5.4 72.0 38 712
367 3.4 NM
Data are means 7 S.E.M. of 3 (3 h, 21 h) or 8 (end) determinations; NM, not measured.
and heart tissue after administration of the low dose during 3h, and about 5–6-fold higher levels at the 5-fold higher dose. Twenty-one hours after discontinuation of the drug, plasma and heart levels had decreased to similar values for both doses, indicating that clearance mechanisms are not saturated at the high dose. After SIT administration for 3 or 4 months, plasma levels (measured at 6 h after end of administration) did not indicate accumulation of SIT over time (Table 4).
4. Discussion The aim of this study was to evaluate whether the genetic diabetic Akita mouse model could reveal beneficial or adverse effects of the anti-diabetic drug SIT. In a previous study, this model detected RGZ-induced weight gain, adiposity and early signs of cardiomyopathy (Hemmeryckx et al., 2013). In the present study, Akita mice were treated with two different doses of the DPP-4 inhibitor SIT, and the effect on body composition, metabolic parameters and cardiac function was analyzed. SIT was previously shown to have a body weight neutrality and anti-hyperglycemic effect (decrease in pre- and postprandial blood glucose and HbA1c levels), to reduce postprandial glucagon levels, while increasing fasting and postprandial plasma insulin and GLP-1 levels, to preserve β-cell mass and to be cardioprotective. The low (10 mg/ kg/day) and high (50 mg/kg/day) doses of SIT are based upon previous experiments in rodents (Ferreira et al., 2010; Goncalves et al., 2012; Mu et al., 2009; Vittone et al., 2012). The rationale for the different time periods (3 versus 4 months) for the low and high dose of SIT stems in the massive induction of plasma levels of the cardiac stress markers endothelin-1 and pro-brain natriuretic peptide by the diet after 4 months of treatment in Akita mice (SIT treated or untreated), masking any effect elicited by SIT treatment. Our main findings are (1) high dose SIT increases fat mass; (2) high dose SIT improves glycemic control; (3) low dose SIT is cardioprotective; and (4) high dose SIT causes LV hypertrophy. 4.1. High dose SIT increases fat mass As observed in other animal models (Lenski et al., 2011; Mu et al., 2009) and humans (Raz et al., 2006; Scott et al., 2007), SIT treatment (either dose) of Akita mice did not affect body weight. Previous studies in rats with doses of 10 mg/kg/day (Reimer et al., 2012) and 30 mg/kg/day (Chen et al., 2011) did not reveal an effect on fat mass; however the effect of SIT treatment on SC or visceral fat mass directly was not investigated. In our study we noticed however that a dose of 50 mg/kg/day SIT induced higher SC and GON fat masses. A high dose SIT may have increased the insulin sensitivity of the adipocytes, as suggested by an increased circulating level of the insulin-sensitizing hormone adiponectin,
leading to an enhanced adipogenic effect of insulin and an increase of the mass of these fat depots. 4.2. High dose SIT improves glycemic control The anti-diabetic action of the DPP-4 inhibitor SIT is reflected by increased concentrations of active GLP-1 and gastric inhibitory peptide (Herman et al., 2011; Mu et al., 2009), lower fasting and postprandial blood glucose and HbA1c levels (Ferreira et al., 2010; Mu et al., 2009) and improved β-cell function, as evidenced by an increased insulin secretion (Ferreira et al., 2010; Mu et al., 2009). In addition, SIT reduces glucagon secretion (Mu et al., 2009) and increases plasma adiponectin levels in diabetic patients (Kubota et al., 2012). In our study, exposure of Akita mice to a low dose of SIT (10 mg/kg/day) for 4 months did not affect fasting blood glucose (indication of current blood glucose level at time of measurement) or HbA1c levels (marker for glycemic control or average blood glucose level over an extended period of time), nor plasma insulin, adiponectin, and GLP-1 levels. In addition, active DPP4 levels were not influenced (data not shown). Since Akita mice are known to display a high level of oxidative stress (Yang and Chen, 2009), and to develop high HbA1c levels, it is conceivable that the 10 mg/kg/day dose was too low to effectively reduce these. In addition, we used a diet containing 19% fat and 1.25% cholesterol to further aggravate the diabetic phenotype and its complications. In a second study with a higher dose (50 mg/kg/ day) of SIT given for 3 months, we indeed observed lower levels of oxidative stress systemically, as evidenced by reduced HbA1c concentrations in the blood, and higher plasma adiponectin levels. In type 2 diabetic patients this negative correlation between adiponectin levels and HbA1c levels exists as well (Stejskal et al., 2003). SIT treatment did, however, not increase fasting plasma insulin levels, probably because of the fact that Akita mice undergo massive apoptosis of pancreatic β-cells due to ER stress, induced by accumulation of mutated pro-insulin (Hong et al., 2007). Furthermore, the diet appears to have masked beneficial effects of SIT on glucose levels. Indeed, blood glucose levels after 3 months exposure to the diet without addition of SIT are much higher than after 4 months. Lenski et al. (2011) previously reported that treatment of diabetic db/db mice for 4 weeks with 16 mg/kg/day SIT added to the R/M–H diet (only 3.3% fat) did not change fasting blood glucose levels, but enhanced the insulin response in a peritoneal glucose tolerance test. One reason could be that before the start of the study, the Akita mice were exposed to standard chow ad libitum. They were fasted for 6 h and blood glucose levels were determined. Subsequently, the mice received every morning a metal cup of 5 g of diet and ate about 50–70% of the diet during the first 3 h. The next morning most (95–100%) of the food was eaten. This means that the animals were in a state of starvation, which resulted in lower blood glucose and HbA1c levels than in the beginning. In addition, we noticed reduced fasting plasma GLP-1 levels in SIT (50 mg/kg/day)-treated Akita versus untreated Akita mice, which may explain the higher blood glucose levels in the treated mice. The reduction in GLP-1 levels may be a defect in nutrient and neuronal stimulation of the intestinal Lcells that are responsible for the basal secretion of GLP-1. However, this remains to be investigated. 4.3. Low dose SIT is cardioprotective At a low dose (10 mg/kg/day), echocardiographic analysis showed that SIT had a beneficial effect on cardiac function, as it improved FS and EF by reducing the diastolic and systolic LV volume and mass. Other preclinical and clinical studies have suggested potential benefits of DPP-4 inhibitors on cardiovascular function (Lenski et al., 2011; Nikolaidis et al., 2004). This may be explained by the protective effect of SIT on GLP-1. It is indeed
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known that elevated GLP-1 levels can improve LV EF and contractile function in patients with acute myocardial infarction (Nikolaidis et al., 2004). Furthermore, SIT prevented myocardial fibrosis in 10 weeks old db/db mice and reduced expression of transforming growth factor β1, 8-hydroxyguanosine (marker of oxidative stress) and advanced glycation end products in myocardium (Lenski et al., 2011). However, as fasting plasma GLP-1 levels were not increased by a low dose of SIT treatment, a role of other endogenous DPP-4 substrates, such as stromal-derived factor-α (Fadini et al., 2010), cannot be excluded.
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Acknowledgments Skillful technical assistance by L. Frederix, A. De Wolf, I. Vorsters, and C. Vranckx is gratefully acknowledged. This study was supported financially by Johnson & Johnson. The Center for Molecular and Vascular Biology, the Division of Cardiovascular Imaging and Dynamics and the Division of Clinical Cardiology are supported by the Programmafinanciering KU Leuven (PF/10/014) and by the Research Foundation-Flanders (FWO; G.0678.10).
References 4.4. High dose SIT causes LV hypertrophy At a higher dose (50 mg/kg/day), SIT did not provide a beneficial effect, as it did not decrease FS nor EF, but instead provoked LV hypertrophy, as evidenced by a higher LVIDd, EDV and a trend towards a higher LV mass, which resulted in an increase in SV and CO. Pharmacokinetic analysis revealed detectable levels of SIT in heart tissue at 3 h after administration, whereas 21 h after discontinuation SIT levels were low, indicating efficient clearance. Chronic SIT exposure did not result in accumulation in plasma. Previous data suggest potential explanations for the induction of LV hypertrophy. Firstly, expansion of plasma volume could lead to LV hypertrophy and increased SV and CO, as an exponent of a dominant compensatory mechanism in chronic stable heart failure (MacIver and Dayer, 2012). In our study, however, plasma volumes were similar for SIT-treated and untreated Akita animals. Secondly, increases in oxidative stress may lead to oxidative myocardial injury and subsequent LV hypertrophy (Takimoto and Kass, 2007). However, high dose SIT treatment of Akita mice for 3 months significantly decreased HbA1c (formed through reactive oxygen species-induced glucose oxidation and subsequent glycation of hemoglobin (Selvaraj et al., 2008)) levels, suggesting a reduction of oxidative stress. Although plasma active DPP4 levels are not influenced by a high dose of SIT (data not shown), fasting plasma GLP-1 levels were significantly lower in SIT-treated versus control animals. As elevated GLP-1 concentrations can improve LV EF and contractile function in patients with acute myocardial infarction (Nikolaidis et al., 2004), decreased levels of GLP-1 may therefore have an adverse effect on cardiac function. Indeed, GLP-1 knockout mice exhibit several cardiac abnormalities including a reduced heart resting rate, an increase in LV end diastolic pressure and wall thickness (Davidson, 2011).
5. Conclusions Treatment of type 1 and type 2 genetic diabetic Akita mice with 10 mg/kg/day SIT predicted some of its beneficial effects such as body weight neutrality and cardioprotective action by improving systolic function. However, metabolically no evidence was found of an improved glycemic control due to a strong impact of the diet. On the other hand, a higher dose of SIT (50 mg/kg/day) showed beneficial as well as adverse side effects. Beneficial effects include body weight neutrality, and improved fasting blood HbA1c levels and plasma adiponectin levels. Adverse side effects include gain of SC and visceral (GON) fat mass and LV hypertrophy, an early sign of heart failure. Thus, the Akita mouse represents a useful preclinical model to reveal beneficial or adverse side effects of anti-diabetic drugs.
Disclosure None.
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