Effect of Sitagliptin and Glimepiride on Glucose Homeostasis and camp Levels in Peripheral Tissues of HFD/STZ Diabetic Rats

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1 Americn Journl of Biomedicl Reserch, 2014, Vol. 2, No. 3, Avilble online t Science nd Eduction Publishing DOI: /jbr Effect of Sitgliptin nd Glimepiride on Glucose Homeostsis nd camp Levels in Peripherl Tissues of HFD/STZ Dibetic Rts Mohmed I Sd 1,*, Mher A Kmel 1, Mervt Y Hnfi 1, Mdih H Helmy 1, Rowid R Sheht 2 1 Deprtment of Biochemistry, Medicl Reserch Institute, Alexndri University, Egypt 2 Deprtment of Phrmcology, Medicl Reserch Institute, Alexndri University, Egypt *Corresponding uthor: m.ibrhim1988@hotmil.com Received August 20, 2014; Revised September 02, 2014; Accepted September 11, 2014 Abstrct Introduction: T2DM is group of metbolic disorders mnifested by hyperglycemi s result of insulin insufficiency nd/or resistnce. The min gol of ntidibetic therpies is to lower glucose levels, nd therefore prevent development of dibetes complictions. DPP-4 inhibitors (e.g. sitgliptin) re reltively new ntidibetic drugs which inhibit the ctivity of DPP-4 enzyme nd therefore prevent rpid degrdtion of incretin hormones. Objective: We investigted effects of sitgliptin on glucose homeostsis, lipid profile, nd insulin signling by determintion of camp levels in peripherl tissues of HFD/STZ dibetic rts, compred to glimepiride. Methods: The experimentl rts were divided into five groups, ech group comprising 10 rts. Group (1) served s the norml control rts nd dministered DMSO (without tretments) s the vehicle. The rest of the groups were rendered dibetic by feeding HFD contining 40% fts for 4 weeks, followed by single I.P. injection of STZ (45 mg/kg of body weight). One week fter STZ injection, the rts with FBG level of 200 mg/dl were considered dibetic. Group (2) served s the dibetic untreted rts nd dministered DMSO (without tretments) s the vehicle. Group (3) served s dibetic rts treted with glimepiride (0.1 mg/kg of body weight). Group (4) nd group (5) served s dibetic rts treted with sitgliptin (10 nd 30 mg/kg of body weight, respectively). Tretments were dissolved in DMSO nd were given orlly for 4 weeks. At the end of the tretment period, the blood, liver nd dipose tissues (White nd brown) were collected for biochemicl nlysis. Results: In norml control rts, the highest content of camp ws observed in BAT. Dibetic rts showed n elevtion in camp levels of liver nd WAT to be 1.3 nd 3.9 fold control vlues, respectively, while in BAT, camp level decresed to be 0.4 fold control vlue. Sitgliptin nd glimepiride significntly decresed camp levels in liver nd WAT. Conclusion: We conclude tht sitgliptin nd glimepiride hve comprble effects on glucose homeostsis. Both drugs hve camp-lowering effect which my suggest their potentil protecting effect ginst vsculr complictions of dibetes. Keywords: dibetes, DPP-4 inhibitors, sitgliptin, glimepiride, camp Cite This Article: Mohmed I Sd, Mher A Kmel, Mervt Y Hnfi, Mdih H Helmy, nd Rowid R Sheht, Effect of Sitgliptin nd Glimepiride on Glucose Homeostsis nd camp Levels in Peripherl Tissues of HFD/STZ Dibetic Rts. Americn Journl of Biomedicl Reserch, vol. 2, no. 3 (2014): doi: /jbr Introduction Type 2 dibetes mellitus (T2DM) is group of metbolic disorders mnifested by hyperglycemi s result of insulin insufficiency nd/or insulin resistnce. Insulin resistnce is the inbility of insulin to exert its ction on cells. The pthophysiology of T2DM is not fully understood nd there re theories link T2DM with insulin signling defects, level of rective oxygen species nd obesity. T2DM could result in deleterious complictions such s crdiovsculr disorders, renl filure, retinopthy nd poor wound heling [1]. Insulin is the min nbolic hormone which secreted from bet (β)-cells of the pncres. It is relesed in response to incresed nutrients level in blood e.g. glucose nd mino cids [2]. It regultes the whole body fuel homeostsis through its ction on liver, skeletl muscles nd dipose tissues. It stimultes glucose uptke, glycogenesis, ftty cid biosynthesis nd dipogenesis, wheres it inhibits glycogenolysis, gluconeogenesis nd lipolysis. Insulin exerts its ctions through mtrix of intercting pthwys, llowing for extensive modultion nd divergence in signl trnsduction, which strts with insulin binding to its cell membrne tyrosine kinse receptors [3]. Importnt components in insulin signling re insulin receptors, insulin receptor substrte proteins (IRS), phosphtidylinositol-3 (PI 3 ) kinse, Akt/protein kinse B (Akt/PKB), nd mitogen ctivted protein kinse (MAPK) [3]. PI 3 kinse is the criticl component of insulin signling, mediting the metbolic effect of insulin through ctivtion of Akt/PKB nd typicl protein kinse C (PKC) pthwys [4].

2 53 Americn Journl of Biomedicl Reserch Adipose tissues ply n importnt role in the development of insulin resistnce nd T2DM. There re two kinds of dipose tissue: white dipose tissue (WAT) nd brown dipose tissue (BAT). WAT is the primry site of energy storge in the form of triglycerides, wheres BAT contins multiloculr dipocytes which contins lrge number of mitochondri nd plys substntil role in non-shivering thermogenesis [5]. Sympthetic nerve endings relese nordrenline in the proximity of brown ft cells, where nordrenline ctivtes G-protein-coupled β-drenergic receptors inititing cscde of metbolic events resulting in the ctivtion of uncoupling protein 1 (UCP1) [6]. Insulin ntgonizes hormone-induced lipolysis - cused by counter-regultory hormones e.g. glucgon minly by ctivtion of phosphodiesterse 3B (PDE3B) vi Akt/PKB tht degrdes intrcellulr camp [3]. Dipeptidyl peptidse-4 inhibitors re reltively new ntidibetic drugs which inhibit the enzymtic ctivity of dipeptidyl peptidse-4 (DPP-4) tht is responsible for rpid degrdtion of the incretin hormones: glucosedependent insulinotropic peptide (GIP) nd glucgon-like peptide-1 (GLP-1) [7]. DPP-4 inhibitors, therefore, cuses stimultion of insulin secretion, inhibition of glucgon secretion nd preservtion of β-cell mss through stimultion of cell prolifertion nd inhibition of poptosis [8]. Sitgliptin, The first DPP-4 inhibitor to be pproved in the USA for the tretment of T2DM, is used nowdys t dose of 100 mg/dy s monotherpy or in combintion with other drugs [7]. Although, the effect of sitgliptin on insulin secretion is well-defined; its effect on insulin ction in the peripherl tissues is not elucidted. The objective of the present study is to investigte the effect of sitgliptin on glucose homeostsis prmeters (e.g. blood glucose level, serum insulin, insulin resistnce using the homeostsis model ssessment), lipid profile, nd insulin signling by determintion of camp levels in liver nd dipose tissues. This study ws performed on high-ft diet (HFD)/streptozotocin (STZ)-induced dibetic rts in which the effect of sitgliptin ws compred to tht of the ntidibetic sulfonylure (glimepiride). 2. Mterils nd Methods 2.1. Experimentl Animls Wistr rts ged 3-4 months were purchsed from the Medicl Technology Center (Alexndri, Egypt). The rts were housed t temperture of 23 ± 1 C with 12/12 h light/drk cycles nd 45 ± 5% humidity with free ccess to wter nd chow diet for week prior to the experiment Experimentl Design The experimentl nimls were divided into five groups, ech group comprising 10 rts detiled s follows: Group (1) served s the norml control rts nd dministered dimethyl sulfoxide (DMSO) s vehicle without ny tretments. The rest of nimls were rendered dibetic by feeding high-ft diet contining 40% fts (HFD) for 4 weeks, followed by single intrperitonel injection of STZ t low dose (45 mg/kg of body weight, dissolved in 0.05 M citrte buffer, ph 4.5, immeditely before use). One week fter injection, fsting blood glucose (FBG) levels were determined from til blood using n Accu- Check Active glucometer (Roche Dignostics, Mnheim, Germny). The rts with FBG levels bove 200 mg/dl were considered s dibetic (Modified Srinivsn, et l. method) [9]. Group (2) served s the dibetic untreted rts nd dministered (DMSO) s vehicle without ny tretments. Group (3) served s dibetic rts treted with glimepiride (0.1 mg/kg of body weight). Group (4) nd group (5) served s dibetic rts treted with sitgliptin (10 nd 30 mg/kg of body weight, respectively). Tretments were dministered dily in DMSO suspension by orl gvge for 4 weeks. The dosge ws djusted every week, ccording to ny chnge in body weight to mintin similr dose per kg body weight of rt over the entire period of study for ech group. FBG level ws mesured every week. At the end of the tretment period, the rts were fsted overnight, nesthetized with diethyl ether nd killed by cervicl decpittion. The blood ws collected for serum seprtion nd biochemicl nlysis. Liver nd dipose tissues (WAT nd BAT) were excised immeditely nd stored t -80 C. WAT ws obtined from viscerl (retroperitonel) dipose depots, while BAT ws obtined from dipose depots in the interscpulr region Orl Glucose Tolernce Test (OGTT) The OGTT ws performed t the end of tretment period (4 weeks). After overnight fsting for 12 hours, the nimls were dministered glucose (2.5 g/kg of body weight) dissolved in wter by orl gvge. Blood glucose levels were determined from the til vein with n Accucheck Active glucometer t 0, 30, 60, 90, nd 120 minutes Determintion of Glucose, Insulin nd Lipid Profile The level of serum glucose ws estimted using n Accu-Check Active glucometer. Serum insulin level ws ssyed using commercil kit (Millipore ) ccording to the mnufcturer's instructions. This ssy is sndwich ELISA bsed on cpture of insulin molecules from smples to the wells of microtitre plte coted by pretitered mount of monoclonl mouse nti-rt insulin ntibodies nd the binding of biotinylted polyclonl ntibodies to cptured insulin, binding of horserdish peroxidse (HRP) to the immobilized biotinylted ntibodies, nd quntifiction of the immobilized ntibody-enzyme conjugtes by monitoring horserdish peroxidse ctivities in the presence of 3,3',5,5'- tetrmethylbenzidine. The enzyme ctivity is mesured spectrophotometriclly by the incresed bsorbnce t 450 nm. Lipid profile ws ssessed by using commercil dignostic kit (Rndox (UK) ) ccording to the mnufcturer's instructions. This kit uses enzymtic methods to determine serum levels of triglycerides, totl cholesterol, HDL-cholesterol. The bsorbnce of the stndrds nd smples were mesured spectrophotometriclly t 546 nm. LDL-cholesterol level ws then clculted from the following eqution: LDL conc = Totl cholesterol conc TG conc ( HDL conc) 5

3 Americn Journl of Biomedicl Reserch Determintion of Homeostsis Model of Insulin Resistnce (HOMA-IR) The insulin resistnce index (IRI) ws evluted by homeostsis model ssessment estimte of insulin resistnce (HOMA-IR) s follows: Fstinginsulin IRI = µiu ( ) xfstingglucose( mmol ) ml L Assy of camp Level The levels of camp in the liver, WAT nd BAT of control nd experimentl groups of rts were determined by DRG Cyclic AMP direct ELISA kit ccording to the mnufcturer's instructions. Tissue smples (liver, WAT nd BAT) were frozen in liquid nitrogen nd stored t - 80 C. Frozen tissues were grinded in stinless steel mortr under liquid nitrogen until it ws fine powder. The liquid nitrogen ws llowed to evporte nd the powdered tissue ws weighed. 1 ml of Smple Diluent ws dded for every 100 mg of tissue nd incubted for 10 minutes on ice, nd then centrifuged t 600 x g t 4 C for 15 minutes. The superntnt ws collected nd the ssy ws done immeditely. A cler microtiter plte coted with n ntibody to cpture sheep IgG ws provided nd neutrlizing plte primer solution ws dded to ll the used wells. Stndrds or diluted smples were pipetted into the primed wells. A camp-peroxidse conjugte ws dded to the stndrds nd smples in the wells. The binding rection ws initited by the ddition of sheep ntibody to camp to ech well. After 2-hour incubtion, the plte ws wshed nd substrte ws dded. After short incubtion, the rection ws stopped nd the intensity of the generted color ws detected in microtiter plte reder t 450 nm. Stndrd plot ws constructed by using stndrd camp, nd then the concentrtions of unknown smples were clculted from the stndrd curve (Figure 1). camp level ws finlly clculted in terms of protein content in ech tissue smple mesured by the modified Lowery, et l. method for totl protein determintion [10]. Figure 1. Stndrd curve of camp. The bbrevitions denote B/B 0: smple or stndrd bound / mximum bound Figure 2. Stndrd curve of totl protein

4 55 Americn Journl of Biomedicl Reserch 2.7. Determintion of Totl Protein Concentrtion A modifiction of the method of Lowery, et l. ws used for the determintion of protein in the smples. The color produced is thought to be due to complex between the lkline copper-phenol regent nd tyrosine nd tryptophn residues of the protein in the smple. The bsorbnce ws red t 695 nm using spectronic 21 spectrophotometer. [10] The protein concentrtion in ech smple ws estimted by referring to stndrd curve (Figure 2) which ws constructed using bovine serum lbumin Sttisticl Anlysis The dt were nlyzed using the one-wy nlysis of vrince (ANOVA) followed by LSD test to compre different groups with ech other (SPSS softwre). Results were expressed s men ± stndrd error (SE) nd vlues of p > 0.05 were considered non-significntly different, while those of p < 0.05 were considered significnt. 3. Results 3.1. Glucose Homeostsis Prmeters Figure 3. Chnge in fsting blood glucose (FBG) levels of studied groups during the tretment period (4 weeks). Vlues re presented s men ± SE (n = 10). The bbrevitions denote Glim: rts treted with glimepiride (0.1 mg/kg), Sit 10: rts treted with sitgliptin (10 mg/kg) nd Sit 30: rts treted with sitgliptin (30 mg/kg). : significntly different from the control group, b: significntly different from the dibetic untreted group, c: significntly different from the Glim group, using ANOVA (LSD), p vlue < 0.05 At the beginning of the study, ll dibetic groups - fter induction of dibetes nd before the dministrtion of tretments - showed significntly higher levels of FBG (hyperglycemi) compred to the control group (Tble 1). Most of treted groups showed time-dependent decrese in FBG during the tretment period. During this time period, ll of treted groups hd significntly lower FBG thn the untreted rts. By the end of the tretment period (fter 4 weeks), glimepiride nd sitgliptin t 10 nd 30 mg/kg showed nerly norml FBG levels (Figure 3). Tble 1. Bseline vlues of glucose homeostsis prmeters nd lipid profile of different studied groups Prmeter Control Dibetic Untreted Glim Sit 10 Sit 30 Fsting blood Glucose (FBG) level (mg/dl) 102.9± ± ± ± ±16.64 Serum Insulin (µiu/ml) 1.64± ± ± ± ±0.26 HOMA-IR 0.4± ± ± ± ±0.39 Triglycerides (mg/dl) 39.5± ± ± ± ±1.25 Totl Cholesterol (mg/dl) 141.8± ± ± ± ±2.75 LDL-Cholesterol (LDL-C) (mg/dl) 90.33± ± ± ± ±3.76 HDL-Cholesterol (HDL-C) (mg/dl) 42.3± ± ± ± ±0.42 Vlues re presented s men ± SE (n = 10). The bbrevitions denote Glim: rts treted with glimepiride (0.1 mg/kg), Sit 10: rts treted with sitgliptin (10 mg/kg) nd Sit 30: rts treted with sitgliptin (30 mg/kg). : significntly different from the control group by ANOVA (LSD), p vlue < 0.05.

5 Americn Journl of Biomedicl Reserch 56 Figure 4. Serum insulin levels (µiu/ml) nd HOMA-IR vlues of different studied groups t the end of the tretment period (4 weeks). Vlues re presented s men ± SE (n = 10). The bbrevitions denote Glim: rts treted with glimepiride (0.1 mg/kg), Sit 10: rts treted with sitgliptin (10 mg/kg) nd Sit 30: rts treted with sitgliptin (30 mg/kg). : significntly different from the control group, b: significntly different from the dibetic untreted group, c: significntly different from the Glim group, d: significntly different from the Sit 10 group, using ANOVA (LSD), p vlue < 0.05 At the strt of the experiment, ll dibetic rts showed higher insulin levels compred to the control group (Tble 1). By the end of the tretment period, ll dibetic groups (untreted nd treted) exhibited significntly higher serum insulin levels compred to the control rts. Dibetic rts treted with glimepiride nd sitgliptin t dose of 10 mg/kg showed significnt lower levels thn the untreted dibetic rts, while rts treted with sitgliptin t dose of 30 mg/kg showed significnt higher level thn the untreted dibetic rts (Figure 4). The insulin resistnce index clculted by the HOMA model (HOMA-IR) using the level of fsting insulin (µiu/ml) nd glucose level (mmol/l) indicted tht ll dibetic groups strted the experiment with significntly higher HOMA-IR vlues compred to the control group (Tble 1). At the end of the tretment period, ll of the treted rts showed significnt decline in the insulin resistnce index compred to the untreted rts with the lest vlue observed in rts treted with glimepiride (Figure 4). Figure 5. Chnge in blood glucose level (mg/dl) during the orl glucose tolernce test (OGTT) of different studied groups done t the end of the tretment period (4 weeks). Vlues re presented s men ± SE (n = 10). The bbrevitions denote Glim: rts treted with glimepiride (0.1 mg/kg), Sit 10: rts treted with sitgliptin (10 mg/kg) nd Sit 30: rts treted with sitgliptin (30 mg/kg). : significntly different from the control group, b: significntly different from the dibetic untreted group, c: significntly different from the Glim group, d: significntly different from the Sit 10 group, using ANOVA (LSD), p vlue < 0.05 From the results of the OGTT constructed t the end of tretment period, it ws cler tht the untreted dibetic rts ws suffering from impired fsting glucose tolernce nd lso impired glucose tolernce during the two hour period of the test (Figure 5), while the treted dibetic rts showed no impirment in the fsting glucose tolernce but showed impired glucose tolernce fter glucose dministrtion which ws lesser extent thn tht observed in the untreted rts (Figure 5). The best response in the OGTT ws ssocited with sitgliptin tretment t dose

6 57 Americn Journl of Biomedicl Reserch of 10 mg/kg followed by glimepiride tretment nd the lest response ws observed with sitgliptin tretment t 30 mg/kg (Figure 5) Lipid Profile At the beginning of the experiment, the bseline vlues of the lipid profile (triglycerides, totl cholesterol, HDL-C nd LDL-C) showed significntly higher levels of triglycerides, cholesterol nd LDL-C nd lower level of HDL-C in the untreted dibetic rts thn the control group (Tble 1). At the end of the tretment period, glimepiride significntly corrected the levels of triglyceride, cholesterol nd LDL-C, while showed no significnt effect on HDL-C (Tble 2). Sitgliptin tretment t dose of 10 mg/kg slightly but significntly decresed the level of triglycerides nd completely normlized the levels of cholesterol nd LDL-C, while the dose of 30 mg/kg hd no effect on triglycerides level nd it decresed the cholesterol nd LDL-C levels. Sitgliptin t the dministered doses showed no effect on the HDL-C level (Tble 2). Tble 2. Lipid profile of different studied groups t the end of the tretment period (4 weeks) Prmeter Control Dibetic Untreted Glim Sit 10 Sit 30 Triglycerides (TGs) (mg/dl) 39.9± ± ± ± ±1.08 b, b, c, c, d Totl Cholesterol (mg/dl) 142.2± ± ± ± ±0.75, b, b, b, c, d LDL-Cholesterol (mg/dl) 91.81± ± ± ± ±0.71, b, b, b, d HDL-Cholesterol (mg/dl) 42.5± ± ± ± ±0.21, b, b, c Vlues re presented s men ± SE (n = 10). The bbrevitions denote Glim: rts treted with glimepiride (0.1 mg/kg), Sit 10: rts treted with sitgliptin (10 mg/kg) nd Sit 30: rts treted with sitgliptin (30 mg/kg). : significntly different from the control group, b: significntly different from the dibetic untreted group, c: significntly different from the Glim group, d: significntly different from the Sit 10 group, using ANOVA (LSD), p vlue < Cyclic Adenosine Monophosphte (camp) Level In the norml control rts, the highest content of camp ws observed in the BAT (Figure 6). The untreted dibetic rts showed gret elevtion in camp levels of the liver nd WAT (12.49±0.6 nd 27.06±4.3, respectively) to be 1.3 nd 3.9 fold the control vlues (9.53±0.7 nd 6.9±0.4, respectively), respectively, while in BAT, the camp level (9.38±0.7) ws decresed to be 0.4 fold the control vlue (21.56±1.3) (Figure 6). Glimepiride treted dibetic rts showed significnt decresed levels of camp in the liver nd WAT, to be ner the control vlue in the dipose tissue nd even lower thn the control vlue in the liver. The BAT of glimepiride treted rts showed significntly higher camp level thn the untreted rts, but lower thn the control rts (Figure 6). Sitgliptin treted dibetic rts showed lower heptic levels of camp thn the untreted rts nd even the control rts t the two doses used. Moreover, the rts showed lower camp content in the WAT compred to the untreted rts; the rts treted with sitgliptin t dose of 30 mg/kg showed no significnt difference from the control rts (Figure 6). Sitgliptin treted rts showed significnt lower level of camp in the BAT compred to the untreted nd the control rts (Figure 6). Figure 6. Cyclic denosine monophosphte (camp) levels (pg/mg protein) in the liver, white dipose tissue (WAT) nd brown dipose tissue (BAT) of different studied groups. Vlues re presented s men ± SE (n = 10). The bbrevitions denote Glim: rts treted with glimepiride (0.1 mg/kg), Sit 10: rts treted with sitgliptin (10 mg/kg) nd Sit 30: rts treted with sitgliptin (30 mg/kg). : significntly different from the control group, b: significntly different from the dibetic untreted group, c: significntly different from the Glim group, d: significntly different from the Sit 10 group, using ANOVA (LSD), p vlue < 0.05

7 Americn Journl of Biomedicl Reserch Discussion T2DM is multiorgn disorder which is chrcterized by erly direct or indirect defects in muscles, heptocytes, dipocytes, nd β-cells [1]. It hs been shown tht dysregultion in insulin signling mechnisms results in glucose intolernce, insulin resistnce nd T2DM [11]. The incresing prevlence of T2DM hs stimulted the development of mny new pproches to sfely tret hyperglycemi. The min gol of these therpies is to reduce glucose levels, nd therefore prevent the development of dibetes complictions. Severl ntidibetic drugs re very well ccepted worldwide in terms of low incidence of dverse effects nd therpeutic efficiency, nd the mechnism of ction for most of these drugs hs been demonstrted. However, individul responses to these medictions cn differ significntly, possibly s result of the heterogeneity of T2DM pthophysiology [12]. This study ws designed to demonstrte sitgliptin effects on glucose homeostsis prmeters, lipid profile nd some insulin signling components in HFD/STZ dibetic rts, compred to the sulfonylure glimepiride. Induction of dibetes ws performed ccording to modified Srinivsn, et l. method [9]; HFD ws combined with single low dose of STZ injected intrperitonelly t 45 mg/kg of body weight. While HFD induces insulin resistnce, the low dose of STZ cuses mild impirment in β-cell function [9]. The HFD/STZ dibetic rts exhibited overt hyperglycemi, dyslipidemi, hyperinsulinemi, nd insulin resistnce (s indicted by incresed HOMA). Therefore, this rt model exhibited fetures of T2DM tht would closely reflect the metbolic chrcteristics of T2DM in humns, nd it could be used for phrmcologicl testing. The higher fsting insulin level ws evident in our model of dibetes, which does not gree with Srinivsn, et l. method [9] in which dibetic rts exhibited nernorml insulin level fter STZ injection. This is could be elucidted s dministrtion of HFD in our model for 4 weeks insted of 2 weeks, excerbted insulin resistnce in dibetic rts even fter dministrtion of low dose of STZ. Therefore, insulin is unble to ct properly on resistnt tissues nd this resulted in poor glucose utiliztion, so β-cells initilly compensted for insulin resistnce by incresing insulin secretion. Multiple orgns contribute to the development of peripherl insulin resistnce, with the mjor contributors being skeletl muscle, liver, nd dipose tissues. Insulin sensitivity is the bility of insulin to lower plsm glucose levels by inhibiting heptic glucose production nd stimulting glucose uptke in skeletl muscle nd dipose tissues. On the contrry, insulin resistnce describes n impired biologicl response to insulin, but there is sufficient vribility in norml sensitivity to insulin tht there is no specific cut-off t which sensitivity ends nd resistnce begins [13]. Moreover, there is no bsolute definition of hyperinsulinemi, since n insulin concentrtion tht is rised for n individul is usully still within the wide rnge of normlity. While hyperinsulinemi my compenste for resistnce to some ctions of insulin, it cn result in overexpression of ctions tht retin norml rectivity to insulin [14]. Sitgliptin nd glimepiride re insulin secretgogues which enhnce insulin secretion from β-cells by different mechnisms. Sitgliptin inhibits DPP-4 enzyme, thus incresing levels of incretin hormones [7]. Therefore, sitgliptin cuses glucose-dependent stimultion of insulin secretion, inhibition of glucgon secretion nd preservtion of β-cell mss through stimultion of cell prolifertion nd inhibition of poptosis [8]. Glimepiride is second-genertion sulfonylure cts directly by binding to the ATP-dependent potssium chnnels (K + ATP) on the β-cells. The closure of these chnnels by sulfonylures results in depolriztion of the β-cells nd successive clcium influx which leds to glucoseindependent insulin relese [15]. Moreover, sulfonylures re reported to inhibit glucgon secretion from pncretic α-cells [16,17]. We found tht glimepiride hd - to some extent - better glucose-lowering effect thn sitgliptin ppered in weekly determined FBG levels. This finding ws in ccord with results from previous studies compred sitgliptin with the sulfonylure glibenclmide in T2DM ptients [18]. Both glucose-utilizing effect nd insulin secretgogue ction of glimepiride nd sitgliptin contributed to lower HOMA-insulin resistnce vlue in dibetic rts. As expected in our model of dibetes (HFD/STZ dibetic model), the disrupted glucose homeostsis is ssocited with derngements in the lipid profile mnifested s incresed triglycerides, totl cholesterol nd LDL-C, s well s decresed HDL-C (collectively clled dyslipidemi). These derngements my be cuse or consequence of the dibetic stte nd insulin resistnce [19]. The dibetic rts treted with glimepiride (0.1 mg/kg) nd sitgliptin (10 mg/kg) showed ner-norml plsm lipid profile. It is reported tht mny T2DM ptients continue to hve bnorml plsm lipid profiles, lthough they hve chieved their glycemic gols [20]. This pttern is - to some extent - consistent with our findings in rts receiving sitgliptin in which incresing drug dose resulted in n bnorml lipid profile mnifested minly by significnt increment of plsm triglycerides level. In line with this, clinicl studies of DPP-IV inhibitors hve demonstrted modest improvements (decrement in levels of totl cholesterol, LDL-C nd triglycerides) in lipid pnels nd not ll studies hve demonstrted significnt benefit [21]. Besides the clssicl effects of sitgliptin nd glimepiride, they hve other wys to meliorte the dibetic stte through their effects on peripherl insulin ctions. It ws documented tht, gluconeogenesis nd heptic glucose production were inhibited by sitgliptin in HFD-induced obese rts due to decresed glycerol vilbility s result of reduced glycerol relese from dipose tissues [22]. Furthermore, studies suggested n insulin sensitizing ction of glimepiride possibly by stimultion of GLUT4 trnsport protein ctivtion nd/or trnsloction in ft nd muscle [23]. camp is n importnt plyer in the regultion of cellulr metbolism nd hormonl ction in the peripherl tissues. Glucgon cts on the liver nd dipose tissues where it binds to its receptors. It ctivtes denylte cyclse thus elevting intrcellulr camp level. It enhnces lipolysis, glycogenolysis s result of phosphorylse stimultion [24], nd gluconeogenesis

8 59 Americn Journl of Biomedicl Reserch through induction of phosphoenolpyruvte crboxykinse (PEPCK) [3]. On the other hnd, it inhibits glucose uptke into dipocytes, thus decresing triglyceride synthesis [2]. Insulin ntgonizes ctbolic ctions of glucgon minly by ctivtion of PDE3B vi Akt/PKB, thus reducing camp levels [3]. Therefore, heptic camp levels could be used s n indictor of glucgon/insulin rtio nd consequently could be used to evlute the efficiency of ntidibetic drugs. The results indicted tht the dibetic stte differentilly ffects camp level ccording to the type of the tissue. There re two kinds of dipose tissue; WAT which plys significnt role in energy storge in the form of triglycerides, nd BAT which plys substntil role in het production [5]. Both WAT nd BAT re innervted by the sympthetic nervous system [25]. In lipolyticlly ctive dipocytes, elevted camp levels result in ctivtion/phosphoryltion of PKA, which ctivtes hormone sensitive lipse (HSL), nd consequently initite lipolysis. Unrestrined lipolysis, which leds to increse circulting free ftty cids in the bsorptive stte, hs been linked to insulin resistnce [26]. In BAT, elevted PKA-ctivity stimultes expression of UCP1 tht uncouples oxidtive phosphoryltion resulting in het production [5]. The dt of camp content in the peripherl tissues clerly indicted higher camp level in BAT thn in the liver nd WAT. It hs been demonstrted tht sympthetic innervtion is more bundnt in BAT thn in WAT [27]. Moreover, the effect of ctecholmines in dipose tissues depends on the type of drenergic receptor (AR); they hve ntilipolytic effect in WAT through the α 2 -ARs which inhibit denylyl cyclse ctivity [28] wheres, the β 3 -ARs re bundntly expressed in BAT which ctivte denylte cyclse [29]. The results indicted tht, in dibetic rts, while the heptic nd WAT contents of camp re gretly elevted, the levels in BAT re depleted. The observed high level of camp in the liver nd WAT could be explined by insulin resistnce stte which fvors the lipolytic pthwy s result of incresing glucgon/insulin rtio, the produced free ftty cids nd glycerol my further exggerte the insulin resistnce in tissues in vicious cycle. On the other hnd, BAT of dibetic rts showed lowered camp which could be explined by the fct tht BAT re more insulin sensitive thn WAT [30] nd my retin insulin sensitivity even under insulin resistnce. This decline in camp in BAT my result in shift in its metbolic cpcity from lipolytic nd ctbolic into lipogenic nd nbolic pthwys which could chnge cells phenotype from brown dipocyte (metboliclly ctive) into white dipocyte (storge) phenotype. 5. Conclusion From the bove discussion, we cn conclude tht the glucose-lowering effects of the two ntidibetic drugs; sitgliptin nd glimepiride re - to some extent - comprble nd re not dependnt only on their insulin secretgogue ction but lso on their effects on peripherl insulin signling components. They hve camp-lowering effect in the liver nd WAT, which my hve protection ginst vsculr complictions of dibetes. The mode of ction nd the moleculr trgets of sitgliptin in peripherl tissues need to be elucidted. Also, the discrepncy between the low nd high doses of sitgliptin needs n explntion. Competing Interests The uthors declre tht they hve no competing interests. Authors' Contribution MIS crried out most experiments nd prticipted in the experimentl design. MAK designed the experiments nd contributed in writing nd revising the mnuscript. MYH nd MHH performed the sttisticl nlysis of dt nd helped in writing the mnuscript. RRS prticipted in dose selection, follow up of the nimls, dt ssembly nd mnuscript revision. All uthors hve red nd pproved the finl mnuscript. List of Abbrevitions T2DM, Type 2 dibetes mellitus; HFD, High-ft diet; STZ, Streptozotocin; IRS, Insulin receptor substrte proteins; MAPK, Mitogen ctivted protein kinse; PKA, Protein kinse A; PKB, Protein kinse B; PKC, typicl protein kinse C; camp, cyclic denosine monophosphte; BAT, Brown dipose tissue; WAT, White dipose tissue; PDEs, Phosphodiesterses; PDE3B, Phosphodiesterse 3B; DPP-4, Dipeptidyl peptidse-4; GIP, Glucosedependent insulinotropic peptide; GLP-1, Glucgon-like peptide-1; K + ATP, ATP-dependent potssium chnnels; PEPCK, phosphoenolpyruvte crboxykinse; AR, Adrenergic receptor; FBG, fsting blood glucose; DMSO, dimethyl sulfoxide; OGTT, Orl glucose tolernce test; IRI, Insulin resistnce index, HOMA-IR, Homeostsis model ssessment estimte of insulin resistnce; ELISA, Enzyme-linked immunosorbent ssy; ANOVA, Anlysis of vrince; LDL-C, Low density lipoprotein-cholesterol; HDL-C, High density lipoprotein-cholesterol. References [1] Lin Y., Sun Z. Current views on type 2 dibetes. J Endocrinol 2010, 204 (1): [2] Brss B.J., Abelev Z., Lio E.P., Poretsky L. Endocrine Pncres. In Principles of Dibetes Mellitus. 2 nd edition. Edited by Poretsky L. New York: Springer; 2010: [3] Wu X., Grvey W.T. Insulin Action. In Textbook of Dibetes. 4 th edition. Edited by Holt R.I.G., Cockrm C.S., Flyvbjerg A., Goldstein R.J. Singpore: Wiley Blckwell; 2010: [4] Cirldi T.P. Cellulr Mechnisms of Insulin Action. In Principles of Dibetes Mellitus. 2 nd edition. Edited by Poretsky L. New York: Springer; 2010: [5] Gest S., Tseng Y.H., Khn C.R. Developmentl origin of ft: trcking obesity to its source. Cell 2007, 131: [6] Sell H., Deshies Y., Richrd D. The brown dipocyte: updte on its metbolic role. Int J Biochem Cell Biol 2004, 36 (11): [7] Drucker D.J., Nuck M.A. The incretin system: glucgon-like peptide-1 receptor gonists nd dipeptidyl peptidse-4 inhibitors in type 2 dibetes. Lncet 2006, 368 (9548):

9 Americn Journl of Biomedicl Reserch 60 [8] Drucker D.J. The biology of incretin hormones. Cell Metb 2006, 3: [9] Srinivsn K., Viswnd B., Asrt L., Kul C.L., Rmro P. Combintion of high-ft diet-fed nd low-dose streptozotocintreted rt: model for type 2 dibetes nd phrmcologicl screening. Phrmcol Res 2005, 52: [10] Lowry O.H., Rosebrough N.J., Frr A.L., Rndll R.J. Protein mesurements with Folin-phenol regent. J Biol Chem 1951, 193: [11] White M.F. Insulin signling in helth nd disese. Science 2003, 302 (5651): [12] Khn S.E., Cooper M.E., Del Prto S. Pthophysiology nd tretment of type 2 dibetes: perspectives on the pst, present, nd future. Lncet 2014, 383 (9922): [13] Pirol L., Johnston A.M., Vn Obberghen E. Modultion of insulin ction. Dibetologi 2004, 47: [14] Reven G.M. Insulin resistnce, the insulin resistnce syndrome, nd crdiovsculr disese. Pnminerv Med 2005, 47: [15] Bryn J., Crne A., Vil-Crriles W.H., Bbenko AP.., Aguilr- Bryn L. Insulin secretgogues, sulfonylure receptors nd K (ATP) chnnels. Curr Phrm Des 2005, 11: [16] Jckson J.E., Bressler R. Clinicl phrmcology of sulphonylure hypoglycemic gents: prt 2. Drugs 1981, 22: [17] Jckson J.E., Bressler R. Clinicl phrmcology of sulphonylure hypoglycemic gents: prt 1. Drugs 1981, 22: [18] Nthn D.M., Buse J.B., Dvidson M.B., Ferrnnini E., Holmn R.R., Sherwin R., Zinmn B.; Americn Dibetes Assocition; Europen Assocition for Study of Dibetes. Medicl mngement of hyperglycemi in type 2 dibetes: consensus lgorithm for the initition nd djustment of therpy: consensus sttement of the Americn Dibetes Assocition nd the Europen Assocition for the Study of Dibetes. Dibetes Cre 2009, 32: [19] Chhil T.J., Ginsberg H.N. Dibetic dyslipidemi. Endocrinol Metb Clin North Am 2006, 35: [20] Moordin A.D. Dyslipidemi in type 2 dibetes mellitus. Nt Clin Prct Endocrinol Metb 2009, 5 (3): [21] Addison D., Aguilr D. Dibetes nd crdiovsculr disese: the potentil benefit of incretin-bsed therpies. Curr Atheroscler Rep 2011, 13 (2): [22] Lu Y.L., Zhou D.Q., Zhi H.L., Wu H., Guo Z.K. Decresed heptic glucose production in obese rts by dipeptidyl peptidse- IV inhibitor sitgliptin. Chin Med J 2012, 125 (10): [23] Müller G., Wied S., Wetekm E.M., Crecelius A., Unkelbch A., Pünter J. Stimultion of glucose utiliztion in 3T3 dipocytes nd rt diphrgm in vitro by the sulphonylures, glimepiride nd glibenclmide, is correlted with modultions of the camp regultory cscde. Biochem Phrmcol 1994, 30 (48): [24] Mgnusson I., Rothmn D., Gerrd D., Ktz L., Shulmn G. Contribution of heptic glycogenolysis to glucose production in humns in response to physiologicl increse in plsm glucgon concentrtion. Dibetes 1995, 44: [25] Bllrd K., Mlmfors T., Rosell S. Adrenergic innervtion nd vsculr ptterns in cnine dipose tissue. Microvsc Res 1974, 8: [26] Boden G. Ftty cid-induced inflmmtion nd insulin resistnce in skeletl muscle nd liver. Curr Dib Rep 2006, 6: [27] Husberg M., Morgn D.A., Mitchell J.L., Sivitz W.I., Mrk A.L., Hynes W.G. Leptin potentites thermogenic sympthetic responses to hypothermi: receptor-medited effect. Dibetes 2002, 51: [28] Lfontn M., Berln M. Ft cell drenergic receptors nd the control of white nd brown ft cell function. J Lipid Res 1993, 34: [29] Sell H., Deshies Y., Richrd D. The brown dipocyte: updte on its metbolic role. Int J Biochem Cell Biol 2004, 36: [30] Orv J., Nuutil P., Lidell M.E., Oikonen V., Noponen T., Viljnen T., Scheinin M., Tittonen M., Niemi T., Enerbck S., Virtnen K.A. Different metbolic responses of humn brown dipose tissue to ctivtion by cold nd insulin. Cell metbolism 2011, 14 (2):