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Life Sciences 90 (2012) 831–837

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Life Sciences

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Coexistence of resistance and increased glucose tolerance in pregnant rats: A physiological mechanism for glucose maintenance

Marcia Aparecida Carrara a, Márcia Regina Batista b, Tiago Ribeiro Saruhashi b, Antonio Machado Felisberto-Junior c, Marcio Guilhermetti a, Roberto Barbosa Bazotte a,⁎

a Department of Pharmacology and Therapeutic, State University of Maringá, Maringá, Paraná-PR 87020–900, Brazil b Department of Clinical Analysis and Biomedicine, State University of Maringá, Maringá, Paraná-PR 87020–900, Brazil c Department of Biology, Faculdade Ingá - UNINGÁ, Maringá, Paraná-PR 87070–000, Brazil

article info abstract

Article history: Aim: The contribution of (IR) and glucose tolerance to the maintenance of blood glucose Received 28 July 2011 levels in non diabetic pregnant Wistar rats (PWR) was investigated. Accepted 22 March 2012 Main methods: PWR were submitted to conventional insulin tolerance test (ITT) and (GTT) using blood sample collected 0, 10 and 60 min after intraperitoneal insulin (1 U/kg) or oral (gavage) Keywords: glucose (1 g/kg) administration. Moreover, ITT, GTT and the kinetics of glucose concentration changes in Pregnancy the fed and fasted states were evaluated with a real-time continuous glucose monitoring system (RT- Gestational CGMS) technique. Furthermore, the contribution of the liver glucose production was investigated. Liver glucose production fi Insulin resistance Key ndings: Conventional ITT and GTT at 0, 7, 14 and 20 days of pregnancy revealed increased IR and glucose tolerance after 20 days of pregnancy. Thus, this period of pregnancy was used to investigate the kinetics of Continuous glucose monitoring system glucose changes with the RT-CGMS technique. PWR (day 20) exhibited a lower (pb0.05) glucose concentra- Glucose tolerance tion in the fed state. In addition, we observed IR and increased glucose tolerance in the fed state (PWR-day 20 vs. day 0). Furthermore, our data from glycogenolysis and gluconeogenesis suggested that the liver glucose production did not contribute to these changes in insulin sensitivity and/or glucose tolerance during late pregnancy. Significance: In contrast to the general view that IR is a pathological process associated with gestational dia- betes, a certain degree of IR may represent an important physiological mechanism for blood glucose mainte- nance during fasting. © 2012 Elsevier Inc. Open access under the Elsevier OA license.

Introduction Thus, the following two questions can be raised: 1. how can there be a simultaneous increase in insulin resistance and a predisposition During early pregnancy there is an anabolic state that is associated to fasting hypoglycemic responses during late pregnancy? 2. Al- with hyperphagia and body fat accumulation (Herrera, 2002). In con- though glucose tolerance is decreased during , trast, during late pregnancy there is a catabolic state associated with what occurs with glucose tolerance during normal pregnancy consid- insulin resistance where not only glucose but also free fatty acids ering the intense glucose utilization by the fetus, which can be 50% of and ketone bodies can serve as important sources of energetic sub- the maternal glucose utilization rate in the basal state (Herrera et al., strates during a fasting state (Herrera et al., 1994; Herrera, 2000). 1994)? During pregnancy, there are two main metabolic concerns. First, These questions are especially relevant when considering that ma- there is possibility of gestational diabetes (ADA, 2011) where the ternal hypoglycemia has the potential to adversely affect the develop- levels of insulin secretion cannot overcome the insulin resistance ing fetus (Kawaguchi et al., 1994; Reece et al., 1994; Johnson, 2008). that occurs in late pregnancy. Second, there is an increased predispo- Moreover, because traditional methods of blood collection provide sition to hypoglycemia which occurs in not only humans (Vadakekut little information about dynamic glucose levels, a real time continu- et al., 2011) but also mice (Reddi et al., 1976), ewes (Schlumbohm ous glucose monitoring system (RT-CGMS) technique was employed and Harmeyer, 2008), bitches (Johnson, 2008) and rats (Leturque et in this study. The RT-CGMS technique provides more detailed data re- al., 1989). garding glucose changes because it is capable of measuring the glu- cose concentration every 5 min, which results in 288 measurements per day (Bode et al., 2004). ⁎ Corresponding author. Tel.: +55 44 3011 5161. Therefore, in this study, we focused on the kinetics of glucose E-mail address: [email protected] (R.B. Bazotte). changes, the participation of insulin resistance, glucose tolerance

0024-3205 © 2012 Elsevier Inc. Open access under the Elsevier OA license. doi:10.1016/j.lfs.2012.03.037 832 M.A. Carrara et al. / Life Sciences 90 (2012) 831–837 and liver glucose production with respect to the maintenance of using a traditional clinical laboratory method (glucose oxidase) blood glucose concentrations during fed and fasted states in pregnant (Bergmeyer and Bernt, 1974). Wistar rats. The female rats were considered hypoglycemic when glucose levels decreased to values below 40 mg/dL. Materials and methods

Materials Conventional glucose tolerance test (GTT)

The glucose sensor device was obtained from Medtronic® (São After 0 (NPWR) 7, 14 or 20 days of pregnancy the rats were food – Saulo, SP, Brazil). Glucose was purchased from Sigma-Aldrich Chemi- deprived (8:00 p.m. 8:00 a.m.). At this time, the rats received oral cal Company (St. Louis, USA). Regular insulin (Humulin®) was (gavage) glucose (1 g/kg) and were killed by decapitation 0, 10 and 60 min after glucose administration. The blood was collected, and obtained from Lilly (São Saulo, SP, Brazil). L-Alanine and L-glutamine were obtained from ICN Biochemical's (Costa Mesa, CA, USA). the blood glucose levels were measured using a conventional colori- metric laboratory method to measure glucose (Bergmeyer and Bernt, 1974). Animals and treatment

A total of 218 female Wistar rats after 0, 7, 14 or 20 days of preg- Real-time continuous glucose monitoring System (RT-CGMS) technique nancy were used in this investigation. The rats were maintained at a constant temperature (23 °C) and under an automatically controlled The kinetics of interstitial glucose changes were determined using photoperiod (12 h light/12 h dark). The protocol for these experi- the RT-CGMS technique. The RT-CGMS device is composed of a glu- ments was approved by the Ethical Committee (058/2008) and com- cose sensor which is a membrane-covered electrode that detects glu- plied with the international law for the protection of the animals. cose levels in the interstitial fluid and sends this information to the transmitter, a transmitter which sends the data to the monitor Conventional insulin tolerance test (ITT) using high frequency radio waves and a monitor which provides readings in real time (Medtronic Guardian® Real-Time CGMS). The After 0 (NPWR) 7, 14 or 20 days of pregnancy the rats were food RT-CGMs system evaluates glucose levels every 10 s, and the results deprived (8:00 p.m.–8:00 a.m.). At this time point the rats received obtained each 5 min represent the average sum of 30 glucose concen- an intraperitoneal injection of regular insulin (1 U/kg) and were tration measurements. The glucose levels (about 280 values per day) killed by decapitation 0, 10 and 60 min after insulin administration. were stored in the monitor. The system measured glucose concentra- The blood was collected and the glucose levels were measured tion in a range of 40–400 mg/dL.

Fig. 1. Glycemic measurements 0, 10 and 60 min after an intraperitoneal injection of insulin (1 U/kg). (A) Non-pregnant rats (0 day); (B) pregnant rats (7 days); (C) pregnant rats (14 days) and (D) pregnant rats (20 days). (a) pb0.05 — 0 min vs. 10 min; (b) pb0.05 — 10 min vs. 60 min; (c) pb0.05 — 0 min vs. 60 min. The data are reported as the mean±SEM of 7–9 animals per group. M.A. Carrara et al. / Life Sciences 90 (2012) 831–837 833

To insert the sensor and install the transmitter, rats were anes- Moreover, a control NPWR (day 0) was submitted to the same exper- thetized with an intraperitoneal injection of sodium thiopental imental procedure. (45 mg/kg). After a suitable level of anesthesia was achieved, the area of skin on the back was shaved, and the sensor was manually Liver perfusion experiments inserted in the interscapular region (subcutaneous tissue). The en- tire procedure took 15–20 min and the attachments of the sensor The in situ liver perfusion technique was performed as previously to the animal's back worked without complications. The monitor described (Nascimento et al., 2008). Gluconeogenesis from L-alanine was kept on the lid of the plastic cage that housed the rat after (5 mM), L-glutamine (5 mM) and glycerol (2 mM) in the livers of . PWR (day 20) and NPWR (day 0) which had fasted for 12 h, were After connecting the transmitter with the monitor, a 2 h initializa- compared. As shown in Fig. 4, after a pre-infusion period (10 min) tion period was required before starting the calibration of the RT- the gluconeogenic substrate was infused during 70 min and submit- CGMS. ted to a post-infusion period (10 min without the gluconeogenic The first and second calibration procedures were performed 2 h substrate) in order to allow the glucose production values to return and 6 h after the installation of the RT-CGMs. Moreover, calibrations to the levels of the pre-infusion period. Samples of the effluent per- were confirmed every 12 h by measuring blood glucose levels with fusion fluid were collected every 5 min and the glucose concentra- a home glucometer (Diagnostics Accu-Chek® Active glucometer). tion was measured (Bergmeyer and Bernt, 1974). The difference For this purpose, a blood droplet was obtained after a small incision between the glucose production during and before the infusion of was made at the tip of the tail. the liver glucose precursor represents the glucose production from During the observation period, the surgery site for the insertion of gluconeogenesis. The area under the curve (AUC) for the infusion pe- the sensor and transmitter did not show any sign of infection or riod of the gluconeogenic substrate was expressed as μmol/g. inflammation. In part of the liver perfusion experiments, the glycogen catabolism Finally, the sensor was removed and the data stored in the monitor in the fed state was measured. This experimental approach permitted were transferred to a receptor (Con Link® Medtronic), which allowed investigation of the basal rate of glycogenolysis and the activation of the study information to be downloaded to a personal computer con- glycogenolysis induced by epinephrine (Lopes et al., 1998). The dif- nected to an on line analysis program (CareLink personal® — ference between the glucose production during and before the infu- Medtronic). sion of epinephrine was calculated by AUC analysis as previously Fig. 3 represents the kinetics for glucose changes in a pregnant rat described. (day 20) during fed and fasted states. For comparative purposes, a control NPWR (day 0) was submitted to the same experimental Statistical analysis procedure. Fig. 4 represents the kinetics for glucose changes of a pregnant rat The results are reported as mean±SEM. The data were evaluat- (day 20) after an intraperitoneal injection of regular insulin (1 U/kg) ed by a Student t test and analysis of variance (one way and two or oral (gavage) glucose administration (1 g/kg). The procedure for ways — ANOVAs) and the difference between means was assessed insulin and glucose administration (starting 8:00 a.m.) were similar by Tukey's test. A P value of less than 0.05 was considered to the previously described conventional ITT or GTT procedures. significant.

Fig. 2. Glycemic measurements 0, 10 and 60 min after oral (gavage) glucose administration (1 g/kg). (A) Non-pregnant rats (0 day); (B) pregnant rats (7 days); (C) pregnant rats (14 days) and (D) pregnant rats (20 days). (a) pb0.05 — 0 min vs. 10 min; (b) pb0.05 — 10 min vs. 60 min. The data are reported as the mean±SEM of 7–9 animals per group. 834 M.A. Carrara et al. / Life Sciences 90 (2012) 831–837

Fig. 3. Kinetics of glucose concentration (mg/dL) on day 0 (●) and day 20 (■) of pregnancy (fed and fasted states). The time of food withdraw is represented by the dashed vertical line. The data are reported as the mean±SEM of 5 measurements.

Results (Fig. 2C) and 60% (Fig. 2D) for the NPWR (day 0), PWR (day 7), PWR (day 14) and PWR (day 21) groups, respectively. Blood glucose concentrations 0, 10 and 60 min after the insulin The kinetics of glucose changes and liver glucose production injection are presented in Fig. 1. The percent decrease in glucose (LGP) were measured 20 days after the start of pregnancy because levels after the insulin injection between 0 and 60 min was 70% of the low percentage of glucose decrease after the insulin injec- (Fig. 1A), 72% (Fig. 1B), 68% (Fig. 1C) and 55% (Fig. 1D) for the tion and the low percentage of glucose increase after the glucose NPWR (day 0), PWR (day 7), PWR (day 14) and PWR (day 21) administration observed during this stage of pregnancy. groups, respectively. The kinetics of the glucose levels measurements according to Fasting blood glucose levels 0, 10 and 60 min after the glucose ad- RT-CGMS showed negligible variations in the NPWR (day 0) fed ministration are presented in Fig. 2. rats (i.e., an approximate mean of 106.7 mg/dL with glucose values The percent increase in glucose levels after the glucose adminis- between 88 and 130 mg/dL). After fasting, the glucose values fluc- tration between 0 and 10 min was 87% (Fig. 2A), 87% (Fig. 2B), 60% tuated at about a mean of 103.3 mg/dL with values between 90

Fig. 4. Glucose levels (mg/dL) on day 0 (●) and 20 (■) of pregnancy after intraperitoneal insulin (1 U/kg) (A) or oral (gavage) glucose (1 g/kg) administration (B). The time of administration are represented by the dashed vertical line. M.A. Carrara et al. / Life Sciences 90 (2012) 831–837 835 and 120 mg/dL. These glycemic changes in the fed and fasted states of the NPWR (day 0) were used here to define the basal physiological condition in the absence of pregnancy. On the other hand, PWR (day 20) showed a lower glucose concentration in the fed and fasted states (PWR — day 20 vs. NPWR). Moreover, after the initiation of fasting, PWR (day 20) showed a transitory increase followed by a discrete decrease of glucose levels (Fig. 3). The values of glucose levels as measured by RT-CGMS after insulin (Fig. 4A) or glucose (Fig. 4B) administration showed less decrease and less increase, respectively, in the fed PWR at day as compared with PWR at day 0. The results of gluconeogenesis and glycogenolysis from liver per- fusion experiments are summarized in Figs. 5 and 6, respectively. PWR (day 20 vs. day 0) exhibited a lower (pb0.05) LGP from L-ala- Fig. 6. Glycogenolysis during the infusion of epinephrine in fed rats on day 0 (●) and nine (Fig. 5A) and L-glutamine (Fig. 5B), and a higher (pb0.05) LGP day 20 (■) of pregnancy. The glucose concentration from the effluent perfusate was from glycerol (Fig. 5C). On the other hand, as shown by Fig. 6, the analyzed every 5 min. The data are reported as the mean±SEM of 3–6 experiments. The comparison (area under curves) between day 0 and day 20 was not statistically basal rates of glycogenolysis (before epinephrine infusion) and the significant (Pb0.05). activation of glycogenolysis induced by epinephrine were similar (day 20 vs. day 0). Discussion

Decreased insulin responsiveness of maternal tissues is a common feature of late pregnancy and occurs in not only human (Hiramatsu et al., 2000; Ehrlich et al., 2011; Schaefer-Graf et al., 2011) but also in several species of mammalians, including rats (Leturque et al., 1986; Ramos and Herrera, 1995; López-Luna et al., 1998). In agreement with these studies, there was less of a decrease in glucose concentra- tions after insulin injection in rats at 20 days of pregnancy when com- pared with NPWR (Fig. 1). However, while insulin resistance in human is associated with a high incidence of gestational diabetes, this pathological change has not been observed in PWR, except when animals have received a diabetogenic diet (Holemans et al., 2004) or drugs (Reece et al., 2006; Campos et al., 2007; Saito et al., 2010). In addition, PWR (day 20) also showed increased glucose tol- erance when compared with NPWR (Fig. 2). Because, insulin resistance (Fig. 1) and increased glucose toler- ance (Fig. 2) were simultaneously observed on day 20 of pregnan- cy, this day was chosen for the investigation of GTT, ITT, and the glucose kinetics using the RT-CGMS technique. This technique per- mits the identification glucose trends with results in real time (Li et al., 2010). However, RT-CGMS devices measure interstitial glu- cose (Mastrototaro, 1999; Tiessen et al., 2002; Keenan et al., 2009), and there is a physiologic lag time between the blood glu- cose and the interstitial glucose levels of approximately 3 to 14 min (Rebrin et al., 1999; Steil et al., 2003). However, taken into account our experimental conditions this lag time did not in- fluence the interpretation of our results. In agreement with previous studies (Lü et al., 2010; Radermecker and Scheen, 2010) we also demonstrated the feasibility of using cur- rent sensor technology in order to achieve glucose monitoring in rats. In other words, late pregnant rats had a lower (pb0.05) glucose con- centration during fed and fasted states (Fig. 3). Interestingly, between 12:00 and 20:00 there was a decrease (pb0.05) of glucose levels among pregnant rats that reached 50 mg/ dL (Fig. 3). A possible explanation for these results is that rats are noc- turnal animals, and between 12:00 and 20:00 there is a low frequency of feeding. Consistent with these results, several reports confirm that during late pregnancy there is an increased predisposition to hypo- glycemia in not only in rats (Leturque et al., 1989) but also human (Vadakekut et al., 2011), mice (Reddi et al., 1976), ewes (Schlumbohm and Harmeyer, 2008) and bitches (Johnson, 2008). Fig. 5. Gluconeogenesis from L-alanine (A), L-glutamine (B) and glycerol (C) in per- In addition, the simultaneous insulin resistance and increased glu- fused livers of 12 h fasted rats on day 0 (●) and day 20 (■) of pregnancy. The effluent cose tolerance demonstrated by conventional ITT (Fig. 1) and GTT perfusate was sampled at 5 min intervals and analyzed for glucose. The data are (Fig. 2) were confirmed by RT-CGMS (Fig. 4). reported as the mean±SEM of 3–6 experiments. All comparison (area under curves) between days 0 and day 20 (to L-alanine, L-glutamine and glycerol) were statistically The advantage of the CGMS technique is the fact that it allows re- significant (Pb0.05). peated investigations of the same rat's insulin sensitivity and glucose 836 M.A. Carrara et al. / Life Sciences 90 (2012) 831–837 tolerance without anesthesia, blood collection or other stressful pro- can be reached. What should be considered a normal value of fasting cedures that can affect glucose levels. To our knowledge this study glucose levels during pregnancy? How can we distinguish physiolog- represents the first demonstration of the coexistence of insulin resis- ical and pathological insulin resistance during pregnancy? The an- tance and increased glucose tolerance during late pregnancy using swers to these questions and establishing a better understanding of the CGMS technique. the coexistence of insulin resistance and increased glucose tolerance During late pregnancy, the presence of insulin resistance in muscle will be necessary before achieving a consensus regarding the values (Barros et al., 2008; Camps et al., 1990)andadiposetissue(Wada of glucose levels for the diagnosis of gestational diabetes. In agree- et al., 2010) is well established. However, the presence of insulin resis- ment with this proposition, several studies have suggest a revaluation tance in liver during late pregnancy is controversial (Davidson, 1984; of the normal values of fasting glucose levels and glucose tolerance Gilbert et al., 1991). Moreover, considering that abnormally elevated tests for the diagnosis of gestational diabetes. For example, the glucose production is the main effect of insulin resistance in the liver, World Health Organization criterion for the diagnosis of gestational the liver's glucose production from gluconeogenesis and glycogenolysis diabetes is a 2-h plasma glucose≥140 mg/dL with a 75-g oral glucose were investigated. tolerance test. However, increased birth weight and the risk of type 2 In the specific case of gluconeogenesis, late pregnant rats exhib- diabetes were observed with an increase of glucose levels above ited higher (pb0.05) glucose production from glycerol and lower 120 mg/dL (Franks et al., 2006). Furthermore, the American Diabetes (pb0.05) glucose production from L-alanine and L-glutamine. Inter- Association position on the fasting value of glucose levels for the diag- estingly, the former results are compatible with the general idea nosis of gestational diabetes has recently changed from ≥95 mg/dL that during late pregnancy the increased catabolism of lipids and (ADA, 2010)to≥92 mg/dL (ADA, 2011). the intensification of gluconeogenesis from glycerol could be a phys- iological mechanism to protect against the loss of protein (Naismith Conflict of interest statement and Morgan, 1976). Furthermore, these results suggest that the change in liver gluconeogenesis during late pregnancy is specific for Nothing to declare. each gluconeogenic substrate and is consistent with the well- established strategy of preferentially using of metabolites from lipol- Acknowledgments ysis during a period of reduced blood amino acids availability (Herrera and Ortega-Senovilla, 2010). Moreover, the increased This research was supported by the CNPq (grant number 563870/ sympatho-adrenal activity responsible for the accelerated mobiliza- 2010-9) and Araucaria Foundation and CAPES. tion of fat deposits that occurs under both fed and fasting conditions (Herrera et al., 1994) cannot be expanded by glucose production from liver glycogen stores because the intensification of hepatic glycogen- Appendix A. Supplementary data olysis promoted by epinephrine was not different between PWR and NPWR groups (Fig. 6). Supplementary data to this article can be found online at http:// Thus, the data shown in Figs. 5 and 6 suggest that the liver glucose dx.doi.org/10.1016/j.lfs.2012.03.037. production was not affected by pregnancy. The concomitant occurrence of insulin resistance and increased References glucose tolerance may appear paradoxical. However, these simulta- neous changes should alter our view of insulin resistance in late preg- American Diabetes Association – ADA. 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