DA SILVA, ADELINO S. R.1,2; PAULI, JOSÉ R.1,3; ROPELLE, EDUARDO R.1; OLIVEIRA, ALEXANDRE G.1; CINTRA, DENNYS E.1; DE SOUZA, CLAUDIO T.1,4; VELLOSO, LÍCIO A.1; CARVALHEIRA, JOSÉ B. C.1; SAAD, MARIO J. A.1
Obesity is associated with insulin resistance and a state of abnormal inflammatory response (8,42). There is convincing evidence that the activation of c-jun N-terminal kinase (JNK) and inhibitor of factor nuclear kappa B (NF-κB) Iκ kinase β (IKKβ) (9,19,44) pathways are related to the reduction of insulin sensitivity. IKKβ and JNK are serine/threonine kinases activated by inflammatory stimuli shown to inhibit insulin signaling through phosphorylation of serine residue on insulin receptor substrate 1 (IRS-1).
Furthermore, the protein tyrosine phosphatases (PTP) are key regulators of a wide variety of signal transduction pathways (41). Several PTP have been shown to modulate insulin signaling activity and, hence, are a therapeutic target for type 2 diabetes (3). Protein tyrosine phosphatase 1B (PTP1B) is an abundant PTP within the skeletal muscle and a major mechanism that negatively regulates (dephosphorylation) insulin receptor (IR) and IRS-1 activities (12,36). Thus, the inhibition of PTP1B activity or PTP1B knockout enhances insulin sensitivity (23,29).
On the other hand, there is a consensus in the literature regarding the beneficial effects of physical exercise on insulin action in insulin-resistant states (17,18). The increased expression and activity of key proteins known to regulate glucose metabolism in skeletal muscle can be used to explain the molecular effects of acute and chronic exercise on glucose uptake enhancement.
However, to date, few studies have examined the effect of acute exercise intensity on the JNK, IKKβ, and PTP1B activities. In one of these investigations, Ropelle et al. (29) verified that a single bout of exercise, in diet-induced obese (DIO) rats, reversed the serine phosphorylation of IRS-1, in parallel with a reduction in JNK and PTP1B activities and IκBα (an inhibitor of NF-κB) degradation in the skeletal muscle. The exercise protocol used by Ropelle et al. (29) and by other studies from our laboratory (14,24) consisted of two 3-h exercise bouts separated by one 45-min rest period. Although this prolonged exercise protocol promotes a substantial improvement in the insulin signaling pathway in DIO rats, it would probably not be possible to submit obese patients to such an acute exercise protocol because of its extensive volume. In addition, the lack of information about the effort intensity performed by DIO rats may be considered as another limitation of this acute session.
The determination of maximal lactate steady state (MLSS), defined as the highest blood lactate concentration and workload that can be maintained over time without continual blood lactate accumulation, has been extensively used to determine the effort intensity performed by rats in swimming (10,11,16,22) and treadmill running (10). Therefore, the main purpose of the present investigation was to compare the effects of an extensive acute exercise protocol consisting of two 3-h exercise bouts, separated by one 45-min rest period with a session of 45 min of swimming at 70% of the MLSS intensity, on insulin sensitivity, insulin signaling, and on different mechanisms of insulin resistance, such as IRS-1 serine phosphorylation, JNK activity, IκBα expression, and PTP1B activity.
Experimental animals and diet.
Male Wistar rats from the University of Campinas Central Animal Breeding Center were used in the experiments. All experiments were approved by the ethics committee of the State University of Campinas. In addition, the present work adheres to the American College of Sports Medicine's animal care standards. The 4-wk-old Wistar rats were divided into four groups: control rats (C; n = 6) fed on standard rodent chow (Table 1), obese rats fed on an obesity-inducing diet for 3 months (DIO; n = 6; Table 1), DIO rats submitted to a single bout of exercise (DIO + EXE; n = 8), and DIO rats submitted to a single bout of exercise at 70% of the MLSS intensity (DIO + MLSS; n = 8).
Exercise protocol 1 (DIO + EXE).
Rats were adapted to swimming for 10 min for 2 d to reduce the water-induced stress without promoting physical training adaptations. The animals swam in groups of three in a 60-cm-depth × 45-cm-diameter cylindrical tank for two 3-h bouts, separated by a 45-min rest period, and the water temperature was maintained at ∼34°C. This exercise protocol was used previously in other studies in our laboratory (14,24,29).
MLSS test in swimming.
The rats' adaptation to the swimming exercise was similarly performed by the DIO + EXE group. After this procedure, the animals were submitted to 25 min of swimming with intensities corresponding to 4.5%, 5.0%, 5.5%, and 6.0% of their body weight. The swimming intensities were controlled by lead fish fixed to the tail of the animals and were performed randomly with a 48-h resting interval.
Blood samples were obtained from a small incision at the tip of the tail in rest and at the 5th, 10th, 15th, 20th, and 25th minutes of each swimming intensity. Blood lactate concentrations were assayed by a portable lactate analyzer (Accutrend Lactate; Roche Diagnostics, Mannheim, Germany). The MLSS was considered as the highest exercise intensity at which the increase in lactacidemia was 1 mM or lower from the 10th to the 25th minute.
Exercise protocol 2 (DIO + MLSS).
The animals were adapted to the swimming exercise as the DIO + EXE rats and swam in groups of three for 40 min at 70% of the MLSS intensity in a 60-cm-depth × 45-cm-diameter cylindrical tank with water temperature maintained at ∼34°C.
Sacrifice of animals.
On the basis of other investigations by our research group (24,29), 16 h after the exercise protocols, the rats were anesthetized with an intraperitoneal injection of sodium thiopental (40 mg·kg−1 body weight). In all experiments, the appropriateness of anesthesia depth was tested by evaluating pedal and corneal reflexes throughout the experimental procedure. After the experimental procedures, the rats were killed under anesthesia (thiopental 200 mg·kg−1) following the recommendations of the National Institutes of Health publication 85-23.
Insulin tolerance test and serum insulin determination.
Sixteen hours after the exercise protocols (24,29), the rats were submitted to an insulin tolerance test (ITT; 9.0 mL·kg−1 of a solution 10−6 mol·L−1 of insulin). Briefly, 9.0 mL·kg−1 of a solution 10−6 mol·L−1 of human recombinant insulin (Humulin R) from Eli Lilly (Indianapolis, IN) was injected (intraperitoneally) in anesthetized rats; thereafter, the blood samples were collected from the tail at 0, 5, 10, 15, 20, 25, and 30 min for serum glucose determination.
The rate constant for serum glucose disappearance (KITT) was calculated using the formula: 0.693/t1/2. The serum glucose t1/2 was calculated from the slope of the least square analysis of the serum glucose concentration during the linear phase of decline (5). Serum glucose level was determined by the colorimetric method using a glucose meter (Advantage; Boehringer Mannheim, Indianapolis, IN). Serum was separated by centrifuging (1100g) for 15 min at 4°C and stored at −80°C until assay. Radioimmunoassay was used to measure serum insulin according to a previous description (35).
Protein analysis by immunoblotting.
As soon as anesthesia was ensured by the loss of pedal and corneal reflexes, the abdominal cavity was opened, the portal vein was exposed, and 0.2 mL of normal saline, with or without insulin (10−6 mol·L−1), was injected. In preliminary experiments, we determined that this dose of insulin could reach peripheral levels that are three to four times higher than the dose that could induce the maximal insulin effect on insulin signaling proteins in the muscle. At 90 s after insulin injection, both portions of gastrocnemius (red and white fibers) were ablated, pooled, minced coarsely, and homogenized immediately in an extraction buffer (1% Triton X-100, 100 mM Tris, pH 7.4, containing 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM PMSF, and 0.1 mg·mL−1 aprotinin) at 4°C with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Westbury, NY), operated at maximum speed for 30 s. The extracts were centrifuged at 9000g and 4°C in a Beckman 70.1 Ti rotor (Palo Alto, CA) for 40 min to remove insoluble material, and the supernatants of these homogenates were used for protein quantification using the Bradford method (7).
Proteins were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol (DTT), run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. These membranes were blocked, probed, and developed as previously described (21,31). The IRβ and IRS-1 were immunoprecipitated from rat muscle, with or without previous insulin infusion. Antibodies used for immunoblotting were antiphosphotyrosine (pY), anti-IR, anti-IRS-1, anti-protein kinase B (Akt), antiphospho [Ser473] Akt, anti-phospho-JNK, and anti-IκBα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antiphosphoserine-IRS-1307 and anti-PTP-1B were from Upstate Biotechnology (Charlottesville, VA), and anti-p85-phosphatidylinositol 3-kinase (PI3-K) was from Cell Signaling Technology (Beverly, MA). Blots were exposed to preflashed Kodak XAR film (Rochester, NY) with Cronex Lightning Plus intensifying screens (Sigma-Aldrich, St. Louis, MO) at 80°C for 12-48 h. Band intensities were quantified by optical densitometry (Scion Image Software; Scion Corp., Frederick, MD) of the developed autoradiographs.
PTP activity assay.
The gastrocnemius muscles were removed and homogenized in the solubilization buffer containing 20 mM Tris (pH 7.6), 5 mM EDTA, 2 mM PMSF, 1 mM EGTA, 130 mM NaCl, 0.1 mg·mL−1 of aprotinin, and 1% Triton X-100. The lysates were centrifuged (15,000g for 25 min at 4°C), and the supernatants were collected for immunoprecipitation, as previously described. Immunoprecipitates were washed in PTP assay buffer containing 100 mM HEPES (pH 7.6), 2 mM EDTA, 1 mM DTT, 150 mM NaCl, and 0.5 mg·mL−1 bovine serum albumin. The pp60c-src C-terminal phosphoregulatory peptide (TSTEPQpYQPGENL; Biomol, Farmingdale, NY) was added to a final concentration of 200 μM in a total reaction volume of 60 μL in a PTP assay buffer for the immunoprecipitation. The reaction was then allowed to proceed for 1 h at 30°C. At the end of the reaction, 40-μL aliquots were placed into a 96-well plate, 100 μL of Biomol Green reagent (Biomol) was added, and absorbance was measured at 630 nm (38).
Where appropriate, the results were expressed as the means ± SEM. Differences among the control, DIO, DIO + EXE, and DIO + MLSS groups were evaluated using one-way ANOVA. When ANOVA indicated significance, a Bonferroni post hoc test was performed.
Physiological and metabolic parameters.
Figure 1 shows comparative data regarding control, DIO, DIO + EXE, and DIO + MLSS groups 16 h after exercise. Rats fed on the high-fat diet for 12 wk (i.e., DIO, DIO + EXE, and DIO + MLSS) had a higher body weight, epididymal fat, and fasting serum insulin than age-matched controls. No significant variations were found in body weight, epididymal fat, and fasting serum insulin in DIO + EXE and DIO + MLSS rats compared with DIO rats. The fasting serum glucose concentrations were similar between the groups; however, the reduction in the glucose disappearance rate (KITT) observed in the DIO group was attenuated in both protocols of exercise in the same magnitude.
FIGURE 1-Characteris...Image Tools
MLSS in DIO rats.
Figure 2 shows the blood lactate concentrations in DIO rats when submitted to four different swimming intensities to identify MLSS. The animals presented stabilization of blood lactate concentrations from the 10th to the 25th minute of exercise with workloads of 4.5%, 5.0%, and 5.5% of their body weight. At 6.0% of body weight, the rats showed a progressive increase in their blood lactate concentrations. Thus, the MLSS corresponded to 5.5% of body weight with mean blood lactate values of 5.59 ± 0.61 mM.
FIGURE 2-Responses o...Image Tools
A single bout of exercise improves insulin signaling in the muscle of DIO rats.
The effect of in vivo intravenous insulin infusion on IR tyrosine phosphorylation was examined in the gastrocnemius muscles of control, DIO, DIO + EXE, and DIO + MLSS rats 16 h after the exercise protocols. The muscles were immunoprecipitated with anti-IR antibody and then blotted with antiphosphotyrosine antibody. Insulin induced an increase in IR tyrosine phosphorylation levels in the muscles of control, DIO, DIO + EXE, and DIO + MLSS rats. IR tyrosine phosphorylation decreased by 3.0-fold in the muscle of DIO rats when compared with the control group after insulin infusion. DIO + EXE and DIO + MLSS increased IR tyrosine phosphorylation by 2.2-fold when compared with DIO rats. No difference was found in IR phosphorylation between exercise protocols (Fig. 3A, upper panel). These results indicate that physical exercise improves insulin-induced IR tyrosine phosphorylation. There were no differences in the basal levels of IR tyrosine phosphorylation (i.e., C−, DIO−, DIO + EXE−, and DIO + MLSS−) or in IR protein levels between the groups (Fig. 3A, lower panel).
FIGURE 3-Insulin sig...Image Tools
After insulin administration, IRS-1 tyrosine phosphorylation decreased by 2.4-fold in the gastrocnemius muscle of DIO rats when compared with the control group after insulin infusion. DIO + EXE and DIO + MLSS increased IRS-1 tyrosine phosphorylation by 2.3- and 2.4-fold, respectively, when compared with DIO rats (Fig. 3B, upper panel). In addition, IRS-1/PI3-K association decreased by 2.2-fold in the muscle of DIO rats when compared with the control group after insulin infusion. DIO + EXE and DIO + MLSS increased IRS-1/PI3-K association by 1.9- and 1.8-fold, respectively, when compared with DIO rats (Fig. 3C). These results demonstrate that the physical exercise improves insulin-induced IRS-1 tyrosine phosphorylation and IRS-1/PI3-K association in the skeletal muscle of DIO rats. There were no differences in basal levels of IRS-1 tyrosine phosphorylation and IRS-1/PI3-K association (i.e., C−, DIO−, DIO + EXE−, and DIO + MLSS− in Figs. 3B and C) or in IRS-1 protein levels between the groups (Fig. 3B, lower panel).
The distal protein of insulin signaling, Akt, was also observed. Akt serine phosphorylation reduced by 2.3-fold in the muscle of DIO rats when compared with the control group after insulin infusion. DIO + EXE and DIO + MLSS increased Akt serine phosphorylation by 2.0- and 1.9-fold, respectively, when compared with DIO rats. No difference was found in Akt phosphorylation between exercise protocols (Fig. 3D, upper panel). These results indicate that physical activity improves the insulin-induced Akt serine phosphorylation in the muscle of DIO rats. There were no differences between the basal levels of Akt serine phosphorylation (i.e., C−, DIO−, DIO + EXE−, and DIO + MLSS−) or in Akt protein levels between the groups (Fig. 3D, lower panel).
A single bout of exercise inhibits JNK activity, IκBα degradation, and serine 307 phosphorylation of IRS-1 in DIO rats.
Next, we sought to determine the JNK serine phosphorylation in the gastrocnemius muscles of control, DIO, DIO + EXE, and DIO + MLSS rats at 16 h after the exercise protocols. JNK phosphorylation was dramatically increased by 4.1-fold in the gastrocnemius muscle of DIO rats when compared with the control animals. DIO + EXE and DIO + MLSS reduced JNK phosphorylation by 2.0- and 1.8-fold, respectively, when compared with DIO rats. No difference was found in JNK phosphorylation between the exercise protocols (Fig. 4A, upper panel). The JNK protein levels were not different between the groups (Fig. 4A, lower panel). Moreover, the DIO rats presented lower levels of IκBα protein expression (i.e., 2.7-fold) in comparison with the control animals. However, in the muscles of the DIO + EXE and DIO + MLSS animals, IκBα protein expression increased by 1.7- and 1.6-fold, respectively, when compared with DIO rats (Fig. 4B).
FIGURE 4-Inflammator...Image Tools
We also monitored the IRS-1 serine phosphorylation at residue serine 307 (Ser307) in the muscle of rats. The IRS-1 serine phosphorylation of the DIO rats increased by 6.4-fold compared with the control animals. However, the IRS-1 serine phosphorylation of the DIO + EXE and DIO + MLSS rats decreased by 1.9-fold when compared with DIO rats (Fig. 4C, upper panel). The IRS-1 protein levels did not differ between the groups (Fig. 4C, lower panel).
Acute exercise-mediated suppression of PTP1B activity in DIO rats.
Thereafter, we investigated the PTP1B activity and expression in the skeletal muscle at 16 h after the exercise protocols. The DIO animals increased the PTP1B activity and expression by 2.2- and 1.9-fold compared with the control rats; however, these responses were reversed in the muscles of DIO + EXE and DIO + MLSS groups in a similar fashion compared with the DIO rats (Figs. 5A and B).
FIGURE 5-Effect of t...Image Tools
Finally, we also monitored the IR/PTP1B association. We observed that the interaction of IR/PTP1B in the muscle of DIO rats was increased (i.e., 2.8-fold) when compared with the control group, and in the DIO + EXE and DIO + MLSS groups, IR/PTP1B association was decreased by 1.8- and 1.6-fold, respectively, when compared with DIO rats (Fig. 5C).
In the present investigation, we demonstrated that obesity induced by high-fat diet leads to an increase in the PTP1B protein level and in the activity and serine phosphorylation of IRS-1. Interestingly, an acute session of different protocols of exercise (DIO + EXE and DIO + MLSS) reversed the behavior of these parameters, concomitantly with the reduction in JNK activity and IκBα degradation and in insulin signaling in the skeletal muscle of obese rats.
In fact, these results were previously described (29); however, the applicability of the acute bout of exercise used by authors and by other studies from our laboratory (14,29) presented some limitations, mainly because of its extensive volume (i.e., 6 h), in obese humans. Thus, the main finding of this study is that an acute bout of exercise, characterized by higher intensity and lower volume (DIO + MLSS), leads to similar responses in the parameters analyzed.
As used in other investigations with humans (26) and rodents (13,16), we determined the MLSS to prescribe the intensity of the acute session of physical exercise. The MLSS is defined as the highest exercise intensity that presents a balance between the production and the removal of the blood lactate and is considered the gold standard protocol in the identification of the metabolic aerobic/anaerobic transition point during exercise (4).
Several reports have shown that the workload and the blood lactate concentration at MLSS, measured in sedentary rats during swimming exercise, are between 4.8% and 6.0% of the body weight and between 5.2 and 6.2 mM of the blood lactate concentration, respectively (10,11,16,22). Thus, the data regarding workload (i.e., 5.5% of body weight) and blood lactate concentration (i.e., 5.59 ± 0.61 mM) at MLSS of the present investigation are within the range of the studies mentioned in the previous paragraphs.
In the current investigation, we first expected that our DIO rats would present lower workload values compared with the sedentary rats fed on commercial chow, as evaluated in the other studies (10,11,16,22). However, we believe that the higher fat mass (i.e., epididymal fat) of our DIO rats allowed them improved floatation during the swimming exercise and, consequently, support similar workloads compared with nonobese Wistar rats. The improved floatation can be considered as a hypothesis to explain the similarity observed in the workloads at MLSS between our DIO rats and the sedentary rats (10,11,16,22).
After determining the intensity of exercise equivalent to MLSS, we compared the effects of two different acute exercise protocols on insulin signaling and on the main negative modulators of this pathway in the gastrocnemius of DIO rats. In the first acute session, the Wistar rats swam during two 3-h bouts, separated by a 45-min rest period. In the second protocol, the rodents swam during 45 min at 70% of the MLSS.
The DIO Wistar rats developed profound insulin resistance with impairment in the muscle insulin signaling compared with the control rats. However, the DIO rats presented high levels of insulin, which is considered one of the main characteristics of obesity, with no change in glucose levels on fasting condition. These results are in accordance with previous studies from our laboratory that used the same experimental model (24,29). The DIO Wistar rats seem to compensate for the increased metabolic load and obesity-induced insulin resistance by increasing insulin secretion from the pancreas, thus maintaining glucose levels similar to the control animals.
Conversely, it is well established that physical exercise can improve the insulin sensitivity of DIO rats. Recently, we and other authors have demonstrated that acute exercise reverses insulin resistance in obese rats in parallel with an improvement in insulin signaling (24,29,39). After acute or chronic exercise, insulin sensitivity is enhanced in insulin-sensitive tissues, such as skeletal muscle, adipose, liver, and hypothalamus (2,14,24,25,28,29). In the present investigation, the DIO rats showed decreased whole-body insulin sensitivity, as indicated by reduced glucose disappearance rate during an ITT. On the other hand, the DIO rats submitted to both acute exercise protocols presented improvements in whole-body insulin sensitivity, which was associated to improvement in insulin signaling in the skeletal muscle.
The serine phosphorylation of IRS proteins is considered to be the major suppression mechanism of IRS-1 activity that contributes to insulin resistance (32,33). Serine-phosphorylated IRS-1 might associate with the IR to block the autophosphorylation reaction; alternatively, the serine-phosphorylated IRS-1 might act indirectly on the IR through an association with an inhibitor that acts on the IR in a stoichiometric or catalytic fashion (20). Thus, the regulation of serine phosphorylation of IR and IRS-1 proteins is directly related to the molecular mechanism of insulin resistance. It is important to mention that independently from the acute exercise protocol (i.e., DIO + EXE and DIO + MLSS), although the IRS-1 serine phosphorylation was reduced, the IR autophosphorylation was increased in the DIO rats after 16 h. In this setting, these data indicate that a high-fat diet mediates insulin resistance, at least in part, by inducing IRS-1 serine phosphorylation and decreasing IRS-1 tyrosine phosphorylation. However, these alterations are inhibited by an acute exercise session.
According to Gao et al. (15), the inflammatory signaling activation (i.e., IκB/NFκB pathway) may also mediate IRS-1 serine phosphorylation. In the present investigation, we observed that the DIO + EXE and DIO + MLSS animals presented diminished IRS-1 Ser307 phosphorylation, concomitantly with increased IκBα. These results are in accordance with Schenk and Horowitz (34), who observed that a single session of exercise during 90 min (45 min of treadmill exercise followed immediately by 45 min of exercise on a cycle ergometer) at approximately 65% of V˙O2peak decreased phosphorylation and activation of JNK and increased abundance of IκBα in the skeletal muscle of insulin-resistant individuals. Interestingly, Sriwijitkamol et al. (37) showed that after 8 wk of aerobic exercise training (the exercise intensity, duration, and frequency were progressively increased up to 70% of V˙O2peak for 45 min four times per week), 50% increases in both IκBα and IκBβ proteins were observed, accompanied by a 40% decrease in tumor necrosis factor-α muscle content and a 37% increase in insulin-stimulated glucose disposal in subjects with type 2 diabetes.
In addition, several investigations have shown that JNK contributes to insulin resistance by phosphorylating IRS-1 at Ser307, and this phosphorylation leads to inhibition of the IRS-1 function (1,27,30). We observed that both acute bouts of exercise inhibited DIO-induced JNK activity and that this inhibition was accompanied by a reduction in IRS-1 serine phosphorylation at Ser307. In comparison, the activity of the JNK intracellular signaling cascade is increased after prolonged running exercise (6,40) but reduced after resistance exercise in older men (43).
Moreover, Myers et al. (23) established, at the molecular level, the ability of PTP1B to negatively regulate IR kinase activity. In addition, the ablation of the PTP1B gene yields mice displaying characteristics that suggest that inhibition of PTP1B function may be an effective strategy for the treatment of diabetes and obesity (12). However, independently from the acute exercise protocol, the PTP1B expression was diminished. This result was accompanied by an increase in the insulin sensitivity in skeletal muscle and was correlated with increases in tyrosyl phosphorylation of IR and IRS-1 and with the reduction in the association of IR-PTP1B in the skeletal muscle.
In conclusion, our study demonstrated that both protocols of exercise increased the insulin sensitivity and increased the insulin-stimulated IR and IRS-1 tyrosine phosphorylation and Akt serine phosphorylation in the muscle of DIO rats by the same magnitude. In parallel, both protocols of exercise also reduced PTP1B activity and IRS-1 serine phosphorylation, with concomitant reductions in JNK and IKK activities in the muscle of DIO rats, in a similar fashion. Thus, our data demonstrate that exercise of low intensity and high volume or exercise of moderate intensity and low volume represents different strategies to restore insulin sensitivity with the same efficacy.
Adelino Sanchez Ramos da Silva and José R. Pauli contributed equally to this work.
The authors thank the technical support provided by Luiz Janeri, Jósimo Pinheiro, and Márcio Alves da Cruz.
This study received financial support from the Fundaão de Amparo a Pesquisa do Estado de São Paulo (process numbers 2006/06961-9 and 2006/06960-2).
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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