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INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS |
University of Melbourne (S.K., S.Z., A.W.T., M.E.D., J.P., S.A.), Department of Medicine, Royal Melbourne Hospital, and Walter and Eliza Hall Institute (R.D., T.W.K.), Parkville, Victoria 3050, Australia
Address all correspondence and requests for reprints to: Sofianos Andrikopoulos, Ph.D., University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. E-mail: . sof{at}unimelb.edu.au
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Abstract |
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Introduction |
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It has been suggested that insulin hypersecretion may cause islet failure via chronic overstimulation and exhaustion of the islet ß-cell. For example, chronic overstimulation-induced impairment in islet function, as a result of a 48-h glucose infusion (6) or a 90% pancreatectomy in rats (7), was prevented by the treatment with the insulin secretion inhibitor diazoxide. Furthermore, treatment of patients with type 2 diabetes with diazoxide or somatostatin resulted in improved glucagon, and tolbutamide induced insulin secretion (8) and restored insulin pulsatility and the insulin/proinsulin ratio (9). These studies suggest a causal link between enhanced and impaired insulin secretion and imply that reducing insulin secretion may be beneficial to islet function in diabetes.
The DBA/2 and related C57BL/KsJ mouse strain, which carries 16% of the DBA/2 genome (10), have a genetic predisposition to pancreatic islet failure. Islet function is grossly impaired in DBA/2 and C57BL/KsJ mice made severely obese and insulin resistant by the expression of the db/db gene (which encodes for a defective leptin receptor molecule) with the absence of both first- and second-phase insulin secretion. Initially, expression of the db/db gene on the DBA/2 or C57BL/KsJ background results in hyperinsulinemia and normoglycemia, followed by a period of declining hyperinsulinemia and hyperglycemia. Eventually, overt hyperglycemia develops in this model as a consequence of decreased insulin production from a reduction in the islet ß-cell population (11, 12, 13). In contrast, leptin deficiency on a C57BL/6 genetic background results in marked obesity and insulin resistance, with only a mild hyperglycemia and hyperinsulinemia. Furthermore, when islets from C57BL/KsJ mice are exposed to a high glucose environment by intrasplenic transplantation in syngeneic donors treated with streptozotocin, they show a depressed rate of cell proliferation and are eliminated within 12 d (14). On the other hand, islets transplanted into normoglycemic donors are retained. Moreover, chronic incubation of DBA/2 mouse islets with glucose concentrations greater than 11.1 mM caused diminished glucose-induced insulin secretion (15). Thus, compared with the resilient C57BL/6 strain, the DBA/2 mouse strain displays susceptibility to pancreatic islet failure when stressed with insulin resistance or high glucose. However, no studies have been published comparing insulin secretory function between the susceptible DBA/2 and control C57BL/6 mouse strains in the absence of the deleterious effects of obesity, insulin resistance, or hyperglycemia. The aim of this study was to determine insulin secretory function in DBA/2 and C57BL/6 control mice, in the absence of any stress. Knowing the secretory behavior in the absence of stress may provide us with information as to why the DBA/2 strain is susceptible and the C57BL/6 is resistant to high glucose-induced ß-cell failure.
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Materials and Methods |
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Animals
Male DBA/2 and C57BL/6 mice were purchased from the Walter and Eliza Hall Institute Animal Research Facility (Kew, Victoria, Australia) and housed in the Department of Medicine Animal Research Facility under a 12-h light, 12-h dark cycle, with a standard laboratory nonpurified diet containing 77% of energy as carbohydrate, 20% protein, and 3% of calories as fat (Barastock Products, Pakenham, Australia), provided ad libitum unless otherwise stated. Animals were studied either at 1 d or at 4 or 10 wk of age, and all procedures described below were approved by the Royal Melbourne Hospital Animal Research Ethics Committee.
Glucose tolerance test
Animals were studied at 0900 h, after an overnight fast of 17 h (food withdrawn at 1600 h the previous day), and were anesthetized with an ip injection (100 mg/kg) of sodium pentobarbitone (Nembutal; Rhone Merieux, Queensland, Australia). A SILASTIC brand catheter (0.012-inch inside diameter, 0.025-inch outside diameter; Dow Corning Corp., Midland, MI), filled with heparinized saline (20 U/ml), was inserted into the right jugular vein, and the animals were allowed to recover from the surgery for 20 min. Animals were kept warm with a heat lamp, and body temperature was monitored using a rectal probe. A bolus of glucose (1 g/kg) was injected through the jugular vein, and 200 µl blood was sampled (through the retroorbital sinus at 0, 2, 5, 10, 15, and 30 min) for plasma glucose and insulin analysis. Blood was immediately centrifuged, the plasma separated, and the red blood cells resuspended in an equal volume of heparinized saline and reinfused into the animal via the jugular vein to prevent anemic shock.
Atropine administration was used to test the effect of blocking parasympathetic nervous system activity on insulin secretion. Mice were anesthetized, and a jugular catheter was inserted as described above, and were injected sc with either normal saline or 10 mg/kg body weight of atropine methyl-nitrate dissolved in normal saline, as previously described (16). After 20 min, an iv glucose tolerance test (IVGTT) was performed as described above.
Insulin secretion in 1-d-old mice was assessed by injection of an ip bolus of glucose (1 g/kg). Blood was collected into EDTA-coated tubes, by decapitation, at 0, 5, or 20 min after the glucose bolus.
Insulin tolerance test
Mice were anesthetized with an ip injection of sodium pentobarbitone (100 mg/kg). Thirty minutes after the induction of anesthesia, a bolus of insulin (1 IU or 0.5 IU/kg body weight; Actrapid; Novo Nordisk Pharma Ltd., North Rocks North Ryde, New South Wales, Australia) was administered ip, and blood was drawn from the retroorbital sinus at 0, 15, 30, 45, and 60 min, using heparinized capillary tubes. Plasma glucose was measured on a Precision Q.I.D. glucometer (MediSense Australia Pty. Ltd., Victoria, Australia).
Islet isolation and culture
Pancreatic islets were isolated from 10-wk-old DBA/2 and C57BL/6 mice, by collagenase digestion, using a modified method of Lacy and Kostianovsky (17) and Gotoh et al. (18). Islets were isolated, using a Ficoll gradient, and hand-picked under a stereomicroscope. Islets were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C in humidified air, 5% CO2.
Insulin secretion assay
Islets were washed twice in Krebs Ringer bicarbonate buffer (KRB) (111 mM NaCl, 4.8 mM KCl, 2.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, pH 7.4), 10 mM HEPES, 2.8 mM glucose, 0.2% BSA fraction V (Sigma, St. Louis, MO), and were preincubated in the same buffer for 90 min at 37 C in humidified air 5%, CO2. Triplicate batches of five islets were transferred to borosilicate tubes containing 1 ml KRB supplemented with glucose concentrations from 2.8–20 mM, and incubated for 60 min at 37 C. Insulin secretion in response to 275 µM tolbutamide and 10 mM methyl-pyruvate was performed over 60 min, in the presence of 2.8 mM glucose. Secretion in response to 2 mM or 10 mM 2-ketoisocaproate was performed over 60 min in the absence of glucose. The medium was collected after gentle centrifugation and stored at -20 C for measurement of insulin.
Glucose utilization
Glucose utilization was determined by measuring the formation of 3H2O from D-[5-3H] glucose (19, 20). Islets were preincubated in KRB (as in the insulin secretion studies) for 30 min before groups of 10 islets were incubated in 40 µl fresh KRB containing 20 mM glucose and 2 µCi D-[5-3H] glucose. After 90 min incubation with gentle shaking at 37 C, 100 µl 1-M HCl was added, to stop the reaction, and incubated for 20 h at 37 C to trap the released 3H2O. The trapped 3H2O and remaining D-[5-3H] glucose in the media were determined by scintillation counting (LS-3000; Beckman Coulter, Inc., Fullerton, CA).
Measurement of glucose phosphorylating activity
Approximately 300 islets were washed with 2.8 mM KRB twice and incubated for another 90 min. Islets were homogenized in ice-cold buffer containing 20 mM K2HPO4, 1 mM EDTA, 5 mM dithiothreitol, and 110 mM KCl. The islet homogenate was then centrifuged at 12,000 x g for 10 min. The supernatant was used for glucokinase and hexokinase determination by a spectrophotometric assay (21). The reaction volume contained 100 µl islet supernatant in 500 µl 50-mM HEPES/HCl, pH 7.6, 100 mM KCl, 7.4 mM MgCl2, 15 mM ß-mercaptoethanol, 0.50 mM NAD+, 0.05% BSA, 0.70 U/ml glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, and 5 mM ATP. The reaction was performed for 90 min, and absorbance was measured at 340 nm. The maximal velocity of glucokinase was calculated by subtracting the maximal velocity of hexokinase, which was determined at a glucose concentration of 0.5 mM, from the total phosphorylating activity, measured at 100 mM glucose.
Glucokinase, hexokinase, and GLUT2 immunoblot analysis
Immunoblot analyses were performed to quantify the glucokinase, hexokinase, and GLUT2 protein level. Approximately 400–500 islets were cultured in 11.1 mM glucose for 24 h. Islets were sonicated, for 20 min at 4 C, in a lysis buffer containing 5% SDS, 80 mM Tris/HCl, pH 6.8, 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 10 µg deoxyribonuclease I, and 0.2 mM N-ethylmaleimide. The lysate was retrieved after centrifugation at 12,000 x g for 10 min at 4 C. Protein content was measured by protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Samples containing 20 µg protein were resolved by electrophoresis through a 10% (for glucokinase and GLUT2) or 7.5% (for hexokinase) polyacrylamide gel. After transfer to polyvinylidene difluoride membrane, glucokinase was immunodetected using a specific antiglucokinase antibody (catalog no. sc-7908; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:500 dilution, followed by incubation with swine antirabbit IgG at 1:2,500 dilution. Hexokinase was immunodetected using a specific antihexokinase antibody (catalog no. sc-6517; Santa Cruz Biotechnology, Inc.) at a 1:500 dilution, followed by incubation with rabbit antigoat IgG at 1:2,500 dilution. GLUT2 was immunodetected using a specific anti-GLUT2 antibody (catalog no. sc-9117; Santa Cruz Biotechnology, Inc.) at a 1:2000 dilution, followed by incubation with swine antirabbit IgG at 1:2,500 dilution. Protein bands were visualized by enhanced chemiluminescence (Renaissance; NEN Life Science Products, Boston, MA) and quantified using scanning densitometry.
Islet insulin content
At the conclusion of the secretion experiments, islets were disrupted by vortexing in acid/ethanol, followed by a freeze-thaw cycle at -20 C and sonication for 20 min in an ice-bath. The lysate was centrifuged at 15,000 rpm for 10 min, and the supernatant was assayed for insulin. Insulin content was calculated as the sum of insulin in the lysate and the media from the secretion experiments.
Pancreatic islet immunohistochemistry
Immunohistochemistry on sections from pancreata was performed as previously described (22). Briefly, pancreata from three DBA/2 and three C57BL/6 mice were excised, fixed in Bouin’s solution, and embedded in paraffin. Sections (5-µm) were treated with 3% hydrogen peroxide in methanol, for 5 min, to block endogenous peroxidase, and blocked with 10% FCS and 2% milk powder in PBS for 20 min. The sections were then treated with primary antibody (guinea-pig antiinsulin or rabbit antiglucagon or rabbit antisomatostatin; DAKO Corp., Santa Barbara, CA) for 30 min, followed by a 10-min wash in PBS, followed with incubation with the appropriate horseradish peroxidase conjugated secondary antibody and developed with 3,3'-diaminobenzidine tetrahydrochloride (Sigma) for 4 min and counterstained with hematoxylin.
Pancreatic islet area determination
Islets from DBA/2 and C57BL/6 mice were isolated as described above and photographed using a Nikon (Tokyo, Japan) FX-35DX camera attached to a Nikon DIAPHOT light microscope. Planar surface area was calculated by measuring the diameter of photographed islets, on a superimposed micron scale using the formula 
r2.
Insulin and glucose measurements
Insulin in plasma and cell media was measured using a rat-specific RIA kit (Linco Research, Inc., El Paso, IL). Plasma glucose was measured using a glucose analyzer (YSI, Inc., Yellow Springs, OH).
Statistical analysis
Data are presented as mean ± SE for the number indicated. Area under the glucose curve (AUCglucose) was determined using the trapezoidal rule. Statistical analysis was performed using the nonparametric Mann-Whitney U test. P < 0.05 was considered significant.
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Results |
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. Fasting plasma glucose was lower in DBA/2, compared with C57BL/6, mice (7.1 ± 0.4 vs. 11.3 ± 0.8 mM, P < 0.05), whereas there was no difference in fasting plasma insulin levels (32.3 ± 11.4 vs. 19.8 ± 5.7 mU/liter in DBA/2 and C57BL/6, respectively). Two minutes after administration of the glucose bolus, plasma insulin levels increased 10-fold in DBA/2 mice, compared with only a 3-fold increase in C57BL/6 mice (P < 0.005, Fig. 1). Thereafter, plasma insulin levels remained significantly higher in DBA/2, compared with C57BL/6, mice. As a result, AUCglucose was significantly lower in the DBA/2 mice (654 ± 73.1 vs. 845 ± 46.7 mM x 30 min, P < 0.05).
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. There was no difference in plasma insulin levels in the presence of atropine in either DBA/2 or C57BL/6 mice, compared with the untreated mice, suggesting that the enhanced secretory rate in DBA/2 mice was not mediated by increased parasympathetic outflow.
To determine whether insulin hypersecretion was an early event, secretion was assessed in 4-wk-old mice and the results shown in Fig. 2. Fasting plasma glucose levels were lower in DBA/2, compared with C57BL/6, mice (7.0 ± 0.3 vs. 10.7 ± 1.1 mM, P < 0.05), whereas plasma insulin levels were not different (45.8 ± 5.6 vs. 32.1 ± 10.9 mU/liter in DBA/2, compared with C57BL/6, respectively). After the glucose bolus, plasma insulin levels were significantly higher at the 2-, 5-, and 10-min time points in DBA/2, compared with C57BL/6, mice. Consequently, plasma glucose levels tended to be lower in DBA/2 mice, with significance attained at the 10- and 15-min time points, post glucose administration (Fig. 2). AUCglucose was also decreased in DBA/2, compared with C57BL/6, mice (579.7 ± 26.0 vs. 678.0 ± 19.5 mM x 30 min, P < 0.05).
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. Basal plasma insulin levels were comparable between the two strains of mice. DBA/2 pups displayed higher plasma insulin levels 5 min after an ip glucose bolus, compared with C57BL/6 pups, indicating that insulin hypersecretion is an early event in the DBA/2 mouse.
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. The fall in plasma glucose levels, in response to both insulin doses, was the same between the two strains of mice, illustrating equal insulin sensitivity (Fig. 4). This and the decreased AUCglucose during the IVGTTs supported the notion that DBA/2 mice were not insulin resistant. Therefore, insulin hypersecretion in DBA/2 mice does not seem to be associated with insulin resistance. In DBA/2 (compared with C57BL/6) mice, at 10 wk of age, fed plasma insulin levels were higher (148.8 ± 11.5 vs. 50.2 ± 5.6 mU/liter, respectively, n = 7, P < 0.05), and fed plasma glucose levels were appropriately lower (10.4 ± 0.5 vs. 12.1 ± 0.4 mM, respectively, n = 7, P < 0.05). Furthermore, insulin hypersecretion in DBA/2 mice was not associated with increased body weight because, at 10 wk of age, body weights were not different between DBA/2 and C57BL/6 mice (24.7 ± 0.4 vs. 25.3 ± 0.6 g, n = 8, respectively).
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. As with the in vivo glucose challenge, isolated islets from DBA/2 mice secreted significantly more insulin in response to a range of glucose concentrations (2.8–20 mM), compared with control C57BL/6 mouse islets (Fig. 5). Islet insulin content was not different between DBA/2 and C57BL/6 mice (2874 ± 538 vs. 3253 ± 559 µU/5 islets, n = 6).
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), indicating normal K+ATP channel and postchannel activity. Insulin secretion in response to methyl-pyruvate was not different between DBA/2 and C57BL/6 mouse islets (Fig. 6A), suggesting that oxidation was not enhanced in DBA/2 mice. However, because it has recently been proposed that methyl-pyruvate can have extramitochondrial effects to induce membrane depolarization (23), insulin secretion was assessed using 2 mM and 10 mM of the mitochondrial substrate 2-ketoisocaproate in the absence of glucose, and the results are shown in Fig. 6B. Insulin secretion at 2 mM 2-ketoisocaproate was the same between the two strains of mice, consistent with the findings of methyl-pyruvate. However, at 10 mM 2-ketoisocaproate, insulin secretion was significantly higher in DBA/2 (compared with C57BL/6) islets, suggesting enhanced glucose oxidation.
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). To find whether the increase in glucose utilization was attributable to enhanced glucose phosphorylation, the activity of both glucokinase and hexokinase was measured. The maximal activity of both glucokinase and hexokinase was increased in DBA/2, compared with C57BL/6, mouse islets (Table 1).
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). There was no difference in either glucokinase or hexokinase protein levels between DBA/2 and C57BL/6 mouse islets (Fig. 7).
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. A mouse liver lysate was used as an internal control and showed an approximately 60-kDa band corresponding to GLUT2. There was a significant increase in GLUT2 protein levels in DBA/2, compared with C57BL/6, mouse islets (Fig. 8).
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, there was no difference between the two strains of mice. Pancreatic islet hormone immunostaining was performed in pancreata from three DBA/2 and three C57BL/6 mice. In agreement with the islet insulin content data and with a recent study (24), no difference was observed in immunostaining for insulin, glucagon, and somatostatin between DBA/2 and C57BL/6 mice (data not shown). Collectively, this data suggests that insulin hypersecretion from DBA/2 mice is not attributable to increased islet size or insulin abundance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report, we show, for the first time, that DBA/2 mice, known to have a predisposition to islet failure, secrete more insulin, both in vivo and in vitro, in response to a glucose challenge (compared with C57BL/6 mice, which are known not to exhibit this susceptibility). This is not attributable to insulin resistance as assessed using the insulin tolerance test. Although the insulin tolerance test is a crude measure of insulin sensitivity and may not have detected insulin resistance in DBA/2 mice, we believe this is unlikely because, at both high and low doses of insulin, the sensitivity in both strains was the same. Moreover, lower fed, fasted, and postchallenge plasma glucose levels also support the notion that insulin sensitivity is normal in DBA/2 mice. We further demonstrate that the higher insulin secretory rate is associated with an accelerated rate of glucose utilization as a result of increased levels of glucokinase and hexokinase activity and enhanced glucose oxidation.
Insulin hypersecretion has also been shown in other animal models of diabetes, including the Zucker (fa/fa) rat (31) and the ventromedial-hypothalamus lesioned rat (32). In fact glucose-mediated insulin hypersecretion is a very early defect in ventromedial-hypothalamus-lesioned animals, occurring within minutes (33). Interestingly, the hypersecretion in these models is mediated via the parasympathetic nervous system, because ligation of the vagus nerves or prior treatment with atropine is able to ameliorate this increase in insulin levels (34, 35, 36). In contrast, this does not seem to be the case in the DBA/2 mouse, because atropine, used at a dose that was previously shown to be effective in the fa/fa rat (16), had no effect in the DBA/2 mouse. This further suggests that, in the DBA/2 mouse, the increase in glucose-induced insulin secretion is by a mechanism intrinsic to the ß-cell.
Increased glucose-mediated insulin release in the DBA/2 mouse was associated with enhanced glucose utilization. This was contributed to by both increased glycolysis and glucose oxidation, as measured by tracer techniques and methyl-pyruvate and 2-ketoisocaproate-mediated insulin release. Our results with the D-[5-3H]-glucose tracer and methyl-pyruvate suggest that insulin hypersecretion in the DBA/2 mouse was associated with increased glycolysis, whereas the use of 2-ketoisocaproate suggested that enhanced glucose oxidation may also contribute to this phenomenon.
Enhanced glucose utilization in DBA/2 mouse islets was associated with increased glucokinase and hexokinase activities. This contributes to insulin hypersecretion at high (20 mM) as well as low (2.8 mM) glucose concentrations from DBA/2 mouse islets. Similar mechanisms have been described in islets from other models of hypersecretion, such as the 20% glucose-infused rat (37), the spontaneously hypertensive rat (SHR) (38), the pregnant rat (39), and partially pancreatectomized mice and rats (40, 41, 42). In all the above cases, except for the SHR, an increase in the catalytic activity of glucokinase and hexokinase was detected, and it explained the increase in insulin secretion at both low and high media glucose concentrations. In the SHR, only the glucokinase activity was up-regulated (there was no change in hexokinase activity), resulting in a leftward shift of the glucose-induced insulin secretion curve, such that the ED50 decreased from 9.6 ± 0.5 mM in the control rats to 6.6 ± 1.0 mM in the SHR, without a change in secretion at low media glucose concentrations (38).
Interestingly, in the present study, we found that glucokinase and hexokinase protein levels were not higher in DBA/2, compared with C57BL/6, mouse islets, despite increased activities of these enzymes. An increase in the activity, but not protein levels, of glucokinase and hexokinase has also been observed in other hyperinsulinemic models mentioned above (37, 38, 39, 40). There are a number of possible explanations for an increase in glucokinase activity without a concomitant increase in protein levels. A study by Noma et al. (43) showed that islet glucokinase was present in a perinuclear area and was translocated in response to glucose to the cytoplasm, where it may exist in a more active state. Similarly, Tiedge et al. (44) showed that, in an immortal ß-cell line, glucose could release glucokinase from a protein-bound inactive state to a diffusible state with high activity. Furthermore, a recent report suggested that the precursor of propionyl-CoA carboxylase ß-subunit could bind to ß-cell glucokinase and increase its activity by 23% (45). Finally, it has been suggested that glucokinase activity can be elevated in the presence of increased GLUT2 protein levels via protein-protein coupling and that this interaction could generate secretory signals that require glucose metabolism (46, 47). It is therefore plausible that the higher GLUT2 protein levels in the DBA/2 mouse islet result in enhanced glucokinase activity and, consequently, increased glucose mediated insulin release.
Although, in this study, we show increased GLUT2 protein levels, we do not believe that this is the cause of insulin hypersecretion in the DBA/2 mouse, for the following reasons: GLUT2 has a high Michaelis-Menten constant for glucose, of approximately 15 mM, which is in the high physiological range and therefore would not be expected to be rate-limiting to consequent glucose utilization. In support of this, it was shown in rat ß-cells that glucose transport can provide a metabolic flux that is two orders of magnitude higher than the actual glycolytic rate and that glucose phosphorylation, not transport, was correlated with glucose sensitivity (48). In addition, glucose was able to stimulate insulin secretion from mouse islets that had a complete absence of GLUT2 (49).
In summary, in this report, we show that the DBA/2 and C57BL/6 mouse strains, which have different susceptibility to islet failure, display different insulin secretory responses to glucose in the absence of any stress (such as insulin resistance or sustained high glucose levels). DBA/2 mice, which are known to develop islet failure, secreted increased amounts of insulin in response to a glucose challenge, from an early age, compared with the resilient C57BL/6 strain. Hypersecretion seems to be secondary to increased glycolytic flux caused by increased levels in islet glucokinase and hexokinase activity and glucose oxidation. It remains to be determined whether the mechanisms responsible for increased hypersecretion are linked to the susceptibility of DBA/2 islets to fail when confronted with insulin resistance.
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Acknowledgments |
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Footnotes |
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Abbreviations: AUCglucose, Area under the glucose curve; IVGTT, iv glucose tolerance test; KRB, Krebs Ringer bicarbonate buffer; SHR, spontaneously hypertensive rat.
Received December 5, 2001.
Accepted for publication February 22, 2002.
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References |
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action on pancreatic beta cells causes class I MHC up-regulation but not diabetes. J Clin Invest 102:1249–1257[Medline]
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) or atropine (
) in DBA/2 mice and saline (
) or atropine (
) in C57BL/6 mice. Animals were 10 wk of age at the time of study. Results are presented as mean ± SE (n = 8–10). *, P < 0.05, compared with C57BL/6 mice.
