Journal of Applied Physiology Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 103: 1764-1771, 2007. First published August 30, 2007; doi:10.1152/japplphysiol.00434.2007
8750-7587/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
103/5/1764    most recent
00434.2007v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, S.
Right arrow Articles by Sung, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, S.
Right arrow Articles by Sung, S. R.

Exercise improves glucose homeostasis that has been impaired by a high-fat diet by potentiating pancreatic β-cell function and mass through IRS2 in diabetic rats

Sunmin Park, Sang Mee Hong, Ji Eun Lee, and So Ra Sung

Department of Food and Nutrition, College of Natural Science, Institutes of Basic Sciences, Hoseo University, Asan-Si, Korea

Submitted 20 April 2007 ; accepted in final form 27 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we investigated the effects of a high-fat diet and exercise on pancreatic β-cell function and mass and its molecular mechanism in 90% pancreatectomized male rats. The pancreatectomized diabetic rats were given control diets (20% energy) or a high-fat (HF) diet (45% energy) for 12 wk. Half of each group was given regular exercise on an uphill treadmill at 20 m/min for 30 min 5 days/wk. HF diet lowered first-phase insulin secretion with glucose loading, whereas exercise training reversed this decrease. However, second-phase insulin secretion did not differ among the groups. Exercise increased pancreatic β-cell mass. This resulted from stimulated β-cell proliferation and reduced apoptosis, which is associated with potentiated insulin or IGF-I signaling through insulin receptor substrate-2 (IRS2) induction. Although the HF diet resulted in decreased proliferation and accelerated apoptosis by weakened insulin and IGF-I signaling from reduction of IRS2 protein, β-cell mass was maintained in HF rats just as much as in control rats via increased individual β-cell size and neogenesis from precursor cells. Consistent with the results of β-cell proliferation, pancreas duodenal homeobox-1 expression increased in the islets of rats in the exercise groups, and it was reduced the most in rats fed the HF diet. In conclusion, exercise combined with a moderate fat diet is a good way to maximize β-cell function and mass through IRS2 induction to alleviate the diabetic condition. This study suggests that dietary fat contents and exercise modulate β-cell function and mass to overcome insulin resistance in two different pathways.

dietary fats; exercise; islet; insulin secretion; insulin signaling; insulin receptor substrate-2

THE MAJOR CHARACTERISTICS of Type 2 diabetes are insulin resistance and insulin deficiency (6). People with insulin resistance do not develop diabetes if they have a sufficient insulin secretion capacity to compensate for insulin resistance. This is related to the recent marked increase in the prevalence of Type 2 diabetes. Asians, including Koreans, do not produce sufficient insulin secretion when insulin resistance is elevated by the increased consumption of fat and simple sugars associated with Western dietary habits (3, 34, 35). In addition, Asian Type 2 diabetic patients are not obese, and their serum insulin concentrations remain at or below the normal range (23). This indicates that nonobese Asians have insufficient insulin secretion capacity when insulin resistance is induced, making them susceptible to Type 2 diabetes. Thus, sufficient insulin secretion capacity contributes to a decrease in the prevalence and progression of Type 2 diabetes.

Furthermore, recent studies on experimental animals have shown that the failure of insulin secretion causes the development of Type 2 diabetes, and this is associated with decreased β-cell expansion through reduced β-cell proliferation and increased apoptosis (37, 39). However, the temporary and modest elevation of insulin resistance has been shown to lead to an expansion of pancreatic β-cells (15, 30, 37), which occurs as a result of hypertrophy and the neogenesis of precursor cells, such as ductal cells (15). Conversely, prolonged and severe insulin resistance has been shown to decrease the proliferation of β-cells as a result of attenuated insulin or IGF-I signaling. Consequently, β-cell expansion with increased insulin resistance cannot be sustained for long periods (37). Thus it is crucial to increase β-cell expansion with hyperplasia to prevent the prevalence and progression of diabetes.

The regulation of β-cell proliferation is complicated and must be coordinated with peripheral and central insulin sensitivity to maintain optimal glucose homeostasis (10, 37). The proliferation is activated by insulin and/or IGF-I signaling of β-cells. Because insulin signaling is constitutively active in β-cells as a result of insulin being secreted when islets are incubated in high-glucose media, IGF-I administration is needed to make insulin and/or IGF-I signaling optimal to study the effects of a high-fat diet and exercise on the signaling. Our previous study (26) showed that basal phosphorylation of insulin receptor substrate-2 (IRS2), Akt, and forkhead transcription factor (FKHR; a member of Forkhead box O subfamily) existed in islets when an islet was incubated in high-glucose media. Additional stimulation of IGF-I potentiated insulin and/or IGF-I signaling by ~25–30%.

Diets high in fat have been well known to increase insulin resistance, which results in the development and/or exacerbation of Type 2 diabetes (19, 32). A high-fat diet [>40 energy percent (E%) from fat] causes hyperinsulinemia with the expansion of islets to compensate for insulin resistance, and this in turn creates transitory normoglycemia (10, 31). However, the consumption of high-fat diets over long terms leads to insufficient insulin release due to β-cell senescence and eventually induces Type 2 diabetes (31); however, the proper consumption of dietary fat may maintain optimum insulin secretion with sufficient β-cell mass. Thus diabetes develops when insulin secretory function fails in a state of severe insulin resistance (6, 10, 38). In addition to high-fat diets (40–50 E% fat), the consumption of low-fat diets (10 E% fat) results in low insulin secretion capacity and pancreatic β-cell mass with high insulin sensitivity, indicating the increased possibility of diabetic prevalence and progression when insulin resistance is elevated (27). A moderate fat diet (20–25 E% fat) is effective in preventing the prevalence and progression of diabetes by improving insulin secretion and action.

Exercise is known to increase insulin sensitivity under normal conditions and to ameliorate impaired insulin action in insulin-resistant humans and animals (28). However, the effect of physical training on β-cell function and mass in patients with Type 2 diabetes has not drawn much attention. Some studies have revealed that regular exercise decreases insulin secretion by insulin secretagogues (7, 18), but others have demonstrated that long-term exercise enhances glucose-stimulated insulin secretion in humans and experimental animals with Type 2 diabetes (8, 9). Therefore, we investigated the effect of dietary fat and exercise on insulin secretion capacity and pancreatic β-cell mass in Sprague-Dawley 90% pancreatectomized (Px) male diabetic rats. In addition, we determined the molecular mechanism to modulate pancreatic β-cell function and mass by modifying insulin and/or IGF-I signaling in the islets of Px diabetic rats. Px rats display mild and nonobese Type 2 diabetes with insulin resistance and insulin deficiency.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and diets.   All surgical and experimental procedures were performed in accordance with the recommendations found in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Hoseo University, Korea.

Male Sprague-Dawley rats, weighing 262 ± 32 g, were housed individually in stainless steel cages in a controlled environment with a constant temperature of 23°C and with alternating 12-h periods of light and darkness. We used the technique of Hosokawa et al. (14) to achieve the 90% pancreatectomy in rats or used a sham pancreatectomy (sham group). After 1 wk of recovery from the surgery procedure, the Px rats included in the experiments showed characteristics of mild diabetes mellitus with random fed serum glucose levels of 9.4–11.8 mM. The sham rats showed no symptoms of diabetes.

One hundred Px diabetic rats were randomly assigned into four groups: 1) a control diet group (20 E% fat); 2) a control diet plus exercise group (EX); 3) a high-fat diet (45 E% fat) group (HF); and 4) a high-fat diet plus exercise group (HFEX). The 20 sham rats were provided with a 20 E% fat diet (the control diet). Semipurified diets for each group were modified based on the AIN-93 formulation for experimental diets (4). The control and HF diet compositions are shown in Table 1. All rats were allowed free access to their respective diets and water for 12 wk. Rats in the EX and HFEX groups ran uphill (15° degree) on a small animal treadmill at 20 m/min for 30 min, 5 days/wk. Serum glucose levels were measured in rats subjected to overnight fasting, and food intake and body weight were measured weekly every Tuesday at 10:00 AM.


View this table:
[in this window]
[in a new window]

  		Table 1. Diet composition

 
Glucose-stimulated insulin secretion.   At the 11th wk of treatment, catheters were surgically implanted into the right carotid artery and left jugular vein of 10 rats from each group, after they were anesthetized with intraperitoneal injections of a ketamine-xylazine mixture (100 mg and 10 mg/kg body wt, respectively). After 5–6 days of implantation, a hyperglycemic clamp was performed in conscious and overnight fasted rats to determine insulin secretion capacity as described in previous studies (2, 25). After clamp, rats were freely provided with food and water for 2 days. The next day, rats were deprived of food for 16 h. The rats were anesthetized with the ketamine-xylazine mixture, and 100 nM insulin was injected through the inferior vena cava of the rats. Rats were killed by decapitation 15 min later. Tissues were rapidly dissected, weighed, frozen in liquid nitrogen, and stored at –70°C until further analyses could be performed.

Serum glucose levels were analyzed with a Glucose Analyzer II (Beckman, Palo Alto, CA). Serum insulin and leptin levels were measured by RIA (Linco Research, St. Charles, MO). At 12 wk, the degree of insulin resistance was estimated by the homeostasis model assessment of insulin resistance (HOMAIR) as described by Matthews et al. (22). Briefly, HOMAIR was calculated by taking into account fasting serum insulin and glucose levels according to the equation HOMAIR = fasting serum insulin (µU/ml) x fasting serum glucose (mmol/l)/22.5. Low HOMAIR values denote normal insulin sensitivity, whereas high values denote insulin resistance. HOMAIR is not the best index for peripheral insulin resistance in insulin-deficient animals such as Px rats. Our previous study (26) revealed that HOMAIR was sufficient to reflect peripheral insulin resistance corresponding to euglycemic hyperinsulinemic clamp in Px rats, although it underestimated insulin resistant states.

Pancreas perfusion.   Five Px rats from each group underwent fasting for 15 h and were anesthetized intraperitoneally with the ketamine-xylazine mixture. The abdominal aorta and portal vein were cannulated for perfusion and sample collection, respectively. The perfusate was composed of Krebs-Ringer bicarbonate buffer containing 3% dextran (Pharmacia Biotech) and 1% fatty acid-free BSA (Sigma) and equilibrated to a pH of 7.4 with 95% O2-5% CO2. Glucose (5 mM) in perfusate buffer was infused for 20 min to stabilize the pancreas in the condition of in situ perfusion. The pancreas was sequentially perfused at 1 ml/min with buffer containing 5 mM glucose (10 min), 19.4 mM glucose (20 min), 5 mM glucose (15 min), and 19.4 mM glucose plus 20 mM arginine (20 min). The perfusion eluate was collected every minute, and the insulin content in each fraction was measured by RIA (Linco). Before or after the perfusion protocol, the pancreas was excised and homogenized, and insulin was extracted by the acid-ethanol protocol (25). The insulin concentration of the pooled supernatant was measured by RIA.

Islet isolation.   Pancreatic islets from 9 or 10 rats of each group were isolated by collagenase digestion at the end of the 12-wk treatment, as described in previous studies (2, 12). Through the pancreatic duct, 3 ml of 1.0 mg/ml collagenase P (Roche) in DMEM-high glucose were injected into the pancreas of rats anesthetized with the ketamine-xylazine mixture. After several steps of isolation, the islets were pooled from 2 or 3 rats from each group. Before islets were lysed, they were administered with 10 nM IGF-I for 10 min to determine insulin/IGF-I signaling cascade.

Immunohistochemistry and islet morphometry.   Five rats from each group were treated with 5-bromo-2-deoxyuridine (BrdU; Roche Molecular Biochemicals, Indianapolis, IN; 100 µg/kg body wt) at the end of the 12-wk experimental period. Six hours postinjection, pancreas samples were prepared and analyzed as previously described (12). After the pancreas was fixed in a 4% paraformaldehyde solution (pH 7.2) and embedded in paraffin blocks, serial 5-µm paraffin-embedded tissue sections were mounted on slides. To prevent the selection of sections with the same islet twice, after rehydration, every sixth or seventh section was selected to determine β-cell area, BrdU incorporation, and apoptosis. The randomly chosen sections were immunostained as previously described (12).

Endocrine β-cells were identified by applying a guinea pig anti-insulin antibody in paraffin-embedded pancreatic sections. β-Cell proliferation was examined by the incorporation of BrdU in β-cells from rats injected with BrdU. This incorporation was determined by performing a double-label immunohistochemistry with anti-insulin (Zymed Laboratories, South San Francisco, CA) and anti-BrdU antibodies (Roche Molecular Biochemicals) on rehydrated paraffin sections. Apoptosis of β-cells was measured with the terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling kit (Roche, Mannheim, Germany) in paraffin sections of the pancreas and counterstained with hematoxylin and eosin to visualize islets (2). Quantification of β-cell area, BrdU-positive cells, and apoptotic body in islets was performed as explained in our group's previous study (2).

RNA isolation and RT-PCR.   Total RNA was isolated from the islets of rats from each group using a monophasic solution of phenol and guanidine isothiocyanate (Trizol reagent; GIBCO BRL, Rockville, MD), followed by extraction and precipitation with isopropyl alcohol. Reverse transcription was performed with superscript III RT, and PCR was performed with high-fidelity Taq DNA polymerase (Invitrogen, Grand Island, NY). The following primers were used to detect rat IRS2, pancreas duodenal homeobox-1 (PDX-1), and cyclophilin proteins: 5'-CCACCTTCTCTGGCAGTTCAG-3' forward and 5'-AAGGGTTGTAGGCCACTTTGG-3' reverse for IRS2, 5'-AGGAAAACAAGAGGACCCGTACT-3' forward and 5'-CGGGAGATGTATTTGTTAAATAAGAATTC-3' reverse for PDX-1, and 5'-CAGACGCCACTGTCGCTTT-3' forward and 5'-TGTCTTTGGAACTTTGTCTGCAA-3' reverse for cyclophilin. Primers were designed to include at least one intron to distinguish the products derived from mRNA and genomic DNA. These experiments were repeated three times for each group.

Immunoblot analysis.   Isolated islets were lysed in 20 mM Tris buffer (pH 7.4) containing 2 mM EDTA, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, and 12 mM {alpha}-glycerol phosphate and protease inhibitors. After 30 min on ice, the lysates were centrifuged for 10 min at 12,000 rpm at 4°C. After protein contents were measured in lysate with a Bio-Rad protein assay kit, equal amounts of protein (30–50 µg) were resolved by SDS-PAGE, and immunoblotting was performed with specific antibodies against IRS1, IRS2 (UBI, Waltham, MA), protein kinase B (Akt; Cell Signaling Technology, Beverly, MA), Ser473 phosphorylation of protein kinase B (Cell Signaling Technology), PDX-1 protein (Santa Cruz Biotechnology, Santa Cruz, CA), glucokinase, GLUT2, and β-actin (Santa Cruz Biotechnology) as previously described (2). The intensity of protein expression was determined with Imagequant TL (Amersham Biosciences, Piscataway, NJ). These experiments were repeated four times for each group.

Statistical analysis.   All results are expressed as means ± SD. Statistical analysis was performed with the SAS statistical analysis program. Significance of dietary fat contents and exercise was determined by two-way ANOVA. Differences among groups with a P < 0.05 were considered statistically significant (per Tukey's test). Differences between Px rats fed with 20 E% fat diets and sham rats were determined by two-sample t-test (at P < 0.05).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Metabolic characteristics.   At the end of the experimental periods, body weight gain was lower in Px diabetic rats than in sham normal rats. However, epididymal fat pads, representing visceral fats, were higher in Px rats (Table 2). Px rats had a greater caloric intake than Sham rats as a result of their diabetes-induced hyperphagia. As we expected, the HF diet resulted in increased body weight in Px rats compared with that shown in control rats, but exercise did not affect body weight (Table 2). The difference in body weight was explained by the caloric intake of Px rats. Epididymal fat pad weight was also higher in the Px rats fed the HF diet. However, the fat pad weight decreased with exercise in both diets. The HF group had considerably increased serum glucose levels for the first 5 wk; however, after that, the increase was minimal (Fig. 1). However, exercise suppressed their elevation rates. Although serum glucose levels were maintained within a normal range in both Px and Sham rats, serum glucose levels taken from Px rats that had fasted overnight were higher than those from sham rats (Table 2). By contrast, fasted serum insulin levels were not significantly different in Px and sham rats. In Px rats, a HF diet resulted in increased fasting serum glucose and insulin levels, whereas exercise reduced these (Table 2). As expected, HOMAIR was higher in Px rats than in sham rats. In addition, in Px rats, HF increased HOMAIR and exercise decreased it (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2. Metabolic parameters at the end of experimental periods

 

Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. Changes of overnight fasting serum glucose levels during experimental periods. During the experimental periods, serum glucose levels were measured weekly in pancreatectomized (Px) diabetic rats on Tuesdays at 10:00 AM after an overnight fast. The sample size in each group was the same as in Table 2, and values are means ± SD. Con, Px diabetic rats given the control diet; HF, Px diabetic rats given the high-fat diet; EX, Px diabetic rats given the control diet + exercise; HFEX, Px diabetic rats given the high-fat diet + exercise; Sham, rats that underwent sham surgery + control diet. *Significant main effect of dietary fats at P < 0.05 by 2-way ANOVA. {dagger}Significant main effect of exercise at P < 0.05 by 2-way ANOVA. {ddagger}Significantly different from Con group at P < 0.05 by 2-sample t-test.

 
Insulin secretion capacity.   Although the hyperglycemic clamp procedure was performed to maintain serum glucose levels at ~12 mM (Table 3), serum insulin levels peaked at 2–5 min and then declined to a low at 10 min, when glucose levels remained elevated and stable in all rats (Fig. 2). This is known as first-phase insulin secretion. Because serum insulin exhibit an ascending curve at 60–120 min, second-phase insulin secretion was defined as the average of serum insulin levels at 60, 90, and 120 min (Fig. 2). Serum insulin levels in the first and second phases represent insulin secretion capacity (2, 14). The serum insulin levels of Px rats were ~52–71% of those in sham rats during first- and second-phase insulin secretion under hyperglycemic clamp (Table 3). The HF diet resulted in attenuated first-phase insulin secretion, whereas exercise reversed this process (Table 3). However, dietary fat contents and exercise did not affect second-phase insulin secretion as we defined it. The area under the curve of insulin markedly increased in Px rats fed the HF diet due to a significant elevation from 10 to 60 min during hyperglycemic clamp. It was then normalized with exercise (Table 3). Furthermore, HF did not cause insulin secretion to significantly fall at 10 min when first-phase insulin secretion ended, whereas HF markedly decreased serum insulin levels at 120 min during hyperglycemic clamp (Fig. 2). This suggests that HF slackened control of insulin secretion causing insufficient insulin secretion when blood glucose was elevated for a long period.


View this table:
[in this window]
[in a new window]

  		Table 3. Insulin secretion capacity during hyperglycemic clamp

 

Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 2. Insulin secretion during hyperglycemic clamp. A hyperglycemic clamp was performed on rats subjected to overnight fasting to determine insulin secretion at the end of experimental periods. During the hyperglycemic clamp procedure to maintain blood glucose levels at 100 mg/dl above fasting levels, serum insulin levels were measured in Con, HF, EX, and HFEX groups of rats. The sample size in each group was the same as in Table 3, and results are expressed as means ± SD. *Significant main effect of dietary fats at P < 0.05 by 2-way ANOVA. {dagger}Significant main effect of exercise at P < 0.05 by 2-way ANOVA. {ddagger}Significantly different from Con group at P < 0.05 by 2-sample t-test.

 
Glucose infusion rates to maintain serum glucose levels 5.5 mM above the baseline were higher by 184% in sham rats than Px diabetic rats, indicating pancreatectomy lessened β-cell function (Table 3). In addition, insulin sensitivity in the hyperglycemic state was found to be lower in sham rats than in Px rats (Table 3). Px rats in the HF group had further decreased glucose infusion rates and insulin sensitivity at hyperglycemic clamp, whereas exercise reversed these traits (Table 3). Thus pancreatectomy attenuated β-cell function and insulin sensitivity at hyperglycemia, and HF impairs this function and exacerbates insulin sensitivity.

In situ pancreatic perfusion.   Along with hyperglycemic clamp, we used in situ pancreatic perfusion to investigate the effects of exercise and HF on insulin secretion in Px rats. Px rats had ~70% less insulin secretion than sham rats when insulin secretion was stimulated with 19.4 mM glucose and 20 mM arginine (Fig. 3A). With the infusion of 5 mM glucose solution, insulin secretion remained at baseline in Px and sham rats; the HF group, however, secreted insulin more than the other Px rat groups (Fig. 3A). In contrast, 19.4 mM glucose solution immediately stimulated insulin secretion in all rats (Fig. 3A). Similar to results obtained in our hyperglycemic clamp study, pancreas perfusion showed the typical pattern of insulin secretion in the first and second phases (Fig. 3A). The increase in first-phase insulin secretion in the HF group was smaller than the increase in the control group; however, the HF group demonstrated an increased second-phase insulin secretion similar to the control group (Fig. 3A). Further stimulation of insulin secretion with glucose plus arginine did not induce insulin secretion in the HF group as much as the other groups (Fig. 3A).


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 3. Glucose- and arginine-stimulated insulin secretion and pancreatic insulin contents at in situ pancreatic perfusion. A: insulin secretion during in situ pancreatic perfusion. During pancreatic perfusion, the pancreas was sequentially perfused at 1 ml/min with Krebs-Ringer bicarbonate buffer plus 3% dextran containing 5 mM glucose (10 min), 16.7 mM glucose (20 min), 5 mM glucose (15 min), and 16.7 mM glucose + 20 mM arginine (20 min). The perfusion eluate was collected every minute, and the insulin content in every minute perfusate was measured by radioimmunoassay in 4 rats in all groups. B: pancreatic insulin content before and after pancreatic perfusion. The insulin content was determined in acid-ethanol extractions of total pancreas from rats from each group before and after pancreatic perfusion protocol. Experiments were repeated in 5 rats from each group, and results are expressed as means ± SD. *Significant main effect of dietary fats at P < 0.05 by 2-way ANOVA. {dagger}Significant main effect of exercise at P < 0.05 by 2-way ANOVA. {ddagger}Significantly different from Con group at P < 0.05 by 2-sample t-test. a,b,cMeans with different superscript letters were different at P < 0.05 by Tukey's test.

 
Exercise had a biphagic effect on insulin secretion. Exercise augmented first-phase insulin secretion at 19.4 mM glucose, but it did not stimulate second-phase insulin secretion (Fig. 3A). In addition, exercise suppressed the basal insulin secretion at 5 mM and second-phase insulin secretion response at 19.4 mM glucose in Px rats fed HF diet (Fig. 3A). Before the perfusion, exercise increased pancreatic insulin contents in both diets, and dietary fat contents did not affect the contents, which was consistent with β-cell mass measured by islet morphometry (Fig. 3B). However, after the perfusion protocol, the HF group showed decreased insulin contents of the pancreas compared with the control group (Fig. 3B). This indicated that Px diabetic rats fed HF diets had reduced insulin secretion as a result of glucose and arginine treatment. This decrease was reversed and restored by exercise (Fig. 3B). These results also support the hypothesis that HF leads to an impairment of the tight regulation of insulin secretion and to a reduction in its secretion capacity. It also affirms the contention that exercise restored this regulation and capacity.

Pancreatic β-cell mass.   The percentage of the pancreatic β-cell area in the total pancreas area of a section was higher in Px rats than in sham rats, since β-cells were rapidly regenerated after pancreatectomy (Table 4). In Px rats, the percentage of the β-cell area was increased by exercise in both diets. This increase was associated with an elevated β-cell number (hyperplasia) resulting from enhanced proliferation and reduced apoptosis. HF maintained the β-cell area by enlarging individual β-cell size, even though HF reduced the overall β-cell number due to decreased proliferation and elevated apoptosis (Table 4). Hypertrophy stemming from HF was caused by increased insulin resistance. Furthermore, both the control and HF groups had generated small β-cell clusters, indicating the onset of new-borne cells from precursor cells, called neogenesis. However, fewer small β-cell clusters were found in the EX and HFEX groups regardless of dietary fats (Table 4). This may be related to the rapid proliferation of β-cells from new small β-cell clusters. Pancreatic β-cell mass, calculated by multiplying the β-cell area by the pancreas weight, was higher in sham rats than in Px rats because of the difference in pancreas weight (Table 4). Because there was no difference in pancreas weight among the groups of Px rats, β-cell mass corresponded to the percentage of the β-cell area in Px rats fed HF and subjected to exercise training (Table 4). If β-cell mass is sufficiently increased by hyperplasia, this can adequately compensate for insulin resistance over a long period. Accordingly, exercise may exhibit a better insulin secretion pattern in hyperglycemic clamp and in situ pancreas perfusion.


View this table:
[in this window]
[in a new window]

  		Table 4. The modulation of islet morphometry

 
Insulin and/or IGF-I signaling in islets.   Dietary fat contents did not alter mRNA levels of IRS2, as shown by real-time PCR, but exercise increased them (Fig. 4). Consistent with the changes in transcription levels, exercise augmented IRS2 protein levels, but HF reduced them greatly, suggesting that HF induced IRS2 degradation (Fig. 5). Because IRS2 protein levels increased, serine phosphorylation of Akt was potentiated with exercise and attenuated because of HF conditions (Fig. 5). This reveals that the changes in IRS2 protein levels were on a level similar to the phosphorylation of Akt. As a result of activating IRS2-Akt signaling, exercise increased the mRNA and protein levels of PDX-1, a transcription factor associated with β-cell growth, whereas the HF diet led to a decrease in the mRNA and protein levels of PDX-1 (Figs. 4 and 5). In addition to the increased expression of PDX-1, exercise increased the expression of glucokinase and GLUT2 and HF diet decreased it (Fig. 5). Because glucokinase and GLUT2 have been known to regulate glucose-stimulated insulin secretion, this explains the loss in the tight regulation of insulin secretion by HF and its restoration through exercise.


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. mRNA levels of insulin receptor substrate-2 (IRS2), pancreas duodenal homeobox-1 (PDX-1), and cyclophilin in islets. Islets were isolated from Con, HF, EX, HFEX, and Sham groups of rats at the end of 12 wk of treatment. AU, arbitrary unit. Total RNA extracted from islets was reverse transcribed and used for PCR using primer sets for full-length of IRS2, PDX-1, and cyclophilin proteins. These experiments were repeated 4 times, and results are expressed as means ± SD. *Significant main effect of dietary fats at P < 0.05 by 2-way ANOVA. {dagger}Significant main effect of exercise at P < 0.05 by 2-way ANOVA. a,b,c,dMeans with different letters are significantly different at P < 0.05 by Tukey's test. {ddagger}Significantly different from Con group at P < 0.05 by 2-sample t-test.

 

Figure 5
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 5. Insulin and/or IGF-I signaling cascade and PDX-1 associated with β-cell mass in islets. Islets were isolated from CON, HF, EX, HFEX, and Sham rats at the end of 12 wk of treatment. Islets were lysed with a lysis buffer, and an equal amount of protein was used for immunoblotting (IB) analysis. The expression of other proteins was determined with specific antibodies by immunoblotting analysis only. These experiments were repeated 4 times.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Many studies have demonstrated that exercise improves glucose homeostasis by enhancing glucose uptake in skeletal muscle and adipose tissues (1, 5, 13). In addition, recent studies have reported that exercise training enhanced hepatic insulin signaling by suppressing hepatic glucose output during hyperinsulinemic clamp states (11, 29). However, not many studies have shown that exercise modulates pancreatic β-cell function and mass in the Type 2 diabetic animal model. To study the antidiabetic effects of HF diet and exercise on pancreatic β-cell function and mass of nonobese diabetic animal models, we chose Px rats, which have characteristics of Type 2 diabetes associated with insulin deficiency and insulin resistance (2, 14). Our present study revealed that HF diet resulted in exacerbated glucose homeostasis by impairing β-cell function and mass via attenuation of insulin/IGF-I signaling in islets of Type 2 diabetic Px rats, whereas exercise training reversed this process. This phenomenon was associated with IRS2 protein levels in islets. The HF diet resulted in decreased IRS2 protein levels without an alteration in mRNA levels, whereas exercise resulted in increased protein levels by promoting mRNA induction. Although HF diet and exercise oppositely regulated insulin/IGF-I signaling, both resulted in increased β-cell mass. However, the mechanism that brought this about was independent. The HF diet resulted in temporarily expanded β-cell mass by hypertrophy and neogenesis, whereas exercise resulted in sustained expansion for a long period, since β-cell mass was elevated by hyperplasia via increased proliferation and decreased apoptosis.

It has been known for some time that the long-term effects of regular exercise are beneficial to patients with Type 2 diabetes (1, 11, 13, 29). Regular aerobic exercise reduces the amount of visceral fat mass and body weight without decreasing lean body mass, and it also ameliorates insulin sensitivity and blood glucose levels. As a result of regular exercise, not only insulin resistance but also glucose-stimulated insulin secretion play important roles in regulating glucose homeostasis (17, 36). However, the effects of exercise on insulin secretion have not been shown to be consistent. It has been well established in in vitro studies that excessive exercise leads to an inhibition of insulin secretion because of the stimulation of the {alpha}2-adrenergic receptor, which increases the presence of either norepinephrine or epinephrine (20, 36). In contrast, excessive exercise training results in increased glucose- and arginine-stimulated insulin secretion somewhat by desensitizing the inhibitory effect of norepinephrine on insulin secretion (8). Consistent with our results, Dela et al. (8) reported that physical training enhances the β-cell response to 18 and 25 mM glucose and arginine in patients with Type 2 diabetes with a moderate secretion capacity of serum C-peptide levels at hyperglycemic clamp. However, regular exercise did not improve β-cell function in patients with diabetes with a low secretory capacity (fasting C peptide = 0.5 pmol/ml; fasting insulin = 69 pM). This suggests that there needs to be some existing insulin secretory reserve capacity to improve β-cell function with regard to exercise training.

It has been known for some time that growth and survival of β-cells are associated with insulin/IGF-I signaling through IRS2 (12, 38) and that the potentiation of insulin/IGF-I signaling enhances hyperplasia but not hypertrophy. This was supported by some studies of gene-manipulated mice (38, 39). IRS2 knockout mice developed severe diabetes as a result of their attenuated glucose-stimulated insulin secretion capacity, which was due to their failure to maintain β-cell mass (38, 39). However, mice with IRS1 deletion did not develop hyperglycemia by improving insulin secretion capacity, even though they developed insulin resistance. In IRS1 knockout mice, IRS2 protein expression increased in islets, which was involved in the induction of β-cell hyperplasia (33, 39). In addition, this was supported by the strong evidence that hyperglycemia did not come about in IRS2 whole body knockout mice with β-cell-specific IRS2 overexpression as a result of their promoting compensatory insulin secretion through expanding β-cell mass (2, 12, 25). This expansion of β-cell mass was due to increased β-cell number (hyperplasia) through proliferation and a reduction of apoptosis as a result of potentiated insulin/IGF-I signaling through IRS2 overexpression in β-cells (2, 12, 25). In addition to the results from gene-manipulated mice, several studies revealed that IRS2 can be induced by the elevation of intracellular cAMP in the liver, islets, and hypothalamus (16, 25). Exendin-4, a glucagon-like peptide-1 receptor agonist, and estrogen-activated cAMP-responding element binding protein led to increased IRS2 expression in the liver and pancreatic islets (16, 25).

The molecular mechanism that the potentiation of insulin/IGF-I signaling through IRS2 induction elevates PDX-1 expression in β-cells has been known for some time (12, 38). PDX-1 plays a crucial role in β-cell proliferation (12, 21). Elevated IRS2 protein potentiates tyrosine phosphorylation in β-cells, which activates Akt, downstream of IRS2 (12, 38). Ser473 phosphorylation of Akt activates Ser256 phosphorylation of FKHR. This phosphorylation of Akt and FKHR elevates PDX-1 expression. PDX-1 expression was remarkably reduced in islets of IRS2 whole body knockout mice through attenuation of insulin/IGF-I signaling, and β-cell-specific IRS2 overexpression reversed the reduction (12). Furthermore, β-cell-specific PDX-1 overexpression in IRS2 whole body knockout mice normalized serum glucose concentration with increased β-cell mass (21). In addition, Ser256 phosphorylation of FKHR contributes to preventing the apoptosis of β-cells, as FKHR plays a proapoptotic role by binding to DNA target sites in the nucleus (41). In the present study, exercise activated the phosphorylation of Akt and FKHR by increasing IRS2 induction in islets, which was associated with increased pancreatic β-cell mass. However, HF diet decreased IRS2 expression and also attenuated the tyrosine phosphorylation of IRS2, which led to a reduction in the phosphorylation of Akt and FKHR, whereas exercise overcame the HF diet effect on insulin/IGF-I signaling. Thus the hyperplasia of β-cells prevents the development and progression of diabetes, and the IRS2-PDX-1 pathway plays an important role in maintaining hyperplasia.

Unlike β-cell proliferation, β-cell neogenesis may not go through an insulin/IGF-I signaling pathway. In our present study, HF diet resulted in increased small β-cell clusters more than exercise training, indicating that HF diet induces the neogenesis of islets from precursor cells compared with moderate fat diets. However, newborn β-cells in the HF group did not grow as fast as those in the moderate fat diet group, where β-cell growth was generated by proliferation through the activation of insulin/IGF-I signaling. Although the hypothesis that insulin/IGF signaling may be enhanced by neogenesis is not excluded, the following results suggest that neogenesis is increased by insulin resistance. In addition, although the HF, EX, and HFEX groups had similar numbers of islets in certain areas, the HF group had smaller size of β-cell clusters than the exercise groups. Exercise potentiated insulin/IGF-I signaling in rats fed a HF diet and newborn β-cells proliferated faster than the nonexercised group. As a result, the number of small β-cell clusters was lower in the nonexercised group.

In conclusion, a HF diet increased peripheral insulin resistance, whereas exercise training reversed this condition. In addition to modulating insulin resistance, HF diet and exercise training affected β-cell function and mass. Both HF diet and exercise increased insulin secretion and β-cell mass; however, the mechanism of each was independent from the other. HF diet increased β-cell mass through hypertrophy and neogenesis as a compensatory mechanism against insulin resistance, whereas exercise elevated β-cell mass by promoting a state of hyperplasia. Hyperplasia resulted from increased β-cell proliferation and decreased apoptosis, which was brought on by potentiating insulin/IGF-I signaling by promoting IRS2 expression in islets. In contrast, HF diet attenuated insulin/IGF-I signaling in islets by decreasing IRS2 protein levels. Furthermore, HF diet delayed and attenuated first-phase insulin secretion with glucose loading, whereas it impaired second-phase insulin secretion, the regulation of which gets out of order. However, exercise training reversed these adverse effects of HF diet. Therefore, regular exercise improved glucose homeostasis not only by attenuating peripheral insulin resistance but also by enhancing β-cell function and mass.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the Korean Science and Engineering Foundation in Korea (R01-2006-000-10389-0).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Park, Dept. of Food and Nutrition, Hoseo Univ., 165 Sechul-Ri Baebang-Myun Asan-Si, Chungnam-Do, 336-795, Korea (e-mail: smpark{at}hoseo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Berggren JR, Hulver MW, Houmard JA. Fat as an endocrine organ: influence of exercise. J Appl Physiol 99: 757–764, 2005.[Abstract/Free Full Text]
  2. Choi SB, Jang JS, Park S. Estrogen and exercise may enhance beta-cell function and mass via IRS2 induction in ovariectomized diabetic rats. Endocrinology 146: 4786–4794, 2005.[Abstract/Free Full Text]
  3. Cockram CS. The epidemiology of diabetes mellitus in the Asia-Pacific region. Hong Kong Med J 6: 43–52, 2000.[Medline]
  4. Committee of the American Institute of Nutrition. Ad hoc committee on standards for nutritional studies. J Nutr 107: 1340–1348, 1997.
  5. Corcoran MP, Lamon-Fava S, Fielding RA. Skeletal muscle lipid deposition and insulin resistance: effect of dietary fatty acids and exercise. Am J Clin Nutr 85: 662–677, 2007.[Abstract/Free Full Text]
  6. DeFronzo RA, Bonadonna RC, Ferrannini E. Pathogenesis of NIDDM: a balanced overview. Diabetes Care 15: 318–353, 1992.[Abstract]
  7. Dela F, Mikines KJ, Tronier B, Galbo H. Diminished arginine-stimulated insulin secretion in trained men. J Appl Physiol 69: 261–267, 1990.[Abstract/Free Full Text]
  8. Dela F, von Linstow ME, Mikines KJ, Galbo H. Physical training may enhance beta-cell function in type 2 diabetes. Am J Physiol Endocrinol Metab 287: E1024–E1031, 2004.[Abstract/Free Full Text]
  9. Farrell PA, Caston AL, Rodd D. Changes in insulin response to glucose after exercise training in partially pancreatectomized rats. J Appl Physiol 70: 1563–1568, 1991.[Abstract/Free Full Text]
  10. Flier SN, Kulkarni RN, Kahn CR. Evidence for a circulating islet cell growth factor in insulin resistant states. Proc Natl Acad Sci USA 98: 7475–7480, 2001.[Abstract/Free Full Text]
  11. Heled Y, Shapiro Y, Shani Y, Moran DS, Langzam L, Barash V, Sampson SR, Meyerovitch J. Physical exercise enhances hepatic insulin signaling and inhibits phosphoenolpyruvate carboxykinase activity in diabetes-prone Psammomys obesus. Metabolism 53: 836–841, 2004.[CrossRef][Web of Science][Medline]
  12. Hennige AM, Burks DJ, Ozcan U, Kulkarni RN, Ye J, Park S, Schubert M, Fisher TL, Dow MA, Leshan R, Zakaria M, Mossa-Basha M, White MF. Upregulation of insulin receptor substrate-2 in pancreatic beta cells prevents diabetes. J Clin Invest 112: 1521–1532, 2003.[CrossRef][Web of Science][Medline]
  13. Holloszy JO. Exercise-induced increase in muscle insulin sensitivity. J Appl Physiol 99: 338–343, 2005.[Abstract/Free Full Text]
  14. Hosokawa YA, Hosokawa H, Chen C, Leahy JL. Mechanism of impaired glucose-potentiated insulin-secretion in diabetic 90 percent pancreatectomy rats. Study using glucagon-like peptide-1 (7-37). J Clin Invest 97: 180–186, 1996.[Web of Science][Medline]
  15. Jetton TL, Lausier J, LaRock K, Trotman WE, Larmie B, Habibovic A, Peshavaria M, Leahy JL. Mechanisms of compensatory beta-cell growth in insulin-resistant rats: roles of Akt kinase. Diabetes 54: 2294–2304, 2005.[Abstract/Free Full Text]
  16. Jhala US, Canettieri G, Screaton RA, Kulkarni RN, Krajewski S, Reed J, Walker J, Lin X, White MF, Montminy M. cAMP promotes pancreatic beta-cell survival via CREB-mediated induction of IRS2. Genes Dev 17: 1575–1580, 2003.[Abstract/Free Full Text]
  17. Kaastra B, Manders RJ, Van Breda E, Kies A, Jeukendrup AE, Keizer HA, Kuipers H, Van Loon LJ. Effects of increasing insulin secretion on acute postexercise blood glucose disposal. Med Sci Sports Exerc 38: 268–275, 2006.
  18. King DS, Staten MA, Kohrt WM, Dalsky GP, Elahi D, Holloszy JO. Insulin secretory capacity in endurance-trained and untrained young men. Am J Physiol Endocrinol Metab 259: E155–E161, 1990.[Abstract/Free Full Text]
  19. Kraegen EW, Clark PW, Jenkins AB, Daley EA, Chisholm DJ, Storlien LH. Development of muscle insulin resistance after liver insulin resistance in high fat fed rats. Diabetes 40: 1397–403, 1991.[Abstract]
  20. Kraemer WJ, Ratamess NA. Hormonal responses and adaptations to resistance exercise and training. Sports Med 35: 339–361, 2005.[CrossRef][Web of Science][Medline]
  21. Kushner JA, Ye J, Schubert M, Burks DJ, Dow MA, Flint CL, Dutta S, Wright CV, Montminy MR, White MF. Pdx1 restores beta cell function in Irs2 knockout mice. J Clin Invest 109: 1193–201, 2002.[CrossRef][Web of Science][Medline]
  22. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28: 412–419, 1985.[CrossRef][Web of Science][Medline]
  23. Min HK. Noninsulin-dependent diabetes mellitus (NIDDM) in Korea. Diabet Med 13: S13–S15, 1996.[Web of Science][Medline]
  24. Muzumdar R, Ma X, Atzmon G, Vuguin P, Yang X, Barzilai N. Decrease in glucose-stimulated insulin secretion with aging is independent of insulin action. Diabetes 53: 441–446, 2004.[Abstract/Free Full Text]
  25. Park S, Dong X, Fisher TL, Dunn S, Omer AK, Weir G, White MF. Exendin-4 promotes IRS2 signaling to mediate pancreatic beta-cell growth and function. J Biol Chem 281: 1159–1168, 2005.[CrossRef][Web of Science][Medline]
  26. Park S, Jang JS, Hong SM. Long-term consumption of caffeine improves glucose homeostasis by enhancing insulinotropic action through islet insulin/IGF-1 signaling in diabetic rats. Metabolism 56: 599–607, 2007.[CrossRef][Web of Science][Medline]
  27. Park S, Park CH, Hong SM. The effect of dietary fat on insulin secretion and pancreatic β-cell mass in 90% pancreatectomized diabetic rats. J Kr Soc Food Sci Nutri 36: 186–193, 2007.
  28. Perrini S, Henriksson J, Zierath JR, Widegren U. Exercise-induced protein Kinase C isoform-specific activation in human skeletal muscle. Diabetes 53: 21–24, 2004.[Abstract/Free Full Text]
  29. Perseghin G, Lattuada G, De Cobelli F, Ragogna F, Ntali G, Esposito A, Belloni E, Canu T, Terruzzi I, Scifo P, Del Maschio A, Luzi L. Habitual physical activity is associated with intrahepatic fat content in humans. Diabetes Care 30: 683–688, 2007.[Abstract/Free Full Text]
  30. Rooman I, Lardon J, Bouwen L. Gastrin stimulates β-cell neogenesis and increases islet mass from transdifferentiated but not from normal exocrine pancreas tissue. Diabetes 51: 686–690, 2002.[Abstract/Free Full Text]
  31. Sone H, Kagawa Y. Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced diabetic mice. Diabetologia 48: 58–67, 2005.[CrossRef][Web of Science][Medline]
  32. Storlien LH, Higgins JA, Thomas TC. Diet composition and insulin action in animal models. Br J Nutr 83: S85–S90, 2000.[Web of Science][Medline]
  33. Tamemoto H, Kadowaki T, Tobe K, Yagi T, Sakura H, Hayakawa T, Terauchi Y, Ueki K, Kaburagi Y, Satoh S, Kadowaki T. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372: 182–186, 1994.[CrossRef][Medline]
  34. The Division of National Nutrition Survey. 1993 National Nutrition Survey Report in Korea. Seoul, Korea: Ministry of Health and Welfare, 1996.
  35. The Expert Panel in Korean Medical Insurance Association. 1992 Annuals for Medical Insurance Statistics. Seoul, Korea: Ministry of Health and Welfare, 1995.
  36. Urano Y, Sakurai T, Ueda H, Ogasawara J, Sakurai T, Takei M, Izawa T. Desensitization of the inhibitory effect of norepinephrine on insulin secretion from pancreatic islets of exercise-trained rats. Metabolism 53: 1424–1432, 2004.[CrossRef][Web of Science][Medline]
  37. Weir GC, Bonner-Weir S. Five stages of evolving β-cell dysfunction during progression to diabetes. Diabetes 53: S16–S21, 2004.[Abstract/Free Full Text]
  38. Withers DJ, Burks DJ, Towery HH, Altamuro SL, Flint CL, White MF. Irs-2 coordinates Igf-1 receptor-mediated β-cell development and peripheral insulin signaling. Nat Genet 23: 32–40, 1999.[Web of Science][Medline]
  39. Withers DJ, Gutierrez JS, Towery H, Burks DJ, Ren JM, Previs S, Zhang Y, Bernal D, Pons S, Shulman GI, Bonner-Weir S, White MF. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391: 900–904, 1998.[CrossRef][Medline]
  40. Zhang X, Gan L, Pan H, Guo S, He X, Olson ST, Mesecar A, Adam S, Unterman TG. Phosphorylation of serine 256 suppresses transactivation by FKHR (FOXO1) by multiple mechanisms. J Biol Chem 277: 45276–45284, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. Rafacho, T. M. Cestari, S. R. Taboga, A. C. Boschero, and J. R. Bosqueiro
High doses of dexamethasone induce increased {beta}-cell proliferation in pancreatic rat islets
Am J Physiol Endocrinol Metab, April 1, 2009; 296(4): E681 - E689.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
103/5/1764    most recent
00434.2007v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, S.
Right arrow Articles by Sung, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, S.
Right arrow Articles by Sung, S. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.