Attenuation of age-related declines in glucagon-mediated signal transduction in rat liver by exercise training

Deborah A. Podolin1,2, Brandon K. Wills1, Ian O. Wood1, Mark Lopez3, Robert S. Mazzeo1, and David A. Roth1,3

1 University of Colorado, Boulder, Colorado 80303; 2 University of New England, Biddeford, Maine 04005; and 3 Collateral Therapeutics, San Diego, California 92121


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study investigated alterations in glucagon receptor-mediated signal transduction in rat livers from 7- to 25-mo-old animals and examined the effects of exercise training on ameliorating these changes. Sixty-six young (4 mo), middle-aged (12 mo), and old (22 mo) male Fischer 344 rats were divided into sedentary and trained (treadmill running) groups. Isolated hepatic membranes were combined with [125I-Tyr10]monoiodoglucagon and nine concentrations of glucagon to determine maximal binding capacity (Bmax) and dissociation constant (Kd). No alterations were found in Bmax among groups; however, middle-aged trained animals had significantly higher glucagon affinity (lower Kd; 21.1 ± 1.8 nM) than did their untrained counterparts (50.2 ± 7.1 nM). Second messenger studies were performed by measuring adenylyl cyclase (AC) specific activity under basal conditions and with four pharmacological stimulations to assess changes in receptor-dependent, G protein-dependent, and AC catalyst-dependent cAMP production. Age-related declines were observed in the old animals under all five conditions. Training resulted in increased cAMP production in the old animals when AC was directly stimulated by forskolin. Stimulatory G protein (Gs) content was reduced with age in the sedentary group; however, training offset this decline. We conclude that age-related declines in glucagon signaling capacity and responsiveness may be attributed, in part, to declines in intrinsic AC activity and changes in G protein [inhibitory G protein (Gi)/Gs] ratios. These age-related changes occur in the absence of alterations in glucagon receptor content and appear to involve both G protein- and AC-related changes. Endurance training was able to significantly offset these declines through restoration of the Gi/Gs ratio and AC activity.

adenylyl cyclase; hepatic membranes; GTP-binding proteins; gluconeogenesis; aging; glucose homeostasis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ONSET OF HYPOGLYCEMIA during sustained submaximal exercise is associated with the induction of fatigue and is due to a mismatch between carbohydrate supply (glucose from the gut, liver, and kidneys) and demand (glucose uptake by working muscle, heart, and brain). The increase in glucose production during exercise is achieved by a combination of hepatic glycogenolysis and gluconeogenesis. These two vital metabolic pathways are regulated by glucagon, norepinephrine, and epinephrine through specific glucagon and alpha - and beta -adrenergic receptors that stimulate intracellular signaling cascades to help maintain blood glucose homeostasis at rest, during fasting and exercise, and on recovery from exercise. Older humans and animals have impaired blood glucose regulation, which has been attributed to decreased glucose utilization by peripheral tissues (19), decreased glucose transport (20, 23), increased insulin resistance (7, 8, 15, 19), and decreased hepatic glucose production (30). Chronic dynamic exercise training has been shown to improve resistance to exercise-induced hypoglycemia, but the mechanisms of this adaptation are controversial. Because glucagon is the primary hormonal regulator of hepatic glucose output, this study sought to investigate the isolated and interactive effects of aging and training on glucagon signal transduction in rat liver.

The ability of glucagon to stimulate hepatic glycogenolysis has been found to either decrease (43) or remain unchanged (16) with increasing age. Aging has also been shown to dramatically impair hepatic gluconeogenic capacity; specifically, our laboratory and others (24, 31, 32) have found age-related declines in glucagon-stimulated hepatic gluconeogenesis. Previous investigations into the mechanism responsible for the aging effect in liver have concluded that, although glucagon-stimulated adenylyl cyclase (AC) activity has been observed to decline with age (5), there appears to be no alteration in glucagon-binding capacity or intrinsic AC activity with age (9).

Endurance training in humans and in animals has consistently resulted in the maintenance of higher blood glucose levels during endurance exercise (12, 44). This has been attributed to enhanced glucose production, a reduction in glucose utilization by the muscle, or both (6, 13, 42). It appears that training not only increases hepatic glycogenolytic (13) and gluconeogenic (25) capacities but may also preserve gluconeogenic function against age-related declines in glucose metabolism (31, 32). Although underlying mechanisms remain unclear, the activities of key gluconeogenic enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK), appear unchanged in response to training (22). Furthermore, training has not been found to affect AC activity, although a net increase in intracellular cAMP may result via a decrease in hepatic phosphodiesterase activity (11, 24).

It was our hypothesis that the mechanisms responsible for the age-related declines in hepatic glucose production, as well as the increased capacity for hepatic glucose output after chronic exercise training, occur as a result of alterations within the glucagon-stimulated signal transduction pathway at regulatory points downstream from the glucagon receptor. With advancing age, decreased responsiveness to the critical counterregulatory hormone glucagon could be due to a decline in stimulatory (Gs) and/or an increase in inhibitory (Gi) protein expression and function. Such changes would result in a net decrease in AC activity per hormone-bound receptor and in turn lead to lower intracellular cAMP concentrations. We predicted that exercise training would increase glucagon-stimulated AC activity across ages because of the relative increase in expression and/or function of Gs and decrease in expression and/or function of Gi, thus contributing to the previously observed enhanced glucoregulatory responsiveness. Therefore, the purpose of our study was to assess the effects of age and training on three potential sites within the glucagon-stimulated signal transduction pathway in the liver: 1) glucagon receptor density and affinity, 2) Gs and Gi protein expression and/or function, and 3) AC specific activity.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Sixty-six male Fischer 344 rats of ages 4, 12, and 22 mo were obtained through the National Institute on Aging (Harlan Sprague Dawley, Indianapolis, IN). Rats were housed in pairs at a temperature of 24°C with a 12:12-h light-dark cycle. Teklad rodent chow and water were provided ad libitum. Protocols were approved by an Institutional Animal Care and Use Committee.

Endurance training. After the 1-wk acclimatization period, all rats performed a graded exercise test on a motor-driven treadmill to determine maximal exercise capacity. Animals from each age group were then pair-matched on the basis of this initial screening and randomly assigned to either a control (sedentary) or an endurance-trained group. Endurance training was conducted as previously described (32). Briefly, training consisted of treadmill running 5 days/wk up a 15% grade for 60 min/day at a speed that elicited 75% maximal capacity (24, 19, and 13.5 m/min for the young, middle-aged, and old animals, respectively) as determined from age group averages from the initial graded exercise test. Age-matched sedentary controls ran 1 day/wk at 75% maximal capacity for 5 min to familiarize them with running and being handled. After the 10-wk training period, a second (final) graded exercise test was performed to determine posttraining maximal capacity. Three days after the final exercise test, endurance capacity was measured by a run to exhaustion at the speed initially eliciting 75% maximal capacity. Exhaustion was determined to be the inability to avoid the shock grid and loss of the righting reflex. After the animals were killed and tissues harvested, citrate synthase activity in homogenized soleus muscle was measured as a marker of training status [mitochondrial oxidative capacity (41)].

Experimental procedures. Three days after the last exercise bout, animals were fasted overnight and anesthetized (Nembutal, 60 mg/kg ip). The abdominal cavity was opened, and the left main lobe was excised and immediately frozen in liquid nitrogen and stored at -70°C for later analyses. Soleus was removed and frozen for citrate synthase analysis.

Liver membrane isolation. In preparation for 125I-labeled glucagon binding, a mixed membrane preparation consisting of the basolateral (sinusoidal) and apical (canalicular) aspects of the hepatocyte membrane was isolated as described by Neville (29). Briefly, 2 g of frozen liver were minced with scissors in an iced beaker and then homogenized at 4°C along with 10 ml of 1 mM phenylmethylsulfonyl fluoride (PMSF) in a large Dounce homogenizer. One milliliter was aliquoted to 6 cryovials and brought up to a 20:1 vol/wt ratio with PMSF. Liver mixture was filtered first through a two-layer cheesecloth and then a four-layered cloth and then was decanted and centrifuged in a Beckman JA-21 at 1,500 g for 10 min. The supernatant was homogenized with 5 ml of 50% sucrose, and the mixture was adjusted to 44%, determined by refractometer. Approximately 6 ml were poured into six centrifuge tubes and overlayed with 3 ml of 42% sucrose. Tubes were loaded into a prechilled swinging bucket rotor (SW-27.1 Beckman) and centrifuged at 98,000 g for 70 min at 4°C. Floating particulate was removed, mixed with 10 ml of PMSF, and pelleted out by centrifugation at 45,000 g for 30 min. The pellet was resuspended in 310 µl of isolation buffer (containing 15 mM Tris · HCl, 300 mM mannitol, 5 mM EGTA, and 1 mM PMSF, pH 7.4) and then aliquoted for protein and enzyme determination of binding capacity.

Preparation for SDS-PAGE and immunoblotting. For each rat, 1 ml of the initial homogenate was decanted into a prechilled minicentrifuge tube. Homogenates were centrifuged at 150,000 g for 15 min at 4°C. The supernatant (S150) was quick-frozen, and pellets (P150) were resuspended using a 22-gauge spinal needle with 950 µl of phosphate buffer (containing 10 mM KH2PO4, 5 mM MgCl2 · 6 H2O, 5 mM EDTA, 2 Na, 1 mM EGTA, and 1 mM PMSF). After resuspension, pellet fractions were quick-frozen and stored with the supernatants at -70°C.

Enzymatic analysis. Enzyme activity was measured in liver homogenates and membrane fractions for evidence of purity and recovery. Na-K-ATPase activity, a basolateral membrane marker, and Mg2+ ATPase, found in the apical plasma membrane, were measured using the modified procedure of Schoner et al. (39). Leucine aminopeptidase and alkaline phosphatase (apical markers) were measured using colorimetric procedures (4, 18). Cytochrome c reductase, a microsomal marker, and succinic dehydrogenase, an inner mitochondrial membrane marker, were measured according to established procedures (2). Protein content was determined by the method of Lowry et al. (27).

125I-glucagon binding. Glucagon receptor binding was measured in individual membrane preparations from each animal, in duplicate, by use of constant radiolabeled glucagon concentrations in the presence of varying concentrations of nonlabeled glucagon. Lyophilized [125I-Tyr10]monoiodoglucagon (2,000 Ci/mmol, Amersham) was dissolved in 1,250 µl of buffer containing 25 mM Na-HEPES (pH 8.0), 5 mM MgCl2, 1 mM EDTA, and 1% BSA to establish a 20 nM radiolabeled concentration. The 20 nM labeled glucagon was then divided into 25-µl aliquots and stored at -4°C for daily use. On each test day, enough labeled glucagon and 900 nM nonlabeled glucagon for each membrane were thawed on ice. Nonlabeled glucagon was serially diluted from 900 nM to produce nine concentrations: 0, 0.28, 0.835, 2.5, 7.5, 22.5, 45.0, 90.0, and 180.0 nM. Each reaction mixture was prepared in a prechilled polypropylene tube and contained 5 µl of buffer, 20 µl of labeled glucagon, 50 µl of varying concentrations of nonlabeled glucagon, and 25 µl of membrane (25 mg protein/ml). Reaction tubes were removed from ice and thoroughly mixed and then placed in a shaking water bath at 37°C for 50 min. These conditions were taken from the literature (9) and confirmed in our laboratory to ensure glucagon-receptor saturation and equilibrium. After incubation, 3 ml of phosphate buffer (25 mM Na-phosphate and 0.1% BSA, pH 8.0) were added to each tube to stop and maintain ligand-receptor binding equilibrium. Rapid vacuum-filtration binding (Cambridge), with filters (Amersham) presoaked in 10% BSA (for 1-2 h), was performed to minimize nonspecific binding to the filter. Immediately after membrane filtration, filters were rinsed with additional (6-10 ml) phosphate buffer, dried, and then removed with forceps and placed into polypropylene tubes and loaded into a Wizard gamma counter for radioactivity determination. Each curve was evaluated to determine the maximal binding capacity (Bmax) and dissociation constant (Kd) with a Hanes-Woolf plot (26).

Glucagon receptors, G proteins, and AC are associated with the basolateral (sinusoidal) plasma membrane of the intact hepatocyte (9, 10, 28). Because membrane preparations were a mixture of both basolateral and apical (canalicular) membranes, Bmax and Kd values representing glucagon binding of basolateral membrane only were calculated by the relative composition of the original membrane
% basolateral<IT>=</IT><FR><NU>Na-K-ATPase activity</NU><DE>(Na-K-ATPase activity<IT>+</IT>Mg-ATPase activity)</DE></FR>
where Na-K-ATPase activity represents the amount of basolateral membrane present, and Mg-ATPase activity represents the amount of apical membrane present (10, 28). The Bmax values were then divided by the percent basolateral contribution to yield the actual glucagon-binding capacity of the glucagon receptor found in the basolateral membrane.

Nonspecific binding was determined by using the value at the highest nonlabeled glucagon concentration (180 nM) to create a linear regression. This regression represented the points at which the binding curve becomes asymptotic, an appropriate method for determining nonspecific binding (26).

Second messenger studies. All biochemical analyses were performed on individual partially purified liver homogenates from each animal in every experimental group. Second messenger studies measured cAMP production in picomoles of cAMP formed per milligram of protein per minute under five conditions: basal (no exogenous stimulation), net stimulation (above each animal's respective basal stimulation) by 1.0 µM glucagon in the presence of 100 µM GTP, 100 µM Gpp(NH)p, 10 mM fluoride ion (AlF; 50 µM aluminum and 10 mM fluoride in water, see Ref. 33), and 200 µM forskolin (all final concentrations), by use of sequential column chromatography as described by Salomon et al. (37) and published previously by our group (21, 33-38). We found that cAMP production under these conditions was linear with respect to time and protein concentration and that IBMX (1.0 mM), adenosine deaminase (5 U/ml), or both in combination had no effect on basal or maximally stimulated cAMP production.

Quantification of Gsalpha and Gialpha 2 by immunoblotting. Assessment of the stimulatory and inhibitory alpha -subunits of the hepatic G proteins, Gsalpha and Gialpha 2, respectively, was conducted using standard SDS-PAGE and immunoblotting techniques, as previously described (21, 33-35). Briefly, all samples were electrophoresed, transferred to nitrocellulose membranes, incubated with purified polyclonal rabbit antisera primary antibodies (Calbiochem, La Jolla, CA), and then identified using goat anti-rabbit IgG conjugated to horseradish peroxidase (no. 13859-012; GIBCO-BRL Life Technologies, Gaithersburg, MD). To quantify hepatic Gsalpha and to ensure equal lane loading, purified fusion proteins were constructed as previously described (21), and both protein standards and sample bands at 45 kDa were quantified. Similar procedures were performed on the 39-kDa band for assessment of Gialpha 2. All gels were poured so that samples from each of the experimental groups were always electrophoresed on the same gels to ensure standardized Western blotting analyses. G proteins were visualized with chemiluminescent solutions A and B (Amersham, Alameda, CA). Image capture analysis and quantification were performed on a Molecular Dynamics Storm 840 (Sunnyvale, CA), and linear regression analysis was performed on the fusion proteins to quantify G protein content of all membrane samples.

Statistics. Data are expressed as mean values ± SE. All rats had successful biochemistry performed unless otherwise reflected in the sample sizes shown in Tables 1 and 2 and Figs. 1-3. Statistical analyses consisted of a two-factor ANOVA using GraphPad-Prism (San Diego, CA). A Fisher post hoc analysis was used in circumstances of significance for multiple comparisons between group means. The null hypothesis was rejected when P < 0.05. 

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Glucagon-binding characteristics in hepatic basolateral membrane fractions from young, middle-aged, and old trained and sedentary animals


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Adenylyl cyclase activity (cAMP production) in liver homogenates measured by four pharmacological stimulators in sedentary and trained rats across age groups



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Representative glucagon-binding curve and Hanes-Woolf plot (inset).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   A: autoradiograms of immunoblots. Purified polyclonal rabbit antisera primary antibodies were used to identify inhibitory G proteins (Gialpha 2). Ten micrograms of purified, mixed rat liver membranes were loaded per lane. Image capture analysis and quantification were performed on a Molecular Dynamics Storm 840, and linear regression analysis was performed on the fusion proteins to quantify G protein content of all membrane samples. YS, young sedentary; YT, young trained; MS, middle-aged sedentary; MT, middle-aged trained; OS, old sedentary; OT, old trained. B: Gialpha 2 content in purified mixed membranes from sedentary and trained young, middle-aged, and old Fischer 344 rats. Sedentary group: n = 10, 10, and 8 for young, middle-aged, and old, respectively.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   A: autoradiograms of immunoblots. Purified polyclonal rabbit antisera primary antibodies were used to identify stimulatory G proteins (Gsalpha ). Ten micrograms of purified mixed rat liver membranes were loaded per lane. Image capture analysis and quantification and linear regression analysis were performed on the fusion proteins (as in Fig. 2). At left, molecular mass marker. B: Gsalpha content in purified mixed membranes from sedentary and trained young, middle-aged, and old Fischer 344 rats. *Significantly different from sedentary age cohort (P < 0.05); dagger significantly different from young and middle-aged sedentary rat livers (P < 0.05). Sedentary group: n = 12, 12, and 7 for young, middle-aged, and old, respectively; trained group: n = 10, 10, and 8 for young, middle-aged, and old, respectively.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal characteristics and training effects. Of the 66 rats that began this study, 59 survived the experimental protocol. All 7 rats that did not survive were from the old group: 2 from the trained group and 5 from the sedentary group. This attrition is expected from previous morbidity and mortality statistics from our laboratory (22, 31, 32, 36). Final body weights were significantly lower in the trained young and middle-aged animals (331 ± 5 and 384 ± 4 g, respectively) compared with their sedentary counterparts (372 ± 5 and 432 ± 6 g, respectively). No difference existed in the old animals' final body weight (368 ± 18 vs. 377 ± 26 g, in the trained and untrained animals, respectively). Training effects were evident in the trained group of all age groups by significantly greater maximal running speeds during the final graded exercise test, time to fatigue during the endurance test, and soleus citrate synthase levels (data shown previously in Ref. 32). Briefly, maximal running speeds were 52, 52, and 53% greater in the trained young, middle-aged, and old animals, respectively, compared with their sedentary counterparts. Endurance times increased 393, 443, and 1,048% in these same groups, whereas soleus citrate synthase levels showed 50, 19, and 31% increases compared with sedentary animals in each age group.

Enzymatic analysis. Enzymology was performed to evaluate viability of membrane preparation, quantify membrane enrichment, and estimate relative contamination of the membrane preparation by other cellular organelles. Hepatocyte enzyme enrichment of six marker enzymes was performed on each liver preparation (data not shown): Na-K-ATPase and Mg-ATPase (markers of the basolateral and apical region, respectively), cytochrome c reductase (a microsomal marker), succinic dehydrogenase (an inner mitochondrial membrane marker), and leucine aminopeptidase and alkaline phosphatase (apical markers). Generally, alkaline phosphatase had the largest enrichment, followed by Na-K-ATPase. These enrichment data reveal that slightly more apical membranes were harvested from the homogenate. Because plasma membrane glucagon receptors are not associated with apical membranes or other cellular organelles, glucagon receptor binding per milligram protein was adjusted to reflect only specific basolateral membrane binding and therefore exclude nonbasolateral membranes of any derivation (see METHODS).

[125I-Tyr10]monoiodoglucagon binding in rat liver. Table 1 shows the results of saturation isotherm experiments obtained from a mean of three isotherms per animal, performed with duplicate points for each of the nine concentrations of glucagon. A representative binding curve is shown in Fig. 1. From the binding curves, a Hanes-Woolf plot (26) was used to determine Bmax and Kd (see inset). Glucagon-binding characteristics are shown in Table 1. There were no significant differences with age or training on maximal glucagon-binding capacity. Middle-aged animals from both sedentary and trained groups demonstrated a twofold greater affinity (lower Kd) than young and old rats. Trained animals, regardless of age, had a greater affinity (1.8-fold) than their sedentary counterparts.

G proteins. Representative autoradiograms of hepatic Gi and Gs proteins are shown in Figs. 2A and 3A, respectively. Hepatic Gialpha 2 was identified at 39 kDa, whereas Gsalpha was located at 45 kDa. Gi protein levels were not altered by age or training status (Fig. 2B). Gs protein content was significantly lower in the old sedentary animals compared with the middle-aged and young groups (Fig. 3B). This reduction was eliminated by training, as the old trained animals demonstrated significantly greater Gs protein content than their sedentary counterparts. As a result, Gs content did not differ across age in the trained group. The ratio of Gi to Gs (Gi/Gs) was significantly greater in old sedentary animals (2.5 ± 0.2) compared with the middle-aged (1.6 ± 0.2) sedentary animals (data not shown). This age-related increase in Gi/Gs ratio represents a 56% increase over the middle-aged animals. Endurance training was able to attenuate the increase in Gi relative to Gs that was seen in the old sedentary animals.

AC activity. Specific activity of AC was measured under five conditions (basal and 4 pharmacological stimulator levels; Table 2). Measuring AC activity under these five conditions allowed us to determine possible transduction sites (receptor, regulatory proteins, catalytic subunit) of signaling impairment, or reduced capacity of activation, in the glucagon-mediated signal transduction pathway. Results in young and middle-aged animals were similar but significantly decreased in both old sedentary and old trained animals. Basal AC activity (no exogenous stimulation) was decreased 43 and 46% in the old sedentary animals compared with young and middle-aged controls, and old trained animals showed a 25% decrease compared with their young counterparts. Basal values from each animal were subtracted from their own pharmacologically stimulated cAMP values to give final net values (21, 33, 35, 36). Glucagon+GTP (receptor-dependent) stimulation resulted in significantly diminished AC activity in the old animals regardless of training status (sedentary: down-arrow 29 and 27% compared with young and middle-aged animals; trained: down-arrow 18 and 21% compared with young and middle-aged animals). This age-related decline in glucagon-stimulated signaling still persists when data are examined in relation to change from basal AC activity (down-arrow 28 and 17% for sedentary and trained animals, respectively). The nonhydrolyzable GTP analog, GppNHp, and AlF were used to bypass the receptor to stimulate Gsalpha protein and showed an age-related decline in cAMP production in the old sedentary animals (down-arrow 48% compared with young and down-arrow 43% compared with middle-aged animals). Old trained animals also displayed an age-related decline in GppNHp-stimulated AC activity (down-arrow 42 and 38% compared with young and middle-aged animals, respectively). AlF G protein stimulation, which occurs at a non-GTPase site, resulted in declines similar to those seen with GppNHp. Forskolin stimulation was used to bypass both the receptor and Gsalpha because it acts directly on the catalytic subunit of AC. Although age-related declines in AC activity were seen in the old animals across training groups, the trained animals had more tempered declines (down-arrow 20 and 14% compared with young and middle-aged animals, respectively) than did the sedentary animals (down-arrow 46% and 43% compared with young and middle-aged animals). Remarkably, forskolin stimulation of AC activity in the livers of old trained animals resulted in a 44% increase compared with their sedentary counterparts.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major finding of this investigation is that alterations in glucagon-stimulated hepatic cAMP production across age and training are evident at several sites along the signal transduction pathway. Aging resulted in significant alterations in the glucagon-binding affinity ratio of the hepatic G proteins, as well as declines in both basal and pharmacologically stimulated cAMP production. Endurance training was able to partially attenuate these age-related declines. Specifically, the increased Gi/Gs ratio observed in old sedentary animals was offset by endurance training. This was primarily accomplished by the training-induced elimination of the Gs protein reduction in old animals. Furthermore, training resulted in increased cAMP production in the old animals when the catalytic subunit of AC was directly stimulated by forskolin. Thus previously observed age-related reductions in hepatic glucagon responsiveness can be partly accounted for by alterations in intrinsic AC activity and G protein ratios. Endurance training is able to diminish these declines through its actions on the glucagon receptor-binding affinity, the Gi/Gs ratio, and the AC activity.

To our knowledge, the present study is the first to demonstrate that aging results in an increased hepatic Gi/Gs protein ratio. Changes in Gi protein content were not significantly different across age or training groups. However, a significant decline in Gs content was observed for old sedentary animals compared with younger groups. This decline was offset by training, as the old trained animals demonstrated Gs content similar to that found in their younger counterparts. This change could have significant physiological consequences and may account for decreases in hormone-stimulated AC activity and in hepatic glucose production with age. Besides alterations found in the present study in G protein ratio, as well as AC activity with age, it is also possible that other protein constituents of hepatocytes may be involved in the observed decline in the ability to maintain blood glucose homeostasis with age. Sastre et al. (38) found that mitochondrial damage, specifically the malate export system in hepatocytes, accompanies increasing age. Another possible factor is the decreased sensitivity of the pancreatic beta -cells to glucose-induced insulin release with age (1).

Our finding that aging results in an intrinsic decline in glucagon-sensitive AC activity differs from Dax et al. (9), who found increased catecholamine-sensitive AC activity with age but no change with glucagon. This difference may be explained by 10-fold glucagon concentrations used (we used 1.0 µM, whereas Dax et al. used 10 µM) and the presence of physiological (100 µM) GTP (Dax et al. did not include GTP). It is also true that total AC activity is governed by the amount of stimulatory and inhibitory input received by AC, so that the increased Gi/Gs ratio may also be responsible for the age-related decline in the catalytic subunit of AC observed in the present study. However, forskolin stimulation of AC, which bypasses both receptor and G proteins, showed age-related declines, thereby suggesting an intrinsic decline in AC activity. Thus the findings of no significant differences in hepatic glucagon-binding capacity (Bmax) with age (in the present study and Ref. 9) suggest that reduced responsiveness to glucagon by liver with advancing age (32) is primarily related to impaired signaling through the regulatory G proteins and the catalytic subunit of AC. Similar findings have been reported with age in adrenergic signaling in heart (36).

Chronic dynamic exercise training in both humans and animals has been associated with increased sensitivity to hepatic hormonal stimulation compared with untrained counterparts. Galbo et al. (17) reported significantly reduced blood levels of glucagon, epinephrine, and norepinephrine during an exercise bout in trained humans compared with untrained controls. These smaller hormonal concentrations resulted in greater rates of hepatic glycogenolysis in the trained individuals, results consistent with an enhanced glucagon sensitization after training. Drouin et al. (14) found a greater glycogenolytic response to glucagon with endurance training in perfused rat livers despite similar liver glycogen levels. These authors implicate increased glycogen phosphorylase concentration and activity for the increased glycogenolytic response to glucagon. We (32) and others (25) have reported that gluconeogenic capacity is also increased in response to glucagon with endurance training. Results from Donovan and Sumida (12) support these findings by demonstrating that training increased hepatic glucose production in glycogen-depleted rats. Most recently, Bergman et al. (3) reported that exercise-trained men increased gluconeogenesis twofold at rest and threefold during absolute and relative exercise intensities. It is clear that the liver is capable of adaptation to chronic dynamic exercise training to enhance hepatic glucose production. We now show that the molecular mechanism for this adaptation occurs, at least in part, in the glucagon-mediated G protein-AC-cAMP signal transduction pathway in aging rat liver.

The present study found that training was able to offset the increased hepatic Gi/Gs ratio seen in the old sedentary animals, as well as significantly increase cAMP production by direct AC stimulation. Our results suggest that these sites within the glucagon signaling pathway are important in the age- and training-induced changes in hepatic glucose production previously observed. In the old animals, GppNHp and AlF stimulation of AC was compromised to nearly the same extent as forskolin, suggesting that changes in AC protein content (and by implication, protein expression), AC integrity or enzymatic function, and/or AC isoform expression may be contributing factors to this apparent lesion at AC (43). There may also be molecular interactions downstream from cAMP that help elucidate alterations in the hormonal signaling cascade in aging rat liver. For example, it is possible that the observed increase in glucose production with training is due to increased intracellular levels of cAMP activating more cAMP-dependent protein kinase (PKA), which is known to have many functions that enhance both glycogenolysis and gluconeogenesis. PKA stimulates phosphorylase kinase and phosphorylase a, while inhibiting glycogen synthase, all of which will favor glycogen breakdown over synthesis. PKA also increases gluconeogenic flux through regulation of gene transcription as it increases levels of PEPCK, glucose-6-phosphatase, and the aminotransferases. Hence, cAMP production has a central role in the activation of PKA and regulation of hepatic glucose production. The glucagon-sensitive signaling pathway, therefore, is a likely locus for age-related declines and training-induced increases in hepatic glucose production.

In summary, we conclude that the mechanisms responsible for age-related declines in hepatic glucose output occur, in part, as a result of alterations within the glucagon-stimulated signal transduction pathway at multiple sites. With advancing age, decreased responsiveness to glucagon appears due to 1) a relative decline in Gs protein expression and 2) decreased AC activity and depressed cAMP production. These changes result in a net decrease in AC activity per hormone-bound receptor, therefore leading to lower rates of cAMP production. Endurance training attenuated the age-related decline in AC activity by restoring Gs protein content and increasing the AC catalytic activity compared with old untrained animals. These adaptations would lead to enhanced hepatic hormonal responsiveness and capacity and improved glucose homeostasis, which may be important for both sedentary and aging people.


    ACKNOWLEDGEMENTS

We acknowledge Eileen Sutherland for sharing the membrane isolation procedures in the laboratory of Dr. Frances Simon, and Margaret H. Niedenthal at Eli Lilly and Company (Indianapolis, IN) for the generous donation of crystalline glucagon (lot no. 258-TN6-46) for these experiments.


    FOOTNOTES

Address for reprint requests and other correspondence: D. A. Podolin, Dept. of Physiology and Pharmacology, Univ. of New England, 11 Hills Beach Rd., Biddeford, ME 04005 (E-mail: dpodolin{at}mailbox.une.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.

Received 25 July 2000; accepted in final form 30 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aizawa, T, Komatsu M, Sato Y, Ishihara F, Suzuki N, Nishii N, Hashizume K, and Yamada T. Insulin secretion by the pancreatic beta cell of aged rats. Pancreas 9: 454-459, 1994[ISI][Medline].

2.   Baron, J, and Tephly TR. Effect of 3-amino-1,2,4-triazole on the stimulation of hepatic microsomal heme synthesis and induction of hepatic microsomal oxidases produced by phenobarbital. Mol Pharmacol 5: 10-20, 1969[Abstract].

3.   Bergman, BC, Horning MA, Casazza GA, Wolfel EE, Butterfield GE, and Brooks GA. Endurance training increases gluconeogenesis during rest and exercise in men. Am J Physiol Endocrinol Metab 278: E244-E251, 2000[Abstract/Free Full Text].

4.   Bessy, OA, Lowry OH, and Brock MJ. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J Biol Chem 164: 321-329, 1946[Free Full Text].

5.   Bitensky, MW, Russell V, and Blanco M. Independent variation of glucagon and epinephrine responsive components of hepatic adenyl cyclase as a function of age, sex, and steroid hormones. Endocrinology 86: 154-159, 1970[ISI][Medline].

6.   Brooks, GA, and Donovan CM. Effect of endurance training on glucose kinetics during exercise. Am J Physiol Endocrinol Metab 244: E505-E512, 1983[Abstract/Free Full Text].

7.   Cotrozzi, G, Relli P, La Villa G, Mazzanti R, Barbagli S, and Buzzelli G. Glucose homeostasis during aging. Arch Gerontol Geriatr Suppl 2: 203-208, 1991.

8.   Davidson, MB, and Karjala RG. Insulin antagonism of glucose transport in muscle from old-obese rat. Metabolism 27, Suppl: 1994-2005, 1978[ISI][Medline].

9.   Dax, EM, Partilla JS, Pineyro MA, and Gregerman RI. beta -Adrenergic receptors, glucagon receptors, and their relationship to adenylate cyclase in rat liver during aging. Endocrinology 120: 1534-1541, 1987[Abstract].

10.   Dixon, BS, Sutherland E, Alexander A, Nibel D, and Simon FR. Distribution of adenylate cyclase and GTP-binding proteins in hepatic plasma membrane. Am J Physiol Gastrointest Liver Physiol 265: G686-G698, 1993[Abstract/Free Full Text].

11.   Dohm, GL, Pennington SS, and Barakat H. Effect of exercise training on adenyl cyclase and phosphodiesterase in skeletal muscle, heart, and liver. Biochem Med 16: 138-142, 1976[ISI][Medline].

12.   Donovan, CM, and Sumida KD. Training improves glucose homeostasis in rats during exercise via glucose production. Am J Physiol Regulatory Integrative Comp Physiol 258: R770-R776, 1990[Abstract/Free Full Text].

13.   Drouin, R, Lavoie C, Bourque J, Ducros F, Poisson D, and Chiasson J-L. Increased hepatic glucose production response to glucagon in trained subjects. Am J Physiol Endocrinol Metab 274: E23-E28, 1998[Abstract/Free Full Text].

14.   Drouin, R, Milot M, Robert G, Massicotte D, Peronnet F, and Lavoie C. Hepatic glucagon sensitivity induced by endurance training: effect mediated by increased glycogenolysis? (Abstract) Med Sci Sports Exerc 3: S224, 2000.

15.   Fink, RI, Kolterman OG, Griffin J, and Olefsky JM. Mechanisms of insulin resistance in aging. J Clin Invest 71: 1523-1535, 1983[ISI][Medline].

16.   Fraeyman, N, and van Ermen A. Influence of aging on the beta- and glucagon-mediated glycogenolysis in rat hepatocytes. Mech Ageing Dev 70: 115-126, 1993[ISI][Medline].

17.   Galbo, H, Richter EA, Holst JJ, and Christensen NJ. Diminshed hormonal responses to exercise in trained rats. J Appl Physiol 43: 953-958, 1977[Abstract/Free Full Text].

18.   Goldberg, JA, and Rutenburg AM. The colorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 11: 283-291, 1958[ISI].

19.   Goodman, MN, Dluz SM, McElaney MA, Belur E, and Ruderman NB. Glucose uptake and insulin sensitivity in rat muscle: changes during 3-96 weeks of age. Am J Physiol Endocrinol Metab 244: E93-E100, 1983[Abstract/Free Full Text].

20.   Hall, JL, Mazzeo RS, Podolin DA, Cartee GD, and Stanley WC. Exercise training does not compensate for an age-related decrease in myocardial GLUT-4 content. J Appl Physiol 76: 328-332, 1994[Abstract/Free Full Text].

21.   Hammond, HK, Roth DA, McKirnan MD, and Ping P. Regional myocardial down-regulation of the inhibitory GTP-binding protein (Gialpha 2) and beta -adrenergic receptors in a porcine model for chronic episodic myocardial ischemia. J Clin Invest 92: 2644-2652, 1993[ISI][Medline].

22.   Horn, DB, Podolin DA, Friedman JE, Scholnick DA, and Mazzeo RS. Alterations in key gluconeogenic regulators with age and endurance training. Metabolism 46: 1-7, 1997.

23.   Kern, M, Dolan PL, Mazzeo RS, Wells JA, and Dohm GL. Effect of aging and exercise on GLUT-4 glucose transporters in muscle. Am J Physiol Endocrinol Metab 263: E362-E367, 1992[Abstract/Free Full Text].

24.   Kmiec, Z, and Mysliwski A. Age-dependent changes of hormone-stimulated gluconeogenesis in isolated rat hepatocytes. Exp Gerontol 18: 173-184, 1983[ISI][Medline].

25.   Lavoie, C, Drouin R, Milot M, Robert G, Massicotte D, and Peronnet F. Hepatic glucagon sensitivity induced by endurance training: effect mediated by increased gluconeogenesis? (Abstract) Med Sci Sports Exerc 32: S224, 2000.

26.   Limbird, LE. Cell Surface Receptors: A Short Course on Theory and Methods. The Hague, the Netherlands: Nijhoff, 1986, p. 63-67.

27.   Lowry, OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951[Free Full Text].

28.   Meier, SL, Sztul ES, Reuben A, and Boyer JL. Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver. J Cell Biol 98: 39-46, 1987.

29.   Neville, DM, Jr. Isolation of an organ specific protein antigen from cell-surface membrane of rat liver. Biochim Biophys Acta 154: 540-552, 1968[ISI][Medline].

30.   Ortiz-Alonso, FJ, Galecki A, Herman WH, Smith MJ, Jacquez JA, and Halter JB. Hypoglycemia counterregulation in elderly humans: relationship to glucose levels. Am J Physiol Endocrinol Metab 267: E497-E506, 1994[Abstract/Free Full Text].

31.   Podolin, DA, Gleeson TT, and Mazzeo RS. Role of norepinephrine in hepatic gluconeogenesis: evidence of aging and training effects. Am J Physiol Endocrinol Metab 267: E680-E686, 1994[Abstract/Free Full Text].

32.   Podolin, DA, Gleeson TT, and Mazzeo RS. Hormonal regulation of hepatic gluconeogenesis: influence of age and training. Am J Physiol Regulatory Integrative Comp Physiol 270: R365-R372, 1996[Abstract/Free Full Text].

33.   Roth, DA, Urasawa K, Helmer GA, and Hammond HK. Down-regulation of cardiac GTP-binding proteins in right atrium and left ventricle in pacing-induced congestive heart failure. J Clin Invest 91: 939-949, 1993[ISI][Medline].

34.   Roth, DA, Urasawa K, Leiber D, Insel PA, and Hammond HK. A substantial proportion of cardiac Gs is not associated with the plasma membrane. FEBS Lett 296: 46-50, 1992[ISI][Medline].

35.   Roth, DA, White CD, Hamilton CD, Hall JL, and Stanley WC. Adrenergic desensitization in left ventricle from streptozotocin diabetic swine. J Mol Cell Cardiol 27: 2315-2325, 1995[ISI][Medline].

36.   Roth, DA, White CD, Podolin DA, and Mazzeo RS. Alterations in myocardial signal transduction due to aging and chronic dynamic exercise. J Appl Physiol 84: 177-184, 1998[Abstract/Free Full Text].

37.   Salomon, Y, Londos C, and Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541-548, 1974[ISI][Medline].

38.   Sastre, J, Pallardo FV, Pla R, Pellin A, Juan G, and O'Conner JE. Aging of the liver: age-related mitochondrial damage in intact hepatocytes. Hepatology 24: 1199-1205, 1996[ISI][Medline].

39.   Schoner, W, Von Ilberge C, Kramer R, and Seubert W. On the mechanism of Na+- and K+-stimulated hydrolysis of adenosine triphosphate. 1. Purification and properties of a Na+- and K+-activated ATPase from ox brain. Eur J Biochem 1: 334-343, 1967[ISI][Medline].

41.   Srere, PA. Citrate synthase. Methods Enzymol 13: 3-5, 1969.

42.   Sumida, KD, Uriales JH, and Donovan CM. Enhanced gluconeogenesis from lactate in perfused livers after endurance training. J Appl Physiol 74: 782-787, 1993[Abstract].

43.   Tsujimoto, A, Tsujimoto G, and Hoffman BB. Age-related change in adrenergic regulation of glycogen phosphorylase in rat hepatocytes. Mech Ageing Dev 33: 167-175, 1986[ISI][Medline].

44.   Winder, WW, Holman RT, and Garhart SJ. Effect of endurance training on liver cAMP response to prolonged submaximal exercise. Am J Physiol Regulatory Integrative Comp Physiol 240: R330-R334, 1981[ISI][Medline].


Am J Physiol Endocrinol Metab 281(3):E516-E523
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society