The Journal of Nutrition Vol. 128 No. 6 June 1998, pp. 927-933

Moderate Dietary Protein and Energy Restriction Modulate cAMP-Dependent Protein Kinase Activity in Rat Liver^1,2,3

L. J. O'Brien, K. D. Levac, and L. E. Nagy⁴

Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Very low protein diets result in a desensitization of hepatic cAMP signaling in rats, which is characterized by a loss of cAMP-dependent protein kinase (PKA) activity and type I regulatory subunit (RI). Here we have tested whether more moderate protein restriction (Trial 1) or energy restriction (Trial 2) also modulates hepatic PKA quantity and activity. In trial 1, weanling rats were allowed free access to diets containing normal protein (15%, AL-NP), moderately restricted protein (12.5%, AL-MP) and low protein (7.5%, AL-LP); in trial 2, rats were allowed free access to diet containing 15% (AL-NP) or 0.5% protein (very low protein, AL-VLP) or were energy restricted by pair-feeding a diet isonitrogenous to AL-NP but at 65% of the energy intake (ER-IN) for 14 d. Body weights were lower (P < 0.05) by d 14 in all restricted groups compared with the AL-NP group. The quantity of cytosolic RI was lower (P < 0.05) in AL-LP and AL-VLP, but not in AL-MP or ER-IN, compared with AL-NP. In contrast, there was no effect of diet on RI in the particulate fraction. RII was not changed by moderate and low protein diets in either the cytosol or particulate fraction. However, type II regulatory subunit (RII) was greater in the cytosol of ER-IN and in the particulate fraction of AL-VLP (P < 0.05) compared with AL-NP. Specific activity of PKA was lower in the cytosol and particulate fraction (P < 0.05) in the AL-VLP and ER-IN groups compared with the AL-NP group. In contrast, specific activity of PKA was maintained in cytosol from AL-LP, but lower in the particulate fraction (P < 0.05) compared with AL-NP. In summary, protein restricted-diets lower RI subunit in the cytosol; however, only in rats fed very low protein diets is this loss of RI associated with lower cytosolic PKA activity. In contrast, energy restriction lowers PKA activity in the cytosol and particulate fractions, independent of signficant reduction in RI or RII subunits. Taken together, these data indicate that moderate protein and energy restrictions have differential effects on activity and quantity of PKA.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

cAMP-dependent protein kinase (PKA)⁵ mediates the effects of cAMP, phosphorylating a variety of key substrates involved in regulating cellular metabolism (Krebs and Beavo 1979), proliferation (Boynton and Whitfield 1983), differentiation (Cho-Chung 1990) and gene transcription (Maurer 1981). In the liver, PKA is also involved in maintaining whole-body glucose homeostasis via the regulation of glycogen metabolism. PKA is a tetrameric holoenzyme complex composed of two regulatory (R) subunits and two catalytic (C) subunits (Beebe and Corbin 1986, Krebs and Beavo 1979). Because intracellular cAMP concentration rises in response to external stimuli, cAMP binds to the R subunits and releases free C subunits from the holoenzyme. These active C subunits phosphorylate target proteins in the cytoplasm and nucleus. There are two major isoforms of the R subunit, RI and RII, which differ in their K_d for cAMP (Cho-Chung 1990), tissue distribution (McKnight et al. 1988), cellular compartmentalization (Meinkoth et al. 1990) and levels of expression during development and thoughout the cell cycle (Cho-Chung and Clair 1993). RI is localized primarily to the cytoplasm (Meinkoth et al. 1990), whereas RII can be localized to specific subcellular compartments including membranes, Golgi apparatus and cytoskeletal elements by A-kinase anchoring proteins (AKAP) (Nigg et al. 1985). This differential localization and expression suggest distinct functions for the different isoforms of PKA, including specific substrate targeting (Scott 1991) and regulation of cell growth (Cho-Chung 1990).

Both the quantity and activity of proteins involved in the hepatic cAMP signaling cascade are sensitive to dietary protein restriction and starvation (Ekanger et al. 1988, Goss et al. 1994, Rozwadowski et al. 1995, Stephen and Nagy, 1996). Consumption of diets very low in protein (0.5 g protein/100 g) by weanling rats for 2 wk resulted in a desensitization of cAMP signal transduction (Goss et al. 1994, Rozwadowski et al. 1995, Stephen and Nagy, 1996). This desensitization was characterized by a decrease in cytosolic RI subunit quantity and total PKA activity (Rozwadowski et al. 1995, Stephen and Nagy, 1996). Losses in PKA activity and RI content were rapid, occurring after only 3 d of feeding the protein-restricted diet, and were prevalent thoughout the 14-d feeding trial. These diet-induced modifications in the cAMP signal transduction pathway disrupt cAMP-controlled cellular functions, including regulation of glutathione concentration in hepatocytes (Goss et al., 1994), cAMP-dependent activation of glycogen phosphorylase and phosphorylation of the cAMP response element binding protein (CREB) (Stephen and Nagy 1997), and are likely to contribute to the profound alterations in liver function observed during protein malnutrition.

The mechanism for the decrease in RI subunit quantity after the consumption of very low protein diets is not known. When rats are provided with very low protein diets, energy intake, as well as protein intake, is compromised, because food intake (g/d) is decreased during wk 2 of feeding (Stephen and Nagy 1996). Thus, diet-induced changes in PKA may be due to the decreased availability of dietary protein, and/or to a reduction in energy availability. Here we have tested whether diets either moderately restricted in dietary protein or energy restricted, but isonitrogenous with control diets, regulate PKA quantity and activity in the liver of weanling rats.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Materials.  Rats were supplied by Harlan Sprague Dawley (Indianapolis, IN). Antibodies were purchased as follows: PKA-RI from Transduction Laboratories (Lexington, KY), PKA-RII from Santa Cruz Biotechnology (Santa Cruz, CA), PKA-C (NT) from Upstate Biotechnology (Lake Placid, NY); peroxidase-labeled anti-mouse-Ig Fab fragment and anti-rabbit immunoglobulin G (IgG) antibodies (IgG-POD) were purchased from Boehringer Mannheim (Dorval, Canada). Protein assay reagent was supplied by BioRad (Mississauga, Canada). Enhanced chemiluminescence (ECL) detection kit and [-³²P]-ATP were purchased from Amersham (Oakville, Canada). Polyvinylidene difluoride (PVDF) membrane was obtained from Gelman Sciences (St. Laurent, Canada). Phosphocellulose sheets and protein kinase inhibitor (PKI (6-22) amide were purchased from GibcoBRL Life Technologies (Burlington, Canada). Kemptide, ATP and cAMP were supplied by Sigma Chemical (St. Louis, MO). Other reagent grade chemicals were supplied by Fisher Scientific (Mississauga, Canada), ICN Biochemicals (St. Laurent, Canada) and Sigma Chemical.

Animals and diets.  Male weanling Wistar rats (initial body weights 40-50 g) were housed individually in closed, plastic-bottomed cages in a temperature- and humidity-controlled room with light from 0800 to 2000 h. Three feeding trials were conducted; feeding trial 1 addressed the effects of moderate protein restriction on PKA; feeding trial 2 was designed to compare the effects of an energy restricted diet, i.e., one that limited growth by restricting dietary carbohydrate rather then protein. The energy-restricted diet provided the same amount of protein, per gram body weight, as the control group. Rats were allowed free access to a casein-based semipurified control diet containing 15% protein for 3 d and then randomly divided into three treatment groups. For trial 1, rats were allowed free access to diets containing normal protein (15%, AL-NP), moderately restricted protein (12.5%, AL-MP) or low protein (7.5%, AL-LP) for 14 d. For feeding trial 2, rats were allowed free access to diets containing 15% (AL-NP) or 0.5% protein (AL-VLP) or energy restricted by pair-feeding a 23% protein diet at 65% of the intake (ER-IN) of the AL-C group. The design of the energy-restricted diet was adapted from Straus and Takemoto (1991) to provide a diet isonitrogenous with that of the AL-NP group. The 65% restriction was chosen on the basis of food intake data from previous experiments (Stephen and Nagy 1996), which showed that rats allowed free access to 0.5% protein diets reduced their intake, on average, to 65% of control intake during wk 2 of feeding. Dietary intakes of control rats were measured, standardized to 100 g per body weight, and rats in the energy-restricted group were given 65% of the intake consumed by the control group during the previous 24-h period. A third feeding trial was conducted to measure PKA activity and included the AL-NP, AL-LP, AL-VLP and ER-IN diets described for feeding trials 1 and 2. Compositions of the diets are shown in Table 1. Rats were allowed free access to water thoughout the feeding trial. The animal care protocol was approved by the University of Guelph Animal Care Committee under the guidelines of the Canadian Council for Animal Care.

Sample preparation.  On d 14 of the feeding trial, rats from all treatment groups were anesthetized with sodium-pentobarbital (0.2 mL/100 g body weight), livers were perfused with saline, removed and immediately frozen in liquid nitrogen. A section of a single lobe was homogenized in 15 mL/g of tissue of ice-cold homogenizing buffer (pH 7.4) containing 25 mmol/L sodium phosphate, 100 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L EGTA, 2 mol/L MgCl₂ and protease inhibitors (20 mL/L phenylmethyl sulfonyl fluoride, 1 mL/L aprotinin, 1 mL/L leupeptin 1 mL/L bestatin and 1 mL/L bacitracin). Homogenates were centrifuged at 50 × g for 5 min at 4°C to separate cellular debris and nuclei. The supernatant was then centrifuged at 100 000 × g for 30 min at 4°C. The resulting supernatant (cytosolic fraction) was removed and the remaining pellet (particulate fraction) was resuspended in 4 mL of homogenizing buffer. Protein concentration was measured with the BioRad protein assay using bovine serum albumin as a standard.

Electrophoresis and Western blotting.  Homogenates, cytosol, particulate and nuclear fractions were sonicated for 20 s, adjusted to 1 g protein/L, diluted 1:1 in Laemmli sample buffer and separated by 10% SDS-PAGE (Laemmli 1970). Proteins were electrophoretically transferred to PVDF membranes and stained with fast green (0.1% fast green stain, 20% methanol, 5% acetic acid) to ensure even protein loading. For detection of the RI, RII and C proteins, PVDF membranes were incubated in blocking buffer containing 5% nonfat dried milk in 10 mmol/L Tris, pH 7.4, 150 mmol/L NaCl and Tween 20 (TBS-T) for up to 2 h. Tween concentrations varied depending on the primary antibody; 0.05% for RII and 0.1% for RI and C. Membranes were then incubated with primary antibody diluted in blocking buffer for 30 min to 1 h as follows: RI (1:250), RII (1:200) and C (1:5000), washed 3 × 10 min in TBS-T and incubated with secondary antibody diluted in blocking buffer for 1 h as follows: anti-mouse-IgG-peroxidase Fab fragments (1:25,000) for RI and anti-rabbit-IgG-peroxidase (1:25,000) for RII and C. Membranes were then washed for 3 × 10 min in TBS-T. All incubations were at room temperature. Immunoreactive proteins from all blots were detected using the enhanced chemiluminescence detection kit and density of immunoreactive bands measured by scanning densitometry. In some experiments, membranes were stripped and reprobed following the procedure outlined by Amersham (Oakville, Canada). The linear range of detection was determined for RI (Stephen and Nagy 1996), RII and C antibodies and experimental samples analyzed within the established linear ranges. Some membranes were also processed without primary antibody to ensure that nonspecific binding of the secondary antibody did not occur. Western blots were conducted in duplicate for each sample and comparisons were made either within a single blot or within a feeding trial by standardizing different blots to a control sample loaded onto every blot.

Cyclic AMP-dependent protein kinase assay.  Activity of PKA was measured using the Kemptide assay (Kemp et al. 1988) in liver cytosol and particulate fractions. Livers were thawed on ice and homogenized in 10 mmol/L potassium phosphate (pH 6.8) containing 1 mmol/L EDTA, 0.1 mmol/L dithiotheitol and protease inhibitors (Stephen and Nagy 1996). Cytosol and particulate fractions were prepared as described above, and samples of the homogenate and particulate fractions were incubated on ice for 1 h in 10% Triton X-100 (Ekanger et al. 1988, Stephen and Nagy, 1996). Activity was measured over 5 min in the absence or presence of 40 mmol/L cAMP after the addition of 10 mL of a [-³²P]ATP/substrate solution (final concentrations of the assay components; 50 mmol/L Kemptide, 10 mmol/L MgCl₂, 0.25 g/L BSA, 50 mmol/L Tris, pH 7.5, and 100 mmol/L [-³²P]ATP). All samples were diluted to two concentrations of protein (0.2 and 0.4 g/L), which were within the established linear range of the assay; PKA activity was determined at both concentrations. Samples were incubated at 30°C for 5 min, 20 mL of the sample was spotted onto phosphocellulose sheets and the reaction was terminated using a phosphoric acid wash. Peptide-incorporated ³²P was counted using a Beckman LS 7800 liquid scintillation spectrometer (Fullerton, CA). Activity in the presence of protein kinase inhibitor peptide [PKI (6-22) amide] was subtracted from total activity to account for nonspecific activity.

Statistical analysis.  Statistical analysis was performed using the General Linear Model procedure of Statistical Analysis System for personal computers (SAS Institute, Cary, NC). PKA activity data were log transformed to generate normal distribution. Statistical comparisons were analyzed by ANOVA; when the F-test indicated a significant effect, differences due to dietary treatment, distribution and/or time were determined using Tukey's multiple comparison test. Statistical significance was defined as P < 0.05. Values are expressed as the mean ± SEM for each group.

	RESULTS

Abstract Introduction Methods Results Discussion References

Growth rates.  Body weights of the weanling rats increased over the 2-wk feeding period with all dietary treatments from feeding trial 1 (P < 0.05)(Fig. 1A). Growth rates were reduced in the AL-LP rats after the first week of feeding relative to AL-NP rats. By d 14, the AL-LP group had grown to 76% of the weight of the AL-NP group (P < 0.05), whereas rats in the AL-MP group had grown to 95% of the weight of the AL-NP group (P < 0.05). Growth was also reduced over the 2-wk feeding trial in weanling rats fed the ER-IN diet (Fig. 1B); by d 14, the rats had grown to 61% of the weight of the AL-NP group (P < 0.05). Body weight decreased in the AL-VLP group during wk 1 relative to their initial weight, but was maintained over wk 2; by d 14, rats had grown to only 32% of the weight of AL-NP group (Fig. 1B).

View larger version (15K): [in this window] [in a new window]   Fig 1. Effect of dietary protein and energy restriction on growth of weanling rats fed diets containing (A) 15% (AL-NP), 12.5% (AL-MP) and 7.5% (AL-LP) protein and (B) 15% (AL-NP), 0.5% (AL-VLP) protein and pair-fed a diet isonitrogenous to the AL-NP group at 65% energy (ER-IN) for 2 wk. Values represent means ± SEM [n = 5; n = 6 for the 15% protein group in (B)]. *Indicates a signficant difference in body weight compared with AL-NP (diet effect, P < 0.05) at each time point.

Trial 1: effect of moderate and low protein diets on hepatic PKA subunit quantity.  Total immunoreactive RI protein was lower in liver homogenates from the AL-LP group, but was not lower in those from the AL-MP group, compared with the AL-NP controls (Table 2). This lower hepatic RI content in the AL-LP group was associated with a 70% loss of RI in the cytosolic fraction, with no difference in the particulate fraction (Table 2). In contrast to the loss in RI protein, total immunoreactive quantity of the RII subunit was not different in the AL-MP or AL-LP group relative to AL-NP controls in any of the liver fractions (Table 2). Total immunoreactive quantity of C subunit in liver homogenate was not different in the AL-MP group, but was greater in the AL-LP group, compared with the AL-NP controls (Table 2). However, this greater C subunit content was not associated with significantly greater C subunit quantity in cytosol, particulate (Table 2) or nuclear extract (data not shown) compared with AL-NP controls.

Trial 2: effect of energy restriction on PKA subunit quantity.  Energy restriction had differential effects on total RI and RII in liver homogenates compared with very low protein diets. Although liver homogenates from AL-VLP rats had lower RI and greater RII quantity (P < 0.05), RI and RII subunit quantities were not different in the ER-IN group compared with the AL-NP or AL-VLP group (Table 2). Rats fed these diets also differed in the subcellular localization of the changes in RII. RII was greater in the particulate fraction in rats fed the AL-VLP diet, whereas in those fed the ER-IN diet, RII was greater in the cytosol compared with AL-NP rats (Table 2). The C subunit contents in homogenate, cytosol and particulate fraction were not different in AL-VLP or ER-IN compared with AL-NP controls (Table 2).

Relative subcellular distribution of PKA subunits.  We compared the distribution of RI, RII and C between the cytosol and particulate fractions in livers from AL-NP, AL-LP, AL-VLP and ER-IN groups (Fig. 2). Consistent with its compartmentalization in the cytosol (Meinkoth et al. 1990), RI was enriched in the cytosolic fraction compared with homogenate, but lower in the particulate fraction (P < 0.05)(Table 3). In contrast, RII was not enriched in either cytosolic or particulate fractions (Table 3). The distribution of the catalytic subunit paralleled both the enrichment of RI in the cytosol and lower RI content in the particulate fraction (Table 3). There was no effect of diet on the distribution of any of the subunits (diet × distribution interaction, P > 0.05).

View larger version (65K): [in this window] [in a new window]   Fig 2. Distribution of the RI, RII and catalytic subunits of cAMP dependent protein kinase in homogenate (H), cytosolic (C) and particulate (P) fractions of the liver from rats fed diets restricted in protein and energy for 2 wk. Immunoreactive RI (A), RII (B) and C (C) proteins from rats provided with 15% (AL-NP), 7.5% (AL-LP) and 0.5% (AL-VLP) protein diets or an isonitrogenous diet energy restricted to 65% of AL-NP (ER-IN) were measured by Western blot. Duplicate Western blots were quantified using scanning densitometry as presented in Table 3. Blots are representative of five rats per treatment group.

PKA activity.  To assess whether the changes in subunit quantity after more moderate protein and/or energy restriction were also associated with changes in PKA activity, activity was measured in cytosol and particulate fractions from rat liver on d 14 of feeding. The specific activity of PKA, measured after activation with 40 mmol/L cAMP, was lower in homogenate from the AL-VLP rats (P < 0.05), but not in AL-LP or ER-IN groups compared with AL-NP controls (see Table 4). Calculated total activity of PKA in the AL-VLP group was only 18% of AL-NP controls (P < 0.05). Total PKA activity was localized predominantly (75-80%) in the cytosolic fraction of all groups. Specific activity of PKA was lower in the cytosol of AL-VLP and ER-IN groups compared with AL-NP controls (P < 0.05). Total activity of PKA was lower in AL-VLP rats; however, because the total cytosolic protein concentration of the ER-IN group was at control levels, total PKA activity was not lower than that of AL-NP controls. Similarly, in the particulate fraction, specific activity of PKA was lower in all restricted groups compared with AL-NP controls (P < 0.05). However, total PKA activity was lowered only in the AL-VLP group (P < 0.05). Thus, total PKA activity was more severely lowered in the AL-VLP groups due to a reduction in both the specific activity of PKA and the protein content of the liver.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Feeding very low protein diets (0.5%) to weanling rats for 14 d decreases PKA activity in the liver (Rozwadowski et al. 1995, Stephen and Nagy 1996). This decrease is associated with an impaired ability for cAMP to activate glycogen phosphorylase in liver homogenates and activate the nuclear transcription factor CREB (Stephen and Nagy 1997), suggesting that a diverse array of PKA-mediated functions are impaired in the liver of rats fed very low protein diets. Decreased PKA activity and function in response to very low protein diets are associated with a lower quantity of the RI subunit of PKA (Rozwadowski et al. 1995, Stephen and Nagy 1996). Here we report that the RI subunit is also responsive to more moderate dietary protein restriction; rats allowed free access to 7.5% protein diets exhibited lower RI quantity. In contrast, hepatic RI was not affected in rats fed an energy-restricted diet that was adequate in protein. Despite the loss of RI, activity of PKA was maintained in the low protein diets, whereas energy restriction resulted in lower PKA activity in cytosol and particulate fractions, independent of reduced RI or RII. These data indicate that RI subunit quantity and PKA activity are differentially responsive to protein and energy restriction.

The quantity of hepatic RI was more responsive to the restriction of dietary protein than to energy restriction. Although low protein and energy-restricted diets reduced growth of weanling male rats to a similar extent (Fig. 1), only the low protein diets reduced cytosolic RI protein (Table 2). The reduction in RI quantity due to protein restriction is very specific; RII and C quantity do not decrease, nor do other proteins involved in cAMP signal transduction, including the stimulatory guanine nucleotide regulatory protein (Stephen and Nagy 1996) and the cAMP response element binding protein (Stephen and Nagy 1997). The RI subunit is very responsive to changes in dietary protein and amino acid availability; changes in quantity may be due to reduced rates of synthesis and/or increased degradation. Feeding very low protein diets to weanling rats reduces total immunoreactive quantity of RI after only 3 d (Stephen and Nagy 1996). This diet-induced loss in RI protein returns to normal with refeeding of a complete diet or amino acid solution (Ekanger et al. 1989) or by culturing hepatocytes from protein-deprived rats in an amino acid-rich medium (Houge et al. 1990). Recovery of RI is paralleled by an increase in RI mRNA (Ekanger et al. 1989, Houge et al. 1990), suggesting that increased amino acid availability regulates transcription of the RI subunit. However, no data are available concerning the effect of decreased amino acid availability on RI transcription.

In addition to possible decreases in RI synthesis, increased degradation may also occur. Dietary protein restriction decreases liver weight and hepatocyte size and volume, as well as total protein and protein synthesis in the liver (Ekanger et al. 1988, reviewed in Hoffer 1994, Torun and Chew 1994). RI is more susceptible to proteolysis than the majority of other liver proteins (Weber and Hilz 1986), which may result in decreased RI concentration during a period of catabolism. In contrast to RI, the RII subunit degrades more slowly than the bulk of other liver proteins (Weber and Hilz 1986). Membrane-anchoring proteins can bind RII subunit dimers alone (Coghlan et al. 1993) or in holoenzyme form (Nigg et al. 1985), thus stabilizing and protecting RII from proteolysis (Otten and McKnight 1989, Weber and Hilz 1986). Therefore, the decrease in the RI:RII ratio with protein restriction may result from increased proteolysis of RI and protection of RII.

The greater quantity of RII in the particulate fraction of livers of rats fed very low protein diets or in the cytosol after energy restriction may be due to increased synthesis or translocation. Translocation of PKA subunits has been proposed as a potential mechanism to regulate the subcellular distribution of PKA and facilitate signal transduction from the plasma membrane to subcellular fractions (reviewed in Jungmann and Russell 1977). However, a net translocation of RII is unlikely in the AL-VLP or ER-IN groups because RII was equally distributed between the cytosolic and particulate fractions (Table 3). Moreover, there was no effect of diet on the distribution of total protein or PKA subunits in the liver (Table 4). Alternatively, greater cytosolic RII in the AL-LP and ER-IN groups may compensate for the loss of RI and preserve overall PKA activity. Overexpression or suppression of one R isoform can be compensated for by a change in the other isoform (Tortora and Cho-Chung 1990). Recently, Cummings and co-workers (1996) reported that RIIb knock-out mice compensate for the loss in RIIb gene in brown adipose tissue by increasing RIa gene expression.

Specific activity of PKA decreases in rats with starvation and feeding very low protein diets (Ekanger et al. 1988, Rozwadowski et al. 1995, Stephen and Nagy 1996) (Table 3); however, this decrease was not associated with a decrease in the amount of immunoreactive C subunit quantity. The lack of association between immunoreactive C subunit quantity and activity suggests that additional factors such as post-translational modifications or interaction with regulatory proteins control the specific activity of PKA after protein and/or energy restriction. One important mechanism for the regulation of C activity is the binding of the heat-stable inhibitor of PKA (PKI); binding of PKI to the C subunit occurs with high affinity and inhibits enzyme activity even in the presence of cAMP (Whitehouse and Walsh 1982). Binding of PKI to the C subunit also protects the subunit from proteolysis (Fantozzi et al. 1992). Although regulation of PKI in the liver has not been studied, our data are consistent with the hypothesis that hepatic PKI expression is enhanced in rats fed very low protein and energy-restricted diets, acting to decrease PKA activity and prevent C degradation.

Cell cycle regulation of PKA has been demonstrated in a variety of cell types (reviewed in Jungmann and Russell 1977, Tortora et al. 1993). Because hepatocytes from weanling rats are continuously proliferating and differentiating, the effect of limiting nutrient availability on the cell cycle and subsequently on PKA subunit expression must be considered. The G1 phase of the cell cycle is very sensitive to limited nutrient availability (Boynton and Whitfield 1983). Increased cAMP may play a role in preventing progression though the cell cycle by promoting cell cycle arrest at the G1/S phase (Vintermyr et al. 1993). We have found that cAMP concentration is 75% greater in freeze-clamped liver of weanling rats fed 0.5% protein diets for 14 d compared with controls (Stephen and Nagy 1997). cAMP-mediated inhibition of cell cycle progression may be a mechanism by which hepatocytes conserve energy for more immediately important liver functions such as gluconeogenesis and protein synthesis (Mellgren et al. 1995). R subunit expression also varies with cell cycle; decreased RI:RII, similar to that observed in rats fed diets restricted in protein and energy, occurs at the G1/S phase of the cell cycle in numerous cell types (reviewed in Jungmann and Russell 1977, Tortora et al. 1993). In addition, PKI levels are low in cells in the G1 phase of the cycle, increase in the cytosol during S phase and shift to the nucleus at G2/M phase (Wen et al. 1995). Cell cycle regulation of PKI suggests specific roles for this inhibitor in regulating PKA activity; for example, PKI may inhibit C activity in the nucleus as cells begin mitosis (Wen et al. 1995). Regulation of PKA activity by PKI may provide a potential mechanism for inhibiting hepatic growth when nutrient availability is reduced and thus shift available resources to maintain critical cellular functions, such as gluconeogenesis. Future research into PKI expression and subsequent regulation of PKA activity in response to dietary protein and energy restriction may help to elucidate the mechanism(s) of regulation of PKA in the liver of malnourished animals.

	ACKNOWLEDGMENTS

The authors thank Laurie Stephen for her advice during the course of these studies and Andrew Aldred for his technical assistance.

	FOOTNOTES

Manuscript received 12 May 1997. Initial reviews completed 26 July 1997. Revision accepted 4 February 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• American Institute of Nutrition Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 1977; 107:1340-1348
• Beebe, S. J. & Corbin, J. D. (1986) The Enzymes: Control by Phosphorylation (Beebe, S. J. & Corbin, J. D., eds.), vol. 17, pp. 43-111. Academic, New York.
• Boynton A. L., Whitfield J. F. The role of cAMP in cell proliferation: a critical assessment of the evidence. Adv. Cyclic Nucl. Res. 1983; 15:193-294
• Cho-Chung Y. S. Role of cAMP receptor proteins in growth, differentiation and suppression of malignancy: new approaches to therapy. Cancer Res. 1990; 50:7093-7100[Abstract/Free Full Text]
• Cho-Chung Y. S., Clair T. The regulatory subunit of cAMP-dependent protein kinase as a target for chemotherapy of cancer and other cellular dysfunctional-related diseases. Pharmacol. Ther. 1993; 60:265-288[Medline]
• Coghlan, V. M., Bereson, S. E., Langeberg, L., Nilaver, G. & Scott, J. D. (1993) A-kinase anchoring proteins; a key to selective activation of cAMP-responsive events? Mol. Cell. Biochem. 127/128: 309-319.
• Cummings D. E., Brandon E. P., Planas J. V., Motamed K., Idzerda R. L., McKnight G. S. Genetically lean mice result from targeted disruption of the RIIb subunit of protein kinase A. Nature (Lond.) 1996; 382:622-626[Medline]
• Ekanger R., Vintermyr O. K., Doskeland S. O. The amount of rat liver cyclic AMP-dependent protein kinase I and II are differentially regulated by diet. Biochem. J. 1988; 256:447-452[Medline]
• Ekanger R., Vintermyr O. K., Houge G., Sand T. E., Scott J. D., Krebs E. G., Eikhom T. S., Chistoffersen T. S., Ogreid D., Doskeland S. O. The expression of cAMP-dependent protein kinase subunits is differentially regulated during liver regeneration. J. Biol. Chem. 1989; 264:4374-4382[Abstract/Free Full Text]
• Fantozzi D. A., Taylor S. S., Howard P. W., Mauer R. A., Meinkoth J. L. Effect of the thermostable protein kinase inhibitor on intracellular localization of the catalytic subunit of cAMP-dependent protein kinase. J. Biol. Chem. 1992; 267:1624-1628
• Goss P., Bray T. M., Nagy L. E. Regulation of hepatocyte glutathione by amino acid precursors and cAMP in protein-energy malnourished rats. J. Nutr. 1994; 124:323-330
• Hoffer, J. L. (1994) Starvation. In: Modern Nutrition in Health and Disease, 8th ed. (Shils, M. E., Olson, J. A. & Shike, M., eds.) vol. 2. pp. 950-976. Lea & Febiger, Malvern, PA.
• Houge, G.,Vintermyr, O. K., Doskeland S. O. The expression of cAMP-dependent protein kinase subunits in primary rat hepatocyte cultures. Cyclic AMP down-regulates its own effector system by decreasing the amount of catalytic subunit and increasing the mRNAs for the inhibitor R subunits of cAMP-dependent protein kinase. Mol. Endocrinol. 1990; 4:481-488[Abstract/Free Full Text]
• Jungmann R. A., Russell D. H. Cyclic AMP-dependent protein kinase, and the regulation of gene expression. Life Sci. 1977; 20:1787-1798[Medline]
• Kemp B. E., Cheng H. C., Walsh D. A. Peptide inhibitors of cAMP-dependent protein kinase. Methods Enzymol. 1988; 159:173-183[Medline]
• Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu. Rev. Biochem. 1979; 48:923-959[Medline]
• Laemmli U. K. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (Lond.) 1970; 227:680-685[Medline]
• Maurer R. A. Transcriptional regulation of the prolactin gene by ergocryptine and cAMP. Nature (Lond.) 1981; 294:94-97[Medline]
• McKnight G. S., Cadd G. G., Clegg C. H., Otten A. D., Correll L. A. Expression of wild-type and mutant subunits of the cAMP dependent protein kinase. Cold Spring Harbor Symp. Quant. Biol. 1988; 53:111-119
• Meinkoth J. L., Ji Y., Taylor S. S., Feramisco J. R. Dynamics of the distribution of cAMP-dependent protein kinase in living cells. Proc. Natl. Acad. Sci. U.S.A. 1990; 87:9595-9599[Abstract/Free Full Text]
• Mellgren G., Vintermyr O. K., Doskeland S. O. Okadaic acid, cAMP, and selected nutrients inhibit hepatocyte proliferation at different stages in G1: modulation of the cAMP effect by phosphatase inhibitors and nutrients. J. Cell. Physiol. 1995; 163:232-240[Medline]
• Nigg E. A., Schafer G., Hilz H., Eppenberger H. M. cAMP dependent protein kinase type II is associated with the Golgi complex and with centrosomes. Cell 1985; 41:1039-1051[Medline]
• Otten A. D., McKnight G. S. Overexpression of type II regulatory subunit of the cAMP dependent protein kinase eliminates type I holoenzyme in mouse cells. J. Biol. Chem. 1989; 264:20255-20260[Abstract/Free Full Text]
• Otten A. D., Parenteau L. A., Doskeland S. O., McKnight G. S. Hormonal activation of gene transcription in ras-transformed NIH-3T3 cells overexpressing RII alpha and beta subunits of the cAMP-dependent protein kinase. J. Biol. Chem. 1991; 266:23074-23082[Abstract/Free Full Text]
• Rozwadowski M., Stephen L., Goss P., Bray T. M., Nagy L. E. Decreased activity of cAMP dependent protein kinase in protein energy malnourished rats. J. Nutr. 1995; 125:401-409
• Scott J. D. Cyclic nucleotide-dependent protein kinases. Pharmacol. Ther. 1991; 50:123-145[Medline]
• Stephen L. L., Nagy L. E. Very low protein diets induce a rapid decrease in hepatic cAMP dependent protein kinase followed by a slower increase in adenylyl cyclase activity in rats. J. Nutr. 1996; 126:1799-1807
• Stephen L. L., Nagy L. E. Very low protein diets decrease cAMP-mediated cytosolic and nuclear responses in isolated rat hepatocytes. J. Nutr. Biochem. 1997; 8:172-180
• Straus, D. S. & Takemoto, C. D. (1991) Specific decrease in liver insulin-like growth factor-I and brain insulin-like growth factor-II gene expression in energy-restricted rats. J.Nutr. 121: 1279-1286.
• Tortura G., Cho-Chung Y. S. Type II regulatory subunit of protein kinase restores cAMP-dependent transcription in a cAMP-unresponsive cell line. J. Biol. Chem. 1990; 265:18067-18070[Abstract/Free Full Text]
• Tortora G., Pepe S., Bianco C., Damiano V., Ruggiero A., Baldassarre G., Corbo C., Cho-Chung Y. S., Bianco A. R., Ciardiello F. Differential effects of protein kinase A subunits on Chinese-hamster ovary cell cycle proliferation. Int. J. Cancer 1994; 59:712-716[Medline]
• Torun, B. & Chew, F. (1994) Protein-energy malnutrition. In: Modern Nutrition in Health and Disease, 8th ed. (Shils, M. E., Olson, J. A. & Shike, M., eds.) vol. 2, pp. 927-949. Lea & Febiger, Malvern, PA.
• Vintermyr O. K., Boe R., Brulamd T., Houge G., Doskeland S. O. Elevated cAMP gives short-term inhibition and long-term stimulation of hepatocyte DNA replication: roles of the cAMP-dependent protein kinase subunits. J. Cell. Physiol. 1993; 156:160-170[Medline]
• Weber W., Hiltz H. cAMP-dependent protein kinase I and II: divergent turnover of subunits. Biochemistry 1986; 25:5661-5667[Medline]
• Wen W., Taylor S. S., Meinkoth J. L. The expression and intracellular distribution of the heat-stable protein kinase inhibitor is cell cycle regulated. J. Biol. Chem. 1995; 270:2041-2046[Abstract/Free Full Text]
• Whitehouse S., Walsh D. A. Purification of a physiological form of the inhibitor protein of the cAMP-dependent protein kinase. J. Biol. Chem. 1982; 257:6028-6032[Abstract/Free Full Text]

0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences