Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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The soluble protein content of brain, liver and leg skeletal muscle, measured per gram wet weight of tissues, did not change in 2 month estivated spadefoot toads, compared with control values. This indicates that, despite the loss of 35.7 % of total body water during the 2 month estivation (11), that the water content of individual organs was yet not perturbed. The maximal activities of 28 metabolic enzymes (listed in Materials and Methods) were assessed in these three tissues of control and 2 months estivated toads. Activities that changed significantly during estivation are shown in Tables 1-3 below.

Table 1. Significant changes in spadefoot toad brain enzyme activities during estivation (Student's t-test, P<0.05); data are U/mg protein, mean (SEM in brackets), n = 3-4.

         Control    2 mo Estivated   Percent change
PFK       57 (1.4)       43 (4.7)     25 % decrease
LDH     3134 (261)     1868 (178)     40 % decrease
CK       256 (9.3)      191 (15)      25 % decrease
G6PDH     88 (12.1)     150 (4.3)     70 % increase
GDH      7.7 (0.57)      24 (6.4)    220 % increase
BDH      8.4 (0.21)    12.4 (1.2)     50 % increase

1. Of 28 enzymes assayed, maximal activities of only 6 changed significantly in brain during 2 months estivation. Hence, the metabolic scope of brain was relatively unaltered by dormancy.

2. Reduced activities of the rate-limiting (PFK) and terminal (LDH) enzymes of glycolysis as well as CK, representing phosphagen metabolism, suggest suppressed energy metabolism during estivation.

3. GDH, an important enzyme of amino acid metabolism rose by 220 % during estivation. Increased GDH correlates with changes in the brain amino acid pool that suggest enhanced protein catabolism during estivation, with amino acids funneled via GDH to produce ammonium ion for urea synthesis. Urea levels rose by 200 mM during the 2 month dormancy.

4. Elevated BDH is consistent with increased ketone body metabolism indicating that during lipid-fueled long-term estivation, ketones may become an important fuel for brain.

Table 2. Significant changes in spadefoot toad liver enzyme activities during estivation (other information as in Table 1).

         Control    2 mo Estivated    Percent change
ME       4.3 (0.6)      10 (0.6)     140 % increase
GAPDH    123 (5.5)      92 (5.9)      25 % decrease
PK       117 (10.9)     91 (2.6)      22 % decrease
LDH     1943 (310)     772 (67)       60 % decrease
FBPase    13 (0.5)     9.7 (1.1)      25 % decrease
IDH      103 (7.4)      50 (8.2)      51 % decrease
GPT      317 (66)      187 (9.0)      41 % decrease
CK       8.7 (0.9)     4.3 (0.4)      51 % decrease
BDH      3.2 (0.3)     1.2 (0.08)     63 % decrease

1. The maximal activities of 9 enzymes changed significantly in liver during 2 months estivation.

2. Only ME activity increased during estivation. ME has an anaplerotic role in providing 4-carbon units (malate made from pyruvate) to the tricarboxylic acid cycle. These can be limiting when fatty acids (providing only 2-carbon acetyl-CoA) are the primary fuel and hence elevated ME would help to maintain optimal TCA cycle function.

3. Activities of three glycolytic enzymes, GAPDH, PK and LDH fell as did the percentage of glycogen phosphorylase in the active form (from 27.6 ± 3.33% to 16.8 ± 1.97%). These changes are consistent with carbohydrate sparing during dormancy.

Table 3. Significant changes in spadefoot toad leg skeletal muscle enzyme activities during estivation (other information as in Table 1).

       Enzymes that increased  %            Enzymes that decreased   %
       Control    Estivated                 Control     Estivated     
PFK     12 (0.2)    18 (0.3)   50    HK      58 (1.3)    28  (2.6)   52
GAPDH   39 (4.2)   233 (13)   500    CL      48 (16)      4  (0.6)   92
MDH-2  102 (13)    304 (28)   200    HOAD    75 (8.3)    22  (3.0)   70
IDH    106 (7.2)   250 (33)   136    FBP    4.8 (0.4)   2.2  (0.2)   54
ME      16 (0.5)    56 (1.6)  250    CPT     36 (1.2)    16  (4.6)   56
GPT     27 (1.5)    36 (4.6)   33    AK    3438 (425)  1914  (232)   44
GDH     17 (0.6)    41 (3.9)  145    MDH-1 1173 (158)   488  (62)    58
COT     36 (3.)     56 (3.5)   56    5'NT   8.4 (0.4)   0.93 (0.1)   89
ALD    872 (80)   1396 (102)   60    BDH    1.4 (0.5)   0.30 (0.05)  79
CS     437 (22)   2419 (273)  450    FAS    1.2 (0.1)   0.16 (0.03)  87
PK    2374 (84)   7527 (775)  217
CK    4121 (378)  8187 (492)   99
G6PDH  1.1 (0.13)  8.8 (1.1)  740
SDH    0.6 (0.04)  1.2 (0.2)  100

1. Enzymes in skeletal muscle were broadly affected during estivation with 14 activities increasing significantly and 10 decreasing.

2. Activities of enzymes of glycolysis (PFK, ALD, GAPDH, PK) increased during estivation but reduced HK and no change in glycogen phosphorylase argue against increased carbohydrate utilization. Perhaps enhanced glycolytic capacity, coupled with elevated CK (producing quick ATP from creatine phosphate) and CS (increasing the capacity for carbohydrate entry into the Krebs cycle), represent an preparatory change to support the high rates of muscular work that will be needed during the frenzied breeding and feeding that occur immediately when toads emerge from estivation.

3. Amino acid oxidation is facilitated in muscle of estivating toads by elevated activities of SDH, GPT and GDH. Elevated GDH, as in brain, suggests increased capacity for NH⁴⁺ production in support of urea synthesis by liver. As in brain, a common suite of changes in free amino acid levels was seen in muscle: glutamate, alanine and valine increased and glutamine decreased. Except for glutamine, the same pattern was seen in liver so it appears that these changes may be characteristic of anuran tissues when poised for urea synthesis from protein catabolism.

4. BDH activity was reduced in muscle of estivating toads and this may suggest that ketones are preferentially oxidized by selected tissues such as brain. Suppressed activities of enzymes involved in fatty acid synthesis (FAS, CL) are consistent with the starved state during estivation.

Table 4. Effect of estivation on cAMP-dependent protein kinase (PKA) in spadefoot toad tissues. Activities are mU (nmol phosphate transferred/min) per gram wet weight at 22°C, means ± SEM; n = 4. * - Significantly different from the corresponding control values, P < 0.05.

          Total PKA       % PKAc          %PKAc	
          mU/gww          Control         Estivated	
Brain     30.2 (4.53)     57.0  (8.0)     25.0  (3.0)*
Liver     9.56 (3.14)     97.0  (5.0)     62.0  (8.0)*
Muscle    6.27 (0.38)     80.0  (6.0)     47.7  (1.0)*

1. Total PKA activity was not affected during estivation but the percentage of PKA present as the active catalytic subunit (%PKAc) was strongly reduced in these 3 organs. %PKAc was also reduced by one-third to one-half in heart, lung, brain and gut of estivating toads (data not shown.

2. PKA is the primary protein kinase involved in the activation of fuel catabolism in vertebrate organs and its target proteins include regulatory enzymes of carbohydrate (GP, PFK, PDH, sometimes PK) and lipid catabolism. A reduced %PKAc is consistent with lower rates of catabolic pathways in estivating toads.

Table 5. Protein kinase C in tissues from control and estivating spadefoot toads. Other information as in Table 4.

          Total Activity, mU/gww        Percent Membrane Bound
          Control       Estivated       Control       Estivated  
Brain     40.0 (1.37)   32.7 (2.67)     45.8 (1.6)    21.5 (3.2)*
Liver     48.5 (6.05)   20.8 (4.00)*     6.6 (5.7)    16.4 (2.4)*
Muscle    0.31 (0.10)   0.17 (0.02)     29.6 (5.6)    29.8 (3.9)

1. Stimuli that elevate phosphatidylserine and Ca²⁺ levels cause translocation of PKC from inactive cytosolic pools to the plasma membrane where it becomes active. Hence, PKC activity state can be evaluated by determining the percentage of enzyme that is membrane-bound.

2. Percent membrane-bound PKC was reduced during estivation in brain and liver and total PKC also dropped by half in liver. The percent membrane-bound PKC also dropped in kidney from 71% in controls to 19 % in estivating toads (data not shown). Muscle, with very low PKC, showed no change in PKC distribution in estivation.

3. As for PKA, a reduced content of active PKC in organs is consistent with lower organ metabolic rate and the suppression of selected metabolic processes that are PKC-activated.

4. Results for both PKA and PKC suggest that a key factor in inducing and maintaining metabolic rate suppression during estivation is control over protein kinase-mediated signal transduction pathways.

Table 6. Protein phosphatase type-1 activities in spadefoot toad tissues. Other information as in Table 4.

         Total activity, mU/mg          % Active
         Control        Estivated       Control     Estivated
Brain    3.90  (0.77)   3.99 (0.03)      21 (3)     16 (1)
Liver    2.78  (0.13)   2.24 (0.08)*     15 (1)     26 (3)*
Muscle   2.33  (0.26)   2.80 (0.77)      20 (3)     13 (3)

Table 7. Protein phosphatase 2A and 2C activities in control and estivated spadefoot toads. Other information as in Table 4.

        PP-2A                       PP-2C
        Control      Estivated      Control       Estivated 
Brain   3.66 (0.48)  2.48 (0.17)    1.56 (0.20)   3.50 (0.25)*
Liver   2.62 (0.24)  1.85 (0.12)*   3.06 (0.43)   2.86 (0.19)
Muscle  4.08 (0.90)  3.08 (0.33)    2.75 (0.19)   4.23 (0.45)*

1. PP-1 exists in two forms - free, active catalytic subunit versus enzyme complexed with other proteins that mask its activity. This association is released by partial digestion with trypsin. This system of PP-1 control obviously occurs in toad tissues as trypsin treatment increased PP-1 activity in all tissues. Active PP-1 ranged from 13-26 % of total in the three tissues.

2. Estivation had little net effect on PP-1 in toad organs; significant changes in liver total and percent active PP-1 were oppositely directed. This lack of consistent changes in PP-1 during estivation suggests that control over the responsiveness of signal transduction pathways during estivation may lie primarily with the protein kinases.

3. Consistent or large changes in PP-2A or PP-2C were also lacking during estivation.

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