Effect of Starvation on Hepatic Glycogen Metabolism and Glucose Homeostasis David E. Goldstein The
effects
glycogen syktems
of
were 24-l
and
changes
plasma.
Fasting
in plasma
I and
subsequent with
and
with
decreases
in
and glycogen
hepatic
decreases in
phosphorylase
E
hr,
Fasting
and
increased
again
at
of glucose
progressive
disposal,
postglucose decreased
insulin basal
synthase
corre-
synthase activi-
phorylase fed rats. ship
rats,
between
phosphorylase
levels
although
were
synthase inactivation
cose in fed or fasted
and and
follow-
in 48-l
consistent
no
hr.
at 48-72
I increments
exaggerated
was
basal
concentrations,
decrements There
levels 120
impairment
decreased
glucagon
were
hr
increased fed
ing glucose
24
levels below
caused
hr-fasted
AMP
decreased
hr. Hepatic
glycogen
which
hepatic
at 96
cyclic
hr,
and
changes by
Hepatic
vein
insulin,
parallel
increases,
increases
were
measure-
in the portal
caused
glucose
in fed
glucose
glucose,
concentrations
concentrations lated
after
ties,
T. Curnow
at 24-48
Enzymic
rats.
cyclic AMP and
hepatic
assessed
simultaneous
of hepatic
concentrations
and
and
the
phosphorylase
20-hr-fasted with
glucagon
on
and
sequentially
before
correlated ments
starvation
synthase
and Randall
seen
clearcut
20phos-
only
in
relation-
activation
and
following
glu-
rats.
FFECTS OF LONG-TERM FASTING on both the basal activities and the responsiveness to glucose administration of the rate-limiting enzyme systems in hepatic glycogen synthesis and degradation are described. These hepatic enzyme systems are, respectively. glycogen synthase and glycogen phosphorylase.’ 4 These hepatic enzymes are acutely responsive to changes in insulin, glucagon, and glucose concentrations.“.’ The hepatic adenylate cyclase cyclic AMP system is thought to mediate some of these etTects.7.8 During long-term fasting, there are signiticant changes in insulin’ and glucagon secretion and in their molar ratios.‘,“.” While some reports have also described basal changes in the hepatic synthase and phosphorylase enzyme systems.“.” there have been no previously reported studies systematically correlating glucoregulatory hormone and cyclic nucleotide changes with fastinginduced alterations in both the responsiveness and basal activities of these enzyme systems. Such studies would provide important new information concerning the control of hepatic glycogen metabolism. Hepatic glycogen metabolism is thought to be important for glucose homeostasis in the fed state and in transition between feeding and fasting.‘“,” A physiologic role for hepatic glycogen metabolism during long-term fasting has
Mefabollsm,
Vol. 27,
No.
3 (March),
1978
316
GOLDSTEIN
AND
CURNOW
not been defined; however, partial reaccumulation of hepatic glycogen during long-term fasting, after initial rapid depletion, has been described.‘6,17 There have been few studies of the enzymatic mechanisms involved. In addition, the potential role of hepatic glycogen metabolism in the well-known glucose intolerance of fasting’* has not been investigated, in spite of reports emphasizing the importance of hepatic glycogen metabolism for glucose disposal.14 Accordingly, we have studied the effects of prolonged starvation in the rat on the basal activities and responsiveness to intravenous glucose administration of these hepatic enzyme systems. Simultaneous measurements of hepatic glycogen and cyclic AMP concentrations and glucose, insulin, and glucagon concentrations in the portal vein plasma were made throughout our study. MATERIALS
AND METHODS
Male Sprague-Dawley rats weighing 150.~200 g were used for all studies. Animals were maintained on Charles River Rat Formula (Agway) and water ad libitum. Rats were housed for I wk prior to studies in individual metabolic cages in a room with controlled temperature and lighting, Initially, I80 rats from a single consignment were studied. Rats were separated into fed and fasted groups. Fasted rats were studied at either 24, 48, 72, 96, or I20 hr following food withdrawal. On each day, both fed and fasted rats were randomly separated into three groups, baseline (time 0), I5 min. and 60 min after glucose injection. Basal and postglucose data were obtained from separate groups of IS fed and 668 fasted rats. Rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and placed on a heating pad to maintain normal body temperatures. In the rats given glucose, the left greater saphenous vein was exposed through a small inguinal incision and 50”; glucose (I g/kg) was injected over IO sec. A U-shaped abdominal incision was made and a 25-gauge needle inserted into the portal vein and passed to the level of the liver, where a 2-ml sample of blood was obtained at times indicated for measurement of glucose, insulin, and glucagon. Blood samples were immediately placed in chilled tubes containing 1.5 mg EDTA and 2000 units kallikrein inhibitor (Trasylol, FBA Pharmaceuticals, New York, N.Y.) in a volume of 0.2 ml. Blood samples were centrifuged at 4°C and the plasma stored at -20°C until assayed. Immediately after blood sampling, a liver lobe was rapidly excised and quick-frozen between liquid nitrogen cooled clamps (- 196°C). Hepatic tissue was stored at -65°C until assayed. Preparation and assay of hepatic tissue for glycogen synthase, glycogen phosphorylase. glycogen, protein, and cyclic AMP, as well as measurement of plasma samples for glucose, insulin, and glucagon, have been described previously in detaik6 Phosphorylase activities reported here were assayed in the presence of 3.3 mM AMP, which has been reported to measure phosphorylase a and a small proportion of phosphorylase h.13”19 Phosphorylase activities were also assayed in representative tissues without AMP and with 0.5 mM caffeine in the absence of AMP,19 and by the low-high-substrate method of Tan and Nuttall.’ No significant qualitative differences were observed between these different assay techniques. Total phosphorylase activities were measured in fed and 72-hr-fasted rats by incubating kinase and by the high-substrate assay.13 homogenates with ATP-Mg2+ and muscle phosphorylase In both fed and fasted rats phosphorylase activities measured in the presence of AMP represented activities. Enzyme activities of total glycogen synthase approximately 75”/, of total phosphorylase (synthase), synthase in the I form (synthase I), and phosphorylase are expressed as units (micromoles of glucose incorporated into glycogen per minute) per gram protein in the 12,000-g supernatant. Plasma glucagon concentrations were determined by radioimmunoassay using Unger’s 30K antibody (Diabetes Research Fund, University of Texas, Southwestern at Dallas) and a charcoal separation technique. In addition, pooled sera from fed and 72-hr-fasted rats were chromatographed on Bio-Gel P30 to compare elution patterns of glucagon immunoreactivity. Differences between mean values for each variable studied were analyzed for significance by Student’s t test for unpaired observations. Correlation coefficients between selected parameters were calculated using standard methods.20
EFFECT
317
OF STARVATION
RESULTS
Basal Changes Mean body weight decreased 32’,(> i 0.6”, after 120 hr of fasting; over the During the study, same time period it increased 26’1,, =t 0.99; in fed controls. mortality was 3 of 120 fasted rats, with one death occurring between 72 and 96 hr and two deaths between 96 and 120 hr. Rats were generally quite active throughout the study, although two 120-hr-fasted rats that were hypoglycemic demonstrated decreased spontaneous activity. Fasted rats tolerated anesthesia well. Figure 1 shows basal values for glucose, insulin, and glucagon concentrations and the molar ratios of insulin to glucagon (I/G) in the portal vein plasma in fed and fasted rats. Glucose concentrations fell from 173 k 5 to 115 + 6 mg/dl by 24 hr (p < 0.01) and then increased at 48-96 hr (p < 0.05 as compared to the 24-hr levels). At 120 hr the mean plasma glucose concentration fell to 24-hr levels because of hypoglycemia in two of eight rats (plasma glucose concentrations of 11 and 62 mg/dl). The mean plasma glucose concentration of the normoglycemic 120-hr-fasted rats was 139 * 7.5 mg/dl, not signiticantly different than levels in 48%96-hr groups. Insulin concentrations
Fig. 1. Effect of fasting on basal values of glucase, insulin, and glucogon and the molar ratios of insulin to glucagon in portal vein plasma. Points and vertical bars, means f SE, respectively, of 15 rats for the fed groups and 8 rats for each fasted group. Asterisks, p < 0.05 as compared to fed rats.
264
1v
2:
+*
0
24 HOURS
48
72 OF
FASIING
96
120
GOLDSTEIN
318
AND
CURNOW
gradually decreased during fasting, reaching very low but detectable levels by 72 hr. Glucagon concentrations decreased significantly at 48872 hr, then increased to fed levels at 96-120 hr. The I/G molar ratios were unchanged through 48 hr, reflecting simultaneously decreasing insulin and glucagon levels. Thereafter they fell sharply, paralleling the decreasing insulin concentrations. The fasting-induced decrements in plasma insulin concentrations described in the present study, have been well documented previously.’ For glucagon, we have no explanation for the discrepancies between the results presented here and those of other reports showing increased plasma glucagon concentrations during fasting.2’ However, in accord with our findings in vivo in the rat, studies of isolated pancreatic islets from fasted rats have shown both decreased insulin of elution patterns and glucagon secretion. 22 In the present study, comparisons of glucagon immunoreactivity on Bio-Gel P30 between pooled sera from fed and 72-hr-fasted rats revealed no differences (data not shown). Figure 2 shows basal values for hepatic glycogen and cyclic AMP concen-
0
24 HOURS
48
72 OF
FASTING
96
120
Effect of fasting on basal values of heFig. 2. patic glycogen and cyclic AMP concentrations and hepatic enzyme activities of total glycogen synthase, the active form of glycogen synthase (glycogen synthase I), and glycogen phosphorylase. Points ond vertical bars, means & SE, respectively, of 15 rats for the fed control group and 8 rats for each fosted group. Asterisks, p c 0.05 as compared to fed rats.
319
EFFECT OF STARVATION
trations and the hepatic enzyme activities of total glycogen synthase, glycogen synthase in the I form, and glycogen phosphorylase in fed and fasted rats. Total hepatic glycogen synthase activities did not change during fasting, but synthase I activities increased significantly except at 48 hr (p = 0.12) and 96 hr (p = 0.13). In contrast, hepatic phosphorylase activities were decreased throughout fasting: activities fell progressively, reaching a plateau at approximately .50”,, of fed levels by 72 hr. Hepatic glycogen concentrations decreased from 20.7 * 0.9 to 0.7 & 0.2 mg/g wet weight by 24 hr, then increased to approximately 25”,, of fed levels between 48 and 120 hr (p < 0.01 as compared to 24-hr levels). The two hypoglycemic 120-hr-fasted rats had no measurable hepatic glycogen. Hepatic cyclic AMP concentrations showed a triphasic pattern with fasting: concentrations increased at 24 48 hr, decreased below fed levels at 96 hr, and increased again at 120 hr. In individual fed and fasted rats. hepatic cyclic AMP levels did not correlate with any measured parameters, including hepatic phosphorylase activities and plasma glucagon concentrations. Efltct of’ Glucose Administration Table I shows the values for glucose, insulin, and glucagon concentrations and the molar ratios of insulin to glucagon in the portal vein plasma in response to intravenous glucose in fed and fasted rats. Glucose concentrations I5 min after glucose administration progressively increased during fasting; in 120-hrfasted rats the increments from baseline were approximately threefold greater than in fed rats. However, glucose concentrations returned to baseline levels
Table 1. Portal Vein Plasma Values Before and After Glucose Administration to Fed and Fasted Rats Fasting (hr) 0 (Fed)
24
48
72
96
120
Plasma glucose, mg/dl 173&5
115 +6
134 zt6
140 zt7
147 +5
15
0
249 f 23*
214 f 27*
271 f
289 f 25*
363 f
60
135 &4*
162 f
153 * 5*
121 f
136 zt9
la*
ll*
16
17*
114 i
18
341 f
16*
112 +7
Plasma insulin, fiU/ml 0
99 l 7
15
167 f
19*
60
145 ziz 13*
71 *22
60 f
a
24 & 8
15 * 1
16 zt6
134 + 25
65 f
14
49 f
12
46 zt 9’
35 * 14
175 i 26*
75 l 12
38 i
18
39 * 7*
35 i
107 + 14
140 i
14
10
Plasma glucagon, pg/ml 199 * 14
175 i
38
15
0
131 i22*
332 f
129
93 + 20
90 &22
60
488 f
294 zt 142
183 i 52
353 f 68*
144*
197 i
33
372 i
158 f 54
11014
882 f
843 i 350
198*
170
Insulin/glucagon molar ratios 10.7 f 2.6
i 5.8
i 4.4
5.0 * 2.2
2.3 i 0.4
2.0 l 0.8
15
48.8 * 12.6*
32.1 f 8.6*
la.3 + 3.7
17 f 6.3
9.5 zt 2.1*
7.0 f 2.5
60
13.6 f 3.1
33.8 * 10.6
12.3 zt 3.1
4.1 f 2.3
1 s * 0.3
3.8 + 1.7
0
Intravenous times
indicated
12.7 f
glucose in
1.5
(1 g/kg) was injected and blood samples were obtained from the portal vein at the
separate groups of fed and fasted rots. Means f SE with 15 rats for each fed group
and 6-8 rats for each fasted group.
lp Fig. 1.
< 0.05 os compared to time 0 within each group. For baseline intergroup statistical comparisons see
320
GOLDSTEIN
Table 2. Hepatic Tissue Values Before and After Glucose Administration Minutes Following
AND
CURNOW
to Fed and Fasted Rats
Fasting (hr) 0 (Fed)
GlUCOS.2
24
48
72
96
120
Hepotic glycogen, mg/g wet wt 0
20.7 f
1.0
0.7 f 0.2
6.1 zk 1.8
3.7 f
6.4 zt 1.1
4.1 zt 1.8
15
19.2 f
1.8
2.1 * 0.5*
3.8 i
0.9
a.2 + o.a*
1.1
8.5 f
1.2
4.5 zt 2.3
60
18.0 zt 1.6
2.7 f 0.89
5.3 zt 1.7
6.0 zt 1.5
7.3 f
1.5
4.8 i
9.2 i
1.4
12.9
f
zt 3.7*
31.1
* 5.0*
1.5
Hepatic glycogen synthose I, percent of total 0
11.7 * 2.8
13.9
* 2.2
zt2.1”
26.4
f 8.1
34.5
*
39.2
+z 8.6*
5.9 f 0.6
13.8
zt 4.1
16.4
zt 2.1
15.4
zk 6.1
7.5 f 0.9
15
11.7
60
11.8 I
1.9
1.9*
47.6
9.6 +z 1.4
2.2
9.2 + 1.0
Hepotic phosphorylase, U/g protein 0
97.2 zt 4.3
64.4 zk .38
50.7 zt 4.3
49.2
zt 4.2
49.1
+ 3.7
49.4
i
15
71.6 zk 4.3*
66.5 zt 7.6
54.1 zk 2.5
50.4
& 7.6
45.4
+ 4.9
49.4
+ 4.2
60
97.3 zt
67.4
58.3
50.7
rt 4.5
64.5
& 5.5
58.8
zt 6.9
Hepatic
cyclic AMP,
8.8
pmoles/mg
j, 5.8
jz 4.2
5.2
wet wt
0
0.16
& 0.01
0.26
i
0.02
0.35
* 0.03
0.20
* 0.03
0.12
f 0.01
0.40
zt 0.10
15
0.17
zt 0.02
0.22
zt 0.02
0.28
i
0.03
0.16
zt 0.04
0.11
l
0.01
0.22
+ 0.03
60
0.22
f 0.04
0.33
+ 0.03*
0.26
z!z 0.04
0.23
zt 0.03
0.19
i
0.03
0.22
zt 0.03
Intravenous glucose (1 g/kg) indicated
in separate
rots for each fasted *p < 0.05
groups
WCIS injected of fed and
and
fasted
blood
samples
rats. Means
obtained
from
the portal
=t SE with 15 rats for each
vein at the times
fed group
and 6-8
group.
(IS compared
to time 0 within
each group.
For baseline
intergroup
statistical
comparisons
see
Fig. 2.
by 60 min except at 24 and 48 hr. In contrast, insulin responses to glucose progressively decreased during fasting. Glucagon concentration decrements after glucose were seen only in fed rats, while I/G molar ratio increments progressively decreased, paralleling the insulin responses. Table 2 shows the values for hepatic glycogen and cyclic AMP concentrations and the enzyme activities of hepatic glycogen synthase in the I form and glycogen phosphorylase in response to glucose administration in fed and fasted rats. Total hepatic glycogen synthase activities did not change after glucose in fed or fasted rats (data not shown). However, synthase I activities were increased significantly from baseline at 1.5 min in all fed and fasted groups except at 24 hr (p < 0.06). The increments were much greater in 48-120-hr-fasted rats than in fed rats, with intermediate values in 24-hr-fasted rats. Enzyme activities returned to baseline levels by 60 min in all study groups. The relation between plasma glucose levels and synthase I activities after glucose administration was examined to determine if the apparent increased synthase responsiveness in 48-120-hr-fasted rats was simply a manifestation of higher plasma glucose levels. In fed and 24-hr-fasted rats there was no correlation between these parameters. However, there was a strong correlation in 48120-hr-fasted rats (p < 0.05). After glucose administration, phosphorylase activities were decreased only in fed rats. However, even in fed rats individual phosphorylase activity decrements did not correlate with either synthase I or plasma glucose increments. Following glucose administration, hepatic glycogen concentrations increased significantly only at 24 and 72 hr of fasting. Hepatic cyclic AMP concentrations
EFFECT
OF
321
STARVATION
did not change after glucose administration, which increases were seen at 60 min (Table 2).
except in 24-hr-fasted
rats.
in
DISCUSSION
In this report we present data depicting the effect of prolonged fasting in the rat on both the basal activities and the response to intravenous glucose of the hepatic glycogen synthase and phosphorylase enzyme systems. Results were correlated with fasting-induced changes in hepatic cyclic AMP levels and other hormonal and nonhormonal factors with known important hepatic actions. The data show striking fasting-induced changes in these hepatic enzyme systems.
By 24 hr of fasting, hepatic glycogen concentrations reached very low levels but increased thereafter to approximately 25”,, of fed levels. Although the physiologic significance of hepatic glycogen reaccumulation is presently unknown. such increases in storage carbohydrate could be important for glucose homeostasis and rapid energy mobilization during prolonged fasting. While the current studies did not address the mechanisms of the initial rapid hepatic glycogen concentration decrements, the subsequent glycogen reaccumulation was associated with changes in hepatic synthase I and phosphorylase activities that would be expected to favor glycogen synthesis. Isolated hepatocytes from fasted rats show similar enzymatic changes in association with rapid glycogen deposition.” The mechanisms of the fasting-promoted enzyme changes are not well understood, but it is possible that the release of inhibition by glycogen of the synthase and phosphorylase phosphatase systems plays a role in the changes seen,“’ since high levels of glycogen, as found in fed rats, are reported to inhibit these synthase-activating and phosphorylase-inactivating enzymes.“,‘h The results indicate that the relationships between the fasting-induced changes in the enzyme activities, plasma insulin, and glucagon concentrations, or their molar ratios, and changes in hepatic cyclic AMP levels are extremely complex and frequently paradoxic. While there are no obvious relationships betwcen hepatic cyclic AMP concentrations and glycogen synthase and phosphorylase activities during fasting, the present studies may not have detected physiologically important changes in subcellular distribution of the cyclic nucleotide that could have mediated some of the changes in enLyme activities.” Gluc,ose ,4 dministration The present data show that during long-term fasting there is exaggerated responsiveness ofthe hepatic synthase enzyme system in vivo to intravenous glucose administration. The mechanism of this altered synthase responsiveness to glucose, which has also been recently observed in isolated hepatocytes,?’ is unknown. However, it is conceivable that the hepatic synthase phosphatase is more responsive to glucose during starvation, when glycogen levels are low.‘” x In accord with this thesis. Curnow et al. 28 have recently shown that glucosemediated activation of hepatic synthase phosphatase could be consistently demonstrated only in fasted rats.
322
GOLDSTEIN
AND CURNOW
Our results indicate that following glucose administration, increases in hepatic synthase I activities throughout fasting are independent of any concomitant decreases in hepatic phosphorylase activities. Synthase activation independent of phosphorylase decrements has also been shown in the rat following oral glucose administration.29 These data suggest that glucose-mediated activation of synthase need not be secondary to glucose effects on phosphorylase, as proposed by Hers and co-workers. ‘,I5 An alternate mechanism for glucose effects on these systems has been suggested by Gilboe and Nuttall. They have shown that glucose may directly promote synthase activation by deinhibiting ATP inhibition of synthase phosphatase. In contrast to the increased responsiveness of the hepatic synthase enzyme system during fasting, plasma insulin responses to glucose were progressively impaired. These data indicate, as previously shown in isolated perfused liver,31 that acute insulin secretion following glucose administration probably does not play an important role in hepatic synthase activation in vivo. The present data demonstrate progressive glucose intolerance with fasting. However, the data indicate that the liver may not play an important role in this phenomena. Neither fed nor fasted rats showed consistent increases in hepatic glycogen levels following intravenous glucose administration. These findings are compatible with recent studies indicating that a major proportion of intravenously administered glucose is taken up by skeletal muscle and that during starvation skeletal muscle glucose uptake is impaired, presumably contributing since increased responsivesignificantly to glucose intolerance. 3z Furthermore, ness of the hepatic synthase enzyme system during fasting would tend to favor hepatic glucose uptake and glycogenesis, it appears unlikely that altered hepatic glycogen metabolism plays any significant role in the glucose intolerance of fasting. In conclusion, the present data depict a systematic assessment of the alterations in the enzyme systems’ rate limiting for hepatic glycogen synthesis and degradation during prolonged fasting in the rat. The results emphasize the complexity of the interrelationships of the hormonal and nonhormonal factors influencing hepatic glycogen metabolism in vivo. Additional studies will be required to clearly define the mechanisms and physiologic significance of these fastinginduced metabolic alterations. ACKNOWLEDGMENT We would like to thank Dr. J. Larner, Dr. F. Murad, and Dr. S. Pohl for reviewing this manuscript. The authors also acknowledge the expert technical assistance of Gwenn H. Clark and Herman Hernandez and the aid of Suellen Lakes and Dana Bolyard in the preparation of this manuscript.
REFERENCES 1. Hers HG, Stalmans W, De Wulf H, et al: The control of glycogen metabolism in the liver, in Fischer EH (ed): Metabolic Interconversion of Enzymes. Berlin. Springer. 197.7. pp 89-98 2. Huijing F: Glycogen metabolism and glycogen-storage diseases. Physiol Rev 55: 609m 658, 1975 3. Hinukuri S, Larner J: Studies on UDPG:
n-1,4-glucan a-4 glucosyltransferase. VII. Conversion of the enzyme from glucose-6-phosphate-dependent to independent form in liver. Biochemistry 3: I783- 1788. 1963 4. Sutherland EW, Cori CF: Effect of hyperglycemic-glycogenolytic factor and epinephrine on liver phosphorylase. J Biol Chem 188:531543.1951 5. Bishop JS, Goldberg ND, Lamer J: In-
EFFECT
323
OF STARVATION
sulin regulation of hepatic glycogen metabolism m the dog. Am J Physiol220:4999506, 1971 6. Curnow RT, Rayfield EJ. George DT, et al: Control of hepatic glycogen metabolism in the rhesus monkey: Effect of glucose, insulin and glucagon administration. Am J Physiol 228:80--88, 1975 7. Exton JH. Lewis SB. Ho RJ, et al: The role of cyclic AMP in the interaction of glucagon and insulin m the control of liver metabolism. Ann NY Acad Sci 185:85-100, 1971 8. Lavine RL. Voyles N. Perrino PV, et al: The erect of fasting on tissue cyclic AMP and plasma glucagon in the obese hyperglycemic mouse. Endocrinology 97:615-620, 1975 9. Grey NJ, Goldring S, Kipnis DM: The effect of fasting, diet, and actinomycin D on insulin secretion in the rat. J Clin Invest 49: 881-889, 1970 10. Fisher M, Sherwin RS, Hendler R. et al: Kinettcs of glucagon in man: Effects of starvation. Proc Nat1 Acad Sci USA 73:1735-1739. I976 I I. Unger RH: Alpha- and beta-ceil interrelationships in health and disease. Metabolism 2358 1-593,1974 12. Curnow RT, Nuttall FQ: Effect of prostaglandin Et administration on the liver glycogen synthase and phosphorylase systems. J Biol Chrm 247:1892 -1898. 1972 13. Tan A. Nuttall FQ: Characteristics of the dephosphorylated form of phosphorylase purltied from rat liver and measurement of its activity tn crude liver preparations. Biochim Biophys Acta 410:45560, 1975 14. Felig P: The liver in glucose homeostasis in normal man and in diabetes, in VallanceOwen J (ed): The Physiological and Biochemical Basis of Dtabetes. London, Medical and Technical Publishmg, 1975 IS. Hers HG: The control of glycogrn metabolism in the liver. Annu Rev Biochem 45: 167m 189. 1976 16. Hershey JM, Orr MD: The removal of glycogen from living muscle. Trans R Sot Can (Set V] 22:151~ 157, 1928 17. Pfliiger E: Ueber den ein Russ einsertrger ernahrting oder nahrungs mangels aug den glykogengchalt des thierischen kiirpers. Pfltigers Arch Ges Physiol I 19:117~126. 1907 18. Cahill GF Jr, Henera MG, Morgan AP, et al: Hormone fuel interrelationships during fasttng. J Clin Invest 45:1751-1769, 1966 19. Stalmans W. Hers HG: The stimuiation ofliver phosphorylase b by AMP, fluoride and sulfate. Eur J Biochem 54:341-350. 1975
20. Snedecor
GW,
Cochran
WG:
Statistical
Methods (ed 6). Ames, lowa. Iowa State Univ Pr, 1967 21. Marliss EB, Aoki TT. Unger RH. et al: Glucagon levels and metabolic effects in fasting man. J Clin Invest 49:2256-2270, 1970 22. Buchanan KD, Vance JE, Williams RH: Effects of starvation on insulin and glucagon release from isolated Islets of Langerhans of the rat. Metabolism l8:155-162, 1969 23. Hue L, Bontemps F, Hers HG: The effect ofglucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations. Biochem J 152: IO551 14. 1975 24. Hutson NY. Brurnley FT. Arsimacopou10s FD. et al: Studies on the a-adrenrrgic acttvation of hepatic glucose output. 1. Studtrs on the ar-adrenergic activation of phosphorylasr and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells. J Biol Chem 251:5200~5208. 1976 25. Abe N. Tsukik S: Factors affectmg the activity of glycogen synthase D phosphatasr from rat liver in vitro. Sci Rep Res lnst Tohoku Univ [Med] 21:17-25. I974 26. Kato K. Bishop JS: Glycogen synthetase-D phosphatase. I. Some new properties ol the partially purified enzyme from rabbit skeletal muscle. J Biol Chem 247:7420- 7429, 1972 27. Terasaki WL. Brooker G: Cardiac adenosme 3’5.monophosphate. Free and bound forms in the isolated rat atrium. J Biol Chem 252:1041-1050, 1977 28. Curnow RT, Rowe JN, Goldstem DE: Role of phosphoprotein phosphatase activjation in the effect of glucose on liver. Diabetes 26 [Suppl 1]:393. 1977 29. Nuttall FQ, Cannon MC. Lamer J: Oral glucose effect on glycogen synthetase and phosphorylase in heart, muscle and liver. Physiol Chem Phys 41497.-515, 1972 30. Gilboe DP, Nuttall FQ: The regulatton of liver glycogen synthetase D phosphatase by ATP and glucose. Biochem Biophys Res Commun 53:164~171, 1973 31. Buschiazzo HJ. Exton JH. Park CR: Effects of glucose on glycogen synthrtasc. phosphorylase and glycogen deposition m the perfused rat liver. Proc Natl Acad Sci USA 65:3833387, 1970 32. Daniel PM. Love ER, Pratt OE: Insultnstimulated entry of glucose into muscle in viva as a major factor tn the regulation of blood glucose. J Physiol 247:273-288, 1975
effects
glycogen syktems
of
were 24-l
and
changes
plasma.
Fasting
in plasma
I and
subsequent with
and
with
decreases
in
and glycogen
hepatic
decreases in
phosphorylase
E
hr,
Fasting
and
increased
again
at
of glucose
progressive
disposal,
postglucose decreased
insulin basal
synthase
corre-
synthase activi-
phorylase fed rats. ship
rats,
between
phosphorylase
levels
although
were
synthase inactivation
cose in fed or fasted
and and
follow-
in 48-l
consistent
no
hr.
at 48-72
I increments
exaggerated
was
basal
concentrations,
decrements There
levels 120
impairment
decreased
glucagon
were
hr
increased fed
ing glucose
24
levels below
caused
hr-fasted
AMP
decreased
hr. Hepatic
glycogen
which
hepatic
at 96
cyclic
hr,
and
changes by
Hepatic
vein
insulin,
parallel
increases,
increases
were
measure-
in the portal
caused
glucose
in fed
glucose
glucose,
concentrations
concentrations lated
after
ties,
T. Curnow
at 24-48
Enzymic
rats.
cyclic AMP and
hepatic
assessed
simultaneous
of hepatic
concentrations
and
and
the
phosphorylase
20-hr-fasted with
glucagon
on
and
sequentially
before
correlated ments
starvation
synthase
and Randall
seen
clearcut
20phos-
only
in
relation-
activation
and
following
glu-
rats.
FFECTS OF LONG-TERM FASTING on both the basal activities and the responsiveness to glucose administration of the rate-limiting enzyme systems in hepatic glycogen synthesis and degradation are described. These hepatic enzyme systems are, respectively. glycogen synthase and glycogen phosphorylase.’ 4 These hepatic enzymes are acutely responsive to changes in insulin, glucagon, and glucose concentrations.“.’ The hepatic adenylate cyclase cyclic AMP system is thought to mediate some of these etTects.7.8 During long-term fasting, there are signiticant changes in insulin’ and glucagon secretion and in their molar ratios.‘,“.” While some reports have also described basal changes in the hepatic synthase and phosphorylase enzyme systems.“.” there have been no previously reported studies systematically correlating glucoregulatory hormone and cyclic nucleotide changes with fastinginduced alterations in both the responsiveness and basal activities of these enzyme systems. Such studies would provide important new information concerning the control of hepatic glycogen metabolism. Hepatic glycogen metabolism is thought to be important for glucose homeostasis in the fed state and in transition between feeding and fasting.‘“,” A physiologic role for hepatic glycogen metabolism during long-term fasting has
Mefabollsm,
Vol. 27,
No.
3 (March),
1978
316
GOLDSTEIN
AND
CURNOW
not been defined; however, partial reaccumulation of hepatic glycogen during long-term fasting, after initial rapid depletion, has been described.‘6,17 There have been few studies of the enzymatic mechanisms involved. In addition, the potential role of hepatic glycogen metabolism in the well-known glucose intolerance of fasting’* has not been investigated, in spite of reports emphasizing the importance of hepatic glycogen metabolism for glucose disposal.14 Accordingly, we have studied the effects of prolonged starvation in the rat on the basal activities and responsiveness to intravenous glucose administration of these hepatic enzyme systems. Simultaneous measurements of hepatic glycogen and cyclic AMP concentrations and glucose, insulin, and glucagon concentrations in the portal vein plasma were made throughout our study. MATERIALS
AND METHODS
Male Sprague-Dawley rats weighing 150.~200 g were used for all studies. Animals were maintained on Charles River Rat Formula (Agway) and water ad libitum. Rats were housed for I wk prior to studies in individual metabolic cages in a room with controlled temperature and lighting, Initially, I80 rats from a single consignment were studied. Rats were separated into fed and fasted groups. Fasted rats were studied at either 24, 48, 72, 96, or I20 hr following food withdrawal. On each day, both fed and fasted rats were randomly separated into three groups, baseline (time 0), I5 min. and 60 min after glucose injection. Basal and postglucose data were obtained from separate groups of IS fed and 668 fasted rats. Rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and placed on a heating pad to maintain normal body temperatures. In the rats given glucose, the left greater saphenous vein was exposed through a small inguinal incision and 50”; glucose (I g/kg) was injected over IO sec. A U-shaped abdominal incision was made and a 25-gauge needle inserted into the portal vein and passed to the level of the liver, where a 2-ml sample of blood was obtained at times indicated for measurement of glucose, insulin, and glucagon. Blood samples were immediately placed in chilled tubes containing 1.5 mg EDTA and 2000 units kallikrein inhibitor (Trasylol, FBA Pharmaceuticals, New York, N.Y.) in a volume of 0.2 ml. Blood samples were centrifuged at 4°C and the plasma stored at -20°C until assayed. Immediately after blood sampling, a liver lobe was rapidly excised and quick-frozen between liquid nitrogen cooled clamps (- 196°C). Hepatic tissue was stored at -65°C until assayed. Preparation and assay of hepatic tissue for glycogen synthase, glycogen phosphorylase. glycogen, protein, and cyclic AMP, as well as measurement of plasma samples for glucose, insulin, and glucagon, have been described previously in detaik6 Phosphorylase activities reported here were assayed in the presence of 3.3 mM AMP, which has been reported to measure phosphorylase a and a small proportion of phosphorylase h.13”19 Phosphorylase activities were also assayed in representative tissues without AMP and with 0.5 mM caffeine in the absence of AMP,19 and by the low-high-substrate method of Tan and Nuttall.’ No significant qualitative differences were observed between these different assay techniques. Total phosphorylase activities were measured in fed and 72-hr-fasted rats by incubating kinase and by the high-substrate assay.13 homogenates with ATP-Mg2+ and muscle phosphorylase In both fed and fasted rats phosphorylase activities measured in the presence of AMP represented activities. Enzyme activities of total glycogen synthase approximately 75”/, of total phosphorylase (synthase), synthase in the I form (synthase I), and phosphorylase are expressed as units (micromoles of glucose incorporated into glycogen per minute) per gram protein in the 12,000-g supernatant. Plasma glucagon concentrations were determined by radioimmunoassay using Unger’s 30K antibody (Diabetes Research Fund, University of Texas, Southwestern at Dallas) and a charcoal separation technique. In addition, pooled sera from fed and 72-hr-fasted rats were chromatographed on Bio-Gel P30 to compare elution patterns of glucagon immunoreactivity. Differences between mean values for each variable studied were analyzed for significance by Student’s t test for unpaired observations. Correlation coefficients between selected parameters were calculated using standard methods.20
EFFECT
317
OF STARVATION
RESULTS
Basal Changes Mean body weight decreased 32’,(> i 0.6”, after 120 hr of fasting; over the During the study, same time period it increased 26’1,, =t 0.99; in fed controls. mortality was 3 of 120 fasted rats, with one death occurring between 72 and 96 hr and two deaths between 96 and 120 hr. Rats were generally quite active throughout the study, although two 120-hr-fasted rats that were hypoglycemic demonstrated decreased spontaneous activity. Fasted rats tolerated anesthesia well. Figure 1 shows basal values for glucose, insulin, and glucagon concentrations and the molar ratios of insulin to glucagon (I/G) in the portal vein plasma in fed and fasted rats. Glucose concentrations fell from 173 k 5 to 115 + 6 mg/dl by 24 hr (p < 0.01) and then increased at 48-96 hr (p < 0.05 as compared to the 24-hr levels). At 120 hr the mean plasma glucose concentration fell to 24-hr levels because of hypoglycemia in two of eight rats (plasma glucose concentrations of 11 and 62 mg/dl). The mean plasma glucose concentration of the normoglycemic 120-hr-fasted rats was 139 * 7.5 mg/dl, not signiticantly different than levels in 48%96-hr groups. Insulin concentrations
Fig. 1. Effect of fasting on basal values of glucase, insulin, and glucogon and the molar ratios of insulin to glucagon in portal vein plasma. Points and vertical bars, means f SE, respectively, of 15 rats for the fed groups and 8 rats for each fasted group. Asterisks, p < 0.05 as compared to fed rats.
264
1v
2:
+*
0
24 HOURS
48
72 OF
FASIING
96
120
GOLDSTEIN
318
AND
CURNOW
gradually decreased during fasting, reaching very low but detectable levels by 72 hr. Glucagon concentrations decreased significantly at 48872 hr, then increased to fed levels at 96-120 hr. The I/G molar ratios were unchanged through 48 hr, reflecting simultaneously decreasing insulin and glucagon levels. Thereafter they fell sharply, paralleling the decreasing insulin concentrations. The fasting-induced decrements in plasma insulin concentrations described in the present study, have been well documented previously.’ For glucagon, we have no explanation for the discrepancies between the results presented here and those of other reports showing increased plasma glucagon concentrations during fasting.2’ However, in accord with our findings in vivo in the rat, studies of isolated pancreatic islets from fasted rats have shown both decreased insulin of elution patterns and glucagon secretion. 22 In the present study, comparisons of glucagon immunoreactivity on Bio-Gel P30 between pooled sera from fed and 72-hr-fasted rats revealed no differences (data not shown). Figure 2 shows basal values for hepatic glycogen and cyclic AMP concen-
0
24 HOURS
48
72 OF
FASTING
96
120
Effect of fasting on basal values of heFig. 2. patic glycogen and cyclic AMP concentrations and hepatic enzyme activities of total glycogen synthase, the active form of glycogen synthase (glycogen synthase I), and glycogen phosphorylase. Points ond vertical bars, means & SE, respectively, of 15 rats for the fed control group and 8 rats for each fosted group. Asterisks, p c 0.05 as compared to fed rats.
319
EFFECT OF STARVATION
trations and the hepatic enzyme activities of total glycogen synthase, glycogen synthase in the I form, and glycogen phosphorylase in fed and fasted rats. Total hepatic glycogen synthase activities did not change during fasting, but synthase I activities increased significantly except at 48 hr (p = 0.12) and 96 hr (p = 0.13). In contrast, hepatic phosphorylase activities were decreased throughout fasting: activities fell progressively, reaching a plateau at approximately .50”,, of fed levels by 72 hr. Hepatic glycogen concentrations decreased from 20.7 * 0.9 to 0.7 & 0.2 mg/g wet weight by 24 hr, then increased to approximately 25”,, of fed levels between 48 and 120 hr (p < 0.01 as compared to 24-hr levels). The two hypoglycemic 120-hr-fasted rats had no measurable hepatic glycogen. Hepatic cyclic AMP concentrations showed a triphasic pattern with fasting: concentrations increased at 24 48 hr, decreased below fed levels at 96 hr, and increased again at 120 hr. In individual fed and fasted rats. hepatic cyclic AMP levels did not correlate with any measured parameters, including hepatic phosphorylase activities and plasma glucagon concentrations. Efltct of’ Glucose Administration Table I shows the values for glucose, insulin, and glucagon concentrations and the molar ratios of insulin to glucagon in the portal vein plasma in response to intravenous glucose in fed and fasted rats. Glucose concentrations I5 min after glucose administration progressively increased during fasting; in 120-hrfasted rats the increments from baseline were approximately threefold greater than in fed rats. However, glucose concentrations returned to baseline levels
Table 1. Portal Vein Plasma Values Before and After Glucose Administration to Fed and Fasted Rats Fasting (hr) 0 (Fed)
24
48
72
96
120
Plasma glucose, mg/dl 173&5
115 +6
134 zt6
140 zt7
147 +5
15
0
249 f 23*
214 f 27*
271 f
289 f 25*
363 f
60
135 &4*
162 f
153 * 5*
121 f
136 zt9
la*
ll*
16
17*
114 i
18
341 f
16*
112 +7
Plasma insulin, fiU/ml 0
99 l 7
15
167 f
19*
60
145 ziz 13*
71 *22
60 f
a
24 & 8
15 * 1
16 zt6
134 + 25
65 f
14
49 f
12
46 zt 9’
35 * 14
175 i 26*
75 l 12
38 i
18
39 * 7*
35 i
107 + 14
140 i
14
10
Plasma glucagon, pg/ml 199 * 14
175 i
38
15
0
131 i22*
332 f
129
93 + 20
90 &22
60
488 f
294 zt 142
183 i 52
353 f 68*
144*
197 i
33
372 i
158 f 54
11014
882 f
843 i 350
198*
170
Insulin/glucagon molar ratios 10.7 f 2.6
i 5.8
i 4.4
5.0 * 2.2
2.3 i 0.4
2.0 l 0.8
15
48.8 * 12.6*
32.1 f 8.6*
la.3 + 3.7
17 f 6.3
9.5 zt 2.1*
7.0 f 2.5
60
13.6 f 3.1
33.8 * 10.6
12.3 zt 3.1
4.1 f 2.3
1 s * 0.3
3.8 + 1.7
0
Intravenous times
indicated
12.7 f
glucose in
1.5
(1 g/kg) was injected and blood samples were obtained from the portal vein at the
separate groups of fed and fasted rots. Means f SE with 15 rats for each fed group
and 6-8 rats for each fasted group.
lp Fig. 1.
< 0.05 os compared to time 0 within each group. For baseline intergroup statistical comparisons see
320
GOLDSTEIN
Table 2. Hepatic Tissue Values Before and After Glucose Administration Minutes Following
AND
CURNOW
to Fed and Fasted Rats
Fasting (hr) 0 (Fed)
GlUCOS.2
24
48
72
96
120
Hepotic glycogen, mg/g wet wt 0
20.7 f
1.0
0.7 f 0.2
6.1 zk 1.8
3.7 f
6.4 zt 1.1
4.1 zt 1.8
15
19.2 f
1.8
2.1 * 0.5*
3.8 i
0.9
a.2 + o.a*
1.1
8.5 f
1.2
4.5 zt 2.3
60
18.0 zt 1.6
2.7 f 0.89
5.3 zt 1.7
6.0 zt 1.5
7.3 f
1.5
4.8 i
9.2 i
1.4
12.9
f
zt 3.7*
31.1
* 5.0*
1.5
Hepatic glycogen synthose I, percent of total 0
11.7 * 2.8
13.9
* 2.2
zt2.1”
26.4
f 8.1
34.5
*
39.2
+z 8.6*
5.9 f 0.6
13.8
zt 4.1
16.4
zt 2.1
15.4
zk 6.1
7.5 f 0.9
15
11.7
60
11.8 I
1.9
1.9*
47.6
9.6 +z 1.4
2.2
9.2 + 1.0
Hepotic phosphorylase, U/g protein 0
97.2 zt 4.3
64.4 zk .38
50.7 zt 4.3
49.2
zt 4.2
49.1
+ 3.7
49.4
i
15
71.6 zk 4.3*
66.5 zt 7.6
54.1 zk 2.5
50.4
& 7.6
45.4
+ 4.9
49.4
+ 4.2
60
97.3 zt
67.4
58.3
50.7
rt 4.5
64.5
& 5.5
58.8
zt 6.9
Hepatic
cyclic AMP,
8.8
pmoles/mg
j, 5.8
jz 4.2
5.2
wet wt
0
0.16
& 0.01
0.26
i
0.02
0.35
* 0.03
0.20
* 0.03
0.12
f 0.01
0.40
zt 0.10
15
0.17
zt 0.02
0.22
zt 0.02
0.28
i
0.03
0.16
zt 0.04
0.11
l
0.01
0.22
+ 0.03
60
0.22
f 0.04
0.33
+ 0.03*
0.26
z!z 0.04
0.23
zt 0.03
0.19
i
0.03
0.22
zt 0.03
Intravenous glucose (1 g/kg) indicated
in separate
rots for each fasted *p < 0.05
groups
WCIS injected of fed and
and
fasted
blood
samples
rats. Means
obtained
from
the portal
=t SE with 15 rats for each
vein at the times
fed group
and 6-8
group.
(IS compared
to time 0 within
each group.
For baseline
intergroup
statistical
comparisons
see
Fig. 2.
by 60 min except at 24 and 48 hr. In contrast, insulin responses to glucose progressively decreased during fasting. Glucagon concentration decrements after glucose were seen only in fed rats, while I/G molar ratio increments progressively decreased, paralleling the insulin responses. Table 2 shows the values for hepatic glycogen and cyclic AMP concentrations and the enzyme activities of hepatic glycogen synthase in the I form and glycogen phosphorylase in response to glucose administration in fed and fasted rats. Total hepatic glycogen synthase activities did not change after glucose in fed or fasted rats (data not shown). However, synthase I activities were increased significantly from baseline at 1.5 min in all fed and fasted groups except at 24 hr (p < 0.06). The increments were much greater in 48-120-hr-fasted rats than in fed rats, with intermediate values in 24-hr-fasted rats. Enzyme activities returned to baseline levels by 60 min in all study groups. The relation between plasma glucose levels and synthase I activities after glucose administration was examined to determine if the apparent increased synthase responsiveness in 48-120-hr-fasted rats was simply a manifestation of higher plasma glucose levels. In fed and 24-hr-fasted rats there was no correlation between these parameters. However, there was a strong correlation in 48120-hr-fasted rats (p < 0.05). After glucose administration, phosphorylase activities were decreased only in fed rats. However, even in fed rats individual phosphorylase activity decrements did not correlate with either synthase I or plasma glucose increments. Following glucose administration, hepatic glycogen concentrations increased significantly only at 24 and 72 hr of fasting. Hepatic cyclic AMP concentrations
EFFECT
OF
321
STARVATION
did not change after glucose administration, which increases were seen at 60 min (Table 2).
except in 24-hr-fasted
rats.
in
DISCUSSION
In this report we present data depicting the effect of prolonged fasting in the rat on both the basal activities and the response to intravenous glucose of the hepatic glycogen synthase and phosphorylase enzyme systems. Results were correlated with fasting-induced changes in hepatic cyclic AMP levels and other hormonal and nonhormonal factors with known important hepatic actions. The data show striking fasting-induced changes in these hepatic enzyme systems.
By 24 hr of fasting, hepatic glycogen concentrations reached very low levels but increased thereafter to approximately 25”,, of fed levels. Although the physiologic significance of hepatic glycogen reaccumulation is presently unknown. such increases in storage carbohydrate could be important for glucose homeostasis and rapid energy mobilization during prolonged fasting. While the current studies did not address the mechanisms of the initial rapid hepatic glycogen concentration decrements, the subsequent glycogen reaccumulation was associated with changes in hepatic synthase I and phosphorylase activities that would be expected to favor glycogen synthesis. Isolated hepatocytes from fasted rats show similar enzymatic changes in association with rapid glycogen deposition.” The mechanisms of the fasting-promoted enzyme changes are not well understood, but it is possible that the release of inhibition by glycogen of the synthase and phosphorylase phosphatase systems plays a role in the changes seen,“’ since high levels of glycogen, as found in fed rats, are reported to inhibit these synthase-activating and phosphorylase-inactivating enzymes.“,‘h The results indicate that the relationships between the fasting-induced changes in the enzyme activities, plasma insulin, and glucagon concentrations, or their molar ratios, and changes in hepatic cyclic AMP levels are extremely complex and frequently paradoxic. While there are no obvious relationships betwcen hepatic cyclic AMP concentrations and glycogen synthase and phosphorylase activities during fasting, the present studies may not have detected physiologically important changes in subcellular distribution of the cyclic nucleotide that could have mediated some of the changes in enLyme activities.” Gluc,ose ,4 dministration The present data show that during long-term fasting there is exaggerated responsiveness ofthe hepatic synthase enzyme system in vivo to intravenous glucose administration. The mechanism of this altered synthase responsiveness to glucose, which has also been recently observed in isolated hepatocytes,?’ is unknown. However, it is conceivable that the hepatic synthase phosphatase is more responsive to glucose during starvation, when glycogen levels are low.‘” x In accord with this thesis. Curnow et al. 28 have recently shown that glucosemediated activation of hepatic synthase phosphatase could be consistently demonstrated only in fasted rats.
322
GOLDSTEIN
AND CURNOW
Our results indicate that following glucose administration, increases in hepatic synthase I activities throughout fasting are independent of any concomitant decreases in hepatic phosphorylase activities. Synthase activation independent of phosphorylase decrements has also been shown in the rat following oral glucose administration.29 These data suggest that glucose-mediated activation of synthase need not be secondary to glucose effects on phosphorylase, as proposed by Hers and co-workers. ‘,I5 An alternate mechanism for glucose effects on these systems has been suggested by Gilboe and Nuttall. They have shown that glucose may directly promote synthase activation by deinhibiting ATP inhibition of synthase phosphatase. In contrast to the increased responsiveness of the hepatic synthase enzyme system during fasting, plasma insulin responses to glucose were progressively impaired. These data indicate, as previously shown in isolated perfused liver,31 that acute insulin secretion following glucose administration probably does not play an important role in hepatic synthase activation in vivo. The present data demonstrate progressive glucose intolerance with fasting. However, the data indicate that the liver may not play an important role in this phenomena. Neither fed nor fasted rats showed consistent increases in hepatic glycogen levels following intravenous glucose administration. These findings are compatible with recent studies indicating that a major proportion of intravenously administered glucose is taken up by skeletal muscle and that during starvation skeletal muscle glucose uptake is impaired, presumably contributing since increased responsivesignificantly to glucose intolerance. 3z Furthermore, ness of the hepatic synthase enzyme system during fasting would tend to favor hepatic glucose uptake and glycogenesis, it appears unlikely that altered hepatic glycogen metabolism plays any significant role in the glucose intolerance of fasting. In conclusion, the present data depict a systematic assessment of the alterations in the enzyme systems’ rate limiting for hepatic glycogen synthesis and degradation during prolonged fasting in the rat. The results emphasize the complexity of the interrelationships of the hormonal and nonhormonal factors influencing hepatic glycogen metabolism in vivo. Additional studies will be required to clearly define the mechanisms and physiologic significance of these fastinginduced metabolic alterations. ACKNOWLEDGMENT We would like to thank Dr. J. Larner, Dr. F. Murad, and Dr. S. Pohl for reviewing this manuscript. The authors also acknowledge the expert technical assistance of Gwenn H. Clark and Herman Hernandez and the aid of Suellen Lakes and Dana Bolyard in the preparation of this manuscript.
REFERENCES 1. Hers HG, Stalmans W, De Wulf H, et al: The control of glycogen metabolism in the liver, in Fischer EH (ed): Metabolic Interconversion of Enzymes. Berlin. Springer. 197.7. pp 89-98 2. Huijing F: Glycogen metabolism and glycogen-storage diseases. Physiol Rev 55: 609m 658, 1975 3. Hinukuri S, Larner J: Studies on UDPG:
n-1,4-glucan a-4 glucosyltransferase. VII. Conversion of the enzyme from glucose-6-phosphate-dependent to independent form in liver. Biochemistry 3: I783- 1788. 1963 4. Sutherland EW, Cori CF: Effect of hyperglycemic-glycogenolytic factor and epinephrine on liver phosphorylase. J Biol Chem 188:531543.1951 5. Bishop JS, Goldberg ND, Lamer J: In-
EFFECT
323
OF STARVATION
sulin regulation of hepatic glycogen metabolism m the dog. Am J Physiol220:4999506, 1971 6. Curnow RT, Rayfield EJ. George DT, et al: Control of hepatic glycogen metabolism in the rhesus monkey: Effect of glucose, insulin and glucagon administration. Am J Physiol 228:80--88, 1975 7. Exton JH. Lewis SB. Ho RJ, et al: The role of cyclic AMP in the interaction of glucagon and insulin m the control of liver metabolism. Ann NY Acad Sci 185:85-100, 1971 8. Lavine RL. Voyles N. Perrino PV, et al: The erect of fasting on tissue cyclic AMP and plasma glucagon in the obese hyperglycemic mouse. Endocrinology 97:615-620, 1975 9. Grey NJ, Goldring S, Kipnis DM: The effect of fasting, diet, and actinomycin D on insulin secretion in the rat. J Clin Invest 49: 881-889, 1970 10. Fisher M, Sherwin RS, Hendler R. et al: Kinettcs of glucagon in man: Effects of starvation. Proc Nat1 Acad Sci USA 73:1735-1739. I976 I I. Unger RH: Alpha- and beta-ceil interrelationships in health and disease. Metabolism 2358 1-593,1974 12. Curnow RT, Nuttall FQ: Effect of prostaglandin Et administration on the liver glycogen synthase and phosphorylase systems. J Biol Chrm 247:1892 -1898. 1972 13. Tan A. Nuttall FQ: Characteristics of the dephosphorylated form of phosphorylase purltied from rat liver and measurement of its activity tn crude liver preparations. Biochim Biophys Acta 410:45560, 1975 14. Felig P: The liver in glucose homeostasis in normal man and in diabetes, in VallanceOwen J (ed): The Physiological and Biochemical Basis of Dtabetes. London, Medical and Technical Publishmg, 1975 IS. Hers HG: The control of glycogrn metabolism in the liver. Annu Rev Biochem 45: 167m 189. 1976 16. Hershey JM, Orr MD: The removal of glycogen from living muscle. Trans R Sot Can (Set V] 22:151~ 157, 1928 17. Pfliiger E: Ueber den ein Russ einsertrger ernahrting oder nahrungs mangels aug den glykogengchalt des thierischen kiirpers. Pfltigers Arch Ges Physiol I 19:117~126. 1907 18. Cahill GF Jr, Henera MG, Morgan AP, et al: Hormone fuel interrelationships during fasttng. J Clin Invest 45:1751-1769, 1966 19. Stalmans W. Hers HG: The stimuiation ofliver phosphorylase b by AMP, fluoride and sulfate. Eur J Biochem 54:341-350. 1975
20. Snedecor
GW,
Cochran
WG:
Statistical
Methods (ed 6). Ames, lowa. Iowa State Univ Pr, 1967 21. Marliss EB, Aoki TT. Unger RH. et al: Glucagon levels and metabolic effects in fasting man. J Clin Invest 49:2256-2270, 1970 22. Buchanan KD, Vance JE, Williams RH: Effects of starvation on insulin and glucagon release from isolated Islets of Langerhans of the rat. Metabolism l8:155-162, 1969 23. Hue L, Bontemps F, Hers HG: The effect ofglucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations. Biochem J 152: IO551 14. 1975 24. Hutson NY. Brurnley FT. Arsimacopou10s FD. et al: Studies on the a-adrenrrgic acttvation of hepatic glucose output. 1. Studtrs on the ar-adrenergic activation of phosphorylasr and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells. J Biol Chem 251:5200~5208. 1976 25. Abe N. Tsukik S: Factors affectmg the activity of glycogen synthase D phosphatasr from rat liver in vitro. Sci Rep Res lnst Tohoku Univ [Med] 21:17-25. I974 26. Kato K. Bishop JS: Glycogen synthetase-D phosphatase. I. Some new properties ol the partially purified enzyme from rabbit skeletal muscle. J Biol Chem 247:7420- 7429, 1972 27. Terasaki WL. Brooker G: Cardiac adenosme 3’5.monophosphate. Free and bound forms in the isolated rat atrium. J Biol Chem 252:1041-1050, 1977 28. Curnow RT, Rowe JN, Goldstem DE: Role of phosphoprotein phosphatase activjation in the effect of glucose on liver. Diabetes 26 [Suppl 1]:393. 1977 29. Nuttall FQ, Cannon MC. Lamer J: Oral glucose effect on glycogen synthetase and phosphorylase in heart, muscle and liver. Physiol Chem Phys 41497.-515, 1972 30. Gilboe DP, Nuttall FQ: The regulatton of liver glycogen synthetase D phosphatase by ATP and glucose. Biochem Biophys Res Commun 53:164~171, 1973 31. Buschiazzo HJ. Exton JH. Park CR: Effects of glucose on glycogen synthrtasc. phosphorylase and glycogen deposition m the perfused rat liver. Proc Natl Acad Sci USA 65:3833387, 1970 32. Daniel PM. Love ER, Pratt OE: Insultnstimulated entry of glucose into muscle in viva as a major factor tn the regulation of blood glucose. J Physiol 247:273-288, 1975
