Liver Regeneration in Normal and Alloxan-induced Diabetic Rats '
ROSEMARY BARRA AND JAMES C. HALL Department ofZoologv and Physiology,Rutgers University, Newark, New Jersey 07102
ABSTRACT The effects of alloxan-induced diabetes on liver regeneration were investigated. Normal and diabetic rats were sacrificed a t eight time periods between 16 hours and 4 weeks following two-thirds partial hepatectomy or sham operation. The results indicate that alloxan-induced diabetes delays but does not prevent liver regeneration following partial hepatectomy. This delay is indicated by a depressed synthesis of RNA, DNA and protein during the first post-operative day and a lack of mitotic figures in the 24-hoursample. In addition, the synthesis of these three cellular constituents did not return to control levels as rapidly in the diabetics. Compared with the sham operated animals, the concentrations of total serum protein remained depressed longer in the diabetic hepatectomized animals. The data indicate that the metabolic alterations associated with alloxan diabetes delay the onset of the regenerative process and prolong the recovery period. It has long been recognized that the mammalian liver enters a phase of compensatory hypertrophy and hyperplasia following partial hepatectomy. The regenerating liver is characterized by an increased rate of DNA, RNA and protein synthesis (Bucher, '63; Leduc, '64; Potter and Barbiroli, '71; Van Lancker, '70) and variations in glycogen and lipid content (Simek et al., '67). Many investigators have reported that the process of hepatic restoration is mediated by humoral agents (Bucher and Swaffield, '73; Fisher e t al., '71; Weinbren et al., '73). Evidence from some laboratories has suggested that a hepatotrophic portal factor is required (Lee et al., '74; Starzl et al., '75; Sgro et al., '73). Starzl et al. ('75) regarded insulin as the most important factor for the initiation of liver regeneration. By using monolayer techniques, Leffert ('74) has demonstrated that insulin acts as an initiator of DNA synthesis. Other experimentors have failed to demonstrate that regeneration is dependent upon a component of portal blood. Bucher and Swaffield ('73) using eviscerated rats observed liver cell proliferation in the absence of portal splanchnic organs. This observation was also supported by the work of Price et al. 1'72) and Max et al. ('72). The liver might also be the J. EXP. ZOOL., 201: 93-100.
site of production for growth promoting factors. LaBrecque and Persch ('75) reported that a stimulator substance is present in the liver of normal weanling and regenerating adult rats. The lack of insulin in the diabetic causes disorders in carbohydrate, fat, protein, and electrolyte metabolism. The diabetic animal is characterized by a decreased utilization of glucose by peripheral tissues, decreased protein synthesis, increased glycogenolysis, mobilization of depot fat and the subsequent development of a fatty liver. The changes in the diabetic resemble some of the adaptations made to starvation. The effects of starvation on the regenerative process have been investigated (MacDonald et al., '63; Montecuccoli et al., '72; Stirling et al., '73). In fasted rats, a significant inhibition of hepatic DNA synthesis has been observed. Stirling et al. ('73) found that, following a starvation period of 48 hours, the initial evidence of proliferation was delayed. We investigated liver regeneration in rats with untreated alloxan induced diabetes to determine if the lack of endogenous insulin would prevent regeneration, and if regenera' Present address: Department of Biochemistry. New Jersey Medical School, Newark, New Jersey 07103.
93
94
ROSEMARY BARRA AND JAMES C. HALL
tion occurred to determine if the metabolic alterations associated with diabetes would affect the course of this rapid growth process. MATERIALS AND METHODS
Animals All experiments were performed on male, Sprague Dawley rats initially weighing 175200 g obtained from the Camm Research Institute (Wayne, New Jersey). The rats were exposed to a 14:lO photoperiod and received Purina rat chow and water ad libitum. They were divided into four experimental groups: normal sham, normal hepatectomized, diabetic sham, and diabetic hepatectomized. Znduction of diabetes Diabetes was induced by a single intravenous injection of a 2% solution of alloxan monohydrate (Matheson Coleman and Bell, East Rutherford, New Jersey) a t a dose rate of 40 mg per kg. Following the injection, the rats were maintained for a t least ten days before being subjected to either partial hepatectomy or sham operation. The rats were considered diabetic when they demonstrated severe glucosuria (Combistix, Ames Co., Elkhart, Indiana) and blood glucose levels above 300 mg% (Glucostat, Worthington, Freehold, New Jersey). Surgical procedures Partial hepatectomies were performed according to the method of Higgins and Anderson ('31), and resulted in the removal of approximately 67%of the total liver mass. Animals from each of the experimental groups were sacrificed by decapitation a t the following time intervals after partial hepatectomy or sham operation: 16 hours, 24 hours, 36 hours, 48 hours, 72 hours, 1 week, 2 weeks and 4 weeks. The operations were performed a t the appropriate time so that all the animals were sacrificed between 9:30 and 1O:OO A.M. Two hours prior to sacrifice, the rats received an intraperitoneal injection of 2 pCi of 6-14Corotic acid and 4 pCi of 2-3H-glycine (New England Nuclear, Cambridge, Massachusetts) per 100 g of body weight.
'70), DNA by the diphenylamine reaction (Burton, '561, and protein by the biuret method (Gornall et al., '49). The concentrations were expressed as mg per g dry weight of liver per 100 g of body weight to account for variations in water content and animal weight a t the eight time periods following partial hepatectomy. The cross contamination among the fractions was found to be less than 5%. A 0.5 ml aliquot of the RNA, DNA and protein extracts was added to 10 ml of scintillation cocktail consisting of 4.0 g PPO and 0.5 g POPOP per liter of toluene and 10%Beckman Bio-Solve. The protein extract was neutralized with 5%perchloric acid prior to addition to the cocktail. The incorporation of isotope was measured in a Beckman LS 100 Liquid Scintillation Counter. Determination of mitotic index A small portion of the liver from the right lateral lobe was fixed in 10% formalin. Paraffin sections 5 p thick were stained with Erhlich's hemotoxylin and eosin (Luna, '68). The mitotic activity was determined by counting the number of parenchymal cells undergoing mitosis in ten randomly selected fields. To correct for variations in cell size, the number of parenchymal cells per field was determined for each experimental condition. The results were expressed as the mitotic index, the number of mitosis per 100,000 cells. Statistical analysis Each experimental group consisted of ten animals for each time period. The mean concentrations and relative specific activities for the four experimental groups a t each time were statistically analyzed for variance and compared for significant differences by the Dtest of Hartley (Snedecor, '59). RESULTS
The diabetic rats consistently displayed extensive glucosuria and blood glucose levels above 300 mg%.The mean body weights of the diabetic sham operated animals were less than the normals a t each time interval (table 1). Following partial hepatectomy both the RNA, DNA and protein determinations normal and diabetic rats decreased in weight. RNA, DNA and protein were extracted from Due to this variation in body weight, the suba 20% liver homogenate by the method of sequent results were calculated per 100 g of Shibko et al. ('67). The RNA concentration body weight. The diabetic liver displayed was determined by the orcinol reaction (Endo, variations in lipid and glycogen content. Com-
95
LIVER REGENERATION IN DIABETIC RATS TABLE 1
Mean body weights of normal and diabetic sham operated and partially hepatectomized rats at time of sacrifice T'
NS2
NH
DS
16 hr 24 hr 36 hr 48 hr 72 hr 1 wk 2 wk 4 wk
196rt7.2 20126.4 206rt 8.3 206rt6.8 21025.3 228k7.5 263rt5.8 352k4.9
188rt5.6 19524.8 202k7.4 194rt 7.1 203t6.9 212k5.5 256rt6.3 34127.2
167rt6.3 163rt 5.5 170f 6.1 166 rt 8.3 15826.3 17127.6 18627.3 205 rt9.4
DHK
153rt5.8 144*7.4 161k9.1 1581- 6.6 152 rt 7.4 156rt5.9 16857.7 187rt8.7
Male, Sprague Dawley rats were exposed to a 14:lO photoperiod and received Purina rat chow and water ad Zibitum. Diabetes was induced by a single i.v. injection of alloxan monohydrate (40 mg/kg). All the animals were maintained for a t least ten days prior to either sham operation or partial hepatectomy. ' T = time following partial hepatectomy or sham operation NS = normal sham operated rats. NH = normal partially hepatectomized rats. DS = diabetic sham operated rats. DH = diabetic partially hepatectomized rats. Mean weight of ten animals in g 2 S . E .
pared with the normal liver, there was an increase in lipid and a decrease in glycogen content (data not shown). In the normal hepatectomized rats, the RNA concentration rapidly increased to a maximum of 90.95 2 4.25 mglg dry weight of liverI100 g of body weight a t 24 hours and returned to a constant level by one week. The relative specific activity of RNA in these animals was greatest in the 16- and 24-hour samples (fig. la). The RNA concentrations and relative specific activities in the diabetic hepatectomized animals followed a different time sequence (fig. lb). The total RNA concentration gradually increased to an initial apex a t 36 hours, decreased and then increased to a maximum of 97.03 rfr 2.08 a t 72 hours. The incorporation of 6-'%-orotic acid also followed a biphasic pattern with peaks a t 24 and 48 hours. In the diabetic hepatectomized group, the synthesis of RNA remained statistically elevated compared with the diabetic shams throughout the 4-week period. The normal sham and hepatectomized animals were not significantly different in either the 2- or 4week samples. The DNA concentrations and specific activities in the normal and diabetic rats are illustrated in figures 2a and 2b respectively. There are two apices of DNA concentration in both the normal and diabetic hepatectomized animals. In the normal hepatectomized group, the maximum concentrations occur in the 24hour (14.69 2 0.89) and 48-hour (14.21 rt 0.74) samples. The DNA concentration peaked later in the diabetic hepatectomized animals,
14.62 I?; 0.72 a t 36 hours and 17.24 rt 0.56 a t 72 hours. The DNA concentration in this group remained statistically elevated through the 2-week sample, while the normal hepatectomized and sham operated animals were not significantly different after 72 hours. There were also two peaks in specific activity. Similar to the biphasic pattern of DNA concentration, the time periods showing the highest rate of incorporation corresponded to the periods of maximum concentration: 24 and 48 hours in the normal hepatectomized animals and 36 and 72 hours in the diabetic. The protein concentrations and the relative specific activities in the four experimental groups are illustrated in figures 3a and 3b. By 16 hours, protein synthesis in the normal hepatectomized rats was elevated compared with the normal shams, and increased to a maximum a t 24 hours. In this group, protein synthesis remained statistically elevated through the one week sample. In the diabetic hepatectomized animals, the protein concentration was below control values at 16 hours, and gradually increased in a biphasic manner. An initial peak of 737.72 rt 11.66 occurred a t 48 hours and a second peak of 703.3 rfr 18.53 a t one week. The incorporation of 2-3H-glycine followed a similar pattern. Protein synthesis remained above control values throughout the experimental period. The decrease in protein concentration and specific activity 72 hours after surgery was a curious feature in the diabetic hepatectomized animals. This is the period when regenerative activity was high as indicated by
96
ROSEMARY BARRA AND JAMES C. HALL 12
4.0
t
z
0
m w 1.0 2
LK
z
Y0 40 LK
W
1 2 T I M E AFTER
01115
0
3 PARTIAL
I
1 TIME
0A"i
HEPATECTOMY
b
2 AFTER
3 '' 1 2 WiiYS 4 PARTIAL HEPATECTOMY
I
Fig. 1 RNA concentrations and relative specific activities a t eight time periods between 16 hours and 4 weeks following partial hepatectomy or sham operation. Each value is the mean of ten experiments. The RNA concentrations are expressed in mg/g dry weight of liver/100 g of body weight. The relative specific activities RNA concentraare expressed as CPM X 10-3/mgRNA. RNA concentrations in sham operated rats 0-0, tions in partially hepatectomized rats 0-0, RNA relative specific activities in sham operated rats A-A, and RNA relative specific activities in partially hepatectomized rats A-A. (a) The above parameters in normal sham and hepatectomized animals. The maximum standard error was 5.7%of the mean. (b) The four parameters in diabetic sham and partially hepatectomized animals. The maximum standard error was 4.6% of the mean values.
1 2 TIME AFTER
"IYS
0
3 PARTIAL HEPATECTOMY
1 2 TIME AFTER
OA"E
b
3 PARTIAL
HEPATECTOMY
Fig. 2 DNA concentrations and relative specific activities a t eight time periods between 16 hours and 4 weeks following partial hepatectomy or sham operation. Each value represents the mean of ten experiments. The DNA concentrations are expressed in mg/g dry weight of liver/100 g of body weight. The relative specific activities are expressed as CPM X lO-Vrng DNA. DNA concentrations in sham operated rats 0-0, DNA concentrations in partially hepatectomized rats 0-0, DNA relative specific activities in sham operated rats A-A, and DNA relative specific activities in partially hepatectomized rats A-A. (a) The above parameters in normal sham and hepatectomized animals. For all the values shown, the standard error was less than 6.0%of the mean. (b) The above values in diabetic sham and partially hepatectomized rats. The maximum standard error was 4.9% of the mean.
the other parameters studied. Protein synthesis increased again in the one week sample while RNA and DNA were gradually returning to control levels. An explanation for this pattern is not evident from the results of this study. I t is doubtful that the increase in protein observed in the 1-week sample is involved in cell replication because of the decrease in RNA and DNA. Mitotic indices were determined for each experimental group and are recorded in table 2. There is little mitotic activity in the normal and diabetic sham operated animals. In the normal hepatectomized animals, mitotic
figures first appeared a t 24 hours and were present a t a high frequency in the 36- and 48hour samples. In the diabetic hepatectomized animals, statistically significant increases in mitotic activity did not occur until 36 hours with the maximum activity present 72 hours after surgery. Table 3 records the serum total protein concentrations in the four experimental groups. The serum protein concentrations decreased in both the normal and diabetic animals in the early hours following partial hepatectomy. In the normal hepatectomized group, they returned to control values by the second
97
LIVER REGENERATION IN DIABETIC RATS
a
TIME AFTER
PARTIAL
HEPATECTOMY
b
TIME AFTER
PARTIAL
HEPATECTOMY
Fig. 3 Protein concentrations and relative specific activities a t eight time periods following partial hepatectomy or sham operation. Each value represents the mean of ten experiments. The protein concentrations are expressed in mg/g dry weight of liver/100 g of body weight. The relative specific activities are expressed as CPM X IOTmg of protein. Protein concentrations in sham operated animals 0-0, protein concentrations in partially hepatectomized rats 0-0, protein relative specific activities in sham operated rats A-A, and protein relative specific activities in partially hepatectomized animals A-A. (a) The above parameters in normal sham and hepatectomized rats. The maximum standard error was 6.0%of the mean values. (b) The four parameters in diabetic sham and partially hepatectomized animals. The maximum standard error was 4.9%of the mean values.
week. However, in the diabetics they remain depressed until the 4-week sample. DISCUSSION
The liver is one of the few mammalian tissues capable of regenerating large amounts of excised or injured tissue. Following partial hepatectomy, the liver enters a phase of compensatory growth characterized by many biochemical and morphological changes. These changes focus on the primary objective of synthesizing the cellular constituents required for replication and for the rapid return of the liver to its pre-operative condition. The results of this investigation of RNA, DNA and protein synthesis indicate that the liver of alloxan induced diabetic animals is capable of synthesizing the necessary components for cell replication. This finding is contrary to the observations of Starzl et al. ('75) who believe that insulin is a primary requirement for regeneration. Insulin does have a positive effect on hepatic proliferation Younger et al. ('66) treated diabetic rats with insulin and observed a marked increase in hepatic activity comparable to the effects of a 60% partial hepatectomy. They suggest that insulin exerts its effect by shifting the synthetic activities of the diabetic animal back towards those present in normal animals. Starzl et al. ('76) investigated the effect of insulin on the livers of dogs following a portacaval shunt. In their system, insulin proved to be a very potent hepatotrophic factor. Since liver is capable of regenerating in the absence
TABLE 2
Mitotic index. The mitotic activity in normal and diabetic rats following sham operation or partial hepatectomy Normal sham ' 9 12 Normal hepatectomized
16 hr 24 hr 36 hr
48 hr 72 hr 1 wk 2 wk 4 wk
122 9 5342 52 8112143 9452188 515rt137 472 9 4 8 2 13 162 7
Diabetic sham ' 9 2 3 Diabetic hepatectomized
16 hr 24 hr 36 hr 48 hr 72 hr
1 wk 2 wk 4 wk
82 5 212 8 1,098rt173 8672214 1,1292169 1383~34 612 6 2 9 2 11
* Average of the sham operated animals from all time periods. ZMitotic index recorded as the number of mitosis per 100,000 parenchymal cells 2S.E.
of insulin, i t is evident that a multifactorial system is involved in this rapid growth process and that insulin by itself does not determine whether liver regeneration can occur. The lack of insulin in the diabetic does effect the time sequence of regeneration. Compared with the normal hepatectomized animals, the diabetics experience a delay in the onset of the regenerative process. In the control animals, the synthetic processes were most active during the first post-operative day which is consistent with the literature (Bucher, '63; Hammarsten et al., '56; Leduc, '64; Potter and Barbiroli, '71). In contrast, the synthesis of RNA, DNA and protein is depressed in the diabetic hepatectomized rats during the first day. In general, maximum activity in the diabetics was seen during the third post-operative day. By this time, the
98
ROSEMARY BARRA AND JAMES C. HALL TABLE 3
Serum protein concentrations. The total serum protein concentrations in normal and diabetic rats following sham operation or partial hepateetomy T'
NS a
16 hr 24 hr 36 hr 48 hr 72 hr 1 wk 2 wk 4 wk
5.11rt0.14 5.47f0.18 5.42f0.14 5.55rt0.14 5.67S0.10 5.80C0.24 5.65rt0.16 5.78rt0.20
DS 6
NH
4.34rt0.22 4.61f0.10 4.4520.26 4.71rt 0.11 4.61rt0.25 4.72rt 0.19 5.53rt0.11 5.70rtO.08
5.38rt0.24 5.72f0.22 5.85f0.15 5.78f0.37 5.83rt0.23 5.67rt0.19 5.60f0.20 5.67rtO.20
DH
4.54rt0.24 4.24f0.17 4.20rt 0.11 4.49rt 0.21 4.4720.17 4.81rt0.17 4.87f0.13 5.65t0.22
T = time following partial hepatectomy or sham operation. NS = normal sham operated rats. NH = normal partially hepatectomized rats. DS = diabetic sham operated rats. "H = diabetic partially hepatectomized rats. I
Every value is the mean of ten experiments recorded in mgZ 2S.E.
synthetic processes in the normal animals were beginning to decrease. The results of this study also suggest a prolongation of the recovery period in the diabetics. The synthesis of RNA, DNA and protein did not return to control levels as rapidly, and the serum protein concentrations remained depressed longer in the diabetic hepatectomized animals. In the early hours following partial hepatectomy, the synthesis of RNA and protein are usually prominent events. The RNA synthesized during this period is involved in the growth of the cell in preparation for division and also acts as a template for the synthesis of proteins involved in DNA replication (Baserga, '68). One possible explanation for a delay in the onset of regeneration in the diabetics is a reduction in the cells' ability to synthesize RNA and protein. The delay in the onset of regeneration and the prolongation of the recovery period may be the result of changes in metabolism associated with the diabetic state. The glycogen and lipid concentrations of the liver are altered in the diabetics with a depletion of glycogen stores and the development of a fatty liver. The significance of glycogen stores in DNA synthesis is uncertain (Baserga, '68).The early depletion of liver glycogen following partial hepatectomy probably supplies some of the energy required for the initial synthetic processes. Due to the decreased glycogen content, the diabetics may lack an important factor involved in the initial regenerative response. This observed response is similar to that described for fasted animals (MacDonald et al., '63; Montecuccoli et al., '72; Stirling et al.,
'73), and supports the possibility that the metabolic state of the animals and not insulin per se is responsible for the delay in regeneration. ACKNOWLEDGMENTS
This study was supported by USPH Grant RR7059. We wish to thank Doctors F. George Zaki and C. Hans Keysser, Director of the Pathology Department of the Squibb Institute for Medical Research for their help and for the use of their facilities. LITERATURE CITED Baserga, R. 1968 Biochemistry of the cell cycle: A review. Cell Tissue Kinet., I: 167-191. Bucher, N. L. R. 1963 Regeneration of mammalian liver. Int. Rev. Cytol., 15: 245-300. Bucher, N. L. R., and M. N. Swaffield 1973 Regeneration of liver in rats in the absence of portal splanchnic organs and a portal blood supply. Cancer Res., 33: 3189-3194. Burton, K. 1956 A study of the condition and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J., 62:315323. Endo, Y. 1970 A simultaneous estimation method of DNA and RNA by the orcinol reaction and a study of the reaction mechanism. 3. Biochem., 67:629-633. Fisher, B., P. Szuch and E. R. Fisher 1971 Evaluation of a humoral factor in liver regeneration utilizing liver transplants. Cancer Res., 31: 322-331. Gornall, A. G., C. J. Baradawill and M. M. David 1949 Determination of serum protein by means of the hiuret reaction. J . Biol. Chem., 177: 751-766. Hammarsten, E., S. Aqvist, E. P. Anderson and N. A. Eliasson 1956 The turnover of polynucleotides and proteins in regenerating rat liver, studies with 15N-glycine. Aeta Chem. Scand., 10: 1568-1575. Higgins, G. M., and R. M. Anderson 1931 Experimental Pathology of the liver. Arch. Pathol., 12: 186-202. LaBrecque, D. R., and L. A. Pesch 1975 Preparation and partial characterization of hepatic regenerative stimulator substance from rat liver. J . Physiol., 248: 273-284. Leduc, E. H. 1964 Regeneration of the liver. In: The
LIVER REGENERATI(3N IN DIABETIC RATS Liver. C. H. Rouiller, ed. Academic Press, New York, p. 63. Lee, S., C. E. Broelsch, J. G. Chandler, C. A. Charters and M. J. Orloff 1974 Liver regeneration following portacaval transposition in rats. Surg. Forum, 25: 391-394. Leffert, H. L. 1974 Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. J. Cell. Biol., 62:792-801. Luna, L. G., ed. 1968 Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. McGraw Hill, New York. MacDonald, R. A., A. E. Rogers and G. S. Pechet 1963 Growth and regeneration of the liver. Ann. N.Y. Acad. Sci., 111: 70-86. Max, M. H., J. B. Price, K. Takeshige and A. B. Voorhees 1972 The role of factors of portal origin in modifying hepatic regeneration. J. Surg. Res., 12: 120-123. Montecuccoli, G., F. Novello and F. Stirpe 1972 Effect of protein deprivation and of starvation on DNA synthesis in resting and regenerating rat liver. J. Nutr., 102: 507512. Price, J. B., K. Takeshige, M. H. Max and A. B. Voorhees 1972 Glucagon as the portal factor modifying hepatic regeneration. Surg., 72: 74-82. Potter, E. R., and B. Barbiroli 1971 DNA synthesis and interaction between controlled feeding schedules and partial hepatectomy in rats. Sci., 172: 738-741. Sgro, J. C., A. C. Charters, J. G. Chandler, D. E. Gramhoit and M. J. Orloff 1973 Site of origin of the hepatotrophic portal blood factor involved in liver regeneration. Surg. Forum, 24: 377-378. Simek, J., V. L. Chemlait, J. Melka, J. Pasderka and Z. Charvat 1967 Influence of protracted infusion of glucose and insulin on the composition and regeneration activity
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of liver after partial hepatectomy in rats. Nature, 213: 910-911. Shibko, S. P., P. Koivestoinin, C. A. Tratnyek, A. R. Newhall and L. Friedman 1967 A method for sequential quantitative separation and determination of protein, RNA, DNA, lipid and glycogen from a single rat liver homogenate or from a subcellular fraction. Anal. Biochem., 19: 514-528. Snedecor, G. W. 1959 Statistical Methods Applied to Experiments in Agriculture and Biology. Iowa State College Press, Ames, Iowa. Starzl, T. E., N. Kashiwage, K. A. Porter, I. Y. Lee and W. J. I. Russell 1975 The effect of diabetes mellitus on portal blood hepatotrophic factors in dogs. Surg. Gyn. and Obst., 140: 549-562. Starzl, T. E., K. A. Porter and C. W. Putnam 1975 Intraportal insulin protects from the liver injury of portacaval shunt in dogs. Lancet, 2: 1241-1242. Starzl, T. E., K. A. Porter, K. Watanabe and C. W. Putnam 1976 Effects of insulin, glucagon, and insulin/glucagon infusions on liver morphology and cell division after complete portacaval shunt in dogs. Lancet, 1: 821-825. Stirling, G. A., J. Laughlin and S. L. A. Washington 1973 The effects of starvation on the proliferative response after partial hepatectomy. Exp. Mol. Pathol., 19: 44-52. Younger, L. R., J. King and D. F. Steiner 1966 Hepatic proliferative response to insulin in severe alloxan diabetes. Cancer Res., 26: 1408-1414. VanLancker, J. L. 1970 Control of DNA synthesis in regenerating rat liver. Fed. Proc., 29: 1439-1442. Weinbren, K., S. Washington and F. Dowling 1973 The proliferative response after partial hepatectomy in hypophysectomized rats bearing portacaval anastomoses. Br. J. Exp. Path., 54: 429-436.
ROSEMARY BARRA AND JAMES C. HALL Department ofZoologv and Physiology,Rutgers University, Newark, New Jersey 07102
ABSTRACT The effects of alloxan-induced diabetes on liver regeneration were investigated. Normal and diabetic rats were sacrificed a t eight time periods between 16 hours and 4 weeks following two-thirds partial hepatectomy or sham operation. The results indicate that alloxan-induced diabetes delays but does not prevent liver regeneration following partial hepatectomy. This delay is indicated by a depressed synthesis of RNA, DNA and protein during the first post-operative day and a lack of mitotic figures in the 24-hoursample. In addition, the synthesis of these three cellular constituents did not return to control levels as rapidly in the diabetics. Compared with the sham operated animals, the concentrations of total serum protein remained depressed longer in the diabetic hepatectomized animals. The data indicate that the metabolic alterations associated with alloxan diabetes delay the onset of the regenerative process and prolong the recovery period. It has long been recognized that the mammalian liver enters a phase of compensatory hypertrophy and hyperplasia following partial hepatectomy. The regenerating liver is characterized by an increased rate of DNA, RNA and protein synthesis (Bucher, '63; Leduc, '64; Potter and Barbiroli, '71; Van Lancker, '70) and variations in glycogen and lipid content (Simek et al., '67). Many investigators have reported that the process of hepatic restoration is mediated by humoral agents (Bucher and Swaffield, '73; Fisher e t al., '71; Weinbren et al., '73). Evidence from some laboratories has suggested that a hepatotrophic portal factor is required (Lee et al., '74; Starzl et al., '75; Sgro et al., '73). Starzl et al. ('75) regarded insulin as the most important factor for the initiation of liver regeneration. By using monolayer techniques, Leffert ('74) has demonstrated that insulin acts as an initiator of DNA synthesis. Other experimentors have failed to demonstrate that regeneration is dependent upon a component of portal blood. Bucher and Swaffield ('73) using eviscerated rats observed liver cell proliferation in the absence of portal splanchnic organs. This observation was also supported by the work of Price et al. 1'72) and Max et al. ('72). The liver might also be the J. EXP. ZOOL., 201: 93-100.
site of production for growth promoting factors. LaBrecque and Persch ('75) reported that a stimulator substance is present in the liver of normal weanling and regenerating adult rats. The lack of insulin in the diabetic causes disorders in carbohydrate, fat, protein, and electrolyte metabolism. The diabetic animal is characterized by a decreased utilization of glucose by peripheral tissues, decreased protein synthesis, increased glycogenolysis, mobilization of depot fat and the subsequent development of a fatty liver. The changes in the diabetic resemble some of the adaptations made to starvation. The effects of starvation on the regenerative process have been investigated (MacDonald et al., '63; Montecuccoli et al., '72; Stirling et al., '73). In fasted rats, a significant inhibition of hepatic DNA synthesis has been observed. Stirling et al. ('73) found that, following a starvation period of 48 hours, the initial evidence of proliferation was delayed. We investigated liver regeneration in rats with untreated alloxan induced diabetes to determine if the lack of endogenous insulin would prevent regeneration, and if regenera' Present address: Department of Biochemistry. New Jersey Medical School, Newark, New Jersey 07103.
93
94
ROSEMARY BARRA AND JAMES C. HALL
tion occurred to determine if the metabolic alterations associated with diabetes would affect the course of this rapid growth process. MATERIALS AND METHODS
Animals All experiments were performed on male, Sprague Dawley rats initially weighing 175200 g obtained from the Camm Research Institute (Wayne, New Jersey). The rats were exposed to a 14:lO photoperiod and received Purina rat chow and water ad libitum. They were divided into four experimental groups: normal sham, normal hepatectomized, diabetic sham, and diabetic hepatectomized. Znduction of diabetes Diabetes was induced by a single intravenous injection of a 2% solution of alloxan monohydrate (Matheson Coleman and Bell, East Rutherford, New Jersey) a t a dose rate of 40 mg per kg. Following the injection, the rats were maintained for a t least ten days before being subjected to either partial hepatectomy or sham operation. The rats were considered diabetic when they demonstrated severe glucosuria (Combistix, Ames Co., Elkhart, Indiana) and blood glucose levels above 300 mg% (Glucostat, Worthington, Freehold, New Jersey). Surgical procedures Partial hepatectomies were performed according to the method of Higgins and Anderson ('31), and resulted in the removal of approximately 67%of the total liver mass. Animals from each of the experimental groups were sacrificed by decapitation a t the following time intervals after partial hepatectomy or sham operation: 16 hours, 24 hours, 36 hours, 48 hours, 72 hours, 1 week, 2 weeks and 4 weeks. The operations were performed a t the appropriate time so that all the animals were sacrificed between 9:30 and 1O:OO A.M. Two hours prior to sacrifice, the rats received an intraperitoneal injection of 2 pCi of 6-14Corotic acid and 4 pCi of 2-3H-glycine (New England Nuclear, Cambridge, Massachusetts) per 100 g of body weight.
'70), DNA by the diphenylamine reaction (Burton, '561, and protein by the biuret method (Gornall et al., '49). The concentrations were expressed as mg per g dry weight of liver per 100 g of body weight to account for variations in water content and animal weight a t the eight time periods following partial hepatectomy. The cross contamination among the fractions was found to be less than 5%. A 0.5 ml aliquot of the RNA, DNA and protein extracts was added to 10 ml of scintillation cocktail consisting of 4.0 g PPO and 0.5 g POPOP per liter of toluene and 10%Beckman Bio-Solve. The protein extract was neutralized with 5%perchloric acid prior to addition to the cocktail. The incorporation of isotope was measured in a Beckman LS 100 Liquid Scintillation Counter. Determination of mitotic index A small portion of the liver from the right lateral lobe was fixed in 10% formalin. Paraffin sections 5 p thick were stained with Erhlich's hemotoxylin and eosin (Luna, '68). The mitotic activity was determined by counting the number of parenchymal cells undergoing mitosis in ten randomly selected fields. To correct for variations in cell size, the number of parenchymal cells per field was determined for each experimental condition. The results were expressed as the mitotic index, the number of mitosis per 100,000 cells. Statistical analysis Each experimental group consisted of ten animals for each time period. The mean concentrations and relative specific activities for the four experimental groups a t each time were statistically analyzed for variance and compared for significant differences by the Dtest of Hartley (Snedecor, '59). RESULTS
The diabetic rats consistently displayed extensive glucosuria and blood glucose levels above 300 mg%.The mean body weights of the diabetic sham operated animals were less than the normals a t each time interval (table 1). Following partial hepatectomy both the RNA, DNA and protein determinations normal and diabetic rats decreased in weight. RNA, DNA and protein were extracted from Due to this variation in body weight, the suba 20% liver homogenate by the method of sequent results were calculated per 100 g of Shibko et al. ('67). The RNA concentration body weight. The diabetic liver displayed was determined by the orcinol reaction (Endo, variations in lipid and glycogen content. Com-
95
LIVER REGENERATION IN DIABETIC RATS TABLE 1
Mean body weights of normal and diabetic sham operated and partially hepatectomized rats at time of sacrifice T'
NS2
NH
DS
16 hr 24 hr 36 hr 48 hr 72 hr 1 wk 2 wk 4 wk
196rt7.2 20126.4 206rt 8.3 206rt6.8 21025.3 228k7.5 263rt5.8 352k4.9
188rt5.6 19524.8 202k7.4 194rt 7.1 203t6.9 212k5.5 256rt6.3 34127.2
167rt6.3 163rt 5.5 170f 6.1 166 rt 8.3 15826.3 17127.6 18627.3 205 rt9.4
DHK
153rt5.8 144*7.4 161k9.1 1581- 6.6 152 rt 7.4 156rt5.9 16857.7 187rt8.7
Male, Sprague Dawley rats were exposed to a 14:lO photoperiod and received Purina rat chow and water ad Zibitum. Diabetes was induced by a single i.v. injection of alloxan monohydrate (40 mg/kg). All the animals were maintained for a t least ten days prior to either sham operation or partial hepatectomy. ' T = time following partial hepatectomy or sham operation NS = normal sham operated rats. NH = normal partially hepatectomized rats. DS = diabetic sham operated rats. DH = diabetic partially hepatectomized rats. Mean weight of ten animals in g 2 S . E .
pared with the normal liver, there was an increase in lipid and a decrease in glycogen content (data not shown). In the normal hepatectomized rats, the RNA concentration rapidly increased to a maximum of 90.95 2 4.25 mglg dry weight of liverI100 g of body weight a t 24 hours and returned to a constant level by one week. The relative specific activity of RNA in these animals was greatest in the 16- and 24-hour samples (fig. la). The RNA concentrations and relative specific activities in the diabetic hepatectomized animals followed a different time sequence (fig. lb). The total RNA concentration gradually increased to an initial apex a t 36 hours, decreased and then increased to a maximum of 97.03 rfr 2.08 a t 72 hours. The incorporation of 6-'%-orotic acid also followed a biphasic pattern with peaks a t 24 and 48 hours. In the diabetic hepatectomized group, the synthesis of RNA remained statistically elevated compared with the diabetic shams throughout the 4-week period. The normal sham and hepatectomized animals were not significantly different in either the 2- or 4week samples. The DNA concentrations and specific activities in the normal and diabetic rats are illustrated in figures 2a and 2b respectively. There are two apices of DNA concentration in both the normal and diabetic hepatectomized animals. In the normal hepatectomized group, the maximum concentrations occur in the 24hour (14.69 2 0.89) and 48-hour (14.21 rt 0.74) samples. The DNA concentration peaked later in the diabetic hepatectomized animals,
14.62 I?; 0.72 a t 36 hours and 17.24 rt 0.56 a t 72 hours. The DNA concentration in this group remained statistically elevated through the 2-week sample, while the normal hepatectomized and sham operated animals were not significantly different after 72 hours. There were also two peaks in specific activity. Similar to the biphasic pattern of DNA concentration, the time periods showing the highest rate of incorporation corresponded to the periods of maximum concentration: 24 and 48 hours in the normal hepatectomized animals and 36 and 72 hours in the diabetic. The protein concentrations and the relative specific activities in the four experimental groups are illustrated in figures 3a and 3b. By 16 hours, protein synthesis in the normal hepatectomized rats was elevated compared with the normal shams, and increased to a maximum a t 24 hours. In this group, protein synthesis remained statistically elevated through the one week sample. In the diabetic hepatectomized animals, the protein concentration was below control values at 16 hours, and gradually increased in a biphasic manner. An initial peak of 737.72 rt 11.66 occurred a t 48 hours and a second peak of 703.3 rfr 18.53 a t one week. The incorporation of 2-3H-glycine followed a similar pattern. Protein synthesis remained above control values throughout the experimental period. The decrease in protein concentration and specific activity 72 hours after surgery was a curious feature in the diabetic hepatectomized animals. This is the period when regenerative activity was high as indicated by
96
ROSEMARY BARRA AND JAMES C. HALL 12
4.0
t
z
0
m w 1.0 2
LK
z
Y0 40 LK
W
1 2 T I M E AFTER
01115
0
3 PARTIAL
I
1 TIME
0A"i
HEPATECTOMY
b
2 AFTER
3 '' 1 2 WiiYS 4 PARTIAL HEPATECTOMY
I
Fig. 1 RNA concentrations and relative specific activities a t eight time periods between 16 hours and 4 weeks following partial hepatectomy or sham operation. Each value is the mean of ten experiments. The RNA concentrations are expressed in mg/g dry weight of liver/100 g of body weight. The relative specific activities RNA concentraare expressed as CPM X 10-3/mgRNA. RNA concentrations in sham operated rats 0-0, tions in partially hepatectomized rats 0-0, RNA relative specific activities in sham operated rats A-A, and RNA relative specific activities in partially hepatectomized rats A-A. (a) The above parameters in normal sham and hepatectomized animals. The maximum standard error was 5.7%of the mean. (b) The four parameters in diabetic sham and partially hepatectomized animals. The maximum standard error was 4.6% of the mean values.
1 2 TIME AFTER
"IYS
0
3 PARTIAL HEPATECTOMY
1 2 TIME AFTER
OA"E
b
3 PARTIAL
HEPATECTOMY
Fig. 2 DNA concentrations and relative specific activities a t eight time periods between 16 hours and 4 weeks following partial hepatectomy or sham operation. Each value represents the mean of ten experiments. The DNA concentrations are expressed in mg/g dry weight of liver/100 g of body weight. The relative specific activities are expressed as CPM X lO-Vrng DNA. DNA concentrations in sham operated rats 0-0, DNA concentrations in partially hepatectomized rats 0-0, DNA relative specific activities in sham operated rats A-A, and DNA relative specific activities in partially hepatectomized rats A-A. (a) The above parameters in normal sham and hepatectomized animals. For all the values shown, the standard error was less than 6.0%of the mean. (b) The above values in diabetic sham and partially hepatectomized rats. The maximum standard error was 4.9% of the mean.
the other parameters studied. Protein synthesis increased again in the one week sample while RNA and DNA were gradually returning to control levels. An explanation for this pattern is not evident from the results of this study. I t is doubtful that the increase in protein observed in the 1-week sample is involved in cell replication because of the decrease in RNA and DNA. Mitotic indices were determined for each experimental group and are recorded in table 2. There is little mitotic activity in the normal and diabetic sham operated animals. In the normal hepatectomized animals, mitotic
figures first appeared a t 24 hours and were present a t a high frequency in the 36- and 48hour samples. In the diabetic hepatectomized animals, statistically significant increases in mitotic activity did not occur until 36 hours with the maximum activity present 72 hours after surgery. Table 3 records the serum total protein concentrations in the four experimental groups. The serum protein concentrations decreased in both the normal and diabetic animals in the early hours following partial hepatectomy. In the normal hepatectomized group, they returned to control values by the second
97
LIVER REGENERATION IN DIABETIC RATS
a
TIME AFTER
PARTIAL
HEPATECTOMY
b
TIME AFTER
PARTIAL
HEPATECTOMY
Fig. 3 Protein concentrations and relative specific activities a t eight time periods following partial hepatectomy or sham operation. Each value represents the mean of ten experiments. The protein concentrations are expressed in mg/g dry weight of liver/100 g of body weight. The relative specific activities are expressed as CPM X IOTmg of protein. Protein concentrations in sham operated animals 0-0, protein concentrations in partially hepatectomized rats 0-0, protein relative specific activities in sham operated rats A-A, and protein relative specific activities in partially hepatectomized animals A-A. (a) The above parameters in normal sham and hepatectomized rats. The maximum standard error was 6.0%of the mean values. (b) The four parameters in diabetic sham and partially hepatectomized animals. The maximum standard error was 4.9%of the mean values.
week. However, in the diabetics they remain depressed until the 4-week sample. DISCUSSION
The liver is one of the few mammalian tissues capable of regenerating large amounts of excised or injured tissue. Following partial hepatectomy, the liver enters a phase of compensatory growth characterized by many biochemical and morphological changes. These changes focus on the primary objective of synthesizing the cellular constituents required for replication and for the rapid return of the liver to its pre-operative condition. The results of this investigation of RNA, DNA and protein synthesis indicate that the liver of alloxan induced diabetic animals is capable of synthesizing the necessary components for cell replication. This finding is contrary to the observations of Starzl et al. ('75) who believe that insulin is a primary requirement for regeneration. Insulin does have a positive effect on hepatic proliferation Younger et al. ('66) treated diabetic rats with insulin and observed a marked increase in hepatic activity comparable to the effects of a 60% partial hepatectomy. They suggest that insulin exerts its effect by shifting the synthetic activities of the diabetic animal back towards those present in normal animals. Starzl et al. ('76) investigated the effect of insulin on the livers of dogs following a portacaval shunt. In their system, insulin proved to be a very potent hepatotrophic factor. Since liver is capable of regenerating in the absence
TABLE 2
Mitotic index. The mitotic activity in normal and diabetic rats following sham operation or partial hepatectomy Normal sham ' 9 12 Normal hepatectomized
16 hr 24 hr 36 hr
48 hr 72 hr 1 wk 2 wk 4 wk
122 9 5342 52 8112143 9452188 515rt137 472 9 4 8 2 13 162 7
Diabetic sham ' 9 2 3 Diabetic hepatectomized
16 hr 24 hr 36 hr 48 hr 72 hr
1 wk 2 wk 4 wk
82 5 212 8 1,098rt173 8672214 1,1292169 1383~34 612 6 2 9 2 11
* Average of the sham operated animals from all time periods. ZMitotic index recorded as the number of mitosis per 100,000 parenchymal cells 2S.E.
of insulin, i t is evident that a multifactorial system is involved in this rapid growth process and that insulin by itself does not determine whether liver regeneration can occur. The lack of insulin in the diabetic does effect the time sequence of regeneration. Compared with the normal hepatectomized animals, the diabetics experience a delay in the onset of the regenerative process. In the control animals, the synthetic processes were most active during the first post-operative day which is consistent with the literature (Bucher, '63; Hammarsten et al., '56; Leduc, '64; Potter and Barbiroli, '71). In contrast, the synthesis of RNA, DNA and protein is depressed in the diabetic hepatectomized rats during the first day. In general, maximum activity in the diabetics was seen during the third post-operative day. By this time, the
98
ROSEMARY BARRA AND JAMES C. HALL TABLE 3
Serum protein concentrations. The total serum protein concentrations in normal and diabetic rats following sham operation or partial hepateetomy T'
NS a
16 hr 24 hr 36 hr 48 hr 72 hr 1 wk 2 wk 4 wk
5.11rt0.14 5.47f0.18 5.42f0.14 5.55rt0.14 5.67S0.10 5.80C0.24 5.65rt0.16 5.78rt0.20
DS 6
NH
4.34rt0.22 4.61f0.10 4.4520.26 4.71rt 0.11 4.61rt0.25 4.72rt 0.19 5.53rt0.11 5.70rtO.08
5.38rt0.24 5.72f0.22 5.85f0.15 5.78f0.37 5.83rt0.23 5.67rt0.19 5.60f0.20 5.67rtO.20
DH
4.54rt0.24 4.24f0.17 4.20rt 0.11 4.49rt 0.21 4.4720.17 4.81rt0.17 4.87f0.13 5.65t0.22
T = time following partial hepatectomy or sham operation. NS = normal sham operated rats. NH = normal partially hepatectomized rats. DS = diabetic sham operated rats. "H = diabetic partially hepatectomized rats. I
Every value is the mean of ten experiments recorded in mgZ 2S.E.
synthetic processes in the normal animals were beginning to decrease. The results of this study also suggest a prolongation of the recovery period in the diabetics. The synthesis of RNA, DNA and protein did not return to control levels as rapidly, and the serum protein concentrations remained depressed longer in the diabetic hepatectomized animals. In the early hours following partial hepatectomy, the synthesis of RNA and protein are usually prominent events. The RNA synthesized during this period is involved in the growth of the cell in preparation for division and also acts as a template for the synthesis of proteins involved in DNA replication (Baserga, '68). One possible explanation for a delay in the onset of regeneration in the diabetics is a reduction in the cells' ability to synthesize RNA and protein. The delay in the onset of regeneration and the prolongation of the recovery period may be the result of changes in metabolism associated with the diabetic state. The glycogen and lipid concentrations of the liver are altered in the diabetics with a depletion of glycogen stores and the development of a fatty liver. The significance of glycogen stores in DNA synthesis is uncertain (Baserga, '68).The early depletion of liver glycogen following partial hepatectomy probably supplies some of the energy required for the initial synthetic processes. Due to the decreased glycogen content, the diabetics may lack an important factor involved in the initial regenerative response. This observed response is similar to that described for fasted animals (MacDonald et al., '63; Montecuccoli et al., '72; Stirling et al.,
'73), and supports the possibility that the metabolic state of the animals and not insulin per se is responsible for the delay in regeneration. ACKNOWLEDGMENTS
This study was supported by USPH Grant RR7059. We wish to thank Doctors F. George Zaki and C. Hans Keysser, Director of the Pathology Department of the Squibb Institute for Medical Research for their help and for the use of their facilities. LITERATURE CITED Baserga, R. 1968 Biochemistry of the cell cycle: A review. Cell Tissue Kinet., I: 167-191. Bucher, N. L. R. 1963 Regeneration of mammalian liver. Int. Rev. Cytol., 15: 245-300. Bucher, N. L. R., and M. N. Swaffield 1973 Regeneration of liver in rats in the absence of portal splanchnic organs and a portal blood supply. Cancer Res., 33: 3189-3194. Burton, K. 1956 A study of the condition and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J., 62:315323. Endo, Y. 1970 A simultaneous estimation method of DNA and RNA by the orcinol reaction and a study of the reaction mechanism. 3. Biochem., 67:629-633. Fisher, B., P. Szuch and E. R. Fisher 1971 Evaluation of a humoral factor in liver regeneration utilizing liver transplants. Cancer Res., 31: 322-331. Gornall, A. G., C. J. Baradawill and M. M. David 1949 Determination of serum protein by means of the hiuret reaction. J . Biol. Chem., 177: 751-766. Hammarsten, E., S. Aqvist, E. P. Anderson and N. A. Eliasson 1956 The turnover of polynucleotides and proteins in regenerating rat liver, studies with 15N-glycine. Aeta Chem. Scand., 10: 1568-1575. Higgins, G. M., and R. M. Anderson 1931 Experimental Pathology of the liver. Arch. Pathol., 12: 186-202. LaBrecque, D. R., and L. A. Pesch 1975 Preparation and partial characterization of hepatic regenerative stimulator substance from rat liver. J . Physiol., 248: 273-284. Leduc, E. H. 1964 Regeneration of the liver. In: The
LIVER REGENERATI(3N IN DIABETIC RATS Liver. C. H. Rouiller, ed. Academic Press, New York, p. 63. Lee, S., C. E. Broelsch, J. G. Chandler, C. A. Charters and M. J. Orloff 1974 Liver regeneration following portacaval transposition in rats. Surg. Forum, 25: 391-394. Leffert, H. L. 1974 Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. J. Cell. Biol., 62:792-801. Luna, L. G., ed. 1968 Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. McGraw Hill, New York. MacDonald, R. A., A. E. Rogers and G. S. Pechet 1963 Growth and regeneration of the liver. Ann. N.Y. Acad. Sci., 111: 70-86. Max, M. H., J. B. Price, K. Takeshige and A. B. Voorhees 1972 The role of factors of portal origin in modifying hepatic regeneration. J. Surg. Res., 12: 120-123. Montecuccoli, G., F. Novello and F. Stirpe 1972 Effect of protein deprivation and of starvation on DNA synthesis in resting and regenerating rat liver. J. Nutr., 102: 507512. Price, J. B., K. Takeshige, M. H. Max and A. B. Voorhees 1972 Glucagon as the portal factor modifying hepatic regeneration. Surg., 72: 74-82. Potter, E. R., and B. Barbiroli 1971 DNA synthesis and interaction between controlled feeding schedules and partial hepatectomy in rats. Sci., 172: 738-741. Sgro, J. C., A. C. Charters, J. G. Chandler, D. E. Gramhoit and M. J. Orloff 1973 Site of origin of the hepatotrophic portal blood factor involved in liver regeneration. Surg. Forum, 24: 377-378. Simek, J., V. L. Chemlait, J. Melka, J. Pasderka and Z. Charvat 1967 Influence of protracted infusion of glucose and insulin on the composition and regeneration activity
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of liver after partial hepatectomy in rats. Nature, 213: 910-911. Shibko, S. P., P. Koivestoinin, C. A. Tratnyek, A. R. Newhall and L. Friedman 1967 A method for sequential quantitative separation and determination of protein, RNA, DNA, lipid and glycogen from a single rat liver homogenate or from a subcellular fraction. Anal. Biochem., 19: 514-528. Snedecor, G. W. 1959 Statistical Methods Applied to Experiments in Agriculture and Biology. Iowa State College Press, Ames, Iowa. Starzl, T. E., N. Kashiwage, K. A. Porter, I. Y. Lee and W. J. I. Russell 1975 The effect of diabetes mellitus on portal blood hepatotrophic factors in dogs. Surg. Gyn. and Obst., 140: 549-562. Starzl, T. E., K. A. Porter and C. W. Putnam 1975 Intraportal insulin protects from the liver injury of portacaval shunt in dogs. Lancet, 2: 1241-1242. Starzl, T. E., K. A. Porter, K. Watanabe and C. W. Putnam 1976 Effects of insulin, glucagon, and insulin/glucagon infusions on liver morphology and cell division after complete portacaval shunt in dogs. Lancet, 1: 821-825. Stirling, G. A., J. Laughlin and S. L. A. Washington 1973 The effects of starvation on the proliferative response after partial hepatectomy. Exp. Mol. Pathol., 19: 44-52. Younger, L. R., J. King and D. F. Steiner 1966 Hepatic proliferative response to insulin in severe alloxan diabetes. Cancer Res., 26: 1408-1414. VanLancker, J. L. 1970 Control of DNA synthesis in regenerating rat liver. Fed. Proc., 29: 1439-1442. Weinbren, K., S. Washington and F. Dowling 1973 The proliferative response after partial hepatectomy in hypophysectomized rats bearing portacaval anastomoses. Br. J. Exp. Path., 54: 429-436.
