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Blood and Liver Metabolites in Fed and Fasted Diabetic Goats1
B l o o d a n d L i v e r M e t a b o l i t e s in F e d a n d Fasted D i a b e t i c G o a t s 1 J. W. SCHWALM = and L. H. SCHULTZ Department of Dairy Science University of Wisconsin Madison 53706 ABSTRACT
Two trials were to study alloxan diabetes in goats. The data were grouped: 1) normal fed goats (10); 2) 48-h fasted goats (5); 3) fed goats sampled 96 h after alloxan treatment (5); and 4) goats treated with alloxan following a 48-h fast and sampled 96 h after alloxan treatment with continued fasting (3). Groups 1 and 4 exhibited the following means: serum insulin 43.9, 16.4, 9.4, and 6.7 btU/ml; blood glucose 55.0, 47.3, 219.6, and 485.6 mg/100 ml; blood ketones 4.3, 2.6, 36.6, and 28.6 rag/100 ml; blood acetate 4.7, 4.0, 42.7, and 4.9 mg/100 ml; plasma-free fatty acids 1.8, 10.0, 14.4, and 40.5 mg/100 ml; and plasma triglyceride 13.3, 7.0, 47.6, and 12.2 mg/100 ml. Liver samples from five fed goats before and 12 days after alloxan treatment exhibited the following means: phosphoIipid 27.5 and 26.1 mg/g; trigiyceride 21.2 and 98.9 mg/g; and percent lipid 7.2 and 14.4. The diabetes was accompanied by fatty liver development and probably reduction in utilization of acetate and triglyceride in the fed animals. INTRODUCTION
Studies on control mechanisms regulating hepatic ketogenesis and esterification in nonruminants (17) have shown that, under normal circumstances, fatty acids are esterified rapidly in the liver and are, therefore, unavailable for ketone body formation. In the ketotic cow, plasma triglycerides are depressed in the face of markedly elevated plasma free fatty acids and
Received June 20, 1975. 1Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by Grant AM-08546 from the National Institutes of Arthritis and Metabolic Diseases. 2Department of Dairy Science, Michigan State University, East Lansing 48824.
ketone bodies (21). The underlying cause of the lowered plasma triglyceride concentration is not clear although Griel and McCarthy (10) have attributed it to impaired hepatic lipoprotein release. That fatty liver development may accompany ketosis has been demonstrated by Saarinen and Shaw (23). They suggested that fatty liver development occurred as a result of negative energy balance and was not a causative factor in ketosis. Kronfeld (12) has suggested that bovine ketosis might be associated with depressed insulin. Meier et al. (17) demonstrated the development of fatty liver in the face of both enhanced hepatic ketogenesis and esterification in the diabetic rat. Since many of the metabolic changes during bovine ketosis are also characteristic of the diabetic state, two trials were undertaken to investigate the effects of insulin insufficiency in fed and fasted diabetic goats. A preliminary report of this work was in abstract (25). EXPERIMENTAL PROCEDURE
Two trials, each involving five female goats, were to investigate the effects of experimentally induced diabetes on circulating blood metabolites, serum insulin, and hepatic lipids. Trial. I involved fed goats and consisted of two periods during which each goat received approximately 300 g grain mixture plus long hay ad libitum daily throughout the entire experiment. Feeding times were at 0800 and 1500. Period I consisted of a control period wherein two blood samples were obtained via jugular venipuncture and duplicate liver samples were taken via laparotomy. When surgical recovery was complete, alloxan was given intravenously at 40 mg/kg (18) to induce experimental diabetes. To minimize decomposition, the prescribed dose of alloxan was dissolved in a phosphate-citrate buffer (pH 4.0). Thereafter, blood samples were drawn at 12-h intervals (0800 and 2000) for 12 days following alloxan administration. The period following alloxan treatment was designated as Period II. At the conclusion of Period II, liver samples were again obtained via laparoto262
METABOLITES IN DIABETIC GOATS my. Because blood acetate was high in Trial I, Trial II was with fasted animals to clarify the source of the acetate. The design of Trial II was: five goats were fed for 2 days (Period I), fasted for 2 days (Period II), and then fasting was continued 4 more days (Period IlI). Three goats received alloxan at the beginning of Period III and two served as control animals. Samples were obtained via jugular venipuncture at 12-h intervals (0800 and 2000) throughout the entire trial. Preliminary feeding and alloxan dosage were the same as in Trial I. Protein-free filtrates were obtained by a Ba(OH)z-ZnSO4 procedure, and blood glucose (GLU) was determined by the method of Somogyi (26). Blood acetoacetate plus acetone (AA+A) and /3-hydroxybutyrate (BHBA) were fractionally distilled as acetone (24), which was then colorimetrically assayed according to Behre and Benedict (4) and Block and Boiling (7). Blood acetate (Ac) was determined by the method of Baumgardt (3). Quantitation of plasma free fatty acids (FFA) and triglycerides (TG) was by procedures of Duncombe (9) and Van Handel (32), respectively. Serum insulin (INS) was determined by radioimmunoassay kit supplied by Amersham/Searle Corporation (1). Phospholipids (PHL) were determined in Trial I according to Beveridge and Johnson (5) and Boltz and Mellon (8) from plasma lipid extracts subjected to thin layer chromatography as described by Varman and Schultz (33). Hepatic lipid extracts were prepared from duplicate homogenized liver samples. Following thin layer chromatography, hepatic free fatty acids, triglycerides, cholesterol esters (CHE), free cholesterol (FCH), and phospholipids were determined according to the methods previously described for plasma. In addition, hepatic total lipids were determined gravimetrically from hepatic lipid extracts. All liver results are expressed on a wet weight basis. Statistical significance was determined by least squares analysis of variance (27). RESULTS A N D DISCUSSION
Results from Trial I are in Tables 1 and 2 while Tables 3 and 4 summarize results of Trial II. Conclusions have been drawn from statistical analysis of the data from each experiment. However, no statistical comparisons were pos-
263
sible between experiments. Nevertheless, for ease of discussion, physiological implications of both trials will be considered together. Normal Fed Vs. Fasted
As in the forepart of Table 1 and during Period I of Table 3, initial serum insulin and blood metabolite measurements were within the accepted ranges representative of normal fed goats (28, 31, 36). Effects of a 48-h fast are in Period II of Tables 3 and 4. Effects of a prolonged fast are reflected by changes in the control group during Period III in Tables 3 and 4. In general, no changes were significant (P<.10) in blood glucose or blood ketone body concentrations as a result of fasting. Martin et aL (15) have shown that in the absence of a glucose drain for production purposes fasted sheep were able to maintain normal blood glucose via gluconeogenesis and glucose-sparing mechanisms. Our fasted goats responded similarly. Even though blood glucose was maintained approximately normal, serum insulin declined during fasting to a basal 15 to 16 /~U/ml. Lewis et al. (14) have shown in rats that insulin suppresses hepatic ketogenesis. We postulate that this basal insulin was sufficient to prevent excessive hepatic ketogenesis in fasted goats; hence, blood ketone bodies were mainmined at low concentration. The primary source of blood acetate in the goat is that which is produced via ruminal fermentation. Since fasting removes the source of exogenous acetate, blood acetate might be expected to decline without endogenous acetate formation. Palmquist (20) and Annison and White (2) have evidence for endogenous acetate production in ruminants a~nd have suggested /3-oxidation of long-chain fatty acids as its source. Knowles et al. (11) observed no change in hepatic acetyl CoA synthetase activity while hepatic acetyI CoA hydrolase activity was increased greatly in starved sheep. However, the latter study indicated that liver mitochondrial fractions did not form acetate from either pyruvate or palmitoyl-(-)-carnitine but instead converted acetate into acetoacetate. They proposed that acetate in the blood of starved sheep is derived from hydrolysis of acetyl CoA. Blood acetate concentration declined during starvation in both studies. Thus, endogenous acetate formation was unable to Journal of Dairy Science Vol. 59, No. 2
264
SCHWALM AND SCHULTZ
TABLE 1. Time course of blood changes a associated with expe ri me nt a l diabetes in fed goats. Day
Time
INS
GLU
KET
FFA
gU/ml Period I 0
am
TG
Ac
PHL
(mg/ 100 ml) b 50.6
36.9
2.4
4.4
12.1
4.5
74.9
Alloxan administration Period II pm
61.8
102.3
2.1
2.9
10.1
8.2
73.5
1
am pm
18.6 12.5
205.0 205.6
3.3 12.6
10.8 7.8
9.8 13.1
5.6 11.7
82.7 80.4
2
am pm
10.9 10.4
210.8 234.7
19.6 26.3
18.5 11.2
19.3 30,9
6.4 30.3
94,4 96.9
3
am pm
10.4 9.4
240.4 219.6
32.7 36.6
27.1 14.4
45.4 47.6
19.9 42.7
118.5 110.8
4
am pm
8.3 9.2
207.7 226.9
40.4 40.8
18.8 15.5
62.6 52.5
22.7 39.2
128.1 124.3
5
am pm
9.0 9.6
223.4 206.9
41.2 40.6
27.0 14.8
60.9 53.6
28.3 31.7
132.8 128.0
6
am pm
8.0 9.8
196.9 200.9
42.6 33.5
23.4 14.9
75.1 60.4
21.6 30.9
132.9 134.3
7
am pm
8.3 9.4
186.1 192.4
34.9 33.5
23.4 15.9
71.1 54.3
22.5 29.0
134.2 122.4
8
am pm
8.6 8.8
177.0 190.5
32.9 35.4
29.5 26.5
71.8 55.4
21.0 24.1
130.0 125.3
9
am pm
10.0 8.7
182.7 197.4
34.1 45.8
24.7 14.9
72.3 59.2
23.7 32.0
121.4 126.9
10
am pm
9.6 9.2
183.6 177.7
45.1 42.8
22.7 15.3
64.5 47.5
28.1 37.7
127.3 127.6
11
am pm
8.6 8.6
172.9 173.8
40.0 37.5
27.9 17.9
57.7 47.6
28.9 36.5
124.1 126.4
12
am
9.2
163.7
35.9
22.4
52.2
26.6
128.5
Inc
Inc
Inc
lnc
lnc
Inc
Change from normal c
Dec
awords abbreviated in table headings are: INS = insulin; GLU = glucose; KET = ketone bodies; FFA = free f atty acids; TG = triglycerides; Ac = acetate; and PHL = phospholipids. bValues represent mean of five female goats. CAll changes within Period II were significant (P<.O1).
TABLE 2. Changes in hepatic lipidsa associated with experime nt a l diabetes in fed goats. % Physiolog4cal state
Obs. b
FFA
FCH
CHE
Normal (prior to alloxan) Diabetic (12 days after alloxan) Change from normal
10 10
.105 .222 NC
1.414 1.231 NC
PHL
TG
Lipid
27.6 26.1 NC
21.2 98.9 Inc c
7.2 14.1 Inc c
(mg/g wet tissue) .362 ~739 NC
awo rd s abbreviated in table headings are: Obs. = n u m b e r of observations; FFA = free fa t t y acids; FCH = free cholesterol; CHE = cholesterol esters; PHL = phospholipids; and TG = triglycerides. bValues represent means of duplicate samples of same 5 female goats before and after alloxan. Cp<.05. Journal of Dairy Science Vol. 59, No. 2
TABLE 3. Changes in b l o o d m e t a b o l i t e means a in fed, fasted, and fasted diabetic goats. C ont rol (2) Day
Time
INS
GLU
KET
pU/ml Period I 0 1
Period II 2 3
Period III 4
Diabetic (3)
FFA
TG
Ac
(mg/1 O0 ml)
INS
GLU
uU/ml
am pm
49,5 26,5
47,6 50.4
.9 3.0
.9 2.8
14.1 11.4
am pm
23,9 26.8
48.0 48.1
3.4 6.6
4.0 1.2
8.8 14.4
am pm
31.6 19.6
47.2 46.8
5.5 3.0
1.1 9.2
18.5 9.2
am pm
7.5 16.4
55.1 46.9
3.2 2.6
16.2 11.2
7.0 6.0
am
17.9
41.4
2.9
16.7
5.4
Feeding period 4.0 57.1 4.6 38.8
< o
FFA
TG
Ac
( m g / l O 0 ml) 50.5 49.6
1.0 3.3
.7 1.1
19.2 13.5
2.8 3.7
43.9 50.3
48.1 53.5
3.6 6.5
1.7 .7
14.9 19.6
3.7 3.2
Fasting period 4.6 34.2 3.9 19.0
47.5 48.3
5.0 2.7
.6 7.3
22.3 10.2
3.6 3.6
49.4 47.5
3.0 2.5
12.5 9.1
8.5 7.6
4.0 4.0
3.2 3.3
3.1 4.0
31.1 16.4
© t~
t~ *q 2.9
17.4
41.3
2.7
16.6
6.5
3.0 Q
No alloxan e-
KET
A llo x an a d m i n i s t r a t i o n
~q
pm
16.0
42.2
2.9
19.8
6.6
4.2
63.9
20,5
2.6
24.3
7.7
3.3
am pm
16.7 15.5
39.5 45.7
3.3 2.3
22.2 18.0
6.3 6.5
4.1 4.3
20.2 11.4
49.1 213.2
3.0 3.9
6.6 13.6
3.7 5.3
4.3 4.0
am pm
15.8 15,2
44.7 50.9
2.0 2.3
27.5 25.5
7.0 6.1
5.2 4.0
8.8 8.5
255.0 409.5
9.3 14.7
31.2 39.1
9.7 7.7
4.3 5.0
am pm
15.5 16,3
49.3 43.6
3.5 2.8
42.1 29.1
6.8 7.1
4.7 5.3
4.9 6.6
381.8 485.6
22.5 28.6
46.5 40.5
10.4 12.2
4.3 4.9
a w o r d s abbreviated in table headings are: INS = insulin; GLU = glucose; KET = k e t o n e bodies; F F A = free f a t t y acids; TG = triglycerides; and Ac = acetate.
bO Ox t~
266
SCHWALM AND SCHULTZ
TABLE 4. Selected analysis of variance on data in Table 3 from fed, fasted, and fasted diabetic goats a. Blood metabolite b Source of variation
INS
GLU
KET
Period I [Control (2) and Treatment (3) both fed] Day/Period I c . . . . . Treatment X day/Period Id . . . . .
FFA
TG
Inc
Dec
Ac
Inc
Period II [Control (2) and Treatment (3) both fasted] Day/Period II c . . . . . Treatment × day/Period IId . . . . . Period Ill [Control (2) fasted and Treatment (3) diabetic fasted] Day/Period IlI c Dec Inc Treatment X day/Period Ill d,e Dec Inc
lnc Inc
Inc ... . . . . . . . . .
Inc
aAll changes P<.10. bWords abbreviated in table headings are: INS = insulin; GLU = glucose; KET = ketone bodies; FFA = free fatty acids; TG = triglycerides; and Ac = acetate. CIndicates changes within each period where those changes represent a composite of all five goats. d • In&cates the changes in Treatment Group (3) relative ;o Control Group (2) within each period. eDifferentiates between effects of fasting and diabetic fasting.
c o m p e n s a t e c o m p l e t e l y for the loss of exogenous acetate. Our results indicated a small but statistically significant rise in c o n c e n t r a t i o n of b l o o d acetate during normal starvation. However, b l o o d acetate was still within the physiological range. Sutherland and R o b i s o n (29) have n o t e d t h a t in rat adipose tissue an insulin deficiency leaves u n o p p o s e d the s t i m u l a t o r y action of the catecholamines on adenyl cyclase, thereby increasing cyclic AMP which in turn leads to excessive adipose triglyceride lipolysis. Our results s u p p o r t this idea in that there was a marked elevation o f plasma free f a t t y acids which was associated with depressed serum insulin during e x t e n d e d fasting. A l t h o u g h hepatic fipids were n o t measured in Trial If, fatty liver has developed in ruminants u n d e r conditions of e x t e n d e d fasting (30). Thus, if fatty liver is developing, this m a y help explain t h e decline in plasma triglycerides. Griel and McCarthy (10) also have suggested that impaired lipoprotein release might be involved in d e v e l o p m e n t of f a t t y liver. Woodside and H e i m b e r g (35) have n o t e d in rats that insulin may be required to permit n o r m a l o u t p u t of hepatic triglycerides. D e v e l o p m e n t of fatty liver was due primarily to increased hepatic triglycefide content. In summary, the changes f r o m normal as a Journal of Dairy Science Vol. 59, No. 2
result of fasting were m a i n t e n a n c e of normal blood glucose and b l o o d k e t o n e bodies, slightly depressed serum insulin and plasma triglycerides, mildly elevated b l o o d acetate, and markedly elevated plasma free fatty acids.
Diabetic Fed Vs. Diabetic Fasted
T h e effects of induction o f e x p e r i m e n t a l diabetes are illustrated for fed alloxanized goats (Table 1) and fasted alloxanized goats (Table 3, Period III, diabetic group). The changes in serum insulin, b l o o d glucose, blood ketone bodies, and plasma free fatty acids were similar in b o t h trials. Serum insulin e x h i b i t e d a characteristic biphasic response in t h a t there was an elevation in insulin f o l l o w e d by a depression. This may be explained by the sudden release of stored insulin as a result of the destructive action of alloxan on the pancreatic /3-cells. Thereafter, serum insulin r e m a i n e d low (about 9 / a U / m l in fed diabetic goats and 5 to 8 / I U / m l in fasted diabetic goats), t h e r e b y indicating successful induction of diabetes. Associated with the depressed insulin was a m a r k e d h y p e r g l y c e m i a a p p r o x i m a t i n g the renal threshold• Sutherland and R o b i s o n (29) and L'age et al. (13) have n o t e d that elevated hepatic cyclic AMP resulting f r o m the unopposed action of glucagon, catecholamines, and
METABOLITES IN DIABETIC GOATS glucocorticoids would cause elevated glycogenolysis and gluconeogenesis. Furthermore, the utilization of glucose by the periphery is impaired markedly under diabetic conditions; hence, the ultimate result is a marked elevation in blood glucose as in our studies. With the impairment in the utilization of glucose, adipose triglyceride lipolysis was accelerated markedly. This would, in turn, lead to a greater supply of plasma free fatty acids being presented to the liver. We postulate that under diabetic conditions, action of insulin is overp o w e r e d m a r k e d l y by glucagon, catecholamines, and glucocorticoids which lead to greatly accelerated hepatic ketogenesis as evidenced by the significant elevation in blood ketone bodies. Whereas insulin was sufficient to prevent hepatic ketogenesis under normal fed or fasted conditions (14), diabetes resulted in insufficient insulin to counteract the effects of glucagon and the glucocorticoids. Differences were evident between fed and fasted diabetic goats in plasma triglycerides and blood acetate. There was a significant rapid rise in plasma triglycerides in fed diabetic goats while fasted diabetic goats showed a nonsignificant upward trend. Possibly the difference in response of the two metabolic states to alloxan may be accounted for by an exogenous source of triglyceride in the fed diabetic goats while all plasma triglyceride in the fasted diabetic goats must have been of endogenous origin. Hepatic esterification mechanisms probably were not depressed during diabetes. In fact, Nikkila and Kekki (19) have evidence in rats to support the idea that hepatic triglyceride output is enhanced under diabetic conditions. Development of fatty liver is indicated by the twofold rise in hepatic lipid content of livers from fed diabetic goats (Table 2). Furthermore, the fivefold elevation in hepatic triglyceride content accounts for nearly all of the rise in hepatic lipid content. No differences were significant in other liver lipid fractions. The accumulation of triglyceride in the liver suggests that there may have been some impairment of lipoprotein release. Weinstein et al. (34) have presented evidence indicating that while hepatic triglyceride output is related inversely to glucagon, it is less sensitive to the action of glucagon that are the mechanisms of glycogenolysis, gluconeogenesis, ureogenesis, and ketogenesis. Meier et al. (17) have demon-
267
strated that plasma glucagon rose markedly in both fed and fasted diabetic rats. Hence, high glucagon may be partially responsible for development of fatty liver. Probably there was also an impairment in triglyceride utilization under diabetic conditions. Studies by Bierman (6) and Mayes (16) have demonstrated that lipoprotein lipase activity is depressed under diabetic and fasting conditions in rats. Thus, accumulation of plasma triglycerides may be associated closely with conditions of insulin insufficiency. As in Table 1, plasma phospholipid was significantly elevated in fed diabetic goats. We suggest this is merely a reflection of elevated plasma triglyceride since phospholipid is an important structural component of the lipoprotein moiety. There was a significant marked rise in blood acetate in fed diabetic goats along with a small but significant rise in blood acetate in fasted diabetic goats. However, no difference between fasted only and fasted diabetic animals (Table 4) indicates that the rise in blood acetate in fasted diabetic goats was simply a consequence of starvation rather than o f diabetes. As previously mentioned, blood acetate is derived primarily from ruminal fermentation in fed ruminants. There may have been some endogenous acetate formation in the fasted diabetic goats as evidenced by small amounts of blood acetate in those animals; acetate utilization is dependent upon glucose utilization. Thus, when glucose utilization is depressed due to insulin insufficiency, acetate utilization also must be affected. Earlier studies of alloxan diabetic sheep led Reid (22) to suggest that impaired utilization of ruminally produced acetate was the reason for accumulation of acetate in the blood. Our study supports this idea. Recent studies by Knowles et al. (11) have shown further that in alloxan diabetic sheep the artery-femoral vein difference of acetate in the hind limb was reduced markedly, thereby indicating impaired acetate utilization during insulin insufficiency. In conclusion, an important role has been demonstrated for insulin in ruminant metabolism. Not only is it involved in regulation of blood glucose, but it is also important in the regulation of adipose tissue metabolism. The results support the concept that insulin is necessary for the utilization of acetate by peripheral tissues and is also important in preventing lipolysis and aiding in utilization of Journal of Dairy Science Vol. 59, No. 2
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plasma triglycerides via its actions on lipoprotein lipase. It appears that a lack of insulin results in an increase in both hepatic ketogenesis and esterification, thereby demonstrating that both metabolic processes can occur simultaneously in the ruminant. ACKNOWLEDGMENTS
The authors express their appreciation to Margaret Iwen for her expert technical assistance and to A. H. Quamme for his assistance in the care and feeding of the experimental animals. Special thanks go to R. H. Stauffacher for performing laparotomies during the study. The assistance of G. E. Shook and G. W. Bodoh in statistical analyses of the data also is acknowledged gratefully. REFERENCES
1 Amersham/Searle Corporation. 1969. Insulin immunoassay kit. Code IM. 49. 2 Annison, E. F., and R. R. White. 1962. Further studies on the entry rates o f acetate and glucose in sheep, with special reference to endogenous production o f acetate. Biochem. J. 84:546. 3 Baumgardt, B. R. 1964. Practical observations on the quantitative analysis of free volatile fatty acids (VFA) in aqueous solution by gas-liquid chromatography. Dept. of Dairy Science, Univ. of WI, Madison. Bull. No. 1. 4 Behre, J. A., and S. R. Benedict. 1926. A colorimetric determination of acetone bodies in blood and urine. J. Biol. Chem. 70:487. 5 Beveridge, J. M. R., and S. E. Johnson. 1949. The determination o f phospholipid phosphorus. Can. J. Res. 27:159. 6 Bierman, E. L. 1972. Insulin and hypertriglyceridemia. Page 129 in Impact of insulin on metabolic pathways. Academic Press, New York. 7 Block, R. J., and D. Boiling. 1951. Page 3 8 i n The determination of amino acids. Rev. ed. Burgess Publ. Co., Minneapolis, MN. 8 Boltz, D. F., and M. G. Mellon. 1947. Determination of phosphorus, germanium, and arsenic by the heteropoly blue method. A n a l Chim. 19:873. 9 Duncombe, W. G. 1964. The colorimetric microdetermination of non-esterified fatty acids in plasma. Clin. Chim. Acta 9:122. 10 Grid, L. C., Jr., and R. D. McCarthy. 1969. Blood serum lipoproteins: A review. J. Dairy Sci. 52:1233. 11 Knowles, S. E., I. G. Jarrett, O. H. Filsell, and F. J. Ballard. 1974. Production and utilization of acetate in mammals. Biocbem. J. 142:401. 12 Kronfeld, D. S. 1971. Hypoglycemia in ketotic cow~ J. Dairy Sci. 54:949. 13 L'age, M., W. Fechner, J. Langholz, and H. Saizmann. 1974. Relationship between plasma corticosterone and the development of ketoacidoJournal of Dairy Science Vol. 59, No. 2
sis in the ailoxan-diabetic rat. Diabetologia 10:131. 14 Lewis, S. B., J. H. Extun, R. J. Ho, and C. R. Park. 1971. Interactions of insulin, glucagon, epinephrine and cyclic AMP in the perfused rat liver. Fed. Proc. 30:1205. 15 Martin, R. J., L. L. Wilson, R. L. Cowan, a n d J . D. Sink. 1973. Effects of fasting and diet on enzyme profiles in ovine liver and adipose tissue. J. Anita. Sci. 36:101. 16 Mayes, P. A. 1969. The role of the liver in fatty acid transport. Proc. Biochem. Soc. 115:45p. 17 Meier, J. M., J. D. McGarry, G. R. Faloona, R. H. Unger, and D. W. Foster. 1972. Studies of the development of diabetic ketosis in the rat. J. Lipid Res. 13:228. 18 Menahan, L. A., L. H. Schultz, and W. G. Hoekstra. 1966. Relationship of ketone body metabolism and carbohydrate utilization to fat mobilization in the ruminant. J. Dairy Sci. 49:957. 19 Nikkila, E. A., and M. Kekki. 1970. Turnover rate of serum triglycerides in diabetes. Diabetologia 6:658. 20 Palmquist, D. L. 1972. Palmitic acid as a source of endogenous acetate and j3-hydroxybutyrate in fed and fasted ruminants. J. Nutr. 102:1401. 21 Radloff, H. D., and L. H. Schultz. 1967. Blood and rumen changes in cows in early stages o f ketosis. J. Dairy Sci. 50:68. 22 Reid, R. L. 1968. The physiolopathology of undernourishment in pregnant sheep, with particular reference to pregnancy toxemia. Adv. Vet. Sci. Comp. Med. 12:164. 23 Saarinen, P., and J. C. Shaw. 1950. Studies on ketosis in dairy cattle. XIII. Lipids and ascorbic acid in the liver and adrenals of cows with spontaneous and fasting ketosis. J. Dairy Sci. 33:515. 24 Schwalm, J. W., R. Waterman, G. E. Shook, and L. H. Schultz. 1972. Blood metabolite interrelationships and changes in mammary gland metabolism during subclinical ketosis. J. Dairy Sci. 55:58. 25 Schwalm, J. W., and L. H. Schultz. 1973. Relationship of insulin concentrations to various blood metabolites in the dairy cow. J. Dairy Sci. 56:672. (Abstr.) 26 Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195:19. 27 Steel, R. G. D., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-HUl Book Co., Inc., New York. 28 Stern, J. S., C. A. Baile, and J. Mayer. 1971. Growth hormone, insulin, and glucose in suckling, weanling, and mature ruminants. J. Dairy Sci. 54:1052. 29 Sutherland, E. W., and G. A. Robison. 1969. The role of cyclic AMP in the control of carbohydrate metabolism. Diebetes 18: 797. 30 Thornton, J. H., and L. H. Schultz. 1975. Unpublished results. 31 Trenlde, A. 1972. Radioimmunoassay of plasma hormones: Review o f plasma insulin in ruminants. J. Dairy Sci. 55:1200. 32 Van Handel, E. 1961. Suggested modifications o f the microdetermination of triglycerides. Clin.
METABOLITES IN DIABETIC GOATS Chem. J. 7:249. 33 Varman, P. N., and L. H. Schultz. 1968. Blood lipids of cows at different stages of lactation. J. Dairy Sci. 51:1971. 34 Weinstein, I., H. A. Klausner, and M. Heimberg. 1973. The effect of concentration of glucagon on output of triglyceride, ketone bodies, glucose, and
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urea by the liver. Biochim. Biophys. Acta 296:300. 35 Woodside, W. F., and M. Heimberg. 1972. Hepatic metabolism of free fatty acids in experimental diabetes. Isr. J. Med. Sci. 8:309. 36 Yamdagni, S., and L. H. Schultz. 1969. Metabolism of 1-14 C palmitic acid in goats in various metabolic states. J. Dairy Sci. 52:1278.
Journal of Dairy Science Vol. 59, No. 2
Two trials were to study alloxan diabetes in goats. The data were grouped: 1) normal fed goats (10); 2) 48-h fasted goats (5); 3) fed goats sampled 96 h after alloxan treatment (5); and 4) goats treated with alloxan following a 48-h fast and sampled 96 h after alloxan treatment with continued fasting (3). Groups 1 and 4 exhibited the following means: serum insulin 43.9, 16.4, 9.4, and 6.7 btU/ml; blood glucose 55.0, 47.3, 219.6, and 485.6 mg/100 ml; blood ketones 4.3, 2.6, 36.6, and 28.6 rag/100 ml; blood acetate 4.7, 4.0, 42.7, and 4.9 mg/100 ml; plasma-free fatty acids 1.8, 10.0, 14.4, and 40.5 mg/100 ml; and plasma triglyceride 13.3, 7.0, 47.6, and 12.2 mg/100 ml. Liver samples from five fed goats before and 12 days after alloxan treatment exhibited the following means: phosphoIipid 27.5 and 26.1 mg/g; trigiyceride 21.2 and 98.9 mg/g; and percent lipid 7.2 and 14.4. The diabetes was accompanied by fatty liver development and probably reduction in utilization of acetate and triglyceride in the fed animals. INTRODUCTION
Studies on control mechanisms regulating hepatic ketogenesis and esterification in nonruminants (17) have shown that, under normal circumstances, fatty acids are esterified rapidly in the liver and are, therefore, unavailable for ketone body formation. In the ketotic cow, plasma triglycerides are depressed in the face of markedly elevated plasma free fatty acids and
Received June 20, 1975. 1Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by Grant AM-08546 from the National Institutes of Arthritis and Metabolic Diseases. 2Department of Dairy Science, Michigan State University, East Lansing 48824.
ketone bodies (21). The underlying cause of the lowered plasma triglyceride concentration is not clear although Griel and McCarthy (10) have attributed it to impaired hepatic lipoprotein release. That fatty liver development may accompany ketosis has been demonstrated by Saarinen and Shaw (23). They suggested that fatty liver development occurred as a result of negative energy balance and was not a causative factor in ketosis. Kronfeld (12) has suggested that bovine ketosis might be associated with depressed insulin. Meier et al. (17) demonstrated the development of fatty liver in the face of both enhanced hepatic ketogenesis and esterification in the diabetic rat. Since many of the metabolic changes during bovine ketosis are also characteristic of the diabetic state, two trials were undertaken to investigate the effects of insulin insufficiency in fed and fasted diabetic goats. A preliminary report of this work was in abstract (25). EXPERIMENTAL PROCEDURE
Two trials, each involving five female goats, were to investigate the effects of experimentally induced diabetes on circulating blood metabolites, serum insulin, and hepatic lipids. Trial. I involved fed goats and consisted of two periods during which each goat received approximately 300 g grain mixture plus long hay ad libitum daily throughout the entire experiment. Feeding times were at 0800 and 1500. Period I consisted of a control period wherein two blood samples were obtained via jugular venipuncture and duplicate liver samples were taken via laparotomy. When surgical recovery was complete, alloxan was given intravenously at 40 mg/kg (18) to induce experimental diabetes. To minimize decomposition, the prescribed dose of alloxan was dissolved in a phosphate-citrate buffer (pH 4.0). Thereafter, blood samples were drawn at 12-h intervals (0800 and 2000) for 12 days following alloxan administration. The period following alloxan treatment was designated as Period II. At the conclusion of Period II, liver samples were again obtained via laparoto262
METABOLITES IN DIABETIC GOATS my. Because blood acetate was high in Trial I, Trial II was with fasted animals to clarify the source of the acetate. The design of Trial II was: five goats were fed for 2 days (Period I), fasted for 2 days (Period II), and then fasting was continued 4 more days (Period IlI). Three goats received alloxan at the beginning of Period III and two served as control animals. Samples were obtained via jugular venipuncture at 12-h intervals (0800 and 2000) throughout the entire trial. Preliminary feeding and alloxan dosage were the same as in Trial I. Protein-free filtrates were obtained by a Ba(OH)z-ZnSO4 procedure, and blood glucose (GLU) was determined by the method of Somogyi (26). Blood acetoacetate plus acetone (AA+A) and /3-hydroxybutyrate (BHBA) were fractionally distilled as acetone (24), which was then colorimetrically assayed according to Behre and Benedict (4) and Block and Boiling (7). Blood acetate (Ac) was determined by the method of Baumgardt (3). Quantitation of plasma free fatty acids (FFA) and triglycerides (TG) was by procedures of Duncombe (9) and Van Handel (32), respectively. Serum insulin (INS) was determined by radioimmunoassay kit supplied by Amersham/Searle Corporation (1). Phospholipids (PHL) were determined in Trial I according to Beveridge and Johnson (5) and Boltz and Mellon (8) from plasma lipid extracts subjected to thin layer chromatography as described by Varman and Schultz (33). Hepatic lipid extracts were prepared from duplicate homogenized liver samples. Following thin layer chromatography, hepatic free fatty acids, triglycerides, cholesterol esters (CHE), free cholesterol (FCH), and phospholipids were determined according to the methods previously described for plasma. In addition, hepatic total lipids were determined gravimetrically from hepatic lipid extracts. All liver results are expressed on a wet weight basis. Statistical significance was determined by least squares analysis of variance (27). RESULTS A N D DISCUSSION
Results from Trial I are in Tables 1 and 2 while Tables 3 and 4 summarize results of Trial II. Conclusions have been drawn from statistical analysis of the data from each experiment. However, no statistical comparisons were pos-
263
sible between experiments. Nevertheless, for ease of discussion, physiological implications of both trials will be considered together. Normal Fed Vs. Fasted
As in the forepart of Table 1 and during Period I of Table 3, initial serum insulin and blood metabolite measurements were within the accepted ranges representative of normal fed goats (28, 31, 36). Effects of a 48-h fast are in Period II of Tables 3 and 4. Effects of a prolonged fast are reflected by changes in the control group during Period III in Tables 3 and 4. In general, no changes were significant (P<.10) in blood glucose or blood ketone body concentrations as a result of fasting. Martin et aL (15) have shown that in the absence of a glucose drain for production purposes fasted sheep were able to maintain normal blood glucose via gluconeogenesis and glucose-sparing mechanisms. Our fasted goats responded similarly. Even though blood glucose was maintained approximately normal, serum insulin declined during fasting to a basal 15 to 16 /~U/ml. Lewis et al. (14) have shown in rats that insulin suppresses hepatic ketogenesis. We postulate that this basal insulin was sufficient to prevent excessive hepatic ketogenesis in fasted goats; hence, blood ketone bodies were mainmined at low concentration. The primary source of blood acetate in the goat is that which is produced via ruminal fermentation. Since fasting removes the source of exogenous acetate, blood acetate might be expected to decline without endogenous acetate formation. Palmquist (20) and Annison and White (2) have evidence for endogenous acetate production in ruminants a~nd have suggested /3-oxidation of long-chain fatty acids as its source. Knowles et al. (11) observed no change in hepatic acetyl CoA synthetase activity while hepatic acetyI CoA hydrolase activity was increased greatly in starved sheep. However, the latter study indicated that liver mitochondrial fractions did not form acetate from either pyruvate or palmitoyl-(-)-carnitine but instead converted acetate into acetoacetate. They proposed that acetate in the blood of starved sheep is derived from hydrolysis of acetyl CoA. Blood acetate concentration declined during starvation in both studies. Thus, endogenous acetate formation was unable to Journal of Dairy Science Vol. 59, No. 2
264
SCHWALM AND SCHULTZ
TABLE 1. Time course of blood changes a associated with expe ri me nt a l diabetes in fed goats. Day
Time
INS
GLU
KET
FFA
gU/ml Period I 0
am
TG
Ac
PHL
(mg/ 100 ml) b 50.6
36.9
2.4
4.4
12.1
4.5
74.9
Alloxan administration Period II pm
61.8
102.3
2.1
2.9
10.1
8.2
73.5
1
am pm
18.6 12.5
205.0 205.6
3.3 12.6
10.8 7.8
9.8 13.1
5.6 11.7
82.7 80.4
2
am pm
10.9 10.4
210.8 234.7
19.6 26.3
18.5 11.2
19.3 30,9
6.4 30.3
94,4 96.9
3
am pm
10.4 9.4
240.4 219.6
32.7 36.6
27.1 14.4
45.4 47.6
19.9 42.7
118.5 110.8
4
am pm
8.3 9.2
207.7 226.9
40.4 40.8
18.8 15.5
62.6 52.5
22.7 39.2
128.1 124.3
5
am pm
9.0 9.6
223.4 206.9
41.2 40.6
27.0 14.8
60.9 53.6
28.3 31.7
132.8 128.0
6
am pm
8.0 9.8
196.9 200.9
42.6 33.5
23.4 14.9
75.1 60.4
21.6 30.9
132.9 134.3
7
am pm
8.3 9.4
186.1 192.4
34.9 33.5
23.4 15.9
71.1 54.3
22.5 29.0
134.2 122.4
8
am pm
8.6 8.8
177.0 190.5
32.9 35.4
29.5 26.5
71.8 55.4
21.0 24.1
130.0 125.3
9
am pm
10.0 8.7
182.7 197.4
34.1 45.8
24.7 14.9
72.3 59.2
23.7 32.0
121.4 126.9
10
am pm
9.6 9.2
183.6 177.7
45.1 42.8
22.7 15.3
64.5 47.5
28.1 37.7
127.3 127.6
11
am pm
8.6 8.6
172.9 173.8
40.0 37.5
27.9 17.9
57.7 47.6
28.9 36.5
124.1 126.4
12
am
9.2
163.7
35.9
22.4
52.2
26.6
128.5
Inc
Inc
Inc
lnc
lnc
Inc
Change from normal c
Dec
awords abbreviated in table headings are: INS = insulin; GLU = glucose; KET = ketone bodies; FFA = free f atty acids; TG = triglycerides; Ac = acetate; and PHL = phospholipids. bValues represent mean of five female goats. CAll changes within Period II were significant (P<.O1).
TABLE 2. Changes in hepatic lipidsa associated with experime nt a l diabetes in fed goats. % Physiolog4cal state
Obs. b
FFA
FCH
CHE
Normal (prior to alloxan) Diabetic (12 days after alloxan) Change from normal
10 10
.105 .222 NC
1.414 1.231 NC
PHL
TG
Lipid
27.6 26.1 NC
21.2 98.9 Inc c
7.2 14.1 Inc c
(mg/g wet tissue) .362 ~739 NC
awo rd s abbreviated in table headings are: Obs. = n u m b e r of observations; FFA = free fa t t y acids; FCH = free cholesterol; CHE = cholesterol esters; PHL = phospholipids; and TG = triglycerides. bValues represent means of duplicate samples of same 5 female goats before and after alloxan. Cp<.05. Journal of Dairy Science Vol. 59, No. 2
TABLE 3. Changes in b l o o d m e t a b o l i t e means a in fed, fasted, and fasted diabetic goats. C ont rol (2) Day
Time
INS
GLU
KET
pU/ml Period I 0 1
Period II 2 3
Period III 4
Diabetic (3)
FFA
TG
Ac
(mg/1 O0 ml)
INS
GLU
uU/ml
am pm
49,5 26,5
47,6 50.4
.9 3.0
.9 2.8
14.1 11.4
am pm
23,9 26.8
48.0 48.1
3.4 6.6
4.0 1.2
8.8 14.4
am pm
31.6 19.6
47.2 46.8
5.5 3.0
1.1 9.2
18.5 9.2
am pm
7.5 16.4
55.1 46.9
3.2 2.6
16.2 11.2
7.0 6.0
am
17.9
41.4
2.9
16.7
5.4
Feeding period 4.0 57.1 4.6 38.8
< o
FFA
TG
Ac
( m g / l O 0 ml) 50.5 49.6
1.0 3.3
.7 1.1
19.2 13.5
2.8 3.7
43.9 50.3
48.1 53.5
3.6 6.5
1.7 .7
14.9 19.6
3.7 3.2
Fasting period 4.6 34.2 3.9 19.0
47.5 48.3
5.0 2.7
.6 7.3
22.3 10.2
3.6 3.6
49.4 47.5
3.0 2.5
12.5 9.1
8.5 7.6
4.0 4.0
3.2 3.3
3.1 4.0
31.1 16.4
© t~
t~ *q 2.9
17.4
41.3
2.7
16.6
6.5
3.0 Q
No alloxan e-
KET
A llo x an a d m i n i s t r a t i o n
~q
pm
16.0
42.2
2.9
19.8
6.6
4.2
63.9
20,5
2.6
24.3
7.7
3.3
am pm
16.7 15.5
39.5 45.7
3.3 2.3
22.2 18.0
6.3 6.5
4.1 4.3
20.2 11.4
49.1 213.2
3.0 3.9
6.6 13.6
3.7 5.3
4.3 4.0
am pm
15.8 15,2
44.7 50.9
2.0 2.3
27.5 25.5
7.0 6.1
5.2 4.0
8.8 8.5
255.0 409.5
9.3 14.7
31.2 39.1
9.7 7.7
4.3 5.0
am pm
15.5 16,3
49.3 43.6
3.5 2.8
42.1 29.1
6.8 7.1
4.7 5.3
4.9 6.6
381.8 485.6
22.5 28.6
46.5 40.5
10.4 12.2
4.3 4.9
a w o r d s abbreviated in table headings are: INS = insulin; GLU = glucose; KET = k e t o n e bodies; F F A = free f a t t y acids; TG = triglycerides; and Ac = acetate.
bO Ox t~
266
SCHWALM AND SCHULTZ
TABLE 4. Selected analysis of variance on data in Table 3 from fed, fasted, and fasted diabetic goats a. Blood metabolite b Source of variation
INS
GLU
KET
Period I [Control (2) and Treatment (3) both fed] Day/Period I c . . . . . Treatment X day/Period Id . . . . .
FFA
TG
Inc
Dec
Ac
Inc
Period II [Control (2) and Treatment (3) both fasted] Day/Period II c . . . . . Treatment × day/Period IId . . . . . Period Ill [Control (2) fasted and Treatment (3) diabetic fasted] Day/Period IlI c Dec Inc Treatment X day/Period Ill d,e Dec Inc
lnc Inc
Inc ... . . . . . . . . .
Inc
aAll changes P<.10. bWords abbreviated in table headings are: INS = insulin; GLU = glucose; KET = ketone bodies; FFA = free fatty acids; TG = triglycerides; and Ac = acetate. CIndicates changes within each period where those changes represent a composite of all five goats. d • In&cates the changes in Treatment Group (3) relative ;o Control Group (2) within each period. eDifferentiates between effects of fasting and diabetic fasting.
c o m p e n s a t e c o m p l e t e l y for the loss of exogenous acetate. Our results indicated a small but statistically significant rise in c o n c e n t r a t i o n of b l o o d acetate during normal starvation. However, b l o o d acetate was still within the physiological range. Sutherland and R o b i s o n (29) have n o t e d t h a t in rat adipose tissue an insulin deficiency leaves u n o p p o s e d the s t i m u l a t o r y action of the catecholamines on adenyl cyclase, thereby increasing cyclic AMP which in turn leads to excessive adipose triglyceride lipolysis. Our results s u p p o r t this idea in that there was a marked elevation o f plasma free f a t t y acids which was associated with depressed serum insulin during e x t e n d e d fasting. A l t h o u g h hepatic fipids were n o t measured in Trial If, fatty liver has developed in ruminants u n d e r conditions of e x t e n d e d fasting (30). Thus, if fatty liver is developing, this m a y help explain t h e decline in plasma triglycerides. Griel and McCarthy (10) also have suggested that impaired lipoprotein release might be involved in d e v e l o p m e n t of f a t t y liver. Woodside and H e i m b e r g (35) have n o t e d in rats that insulin may be required to permit n o r m a l o u t p u t of hepatic triglycerides. D e v e l o p m e n t of fatty liver was due primarily to increased hepatic triglycefide content. In summary, the changes f r o m normal as a Journal of Dairy Science Vol. 59, No. 2
result of fasting were m a i n t e n a n c e of normal blood glucose and b l o o d k e t o n e bodies, slightly depressed serum insulin and plasma triglycerides, mildly elevated b l o o d acetate, and markedly elevated plasma free fatty acids.
Diabetic Fed Vs. Diabetic Fasted
T h e effects of induction o f e x p e r i m e n t a l diabetes are illustrated for fed alloxanized goats (Table 1) and fasted alloxanized goats (Table 3, Period III, diabetic group). The changes in serum insulin, b l o o d glucose, blood ketone bodies, and plasma free fatty acids were similar in b o t h trials. Serum insulin e x h i b i t e d a characteristic biphasic response in t h a t there was an elevation in insulin f o l l o w e d by a depression. This may be explained by the sudden release of stored insulin as a result of the destructive action of alloxan on the pancreatic /3-cells. Thereafter, serum insulin r e m a i n e d low (about 9 / a U / m l in fed diabetic goats and 5 to 8 / I U / m l in fasted diabetic goats), t h e r e b y indicating successful induction of diabetes. Associated with the depressed insulin was a m a r k e d h y p e r g l y c e m i a a p p r o x i m a t i n g the renal threshold• Sutherland and R o b i s o n (29) and L'age et al. (13) have n o t e d that elevated hepatic cyclic AMP resulting f r o m the unopposed action of glucagon, catecholamines, and
METABOLITES IN DIABETIC GOATS glucocorticoids would cause elevated glycogenolysis and gluconeogenesis. Furthermore, the utilization of glucose by the periphery is impaired markedly under diabetic conditions; hence, the ultimate result is a marked elevation in blood glucose as in our studies. With the impairment in the utilization of glucose, adipose triglyceride lipolysis was accelerated markedly. This would, in turn, lead to a greater supply of plasma free fatty acids being presented to the liver. We postulate that under diabetic conditions, action of insulin is overp o w e r e d m a r k e d l y by glucagon, catecholamines, and glucocorticoids which lead to greatly accelerated hepatic ketogenesis as evidenced by the significant elevation in blood ketone bodies. Whereas insulin was sufficient to prevent hepatic ketogenesis under normal fed or fasted conditions (14), diabetes resulted in insufficient insulin to counteract the effects of glucagon and the glucocorticoids. Differences were evident between fed and fasted diabetic goats in plasma triglycerides and blood acetate. There was a significant rapid rise in plasma triglycerides in fed diabetic goats while fasted diabetic goats showed a nonsignificant upward trend. Possibly the difference in response of the two metabolic states to alloxan may be accounted for by an exogenous source of triglyceride in the fed diabetic goats while all plasma triglyceride in the fasted diabetic goats must have been of endogenous origin. Hepatic esterification mechanisms probably were not depressed during diabetes. In fact, Nikkila and Kekki (19) have evidence in rats to support the idea that hepatic triglyceride output is enhanced under diabetic conditions. Development of fatty liver is indicated by the twofold rise in hepatic lipid content of livers from fed diabetic goats (Table 2). Furthermore, the fivefold elevation in hepatic triglyceride content accounts for nearly all of the rise in hepatic lipid content. No differences were significant in other liver lipid fractions. The accumulation of triglyceride in the liver suggests that there may have been some impairment of lipoprotein release. Weinstein et al. (34) have presented evidence indicating that while hepatic triglyceride output is related inversely to glucagon, it is less sensitive to the action of glucagon that are the mechanisms of glycogenolysis, gluconeogenesis, ureogenesis, and ketogenesis. Meier et al. (17) have demon-
267
strated that plasma glucagon rose markedly in both fed and fasted diabetic rats. Hence, high glucagon may be partially responsible for development of fatty liver. Probably there was also an impairment in triglyceride utilization under diabetic conditions. Studies by Bierman (6) and Mayes (16) have demonstrated that lipoprotein lipase activity is depressed under diabetic and fasting conditions in rats. Thus, accumulation of plasma triglycerides may be associated closely with conditions of insulin insufficiency. As in Table 1, plasma phospholipid was significantly elevated in fed diabetic goats. We suggest this is merely a reflection of elevated plasma triglyceride since phospholipid is an important structural component of the lipoprotein moiety. There was a significant marked rise in blood acetate in fed diabetic goats along with a small but significant rise in blood acetate in fasted diabetic goats. However, no difference between fasted only and fasted diabetic animals (Table 4) indicates that the rise in blood acetate in fasted diabetic goats was simply a consequence of starvation rather than o f diabetes. As previously mentioned, blood acetate is derived primarily from ruminal fermentation in fed ruminants. There may have been some endogenous acetate formation in the fasted diabetic goats as evidenced by small amounts of blood acetate in those animals; acetate utilization is dependent upon glucose utilization. Thus, when glucose utilization is depressed due to insulin insufficiency, acetate utilization also must be affected. Earlier studies of alloxan diabetic sheep led Reid (22) to suggest that impaired utilization of ruminally produced acetate was the reason for accumulation of acetate in the blood. Our study supports this idea. Recent studies by Knowles et al. (11) have shown further that in alloxan diabetic sheep the artery-femoral vein difference of acetate in the hind limb was reduced markedly, thereby indicating impaired acetate utilization during insulin insufficiency. In conclusion, an important role has been demonstrated for insulin in ruminant metabolism. Not only is it involved in regulation of blood glucose, but it is also important in the regulation of adipose tissue metabolism. The results support the concept that insulin is necessary for the utilization of acetate by peripheral tissues and is also important in preventing lipolysis and aiding in utilization of Journal of Dairy Science Vol. 59, No. 2
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SCHWALM AND SCHULTZ
plasma triglycerides via its actions on lipoprotein lipase. It appears that a lack of insulin results in an increase in both hepatic ketogenesis and esterification, thereby demonstrating that both metabolic processes can occur simultaneously in the ruminant. ACKNOWLEDGMENTS
The authors express their appreciation to Margaret Iwen for her expert technical assistance and to A. H. Quamme for his assistance in the care and feeding of the experimental animals. Special thanks go to R. H. Stauffacher for performing laparotomies during the study. The assistance of G. E. Shook and G. W. Bodoh in statistical analyses of the data also is acknowledged gratefully. REFERENCES
1 Amersham/Searle Corporation. 1969. Insulin immunoassay kit. Code IM. 49. 2 Annison, E. F., and R. R. White. 1962. Further studies on the entry rates o f acetate and glucose in sheep, with special reference to endogenous production o f acetate. Biochem. J. 84:546. 3 Baumgardt, B. R. 1964. Practical observations on the quantitative analysis of free volatile fatty acids (VFA) in aqueous solution by gas-liquid chromatography. Dept. of Dairy Science, Univ. of WI, Madison. Bull. No. 1. 4 Behre, J. A., and S. R. Benedict. 1926. A colorimetric determination of acetone bodies in blood and urine. J. Biol. Chem. 70:487. 5 Beveridge, J. M. R., and S. E. Johnson. 1949. The determination o f phospholipid phosphorus. Can. J. Res. 27:159. 6 Bierman, E. L. 1972. Insulin and hypertriglyceridemia. Page 129 in Impact of insulin on metabolic pathways. Academic Press, New York. 7 Block, R. J., and D. Boiling. 1951. Page 3 8 i n The determination of amino acids. Rev. ed. Burgess Publ. Co., Minneapolis, MN. 8 Boltz, D. F., and M. G. Mellon. 1947. Determination of phosphorus, germanium, and arsenic by the heteropoly blue method. A n a l Chim. 19:873. 9 Duncombe, W. G. 1964. The colorimetric microdetermination of non-esterified fatty acids in plasma. Clin. Chim. Acta 9:122. 10 Grid, L. C., Jr., and R. D. McCarthy. 1969. Blood serum lipoproteins: A review. J. Dairy Sci. 52:1233. 11 Knowles, S. E., I. G. Jarrett, O. H. Filsell, and F. J. Ballard. 1974. Production and utilization of acetate in mammals. Biocbem. J. 142:401. 12 Kronfeld, D. S. 1971. Hypoglycemia in ketotic cow~ J. Dairy Sci. 54:949. 13 L'age, M., W. Fechner, J. Langholz, and H. Saizmann. 1974. Relationship between plasma corticosterone and the development of ketoacidoJournal of Dairy Science Vol. 59, No. 2
sis in the ailoxan-diabetic rat. Diabetologia 10:131. 14 Lewis, S. B., J. H. Extun, R. J. Ho, and C. R. Park. 1971. Interactions of insulin, glucagon, epinephrine and cyclic AMP in the perfused rat liver. Fed. Proc. 30:1205. 15 Martin, R. J., L. L. Wilson, R. L. Cowan, a n d J . D. Sink. 1973. Effects of fasting and diet on enzyme profiles in ovine liver and adipose tissue. J. Anita. Sci. 36:101. 16 Mayes, P. A. 1969. The role of the liver in fatty acid transport. Proc. Biochem. Soc. 115:45p. 17 Meier, J. M., J. D. McGarry, G. R. Faloona, R. H. Unger, and D. W. Foster. 1972. Studies of the development of diabetic ketosis in the rat. J. Lipid Res. 13:228. 18 Menahan, L. A., L. H. Schultz, and W. G. Hoekstra. 1966. Relationship of ketone body metabolism and carbohydrate utilization to fat mobilization in the ruminant. J. Dairy Sci. 49:957. 19 Nikkila, E. A., and M. Kekki. 1970. Turnover rate of serum triglycerides in diabetes. Diabetologia 6:658. 20 Palmquist, D. L. 1972. Palmitic acid as a source of endogenous acetate and j3-hydroxybutyrate in fed and fasted ruminants. J. Nutr. 102:1401. 21 Radloff, H. D., and L. H. Schultz. 1967. Blood and rumen changes in cows in early stages o f ketosis. J. Dairy Sci. 50:68. 22 Reid, R. L. 1968. The physiolopathology of undernourishment in pregnant sheep, with particular reference to pregnancy toxemia. Adv. Vet. Sci. Comp. Med. 12:164. 23 Saarinen, P., and J. C. Shaw. 1950. Studies on ketosis in dairy cattle. XIII. Lipids and ascorbic acid in the liver and adrenals of cows with spontaneous and fasting ketosis. J. Dairy Sci. 33:515. 24 Schwalm, J. W., R. Waterman, G. E. Shook, and L. H. Schultz. 1972. Blood metabolite interrelationships and changes in mammary gland metabolism during subclinical ketosis. J. Dairy Sci. 55:58. 25 Schwalm, J. W., and L. H. Schultz. 1973. Relationship of insulin concentrations to various blood metabolites in the dairy cow. J. Dairy Sci. 56:672. (Abstr.) 26 Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195:19. 27 Steel, R. G. D., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-HUl Book Co., Inc., New York. 28 Stern, J. S., C. A. Baile, and J. Mayer. 1971. Growth hormone, insulin, and glucose in suckling, weanling, and mature ruminants. J. Dairy Sci. 54:1052. 29 Sutherland, E. W., and G. A. Robison. 1969. The role of cyclic AMP in the control of carbohydrate metabolism. Diebetes 18: 797. 30 Thornton, J. H., and L. H. Schultz. 1975. Unpublished results. 31 Trenlde, A. 1972. Radioimmunoassay of plasma hormones: Review o f plasma insulin in ruminants. J. Dairy Sci. 55:1200. 32 Van Handel, E. 1961. Suggested modifications o f the microdetermination of triglycerides. Clin.
METABOLITES IN DIABETIC GOATS Chem. J. 7:249. 33 Varman, P. N., and L. H. Schultz. 1968. Blood lipids of cows at different stages of lactation. J. Dairy Sci. 51:1971. 34 Weinstein, I., H. A. Klausner, and M. Heimberg. 1973. The effect of concentration of glucagon on output of triglyceride, ketone bodies, glucose, and
269
urea by the liver. Biochim. Biophys. Acta 296:300. 35 Woodside, W. F., and M. Heimberg. 1972. Hepatic metabolism of free fatty acids in experimental diabetes. Isr. J. Med. Sci. 8:309. 36 Yamdagni, S., and L. H. Schultz. 1969. Metabolism of 1-14 C palmitic acid in goats in various metabolic states. J. Dairy Sci. 52:1278.
Journal of Dairy Science Vol. 59, No. 2