- Email: [email protected]
Autoregulation of Alimentary and Hepatic Ketogenesis in Sheep
Autoregulation of A l i m e n t a r y and Hepatic Ketogenesis in Sheep R. N. HEITMANN and J. M. FERNANDEZ 1 Department of Animal Science University of Tennessee Knoxville 37901
ABSTRACT
INTRODUCTION
To determine the mechanism of autoregulation of ketogenesis, ~3-hydroxybutyrate was infused into 5 normal; 3 diabetic, insulin-treated; and 3 diabetic, untreated anesthetized sheep. Net flux of fatty acids, acetoacetate, ~-hydroxybutyrate, and insulin were measured across splanchnic tissues by multiplying venoarterial differences by blood flow. ~3-hydroxybutyrate depressed fatty acid concentrations and hepatic uptake. This decrease in hepatic uptake was not due solely to decreased concentrations, because hepatic extraction decreased 40% in normal and insulin-treated sheep. Portaldrained visceral release of acetoacetate was increased by 13-hydroxybutyrate infusion in normal and insulin-treated sheep, but this was associated with even larger increases in hepatic uptake, resulting in decreased total splanchnic release. Portal-drained viscera switched from release to uptake of fl-hydroxybutyrate in both normal and insulin-treated animals, but hepatic release increased slightly in normal sheep, fi-hydroxybutyrate increased insulin concentration, pancreatic production, and hepatic uptake. Because effects of ketone infusion on net fluxes of fatty acids, acetoacetate, and ~3-hydroxybutyrate were similar in normal and diabetic, insulin-treated sheep but were diminished or totally absent in diabetic, untreated animals, the mechanism of autoregulation of ketogenesis may be mediated at the insulin receptor or at the site of hepatic fatty acid uptake.
Rumen epithelium has a ketogenic pathway similar to the hepatocyte (18). Substrates for alimentary ketogenesis are acetate and butyrate from rumen fermentation, whereas liver relies mainly on endogenously produced circulating free fatty acids (FFA) from adipose lipolysis. Due to rumen volume and rate of fermentative volatile fatty acid production, alimentary ketogenesis in the fed ruminant is at least as quantitatively important as hepatic ketogenesis (2, 15). In conditions of insufficient energy intake or accelerated lipolysis during late pregnancy or early lactation, the liver becomes the major ketogenic organ (15). However, in many cases of pregnancy, lactation, and even diabetes, clinical ketoacidosis does not develop. Earlier work indicated that some autoregulation of ketogenesis occurred in the dog (20) by a direct stimulation of 13-hydroxybutyrate on pancreatic insulin production, thereby limiting lipolysis and in the rat (5) by preventing the activation of hormone-sensitive lipase in the adipocyte. No direct evidence has been presented in intact animals demonstrating the ability of ketone bodies to slow their own rate of alimentary and hepatic production or the mechanisms involved. Such studies would be beneficial in explaining the etiology of pregnancy acetonemia and lactation ketosis and may possibly have implications for the development of diabetic ketoacidosis. Therefore, in this study, /3-hydroxybutyrate was infused into normal, 1-d fasted sheep at rates approximating maximum utilization (4). Changes in concentrations and net fluxes of FFA, acetoacetate, /3-hydroxybutyrate, and insulin were measured across the portal-drained viscera and liver. In addition, a series of infusion experiments were conducted on diabetic, insulin-treated sheep to determine if an increase in pancreatic production of insulin was required for autoregulation of ketogenesis. A final set of experiments were conducted on diabetic, untreated sheep to
Received July 12, 1985. 1Department of Animal Science, North Carolina State University, Raleigh 27650. 1986 J Dairy Sci 69:1270-1281
1270
AUTOREGULATION OF KETOGENESIS determine if autoregulation of ketogenesis could occur in the total absence of insulin. MATERIALS AND METHODS Animals and Procedures
Nonpregnant, nonlactating, sheared Suffolk crossbred ewes weighing 55 to 65 kg were housed in individual indoor 1.8 × 3.0-m pens at 19 to 25°C and natural lighting. The ewes were fed 800 g of commercially prepared alfalfa pellets per day in two 400-g aliquots at 0800 and 1700 h. Water and trace mineralized salt were available ad libitum. All ewes were accustomed to laboratory personnel and never had to be restrained. Five normal; 3 diabetic, insulin-treated; and 3 diabetic, untreated ewes were used. Diabetes was induced pharmacologically by a single 50 mg/kg intravenous dose of alloxan. Treated ewes received sufficient subcutaneous doses (270 U) of insulin (Iletin-40) daily to maintain their blood glucose within normal physiological range (50 to 60 mg/dl). Untreated ewes were withdrawn from insulin for 3 d prior to the experimental period. On experimental days, catheters were implanted into the portal, hepatic, mesenteric veins, caudal vena cava, and caudal aorta. Experiments were initiated immediately following surgery and while the ewe was still under sodium pentobarbital-induced anesthesia, pAminohippuric acid (PAH) was infused (1.5% at .764 ml/min) into a mesenteric vein for a 1-h equilibration period. Three 23-ml blood samples were withdrawn simultaneously from the caudal aorta and hepatic and portal veins at 15-min intervals. Following this c o n t r o l period, 23 ml of 17 to 20%/3-hydroxybutyrate were pulse dosed via the caudal vena cava and then infused at .4 g/h/kg. 7s. This infusion rate approximated the maximum rate of /3-hydroxybutyrate utilization for sheep reported by Bergman and Kon (4). Four sets of infusion period blood samples (23 ml) were withdrawn simultaneously from the caudal aorta and hepatic and portal veins at 30-min intervals beginning 30-min following initiation of /~-hydroxybutyrate infusion. Catheters were maintained with a 6% Na2 ethylenediaminetetraacetate (EDTA) solution between samples. All samples were immersed immediately into an ice bath and treated in accordance with assay
1271
procedures described subsequently. Anesthesia was withdrawn and the ewe was allowed to recover. All ewes recovered successfully and without complications. Chemical Methods
Blood packed cell volume; concentrations of whole blood /3-hydroxybutyrate, acetoacetate, and PAH; and plasma F F A and insulin were determined. p-Aminohippuric acid was determined as described by Kaufman and Bergman (16). Four milliliters of freshly sampled blood were added to an equal volume of cold 1 M HC104 in preparation for acetoacetate and /3-hydroxybutyrate analysis. The tubes were vortexed, centrifuged at 4°C and 1500 × g for 25 min, and buffered to a final pH of 6.4 to 7.6 with cold 1 M KOH. Samples were analyzed immediately using the enzymatic assay of Williamson and Mellanby (28). Insulin was determined by using a standard double antibody radioimmunoassay technique (29) with giunea pig antibovine insulin as the first antibody and goat antiguinea pig immunoglobulin G as the second antibody. Intraassay errors of greater than 5% were rejected and, in the few cases where this occurred, the samples were reanalyzed. Interassay errors, as measured by a pooled ewe sample, were always less than 10% and usually less than 7%. Plasma F F A were analyzed by a modification of the microtitiration technique of Trout et al. (25). Briefly, 2 ml of plasma were added to 50-ml separatory funnels containing 10 ml Of extraction mixture (2-propanol/heptane/1 × H2 SO4, 40:10:1, vol/vol/vol, shaken vigorously for 30 s and vented. Four milliliters of distilled water and 6 ml of heptane were added and the flasks shaken for an additional 2 min. The precipitate was removed and 1 ml of .05% H2SO4 was added to the remaining organic solvent layer and the flasks were shaken for a final 2 min. The acid layer was removed and 5 ml of the organic solvent laver were added to 1 ml of .01% t h y m o l blue in 90% ethanol indicator mixture and titrated with .0018N NaOH under CO2. Calculations
Whole blood flow rates through the portaldrained viscera and liver were calculated by the Journal of Dairy Science Vol. 69, No. 5, 1986
1272
HEITMANN AND FERNANDEZ
indicator-dilution method using the equation: IPAH F = c~AH _ C PAH where F equals whole blood flow through the tissue in liters per minute, IPAH is the infusion rate of PAH in optical density (OD) units per minute, and C PAH and CPAH are the PAH OD units per liter in the arterial input and venous drain. Portal and hepatic vein flows were measured directly and hepatic arterial flow was calculated from the difference between the two. Plasma flow were obtained by subtracting that portion of the flow represented by the packed cell volume. Net fluxes of acetoacetate, fl-hydroxybutyrate, FFA, and insulin across the portaldrained viscera, liver, and total spanchnic bed were calculated using the following equations: Portal-drained viscera flux = PF × (Cp -- Ca) Hepatic flux = PF × (Ch -- Cp) + AF × (Ch -- Ca) Total splanchnic flux = HF × (Ch - Ca) where PF, AF, and HF are the whole blood or plasma flow rates (L/rain) in the portal vein, hepatic artery, and hepatic vein; and Cp, Ca, and Ch are the concentrations of acetoacetate (/aM) or /3-hydroxybutyrate (/z44) in whole blood or FFA (/aM) or insulin (mU/L) in the plasma. Positive flux indicated net release and negative flux indicated net uptake of the metabolite by that specific tissue. Extraction ratios for each metabolite and each tissue were calculated. Extraction ratios are that fraction of the metabolite presented to the tissue (tissue blood flow multiplied by the concentration in the arterial input) that is taken up by the tissue (net flux). Significant changes in extraction ratios between treatments are indicative that changes in net flux with treatment are at least in part due to some physiological mechanism and not due solely to a change in metabolite concentration. Statistical Analysis
All data were analyzed by a two-way analysis of variance to detect differences due to ~-hydroxybutyrate infusion. In addition, a Duncan's new multiple range test was used to determine differences among normal; diabetic, Journal of Dairy Science Vol. 69, No. 5, 1986
insulin-treated; and diabetic, untreated groups. Because no changes in blood flow rates occurred during i n f u s i o n of ~-hydroxybutyrate, an average blood flow for the entire experimental period was used to test for treatment differences. Concentrations and flux rates for all metabolites reached new near steady-state during infusion (i.e., slope of concetnration or rate of flux over time during the control or infusion periods was not different from zero. Consequently, averages of the control and infusion periods were used to test for significant differences. R ESU LTS Blood Flow Rates
Whole blood flow rates for the portal and hepatic veins and portal to hepatic ratios are in Table 1. In normal, 1-d fasted sheep, portal flow was 1.7 L/min and approximately 70% that of hepatic flow. This is in close agreement with previously reported data on fed and 3-d fasted sheep (12). However, there was a 55% decrease (P<.05) in portal flow and 37% decrease (P<.01) in portal to hepatic ratio in diabetic, insulin-treated sheep. This is contrary to previous work that reported no change in blood flows between normal and insulin-treated, alloxanized sheep (8). No satisfactory physiological explanation has been found to explain this occurrence. However, diabetic untreated sheep had flow rates simialr to normal animals. Free Fatty Acid Metabolism
Arterial plasma FFA concentrations and venoarterial differences are in Table 2. Infusion of ~-hydroxybutyrate lowered F F A concentrations by 39, 42, and 11% in normal; diabetic, insulin-treated; and diabetic, untreated sheep (P<.05). The 36% increase in preinfusion F F A concentrations between normal and diabetic, insulin-treated sheep suggests that, although these sheep were euglycemic, some lipolysis was occurring. Increase in FFA in untreated ewes was more than twice that in normal ewes during preinfusion and is demonstrative of the high rate of lipolysis characteristic of uncontrolled diabetes. Most importantly, the venoarterial differences were, for the most part, different from zero (P<.01), indicating significant tissue uptake or release.
AUTOREGULATION OF KETOGENESIS
1273
TABLE 1. Whole blood flow rates (L/min) in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, untreated (DUT) 1-d fasted sheep.
Sheep
No. experiments
Portal
NOR DIT DUT
5 3 3
1.66 .76 1.35
Portal I hepatic
Hepatic SE
X
SE
X
SE
.30 .31" .26
2.38 1.62 2.02
.38 .50 .15
.70 .44 .69
.02 .05** .18
*Significantly different from NOR (P<.05). **Significantly different from NOR (P<.01).
Plasma F F A n e t fluxes a n d h e p a t i c e x t r a c t i o n ratios are in T a b l e 3. T h e r e were n o significant effects of t 3 - h y d r o x y b u t y r a t e infusion o n p o r t a l - d r a i n e d visceral fluxes. However, infusion of 13-hydroxybutyrate decreased ( P < . 0 5 ) h e p a t i c u p t a k e o f F F A in all t h r e e t r e a t m e n t s . A l t h o u g h this decrease was q u a n t i t a t i v e l y similar for all t r e a t m e n t s (176, 186, a n d 166 / 2 m o l / m i n in n o r m a l , t r e a t e d a n d u n t r e a t e d ewes), t h e r e was a m u c h g r e a t e r q u a l i t a t i v e decrease in n o r m a l a n d t r e a t e d (62 a n d 66%) t h a n in u n t r e a t e d s h e e p (34%).
Significantly, h e p a t i c F F A e x t r a c t i o n ratios also were d e c r e a s e d ( P < ° 0 1 ) w i t h /3-hydroxyb u t y r a t e i n f u s i o n in n o r m a l a n d t r e a t e d b u t n o t in u n t r e a t e d ewes. H e p a t i c u p t a k e o f F F A increased ( P < . 0 5 ) in u n t r e a t e d d i a b e t e s ; h o w ever, this was solely d u e t o increased c o n c e n t r a t i o n s because e x t r a c t i o n ratios a m o n g g r o u p s were similar. As a result o f t h e s e changes in liver m e t a b o l i s m , t o t a l s p l a n c h n i c u p t a k e of F F A d e c r e a s e d ( P < . 0 5 ) in n o r m a l a n d t r e a t e d b u t n o t u n t r e a t e d ewes w i t h i n f u s i o n of t3-hyd r o x y b u t y r a t e ; h o w e v e r , t o t a l s p l a n c h n i c up-
TABLE 2. Effects of /3-hydroxybutyrate (BOHB) on free fatty acid (FFA) concentrations and venoarterial differences in normal (NOR), diabetic, insulin-treated (DIT); and diabetic, untreated (DUT), 1-d fasted sheep.
Sheep 1
Hepaticartery
Artery
Hepaticportal
Portalartery
(Izmol FFA/L) SE
X
SE
X
SE
X
SE
NOR (5) Control BOHB infusion
872 528
83 55*
-132 --31
32** 18
-203 --93
19"* 13"*
71 62
18"* 12"*
DIT (3) Control BOHB infusion
t186 684
1054~ 33*
-258 54
41"* 14"*
-270 --123
33** 18"*
12 69
22
DUT (3) Control BOHB infusion
1933 1719
-285 --217
49** 22**
-333 --234
53** 55**
49 17
24 45
92 ~ 85 *'*
14"*
1 Number of experiments are in parentheses. * Significantly different from control (P<.05). ** Significantly different from 0 (P<.01). Slgmficantly different from corresponding NOR (P<.O1). Journal of Dairy Science Vol. 69, No. 5, 1986
1274
HEITMANN AND FERNANDEZ
TABLE 3. Effects of /3-hydroxybutyrate (BOHB) on portal-drained visceral, hepatic and total splanchnic net fluxes, and hepatic extraction ratios of free fatty acids (FFA) in normal (NOR); diabetic; insulin-treated (DIT); and diabetic untreated (DUT), 1-d fasted sheep.
Sheep 1
Portaldrained viscera
Hepatic extraction ratio
Liver
Total splanchnic
(#mol FFA/min) SE
.~
SE
X
SE
X
SE
97 76
33 19
-284 --108
31 19"
.18 .11
.02 .02**
-187 --33
50 31"
DIT (3) Control BOHB infusion
3 32
9* 10
-280 --94
49 22*
.21 .12
.03 .03"*
-276 --62
50 16"
DUT (3) Control BOHB infusion
43 --10
27 43*
-485 --319
82* 66"*
.16 .12
.03 .02
-442 --329
81 + 38 +
NOR (5) Control BOHB infusion
1Number of experiments are in parentheses. *Significantly different from control (P<.05). **Significantly different from control (P<.01 ). *Significantly different from corresponding NOR (P<.05),
take of F F A increased in untreated diabetes relative to the normal and insulin-treated sheep.
Ketone Body Metabolism
Whole blood acetoacetate and l~-hydroxybutyrate concentrations and venoarterial differences are reported in Table 4. Untreated diabetes increased acetoacetate and/3-hydroxybutyrate concentration 11- and 24-fold. As with FFA, /3-hydroxybutyrate but not acetoacetate concentrations were elevated in treated animals compared with normal animals during preinfusion (P<.01), although both groups exhibited euglycemia. Venoarterial differences for both acetoacetate and /3-hydroxybutyrate, for the most part, were different from zero (P<.05) in normal and diabetic, insulin-treated sheep. This effect was lost in untreated animals due to the high acetoacetate and fl-hydroxybutyrate and a subsequent loss in assay sensitivity at these elevated concentrations. Net fluxes for acetoacetate and fl-hydroxybutyrate are in Table 5. /3-Hydroxybutyrate infusion increased portal-drained visceral release of acetoacetate by 43 and 59 #mol/min but switched from a condition of release to one of Journal of Dairy Science Vol. 69, No. 5, 1986
uptake of ~-hydroxybutyrate (137 vs. - 3 9 4 and 9 vs. - 2 7 8 /amol/min) in normal and diabetic, insulin-treated sheep (P<.05). These effects were not significant in untreated animals, although trends were similar. Hepatic uptake of acetoacetate increased by 76 and 190/~mol/min (P<.05) with fl-hydroxybutyrate infusion in normal and treated sheep. However, hepatic release of/3-hydroxybutyrate increased (P<.05) in normal animals. As with portal-drained visceral metabolism, these effects were not significant in untreated animals although similar trends were noted. Untreated diabetes decreased portal-drained visceral but increased hepatic release of acetoacetate. Conversely, diabetes tended to increase ketogenesis of /3-hydroxybutyrate in both these splanchnic tissues. Total splanchnic metabolism, as reflected by a combination of portal-drained visceral and hepatic rates, demonstrated decreased overall ketogenesis of acetoacetate (P<.05) in normal and treated sheep with ~-hydroxybutyrate infusion and showed a similar trend ( P < . I ) for fl-hydroxybutyrate in normal animals with the ketone infusion. Untreated diabetes increased total splanchnic ketogenesis of both ketones (P<.05).
412 419
DUT (3) Control BOHBinfusion
53* 44*
9 15"*
13,771 19,537
1023 4321
565 4105
0o Ox
t/i
O
Z
O~
O
<
Slgmficantly different from corresponding NOR (P<.01).
* Significantly different from 0 (P<.05).
* * Significantly different from control (P<.O 1).
2033 ~ 1701 **,~
81 ~ 249 * ~ t
46 352**
W
1 Number of experiments are in parentheses.
46 213
DIT (3) Control BOHB infusion
5 14"*
G~
e~
36 196
SE
X
SE
.X
NOR (5) Control BOHB infusion
Sheep ~
3-hydroxybutyrate
Acetoacetate
Artery
9 8*
5 8*
SE
29 17 11 4*
16 -67
-2 -25
X
Acetoacetate
754 822
481 353
244 105
X
400 316"
46* 77*
41" 111
SE
X
46 2
6 -143 32 4
11 15"
5* 6*
SE
422 615
426 850
155 370
X
381 291"
45* 149"
25* 57*
SE
3-hydroxybutyrate
Hepatic-portal Acetoacetate
-15 63
(#mol/L)
3-hydroxybutyrate
Hepatic-artery
4* 5*
SE
- 1 7 15 10 4*
10 5 77 10"
13 39
X
Acetoacetate
331 207
55 -497
89 --264
X
311 309
38 204*
22* 71"
SE
3-hydroxybutyrate
Portal-artery
TABLE 4. Effects of 3-hydroxybutyrate (BOHB) on acetoacetate and 3-hydroxybutyrate concentrations and venoarterial differences in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, untreated (DUT), 1-d fasted sheep.
bO ~q
~q O C3 t~ Z
Z O
5
.q
70
o
O
00
t.n
Z
G~ xo
O
<
~7 ,<
4 63
9 12
DIT (3) Control BOHB infusion
DUT (3) Control BOHB infusion
14' 6*
6 17"
9 10"
SE
:L
.
.
450 227
9 --278 471 435*
26* 102"
28 94*
SE
Significantly different from corresponding NOR(P<.05).
.
X
t3-hydroxybutyrate
137 --394
* Significantly different from control (P<.05).
1 Number of experiments are in parentheses.
25 68
'X
NOR (5) Control BOHB infusion
Sheep 1
Acetoacetate
Portal-drained viscera
72 13
13 -177
-36 -112
X
Acetoacetate
50* 8*
13 38*
14 13"
SE
1138 1445
749 881
395 679
R
788* 563*
126' 100
51 127"
SE
/3-hydroxybutyrate O~mol/min)
Liver
62 25
17 -114
-11 44
.X
Acetoacetate
36* 8*
11 23*,*
12 16"
SE
1588 1672
758 602
532 285
859* 681'
113 140
65 191
SE
13-hydroxybutyrate
Total splanchnic
TABLE 5. Effects of ~3-hydroxybutyrate (BOHB) on portal-drained visceral, hepatic, and total splanchnic net fluxes of acetoacetate and in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, u n t r e a t e d (DUT), 1-d fasted sheep.
N
> Z
Z
Z
.q Gx
AUTOREGULATION OF KETOGENESIS Insulin Metabolism
Insulin concentration and venoarterial differences are in Table 6. Concentrations were lower (P<.01) in diabetic than in normal animals. Although insulin concentrations in diabetic ewes were very low, they were different from zero (P<.05), suggesting some viable beta cells may remain or perhaps some crossreactivity in the assay. Only portal-arterial and hepatic-portal venoarterial differences in normal sheep were different from zero (P<.01). Importantly, the portal-arterial differences in both groups of diabetic animals were not different from zero, indicating that pancreatic beta cell function was indeed inoperative. Infusion of ~-hydroxybutyrate increased arterial insulin slightly (P<.I) in normal ewes but had no effect on diabetic ewes. Net flux of insulin are in Table 7. Infusion of J3-hydroxybutyrate in normal animals increased pancreatic insulin production (P<.05), as represented by portal-drained visceral release, by 11 mU/min. However, there was a concomitant increase of hepatic insulin extraction of 10 mU/min, resulting in no change in total splanchnic release. This is of great importance when considering site of sampling to measure blood insulin profiles. Blood withdrawn peri-
1277
pherally, i.e., jugular or cephalic, would not detect these marked changes occurring in pancreatic production and hepatic uptake. The liver of normal animals was extracting 67 to 75% of the portal production and 13 to 17% of the total circulating insulin. These values are slightly higher than those of 50 and 8% reported by Brockman and Bergman (9) in normal sheep.
DISCUSSION
Net fluxes of ketone bodies, FFA, and insulin across the major splanchnic beds in ruminants have been studied previously under various conditions mainly in the laboratories of Bergman for the ovine [reviewed in (3)1 and Baird for the bovine [reviewed in (1)]. In addition, some preliminary work demonstrating the capacity of ~-hydroxybutyrate to lower circulating F F A concentrations has been done in the dog (20) and rat (5). However, the present work is an initial effort to obtain direct evidence that elevated ketone bodies can inhibit ketogenesis in intact sheep as well as depress plasma FFA concentrations and to determine the mechanism of action involved for this proposed negative feedback. As such, this work
TABLE 6. Effects of/~-hydroxybutyrate (BOHB) on insulin concentrations and venoarterial differences in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, untreated (DUT), 1-d fasted sheep.
Sheep 1
Hepaticartery
Artery
Hepaticportal
Portalartery
X
(#U/ml) NOR (5) Control BOHB infusion DIT (3) Control BOHB infusion DUT (3) Control BOHB infusion
SE
X
SE
X
SE
SE
38
5
4
2
-16
4**
20
4**
49
45"
4
2
-27
4**
31
4**
22 22
2+
--3
1"*
--1
I
2~
-3
1"*
--2
1
--i
1
3 3
0* 1#
-1 --0
0 1
-1 -0
1 1
1 -0
1 1
2
1
1Number of experiments are in parentheses. * * Significantly different
from 0 (P<.o1). Sigmflcantly different from control (P<.I). *Significantly different from corresponding NOR (P<.01). t
•
.
Journal of Dairy Science Vol. 69, No. 5, 1986
1278
HEITMANN AND FERNANDEZ
TABLE 7. Effects of ~-hydroxybutyrate (BOHB) on portal-drained visceral, hepatic, and total splanchnic net fluxes of insulin in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, untreated (DUT), 1-d fasted sheep. 1 Portal
Sheep
drained viscera
Total splanchnic
Liver (mU/min)
NOR (5) Control BOHB infusion DIT (3) Control BOHB infusion DUT (3) Control BOHB infusion
SE
X
SE
X
SE
25 36
5 5*
-17 --27
5 4*
8 9
4 4
-1 -1
0 ~: O#
-2 -3
1~: 1'
-3 3
1* 1#
1* 1*
-2 -O
1* 1#
-1 -0
1t 1#
1 O
I Number of experiments are in parentheses. *Significantly different from control (P<.05). *Significantly different from corresponding NOR (P<.05).
may explain the etiology of pregnancy acetonemia and lactation ketosis and may have possible implications in the role of developing diabetic ketoacidosis. There are two current hypotheses. First, autoregulation of ketogenesis may be mediated at the pancreatic level by increased insulin production in response to elevated ketones (20), thereby increasing adipocyte lipogenesis and inhibiting lipolysis, which in turn decreases hepatic ketogenic precursor supply. Second, the ketone may act directly at the adipocyte by decreasing the activity of hormone-sensitive lipase (5). These studies were conducted on sodium pentobarbital-anesthetized sheep and immediately following abdominal surgery. Blood flow rates through the major splanchnic tissues were in very close agreement with measurements on unanesthetized sheep (7, 8, 9, 12, 15). Concentrations of acetoacetate (36 /~/), /3-hydroxybutyrate (565 gA//), and insulin (38 /~U/ml) in normal, preinfused animals are well within the physiological ranges for sheep reported by numerous researchers as summarized by Lindsay and Lea, (19). Plasma FFA (872 MM) were slightly elevated relative to fed ruminants (1, 15, 16, 19) but were in close Journal of Dairy Science Vol. 69, No. 5, 1986
agreement with concentrations reported in 1-d fasted sheep (13). In addition, net fluxes of normal, preinfused animals in the portaldrained viscera and liver for acetoacetate (25 and - 3 6 ~tmol/min), /3-hydroxybutyrate (137 and 395 ~mol/min), FFA (97 a n d - 2 8 4 / . t m o l / min), and insulin (25 and - 1 7 /lmol/min) are similar to previously reported values on conscious sheep (7, 9, 15). Finally, no differences were noted in portal and hepatic metabolism of insulin in dogs awake and dogs anesthetized with sodium-pentobarbitol (14). Portal-drained visceral FFA fluxes were small but generally exhibited net release. Because FFA absorption across the gut is mostly lymphatic, this was to be expected, and the significant net release may have represented some omental lipolysis. Conversely, there were high rates of F F A uptake in liver in the preinfusion periods of all three groups of animals. Untreated diabetes increased hepatic uptake by 70%, however, this was due solely to increased F F A concentrations, because the hepatic extraction ratios did not change and were between 16 to 21%. Rates of F F A metabolism in the portal-drained viscera and liver and the hepatic extraction ratios in normal, preinfused sheep were similar to those reported for non-
AUTOREGULATION OF KETOGENESIS pregnant, fed sheep (15). Infusion of /3-hyd r o x y b u t y r a t e at the reported rate of maximum utilization in sheep (4) had no effects on portal-drained visceral F F A metabolism. Hepatic F F A uptake was reduced by 62 and 66% in normal and diabetic, insulin-treated ewes but only 34% in diabetic, untreated ewes. Most important, infusion of /3-hydroxybutyrate decreased the hepatic extraction ratios of F F A in normal and diabetic, insulin-treated animals but not in untreated animals, implying the involvement of some physiological mechanism at the hepatocyte. This was surprising, because it has been reported that hepatic extraction (26) and oxidation (6) of F F A is concentration dependent. Because hepatic extraction ratios decreased in normal and diabetic insulin-treated ewes but not diabetic, untreated ewes, it may be concluded that insulin may be involved in this altered hepatic F F A metabolism. Sheep hepatocytes have insulin receptors (11), and these increased in number but showed no change in binding kinetics with progressive lactation. As a result, hepatic insulin binding increased and circulating concentrations decreased. Similar results have been reported for the lactating goat hepatocyte (10). According to McGarry and Foster's bihormonal theory (22), insulininduced malonyl-coenzyme A (CoA) concentrations could inhibit fatty acid oxidation by inhibiting mitochondrial translocation of longchain acyl-CoA via carnitine acyltransferase I. However, Prior and Smith (24) have recently demonstrated that acetyl-CoA carboxylase in the alloxan-diabetic steer adipocyte does not require insulin for activation. Infusion of /3-hydroxybutyrate decreased F F A in plasma in all three groups of sheep. However, the effect of/3-hydroxybutyrate was much more pronounced in normal and diabetic insulin-treated sheep (40% depression) relative to diabetic untreated animals (10% depression) even though/3-hydroxybutyrate has been shown to be more efficacious in lowering elevated concentrations (13). Because /3-hydroxybutyrate both lowered circulating plasma F F A and decreased hepatic uptake, there must have been a concomitant decrease in lipolysis at the adipocyte. Both acetoacetate and /3-hydroxybutyrate are released by portal-drained viscera, but acetoacetate is removed and/3-hydroxybutyrate released by the liver of normal, preinfused
1279
animals. This is typical of data reported for both ovine (15) and bovine (2) animals. Hepatic uptake of acetoacetate may seem paradoxical since the rate-limiting enzyme of ketone body utilization, 3-oxoacid-CoA transferase, is not present in significant quantity in the liver. However, ovine liver does contain a/3-hydroxybutyrate dehydrogenase of sufficient activity, .6 to 1.0 /~mol/min/g (17, 27), to account for all the acetoacetate uptake observed in the current study to be metabolized to /~-hydroxybutyrate. Untreated diabetes tended to increase portal-drained visceral total ketone release threefold. In addition, infusion of fl-hydroxybutyrate caused the portal-drained viscera to switch from release to uptake of /3-hydroxybutyrate but to increase acetoacetate release slightly. The net effect of /3-hydroxybutyrate infusion was to completely inhibit net alimentary ketogenesis. Although the same trends were in diabetic, untreated sheep, these effects were not significant. The portal-drained viscera always had small net rates of release of F F A ; however, there may have been substantial unidirectional rates of uptake occurring that were not measureable by this model. Therefore, the loss of effects of /3-hydroxybutyrate on alimentary ketone flux with the absence of insulin may have been due to an altered F F A metabolism. Because hepatic F F A uptake is depressed markedly in normal and diabetic, insulintreated sheep following infusion of fl-hydroxybutyrate, it would be expected that hepatic ketogenesis was decreased in these animals as well. Indeed, acetoacetate uptake increased markedly, but, paradoxically, release of 13-hyd r o x y b u t y r a t e increased. Once again, these effects were totally absent in diabetic, untreated sheep. The net effect of /3-hydroxybutyrate infusion on the total splanchnic tissue was to increase acetoacetate uptake in normal and diabetic, insulin-treated sheep and to tend ( P < . I ) to decrease/3-hydroxybutyrate release in normal animals. Total ketone release was depressed in both normal and treated but not untreated sheep. The effects of/3-hydroxybutyrate infusion at rates simulating maximum ketone utilization (4) on the net portal-drained visceral and hepatic fluxes of F F A , acetoacetate, and l~-hydroxybutyrate were similar in normal and diabetic, insulin-treated sheep but were diminJournal of Dairy Science Vol. 69, No. 5, 1986
1280
HEITMANN AND FERNANDEZ
ished f o r F F A a n d t o t a l l y a b s e n t f o r acetoacetate and / 3 - h y d r o x y b u t y r a t e in diabetic, u n t r e a t e d animals. T h e r e f o r e , it is suggested t h a t t h e m e c h a n i s m o f a u t o r e g u l a t i o n of k e t o g e n e s i s m a y n o t be t o t a l l y d u e to i n c r e a s e d p a n c r e a t i c insulin p r o d u c t i o n b u t m a y b e m e d i a t e d at t h e h e p a t o c y t e insulin r e c e p t o r b y a f f e c t i n g e i t h e r r e c e p t o r n u m b e r s of affinity. Whereas t h e r e is a decreased a d i p o c y t e insulin r e c e p t o r a f f i n i t y in d i a b e t i c k e t o a c i d o s i s , this a p p e a r s to b e d u e to t h e acidosis itself, pred o m i n a n t l y an increased r a t e o f insulin diss o c i a t i o n (21). F u r t h e r m o r e , it has b e e n dem o n s t r a t e d t h a t t 3 - h y d r o x y b u t y r a t e , a d d e d in c o n c e n t r a t i o n s similar t o t h o s e in d i a b e t i c ketoacidosis, can r e s t o r e normal binding k i n e t i c s t o insulin r e c e p t o r s o f h u m a n l y m p h o cytes (23). It is still possible t h a t k e t o n e s m a y act d i r e c t l y at t h e a d i p o c y t e b y i n h i b i t i n g h o r m o n e - s e n s i t i v e lipase a n d decreasing lipolysis or b y s t i m u l a t i n g l i p o p r o t e i n lipase a n d increasing lipid u p t a k e a n d r e e s t e r i f i c a t i o n , t h e r e b y l o w e r i n g circulating F F A c o n c e n t r a tions. It m u s t be e m p h a s i z e d t h a t t h e s e a n i m a l s were o n l y f a s t e d 1 d, w h i c h is n o t a p r o l o n g e d fast for a r u m i n a n t . I n d e e d rates of F F A a n d k e t o n e flux in t h e n o r m a l 1-d f a s t e d ewes were very similar t o t h o s e o f f e d r u m i n a n t s and, t h e r e f o r e , t h e ewes were n o t in a n a c c e l e r a t e d k e t o g e n i e m o d e . It w o u l d b e m o s t b e n e f i c i a l t o repeat these experiments on animals during energy d r a i n i n g physiological c o n d i t i o n s such as p r o l o n g e d fasting, p r e g n a n c y , or l a c t a t i o n . ACKNOWLEDGMENTS
T h e a u t h o r s w o u l d like to t h a n k J o a n H e m b r e e a n d A a r o n Cole for assistance d u r i n g t h e surgical p h a s e a n d Chris R e y n o l d s f o r assistance w i t h t h e insulin assay o f this study.
REFERENCES
1 Baird, G. D. 1977. Aspects of ruminant intermediary metabolism in relation to ketosis. Biochem. Rev. 5:819. 2 Baird, G. D., H. W. Symonds, and R. Ash. 1975. Some observations on metabolite production and utilization in vivo by the gut and liver of adult dairy cows. J. Agric. Sci., Camb. 85:281. 3 Bergman, E. N. 1971. Hyperketonemia ketogenesis and ketone body metabolism. J. Dairy Sci. 54:936. 4 Bergman, E. N., and K. Kon. 1964. Acetoacetate turnover and oxidation rates in ovine pregnancy ketosis. Am. J. Physiol. 206:449. Journal of Dairy Science Vol. 69, No. 5, 1986
5 Bjorntorp, P. 1966. Effect of ketone bodies on lipolysis in adipose tissue in vitro. J. Lipid Res. 7:621. 6 Boyd, R. O., R. A. Britton, H. Knoche, D. B. Moser, E. R. Pen, Jr., and R. K. Johnson. 1982. Oxidation rates of major fatty acids in fasting neonatal pigs. J. Anita. Sci. 55:95. 7 Brockman, R. P. 1976. Effect of glucagon and insulin on lipolysis and ketogenesis in sheep. Can. J. Comp. Med. 40:166. 8 Brockman, R. P., and E. N. Bergman. 1975. Effect of glucagon on plasma alanine and glutamine metabolism and hepatic gluconeogenesis in sheep. Am. J. Physiol. 228:I627. 9 Brockman, R. P., and E. N. Bergman. 1975. Quantitative aspects of insulin secretion and its hepatic and renal removal in sheep. Am. J. Physiol. 229:1338. 10 Gill, R. D., and I. C. Hart. 1979. The effect of dietary composition on the binding of insulin and glucagon to goat hepatocytes. Biochem. Soc. Trans. 7:910. 11 Gill, R. D., and I. C. Hart. 1980. Properties of insulin and glucagon receptors on sheep hepatocytes: a comparison of hormone binding and plasma hormones and metabolites in lactating and non-lactating ewes. J. Endocrinol. 84:234. 12 Heitmann, R. N., and E. N. Bergman. 1978. Glutamine metabolism, interorgan transport, and glucogenicity in the sheep. Am. J. Physiol. 234: E197. 13 Heitmann, R. N., and D. R. Metzler. 1983. Effects of fl-hydroxybutyrate on plasma free fatty acids in mature wethers. Fed. Proc. 45 : 532. 14 Ishida, T., R. M. Lewis, C. J. Hartley, M. L. Entman, and J. B. Field. 1983. Comparison of hepatic extraction of insulin and glucagon in conscious and anesthetized dogs. Endocrinology 112:1098. 15 Katz, M. L., and E. N. Bergman. 1969. Hepatic and portal metabolism of glucose, free fatty acids, and ketone bodies in the sheep. Am. J. Physiol, 216-953. 16 Kaufman, C. F., and E. N. Bergman. 1971. Renal glucose, free fatty acid, and ketone body metabolism in the unanesthetized sheep. Am. J. Physiol. 221:967. 17 Koundakjian, P. P., and A. M. Snoswell. 1970. Ketone body and fatty acid metabolism in sheep tissues. 3-Hydroxybutyrate dehydrogenase, acetoplasmic enzyme in sheep liver and kidney. Biochem. J. 119:49. 18 Leighton, B., A. R. Nicholas, and C. I. Pogson. 1983. The pathway of ketogenesis in rumen epithelium of sheep. Biochem. J. 216:769. 19 Lindsay, D. B., and W.M.F. Leat. 1975. Carbohydrate and lipid metabolism in the blood of sheep: Composition and function. Pages 4 5 - 6 2 in The blood of sheep. M. H. Blunt, ed. SpringerVerlag, New York, NY. 20 Madison, L. L., D. Mebane, R. H. Unger, and A. Lochner. 1964. The hypoglycemic action of ketones. II. Evidence for a stimulatory feedback of ketones on the pancreatic beta ceils. J. Clin. Invest. 43:408. 21 Marshall, S., D. A. Podlecki, and J. M. Olefski. 1983. Low pH accelerates dissociation of receptor
A U T O R E G U L A T I O N OF KETOGENESIS b o u n d insulin. Endocrinology. 113 : 37. 22 McGarry, J. D., G. P. Mannaerts, and D. W. Foster. 1977. A possible role for malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis. J. Clin. Invest. 60:265. 23 Misbin, R. I., A. J. Pulkkinen, S. A. Lofton, and T. J. Merimee. 1978. Ketoacids and the insulin receptor. Diabetes 27:539. 24 Prior, R. L., and S. P. Smith. 1982. Hormonal effects on partitioning of nutrients for tissue growth: role of insulin. Fed. Proc. 40:2545. 25 Trout, D. L., E. H. Estes, and S. J. Freidberg. 1960. Titration of free f a t t y acids of plasma: a s t u d y of current m e t h o d s and a new modification. J. Lipid Res. 1:199.
1281
26 Van Harken, D. R., C. W. Dixon, and M. Heimberg. 1969. Hepatic lipid metabolism in experimental diabetes. V. The effect of concentration of oleate on metabolism of tryglycerides and on ketogenesis. J. Biol. Chem. 244:2278. 27 Watson, H. R., and D. B. Lindsay. 1972. 3-Hyd r o y x b u t y r a t e dehydrogenase in tissues from normal and ketonaemic sheep. Biochem. J. 128:53. 28 Williamson, D. H., and J. Mellanby. 1965. Acetoacetate and D-(-)-fl-hydroxybutyrate. Pages 4 5 4 461 in Methods of enzymatic analysis. 2nd ed. H. V. Bergmeyer, ed. Academic Press, New York, NY. 29 Yalow, R. S. 1976. Methods in radioimmunoassay of peptide h o r m o n e s . North Holland Publishing Co., A m s t e r d a m .
Journal o f Dairy Science Vol. 69, No. 5, 1986
ABSTRACT
INTRODUCTION
To determine the mechanism of autoregulation of ketogenesis, ~3-hydroxybutyrate was infused into 5 normal; 3 diabetic, insulin-treated; and 3 diabetic, untreated anesthetized sheep. Net flux of fatty acids, acetoacetate, ~-hydroxybutyrate, and insulin were measured across splanchnic tissues by multiplying venoarterial differences by blood flow. ~3-hydroxybutyrate depressed fatty acid concentrations and hepatic uptake. This decrease in hepatic uptake was not due solely to decreased concentrations, because hepatic extraction decreased 40% in normal and insulin-treated sheep. Portaldrained visceral release of acetoacetate was increased by 13-hydroxybutyrate infusion in normal and insulin-treated sheep, but this was associated with even larger increases in hepatic uptake, resulting in decreased total splanchnic release. Portal-drained viscera switched from release to uptake of fl-hydroxybutyrate in both normal and insulin-treated animals, but hepatic release increased slightly in normal sheep, fi-hydroxybutyrate increased insulin concentration, pancreatic production, and hepatic uptake. Because effects of ketone infusion on net fluxes of fatty acids, acetoacetate, and ~3-hydroxybutyrate were similar in normal and diabetic, insulin-treated sheep but were diminished or totally absent in diabetic, untreated animals, the mechanism of autoregulation of ketogenesis may be mediated at the insulin receptor or at the site of hepatic fatty acid uptake.
Rumen epithelium has a ketogenic pathway similar to the hepatocyte (18). Substrates for alimentary ketogenesis are acetate and butyrate from rumen fermentation, whereas liver relies mainly on endogenously produced circulating free fatty acids (FFA) from adipose lipolysis. Due to rumen volume and rate of fermentative volatile fatty acid production, alimentary ketogenesis in the fed ruminant is at least as quantitatively important as hepatic ketogenesis (2, 15). In conditions of insufficient energy intake or accelerated lipolysis during late pregnancy or early lactation, the liver becomes the major ketogenic organ (15). However, in many cases of pregnancy, lactation, and even diabetes, clinical ketoacidosis does not develop. Earlier work indicated that some autoregulation of ketogenesis occurred in the dog (20) by a direct stimulation of 13-hydroxybutyrate on pancreatic insulin production, thereby limiting lipolysis and in the rat (5) by preventing the activation of hormone-sensitive lipase in the adipocyte. No direct evidence has been presented in intact animals demonstrating the ability of ketone bodies to slow their own rate of alimentary and hepatic production or the mechanisms involved. Such studies would be beneficial in explaining the etiology of pregnancy acetonemia and lactation ketosis and may possibly have implications for the development of diabetic ketoacidosis. Therefore, in this study, /3-hydroxybutyrate was infused into normal, 1-d fasted sheep at rates approximating maximum utilization (4). Changes in concentrations and net fluxes of FFA, acetoacetate, /3-hydroxybutyrate, and insulin were measured across the portal-drained viscera and liver. In addition, a series of infusion experiments were conducted on diabetic, insulin-treated sheep to determine if an increase in pancreatic production of insulin was required for autoregulation of ketogenesis. A final set of experiments were conducted on diabetic, untreated sheep to
Received July 12, 1985. 1Department of Animal Science, North Carolina State University, Raleigh 27650. 1986 J Dairy Sci 69:1270-1281
1270
AUTOREGULATION OF KETOGENESIS determine if autoregulation of ketogenesis could occur in the total absence of insulin. MATERIALS AND METHODS Animals and Procedures
Nonpregnant, nonlactating, sheared Suffolk crossbred ewes weighing 55 to 65 kg were housed in individual indoor 1.8 × 3.0-m pens at 19 to 25°C and natural lighting. The ewes were fed 800 g of commercially prepared alfalfa pellets per day in two 400-g aliquots at 0800 and 1700 h. Water and trace mineralized salt were available ad libitum. All ewes were accustomed to laboratory personnel and never had to be restrained. Five normal; 3 diabetic, insulin-treated; and 3 diabetic, untreated ewes were used. Diabetes was induced pharmacologically by a single 50 mg/kg intravenous dose of alloxan. Treated ewes received sufficient subcutaneous doses (270 U) of insulin (Iletin-40) daily to maintain their blood glucose within normal physiological range (50 to 60 mg/dl). Untreated ewes were withdrawn from insulin for 3 d prior to the experimental period. On experimental days, catheters were implanted into the portal, hepatic, mesenteric veins, caudal vena cava, and caudal aorta. Experiments were initiated immediately following surgery and while the ewe was still under sodium pentobarbital-induced anesthesia, pAminohippuric acid (PAH) was infused (1.5% at .764 ml/min) into a mesenteric vein for a 1-h equilibration period. Three 23-ml blood samples were withdrawn simultaneously from the caudal aorta and hepatic and portal veins at 15-min intervals. Following this c o n t r o l period, 23 ml of 17 to 20%/3-hydroxybutyrate were pulse dosed via the caudal vena cava and then infused at .4 g/h/kg. 7s. This infusion rate approximated the maximum rate of /3-hydroxybutyrate utilization for sheep reported by Bergman and Kon (4). Four sets of infusion period blood samples (23 ml) were withdrawn simultaneously from the caudal aorta and hepatic and portal veins at 30-min intervals beginning 30-min following initiation of /~-hydroxybutyrate infusion. Catheters were maintained with a 6% Na2 ethylenediaminetetraacetate (EDTA) solution between samples. All samples were immersed immediately into an ice bath and treated in accordance with assay
1271
procedures described subsequently. Anesthesia was withdrawn and the ewe was allowed to recover. All ewes recovered successfully and without complications. Chemical Methods
Blood packed cell volume; concentrations of whole blood /3-hydroxybutyrate, acetoacetate, and PAH; and plasma F F A and insulin were determined. p-Aminohippuric acid was determined as described by Kaufman and Bergman (16). Four milliliters of freshly sampled blood were added to an equal volume of cold 1 M HC104 in preparation for acetoacetate and /3-hydroxybutyrate analysis. The tubes were vortexed, centrifuged at 4°C and 1500 × g for 25 min, and buffered to a final pH of 6.4 to 7.6 with cold 1 M KOH. Samples were analyzed immediately using the enzymatic assay of Williamson and Mellanby (28). Insulin was determined by using a standard double antibody radioimmunoassay technique (29) with giunea pig antibovine insulin as the first antibody and goat antiguinea pig immunoglobulin G as the second antibody. Intraassay errors of greater than 5% were rejected and, in the few cases where this occurred, the samples were reanalyzed. Interassay errors, as measured by a pooled ewe sample, were always less than 10% and usually less than 7%. Plasma F F A were analyzed by a modification of the microtitiration technique of Trout et al. (25). Briefly, 2 ml of plasma were added to 50-ml separatory funnels containing 10 ml Of extraction mixture (2-propanol/heptane/1 × H2 SO4, 40:10:1, vol/vol/vol, shaken vigorously for 30 s and vented. Four milliliters of distilled water and 6 ml of heptane were added and the flasks shaken for an additional 2 min. The precipitate was removed and 1 ml of .05% H2SO4 was added to the remaining organic solvent layer and the flasks were shaken for a final 2 min. The acid layer was removed and 5 ml of the organic solvent laver were added to 1 ml of .01% t h y m o l blue in 90% ethanol indicator mixture and titrated with .0018N NaOH under CO2. Calculations
Whole blood flow rates through the portaldrained viscera and liver were calculated by the Journal of Dairy Science Vol. 69, No. 5, 1986
1272
HEITMANN AND FERNANDEZ
indicator-dilution method using the equation: IPAH F = c~AH _ C PAH where F equals whole blood flow through the tissue in liters per minute, IPAH is the infusion rate of PAH in optical density (OD) units per minute, and C PAH and CPAH are the PAH OD units per liter in the arterial input and venous drain. Portal and hepatic vein flows were measured directly and hepatic arterial flow was calculated from the difference between the two. Plasma flow were obtained by subtracting that portion of the flow represented by the packed cell volume. Net fluxes of acetoacetate, fl-hydroxybutyrate, FFA, and insulin across the portaldrained viscera, liver, and total spanchnic bed were calculated using the following equations: Portal-drained viscera flux = PF × (Cp -- Ca) Hepatic flux = PF × (Ch -- Cp) + AF × (Ch -- Ca) Total splanchnic flux = HF × (Ch - Ca) where PF, AF, and HF are the whole blood or plasma flow rates (L/rain) in the portal vein, hepatic artery, and hepatic vein; and Cp, Ca, and Ch are the concentrations of acetoacetate (/aM) or /3-hydroxybutyrate (/z44) in whole blood or FFA (/aM) or insulin (mU/L) in the plasma. Positive flux indicated net release and negative flux indicated net uptake of the metabolite by that specific tissue. Extraction ratios for each metabolite and each tissue were calculated. Extraction ratios are that fraction of the metabolite presented to the tissue (tissue blood flow multiplied by the concentration in the arterial input) that is taken up by the tissue (net flux). Significant changes in extraction ratios between treatments are indicative that changes in net flux with treatment are at least in part due to some physiological mechanism and not due solely to a change in metabolite concentration. Statistical Analysis
All data were analyzed by a two-way analysis of variance to detect differences due to ~-hydroxybutyrate infusion. In addition, a Duncan's new multiple range test was used to determine differences among normal; diabetic, Journal of Dairy Science Vol. 69, No. 5, 1986
insulin-treated; and diabetic, untreated groups. Because no changes in blood flow rates occurred during i n f u s i o n of ~-hydroxybutyrate, an average blood flow for the entire experimental period was used to test for treatment differences. Concentrations and flux rates for all metabolites reached new near steady-state during infusion (i.e., slope of concetnration or rate of flux over time during the control or infusion periods was not different from zero. Consequently, averages of the control and infusion periods were used to test for significant differences. R ESU LTS Blood Flow Rates
Whole blood flow rates for the portal and hepatic veins and portal to hepatic ratios are in Table 1. In normal, 1-d fasted sheep, portal flow was 1.7 L/min and approximately 70% that of hepatic flow. This is in close agreement with previously reported data on fed and 3-d fasted sheep (12). However, there was a 55% decrease (P<.05) in portal flow and 37% decrease (P<.01) in portal to hepatic ratio in diabetic, insulin-treated sheep. This is contrary to previous work that reported no change in blood flows between normal and insulin-treated, alloxanized sheep (8). No satisfactory physiological explanation has been found to explain this occurrence. However, diabetic untreated sheep had flow rates simialr to normal animals. Free Fatty Acid Metabolism
Arterial plasma FFA concentrations and venoarterial differences are in Table 2. Infusion of ~-hydroxybutyrate lowered F F A concentrations by 39, 42, and 11% in normal; diabetic, insulin-treated; and diabetic, untreated sheep (P<.05). The 36% increase in preinfusion F F A concentrations between normal and diabetic, insulin-treated sheep suggests that, although these sheep were euglycemic, some lipolysis was occurring. Increase in FFA in untreated ewes was more than twice that in normal ewes during preinfusion and is demonstrative of the high rate of lipolysis characteristic of uncontrolled diabetes. Most importantly, the venoarterial differences were, for the most part, different from zero (P<.01), indicating significant tissue uptake or release.
AUTOREGULATION OF KETOGENESIS
1273
TABLE 1. Whole blood flow rates (L/min) in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, untreated (DUT) 1-d fasted sheep.
Sheep
No. experiments
Portal
NOR DIT DUT
5 3 3
1.66 .76 1.35
Portal I hepatic
Hepatic SE
X
SE
X
SE
.30 .31" .26
2.38 1.62 2.02
.38 .50 .15
.70 .44 .69
.02 .05** .18
*Significantly different from NOR (P<.05). **Significantly different from NOR (P<.01).
Plasma F F A n e t fluxes a n d h e p a t i c e x t r a c t i o n ratios are in T a b l e 3. T h e r e were n o significant effects of t 3 - h y d r o x y b u t y r a t e infusion o n p o r t a l - d r a i n e d visceral fluxes. However, infusion of 13-hydroxybutyrate decreased ( P < . 0 5 ) h e p a t i c u p t a k e o f F F A in all t h r e e t r e a t m e n t s . A l t h o u g h this decrease was q u a n t i t a t i v e l y similar for all t r e a t m e n t s (176, 186, a n d 166 / 2 m o l / m i n in n o r m a l , t r e a t e d a n d u n t r e a t e d ewes), t h e r e was a m u c h g r e a t e r q u a l i t a t i v e decrease in n o r m a l a n d t r e a t e d (62 a n d 66%) t h a n in u n t r e a t e d s h e e p (34%).
Significantly, h e p a t i c F F A e x t r a c t i o n ratios also were d e c r e a s e d ( P < ° 0 1 ) w i t h /3-hydroxyb u t y r a t e i n f u s i o n in n o r m a l a n d t r e a t e d b u t n o t in u n t r e a t e d ewes. H e p a t i c u p t a k e o f F F A increased ( P < . 0 5 ) in u n t r e a t e d d i a b e t e s ; h o w ever, this was solely d u e t o increased c o n c e n t r a t i o n s because e x t r a c t i o n ratios a m o n g g r o u p s were similar. As a result o f t h e s e changes in liver m e t a b o l i s m , t o t a l s p l a n c h n i c u p t a k e of F F A d e c r e a s e d ( P < . 0 5 ) in n o r m a l a n d t r e a t e d b u t n o t u n t r e a t e d ewes w i t h i n f u s i o n of t3-hyd r o x y b u t y r a t e ; h o w e v e r , t o t a l s p l a n c h n i c up-
TABLE 2. Effects of /3-hydroxybutyrate (BOHB) on free fatty acid (FFA) concentrations and venoarterial differences in normal (NOR), diabetic, insulin-treated (DIT); and diabetic, untreated (DUT), 1-d fasted sheep.
Sheep 1
Hepaticartery
Artery
Hepaticportal
Portalartery
(Izmol FFA/L) SE
X
SE
X
SE
X
SE
NOR (5) Control BOHB infusion
872 528
83 55*
-132 --31
32** 18
-203 --93
19"* 13"*
71 62
18"* 12"*
DIT (3) Control BOHB infusion
t186 684
1054~ 33*
-258 54
41"* 14"*
-270 --123
33** 18"*
12 69
22
DUT (3) Control BOHB infusion
1933 1719
-285 --217
49** 22**
-333 --234
53** 55**
49 17
24 45
92 ~ 85 *'*
14"*
1 Number of experiments are in parentheses. * Significantly different from control (P<.05). ** Significantly different from 0 (P<.01). Slgmficantly different from corresponding NOR (P<.O1). Journal of Dairy Science Vol. 69, No. 5, 1986
1274
HEITMANN AND FERNANDEZ
TABLE 3. Effects of /3-hydroxybutyrate (BOHB) on portal-drained visceral, hepatic and total splanchnic net fluxes, and hepatic extraction ratios of free fatty acids (FFA) in normal (NOR); diabetic; insulin-treated (DIT); and diabetic untreated (DUT), 1-d fasted sheep.
Sheep 1
Portaldrained viscera
Hepatic extraction ratio
Liver
Total splanchnic
(#mol FFA/min) SE
.~
SE
X
SE
X
SE
97 76
33 19
-284 --108
31 19"
.18 .11
.02 .02**
-187 --33
50 31"
DIT (3) Control BOHB infusion
3 32
9* 10
-280 --94
49 22*
.21 .12
.03 .03"*
-276 --62
50 16"
DUT (3) Control BOHB infusion
43 --10
27 43*
-485 --319
82* 66"*
.16 .12
.03 .02
-442 --329
81 + 38 +
NOR (5) Control BOHB infusion
1Number of experiments are in parentheses. *Significantly different from control (P<.05). **Significantly different from control (P<.01 ). *Significantly different from corresponding NOR (P<.05),
take of F F A increased in untreated diabetes relative to the normal and insulin-treated sheep.
Ketone Body Metabolism
Whole blood acetoacetate and l~-hydroxybutyrate concentrations and venoarterial differences are reported in Table 4. Untreated diabetes increased acetoacetate and/3-hydroxybutyrate concentration 11- and 24-fold. As with FFA, /3-hydroxybutyrate but not acetoacetate concentrations were elevated in treated animals compared with normal animals during preinfusion (P<.01), although both groups exhibited euglycemia. Venoarterial differences for both acetoacetate and /3-hydroxybutyrate, for the most part, were different from zero (P<.05) in normal and diabetic, insulin-treated sheep. This effect was lost in untreated animals due to the high acetoacetate and fl-hydroxybutyrate and a subsequent loss in assay sensitivity at these elevated concentrations. Net fluxes for acetoacetate and fl-hydroxybutyrate are in Table 5. /3-Hydroxybutyrate infusion increased portal-drained visceral release of acetoacetate by 43 and 59 #mol/min but switched from a condition of release to one of Journal of Dairy Science Vol. 69, No. 5, 1986
uptake of ~-hydroxybutyrate (137 vs. - 3 9 4 and 9 vs. - 2 7 8 /amol/min) in normal and diabetic, insulin-treated sheep (P<.05). These effects were not significant in untreated animals, although trends were similar. Hepatic uptake of acetoacetate increased by 76 and 190/~mol/min (P<.05) with fl-hydroxybutyrate infusion in normal and treated sheep. However, hepatic release of/3-hydroxybutyrate increased (P<.05) in normal animals. As with portal-drained visceral metabolism, these effects were not significant in untreated animals although similar trends were noted. Untreated diabetes decreased portal-drained visceral but increased hepatic release of acetoacetate. Conversely, diabetes tended to increase ketogenesis of /3-hydroxybutyrate in both these splanchnic tissues. Total splanchnic metabolism, as reflected by a combination of portal-drained visceral and hepatic rates, demonstrated decreased overall ketogenesis of acetoacetate (P<.05) in normal and treated sheep with ~-hydroxybutyrate infusion and showed a similar trend ( P < . I ) for fl-hydroxybutyrate in normal animals with the ketone infusion. Untreated diabetes increased total splanchnic ketogenesis of both ketones (P<.05).
412 419
DUT (3) Control BOHBinfusion
53* 44*
9 15"*
13,771 19,537
1023 4321
565 4105
0o Ox
t/i
O
Z
O~
O
<
Slgmficantly different from corresponding NOR (P<.01).
* Significantly different from 0 (P<.05).
* * Significantly different from control (P<.O 1).
2033 ~ 1701 **,~
81 ~ 249 * ~ t
46 352**
W
1 Number of experiments are in parentheses.
46 213
DIT (3) Control BOHB infusion
5 14"*
G~
e~
36 196
SE
X
SE
.X
NOR (5) Control BOHB infusion
Sheep ~
3-hydroxybutyrate
Acetoacetate
Artery
9 8*
5 8*
SE
29 17 11 4*
16 -67
-2 -25
X
Acetoacetate
754 822
481 353
244 105
X
400 316"
46* 77*
41" 111
SE
X
46 2
6 -143 32 4
11 15"
5* 6*
SE
422 615
426 850
155 370
X
381 291"
45* 149"
25* 57*
SE
3-hydroxybutyrate
Hepatic-portal Acetoacetate
-15 63
(#mol/L)
3-hydroxybutyrate
Hepatic-artery
4* 5*
SE
- 1 7 15 10 4*
10 5 77 10"
13 39
X
Acetoacetate
331 207
55 -497
89 --264
X
311 309
38 204*
22* 71"
SE
3-hydroxybutyrate
Portal-artery
TABLE 4. Effects of 3-hydroxybutyrate (BOHB) on acetoacetate and 3-hydroxybutyrate concentrations and venoarterial differences in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, untreated (DUT), 1-d fasted sheep.
bO ~q
~q O C3 t~ Z
Z O
5
.q
70
o
O
00
t.n
Z
G~ xo
O
<
~7 ,<
4 63
9 12
DIT (3) Control BOHB infusion
DUT (3) Control BOHB infusion
14' 6*
6 17"
9 10"
SE
:L
.
.
450 227
9 --278 471 435*
26* 102"
28 94*
SE
Significantly different from corresponding NOR(P<.05).
.
X
t3-hydroxybutyrate
137 --394
* Significantly different from control (P<.05).
1 Number of experiments are in parentheses.
25 68
'X
NOR (5) Control BOHB infusion
Sheep 1
Acetoacetate
Portal-drained viscera
72 13
13 -177
-36 -112
X
Acetoacetate
50* 8*
13 38*
14 13"
SE
1138 1445
749 881
395 679
R
788* 563*
126' 100
51 127"
SE
/3-hydroxybutyrate O~mol/min)
Liver
62 25
17 -114
-11 44
.X
Acetoacetate
36* 8*
11 23*,*
12 16"
SE
1588 1672
758 602
532 285
859* 681'
113 140
65 191
SE
13-hydroxybutyrate
Total splanchnic
TABLE 5. Effects of ~3-hydroxybutyrate (BOHB) on portal-drained visceral, hepatic, and total splanchnic net fluxes of acetoacetate and in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, u n t r e a t e d (DUT), 1-d fasted sheep.
N
> Z
Z
Z
.q Gx
AUTOREGULATION OF KETOGENESIS Insulin Metabolism
Insulin concentration and venoarterial differences are in Table 6. Concentrations were lower (P<.01) in diabetic than in normal animals. Although insulin concentrations in diabetic ewes were very low, they were different from zero (P<.05), suggesting some viable beta cells may remain or perhaps some crossreactivity in the assay. Only portal-arterial and hepatic-portal venoarterial differences in normal sheep were different from zero (P<.01). Importantly, the portal-arterial differences in both groups of diabetic animals were not different from zero, indicating that pancreatic beta cell function was indeed inoperative. Infusion of ~-hydroxybutyrate increased arterial insulin slightly (P<.I) in normal ewes but had no effect on diabetic ewes. Net flux of insulin are in Table 7. Infusion of J3-hydroxybutyrate in normal animals increased pancreatic insulin production (P<.05), as represented by portal-drained visceral release, by 11 mU/min. However, there was a concomitant increase of hepatic insulin extraction of 10 mU/min, resulting in no change in total splanchnic release. This is of great importance when considering site of sampling to measure blood insulin profiles. Blood withdrawn peri-
1277
pherally, i.e., jugular or cephalic, would not detect these marked changes occurring in pancreatic production and hepatic uptake. The liver of normal animals was extracting 67 to 75% of the portal production and 13 to 17% of the total circulating insulin. These values are slightly higher than those of 50 and 8% reported by Brockman and Bergman (9) in normal sheep.
DISCUSSION
Net fluxes of ketone bodies, FFA, and insulin across the major splanchnic beds in ruminants have been studied previously under various conditions mainly in the laboratories of Bergman for the ovine [reviewed in (3)1 and Baird for the bovine [reviewed in (1)]. In addition, some preliminary work demonstrating the capacity of ~-hydroxybutyrate to lower circulating F F A concentrations has been done in the dog (20) and rat (5). However, the present work is an initial effort to obtain direct evidence that elevated ketone bodies can inhibit ketogenesis in intact sheep as well as depress plasma FFA concentrations and to determine the mechanism of action involved for this proposed negative feedback. As such, this work
TABLE 6. Effects of/~-hydroxybutyrate (BOHB) on insulin concentrations and venoarterial differences in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, untreated (DUT), 1-d fasted sheep.
Sheep 1
Hepaticartery
Artery
Hepaticportal
Portalartery
X
(#U/ml) NOR (5) Control BOHB infusion DIT (3) Control BOHB infusion DUT (3) Control BOHB infusion
SE
X
SE
X
SE
SE
38
5
4
2
-16
4**
20
4**
49
45"
4
2
-27
4**
31
4**
22 22
2+
--3
1"*
--1
I
2~
-3
1"*
--2
1
--i
1
3 3
0* 1#
-1 --0
0 1
-1 -0
1 1
1 -0
1 1
2
1
1Number of experiments are in parentheses. * * Significantly different
from 0 (P<.o1). Sigmflcantly different from control (P<.I). *Significantly different from corresponding NOR (P<.01). t
•
.
Journal of Dairy Science Vol. 69, No. 5, 1986
1278
HEITMANN AND FERNANDEZ
TABLE 7. Effects of ~-hydroxybutyrate (BOHB) on portal-drained visceral, hepatic, and total splanchnic net fluxes of insulin in normal (NOR); diabetic, insulin-treated (DIT); and diabetic, untreated (DUT), 1-d fasted sheep. 1 Portal
Sheep
drained viscera
Total splanchnic
Liver (mU/min)
NOR (5) Control BOHB infusion DIT (3) Control BOHB infusion DUT (3) Control BOHB infusion
SE
X
SE
X
SE
25 36
5 5*
-17 --27
5 4*
8 9
4 4
-1 -1
0 ~: O#
-2 -3
1~: 1'
-3 3
1* 1#
1* 1*
-2 -O
1* 1#
-1 -0
1t 1#
1 O
I Number of experiments are in parentheses. *Significantly different from control (P<.05). *Significantly different from corresponding NOR (P<.05).
may explain the etiology of pregnancy acetonemia and lactation ketosis and may have possible implications in the role of developing diabetic ketoacidosis. There are two current hypotheses. First, autoregulation of ketogenesis may be mediated at the pancreatic level by increased insulin production in response to elevated ketones (20), thereby increasing adipocyte lipogenesis and inhibiting lipolysis, which in turn decreases hepatic ketogenic precursor supply. Second, the ketone may act directly at the adipocyte by decreasing the activity of hormone-sensitive lipase (5). These studies were conducted on sodium pentobarbital-anesthetized sheep and immediately following abdominal surgery. Blood flow rates through the major splanchnic tissues were in very close agreement with measurements on unanesthetized sheep (7, 8, 9, 12, 15). Concentrations of acetoacetate (36 /~/), /3-hydroxybutyrate (565 gA//), and insulin (38 /~U/ml) in normal, preinfused animals are well within the physiological ranges for sheep reported by numerous researchers as summarized by Lindsay and Lea, (19). Plasma FFA (872 MM) were slightly elevated relative to fed ruminants (1, 15, 16, 19) but were in close Journal of Dairy Science Vol. 69, No. 5, 1986
agreement with concentrations reported in 1-d fasted sheep (13). In addition, net fluxes of normal, preinfused animals in the portaldrained viscera and liver for acetoacetate (25 and - 3 6 ~tmol/min), /3-hydroxybutyrate (137 and 395 ~mol/min), FFA (97 a n d - 2 8 4 / . t m o l / min), and insulin (25 and - 1 7 /lmol/min) are similar to previously reported values on conscious sheep (7, 9, 15). Finally, no differences were noted in portal and hepatic metabolism of insulin in dogs awake and dogs anesthetized with sodium-pentobarbitol (14). Portal-drained visceral FFA fluxes were small but generally exhibited net release. Because FFA absorption across the gut is mostly lymphatic, this was to be expected, and the significant net release may have represented some omental lipolysis. Conversely, there were high rates of F F A uptake in liver in the preinfusion periods of all three groups of animals. Untreated diabetes increased hepatic uptake by 70%, however, this was due solely to increased F F A concentrations, because the hepatic extraction ratios did not change and were between 16 to 21%. Rates of F F A metabolism in the portal-drained viscera and liver and the hepatic extraction ratios in normal, preinfused sheep were similar to those reported for non-
AUTOREGULATION OF KETOGENESIS pregnant, fed sheep (15). Infusion of /3-hyd r o x y b u t y r a t e at the reported rate of maximum utilization in sheep (4) had no effects on portal-drained visceral F F A metabolism. Hepatic F F A uptake was reduced by 62 and 66% in normal and diabetic, insulin-treated ewes but only 34% in diabetic, untreated ewes. Most important, infusion of /3-hydroxybutyrate decreased the hepatic extraction ratios of F F A in normal and diabetic, insulin-treated animals but not in untreated animals, implying the involvement of some physiological mechanism at the hepatocyte. This was surprising, because it has been reported that hepatic extraction (26) and oxidation (6) of F F A is concentration dependent. Because hepatic extraction ratios decreased in normal and diabetic insulin-treated ewes but not diabetic, untreated ewes, it may be concluded that insulin may be involved in this altered hepatic F F A metabolism. Sheep hepatocytes have insulin receptors (11), and these increased in number but showed no change in binding kinetics with progressive lactation. As a result, hepatic insulin binding increased and circulating concentrations decreased. Similar results have been reported for the lactating goat hepatocyte (10). According to McGarry and Foster's bihormonal theory (22), insulininduced malonyl-coenzyme A (CoA) concentrations could inhibit fatty acid oxidation by inhibiting mitochondrial translocation of longchain acyl-CoA via carnitine acyltransferase I. However, Prior and Smith (24) have recently demonstrated that acetyl-CoA carboxylase in the alloxan-diabetic steer adipocyte does not require insulin for activation. Infusion of /3-hydroxybutyrate decreased F F A in plasma in all three groups of sheep. However, the effect of/3-hydroxybutyrate was much more pronounced in normal and diabetic insulin-treated sheep (40% depression) relative to diabetic untreated animals (10% depression) even though/3-hydroxybutyrate has been shown to be more efficacious in lowering elevated concentrations (13). Because /3-hydroxybutyrate both lowered circulating plasma F F A and decreased hepatic uptake, there must have been a concomitant decrease in lipolysis at the adipocyte. Both acetoacetate and /3-hydroxybutyrate are released by portal-drained viscera, but acetoacetate is removed and/3-hydroxybutyrate released by the liver of normal, preinfused
1279
animals. This is typical of data reported for both ovine (15) and bovine (2) animals. Hepatic uptake of acetoacetate may seem paradoxical since the rate-limiting enzyme of ketone body utilization, 3-oxoacid-CoA transferase, is not present in significant quantity in the liver. However, ovine liver does contain a/3-hydroxybutyrate dehydrogenase of sufficient activity, .6 to 1.0 /~mol/min/g (17, 27), to account for all the acetoacetate uptake observed in the current study to be metabolized to /~-hydroxybutyrate. Untreated diabetes tended to increase portal-drained visceral total ketone release threefold. In addition, infusion of fl-hydroxybutyrate caused the portal-drained viscera to switch from release to uptake of /3-hydroxybutyrate but to increase acetoacetate release slightly. The net effect of /3-hydroxybutyrate infusion was to completely inhibit net alimentary ketogenesis. Although the same trends were in diabetic, untreated sheep, these effects were not significant. The portal-drained viscera always had small net rates of release of F F A ; however, there may have been substantial unidirectional rates of uptake occurring that were not measureable by this model. Therefore, the loss of effects of /3-hydroxybutyrate on alimentary ketone flux with the absence of insulin may have been due to an altered F F A metabolism. Because hepatic F F A uptake is depressed markedly in normal and diabetic, insulintreated sheep following infusion of fl-hydroxybutyrate, it would be expected that hepatic ketogenesis was decreased in these animals as well. Indeed, acetoacetate uptake increased markedly, but, paradoxically, release of 13-hyd r o x y b u t y r a t e increased. Once again, these effects were totally absent in diabetic, untreated sheep. The net effect of /3-hydroxybutyrate infusion on the total splanchnic tissue was to increase acetoacetate uptake in normal and diabetic, insulin-treated sheep and to tend ( P < . I ) to decrease/3-hydroxybutyrate release in normal animals. Total ketone release was depressed in both normal and treated but not untreated sheep. The effects of/3-hydroxybutyrate infusion at rates simulating maximum ketone utilization (4) on the net portal-drained visceral and hepatic fluxes of F F A , acetoacetate, and l~-hydroxybutyrate were similar in normal and diabetic, insulin-treated sheep but were diminJournal of Dairy Science Vol. 69, No. 5, 1986
1280
HEITMANN AND FERNANDEZ
ished f o r F F A a n d t o t a l l y a b s e n t f o r acetoacetate and / 3 - h y d r o x y b u t y r a t e in diabetic, u n t r e a t e d animals. T h e r e f o r e , it is suggested t h a t t h e m e c h a n i s m o f a u t o r e g u l a t i o n of k e t o g e n e s i s m a y n o t be t o t a l l y d u e to i n c r e a s e d p a n c r e a t i c insulin p r o d u c t i o n b u t m a y b e m e d i a t e d at t h e h e p a t o c y t e insulin r e c e p t o r b y a f f e c t i n g e i t h e r r e c e p t o r n u m b e r s of affinity. Whereas t h e r e is a decreased a d i p o c y t e insulin r e c e p t o r a f f i n i t y in d i a b e t i c k e t o a c i d o s i s , this a p p e a r s to b e d u e to t h e acidosis itself, pred o m i n a n t l y an increased r a t e o f insulin diss o c i a t i o n (21). F u r t h e r m o r e , it has b e e n dem o n s t r a t e d t h a t t 3 - h y d r o x y b u t y r a t e , a d d e d in c o n c e n t r a t i o n s similar t o t h o s e in d i a b e t i c ketoacidosis, can r e s t o r e normal binding k i n e t i c s t o insulin r e c e p t o r s o f h u m a n l y m p h o cytes (23). It is still possible t h a t k e t o n e s m a y act d i r e c t l y at t h e a d i p o c y t e b y i n h i b i t i n g h o r m o n e - s e n s i t i v e lipase a n d decreasing lipolysis or b y s t i m u l a t i n g l i p o p r o t e i n lipase a n d increasing lipid u p t a k e a n d r e e s t e r i f i c a t i o n , t h e r e b y l o w e r i n g circulating F F A c o n c e n t r a tions. It m u s t be e m p h a s i z e d t h a t t h e s e a n i m a l s were o n l y f a s t e d 1 d, w h i c h is n o t a p r o l o n g e d fast for a r u m i n a n t . I n d e e d rates of F F A a n d k e t o n e flux in t h e n o r m a l 1-d f a s t e d ewes were very similar t o t h o s e o f f e d r u m i n a n t s and, t h e r e f o r e , t h e ewes were n o t in a n a c c e l e r a t e d k e t o g e n i e m o d e . It w o u l d b e m o s t b e n e f i c i a l t o repeat these experiments on animals during energy d r a i n i n g physiological c o n d i t i o n s such as p r o l o n g e d fasting, p r e g n a n c y , or l a c t a t i o n . ACKNOWLEDGMENTS
T h e a u t h o r s w o u l d like to t h a n k J o a n H e m b r e e a n d A a r o n Cole for assistance d u r i n g t h e surgical p h a s e a n d Chris R e y n o l d s f o r assistance w i t h t h e insulin assay o f this study.
REFERENCES
1 Baird, G. D. 1977. Aspects of ruminant intermediary metabolism in relation to ketosis. Biochem. Rev. 5:819. 2 Baird, G. D., H. W. Symonds, and R. Ash. 1975. Some observations on metabolite production and utilization in vivo by the gut and liver of adult dairy cows. J. Agric. Sci., Camb. 85:281. 3 Bergman, E. N. 1971. Hyperketonemia ketogenesis and ketone body metabolism. J. Dairy Sci. 54:936. 4 Bergman, E. N., and K. Kon. 1964. Acetoacetate turnover and oxidation rates in ovine pregnancy ketosis. Am. J. Physiol. 206:449. Journal of Dairy Science Vol. 69, No. 5, 1986
5 Bjorntorp, P. 1966. Effect of ketone bodies on lipolysis in adipose tissue in vitro. J. Lipid Res. 7:621. 6 Boyd, R. O., R. A. Britton, H. Knoche, D. B. Moser, E. R. Pen, Jr., and R. K. Johnson. 1982. Oxidation rates of major fatty acids in fasting neonatal pigs. J. Anita. Sci. 55:95. 7 Brockman, R. P. 1976. Effect of glucagon and insulin on lipolysis and ketogenesis in sheep. Can. J. Comp. Med. 40:166. 8 Brockman, R. P., and E. N. Bergman. 1975. Effect of glucagon on plasma alanine and glutamine metabolism and hepatic gluconeogenesis in sheep. Am. J. Physiol. 228:I627. 9 Brockman, R. P., and E. N. Bergman. 1975. Quantitative aspects of insulin secretion and its hepatic and renal removal in sheep. Am. J. Physiol. 229:1338. 10 Gill, R. D., and I. C. Hart. 1979. The effect of dietary composition on the binding of insulin and glucagon to goat hepatocytes. Biochem. Soc. Trans. 7:910. 11 Gill, R. D., and I. C. Hart. 1980. Properties of insulin and glucagon receptors on sheep hepatocytes: a comparison of hormone binding and plasma hormones and metabolites in lactating and non-lactating ewes. J. Endocrinol. 84:234. 12 Heitmann, R. N., and E. N. Bergman. 1978. Glutamine metabolism, interorgan transport, and glucogenicity in the sheep. Am. J. Physiol. 234: E197. 13 Heitmann, R. N., and D. R. Metzler. 1983. Effects of fl-hydroxybutyrate on plasma free fatty acids in mature wethers. Fed. Proc. 45 : 532. 14 Ishida, T., R. M. Lewis, C. J. Hartley, M. L. Entman, and J. B. Field. 1983. Comparison of hepatic extraction of insulin and glucagon in conscious and anesthetized dogs. Endocrinology 112:1098. 15 Katz, M. L., and E. N. Bergman. 1969. Hepatic and portal metabolism of glucose, free fatty acids, and ketone bodies in the sheep. Am. J. Physiol, 216-953. 16 Kaufman, C. F., and E. N. Bergman. 1971. Renal glucose, free fatty acid, and ketone body metabolism in the unanesthetized sheep. Am. J. Physiol. 221:967. 17 Koundakjian, P. P., and A. M. Snoswell. 1970. Ketone body and fatty acid metabolism in sheep tissues. 3-Hydroxybutyrate dehydrogenase, acetoplasmic enzyme in sheep liver and kidney. Biochem. J. 119:49. 18 Leighton, B., A. R. Nicholas, and C. I. Pogson. 1983. The pathway of ketogenesis in rumen epithelium of sheep. Biochem. J. 216:769. 19 Lindsay, D. B., and W.M.F. Leat. 1975. Carbohydrate and lipid metabolism in the blood of sheep: Composition and function. Pages 4 5 - 6 2 in The blood of sheep. M. H. Blunt, ed. SpringerVerlag, New York, NY. 20 Madison, L. L., D. Mebane, R. H. Unger, and A. Lochner. 1964. The hypoglycemic action of ketones. II. Evidence for a stimulatory feedback of ketones on the pancreatic beta ceils. J. Clin. Invest. 43:408. 21 Marshall, S., D. A. Podlecki, and J. M. Olefski. 1983. Low pH accelerates dissociation of receptor
A U T O R E G U L A T I O N OF KETOGENESIS b o u n d insulin. Endocrinology. 113 : 37. 22 McGarry, J. D., G. P. Mannaerts, and D. W. Foster. 1977. A possible role for malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis. J. Clin. Invest. 60:265. 23 Misbin, R. I., A. J. Pulkkinen, S. A. Lofton, and T. J. Merimee. 1978. Ketoacids and the insulin receptor. Diabetes 27:539. 24 Prior, R. L., and S. P. Smith. 1982. Hormonal effects on partitioning of nutrients for tissue growth: role of insulin. Fed. Proc. 40:2545. 25 Trout, D. L., E. H. Estes, and S. J. Freidberg. 1960. Titration of free f a t t y acids of plasma: a s t u d y of current m e t h o d s and a new modification. J. Lipid Res. 1:199.
1281
26 Van Harken, D. R., C. W. Dixon, and M. Heimberg. 1969. Hepatic lipid metabolism in experimental diabetes. V. The effect of concentration of oleate on metabolism of tryglycerides and on ketogenesis. J. Biol. Chem. 244:2278. 27 Watson, H. R., and D. B. Lindsay. 1972. 3-Hyd r o y x b u t y r a t e dehydrogenase in tissues from normal and ketonaemic sheep. Biochem. J. 128:53. 28 Williamson, D. H., and J. Mellanby. 1965. Acetoacetate and D-(-)-fl-hydroxybutyrate. Pages 4 5 4 461 in Methods of enzymatic analysis. 2nd ed. H. V. Bergmeyer, ed. Academic Press, New York, NY. 29 Yalow, R. S. 1976. Methods in radioimmunoassay of peptide h o r m o n e s . North Holland Publishing Co., A m s t e r d a m .
Journal o f Dairy Science Vol. 69, No. 5, 1986