The effect of diet on tumor necrosis factor stimulation of hepatic lipogenesis

The Effect of Diet on Tumor Necrosis Factor Stimulation Kenneth

R. Feingold,

Mounzer

Soued,

of Hepatic Lipogenesis

M. Kerrela Serio, Saleh Adi, Arthur H. Moser,

and Carl Grunfeld

Previous studies have demonstrated that tumor necrosis factor (TNF) acutely increases serum triglyceride levels and stimulates hepatic lipid synthesis. In this study, we determined the effects of TNF on serum lipid levels and hepatic lipid synthesis in animals whose diets and feeding conditions were varied to induce changes in baseline serum lipid levels and/or rates of hepatic lipid synthesis. In animals studied at both the nadir and peak of the diurnal cycle of hepatic lipid synthesis, TNF acutely increases serum triglyceride levels, stimulates hepatic fatty acid synthesis, and increases the quantity of newly synthesized fatty acids found in the serum. Similarly, in animals ingesting either high-sucrose or cholesterol-enriched diets, TNF induces the characteristic rapid increase in serum triglyceride levels, hepatic fatty acid synthesis, and quantity of labeled fatty acids in the serum. In animals fed a diet high in triglycerides, using either corn oil or lard, TNF stimulates hepatic fatty acid synthesis and increases the quantity of newly synthesized fatty acids in the serum, but serum triglyceride levels do not change. However, TNF inhibits gastric emptying, which results in a marked decrease in fat absorption in TNF-treated animals. It is likely that a decrease in the dietary contribution to serum triglyceride levels during high-triglyceride feeding counterbalances the increased hepetic contribution induced by TNF treatment. In animals fasted before TNF administration there was no acute change in either serum lipid levels, hepatic fatty acid synthesis, or the quantity of labeled fatty acids in the serum. Thus, TNF stimulates hepatic fatty acid synthesis and increases serum triglyceride levels under many diverse dietary conditions, suggesting that there is a strong linkage between the immune system and lipid metabolism that is independent of most dietary manipulations end may be of fundamental importance in the body’s response to infection. @ 1990 by W.B. Saunders Company.

I

NFECTION

BY BACTERIA, viruses, or parasites leads that eliminates the infectious agent and induces immunity against future challenges. Additionally, infection can result in multiple disturbances in intermediary metabolism; hyperlipidemia is one such metabolic aberration.le5 The response to infection involves many cell types and is mediated by cytokines.6-8 These cytokines coordinate the immune response and also have a large number of other biological effects. Beutler and Cerami have proposed that the cytokine, tumor necrosis factor (TNF), is responsible for the hyperlipidemia that occurs in association with infections.’ Our laboratory has demonstrated that the administration of purified TNF to intact rats results in a rapid increase in serum triglyceride levels.” In our studies, 2 hours after TNF administration, serum triglyceride concentrations were increased and remained elevated for at least 17 hours. Serum cholesterol concentrations also increase in response to TNF administration.” A number of laboratories have shown that TNF decreases the activity of lipoprotein lipase in cultured fat cells,““3 and Semb et al have shown that TNF decreases adipose tissue lipoprotein lipase activity in vivo.14 A decrease in adipose tissue lipoprotein lipase activity could lead to the decreased clearance of triglyceriderich lipoproteins and thereby result in hyperlipidemia. We have observed that hepatic lipid synthesis is also stimulated following TNF administration.” The de novo synthesis of fatty acids in the liver is increased 1 to 2 hours following TNF administration, an effect that persists for over 17 hours. Additionally, this acute increase in hepatic fatty acids is associated with an increased quantity of labeled fatty acids in the serum of TNF-treated animals.” Hepatic cholesterol synthesis is also increased 16 to 17 hours following TNF treatment. lo Thus, TNF administration increases both hepatic fatty acid and cholesterol synthesis. Recent data indicate that TNF increases serum triglyceride levels in vivo by stimulating hepatic lipogenesis and very-low-density lipoprotein (VLDL) production, rather to a complex host response

Metabolism, Vol39,

No 6 (June), 1990: pp 623-632

than by inhibiting adipose lipoprotein lipase (LPL) and triglyceride clearance. First, the time course of the increase in serum triglycerides parallels the increase in de novo hepatic lipogenesis.” Second, the increase in serum triglycerides precedes the decrease in LPL activity in the epididymal fat pad; in addition, little or no decrease is seen in LPL in multiple other sites of adipose tissue or muscle.‘4~‘5 Third, TNF is just as effective at increasing serum triglyceride levels in streptozotocin-diabetic rats in whom adipose tissue LPL activity is markedly reduced secondary to the diabetes. Most important, no further decrease in LPL is seen after TNF treatment of diabetic animals, yet TNF still stimulates de novo hepatic fatty acid synthesis in diabetic animals without inducing changes in serum insulin levels.16 Fourth, TNF treatment does not decrease the clearance of triglyceride-rich lipoproteins from the circulationt6.” Fifth, total hepatic triglyceride production is increased in TNF-treated animals as measured by the incorporation of ‘H-glycerol into hepatic and serum triglyceride.‘* Finally, the increase in hepatic lipogenesis is in turn reflected by an increase in the hepatic VLDL production rate as measured by the Triton WR-1339 technique.“,‘* Recent studies have demonstrated that the increase in serum triglyceride levels induced by TNF

From the Department of Medicine, University of California, San Francisco, and the Metabolism Section, Medical Service, Veterans Administration Medical Center, San Francisco, CA. Supported by grants from the Veterans Administration, the National Institutes of Health (DK40990), and the University of California University Wide Task Force on AIDS. Dr Grunfeld is a recipient of a Clinical Investigator Award from the Veteran’s Administration. Address reprint requests to Kenneth R. Feingold, MD, VA Medical Center, Metabolism Section (I I IF), 4150 Clement St. San Francisco, CA 94121. Q1990 by W.B. Saunders Company. 0026-0495/90/3906-0014%03.00/0

623

624

FEINGOLD ET AL

is due to an increase in large VLDL particles that are similar in lipid and apoprotein content to particles present in control animals (submitted for publication, R. Krauss, C. Grunfeld, and K.R. Feingold). In our previous studies, the effect of TNF on serum lipid levels and hepatic lipid synthesis was determined only in animals fed standard rat chow ad libitum.” It is well recognized that dietary manipulations can have marked effects on hepatic lipid synthesis and serum lipid levels.‘9*20 Whether manipulations that alter baseline lipid metabolism would affect the response to TNF administration is unknown. In the present report, we have determined the effect of TNF on serum lipid levels and hepatic lipid synthesis in animals whose diets and feeding conditions were varied to induce changes in baseline serum lipid levels and/or rates of hepatic lipid synthesis.

chloroform, and the cholesterol separated by TLC. Plates were developed in ethyl acetate: benzene (1:5) and the band corresponding to a standard of cholesterol was counted by liquid scintillation. The window settings of the scintillation counter were adjusted so that less than 0.2% of the tritium counts were recorded in the 14Cwindow and approximately 10% of the 14Ccounts in the tritium window. Incorporation was corrected for spillover of tritium, spillover of 14C, background, and recovery of internal standard. After acidifying the saponified material to a pH less than 2 with concentrated hydrochloric acid, the fatty acids were extracted three times with petroleum ether. The extract was dried, dissolved in chloroform, and an aliquot counted as described above. The specific activity of the tritiated water was determined individually for each animal at the end of the experiment. The validity of our methodology for measuring lipid synthesis has been demonstrated in earlier publications.23~24 Lipid Absorption

‘H,O (1 Ci/g) was purchased from ICN Radiochemicals. 26-14C cholesterol, 14Coleic acid, and ‘H triolein (labeled in the fatty acids) were purchased from New England Nuclear (Boston, MA). The thin-layer chromatography (TLC) polygram Sil G plates were purchased from Brinkmann Instruments. Ready Safe scintillation fluid was purchased from Beckman. Human TNF-a with a specific activity of 5 x 10’ U/mg produced by recombinant DNA technology was kindly provided by Genentech.

Immediately following TNF or saline injection, the animals were administered 7.5 PCi of ‘H triolein (labeled in the fatty acids) in 1 mL corn oil intragastrically via an oral catheter. The catheter was immediately removed and 2 hours later the animals were anesthetized, a blood specimen obtained, the animals killed and the organs removed. The liver, stomach, small intestine, serum, and carcass (all tissues not individually studied) were saponified as described above. After cooling, an internal standard of ‘% oleic acid was added. After acidifying the saponified material to a pH less than 2 with concentrated hydrochloric acid the labeled lipid was extracted three times with petroleum ether and an aliquot counted as described above.

Animal Procedures

Serum Chemistries

Male Sprague-Dawley rats (approximately 200 g) were purchased from Bantin Kingman Animal Vendors. Unless otherwise indicated, the animals were maintained on a reverse 12-hour light cycle (3 AM to 3 PM dark, 3 PM to 3 AM light). In our usual experiments, animals are studied at 9 AM (actual time), which corresponds to l2:OO midnight in a reverse light cycle animal room. Animals were fed food and water ad libitum unless indicated otherwise. In these studies we used several different diets: (1) our standard rat chow diet (Simonsen), which contains 24% protein, 6% fat, 6% ash, 3.5% fiber, and 60% complex carbohydrate all derived from ground wheat, soybean meal, and rice bran. Where indicated, 5% cholesterol was added to this standard rat chow. (2) A high sucrose diet consisting of 20% vitamin-free casein, 4% salt Hegsted, 76% sucrose, and 22 g/kg of a vitamin mixture (ICN). (3) A high-fat diet consisting of 30% corn oil or tocopherol-free lard (ICN), 20% vitamin-free casein, 4% salt Hegsted, 46% sucrose, and 22 g/kg of a vitamin mixture. Animals were injected via the tail vein with 25 pegof TNF in 0.5 mL of 0.9% saline or saline alone. This dose is approximately one-fourth of that shown to produce tumor necrosis in viva” and is the optimal dose for increasing hepatic lipid synthesis in rats.” After TNF injection, all animals were fasted because TNF administration induces anorexia.”

Serum triglyceride levels were measured using Sigma Diagnostic Kit no. 405 (St Louis, MO) after extraction with Dole’s reagent. Serum cholesterol levels were measured by using Sigma Diagnostic Kit no. 351. Serum glucose levels were determined using a YSI glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH).

METHODS

Materials

Lipogenesis At the time indicated after TNF administration, animals were injected intraperitoneally (IP) with tritiated water (50 mCi). One hour later, animals were anesthetized, weighed, and a blood specimen obtained. Livers and small intestines were removed, weighed, and the lipid saponified by refluxing overnight in a solution of 45% KOH, water, and 70% ethyl alcohol (2:1:5). After cooling, internal standards of 14Ccholesterol and 14Coleic acid were added before extracting the nonsaponifiable material three times with 25 mL of petroleum ether. The petroleum ether extract was dried, dissolved in

Statistics Statistical differences were determined by using the one- or two-tailed Student’s t test. The one-tailed Student’s t test was used only when the direction of change expected was known based on previous experiments. As in all of our previous studies, we have found a variation in the absolute rates of lipid synthesis from month to month and therefore we only compare animals that are studied simultaneously under identical conditions. The individual experiments are presented in separate tables. RESULTS

Effect of Light Cycle

It is well recognized that both hepatic fatty acid and cholesterol synthesis undergo a diurnal rhythm with maximal synthesis occurring at approximately 12:00 midnight and the nadir of synthesis at approximately 12:00 noon.25,26 Therefore, we first compared the rate of lipid synthesis in animals on a reversed light cycle studied at the equivalent of 12:00 midnight (peak of synthesis) to animals on a normal light cycle studied at 12:OO noon (nadir of synthesis). The incorporation of ‘H,O into fatty acids and cholesterol was increased 2.5 and 2.6-fold, respectively, in the livers of animals studied at midnight (maximal synthesis) as compared with noon (nadir of synthesis). (Fatty acids: midnight

DIET AND TNF-INDUCED

625

LIPOGENESIS

(n = 5) 4.48 -t .33 v midday (n = 5) 1.75 + .17, P < .OOl; cholesterol: midnight 3.52 + .63 v midday 1.43 + .20 rmole 3H,0 incorporated per hour per gram, P -C .02.) In our previous studies of the acute effects of TNF on hepatic lipid synthesis we only studied animals at the equivalent of midnight, the point in the diurnal cycle where hepatic lipid synthesis is maximal. The effect of TNF administration on serum lipid levels and hepatic lipid synthesis at the nadir of synthesis (noon) is shown in Table 1. As seen in our studies performed at midnight (peak of synthesis), we observed that TNF treatment acutely (1 to 2 hours after TNF administration) increases serum triglyceride levels (164%) without affecting serum cholesterol concentrations. Serum glucose concentrations were decreased in the TNF-treated animals as seen in our previous studies. Moreover, hepatic fatty acid synthesis was increased 65% in TNF-treated animals but hepatic cholesterol was not significantly altered acutely. These observations are similar to those previously reported in animals studied at midnight and indicate that the diurnal cycle of lipid synthesis does not alter the effects of TNF on lipid metabolism.

Effects of Variations in Diet High-sucrose diet. In prior studies, we determined the effect of TNF on lipid metabolism in animals fed standard rat chow ad libitum.” It is well recognized that changes in diet can effect serum lipid levels and hepatic lipid synthesis. Previous studies by other laboratories have demonstrated that hepatic fatty acid synthesis is regulated by the quantity of fat in the diet with increased dietary fat intake resulting in a decrease in hepatic fatty acid synthesis.20~27Additionally, high-sucrose diets have been shown to stimulate hepatic fatty acid synthesis and hepatic VLDL production.‘9,20.27 Table 2 presents data on serum lipid levels and hepatic lipid synthesis in animals fed a high-sucrose diet alone or a high-sucrose diet to which 30% corn oil was added (high-fat diet). The serum glucose and triglyceride levels in the fed state were similar on both diets, but the addition of corn oil significantly decreased the serum cholesterol levels, an effect previously described for corn oil and other polyunsaturated fats.‘9’2’The addition of corn oil to the diet also suppresses hepatic fatty acid synthesis (76%). Hepatic cholesterol synthesis is similar in both dietary conditions, but it should be noted that the rate of cholesterol synthesis is relatively suppressed compared with chow-fed animals. Previous stud-

ies by this and other laboratories have shown that dietary sucrose suppresses hepatic cholesterol synthesis.29,30 Thus, the high-sucrose diet represents an experimental model in which hepatic fatty acid synthesis is relatively stimulated, and the high-fat diet represents a dietary model in which hepatic fatty acid synthesis is relatively suppressed compared with the sucrose diet. In comparison to chow-fed animals, combined fat and sucrose feeding results in a stimulation of hepatic fatty acid synthesis and a decrease in hepatic cholesterol synthesis. (Fatty acids: chow-fed (n = 5) 3.5 + .41 v fat/sucrose-fed (n = 5) 6.3 + .43, P -C .Ol; cholesterol: chow-fed 1.69 + .23 v fat/sucrose-fed 0.67 + .07 pmol 3H20 incorporated per hour per gram, P -C .Ol.) The increase in hepatic fatty acid synthesis in the fat/sucrose-fed animals is most likely due to the large quantity of sucrose in the fat diet. Additionally, studies by Hill et al*’ and by our laboratory (unpublished observations) have demonstrated that small amounts of fat, similar to that present in rat chow, cause a maximal fat-induced suppression of hepatic fatty acid synthesis. Table 3 presents data on the acute (1 to 2 hours after TNF administration) effect of TNF on serum lipid levels and hepatic lipid synthesis in animals fed a high-sucrose diet. As in animals fed standard rat chow, TNF administration acutely increases serum triglyceride levels (192%) and stimulates hepatic fatty acid synthesis (93%). Neither serum cholesterol levels nor hepatic cholesterol synthesis are acutely affected by TNF administration, an observation similar to prior observations in chow-fed animals. As seen in our previous experiments, serum glucose levels are acutely decreased following TNF treatment. These observations indicate that TNF increases serum triglyceride levels and stimulates hepatic fatty acid synthesis to the same extent in rats fed a high-sucrose diet as in rats fed standard rat chow despite the increased baseline hepatic lipogenesis in the sucrose-fed rats. High-fat diet. Table 4 presents data on the acute effect of TNF on serum lipid levels and hepatic lipid synthesis in animals fed a high-fat diet (sucrose diet to which 30% corn oil is added). As seen with previous studies, TNF administration stimulates hepatic fatty acid synthesis (62%) in rats fed a high-fat diet. However, in contrast to previous observations, serum triglyceride levels were not increased by TNF treatment in animals fed a high-fat diet. As seen previously, serum cholesterol levels and hepatic cholesterol synthesis are

Table 1. Acute Effects of TNF in Animals Studied at the Nadir of Synthesis (12:OCl Noon)

Weight (g) Liver

GIWWE

t 2.9

9.7 & .26

192 * 13

+ 2.4

10.4 + .40 NS

Total Body

LipidSynthesis (vml/h/g)

Serum(mg/dL) Triglyceride

Cholesterol

Fatty Acids

Cholesterol

Saline (n = 5)

246

47 + 6

53 t 5

5.09

? .47

2.79

143 * 9

124 + 26

43 * 1

8.38

* .44

3.45

P < .02

P < ,025

NS

+ .15

TNF (n = 5)

253

NS

P < ,001

+ .30 NS

NOTE. Values are mean + SE. Animals were maintained on a normal 12-hour light cycle (8 AM to 8 PM light, 8 PMto 6 AM dark). At 11 AM of the day of the study. the animals were injected intravenously (IV) with TNF (25 rg/200

g) or saline. One hour later the animals were injected IP with 50 mCi of

tritiated water. After another hour, the animals were killed and the liver removed, weighed, and saponified in a KOH-ethanol solution. The incorporation of ‘H,O into fatty acids and cholesterol was determined after petroleum ether extraction as described in the Methods.

626

FEINGDLD ET AL

Table 2. The Effect of Corn Oil on Serum Lipids and Hepatic Lipid Synthesis

Total Body

LipidSynthesis (amol/h/g)

Serum(mg/dL)

Weight (a) Liver

GlUCOse

Triglyceride

Cholesterol

Fatty Acids

Cholesterol

High-sucrose diet (n = 5)

269 + 6

14.2 + 0.87

169 k 8

120 f 11

276 + 6

12.2 + 0.38

166 * 7

115 * 10

NS

NS

NS

NS

107 * 7

23.7

f 3.42

0.853

5.6 k 0.64

0.684

f 0.260

High-sucrose + corn oil diet (n = 5)

82 + 3 Pi

.Ol

PC .OOl

+ .043 NS

NOTE. Values are mean f SE. Animals maintained on a reverse 12-hour light cycle were fed either a high-sucrose diet or a high-sucrose diet plus corn oil (high-fat diet) for 4 days. Fatty acid and cholesterol synthesis was determined as described in the Methods.

not acutely affected by TNF administration. Similar to the results described above, serum glucose concentrations are acutely decreased in the TNF-treated animals. These results indicate that in animals fed a high-fat corn oil diet, TNF treatment stimulates hepatic fatty acid synthesis but this does not result in an increase in serum triglyceride levels. To determine if the effect of the high-fat diet was secondary to the type of fatty acid fed, we performed identical studies using a high-fat diet containing lard. As shown in Table 5, the results are similar to those described with a high-fat corn oil diet. Namely, hepatic fatty acid synthesis is acutely stimulated (39%) following TNF administration, but serum triglyceride levels are not increased. Additionally, TNF administration acutely decreases serum glucose levels as observed in our other experiments. Thus, these results indicate that the effect of a high-fat diet on the response to TNF administration is not specific to the type of fat ingested. It should be recognized that animals were allowed free access to food in the previous experiments until the administration of TNF. Therefore, it is likely that serum triglyceride levels are influenced by the continued absorption of dietary triglycerides. Since TNF decreases gastric emptying and slows small intestinal motility, *’ it is possible that treatment with TNF decreases fat absorption, with a resulting decreased contribution of dietary fat to serum triglyceride levels. To test this hypothesis, we determined the effect of TNF administration on fat absorption (Table 6). The quantity of labeled lipid localized to the stomach 2 hours after isotope administration is increased over threefold in the TNF-treated animals. In contrast, the amount of labeled lipid in the small intestine, liver, and carcass is decreased in TNF-treated animals as compared with the control animals. Also of note, is that 2 hours following TNF and isotope

administration the quantity of labeled lipid in the serum is 5.4-fold higher in controls compared with TNF-treated animals. These results demonstrate that TNF treatment decreases gastric emptying, which results in the decreased absorption of orally administered lipids. High-cholesterol diet. Numerous studies have shown that cholesterol ingestion suppresses hepatic cholesterol synthesis.3’,32 The acute effect of TNF on serum lipid levels and hepatic lipid synthesis in cholesterol-fed animals is shown in Table 7. As observed with other diets, TNF administration acutely increases serum triglyceride levels (130%) and hepatic fatty acid synthesis (57%) and decreases serum glucose concentrations (22%). Both serum cholesterol levels and hepatic cholesterol synthesis are not acutely affected by TNF administration. Thus, the acute effects of TNF on hepatic lipid synthesis are not altered by the ingestion of a high-cholesterol diet. Of greater significance are the effects of cholesterol ingestion on serum lipid and hepatic lipid synthesis 16 to 17 hours following TNF administration, the time period in which our previous studies have demonstrated that TNF administration increases serum cholesterol levels and stimulates hepatic cholesterol synthesis. As shown in Table 8, serum cholesterol and triglyceride concentrations are increased 93% and 38%, respectively, in TNF-treated animals fed a high-cholesterol diet. Cholesterol synthesis in the liver is markedly decreased in this experiment, most probably secondary to the combination of a high-cholesterol diet and fasting for 16 hours. Of particular note is that hepatic cholesterol synthesis is not stimulated 16 to 17 hours following TNF treatment. These results demonstrate that the increase in serum cholesterol levels can occur in the absence of stimulation of hepatic cholesterol synthesis when adequate dietary cholesterol is provided.

Table 3. Acute Effects of TNF in Animals Fed a High-Sucrose

Weight (8) Total Body

Diet LipidSyntheses lpmollhlgl

SerumImg/dL) Liver

GlUCOSe

Triglyceride

Cholesterol

Fatty Acids

Cholesterol

Saline (n = 5) TNF (n = 5)

271 +3

14.1 * .74

212 + 14

87 + 2

103 f 5

17.5 t 1.54

1.40 + 0.52

280 + 5 NS

14.4 + .59 NS

150 + 6 P< .Ol

254 + 32 PC .Ol

100 + 9 NS

33.7

1.17 i 0.29

* 0.70

P < ,001

NS

NOTE. Values are mean + SE. Animals maintained on a reverse 12-hour light cycle were fed a high-sucrose diet for 4 days. On the morning of the day of the study, the animals were injected IV with TNF (25 pg/200

g) and studied es described in Table 1 and Methods.

DIET AND TNF-INDUCED

627

LIPOGENESIS

Table 4. Acute Effects of TNF in Animals Fed a High-Fat Corn Oil Diet

Weight fg) Total Bodv

LipidSynthesis twnollhlgl

Serum(mg/dL) Liver

GhJCOSe

Triglvceride

Cholesterol

Fatty Acids

Cholesterol

Saline (n = 5)

217

+ 2

9.9 * .31

175 + 7

216 k 3

10.5 + .58

140 + 5

NS

NS

85 + 5

82 t 4

8.90

+ 0.74

0.610

82 i 3

14.44

+ 1.46

0.769

+ .056

TNI’ hl = 5)

P<

.Ol

92r

16 NS

NS

P<.Ol

2 ,094 NS

NOTE. Values are mean + SE. Animals maintained on a reverse 12-hour light cycle were fed a high-fat diet (30% cwn oil, 46% sucrose) for 4 days. On the morning of the day of the study, the animals were injected IV with TNF (25 pg/200

Efleci of Fasting Before TNF Administration

It is widely recognized that fasting leads to a decrease in hepatic fatty acid and cholesterol synthesis.20~3”-35In our laboratory, animals fasted for 24 hours showed a 29% decrease in fatty acid synthesis and a 56% decrease in cholesterol synthesis. (Fatty acids: fed (n = 5) 2.82 + .17 v fasted (n = 5) 2.02 + .28, P < .05; cholesterol: fed 2.21 t .I3 v fasted 0.97 + .06 pmol ‘H,O incorporated per hour per gram, P -C .OOl.) In addition, serum triglyceride levels are markedly decreased in the fasted animals (fed 62 + 5.0 v fasted 20 t 2.7 mg/dL, P < .OOl), whereas serum cholesterol concentrations are unchanged. Table 9 presents data on the acute effect of TNF administration on serum lipid levels and hepatic lipid synthesis in animals that had been fasted for 24 hours before TNF administration. No significant increase in serum triglyceride levels was seen in the TNF-treated animals. Moreover, hepatic fatty acid synthesis is similar in the control and TNF-treated animals. As in our previous short-term experiments, serum cholesterol concentrations and hepatic cholesterol synthesis are not acutely increased in TNF-treated animals. Also of note is that in contrast to our previous studies TNF administration did not produce a decrease in serum glucose levels. Thus, neither the typical elevation in serum triglyceride levels that occurs acutely following TNF administration nor the characteristic acute increase in hepatic fatty acid synthesis induced by TNF are observed in fasting animals. ‘H,O Labeling of Serum Fatty Acids

Figure 1 shows the quantity of 3H20 fatty acids in the serum of control and TNF-treated animals fed different diets or studied at different points in the diurnal cycle of hepatic lipid synthesis. In these studies, animals were administered

g) and studied as described in Table 1 and Methods.

‘H,O 1 hour after TNF treatment and serum was obtained 1 hour later (2 hours after TNF administration). The data on animals fed standard rat chow are taken from a previous publication by this laboratory and are shown here to facilitate comparisons.” In control animals, the quantity of labeled fatty acids in the serum is greatest in the animals fed a high-sucrose diet and follows a pattern that one would expect based on the literature, ie, high-sucrose greatest followed by chow-fed studied at midnight, chow-fed studied at midday, high-fat corn oil diet, high-fat lard diet, and fasted.‘9,20 (High sucrose 0.97 k .26, chow-fed midnight 0.48 k .08, chow-fed midday 0.31 + .04, high-fat corn oil 0.24 * .04, high-fat lard 0.205 i .03, fasted 0.097 it .005 pmol ‘H,O/mL serum.) Of greater importance is that with the exception of the fasted animals, TNF treatment uniformly results in a marked increase in the quantity of labeled fatty acids in the serum (chow-fed midnight increased 3.8-fold, chow-fed midday 4.1-fold, high-sucrose 8.2-fold, high-fat corn oil 2.4-fold, and high-fat lard 2.7-fold). The quantity of labeled cholesterol in the serum is similar in control and TNF-treated animals (data not shown). These results demonstrate that TNF treatment results in an increased quantity of newly synthesized fatty acids in the serum under every condition in which TNF stimulates hepatic lipogenesis. Effect of TNF on Small Intestinal Lipid Synthesis

In addition to the liver, the other major source of circulating lipids is the small intestine. In previous studies, we demonstrated that small intestinal fatty acid and cholesterol synthesis is unchanged 16 hours following TNF administration to chow-fed animals.” The acute effect of TNF administration on fatty acid and cholesterol synthesis in the small intestine is shown in Fig 2. Regardless of diet, fatty acid

Table 5. Acute Effects of TNF in Animals Fed a High-Fat Lard Diet LiprdSynthesis Weight fg)

bmollh/gl

Serum (mg/dL) GlUClXe

Triglyceride

Cholesterol

Fattv Acids

Cholestsml

Total Body

Liver

262 + 5

9.8 + .45

208 f 6

84 + 17

79 +I 7

2.77

c 0.28

0.217

265 k 4

10.1 f .31

174 + 7

104 * 15

63 r 6

3.86

f 0.40

0.356

NS

NS

NS

NS

Saline (n = 5)

k ,035

TtWF hl = 4)

Pi

.Ol

P<

.05

-r ,113 NS

NOTE. Values are mean + SE. Animals maintained on a reverse 1P-hour light cycle were fed a high-fat diet (30% lard, 46% sucrose) for 4 days. On the morning of the day of the study, the animals were injected IV with TNF (25 pg/200

g) and studied as described in Table 1 and Methods.

FEINGDLD ET AL

628

Table 6. The Effect of TNF Administration

on Lipid Absorption serum Total

Stomach

Small Intestine

Liver

CWCBS

per ml

(dcm x 1.000)

ldpm x 1,000)

(dpm x 1.0001

tdpm x 1,000)

(dpm x 1,000)

(dpm x 1,000)

Control (n = 5)

3,740

* 505

9,004

+ 360

863 f 55

+ 874

5,339

+ 1,228

281 + 40

792 * 264

11 *4

P < ,001

P < ,001

PC

4,056

57 i

? 201

17

17,702

+ 384

18,194

+ 786

TNF (n = 5)

Il.771 PC

,001

P<

.05

.05

NS

NOTE. Values are mean + SE. Animals were maintained on a reverse 12-hour light cycle and were fed standard rat chow. On the day of the study, the animals were injected IV with TNF (26 pg/200 oil intragastrically.

g) or saline. Immediately following this, the animals were administered 7.6 $i

or 3H triolein in 1 mL corn

Two hours later, the animals were killed and the tissues saponified in a KOH-ethanol solution. The labeled lipid present in each tissue

was quantitated as described in the Methods. The values given for the stomach, small intestine, liver, and carcass represent the total counts in the entire organ. The values given for the serum are on a per milliliter basis.

synthesis in the small intestine is similar in control and TNF-treated animals (Fig 2A). These results suggest that the increase in labeled fatty acids in the circulation following TNF treatment is due to increased hepatic fatty acid synthesis. As expected, fasting resulted in a decrease in small intestinal fatty acid synthesis. In chow-fed, sucrose-fed, and fat-fed animals, TNF administration did not significantly alter cholesterol synthesis in the small intestine (Fig 2B). In the fasted animals, small intestinal cholesterol synthesis is decreased and TNF administration resulted in a quantitatively small but statistically significant increase in cholesterol synthesis. DISCUSSION

Studies by this laboratory have demonstrated that the administration of TNF to intact rats results in an increase in both serum triglyceride and cholesterol levels.“’ Serum triglyceride levels increase within 2 hours following TNF treatment and remain elevated for at least 17 hours. Serum cholesterol levels also increase following TNF administration, but this response occurs after a considerable delay.” The rapid increase in serum triglyceride levels is accounted for by an increase in normal VLDL particles, whereas the late increase in serum cholesterol and triglyceride levels is due to an increased number of triglyceride enriched LDL particles (submitted for publication, R. Krauss, C. Grunfeld, and K.R. Feingold). In conjunction with this increase in circulating lipid levels we have observed that TNF administration stimulates both hepatic fatty acid and cholesterol synthesis.” Fatty acid synthesis in the liver is increased 1 to 2 hours following TNF administration, an effect that persists for an extended period

of time. Moreover, this acute increase in hepatic fatty acid synthesis is associated with an increased quantity of labeled fatty acids in the serum of TNF-treated animals.” In contrast, hepatic cholesterol synthesis is increased only 16 to 17 hours following TNF treatment. The TNF-induced increases in serum lipid levels and hepatic lipid synthesis were initially found in animals fed a rat chow diet ad libitum who were studied at the peak point in the diurnal cycle of lipid synthesis (12:00 midnight). It is well recognized that the diurnal rhythm and dietary manipulations can have marked effects on hepatic lipid synthesis.‘9,20~25*26 Whether such alternations would affect the ability of TNF to stimulate lipid synthesis in the liver and increase serum lipid concentrations was previously unknown. In the present study, we determined the effect of TNF administration on circulating lipid levels and hepatic lipid synthesis in animals under experimental conditions that would alter baseline levels of serum lipids and/or hepatic lipid synthesis. In animals studied at the nadir of the diurnal cycle of hepatic lipid synthesis (12:00 noon),25*26we observed that TNF administration rapidly increases serum triglyceride levels, stimulates hepatic fatty acid synthesis, and increases the quantity of newly synthesized fatty acids in the serum. Similarly, in animals ingesting a high-sucrose diet, which increases baseline hepatic fatty acid synthesis,‘9~20~27 TNF acutely increases serum triglyceride levels, stimulates hepatic fatty acid synthesis, and increases the quantity of newly synthesized fatty acids in the serum. Additionally, in animals fed a high-cholesterol diet, the characteristic rapid increase in serum triglyceride concentrations and hepatic fatty acid synthesis is also observed. As in our prior studies, in all three of these models TNF administration did not acutely alter

Table 7. Acute Effects of TNF in Animals Fed a High-Cholesterol

LipidSynthesis (pmollhlgj

Serum(mg/dLj

Weight (gj Total Body

Diet

Liver

GlUCOSe

Triglvceride

Cholesterol

Fatty Acids

Cholesterol

Saline (n = 5)

310 + 7.8

13.9 + .40

301 + 4.2

12.8 f .32

NS

NS

191 + 7

117 + 14

70 f 8

5.64

_t ,467

0.375

149 + 4

238

61 t4

8.88

+ ,585

0.426

* ,057

TNF hl = 5)

P<

,001

PC

r 39 .Ol

NS

PC

.Ol

* ,047 NS

NOTE. Values are mean ? SE. Animals maintained on a reverse 12-hour light cycle ware fed our standard rat chow to which 5% cholesterol was added for 4 days. On the morning of the day of the study, the animals were injected IV with TNF (25 pg/200 Methods.

gj and studied as described in Table 1 and

DIET AND TNF-INDUCED

LIPOGENESIS

629

Table 8. Chronic Effecta of TNF in Animals Fed a High-Cholesterol

Diet Lipid Synthesis

Weight (g)

Serum (mg/dL)

Liver

Total Body

GlUW~

Triglycsrids

(pmollhlg)

Cholesterol

Fatty Acids

Cholesterol

Saline In = 5)

210 f 4.3

7.1 k .23

122 + 8

210 + 2.1

a.0 + .21

119 * 4

42 k 3

54 k 6

1.83 f .120

,064

58 * 4

104 k 16

1.81 r ,180

,077

_+ ,005

TNF In = 5)

P<

NS

.Ol

NS

P-G

Pi

.Ol

.Ol

NS

‘- ,003 NS

NOTE. Values are mean i SE. Animals maintained on a reverse 12-hour light cycle were fed our standard rat chow to which 5% cholesterol was added for 4 days. The evening before study, the animals were injected IV with TNF (25 pg/200 morning (16 hours later), the animals were injected IP with 50 mCi of ‘Ii,0

either serum cholesterol levels or hepatic cholesterol synthesis. These findings indicate that the previously described acute effects of TNF on serum triglyceride levels and hepatic fatty acid synthesis can occur under a variety of diverse experimental conditions, suggesting that this response is a generalized phenomenon. In animals fed a high-fat diet, either corn oil or lard, which decreases hepatic fatty acid synthesis,20,27TNF administration acutely stimulates hepatic fatty acid synthesis and increases the quantity of newly synthesized fatty acids in the serum, but serum triglyceride levels do not change. In fed animals, serum triglyceride levels are a reflection of both hepatic VLDL production and intestinal chylomicron production. When animals are fed a high-fat diet (30% corn oil or 30% lard), it is likely that intestinally derived chylomicrons make a significant contribution to total serum triglyceride levels. In contrast, in animals fed standard rat chow, which has very little fat, or sucrose, which has no fat, the hepaticderived VLDL accounts for a major portion of circulating triglycerides. Unfortunately, in the rat it is difficult to precisely determine the origin of circulating lipoproteins, because, in contradistinction to most other species, rat liver is capable of synthesizing both apoprotein B-100 and apoprotein I3-48.36,37Therefore, the usual criteria for determining the source of origin of a triglyceride-rich lipoprotein (ie, apoprotein B- 100 = liver origin, apoprotein B-48 = intestinal origin) is not valid in rats. In the present study, we demonstrated that the absorption of orally administered lipid is inhibited by TNF treatment. The quantity of labeled lipid in the serum was decreased by more than 80% in TNF-treated animals 2 hours following oral isotope administration. Similarly, marked decreases in the amount of labeled lipid in the liver and carcass were also

g) or saline. After the injection the animals were fasted. The next

and fatty acid and cholesterol synthesis determined.

observed. In contrast, the quantity of labeled lipid remaining in the stomach was increased over threefold in the TNFtreated animals. This demonstrates that there is a decreased gastric emptying in TNF-treated animals, which results in a marked decrease in lipid absorption. Patton et al have previously shown that TNF decreases gastric emptying and motility in the proximal small intestine,22 and the present study suggests that decreased gastrointestinal motility may have physiological and metabolic implications. In animals fed the standard rat chow diet, which has a low fat content, or the sucrose diet, which has no fat, the contribution of the dietary fat to circulating lipid levels is small or absent and the gastrointestinal effects of TNF would not be expected to significantly effect serum lipid concentrations. In contrast, in animals fed a high-fat diet, a decrease in fat absorption following TNF administration could be anticipated to effect serum lipid levels. Thus, we hypothesize that in animals fed a high-fat diet, both hepatic and intestinally derived lipoproteins make a significant contribution to serum triglyceride levels. Following TNF administration, the hepatic contribution increases while the intestinal contribution decreases, resulting in only a small but not statistically significant increase in serum triglyceride levels. TNF had no significant effect on de novo fatty acid synthesis in the small intestine regardless of diet. Limiting nutrient intake results in a decrease in hepatic lipid synthesis.20,33-3s Thus, decreasing gastric emptying would be expected to decrease hepatic lipid synthesis. However, TNF administration increases hepatic lipid synthesis despite its effect on gastric emptying. In fact, the TNF-induced stimulation of hepatic lipid synthesis might be partially suppressed by the gastrointestinal effects of this cytokine. It should be recognized that we have found a single

Table 9. Acute Effects of TNF in Fasted Animals Lipid Synthesis Weight fgl Total Body

(f.tmol/h/g)

Serum (mg/dL) Liver

GlUCOS0

Triglyceride

Cholesterol

Fatty Acids

Cholesterol

Saline (n = 5)

233

+ 11.5

7.51

234 k 10.9

6.09

+ .41

134 + 6

16 f 3

63 + 6

3.06

+ .26

129 f 6

22 k 2

53 f 4

2.46

NS

NS

NS

+ .30

1.08 + ,095

+ .48

1.38 i ,134

TNF h-l = 5)

NS

NS

NS

NS

NOTE. Values are mean + SE. Animals maintained on a reverse 1P-hour light cycle were fed our standard rat chow. Twenty-four hours before study, the animals were denied access to chow. On the morning of the day of the study, the animals were injected IV with TNF (25 fig/200 described in Table 1 and Methods.

g) end studied as

630

FEINGOLD ET AL

0

control B3TNF

PEAK

NADIR SUCROSE

Cg;N

LARD

FASTED

Fig 1. Labeled fatty acids in the serum. Values are mean ? SE. Animals were injected intravenously with TNF (25 pg/2CKl g) or saline. One hour later, the animals were injected IP with 50 mCi of 3H,0. After 1 hour, blood was obtained and the serum separated by centrifugation. The quantity of ‘H fatty acids was determined after saponification and petroleum ether extraction. Peak = standard rat chow (reverse light cycle); Nadir = standard rat chow (normal light cycle): Sucrose = high-sucrose diet; corn oil = high-fat corn oil/sucrose diet; lard = high-fat lard/sucrose diet; Fasted = fasted for 24 hours before study. N = 5 for all groups, except the TNF-treated high-fat lard diet group, where N = 4. Open bars are the saline-treated animals and the hatched bars are the TNF-treated animals. In groups l-5, the TNF-treated group is statistically increased in comparison with the matched controls.

characteristic increase in serum triglyceride levels in response to TNF, providing further support for our hypothesis, discussed in detail at the beginning of this report, that the TNF-induced increase in circulating lipid levels is primarily due to an increase in hepatic lipid synthesis. In contrast to our observations in animals fed rat chow, which contains very little cholesterol, in animals fed a high-cholesterol diet, we observed that serum cholesterol

experimental situation in which the characteristic effects of TNF on lipid metabolism are not produced. In animals fasted for 24 hours before TNF treatment, there were no changes in either serum lipid levels, hepatic fatty acid synthesis, or the quantity of labeled fatty acids in the serum following TNF treatment. It is noteworthy that in fasted animals, the absence of an increase in hepatic fatty acid synthesis following TNF treatment is associated with the absence of the

e

A

8

40 : 0 30

I

I kig

L

0 f

f P o 20 5

$4 ;

10 0

CHOW

SUCROSE

FAT

FASTED

CHOW

S

:OSE

FAT

FASTED

Fig 2. Effect of TNF on lipid synthesis in the small intestine. Animals were injected intravenously with TNF (26 ~g/200 g) or saline. One hour later, the animals were injected IP with 50 mCi of ‘H,O. After 1 hour, the animals were killed and the small intestine removed, weighed, and saponified in a KOH-ethanol solution. The incorporation of tritieted weter into (Al fatty acids end (B) cholesterol was determined after petroleum ether extraction as described in the Methods. Values are mean + SE. Open bars are the saline-treated animels and the hatched bars are the TNF-treated animals. N = 5 for all groups. Chow refers to stenderd rat chow. sucrose to the high-sucrose diet, fat to the corn oil/sucrose diet, and the fasted group was without food for 24 hours before study.

DIET AND TNF-INDUCED

631

LIPOGENESIS

levels are increased 17 hours following TNF treatment but that hepatic cholesterol synthesis is unchanged. These data suggest that the stimulation of hepatic cholesterol synthesis induced by TNF administration is a secondary response and can be prevented by providing an exogenous source of cholesterol. On the other hand, the acute increase in hepatic fatty acid synthesis following TNF administration appears to be a primary response, because providing the liver with large quantities of exogenous fatty acids (high-fat diet) does not inhibit the characteristic increase in fatty acid synthesis induced by TNF. Last, in almost all the experimental circumstances studied, the acute administration of TNF results in a decrease in serum glucose levels. The only exception is when the animals are studied in the fasting state. As discussed above in regard to fat absorption, the physiological effects of TNF on gastrointestinal function can affect nutrient absorption. A possible mechanism for the TNF-induced decrease in serum glucose levels is reduced glucose absorption by the small intestine, which could explain the decrease in serum glucose

levels in fed animals and the absence of an effect in fasting animals. Alternatively, studies have demonstrated that TNF administration produces an increase in serum insulin levels, which could lower serum glucose leve1s3* There is no clear link between the effect of TNF on glucose and lipid metabolism, as TNF is also capable of increasing serum triglycerides and hepatic fatty acid synthesis in insulinopenic diabetic ratsI In summary, the present study demonstrates that TNF administration is capable of acutely stimulating hepatic fatty acid synthesis under many diverse dietary conditions, suggesting that there is a strong linkage between the immune system and lipid metabolism that may be of importance in the response to infection and/or inflammation.

ACKNOWLEDGMENT

We thank Dr J. Patton of Genentech, Inc and Dr Marvin D. Siperstein for their continued interest in our work. We thank Pam Herranz and Maggie Joe for the excellent editorial assistance.

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