Molecular and Cellular Biochemistry 112: 53-59, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Effects of tumour necrosis factor(cachectin) on glucose metabolism in the rat Intestinal absorption and isolated enterocyte metabolism Joan Arb6s, Francisco J. L6pez-Soriano, Neus Carb6 and Josep M. Argilds Departament de Bioqu[mica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071-Barcelona, Spain Received 16 October 1991, accepted 15 January 1992

Abstract Intravenous administration of a single dose (20/xg) of recombinant tumour necrosis factor-c~ (TNF, cachectin) to rats decreased the rate of intestinal glucose absorption. In vivo, the oxidation of [U-14C]glucose to 14C02 was significantly increased by the cytokine. In addition, [~4C]lipid accumulation from [U-14C]glucose was increased both in liver and brown adipose tissue of the TNF-injected animals. The decrease observed in intestinal glucose absorption was not associated with changes in intestinal metabolism. There was no difference in glucose metabolism by isolated enterocytes from either control or TNF-injected rats whether in the absence or presence of different concentrations of the cytokine in the incubation medium. In contrast, tumour necrosis factor altered the rate of gastric emptying as measured by the gastrointestinal distribution of 3[H]inulin following an intragastric glucose load. These results suggest that the cytokine profoundly alters glucose metabolism by increasing its whole-body oxidation rate and delaying intestinal absorption through a reduced gastric emptying. (Mol Cell Biochem 112" 53-59, 1992

Key words." tumour necrosis factor-a, glucose, intestine, rat

Introduction The cytokine tumour necrosis factor-a (TNF, cachectin), released by macrophages in response to invasive stimuli, has been shown to have a wide variety of effects on lipid and amino acid metabolism [7]. It has been demonstrated that TNF decreases the activity of lipoprotein lipase in adipose tissue in vivo [8, 30] and in cultured 3T3-L1 adipose cells [17, 27], where it also inhibits the synthesis and storage of fatty acids [23-25]. In addition, TNF stimulates hepatic lipid synthesis [9,

10], this fact having been postulated to be the main cause for the hypertriglyceridaemia that follows TNF administration [5]. Regarding protein and amino acid metabolism, TNF has been shown to increase liver uptake of amino acids in vivo [2, 4, 28, 36], although no effects were observed with isolated hepatocytes in the presence of the cytokine [36]. In addition, muscle proteolysis is enhanced by TNF [26] and synergistically augmented by the addition

Address for offprints." J.M. Argil6s, Departament de Bioqufmica i Fisiologia, Unitat de Bioqufmica i Biologia Molecular B, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071-Barcelona, Spain

54 of interleukin-1 [11]; however, despite these changes in vivo, recombinant TNF had no direct effect on muscle preparations in vitro [13] even when the animals were pre-treated with TNF in vivo [22]. The reported effects of TNF on glucose metabolism have been variable and not so clear cut. Recombinant TNF stimulated glucose uptake, lactate formation and ' glycogen breakdown in myotubes of the L6 muscle cell line, the increased uptake being dependent on the synthesis of new glucose transporters [19]. However, TNF did not stimulate glucose utilization by rat epitrochlearis muscle [21] or its conversion to lactate by hemidiaphragm muscle [29]. Similarly, glucose utilization was not increased by TNF in isolated spleen cells or alveolar macrophages [21] suggesting that its action in vivo may not be direct. TNF did increase glycolysis and its positive effector, fructose 2,6-bisphosphate, in cultured reumathoid synovial cells, although high concentrations were required [34]. Little is known of the effects of TNF in vivo on glucose metabolism. We have therefore studied the effects of acute TNF administration on the gastrointestinal absorption and utilization of dietary glucose in the rat.

Experimental Animals Female Wistar rats, weighing 150-180 g, were fed ad libitum on a chow diet (Panlab, Barcelona, Spain) consisting of (by wt.) 54% carbohydrate, 17% protein and 5% fat (the residue was non-digestible material; with free access to drinking water, and were maintained at an ambient temperature of 22_+ 2°C with a 12hlight/12 h-dark cycle (lights on from 08:00 h).

Biochemicals All enzymes and coenzymes were either purchased from Boehringer Mannheim S.A. (Barcelona, Spain) or Sigma Chemical Co. (St. Louis, U.S.A.) [U-14C]glucose ( s ~ c activity 270mCJmmol) and [3H]inulin (specific activity 4C~/mmol) were purchased from Amersham (Bucks, U.K.). Human recombinant tumour necrosis factor-ct was kindly given by BASF/Knoll AG (Ludwigshafen, Germany) and Dainippon Pharmaceuticals Co. (Osaka, Japan).

Measurement of glucose oxidation and tissue incorporation of the tracer At 1 h after the intravenous administration (tail vein) of a single dose of tumour necrosis factor-c~ (20/zg in 0.5 ml of 0.9% NaC1) or saline, the metabolic fate of an orally administered [U-14C]glucose load was examined. During this period they had free access to food and drink. About 4 mmoles (1/zCi) of [U-14C]glucose per rat was given enterally by gastric intubation, without anaesthetic but with minimal stress to the animal. Expired CO2 was then collected every 20 minutes for 2 h by absorption in Lumasorb (Lumac, Holland) and the rate of 14CO2 production was estimated by counting radioactivity in a sample of Lumasorb. After the collection period, the animals were killed. Arterial blood was collected in a heparinized syringe. The gastrointestinal tract (plus contents) was homogenized in 150 ml of 3% (w/v) HCIO4. Samples were taken of liver, perigenital adipose tissue, brown adipose tissue, skeletal muscle, brain and carcass. Carcass (minus liver and intestinal tract) was finely minced by means of an electric blender. Tissues were saponified and the lipid was extracted [33]. The extracted fatty acids were dissolved in 8 ml of liquid scintillation fluid and hence determination of p4C]lipid formation. [U-14C]glucoseabsorption was calculated by subtracting total gastrointestinal radioactivity from that administered.

Blood metabolites, plasma insulin and tissular glycogen content Whole-blood glucose was determined by the method of Slein (1963) and lactate by the method of Hohorst (1963). Plasma insulin was determined by radioimmunoassay with a rat insulin standard [1]. Liver and skeletal muscle glycogen content was assessed after ethanol precipitation [14] followed by hydrolysis and glucose estimation [31]. The incorporation of the tracer into tissue glycogen was estimated, after precipitation and hydrolysis of the polysaccharide, by means of hquid scintillation counting. It has to be pointed out that [14C]glucose incorporation into lipid or glycogen is not necessarely a measurement of total synthetic rate since it can be influenced by changes in blood flow or pool sizes.

55

Isolation and incubation of enterocytes It was carried out following a modification of the method by Wafford et al. (1979). After killing the animal by decapitation, the intestine was removed and flushed with calcium free Krebs-Henseleit solution [18]. After ligaIing one end of the small intestine, the lumen was filled with calcium-free Krebs-Henseleit solution containing 5 mM EDTA. The other end was ligated and the intestine shaken for about 20min at 37°C in a flask containing well-oxygenated calcium-free medium. After incubation the intestine was drained and then rinsed with a medium containing no EDTA to remove loose cells, EDTA and mucus. The intestine was refilled with Krebs-Henseleit solution containing calcium and 5 mM dithiothreitol and patted with the finger tips for 1 rain. After this operation, it was emptied and the cell suspension obtained. The viability of the cells was determined before and after the different incubations using the Trypan Blue exclusion method; any cell preparation that contained less than 85% viable cells, or where cell clumping was present, was excluded. In the different incubations, either 30raM [U14C]glucose or 2 mM [U-14C]lactate were used as substrates. TNF was added to the incubation medium at either 0, 25 or 50 nM concentrations. The cells were dispended in specially designed flasks that contained a centre well, in a total volume of Krebs-Henseleit bicarbonate buffer pH7.4 Flasks were sealed with rubber stoppers and incubated for 20 min in a thernaostaticallycontrolled bath (37 ° C) with a shaking device (50 cycles/ min). At the end of the incubation, hyamine hydroxide (0.2 ml) was added to the centre well and the reaction was stopped by the addition of 60% (w/v) perchloric acid solution (0.2 ml) to the reaction medium. The wells were counted for radioactivity in order to assess the amount of the substrate that was oxidised to ~4CO2 during the incubation time. The incubation medium was centrifuged for 10 rain at 3000 g, the pellet being suspended in chloroform/metanol (2:1) and hence lipid extraction. After the extraction, the samples were filtered using Whatman paper no. 1 and later mixed with liquid scintillation fluid before counting and thus estimating [14C]lipid synthesis. The filter paper was also placed in liquid scintillation vials that contained 8 ml of liquid scintillation fluid and its radioactivity estimated. This fraction represented the radioactivity retained in the cells which was not associated with the lipid fraction (hydrosoluble radioactive fraction, HRF). An aliquot of the incubation medium - after centrifugation - was

used for lactate estimation [16] in those incubations where glucose was used as substrate.

Estimation of gastric emptying To see if the striking effects of TNF on intestinal glucose absorption were related to altered gastric emptying, the distribution of [3H]inulin along the gastrointestinal tract was studied. The animals were either injected with saline or TNF and after l h they were given an intragastric load of glucose (4 mmoles containing a tracer amount of 3H-inulin (1/xCi). 2 h later, they were sacrificed and the gastrointestinal tract extracted. This was divided into stomach and the intestine (and contents). The intestine was cut into six equivalent fragments. These were mixed with 3% (w/v) perchloric acid and homogenized in a Waring Blender. After centrifugation, samples of the supernatants were used for total radioactivity estimation.

Results and discussion Absorption, oxidation and tissue incorporation of an oral [UJ4C]glucose load Treatment with TNF decreased by 32% the intestinal absorption of glucose, therefore all results concerning [U-14C]glucose handling have been expressed as % of absorbed dose (Table 1). Similar effects of TNF acute administration upon intestinal absorption were obtained following lipid [8] or amino acid [3] intragastric administration to rats. Administration of TNF increased oxidation of [U-'4C]glucose to 14CO2 by 36% over a 2 h period (Table 1). These results are in agreement with those of Mdsz~iros et al. (1988) who reported an increase of 24% in the turnover rate of plasma glucose following TNF infusion in the rat. Using the 2deoxyglucose tracer technique, M6szfiros et al. (1988) have shown that glucose utilization is increased in the macrophage-rich tissues rather than brain or muscle. No changes were observed in the [14C]incorporation of glucose into either liver or muscle glycogen (Table 1). TNF increased the incorporation of the tracer into liver lipid (5 fold). This result is in agreement with those of Feingold and Grunfeld (1987) which proposed that the cytokine stimulates hepatic lipogenesis in vivo by apparently raising the levels of citrate in the liver, this being followed by an increase in fiver acetyl-CoA car-

56

altered by TNF treatment. The cytokine tended to increase plasma insulin concentrations leading to a significant increase in the insulin/glucose ratio. These results are in contrast with a previous report showing that administration of high doses (600txg/kg body wt) of recombinant human TNF to rats resulted in hyperglycaemia and hyperlactaemia [35]. These reported changes could in part be due to the accompanying hypotension. The maintenance of the plasma insulin levels in the TNF-treated rats suggests that changes in the concentration of the hormone are not involved in the alterations in liver [14C]lipid incorporation. Although TNF treatment caused no alterations in the muscular glycogen content, it reduced the liver content by 50% despite the lack of effect on the incorporation of the tracer into the [14C]glycogen fraction. Rofe et al. (1987), using isolated hepatocytes from fed rats, reported that the presence of TNF (1.2 riM) in the incubation medium had no effect on glycogenolysis.

boxylase and fatty acid synthase activities [15]. In addition, TNF increased the [~4C]incorporation into brown adipose tissue lipid by nearly three fold (Table 1). This is the first report suggesting that TNF may possibly enhance lipogenesis in this tissue. In spite of this, the effects of the cytokine on brown adipose tissue have previously been studied by Coombes et al. (1987) showing that TNF stimulates brown adipose tissue thermogenic activity in young rats, this effect being partly responsible for the increases in body temperature and metabolic rate associated with TNF treatment.

Blood metabolites, plasma insulin and tissular glycogen content

TNF administration caused a tendency to hypoglycaemia although the difference was not statistically significant (Table 2). Blood lactate concentrations were not

Table 1. Effects of tumour necrosis factor-a on the absorption and metabolic fate of orally administered [U-~4C]glucose to rats. For full details see the Experimental section. 14CO2 production was calculated during the course of the 2 h after glucose administration. The results are mean values + S.E.M. for five different animals. Incorporation is expressed both as a percentage of absorbed dose per g or ml (a) and per total tissue (b). Total skeletal muscle [12], and brown adipose tissue [32] were calculated assuming previously reported values. Plasma volume was considered to represent 4% of body weight. TNF-treated values that are significantly different by Student's t test from their corresponding control values are indicated by: * p < 0 . 0 5 , * * p < 0 . 0 1 , ***p < 0 . 0 0 1 .

Measurement

Absorption (% administered dose) ~4CO2 production (% of absorbed dose) Incorporation into tissular [~4C]glycogen Liver Skeletal muscle Incorporation into tissular [14C]lipid Liver Skeletal muscle White adipose tissue Brown adipose tissue Carcass Plasma

Treatment Control

TNF-c~

76.7_+ 2.25

52.1 _+ 3.29**

34.1 _+ 1.69

46.4 _+ 4.53*

a b a b

0.247 _+ 0.046 1.89~)_+ 0.351 0.026 +_ 0.004 2.184_+ 0.336

0.179 _+ 0.028 1.326_+ 0.207 0.037 _+ 0.003 3.234_+ 0.262

a b a b a b a b a b a b

0.025 _+ 0.fV~2 0.191 + 0.013 0.004 -+ 0.001 0 . 3 3 6 + 0.113 0.100 _+ 0.040 1.700_+ 0.651 0.372 _+ 0.010 0.498 _+ 0.013 0 . 0 0 9 + 0.006 1.560 _+ 0.%3 0.017 _+ 0.003 0.138_+ 0.027

0.135 _+ 0.003*** 1.000+ 0.024*** 0.005 _+ 0.001 0.437_+ 0.065 0.09(I _+ 0.030 1.770_+ 0.630 1.030 _+ 0.025*** 1.340 _+ 0.032*** 0.011_+ 0.003 1.960 _+ 1.430 0.024 _+ 0.006 0.190_+ 0.044

57 Table 2. Effects of tumour necrosis factor-a on blood glucose and lactate and on tissular glycogen content. For full details see the Experimental section. The results are mean values + S.E.M. with the number of animals indicated in parentheses. For the calculation of insulin/glucose ratios, insulin was expressed as/xunitsdml of plasma. Values that are significantly different by Student's t test from control values are indicated by * p < 0.05. Measurement

Treatment Control

Blood metabolite 0xmolesJml) Glucose Lactate Insulin Insulin-glucose ratio Glycogen content (g/100g organ weight) Liver Skeletal muscle

7.20+ (4) 1.95+ (4) 31.0-+ (4) 4.31 + (4)

TNF~

0.23 0.18 4.62 1.34

3.86 -+ 0.90 (4) 0.42_+ 0.22 (5)

6.60_+ (9) 1.75± (9) 110 _+ (5) 16.5 _+ (5)

0.25

both glucose and lactate by isolated enterocytes from control and TNF-injected animals. The results (Table 3) show that neither the presence of 25 or 50 nM concentrations of the cytokine in the medium caused any significant changes in 14CO2formation, [14C]lipid synthesis or lactate released by the cells. Pre-treatment of the animals with TNF did not result in any changes, either (Table 3). The observed lack of effects of the cytokine on isolated enterocytes could therefore not explain the diminished absorption observed in vivo.

Effects of TNF on gastric emptying

0.13 31.1 4.47*

1.93 _+ 0.34* (11) 0.31+ 0.05 (11)

Effects of TNF on isolated enterocytes Bearing in mind the effect of the cytokine upon glucose absorption we decided to focus our study on the mechanism by which TNF caused this effect. For this reason, we examined the role of the cytokine in the utilization of

In order to find out the reason for the diminished gastrointestinal absorption of the tracer, we measured gastric emptying. Patton et al. (1987) had previously described that the cytokine inhibited gastric emptying. Using [3H]inulin, a non-metabolizable, non-absorbable compound, we determined the distribution of 3H-radioactivity in the gastrointestinal tract following an intragastric glucose load (4 mmoles) containing 1 txCi of the tracer. TNF treatment resulted in alterations in the distribution of the tracer (Fig. 1). The cytokine significantly increased the amount of radioactivity retained in the stomach of the animals (2 fold) (Fig. 1). It can thus be concluded that the action of the cytokine on intestinal glucose absorption is not based on changes in glucose uptake by the absorptive ceils, but rather by an altered gastrointestinal transit. A similar observation had been previously made following the effects of an acute administration of interleukin-1 upon [14C]lipid

Table 3. Effects of tumour necrosis factor-ct on the metabolism of [U-14C]glucoseand [UN4C]lactate by isolated enterocytes from control and TNF-injected rats. For full details see the Experimental section. The results are mean values + S.E.M. of six individual cell isolations expressed as txmoles of substrate transformed/h per g of dry weight. Lactate released into the medium is expressed as/xmoles/h per g of dry weight. [14C]HRF indicates the radioactivity remaining in the hydrosoluble fraction assessed as indicated in the Experimental section. Treatment

Substrate

Additions

14CO2 formation

[J4C]lipid synthesis

[14C]HRF

lactate released

Control

[U-14C]glucose

none TNF25nM TNF50nM none TNF 25 nM TNF 50nM none TNF 25riM TNF50nM none TNF25nM TNF50nM

16.8_+ 3.91 15.8 + 3.33 24.9_+ 5.68 5.28 _+ 2.46 4.08 + 1.77 4.16 _+ 0.90 18.3 _+ 2.89 14.1 + 2.15 15.0 + 3.87 6.00 + 0.85 5 . 3 9 ± 0.66 4.79__ 1.05

23.2_+ 34.7-+ 34.5+ 2.06-+ 1.93 + 2.72 _+ 25.0 ± 21.5 ± 27.1 + 2.00 + 2.06_+ 1.62_+

5.14_+ 0.60 7.02+ 1.21 6.97-+ 1.09 0.37 + 0.06 0.33 ± 0.08 0.31 _+ 0.03 6.23 + 2.04 5.54 _+ 1.28 7.01 ± 1.70 0.35 _+ 0.05 0,34+ 0.04 0.32_+ 0.04

21.1+ 19.1_+ 17.2-+ 13.3 _+ 12.0 ± 12.0± -

[UN4C]lactate

TNF-a

[U-14C]glucose

[UN4C]lactate

5.59 7.43 8.95 0.56 0.36 0.79 5.66 3.59 5.43 0.37 0.23 0.18

7.88 7.93 6.35

1.66 0.54 2.51

58

i00

0

I ]

CONTROL

......

TNF

70

50 Z:

T ik,

20

ST

I

12

13

14

15

16

Fig. 1. Effects of TNF-(~ on the distribution of [3H]inulin in the gastrointestinal tract following orally administered glucose. Deproteinized samples of stomach (ST) and intestine (I1-16) of control and TNF-injected rats, 2 h after the administration of the tracer were used. The intestine was divided into six equivalent segments and the amount of label retained calculated for each of them. The results are mean values + S.E.M. for five different animals in each group. For full details see the Experimental section.

absorption in the rat [2]. Profound gastrointestinal-tract disfunction, with even macroscopic haemorrhagic necrosis of the small bowel, has been reported in rats in response to high doses of TNF [35]. This effect may account for many of the metabolic derangements noted in previously reported studies, as recorded food intake or even gastric gavage may be an unreliable index of metabolic substrate availability to the animal in vivo. In our experiments (0.1 mg of TNF/kg body wt), the bowel appeared macroscopically normal and no rats died.

2.

3.

4.

5.

Acknowledgements 6.

This work was supported by grant 0663/90 from the Fondo de Investigaciones Sanitarias de la Seguridad Social (F.I.S.S.S.) from the Ministry of Health, Spanish Government. We thank BASF/Knoll AG. (Ludwigshafen, Germany) and Dainippon Pharmaceutical Co. (Osaka, Japan) for a generous sample of human recombinant TNF-ct.

7.

8.

9.

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