Clofibrate improves glucose tolerance in fat-fed rats but decreases hepatic glucose consumption capacity

Journal of Hepatology 37 (2002) 425–431 www.elsevier.com/locate/jhep

Clofibrate improves glucose tolerance in fat-fed rats but decreases hepatic glucose consumption capacity Lori A. Gustafson 1, Folkert Kuipers 2, Coen Wiegman 2, Hans P. Sauerwein 3, Johannes A. Romijn 4, Alfred J. Meijer 1,* 1

Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands 2 Center for Liver, Digestive and Metabolic Diseases, University Hospital of Groningen, Groningen, The Netherlands 3 Metabolism Unit, Department of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 4 Department of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands

Background/Aims: High-fat (HF) diets cause glucose intolerance. Fibrates improve glucose tolerance. We have tried to obtain information on possible hepatic mechanisms contributing to this effect. Methods: Rats were fed a HF diet, isocaloric with the control diet, for 3 weeks without or with clofibrate. Several parameters related to liver glucose and glycogen metabolism were measured. Results: Clofibrate prevented the induction of glucose intolerance by 3 weeks HF feeding. Improved glucose tolerance by clofibrate was not due to increases in glucose phosphorylation or glycolysis in the liver, since both the HF diet and clofibrate suppressed glucokinase and pyruvate kinase activities with no effect on glucose 6-phosphatase. Clofibrate decreased glycogen storage in both control and HF rats. Clofibrate, with and without HF feeding, inhibited weight gain during the experimental period. Body temperature was significantly elevated by clofibrate, indicative of an increased basal metabolic rate. The capacity of liver mitochondria to oxidize long-chain fatty acids increased by clofibrate treatment. Mitochondria did not show uncoupling. Conclusions: Clofibrate does not improve glucose tolerance by improving hepatic glucose or glycogen metabolism. Peripheral glucose oxidation may be facilitated by increased energy dissipation. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Glycogen; Glucose; Clofibrate; High-fat diet; Insulin

1. Introduction Type 2 diabetes mellitus (DM2) is characterized by glucose intolerance, hyperinsulinemia and dyslipidemia [1–3]. Although the pathogenesis of DM2 is incompletely understood, evidence is accumulating which supports the notion that DM2 results from abnormalities in fat metabolism rather than in carbohydrate metabolism [2,4,5]. This point of view is not new; Randle et al. [6] proposed almost 40 years ago that substrate competition in muscle could be the cause of glucose intolerance, i.e. elevated free fatty acid Received 22 August 2001; received in revised form 10 June 2002; accepted 11 June 2002 * Corresponding author. Tel.: 131-20-566-5154; fax: 131-20-691-5519. E-mail address: [email protected] (A.J. Meijer). Abbreviations: AUC, area under the curve; DM2, type 2 diabetes mellitus; HF, high fat; G6P, glucose 6-phosphate

concentrations result in enhanced fatty acid oxidation at the expense of carbohydrate oxidation. Fibrates have been used successfully to treat hyperlipidemia [7,8], also in patients with DM2 [9]. The action of fibrates on lipid metabolism is mediated by the activation of peroxisome proliferator-activated receptor a (PPARa) [7,10,11]. Since fibrate treatment stimulates hepatic fatty acid oxidation [12] without stimulating skeletal muscle fatty acid oxidation [13], the Randle hypothesis would predict that an increase in hepatic fatty acid oxidation improves peripheral glucose tolerance. Indeed, in man, treatment with fibrates has been shown to increase glucose tolerance and insulin sensitivity in patients with DM2 [14,15]. Recent research indicates that PPARa activation also improves insulin sensitivity in two different animal models of insulin resistance, obese fa/fa Zucker rats and high fat (HF)-fed C57BL/6 mice [16].

0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S 0168-827 8(02)00212-X

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It was the aim of this study to examine how the PPARa activator clofibrate affects liver glucose utilizing enzymes in chow-fed and HF-fed rats. 2. Experimental procedures 2.1. Materials A standard chow (60% of weight from carbohydrate, 22% from protein and 8% from fat) and an isocaloric HF chow (25% of weight from carbohydrate, 32% from protein and 25% from fat) were obtained from Hope Farms (Woerden, The Netherlands). Clofibrate was obtained from Aldrich (Steinheim, Germany), dissolved in ethanol and then mixed thoroughly through the ground chow. The ethanol was allowed to evaporate for a period of 3 days under a ventilated hood.

from glucose at 278C was coupled to its oxidation by glucose 6-phosphate dehydrogenase (Leuconostoc mesenteriodes, NAD 1 dependent) and NAD 1. Pyruvate kinase activity was also determined in the same postmicrosomal supernatant using a spectrophotometric assay similar to that described in Ref. [17], which contained 100 mM triethanolamine (pH 7.6), 13 mM KCl, 3 mM MgSO4, 0.5 mM phosphoenolpyruvate, 5 mM ADP, 0.2 mM NADH and 20 U lactate dehydrogenase, and whereby 10 mM fructose 1,6-bisphosphate (for full activation of the enzyme) was also present. Glucose 6-phosphatase activity was determined from tissue samples sonicated in buffer containing 250 mM sucrose and 10 mM Na–HEPES (pH 7.4, buffer A) based on the production of Pi from 40 mM glucose 6-phosphate, as described in Ref. [17]. AcylCoA oxidase activity was determined as described by Vamecq [23] in tissue samples sonicated in buffer A in an assay medium containing 0.1 mM palmitoylCoA, 100 mM Na–Mops (pH 7.6), 1 mM homovanillate, 0.05 mg/ml horseradish peroxidase type II, 5 mM FAD, 0.12 mg/ml bovine serum albumin and 0.06% Triton X-100.

2.4. Mitochondrial determinations 2.2. Animals Animal treatment was in compliance with the guidelines of the animal care and use committee of the Academic Medical Center, Amsterdam, The Netherlands. Male Wistar rats weighing 240–280 g were housed in pairs and given ad libitum access for a 3-week period to four different isocaloric diets: standard chow, HF chow, standard chow combined with 1% (w/w) clofibrate and HF chow combined with 1% clofibrate. Body weight and food intake were measured daily at 10.00 a.m. After 21 days on the diet, the animals were anesthetized between 9 a.m. and 11 a.m. with 0.5 ml nembutal (30 mg sodium pentobarbital), whereupon rectal temperature was measured within 5 min of full anesthesia using a thermoprobe. A laparotomy was then performed and 1 ml of blood was drawn from the portal vein for the measurement of glucose, insulin and glucagon. Immediately thereafter, a lobe from the liver was removed and placed quickly in liquid nitrogen.

2.3. Liver determinations All determinations were performed with tissue samples that had been ground in liquid nitrogen and briefly sonicated in the appropriate buffer (see subsequently) to ensure homogeneity. Glycogen content was measured in the tissue sample (100 mg) by extracting it with 1 ml 0.1 M KOH for 45 min at 958C to destroy residual glucose. The samples were brought to pH 4.5 by the addition of 3 M acetic acid, incubated at 408C for 2 h with amyloglucosidase to degrade glycogen to glucose and then a standard spectrophotometric glucose assay was performed at pH 7.4 (with hexokinase, glucose 6-phosphate dehydrogenase, NADP 1 and ATP) [17]. The glycogen metabolizing enzymes, glycogen synthase (GS) and glycogen phosphorylase (Pa), were determined from tissue samples dissolved with buffer containing 50 mM glycylglycine, pH 7.0, 75 mM NaF, 3 mM EDTA, 0.5% glycogen and 0.1% Triton X-100. GS activity was measured with UDP-[ 14C]glucose and glycogen (pH 7.2) for 20 min at 378C as previously described by Lavoinne et al. [18], in the presence of 6 mM glucose 6phosphate (for GSa 1 b) or 7.5 mM Na2SO4 (for GSa). Pa activity was measured with [ 14C]glucose-1-phosphate and glycogen (pH 6.8) for 20 min at 378C as previously described by Hue et al. [19], in the presence of caffeine for Pa or AMP and 1,2-dimethoxyethane for Pa 1 b [20]. Plasma glucose was determined with the hexokinase method (see above) in HClO4 deproteinized and neutralized plasma samples. Insulin and glucagon were determined as described in Ref. [21]. Plasma free fatty acid concentration (abdominal aorta) was determined with the NEFAC kit (Wako Chemical Gmbh, Neuss, Germany), using acylCoA synthetase and ascorbate oxidase. Glucokinase activity was determined from tissue samples sonicated in buffer containing 50 mM Na–HEPES (pH 7.4), 100 mM KCl, 1 mM EDTA, 5 mM MgCl2 and 2.5 mM dithioerythritol. Homogenates were centrifuged at 100,000 £ g for 25 min and the post-microsomal supernatant was used for the spectrophotometric continuous assay as described by Davidson and Arion [22], whereby the formation of glucose 6-phosphate

Liver mitochondria were prepared by differential centrifugation using 250 mM mannitol, 5 mM Na–HEPES (pH 7.0) and 0.5 mM EGTA as the isolation medium [24]. Mitochondria (2.5–3 mg protein) were incubated in a medium (2.5 ml) containing 15 mM KCl, 50 mM Tris–HCl (pH 7.4), 2 mM EDTA, 5 mM MgCl2, 10 mM KPi and either 7 mM K-succinate, 7 mM K-glutamate, 0.2 mM oleate plus 1 mM carnitine plus 0.65% (w/v) fatty acid-free serum albumin, or 0.2 mM palmitate plus 1 mM carnitine plus 0.65% (w/v) fatty acid-free serum albumin. State 3 respiration was initiated by the addition of an excess of ADP (1–2 mM). Oxygen consumption was measured at 338C with a Clarke-type electrode. Mitochondrial protein was determined according to the method of Lowry et al. [25].

2.5. Intravenous glucose tolerance test Intravenous glucose tolerance tests (IVGTT) were performed in fasted rats as described in Ref. [26]. Briefly, at t ¼ 0, an intravenous glucose infusion (5 mg/min for 20 min) was started and blood samples were withdrawn at t ¼ 1, 3, 5, 10, 15 and 20 min. The infusion was terminated after sampling at t ¼ 20 min, and additional blood samples were taken at t ¼ 25, 30, 35 and 40 min. All of the IVGTTs were performed between 9.00 and 11.00 h, i.e. in the light period.

2.6. Analysis of the data Values are expressed, where appropriate, as per gram wet mass of liver and as means ^ standard errors. Statistical differences between the experimental groups were analyzed with the Kruskal–Wallis non-parametric ANOVA, with multiple comparisons of groups [27]. Differences were considered to be significant if P , 0:05.

3. Results 3.1. Food intake Food intake was not significantly different among the four groups (22 ^ 1, 23 ^ 1, 22 ^ 1 and 21 ^ 2 g/day, for the standard, HF, 1% clofibrate and HF 1 1% clofibrate, respectively; n ¼ 4 in each group), in agreement with existing literature [28,29]. 3.2. Weight gain A 3-week feeding period of the HF diet did not cause excessive weight gain when compared to the chow-fed group (Fig. 1). Treatment with clofibrate, with or without

L.A. Gustafson et al. / Journal of Hepatology 37 (2002) 425–431

Fig. 1. Weight gain (g) during the 3 week diet for the standard chow fed (V, n ¼ 7), HF fed (B, n ¼ 7), the standard chow fed 1 clofibrate (O, n ¼ 4) and HF fed 1 clofibrate (X, n ¼ 6) diet groups. *P , 0.05 vs. standard chow, #P , 0.05 vs. HF diet.

the HF diet, however, significantly ðP , 0:05Þ decreased the amount of weight gained, as early as after 1 week of treatment, and continuing for the entire 3-week treatment period. 3.3. Blood parameters Post-prandial portal glucose concentration was 12.5 ^ 0.3 mM for the chow-fed group (Table 1). This value was not significantly altered by either the HF diet or by clofibrate treatment. The portal insulin and glucagon concentrations were also not affected by any of the diets, with the exception that in the HF 1 clofibrate group glucagon was significantly lower than in the chow-fed group. 3.4. Glucose tolerance and insulin response Blood glucose concentrations during the IVGTT were significantly elevated by the HF diet (Table 2). The area under the curve was increased by 63% by the HF diet, whereas the addition of clofibrate to the diet decreased the area under the curve to such a degree that the difference with the chow-fed group was no longer significant. The insulin response to the intravenous glucose was significantly suppressed by HF feeding, when compared to the control group, irrespective of the absence or presence of clofibrate. 3.5. Glucose- and glycogen-metabolizing enzymes When compared to the chow-fed animals, the HF diet, as well as clofibrate treatment, had similar results on the

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glucose- and glycogen-metabolizing enzymes. Activity of the glucose-phosphorylating enzyme, glucokinase, was reduced by 43% by the HF diet while clofibrate and HF 1 clofibrate reduced glucokinase activity by 53 and 69%, respectively (Table 3). The activity of the glucose-producing enzyme, glucose 6-phosphatase, was, however, unchanged by any of the treatments. The glycolytic enzyme pyruvate kinase was severely reduced by all three treatments: HF reduced pyruvate kinase by 61% while clofibrate and HF 1 clofibrate reduced pyruvate kinase activity by 81 and 91%, respectively. Post-prandial liver glycogen content was reduced by all three treatments (Table 4). The glycogen content in the chow-fed rat was 288 ^ 27 mmol/g wet mass, which was decreased by 41% by HF feeding, 68% by clofibrate treatment alone and 59% by HF 1 clofibrate. Concomitant with the lower glycogen contents, the active form of phosphorylase (Pa), as well as the total amounts of glycogen synthase (GSa 1 b) and phosphorylase (Pa 1 b) were significantly reduced by clofibrate treatment, with or without the HF diet. GSa showed a tendency to decrease at the lower glycogen levels, but this did not reach significance. Intracellular glucose 6-phosphate was not significantly different for any of the four groups. 3.6. Fatty acid oxidation The activity of peroxisomal acylCoA oxidase in liver homogenates was increased 8-fold by treatment with clofibrate as expected, and 13-fold by HF 1 clofibrate, whereas the HF diet alone had no effect (data not shown). In freshly isolated liver mitochondria, carnitine-dependent state 3 respiration in the presence of either oleate or palmitate was greatly elevated in the mitochondria obtained from clofibrate-treated livers whereas HF feeding alone only slightly increased state 3 respiration with these fatty acids (Table 5). As controls, we also studied the oxidation of glutamate and of succinate. State 3 respiration with glutamate as substrate was not significantly influenced by clofibrate nor the HF diet. Succinate oxidation slightly decreased by HF feeding in the absence, but not in the presence, of clofibrate. Irrespective of the substrates used, state 4 respiration (ADP absent) was not affected by any of the dietary interventions (not shown). This indicates that the coupling between liver mitochondrial oxidation and ATP synthesis was not affected.

Table 1 Non-fasting plasma glucose, insulin and glucagon in the portal vein a Diet

n

Glucose (mM)

Insulin (pmol/l)

Glucagon (ng/l)

Chow Chow 1 clofibrate HF HF 1 clofibrate

4 4 4 7

12.5 ^ 0.3 11.1 ^ 1.0 11.2 ^ 0.6 11.3 ^ 0.4

533 ^ 151 243 ^ 120 295 ^ 79 334 ^ 59

68 ^ 14 48 ^ 14 87 ^ 32 31 ^ 4* #

a

Data are means ^ SE for which the number of animals (n) in each experimental group is as indicated. *P , 0:05 vs. standard chow; #P , 0:05 vs. HF diet.

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Table 2 Glucose tolerance test a Diet

n

Chow Chow 1 clofibrate HF HF 1 clofibrate a

7 4 7 7

AUC Insulin

Glucose

99 ^ 9 71 ^ 20 70 ^ 8* 55 ^ 14*

199 ^ 46 295 ^ 99 325 ^ 64* 275 ^ 126

Data are means ^ SE. *P , 0:05 vs. standard chow.

3.7. Plasma free fatty acid concentration Plasma free fatty acid concentrations were significantly reduced by clofibrate in rats fed the standard diet (0.20 ^ 0.01 mM, standard diet; 0.10 ^ 0.02 mM, 1 clofibrate; P , 0:05) but not in the rats fed the HF diet (0.41 ^ 0.01 mM, HF diet; 0.38 ^ 0.02 mM, HF 1 clofibrate) (n ¼ 4 in each group; data not shown). 3.8. Body temperature In order to examine the effect of a HF diet or clofibrate, and thus of enhancement of hepatic mitochondrial and peroxisomal fatty acid oxidation (reviewed in Ref. [11]), on basal metabolism, core body temperature was measured shortly after the induction of anesthesia. The HF-fed animals as well as the clofibrate-treated animals exhibited a significant elevation in body temperature, from 36.0 ^ 0.18C for the control group to 36.7 ^ 0.18C for the clofibrate group and to 36.8 ^ 0.18C for the HF group (Fig. 2). The group fed the combination of a HF diet plus clofibrate exhibited the greatest elevation in core body temperature: 37.3 ^ 0.18C.

4. Discussion The primary objective of this study was to examine the hepatic mechanisms possibly involved in the improvement of glucose intolerance by PPARa activation in rats. The results confirm that 3 weeks of HF feeding induced glucose intolerance, which was not present in rats on clofibrate. Analysis of different parameters of hepatic glucose metaboTable 3 Glucose metabolizing enzymes a Diet

n

Glucokinase (mmol/min/g)

G6Pase

Pyruvate kinase

Chow Chow 1 clofibrate HF HF 1 clofibrate

7 4 7 6

2.47 ^ 0.23 1.15 ^ 0.10* 1.40 ^ 0.05* 0.77 ^ 0.10* #

16.5 ^ 0.6 16.5 ^ 1.7 15.1 ^ 0.5 17.7 ^ 1.2

96 ^ 10 18 ^ 2* 38 ^ 3* 9 ^ 1* #

a

Data are means ^ SE. *P , 0:05 vs. standard chow; #P , 0:05 vs. HF diet.

lism and of enzymes involved in hepatic glucose metabolism did not give any indication of increased hepatic glucose consumption upon clofibrate treatment. Clearly, clofibrate treatment must have resulted in increased extrahepatic glucose consumption. It may be argued that the anesthesia and surgical stress may have influenced our data. Although this can never be excluded it must be stressed that the experimental procedures were identical for each of the four groups. Competition between fatty acids and glucose for oxidation in muscle as an underlying mechanism for disturbed glucose metabolism in DM2, as proposed in the Randle hypothesis [6], would predict an improvement of peripheral glucose tolerance by PPARa activation. This is because PPARa is predominantly expressed in the liver and less so in muscle [7]. Increased fatty acid oxidation in the liver would drain fatty acids from the circulation, thereby relieving glucose oxidation in the muscle from competition with fatty acids for oxidation. Indeed, variation in plasma free fatty acid concentration directly influences skeletal muscle glucose consumption [30,31] and fibrate treatment is known to decrease plasma triglyceride and free fatty acid concentrations [7,32]. In our experiments we also observed a significant reduction in plasma free fatty acid concentration by clofibrate in rats fed the standard diet, but not in the rats fed the HF diet, presumably because an increase in lipoprotein lipase [7,32] can compensate for increased hepatic fatty acid oxidation under these conditions. Thus, a decreased competition between free fatty acids and glucose for oxidation is unlikely to explain the increased glucose tolerance by clofibrate feeding to HF-fed rats. In our model of mild glucose intolerance induced by HF feeding in the rat, clofibrate significantly improved glucose intolerance. The area under the insulin curve during an intravenous glucose tolerance test slightly decreased by HF feeding. Clofibrate did not significantly affect the area under the insulin curve, whether added to the standard diet or to the HF diet (Table 2). Taken together, these data suggest a slightly increased insulin sensitivity, in agreement with existing literature (cf. Section 1). Glucokinase and glucose 6-phosphatase play important roles in the regulation of hepatic glucose output and dysregulation of the glucose/glucose 6-phosphate cycle may contribute to increased hepatic glucose production in DM2 [33–35]. In our study the HF diet decreased the capacity of hepatic glucokinase (cf. Refs. [36,37]) while having no effect on glucose 6-phosphatase. The lack of effect on glucose 6phosphatase is in agreement with Oakes et al. [36]. Minassian et al. [37], on the other hand, reported a decline in glucose 6phosphatase, although the glucokinase/glucose 6-phosphatase ratio also decreased upon HF feeding in their studies. The decrease in both glucokinase and pyruvate kinase (by 40 and 60%, respectively; Table 3) after HF feeding suggests a decline in glycolysis. This agrees with the notion that the glycolytic pathway can be up- or downregulated, depending upon glucose exposure [38].

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Table 4 Glycogen metabolism a Diet

n Glucose 6-phosphate Glycogen (mmol/g) content

Chow Chow 1 clofibrate HF HF 1 clofibrate

7 4 7 6

a

0.54 ^ 0.08 0.45 ^ 0.11 0.40 ^ 0.04 0.51 ^ 0.08

288 ^ 27 92 ^ 15* 171 ^ 15* 118 ^ 3* #

Glycogen synthase (a) Glycogen synthase (mmol/min/g) (a 1 b)

Glycogen phosphorylase (a)

Glycogen phosphorylase (a 1 b)

0.30 ^ 0.03 0.23 ^ 0.04 0.26 ^ 0.03 0.19 ^ 0.01

32.4 ^ 5.6 9.2 ^ 1.2* 28.7 ^ 3.3 6.4 ^ 1.1* #

48.4 ^ 6 11.2 ^ 1* 38.2 ^ 3 9.7 ^ 1* #

2.2 ^ 0.1 0.9 ^ 0.1* 1.9 ^ 0.1 0.8 ^ 0.1* #

Data are means ^ SE. *P , 0:05 vs. standard chow; #P , 0:05 vs. HF diet.

Independent of the fat content of the diet, clofibrate treatment strongly reduced glucokinase and pyruvate kinase activities (Table 3). By contrast, glucose 6-phosphatase activity was not affected by clofibrate (Table 3). The changes in the glucose 6-phosphate producing and utilizing enzymes were such that the intrahepatic glucose 6-phosphate levels remained similar for all treatment groups (Table 4). This supports the notion that the concentration of glucose 6-phosphate is strongly buffered [39,40]. The reduction of the glycolytic enzymes was not related to a lower portal glucose concentration nor was there a clear relationship with portal vein insulin and glucagon concentrations (Table 1). The combination of HF and clofibrate resulted in the greatest decrease in glucokinase and pyruvate kinase activities, which were significantly lower than in the livers of clofibrate-treated control rats. This observation lends support to the postulation that the decrease in glycolytic flux is related to enhanced fatty acid oxidation since the combination of HF plus clofibrate clearly showed the highest peroxisomal acylCoA oxidase activity and rate of mitochondrial b-oxidation (Table 5). The glycogen content of the livers paralleled glucokinase and pyruvate kinase activities but not the activity of glycogen synthase and phosphorylase: the activity of the latter two enzymes was unaffected by the HF diet but greatly decreased after clofibrate feeding (Table 4). In general, the influence of isocaloric HF feeding or clofibrate treatment on the glucose- and glycogen-metabolizing enzymes suggests a situation in which hepatic fatty acid oxidation is enhanced and hepatic glucose consumption is diminished. These results are in clear contrast with those found in obese Zucker rats. This leptin receptor-deficient

model of diabetes through overfeeding exhibits very high levels of glucokinase activity and glycolytic flux [41], along with extremely high glycogen levels [42]. Isocalorically HF-fed rats with glucose intolerance, however, have completely opposite characteristics. This emphasizes that caution should be used when comparing various models of diabetes. An important observation is that clofibrate treatment as well as HF feeding which, like clofibrate, activates PPARa [43], increased body temperature significantly. Clofibrate has been reported to be thyromimetic [28] and PPARa activators, in general, have been shown to induce malic enzyme and other enzymes which are classically considered to be thyroid hormone-dependent in their expression [44]. Indeed, fasted PPARa-null mice show hypothermia and a lower metabolic rate when compared to fasted wild-type mice [45], whereas treatment of rats with fenofibrate increased the resting metabolic rate [46]. Since state 4 respiration of liver mitochondria was not affected by clofibrate treatment (not shown), in agreement with similar observations with fenofibrate [46], increased energy expenditure must have been extrahepatic of origin. A possibility is that clofibrate, like the PPARa-activator WY 14,643 [47], induces expression of UCP3, a gene encoding a mitochondrial uncoupling protein that is predominantly expressed in muscle. Indeed, it has recently been shown that over-expression of the mitochondrial uncoupling protein in skeletal muscle prevents diet-induced obesity and insulin resistance in mice [48]. In summary, this study sought to clarify hepatic mechanisms whereby clofibrate improves glucose intolerance induced by HF feeding. The reductions in hepatic glucokinase, pyruvate kinase and glycogen storage, combined with

Table 5 Mitochondrial respiration a Diet

n

Succinate Glutamate (natom O/min/mg mitochondrial protein)

Oleate 1 carnitine

Palmitate 1 carnitine

Chow Chow 1 clofibrate HF HF 1 clofibrate

4 3 3 3

148.5 ^ 13.4 140.3 ^ 18.0 105.0 ^ 13.2* 144.9 ^ 4.3

52.6 ^ 6.2 147.6 ^ 14.1* 79.9 ^ 8.9* 202.0 ^ 0.6* #

41.2 ^ 6.8 103.9 ^ 3.0* 72.1 ^ 8.7* 119.4 ^ 17.1* #

a

88.0 ^ 11.2 99.5 ^ 14.6 82.1 ^ 12.5 108.0 ^ 5.4

Data are means ^ SE. *P , 0:05 vs. standard chow; #P , 0:05 vs. HF diet.

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[10]

[11]

[12] [13] Fig. 2. Diet effects on core body temperature of anesthetized rats. Chow, standard chow fed (n ¼ 7); clo, standard chow fed 1 clofibrate (n ¼ 4); HF, HF fed (n ¼ 7); HF 1 clo, HF fed 1 clofibrate (n ¼ 6). *P , 0.05 vs. standard chow, #P , 0.05 vs. HF diet, $P , 0.05 vs. clofibrate.

[14]

[15]

the lack of effect of glucose-stimulated insulin secretion observed after clofibrate treatment, negatively rather than positively affect whole-body glucose tolerance, and are similar to the effects of HF feeding. A possible explanation for the ability of clofibrate to increase glucose tolerance induced by a HF diet would have been that an increase in hepatic mitochondrial fatty acid oxidation drains free fatty acids from the circulation and decreases the competition between glucose and fatty acids for oxidation in the periphery but direct evidence supporting this mechanism could not be obtained. Peripheral glucose oxidation may be facilitated by increased energy dissipation. Acknowledgements The authors are grateful to R. Havinga for excellent technical assistance. This study was supported by a grant (nr. 96.604) from the Dutch Diabetes Fund.

[16]

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[21]

[22]

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