Acute Increase in Food Intake After Intraperitoneal Injection of Metformin in Rats

Physiology & Behavior, Vol. 67, No. 5, pp. 685–689, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99/$–see front matter

PII S0031-9384(99)00122-5

Acute Increase in Food Intake After Intraperitoneal Injection of Metformin in Rats E. DEL PRETE,1 T. A. LUTZ AND E. SCHARRER Institute of Veterinary Physiology, Winterthurerstr. 260, 8057 Zürich, Switzerland Received 8 February 1999; Accepted 10 June 1999 DEL PRETE, E., T. A. LUTZ AND E. SCHARRER. Acute increase in food intake after intraperitoneal injection of metformin in rats. PHYSIOL BEHAV 67(5) 685–689, 1999.—We studied the effect of intraperitoneal injection of different doses of the antihyperglycemic agent metformin on food intake and plasma metabolites (glucose, free fatty acids, b-hydroxybutyrate) in rats fed a high-fat (HF) or a high-carbohydrate (HC) diet. Unexpectedly, metformin, at a dose of 120 mg/kg b.wt. stimulated food intake in both HF- and HC-fed rats, without affecting blood glucose level. This result is in contrast with the hitherto performed studies that found an anorectic effect of metformin in rodents. It is postulated that the hyperphagic effect of metformin might be related to reduced energy availability to hepatic metabolic sensors controlling food intake, because metformin’s known inhibitory effect on oxidative phosphorylation mainly affects the hepatoportal area, and blockade of oxidative phosphorylation in this area has been shown to stimulate feeding. © 1999 Elsevier Science Inc. Metformin

Intraperitoneal injection

Rats

Hyperphagic effect

involvement of neuropeptide Y (15). Unspecific, possibly even toxic effects of metformin may also be involved in the anorectic effect of metformin (15). The purpose of the present study was, therefore, to further characterize the effect of metformin on feeding in rats. With regard to its effect on glucose metabolism, we wanted to investigate whether the carbohydrate content of the diet influences metformin’s effect on feeding behavior. We, therefore, tested the effect of intraperitoneally injected metformin on food intake in normal rats fed a high-fat, carbohydrate-free (HF), or a high-carbohydrate, low-fat (HC) diet. The influence of metformin on plasma metabolites (glucose, free fatty acids, b-hydroxybutyrate) was also investigated. Unexpectedly metformin stimulated food intake in these experiments without affecting blood glucose level.

METFORMIN (dimethylbiguanide) is an antihyperglycemic agent used in the treatment of type 2 diabetes mellitus (2,3). The antihyperglycemic effect of metformin has been attributed mainly to decreased hepatic glucose output and enhanced peripheral glucose uptake (4). In obese Zucker rats, parenteral or oral metformin administration produced an inhibition of food intake (13–15). A mild and transient anorectic effect of a large parenteral dose of metformin was also observed in lean and obese mice (1). Furthermore, it has been recently shown that in obese nondiabetic humans (11) or in obese subjects with noninsulin-dependent diabetes mellitus (8) metformin administration was useful to inhibit food intake and to lower body weight and body fat. The mechanism for the food intake-reducing effect of metformin is not clear. It has been proposed that the amelioration of peripheral insulin resistance by metformin can indirectly affect body energy balance and caloric requirements, resulting in reduced food intake (14), or that metformin might exert a transient anorectic effect by modulating insulin target cells in the hypothalamus (1,8,10). A direct effect of metformin on central neurotransmitters involved in the regulation of feeding behavior has also been proposed (14), although there is lack of evidence for the

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MATERIALS AND METHODS

Animals, Housing Conditions, and General Procedures The experiments were performed with male rats (ZUR:SD rats; Institut für Labortierkunde, Universität Zürich) housed individually in a temperature-controlled (21 6 18C) colony

whom requests for reprints should be addressed. E-mail: delprete @vetphys.unizh.ch

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DEL PRETE, LUTZ AND SCHARRER TABLE 1 COMPOSITION OF THE HC (HIGH-CARBOHYDRATE, LOW-FAT) AND HF (HIGH-FAT, CARBOHYDRATE-FREE) DIETS

Casein* Corn startch† Soybean oil‡ Beef tallow§ Lard§ Vitamin premix¶ Mineral premix¶ Diluent# BHTi Total

HC

HF

13.00 76.67 3.33 — — 3.00 4.00 — 0.003 100.00

13.00 — 3.54 23.54 12.92 3.00 4.00 39.96 0.04 100.00

*Zentral-schweizerischer Milchverband Luzern, Dagmersellen, Switzerland. †Blattman, Wädenswil, Switzerland. ‡Sais, Horn, Switzerland. §Häute- und Fettwerk AG, Zürich, Switzerland. ¶Kliba Mühlen, Kaiseraugst, Switzerland. #Polyethylene powder (Lupolen, Albisplastik GmbH, Hamburg, Germany). i2,6-Di-tert-Butyl-p-cresol (antioxidant), Sigma, Buchs, Switzerland.

room and kept on a 12-h light–dark cycle. The composition of the high-fat, carbohydrate-free (HF) and the high-carbohydrate, low-fat (HC) diets is shown in Table 1. The diets were isoenergetic (16.5 kJ/g). The rats were adapted to the diets for at least 2 weeks. They were also adapted to the test procedure [weighing of food cups to measure cumulative food intake, and intraperitoneal (i.p.) injections]. The body weight of the rats was 777–917 g (age: 5–9 months). The groups of rats were matched for baseline food intake and body weight. Isotonic solutions of metformin (1,1-dimethylbiguanide, hydrochloride; Sigma Chemie, Buchs, Switzerland) (60, 100, 120, and 200 mg metformin per kg body weight) were freshly prepared immediately prior to injection. Injection of saline served as control. In Experiment 1, rats were injected i.p. in the middle of both the light and the dark phase. In Experiment 2, rats were injected in the middle of the dark phase. Experiments 3–6 were performed in the middle of the light phase. The injection volume was 3.8 mL/kg b.wt. (with exception of Experiment 4, 6.3 mL/ kg). Food intake (Experiments 1–4) was determined by measuring the weight of the food containers (60.1 g). Food was offered in relatively spill-resistant feeding cups. In the cases in which food was spilled outside the feeding cups, spillage was picked up with newspapers and accounted for. Cumulative food intake was measured 2, 4, and 6 h after injection. Effect of Metformin on Food Intake In Experiment 1 a total of 32 HF-fed rats was injected with 120 mg/kg metformin or saline. One rat of the saline group injected in the middle of the light phase was retrospectively excluded from the experiment because of anorexia and coprostasis and was euthanized after 1 week. In Experiment 2 a total of 16 HC- and 15 HF-fed rats was injected with 120 mg/kg metformin (n 5 8 for each diet) or saline. Four days later the rats were injected in counterbalanced order and every rat served as its own control. In Experiment 3 and 4 the effect of different doses of met-

FIG. 1. (Experiment 1): effect of metformin (120 mg/kg, i.p.) on cumulative food intake in HF-fed rats. Injection in the middle of the light (top) or dark (bottom) phase. **p , 0.01; ***p , 0.001 (unpaired Student’s t-test).

formin on food intake was investigated. A total of 16 HC- and 15 HF-fed rats was injected with 60- or 120 mg/kg metformin or saline (Experiment 3) or with 100- or 200 mg/kg metformin or saline (Experiment 4) (HF-rats: n 5 5 each group; HC-rats: n 5 5, 5, and 6). Experiment 3 was performed in three trials, with 6- and 3-day time intervals, to have each rat to act as its own control.

Effect of Metformin on Plasma Glucose, Free Fatty Acids, and b-Hydroxybutyrate For the measurement of the plasma metabolites (glucose, free fatty acids, and b-hydroxybutyrate) a total of 20 HF-fed rats (Experiment 5) or of 12 HC- and 13 HF-fed rats (Experi-

STIMULATION OF FEEDING AFTER METFORMIN

FIG. 2. (Experiment 2): effect of metformin (120 mg/kg, i.p.) on cumulative food intake in HC (top)- and HF (bottom)-fed rats. Injection in the middle of the dark phase. Two and 4 h after injection: effect of metformin: p , 0.01 [fully factorial (M)ANOVA]. *p , 0.05; **p , 0.01 (paired Student’s t-test). Six hours after injection: effect of metformin: p , 0.05, effect of the diet: p , 0.01 [fully factorial (M)ANOVA]. p , 0.05, HC saline versus HF saline (post hoc Bonferroni).

ment 6) was injected with 120 mg/kg metformin or saline (n 5 10, Experiment 5; n 5 6 or 7, Experiment 6). One (Experiment 5) or 2 h (Experiment 6) after injection (the rats were food deprived during this period of time) blood was drawn under ether anesthesia from the venous retrobulbar plexus of the eye (Experiment 5) or from the aorta before exsanguination (Experiment 6) into sodium fluoride tubes for enzymatic determination (glucose: Gluc HK, Hoffmann–LaRoche, Basel, Switzerland; free fatty acids: NEFA C, Wako Chemicals GmbH, Neuss, Germany; b-hydroxybutyrate: b-HB, Sigma Chemie, Buchs, Switzerland) on an automatic analyser (Cobras Mira, Hoffmann–LaRoche, Basel). Statistics Results are presented as means 6 SEM. The results were statistically evaluated using the unpaired Student’s t-test (Ex-

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FIG. 3. (Experiment 3): effect of metformin (60- and 120 mg/kg, i.p.) on cumulative food intake in HC (top)- and HF (bottom)-fed rats. Injection in the middle of the light phase. Two and 4 h after injection: effect of metformin: p , 0.001 [fully factorial (M)ANOVA]. ##p , 0.01; ###p , 0.001, 120 mg/kg versus 60 mg/kg; 111p , 0.001, 120 mg/kg versus saline (post hoc Bonferroni).

periment 1 and 5) or an analysis of variance (ANOVA) with the Bonferroni post hoc test (Experiment 6) or when appropriate a fully factorial (M)ANOVA with the paired Student’s t-test (Experiment 2, because there was no effect of the diet on food intake) or the Bonferroni test (Experiment 3) as post hoc test. RESULTS

Effect of Metformin on Food Intake The injection of 120 mg/kg metformin in HF-fed rats stimulated food intake, both in the light phase and in the dark phase (Experiment 1, Fig. 1). Also, the injection of 120 mg/kg metformin in the middle of the dark phase significantly stimulated feeding in the HC- and the HF-fed rats (Experiment 2, Fig. 2). Cumulative food intake was significantly increased un-

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DEL PRETE, LUTZ AND SCHARRER TABLE 2 EFFECT OF METFORMIN (120 mg/kg) ON PLASMA GLUCOSE, FFA, AND b-BH IN HF-FED RATS 1 H AFTER INJECTION IN THE MIDDLE OF THE LIGHT PHASE

Glucose (mmol/L) FFA (mmol/L) b-HB (mmol/L)

Saline (n 5 10)

Metformin (n 5 10)

7.2 6 0.2 0.19 6 0.02 546 6 79

7.4 6 0.2 0.18 6 0.01 515 6 26

Values are means 6 SEM. FFA: free fatty acids, b-HB: b-hydroxybutyrate, HF: highfat diet.

til the fourth hour after injection (Fig. 2). In the middle of the light phase (Experiment 3) the injection of 120 mg/kg metformin in HC-fed rats significantly stimulated food intake 2 and 4 h after injection when compared to saline injected rats and to rats injected with 60 mg/kg metformin (Fig. 3). In HFfed rats the stimulating effect of 120 mg/kg metformin in the light phase was significant until the second hour (when compared to saline control rats) and until the fourth hour (when compared to the rats injected with 60 mg/kg metformin) after injection. Experiment 4 was stopped 2 h after injection because following injection of 200 mg/kg metformin four HCfed rats and one HF-fed rat became comatose, and at times had convulsions. Three of these rats died a few hours after injection, and the other two were euthanized. Effect of Metformin on Plasma Glucose, Free Fatty Acids, and b-Hydroxybutyrate The injection of 120 mg/kg metformin in the middle of the light phase did not significantly influence plasma concentra-

TABLE 3 EFFECT OF METFORMIN (120 mg/kg) ON PLASMA GLUCOSE, FFA, AND b-HB IN HF-OR HC-FED RATS 2 H AFTER INJECTION IN THE MIDDLE OF THE LIGHT PHASE

HF-fed rats Glucose (mmol/L) FFA (mmol/L) b-HB (mmol/L) HC-fed rats Glucose (mmol/L) FFA (mmol/L) b-HB (mmol/L)

Saline (n 5 6)

Metformin (n 5 7)

7.9 6 0.2 0.14 6 0.02 487 6 52* (n 5 6) 6.8 6 0.4 0.10 6 0.02 82 6 6

6.9 6 0.3 0.16 6 0.02 650 6 76* (n 5 6) 7.1 6 0.1 0.17 6 0.01 142 6 14

Values are means 6 SEM. FFA: free fatty acids, b-HB: b-hydroxybutyrate, HF: high-fat diet, HC: high-carbohydrate diet. *Significantly (p , 0.001, post hoc Bonferroni) different from corresponding value of HC-fed rats [ANOVA: F(1, 23) 5 30.07, p , 0.001].

tion of glucose, free fatty acids, and b-hydroxybutyrate measured 1 h later in HF-fed rats (Table 2) or 2 h later in HF- and HC-fed rats (Table 3). Both saline and metformin-injected HF-fed rats showed a significant increase in plasma concentration of b-hydroxybutyrate when compared to the corresponding value in the HC-fed rats (Table 3). This confirms previous observations (7).

DISCUSSION

The present study demonstrates for the first time an acute stimulating effect of metformin on food intake. In contrast, other studies performed to investigate the effect of metformin on food intake in rodents (1,14,15) or humans (8,11) found an anorectic effect of metformin. A single subcutaneously injected metformin dose (300 mg/kg) was shown to significantly reduce food intake in obese Zucker rats (15). Also, chronic oral metformin treatment has been shown to significantly reduce cumulative food intake in genetically obese Zucker rats (14,15). The results of the present study show a new aspect of metformin’s effect on feeding behavior in rats. The acute stimulatory effect of metformin on food intake following intraperitoneal injection was effective in both rats fed a carbohydrate-free, high-fat diet and a high-carbohydrate diet. This finding argues against a role of the carbohydrate content in the diet in the hyperphagic effect of metformin. The finding that metformin did not influence plasma glucose suggests that the stimulation of eating by metformin was not coupled to acute glucose deprivation, which is known to trigger feeding (9,17). This is in agreement with the antihyperglycemic but not hypoglycemic effect attributed to metformin (4). It is very unlikely that the small and transient increase in blood glucose resulting from anesthesia has masked an effect of metformin on blood glucose, because variability and mean values of blood glucose were similar in control rats and metformin-treated rats. Also, the time of injection did not influence the hyperphagic effect of metformin, which was evident in both the active and the inactive phase of the rats. Because biguanides injected intraperitoneally bind to biologic membranes and produce an inhibition of oxidative phosphorylation in particular in the liver (3,5) and a partial blockade of oxidative metabolism in the hepatoportal area has been shown to stimulate food intake (6,12,16) metformin’s hyperphagic effect might be related to reduced energy availability to peripheral metabolic sensors controlling food intake. This hypothesis remains to be tested in further investigations. With regard to the symptoms (coma, convulsions) in five rats after injection of 200 mg/kg metformin, it is possible that at this dose metformin induced hypoglycemia or had unspecific toxic effects. In conclusion, the present study in which metformin was injected intraperitoneally, is not in accordance with the results of the hitherto performed studies that showed a suppressive effect of metformin on food intake after oral or subcutaneous administration.

ACKNOWLEDGEMENTS

The authors are grateful to Mrs. Barbara Schneider for expert technical assistance.

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