1Institute of Animal Sciences, Eidegnossische Technische Hochschule Zurich, Schwerzenbach; 2Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland; and 3Deptartment of Cell and Molecular Biology, Biomedical Center, Lund University, Lund, Sweden
Submitted 12 August 2004 ; accepted in final form 9 February 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
body weight; appetite; glutathione; peroxisome proliferator-activated receptors; uncoupling protein-2
One aim of this study was to expand on previous investigations showing that enterostatin resulted in a decrease in body weight associated with a decreased intake of a high-fat (HF) diet (21). Using ad libitum-fed rats that had a choice of HF and low-fat (LF) diets, we studied the effect of chronic administration of enterostatin through intraperitoneally implanted osmotic minipumps on food intake and body weight. We also examined whether enterostatin changed certain metabolic parameters in the postprandial phase, including the relationship between expression of the peroxisome proliferator-activated receptors (PPARs) (10), which can regulate UCP2 expression and metabolism in various tissues. More specifically, we chose epididymal fat pad, liver, and pancreas. Finally, because enterostatin has been shown to influence insulin secretion, we measured plasma concentrations of insulin and amylin, two hormones that are cosynthesized in and cosecreted by pancreatic -cells and are involved in energy balance.
![]() |
METHODS AND MATERIALS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Male Sprague-Dawley rats (200225 g body wt at the start of the experiment, n = 5) from our own breeding facility (Schwerzenbach, Zurich, Switzerland) were caged individually in a temperature controlled room with a 12:12-h dark-light cycle with the lights off at 0700. All procedures were approved by the Canton of Zurich's Animal Use and Care Committee. The rats had access to both high HF and LF diets every day for 6 h (0930 to 1530) (15). Diets were obtained from Kliba (Kaiseraugst, Switzerland). The LF diet (Kliba no. 2011) consisted of 77% (wt/wt) corn starch, 16% casein, 1% dextrose, 4% soya oil, and 5% other additives (vitamins, minerals), and had an energy density of 14.1 MJ/kg. The HF diet (Kliba No. 2114) contained 45% corn starch, 16% casein, 22% beef extract, 12% pig fat, 4% soya oil, and 5% other additives and had an energy density of 14.9 MJ/kg. After 2 wk of adaptation to the dietary choice and the feeding schedule, rats were randomly divided into two groups. They were anesthetized with isoflurane (Abbott Laboratories, Abbott Park, IL), a small incision was made in the abdomen, and a mini osmotic pump (Alzet type 2001; Charles River Laboratories, Sulzfeld, Germany) was inserted. One group received a pump delivering enterostatin (APGPR; Ferring, Malmoe, Sweden), 80 µg/h (160 nmol/h in 1 µl/h) dissolved in vehicle (0.9% saline containing 0.3% BSA) for 5 days, whereas control rats received pumps containing only the vehicle. Body weight and food intake were recorded daily. Our automated system for food intake measurements (15) allows for continuous recordings of meal size and meal frequency. On the basis of previous studies (15), we used the following minimum criteria to define a meal: meal size, 0.3 g; meal duration, 1 min; intermeal interval, 15 min. After the food access period (15301630) on infusion day 5, rats were decapitated; blood was collected; and liver, WAT, (epididymal fat pads) and pancreas were removed and frozen immediately in liquid nitrogen and stored at 80°C.
Blood was collected in EDTA- or heparin/NaF-coated tubes on ice and centrifuged at 4°C at 3,000 rpm in a microcentrifuge. Standard enzymatic tests were used to determine plasma concentrations of lactate, glucose, triglycerides, and free fatty acids (FFA). Rat insulin (Linco, St. Charles, MO) and amylin (16) were determined by radioimmunoassay.
Liver, WAT (epididymal fat pads), and pancreas were analyzed for UCP2 and PPAR expression (PPAR, PPAR
, PPAR
1 and PPAR
2). Total RNA was isolated as previously described (2). The RNA was then treated with DNase, after which it was reverse transcribed (Promega). Thereafter, we ran a PCR (Invitrogen) and separated the product by gel electrophoresis containing ethidium bromide. The bands were then quantified using the Scion Image program (Scion, Frederick, MD). Each sample was normalized with its glyceraldehyde-3-phosphate dehydrogenase (GAPDH) value. The primers used were as follows: UCP2 [sense 5'-TAC CAG AGC ACT GTC GAA GCC-3', antisense 5'-AGT CCC TTT CCA GAG GCC C-3' (24)], PPAR
[sense 5'-TGC ATG TCC GTG GAG ACC GTC AC-3', antisense 5'-GGT CAT CAA GAA GAC CGA GT-3' (26)], PPAR
[sense 5'-AGTTCTTGCGCAGTATCCG-3', antisense 5'-AGTGTTGTGAGTGGCTCTAG-3', (26)], PPAR
1 [sense 1 5'-TATGCTGTTATGGGTGAAAC-3', antisense 1 5'-TGGTAATTTCTTGTGAAGTGCTC-3', (26)], PPAR
2 [sense 2 5'-TGATATCGACCAGCTGAACC-3', antisense 2 5'-GTCCTCTCAGCTGTTCGCCA-3' (12)] and GAPDH [sense 5'-TGA AGG TCG GTG TCA ACG GAT TTG GC-3', antisense 5'-CAT GTA GGC CAT GAG GTC CCA CCA C-3' (22)] as reference control. All values for the expression of a gene were normalized with GAPDH for presentation.
Metabolite Measurements
Glutathione levels. Tissue was homogenized (3 g tissue/10 ml) in 10 mM Tris, pH 7.4, from control and enterostatin rats (n = 5/group). Protein was precipitated by metaphosphoric acid, and the supernatant was used to quantify GSH. Total GSH levels were measured using a method based on the formation of a chromophoric product resulting from the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Sigma Chemicals) with GSH. The absorbance was immediately measured at 412 nm. Glutathione contents were calculated using a calibration curve established with standard samples (7).
Malondialdehyde. To measure lipid peroxidation, tissues were homogenized (3 g tissue/10 ml buffer) in 10 mM Tris buffer (pH 7.4) containing 5 mM butylated hydroxytoluene. The homogenate was centrifuged at 4°C to remove cellular debris. Aliquots were then taken for determination of malondialdehyde (MDA) using an acid extraction procedure. Absorbance was measured at 586 nm, and corrections were made for sample and reagent blanks. Concentrations were then determined using a standard curve (7).
Data Analysis
All data are presented as means ± SE. Statistical analysis was performed using a Mann-Whitney nonparametric ANOVA. A P value <0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Continuous intraperitoneal enterostatin infusion reduced body weight (P < 0.01) over the 5-day period (Fig. 1A). Enterostatin decreased HF diet intake (P < 0.02) over the 5-day period but had no effect on LF diet intake (Fig. 1B). The number of meals during the feeding period was the same in both groups, with the last meal eaten between 1400 and 1520. Animals from both groups ate on average two HF and two LF meals during the feeding period (data not shown).
|
Continuous intraperitoneal enterostatin infusion resulted in elevated UCP2 expression in liver and epididymal fat pad (WAT), whereas UCP2 expression in the pancreas was decreased by enterostatin (P < 0.001; Fig. 2A). Also, enterostatin increased MDA in liver and WAT (P < 0.001) but not in the pancreas (Fig. 2B). In comparison, enterostatin reduced GSH in liver and WAT substantially (P < 0.001) but to a much smaller extent in the pancreas (Fig. 2C). The decrease in the antioxidant GSH was inversely associated with UCP2 expression.
|
Enterostatin increased circulating levels of insulin (P < 0.02), amylin (P < 0.002), glucose (P < 0.005), and lactate (P < 0.002), whereas plasma FFA (P < 0.01) and triglycerides (P < 0.006) were reduced by enterostatin (Fig. 3, AD).
|
Enterostatin increased PPAR expression in liver (P < 0.01) and pancreas (P < 0.01) but not in WAT (Fig. 4A). PPAR
was markedly elevated by enterostatin in WAT and pancreas and to a lesser extent in the liver (P < 0.01 for all tissues; Fig. 4B). PPAR
1 was elevated by enterostatin in all tissues examined (P < 0.01 for all tissues; Fig. 4C). PPAR
2 was increased in pancreas (P < 0.01) and, to a lesser extent, in WAT (P < 0.05) but decreased in liver (P < 0.01; Fig. 4D).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The observed changes in circulating pancreatic hormone levels support the idea that enterostatin-induced changes in UCP2 in the pancreas may contribute to metabolic regulation. Results from UCP2 knockout mice suggest that UCP2 is a negative regulator of insulin (27). Thus, with a reduction of UCP2 in the pancreas, one might expect an increase in circulating insulin, and this is what we actually found in the rats treated with enterostatin. The enterostatin effect did not seem to be specific for insulin, as amylin was also increased, consistent with the fact that amylin is always cosecreted with insulin (16). Previous studies have shown that enterostatin acutely decreases rather than increases the plasma insulin level (18). Yet insulin was not measured postprandially in that acute study, whereas we looked at the effect of a continuous enterostatin administration at 12 h after the 6-h feeding period. The two apparently contradictory results may in fact be compatible, because enterostatin has been shown to initially decrease and later increase intracellular ATP in insulin-secreting INS-1 cells in vitro (4). A chronic increase in ATP would favor insulin release (4). Whether the observed increase in circulating insulin and amylin in response to enterostatin is due to increased transcription, translation, or secretion of the two proteins remains to be determined.
Although the increase in circulating insulin and amylin levels may be mainly due to increased secretion, a reduced degradation rate of the two proteins might contribute, based on the fact that hepatic GSH expression was reduced by enterostatin. Hepatic GSH is required for insulin degradation (6). Whether the reducing effect of enterostatin on food intake is mediated by the elevated concentrations of circulating amylin and insulin remains to be determined. Pancreatic GSH levels could also influence insulin secretion by a direct effect (5, 19). Finally, the increase in circulating insulin and amylin levels may, in part, reflect a response to the hyperglycemia induced by chronic enterostatin.
We found that one class of transcription factors, the PPARs, was altered by chronic enterostatin in pancreas, liver and WAT, as would be expected from a substance that alters lipid levels. An increase in PPAR expression in the pancreas may also contribute to an increase in insulin release (11), as could increased expression of PPAR
1 and -
2 (12). PPAR expression was also altered in other tissue, suggesting that both carbohydrate and lipid metabolism were altered by chronic enterostatin administration. Increased PPAR
expression in the liver could implicate augmented lipid catabolism, as would an increased expression of PPAR
1 and PPAR
2 in the liver. Alteration of PPAR
1 and PPAR
2 expression in the WAT tissue may also indicate changes in fat deposition, i.e., adipogenesis (17, 25).
Enterostatin has been shown to increase sympathetic nervous system activity (19) and UCP1 expression in brown adipose tissue (BAT) (21). This suggests that enterostatin increases metabolic rate and enhances lipolysis, which could contribute to the decrease in body weight. It has previously been shown that epididymal fat mass is reduced by enterostatin (21). An increase in adipocyte conversion of glucose to lactate could also be a way to diminish fat stores, and this is consistent with our findings of an increased plasma lactate level in response to enterostatin. An enhanced white adipocyte anaerobic metabolism has been shown to occur with an increase in circulating insulin (8, 23). The increase in lactate could, in turn, fuel gluconeogenesis and, hence, contribute to the observed increase in plasma glucose levels (8). Yet, whether the lactate in this model is really derived from adipose tissue remains to be demonstrated.
The finding that enterostatin increased circulating levels of insulin, amylin, glucose, and lactate, whereas it reduced FFA and triglycerides, could reflect a shift from oxidative to anaerobic metabolism. This switch may favor the reduction of mitochondrial oxidative metabolism and therefore decrease mitochondrial ROS, particularly in tissues where UCP2 expression is increased, such as adipose tissue. Enterostatin could decrease body fat by increasing BAT UCP1 activity, and it may also shift adipose tissue metabolism from fat storage to release, as is suggested by altered PPAR expression in tissues involved in lipid metabolism.
Overall, our studies suggest that chronic enterostatin can reduce body fat by attenuating high-fat intake and by altering metabolism, in particular by favoring anaerobic metabolism as seen by the increase in lactate levels and the increase in glucose levels possibly due to an increased gluconeogenesis. The circulating levels of glucose, lactate, and FFA after enterostatin treatment are more consistent with an action of amylin than of insulin (9). Whether amylin, alone or together with nonhypoglycemic circulating insulin, contributes to the observed food intake reduction needs to be examined. Furthermore, enterostatin can differentially modify tissue and mitochondria oxidative state, as seen by changes in UCP2. Whether enterostatin can modify postprandial insulin and amylin levels directly merits further investigation, as does the role of enterostatin in PPAR expression and the way lipid metabolism is modified. In conclusion, we have shown that enterostatin alters energy balance and may play a role in energy partitioning between glucose and fat metabolism.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |