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The Journal of Nutrition Vol. 129 No. 1 January 1999,
pp. 46-50
Tezukayama Gakuin College, Sakai, Osaka 590-0113, and * Takeda Chemical Industries, Ltd., Osaka 532-8686, Japan
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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In a previous study of glucose tolerance, plasma insulin levels were greatly elevated in genetically obese Wistar fatty rats but not lean rats fed a diet containing polyunsaturated fatty acids. In the present study, triacylglycerol-regulation of levels of circulating insulin and glucagon-like peptide-1 (7-36) (GLP-1) has been investigated in these rats. In the glucose tolerance test, the two plasma insulin peaks appeared in obese and lean rats intubated with glucose + corn oil, at 15- 30 min and 4 h, whereas only the first peak appeared in rats intubated with glucose alone, although the glucose response did not differ. After intubation of corn oil only, the insulin peak at 15 min was not detected but the peak at 4h was large. The two plasma GLP-1 peaks appeared 15 min and 4 h after intubation of glucose + corn oil similarly to the insulin responses, although the first peak was small and the second peak was very large. A small peak at 15 min was not significant in rats intubated glucose alone and no peak was seen at 4 h. The GLP-1 concentrations were significantly higher in the following order: portal vein > inferior vena cava > tail vein. The plasma GLP-1 increment in response to oral triacylglycerols was significantly higher in obese rats than in lean rats as was the insulin increment. Thus, oral triacylglycerols (possibly polyunsaturated) appeared to act at the gut lumen to stimulate GLP-1 secretion, which may be responsible for the second (4 h) insulin peak.
KEY WORDS: triacylglycerol-regulation · insulin · glucagon-like peptide-1 · obese · rats
We previously found that plasma insulin concentrations were significantly higher in Wistar fatty rats fed diets containing corn or fish oils than in those fed diets with saturated beef tallow, particularly in those fed soybean protein-fed groups (Iritani et al. 1997 The intestine plays an important role in the regulation of endocrine pancreatic secretion. The enteroinsular axis is regulated by neural and endocrine pathways in addition to the direct stimulation of insulin secretion by glucose (Creutzfeldt and Ebert 1993 Hoyt et al. (1996) Materials.
The insulin assay kit (RIA) was obtained from Eiken Chemical Company (Tokyo, Japan). The GLP-1 assay kit (RIA) was from Penninsula Co. (Belmont, CA). Most other reagents were obtained from Nacalai tesque (Kyoto, Japan), Wako (Osaka, Japan) and Sigma (St. Louis, MO).
Animals.
Female Wistar fatty rats (fa/fa, fa/Fa) and their lean littermates (Fa/Fa) (Ikeda et al. 1981 Analyses.
Plasma glucose concentrations were determined by the glucose-oxidase method (Werner et al. 1970 Statistical analysis.
Two- or three-way ANOVA was followed by inspection of all differences between pairs of means by using the least significant difference test (Snedecor and Cochran 1967
Effects of corn oil intubation on glucose tolerance test in obese and lean rats.
When starved Wistar fatty rats were intubated with glucose + corn oil, two insulin peaks appeared in the plasma of obese and lean rats at 15-30 min and 4 h (Fig. 1 C and D). Only the first peak appeared in rats intubated with glucose alone. The magnitude of the first insulin peak in rats given glucose + corn oil did not differ from that of rats given only glucose. The second peak began to form at 3 h in rats intubated with glucose + corn oil, and reached its maximum at 4 h after the intubation. Although insulin peaks were observed in both genotypes, the concentrations were significantly higher in the obese rats than in the lean. Increments of plasma glucose concentration were not significantly different in rats intubated with glucose + corn oil or glucose alone, with a peak detected at 15-30 min (Fig. 1 A and B).
Effects of glycerol or fatty acid intubation on plasma glucose and insulin concentrations in obese and lean rats.
When both obese and lean rats were intubated with glycerol or ethyl linoleate at the molar weight equivalent to that in the intubated corn oil, the plasma glucose concentration was not increased by the linoleate intubation, but was slightly increased by glycerol intubation after 30-60 min (P < 0.05; Fig. 2). The plasma insulin concentration was not increased by linoleate intubation, but was slightly increased by glycerol intubation after 4 h in obese rats (P < 0.05). This was in contrast to the marked increase in plasma insulin concentration 4 h after corn oil (triacylglycerols) intubation (Fig. 1).
Effects of corn oil intubation on plasma GLP-1 concentrations.
Although the plasma GLP-1 concentration in the tail vein of normal (commercially available) Wistar rats was too low to be measured, the concentration in the portal vein was high enough to be measured. Therefore, the time course of changes in the plasma GLP-1 concentration in the portal vein of the normal rats after intubation with glucose or glucose + corn oil was examined. The plasma GLP-1 concentration in the portal vein reached a peak 3-4 h after intubation with glucose + corn oil, and the concentration was significantly higher than that before the intubation (Fig. 3 A). However, the GLP-1 concentration did not increase, but decreased 3 h after the intubation of glucose alone. The plasma insulin concentration in the portal vein of the rats markedly increased at 30 min after intubation with glucose + corn oil, then decreased, began to increase again at 3 h and reached a large peak after 4 h (Fig. 3 C). However, the insulin concentration markedly increased 15-30 min after the intubation of glucose alone, but did not increase any more. The change in the plasma GLP-1 and insulin concentrations in the inferior vena cava was similar to those in the portal vein after the intubation, but were much lower (Fig. 3 Band D) .
Effects of corn oil intubation on plasma GLP-1 concentration in obese rats.
The plasma GLP-1 concentrations in the portal veins of obese and lean rats were measured at 15 and 30 min and 4 h after the intubation of glucose or glucose + corn oil (Table 1). The GLP-1 concentrations at the peaks were significantly higher in the obese rats than in the lean at 15 and 30 min and 4 h after the intubation of glucose + corn oil. In the obese rats, the glucose + corn oil intubation increased the plasma GLP-1 concentrations to seven times of those of starved rats at 15 min and 1.8 times at 4 h. In contrast, in the lean rats the GLP-1 concentrations at 15 min and 4 h after the glucose + corn oil intubation did not differ and were about 1.5 times those of starved rats. The intubation of glucose alone significantly elevated the GLP-1 concentrations at 15 and 30 min in the obese rats, but did not do so in the lean rats.
Plasma GLP-1 concentrations in teil vein, portal vein and inferior vena cava of obese rats.
The GLP-1 concentrations were compared in veins of obese rats at 15 and 30 min and 4 h after intubation of glucose + corn oil. The GLP-1 concentrations in veins generally were higher in the following order: portal vein > inferior vena cava > tail vein (Fig. 4).
Two insulin concentration peaks appeared in the plasma of obese and lean rats intubated with glucose + corn oil after 15 min and 4 h, whereas only the first peak appeared in rats intubated with glucose only. In rats intubated with corn oil only, the insulin peak at 15-30 min was not detected, but the 4 h peak was large. Therefore, the increment of insulin release at 4 h appears to be due to the oral administration of corn oil. The insulin increment was always greater in the obese rats than in the lean. The first insulin release was larger in the lean rats after the intubation of glucose + corn oil than after that of corn oil only, whereas it did not differ in the obese rats. It may be because glucose stimulated the first insulin release at 15 min, which stimulated the second insulin release by intubation with glucose + corn oil in the lean rats. We previously found that dietary polyunsaturated triacylglycerols, such as those in corn and fish oils, greatly increased the plasma insulin concentrations in comparison with dietary saturated fat, particularly in obese Wistar fatty rats, whereas the glucose concentrations were not changed (Iritani et al. 1997
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). However, plasma glucose concentrations were not significantly affected by dietary protein or triacylglycerols. A similar result was observed in a glucose tolerance test. Wistar fatty rats with non-insulin-dependent diabetes mellitus (NIDDM)4 are obese, hyperphagic, hyperinsulinemic and hypertriglyceridemic, and have impaired glucose tolerance (Ikeda et al. 1981, Matsuo et al. 1984).
). The circulating levels of the incretin gastric polypeptide, glucose-dependent insulinotropic polypeptide (GIP), are elevated in some obese groups of humans as well as in the obese hyperglycemic mouse and rat (Creutzfeldt et al. 1978, Ebert et al. 1979, Flatt et al. 1984, Pedersong et al. 1991, Salera et al. 1982). Moreover, the intestinal products of the proglucagon gene, glucagon-like peptide (GLP-1) (7-36) and GLP-1 (7-37), were shown to contribute significantly to the overall insulin response to oral glucose and are now considered to be important incretins (Habener 1993). In addition, circulating levels of GLP-1 were found to be elevated in obese patients (Fukase et al. 1993). Recently, it was also demonstrated that GLP-1 has antidiabetogenic actions to promote insulin secretion, reduce glucagon secretion and reduce plasma glucose in type II diabetic patients (Gutniak et al. 1992, Nathan et al. 1992). Jia et al. (1995) found significant increases in GLP-1 levels in response to glucose in obese Zucker rats.
found that proglucagon mRNA concentrations in the jejunum were 40% lower due to food-deprivation and with refeeding increased to levels similar to those of rats with free access to the diet, similar to changes in insulin concentration. Plasma enteroglucagon and GLP-1 levels correlated with jejunal proglucagon. They reported also that intrajejunal infusion of long-chain triacylglycerols resulted in a greater abundance of jejunal proglucagon mRNA than infusion of saline, whereas the infusion did not significantly increase plasma enteroglucagon or GLP-1 concentrations. Other groups also reported that oral triacylglycerols stimulated the release of GLP-1 (Herrmann et al. 1995, Hoyt et al. 1996, Jia et al. 1995). GLP-1 is released into the circulation after eating and causes a potent stimulation of insulin secretion from the pancreas (Fehmann et al. 1992, Gutniak et al. 1992, Kreymann et al. 1987, Nathan et al. 1992). To investigate triacylglycerol regulation of insulin secretion, we have examined the circulating levels of insulin and GLP-1 (7-36) after the intubation of glucose and/or corn oil in Wistar fatty rats.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
, Matsuo et al. 1984) at 11 wk of age were obtained from Takeda Chemical Industries, Ltd. (Osaka, Japan). Rats were housed in wire-bottomed cages in a temperature-controlled room (24°C) under an automatic lighting schedule (0800-2000 h), and had free access to water and commercially available non-purified diet (No. MF, Oriental Shiryou Co., Osaka, Japan). The diet contained (g/100 g) crude protein (24.0), crude fat (5.1), crude ash (6.2), crude fiber (3.2), crude carbohydrate (54.5), water (7.0) and vitamins.
). Plasma insulin concentrations were measured by a two-antibody system radioimmunoassay according to the method of Morgan and Lazarow (1963). Plasma GLP-1 concentrations from the portal vein, inferior vena cava or tail vein were measured by a two-antibody system radioimmunoassay according to the supplier's method information (Dwenger 1984, Patrono and Peskar 1987).
). Data for which variances are unequal were logarithmically transformed before ANOVA. Untransformed data are shown in Table 1 and Figures 1-3. Differences were considered significant at P < 0.05.
View this table:
Table 1.
Plasma glucagon-like peptide-1 concentrations in portal vein of obese and lean rats after intubation of glucose or glucose + corn oil1,2
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Fig 1.
Effects of corn oil intubation on plasma glucose and insulin concentrations of obese and lean rats in glucose tolerance tests. In the oral glucose tolerance test, rats received 3 g glucose /kg body weight, 5 g corn oil/kg body weight, or both after being deprived of food for 20 h. ANOVA for glucose concentrations (A and B): G (genotype) (P < 0.001), I (intubation) (P < 0.001), T (time) (P < 0.001), G × I (P < 0.05), I × T (P < 0.001): for insulin concentrations were (C and D): G (P < 0.001), I (P < 0.001), T (P < 0.001), G × I (P < 0.001), G × T (P < 0.05), I × T (P < 0.001). Values are means ± SD, n = 4.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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Fig 2.
Effects of glycerol or fatty acid intubation on plasma glucose and insulin concentrations of obese and lean rats. The 20-h starved rats were intubated with glycerol or ethyl linoleate of the molar weight equivalent to that of 5 g corn oil/kg body weight. ANOVA for glucose concentrations (A and B): G (genotype) (P < 0.001), T (time) (P < 0.001); for insulin concentrations were (C and D): G (P < 0.001). Values are means ± SD, n = 4. Means for glucose (A and B) or insulin (C and D) with different letters are significantly different (P < 0.05).
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Fig 3.
Time courses of plasma glucagon-like peptide-1 concentrations in portal vein and inferior vena cava after intubation of starved rats with glucose or glucose + corn oil. The starved normal (commercially available) Wistar rats were intubated with glucose or glucose + corn oil by the method in Fig. 1. ANOVA for GLP-1 concentrations (A and B): V (vein) (P < 0.001), I (intubation) (P < 0.001), T (time) (P < 0.001), I × T (P < 0.001), V × I (P < 0.05); for insulin concentrations (C and D): I (P < 0.001), V (P < 0.001), T (P < 0.01), V × I (P < 0.05), V × T (P < 0.001), I × T (P < 0.001). Values are means ± SD, n = 3. Means for GLP-1 (A and B) or insulin (C and D) with different letters are significantly different (P < 0.05).
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Fig 4.
Plasma glucagon-like peptide-1 concentrations in portal vein, inferior vena cava and tail vein of obese rats after intubation with glucose + corn oil. The starved (20 h) obese rats were intubated with glucose + corn oil. Blood samples were taken from the tail vein first, from the portal vein next and then from the inferior vena cava. ANOVA for GLP-1 concentrations: V (vein) (P < 0.001), T (time) (P < 0.001), V × T (P < 0.001). Means with different letters are significantly different (P < 0.05). Values are means ± SD, n = 3.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). Therefore, the great increment of insulin concentration after intubation with corn oil is likely due to polyunsaturated triacylglycerols.
). Ethyl-linoleate should be absorbed and highly incorporated into the phospholipids. Therefore, ethyl-linoleate could be used instead of linoleic acid in this experiment.
reported that intrajejunal infusion of long chain triacylglycerols resulted in a greater abundance of jejunal proglucagon mRNA than did an infusion of saline, whereas the infusion did not significantly increase the enteroglucagon or GLP-1 concentration in the inferior vena cava plasma. In our present experiment, however, oral triacylglycerols elevated plasma GLP-1 concentrations markedly in the portal vein but only slightly in the inferior vena cava. The different results in both experiments may be due to the different method of triglyceride-treatment or blood sampling from different parts of veins.
reported that significant increases in plasma GLP-1 levels in the tail vein in response to an oral glucose load were observed only in obese rats, 10 and 20 min after administration. There was a small increase in the mean GLP-1 level in response to glucose in the lean animals, although values did not differ significantly (Jia et al. 1995). In the present experiment, however, we observed that the GLP-1 concentration in the portal vein was significantly elevated in response to glucose, even in lean rats. The GLP-1 concentrations were very high in the portal vein, lower in the inferior vena cava and even lower in the tail vein. This may be because the half-lives of GLP-1 and its metabolites are short.
reported that oral administration of glucose to humans induced a biphasic GLP-1 release peaking at 30-60 min and a biphasic GIP release peaking at 5 and 45 min. The increases roughly paralleled the insulin release peaking at 15-30 min. After oral administration of fat, a strong and long-lasting (>120 min) increase of GLP-1 plasma levels occurred. In addition, as a result of intestinal infusion study, they hypothesized that the GLP-1 release from L cells is triggered by nervous reflexes and by putative humoral factor(s) being released from the jejunum in addition to nutrient stimuli acting at the luminal surface of the gut. Knapper et al. (1996) reported that, in pigs, dual nutrient infusion of glucose + fat was a strong stimulus for both GIP and GLP-1 secretion. Plasma triacylglycerols were elevated following fat or glucose + fat infusion.
, Hoyt et al. 1996, Jia et al. 1995). Therefore, the GLP-1 secretion should be triggered with some factors released by triacylglycerol stimulation at the luminal surface of the gut. The GLP-1 secretion appeared to be responsible for the second insulin secretion.
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FOOTNOTES |
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Manuscript received 17 June 1998. Initial reviews completed 1 August 1998. Revision accepted 25 September 1998.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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