Effect of adrenalectomy on the activity of small intestine enzymes in monosodium glutamate obese rats

It is well known that adrenalectomy (ADX) reverses the eating and energy balance disturbances in a variety of models of obesity associated with elevated food intake. We have previously demonstrated enhanced functional activity in the small intestine of neonatally monosodium glutamate-treated (MSG) obese rats despite the absence of overeating and we concluded that these changes might also contribute to the development of MSG obesity. The objective of the present experiments was to investigate whether ADX would affect the small intestinal functions and whether their changes would counteract attenuation or prevention of obesity development in MSG rats. Therefore the investigation was carried out in MSG-obese Wistar male rats and untreated intact rats adrenalectomized on day 40, as well as in lean littermates of MSG rats and intact rats subjected to Sham-ADX surgery. All animals had free access to a standard pellet diet after weaning. At the age of 80 days, body mass, body fat content and food consumption as well as changes of the brush-border-bound duodenal and jejunal alkaline phosphatase (AP), the dipeptidyl(amino)peptidase IV (DPP IV) and maltase activity were measured. During the postoperative period, ADX resulted in a significant decrease of mass gain in both MSG and control rats (P<0.05). ADX fully prevented the development of obesity in MSG rats (significantly decreased epididymal+retroperitoneal fat pad mass, P<0.05) and increased mean daily food intake (P<0.001). These effects were only minimal in the ADX controls suggesting that enhanced adrenal secretion is involved in the expression of MSG obesity and its complications. The AP activity in obese MSG rats was increased by about 21 % (P<0.01) in both intestinal segments when compared to the lean controls, whereas no parallel variations in the activities of DPP IV and maltase were observed in the intestinal parts mentioned. In MSG rats, ADX significantly reduced the AP activity in the duodenum and jejunum (P<0.01). A similar tendency was also seen in the DPP IV activity of adrenalectomized MSG rats as well as in lean control rats. Nevertheless, no significant effect of adrenal withdrawal on maltase activity was found. These results indicate that the decrease of enzyme activities in the small intestine and the different effectiveness of nutrient absorption might be a significant factor preventing the development of excess adiposity in glutamate-treated rats. This information contributes to a better understanding of the importance of small intestinal function for the development of obesity and its maintenance in later life.     

Glucose tolerance and insulin action in monosodium glutamate (MSG) obese exercise-trained rats

The present study was designed to evaluate the effects of chronic aerobic exercise (swimming, 1h/day, 5 days/week, with an overload of 5% body weight) on glucose metabolism in obese male Wistar rats. Hypothalamic obesity was induced through administration of monosodium glutamate (MSG) at 4 mg/g of body weight every other day from birth to 14 days old. Fourteen weeks after drug administration, the rats were separated into two groups: MSG-S (sedentary) and MSG-T (swimming for 10 weeks). Rats of the same age and strain, receiving saline in place of MSG, were used as control (C), and subdivided into two groups: C-S and C-T. At the end of the experimental period, an oral glucose tolerance test was performed and serum glucose (AG) and insulin (AI) were evaluated. A constant for serum glucose decrease (Kitt) in response to exogenous insulin was calculated. Soleus muscle strips and adipose tissue samples were incubated and insulin stimulated glucose uptake determined. No differences were observed in AG among the 4 groups. MSG-S rats showed higher Al (418%) and lower Kitt (92.3%) than C-S rats. T-rats showed higher glucose uptake by muscle (224.0%) and adipose tissues (94.1%) than S-rats. Among trained rats, glucose uptake by muscle was higher in MSG-T (5.4%) than in C-T, while the opposite was observed in adipose tissue (39% higher in C-T). Chronic aerobic exercise was able to improve glucose tolerance and reduce insulin resistance in MSG-obese rats. These effects were associated to an increase in glucose uptake by muscle and adipose tissue in response to insulin.     

Elevated AT1 receptor protein but lower angiotensin II-binding in adipose tissue of rats with monosodium glutamate-induced obesity.

Age-related hypertrophy of adipose tissue has been associated with a significant decrease in the number of angiotensin II receptors. The aim of this study was to investigate the characteristics of angiotensin II receptors in hypertrophic adipose tissue in animal obesity model using rats postnatally treated with monosodium glutamate. Angiotensin II is known to induce hypertrophy in several tissues of the cardiovascular system and might do the same in fat tissue. The expression and binding properties of angiotensin II AT(1) receptors in epididymal fat tissue of adult rats were studied using membrane-binding, RT-PCR, and immunoblotting. The amount of AT(1) receptor mRNA did not differ significantly between obese and control rats. Despite that glutamate-treated rats displayed approximately 4-times more AT(1) receptor immunoreactive protein content in fat tissue cell membranes than the controls did. In contrast, binding experiments showed a significant (40.3 +/- 6.2 %) decrease of (125)I-Sar(1)-Ile(8)-angiotensin II-binding to fat tissue cell membranes in obese rats compared to controls. In conclusion, the present study provides evidence for the low binding properties associated with an accumulation of AT(1) receptor protein in cell membranes of the fat tissue of rats with glutamate-induced obesity. Discrepancies among angiotensin II-binding, AT(1) receptor protein, and AT(1) receptor mRNA levels indicate a possible defect in the receptor protein, which remains to be identified. The results obtained support a role of angiotensin II and AT(1) receptors in the pathogenesis of obesity.     

Lipid metabolism of monosodium glutamate obese rats after partial removal of adipose tissue

We analyzed the effects of partial fat pad removal on retroperitoneal and epididymal fat depots and carcass metabolism of control (C) and MSG-obese (M) rats. Three-month-old C and M male Wistar rats were submitted to either partial surgical excision of epididymal and retroperitoneal fat tissue (lipectomy, L) or sham surgery (S) and studied after 7 or 30 days. Retroperitoneal and epididymal tissue re-growth after lipectomy was not observed, as indicated by the low pads weight of the L groups. The lipolysis rate was stimulated in LC7 and LM7, probably due to surgical stress and low insulin levels. In LM7, but not in LC7, in vivo lipogenesis rate increased in retroperitoneal and epididymal fat tissue, as did the diet-derived lipid accumulation in epididymal fat tissue. Although these local increases were no longer present in LM30, this group showed a large increase in the percentage of small area adipocytes in both pads as well as increased carcass lipogenesis rate. The present data showed that the partial removal of fat depots affected the metabolism of control and MSG-obese rats differently. In the obese animals only, it stimulated both local and carcass lipogenesis rate as well as adipocyte differentiation, i.e. responses likely to favor excised tissue re-growth and/or compensatory growth of non-excised depots.     

Low number of insulin receptors but high receptor protein content in adipose tissue of rats with monosodium glutamate-induced obesity

In order to better understand the mechanisms leading to insulin resistance, the number of fat tissue insulin receptors, their affinity and insulin receptor protein in rats with monosodium glutamate-induced obesity were studied. Obese rats displayed significantly lower number of insulin receptors with high affinity. Surprisingly, the amount of insulin receptor protein was significantly elevated in these animals. The same relations have been already reported for angiotensin II binding and AT1 receptor protein in the same model of obesity. Therefore we suggest an existence of general defect of adipocyte cell membrane in monosodium glutamate-induced obesity characterized by the presence of high quantity of impaired receptor protein.     

Effect of fasting and refeeding on duodenal alkaline phosphatase activity in monosodium glutamate obese rats

In the present work the effects of fasting and refeeding on fat pad weight and alkaline phosphatase activity in the brush border of individual duodenal enterocytes have been evaluated in male Wistar rats with obesity induced by monosodium glutamate (MSG) treatment during the early postnatal period. Neonatal rats were treated subcutaneously with MSG (2 mg/g b.w.) or saline (controls) for 4 days after birth. At 4 months of age, two types of experiments were performed. In the first experiment rats, were submitted to 3 or 6 days lasting food deprivation. In the second experiment the rats were refed for 3 or 6 days ad libitum or restrictedly (60% of pre-fasting intake) after a 6 day-fasting period. Fasting and refeeding influenced the body fat and function of the duodenum in MSG-treated rats differently as compared to the controls. However, alkaline phosphatase activity and the weight of epididymal and retroperitoneal fat depots were significantly increased in MSG obese rats (P<0.001) during all the periods examined. While 3 days of food deprivation resulted in both groups in a similar loss of adipose tissue weight and alkaline phosphatase activity, the decrements of these parameters after 6 days of fasting were lower in obese rats suggesting that their capacity to spare body fat stores was enhanced. After 3 days of ad libitum refeeding, a more marked adaptational increase of food consumption and also a significantly increased alkaline phosphatase activity above the pre-fasting level (P<0.01) was observed in the MSG-treated rats. Consequently, a more rapid body fat restoration was demonstrated in these animals. Refeeding of rats at 60% of the pre-fasting intake level resulted in a significant increase of alkaline phosphatase activity in both the MSG and control group; moreover, as food restriction continued, MSG-treated rats tended to further increase the enzyme activity. Our results revealed that MSG treatment of neonatal rats may significantly change the intestinal functions. Permanently increased alkaline phosphatase activity observed in MSG obese rats during all investigated periods suggests that this functional alteration is probably not a consequence of actual nutritional variation but could be a component of regulatory mechanisms maintaining their obesity at critical values.     

Insulin secretion in monosodium glutamate (MSG) obese rats submitted to aerobic exercise training

The present study was designed to evaluate the effects of aerobic exercise training on glucose tolerance and insulin secretion of obese male Wistar rats (monosodium glutamate [MSG] administration, 4 mg/g-body weight, each other day, from birth to the 14th day). Fourteen weeks after the drug administration, the rats were separated into two groups: MSG-S (sedentary) and MSG-T (T = swimming, 1 h/day, 5 days/week, with an overload of 5% body weight for 10 weeks). Rats of the same age and strain injected with saline were used as control (C) and subdivided into two groups: C-S and C-T. Insulin and glucose responses during an oral glucose tolerance test (GTT) were evaluated by the estimation of the total areas under serum insulin (AI) and glucose (AG) curves. Glucose-induced insulin secretion by isolated pancreatic islets was also evaluated. MSG-S rats showed higher AI than C-rats while MSG-T rats presented lower AI than MSG-S rats. No differences in AG were observed among the 4 groups. Pancreatic islets from MSG-rats showed higher insulin secretion in response to low (2.8) and moderate (8.3 mM) concentrations of glucose than those from their control counterparts and no differences were observed between MSG-S and MSG-T rats. These results provide evidences that the hyperinsulinemia at low or moderate glucose concentrations observed in MSG-obese rats is, at least in part, a consequence of direct hypersecretion of the B cells and that chronic aerobic exercise is able to partially counteract the hyperinsulinemic state of these animals without disrupting glucose homeostasis.     

APM/CD13 and FOS in the hypothalamus of monosodium glutamate obese and food deprived rats

Protein (western blotting) and gene (PCR) expressions, catalytic activity of puromycin-insensitive membrane-bound neutral aminopeptidase (APM/CD13) and in situ regional distribution of CD13 and FOS immunoreactivity (ir) were evaluated in the hypothalamus of monosodium glutamate obese (MSG) and/or food deprived (FD) rats in order to investigate their possible interplay with metabolic functions. Variations in protein and gene expressions of CD13 relative to controls coincided in the hypothalamus of MSG and MSG-FD (decreased 2- to 17-fold). Compared with controls, the reduction of hypothalamic CD13 content reflected a negative balance in its regional distribution in the supraoptic, paraventricular, periventricular and arcuate nuclei. CD13-ir increased in the supraoptic nucleus in MSG (2.5-fold) and decreased in the paraventricular nucleus (2-fold) together with FOS-ir (1.5-fold) in FD. In MSG-FD, FOS-ir decreased (7-fold) in the paraventricular nucleus, while CD13-ir decreased in the periventricular (5.6-fold) and the arcuate (3.7-fold) nuclei. It was noteworthy that all these changes of CD13 were not related to catalytic activity of APM. Data suggested that hypothalamic CD13 plays a role in the regulation of energy metabolism not by means of APM enzyme activity.     

Examination of brown adipose tissue thermogenesis of glutamate-induced obese rats by differential temperature measurements.

Differential temperature measurement between interscapular brown adipose tissue (BAT, Tbat), rectum (Trect) and a subcutaneous point in the back left of the vertebral column (Tsc) was useful for examination of BAT-thermogenesis in glutamate-induced obese Wistar-rats. Positive temperature gradients Tbat-Tsc pointed to a basal BAT-thermogenesis, whereas negative temperature gradients Tbat-Trect did not indicate that heat production in lean and obese rats. One may conclude from this, that inclusion of subcutaneous points outside the BAT improves sensitivity of differential temperature measurements for BAT-thermogenesis. Basal temperatures Tbat, Trect and Tsc were reduced in obese rats compared to lean rats, although thermoinsulation of obese rats is improved on account of their high fat content. This points to a diminished heat production in obese rats. Cold exposure at 4 degrees C elicited an increase of temperature gradients Tbat-Trect in lean as well as in obese rats, with positive values found only in lean rats. However, positive values Tbat-Tsc were calculated for both groups. Increases were noted only in lean rats. Injection of noradrenaline (0.5 mg/kg i.m.) was followed by positive temperature gradients Tbat-Trect and increased positive values for Tbat-Tsc, pointing to a remarkable activation of BAT-thermogenesis in lean and obese rats. These findings confirm, that glutamate-induced obese rats preserved the ability to activate BAT-thermogenesis. There were, however, hints of reduced heat production in BAT of obese rats, thus contributing to obesity despite normophagia.     

Decreased epoxygenase and increased epoxide hydrolase expression in the mesenteric artery of obese Zucker rats

Previous studies suggest that epoxyeicosatrienoic acids (EETs) are vasodilators of the mesenteric artery; however, the production and regulation of EETs in the mesenteric artery remain unclear. The present study was designed 1) to determine which epoxygenase isoform may contribute to formation of EETs in mesenteric arteries and 2) to determine the regulation of mesenteric artery cytochrome P-450 (CYP) enzymes in obese Zucker rats. Microvessels were incubated with arachidonic acid, and CYP enzyme activity was determined. Mesenteric arteries demonstrate detectable epoxygenase and hydroxylase activities. Next, protein and mRNA expressions were determined in microvessels. Although renal microvessels express CYP2C23 mRNA and protein, mesenteric arteries lacked CYP2C23 expression. CYP2C11 and CYP2J mRNA and protein were expressed in mesenteric arteries and renal microvessels. In addition, mesenteric artery protein expression was evaluated in lean and obese Zucker rats. Compared with lean Zucker rats, mesenteric arterial CYP2C11 and CYP2J proteins were decreased by 38 and 43%, respectively, in obese Zucker rats. In contrast, soluble epoxide hydrolase mRNA and protein expressions were significantly increased in obese Zucker rat mesenteric arteries. In addition, nitric oxide-independent dilation evoked by acetylcholine was significantly attenuated in mesenteric arteries of obese Zucker rats. These data suggest that the main epoxygenase isoforms expressed in mesenteric arteries are different from those expressed in renal microvessels and that decreased epoxygenases and increased soluble epoxide hydrolase are associated with impaired mesenteric artery dilator function in obese Zucker rats.     

Decreased rate of fatty acid synthesis in brown adipose tissue of hypothalamic obese rats

Intranuclear coinjection of the late SV40 KpnI/BclI DNA fragment and the early promotor/enhancer HpaII/BglI DNA segment into permissive monkey and non-permissive mouse cells allows late SV40 gene expression without T-antigen synthesis and DNA replication. These conditions were chosen to analyse the effect of DNA methylation on V-antigen synthesis detached from the process of DNA replication. We found that in vitro methylation of a single cytosine nucleotide proximal to the major late mRNA cap site by the HpaII methylase does not block capsid protein synthesis. This result is in contrast to reported data obtained in Xenopus laevis oocyte injection experiments [(1982) Proc. Natl. Acad. Sci. USA 79, 5142-5146].     

Insulin secretion and acetylcholinesterase activity in monosodium l-glutamate-induced obese mice.

OBJECTIVE: Pancreatic islets isolated from mice treated neonatally with monosodium L-glutamate (MSG) were used to study insulin secretion. MATERIALS AND METHODS: Total acetylcholinesterase (AchE) activity of tissue extract was measured as a cholinergic activity marker. Obesity recorded in 90-day-old MSG mice (OM) by Lee index reached 366.40 +/- 1.70, compared to control mice (CM) 324.40 +/- 1.10 (p < 0.0001). Glucose 5.6 mM induced insulin secretion of 36 +/- 5 pg/15 min from islets of CM and 86 +/- 13 from OM (p < 0.001). When glucose was raised to 16.7 mM, islets from OM secreted 1,271 +/- 215 and 1,017 +/- 112 pg/30 min to CM. AchE activity of pancreas from OM was 0.64 +/- 0.02 nmol of substrate hydrolyzed/min/mg of tissue and 0.52 +/- 0.01 to CM (p < 0.0001). Liver of obese animals also presented increase of AchE activity. RESULTS: These indicate that OM insulin oversecretion in low glucose may be attributed, at least in part, to an enhancement of parasympathetic tonus.     

The neuroprotective effects of PACAP in monosodium glutamate-induced retinal lesion involve inhibition of proapoptotic signaling pathways

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present in the retina and exert several distinct functions. PACAP has well-known neuroprotective effects in neuronal cultures in vitro and against different insults in vivo. Recently we have shown that PACAP is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. In the present study we investigated the possible signal transduction pathways involved in the protective effect of intravitreal PACAP administration against apoptotic retinal degeneration induced by neonatal MSG treatment. MSG induced activation of proapoptotic signaling proteins and reduced the levels of antiapoptotic molecules in neonatal retinas. Co-treatment with PACAP attenuated the MSG-induced activation of caspase-3 and JNK, inhibited the MSG-induced cytosolic translocation of apoptosis inducing factor (AIF) and cytochrome c, and increased the level of phospho-Bad. Furthermore, PACAP treatment alone decreased cytosolic AIF and cytochrome c levels, while PACAP6-38 increased cytochrome c release, caspase-3 and JNK activity and decreased phospho-Bad activity. In summary, our results show that PACAP treatment attenuated the MSG-induced changes in apoptotic signaling molecules in vivo and suggest that also endogenously present PACAP has neuroprotective effects. These results may have further clinical implications in reducing glutamate-induced excitotoxicity in several ophthalmic diseases.     

Effect of cholecystokinin on feeding is attenuated in monosodium glutamate obese mice

Treatment of newborn mice with monosodium glutamate (MSG) is neurotoxic for hypothalamic arcuate nucleus (ARC) and causes obesity. In the MSG-treated 16-week-old NMRI mice, we detected specific ablation of ARC neuronal cells, 8 times higher fat to body mass ratio but unchanged body mass compared to controls, advanced hyperglycemia and hyperinsulinemia--both more pronounced in males, and hyperleptinemia--more severe in females. After fasting, the MSG-treated mice showed attenuated food intake compared to controls. Cholecystokinin octapeptide, which decreased food intake in a dose-dependent manner in 24 h fasted controls, did not significantly affect food intake in the MSG-treated animals. We propose that the obesity-related changes in the feeding behavior of the MSG-treated obese mice were the result of missing leptin and insulin receptors in ARC and consequent altered neuropeptide signaling. This makes the MSG model suitable for clarifying generally the central control of food intake.     

Decreased plasma leptin levels in lean and obese Zucker rats after treatment with the serotonin reuptake inhibitor fluoxetine.

Leptin inhibits feeding, stimulates thermogenesis and decreases body weight. Serotonin reduces food intake when injected into the hypothalamus and may interact with other neurotransmitters in the control of appetite. We therefore investigated the effects of the serotonergic drug fluoxetine, which inhibits serotonin reuptake, on food intake and plasma leptin levels in lean and obese Zucker rats. Fluoxetine significantly reduced food intake in lean and obese rats, both acutely after a single injection (10 mg/kg) and during continuous subcutaneous infusion (10 mg/kg/day for 7 days). Plasma leptin levels were reduced after both 4 hours and 7 days of fluoxetine administration in lean and after 7 days in fatty rats (all p<0.01). We have previously suggested that serotonin may decrease food intake by inhibiting neuropeptide Y neurones, and we further suggest that it also inhibits leptin, possibly by an effect on white adipose tissue.     

Type 2 diabetes mellitus in obese mouse model induced by monosodium glutamate.

The number of diabetic patients is increasing every year, and new model animals are required to study the diverse aspects of this disease. An experimental obese animal model has reportedly been obtained by injecting monosodium glutamate (MSG) to a mouse. We found that ICR-MSG mice on which the same method was used developed glycosuria. Both female and male mice were observed to be obese but had no polyphagia, and were glycosuric by 29 weeks of age, with males having an especially high rate of incidence (70.0%). Their blood concentrations of glucose, insulin, total cholesterol, and triglycerides were higher than in the control mice at 29 weeks. These high concentrations appeared in younger males more often than in females, and were severe in adult males. Also, the mice at 54 weeks of age showed obvious obesity and increased concentrations of glucose, insulin, and total cholesterol in the blood. The pathological study of ICR-MSG female and male mice at 29 weeks of age showed hypertrophy of the pancreatic islet. This was also observed in most of these mice at 54 weeks. It was recognized as a continuation of the condition of diabetes mellitus. From the above results, these mice are considered to be useful as new experimental model animals developing a high rate of obese type 2 (non-insulin dependent) diabetes mellitus without polyphagia.     

Discrimination between the tastes of sucrose and monosodium glutamate in rats

Conditioned taste aversion studies have demonstrated that rats conditioned to avoid monosodium glutamate (MSG) with amiloride added to reduce the intensity of the sodium component of MSG taste, will generalize an aversion for MSG to sucrose and vice versa. This suggests that taste transduction for sodium, sucrose and MSG may intersect at some point. Generalization of conditioned taste aversion indicates that two substances share similar taste features, but it does not reveal the extent of their differences. In this study, we tested how well rats can discriminate sucrose and MSG under a variety of conditions. Water-deprived rats were trained on a combination of water reinforcement and shock avoidance to discriminate between MSG and sucrose, both with and without amiloride, and with and without equimolar NaCl in all solutions. In the absence of amiloride, rats reliably distinguished between MSG and sucrose down to 10 mM solutions. However, they could correctly identify solutions only above 50 mM in the presence of amiloride, equimolar sodium chloride, or both. These results suggest that gustatory stimulation by MSG and sucrose interact somewhere in taste transduction, perhaps within taste receptor cells or gustatory afferent pathways.     

Rats with monosodium glutamate-induced obesity and insulin resistance exhibit low expression of Galpha(i2) G-protein

In order to test the potential role of inhibitory G-proteins in mechanisms of insulin resistance in adipose tissue of obese animals we determined the content of Galpha(i1) and Galpha(i2) proteins and an extent of protein tyrosine phosphorylation in epididymal fat tissue cell membranes using immunoblot. Monosodium glutamate-induced obese rats displayed adipose tissue hypertrophy, elevated levels of insulin, leptin and slightly elevated serum glucose. We found significantly decreased protein content of Galpha(i2) in adipose tissue plasma membranes of obese rats. This was in accordance with lower protein tyrosine phosphorylation noticed in adipose tissue cell homogenate of glutamate-treated animals. Our results confirm the role of Galpha(i2) in development of insulin resistance by crosstalk between the reduced level of inhibitory G-protein and insulin receptor mediated most likely by activation of phosphotyrosine protein dephosphorylation.