Effect of Experimental Diabetes and Vagotomy on Gastric Emptying in Rats
NALIN MEHTA*, SUSHEEL AVELIATH AND D. P. THOMBRE
Department of Physiology,
JIPMER, Pondicherry – 605 006.
(Received on May 4, 2002)

Abstract: The present study was performed to evaluate the rate of whole stomach emptying (WSE) in streptozotocin (STZ) induced rat model of diabetes mellitus by assessing the effect of bilateral gastric vagotomy in control as well as in experimental animals. Our results revealed that delayed emptying of solid foods by the stomach is demonstrated within 5 days of induction of diabetes in rats. After bilateral vagotomy, the later stages of WSE slowed down because of the diabetes state, most probably due to the direct effect of diabetes on the gastric antrum.

Key words:     Vagotomy                                            Whole stomach emptying

proximal stomach                                  distal stomach              

STZ induced diabetes mellitus

Introduction
Methods
Results
Discussion
References

INTRODUCTION

There are conflicting reports about disturbances in gastrointestinal functions in insulin dependent diabetes (IDDM). Gastroparesis (1,2,3,4,5) as well as normal gastric emptying in diabetes (6) have been documented. Rapid gastric emptying too has been encountered though it has been found to occur much less frequently than delayed emptying (7, 8). Delayed gastric emptying in IDDM (9) of solids has been reported (1, 2) but this does not seem to affect liquid foods (1); in fact, liquids at times show accelerated emptying (10). The pathophysiology of this disorder, however, is poorly understood (11,12). Besides microangiopathy, electrolyte imbalance and altered hormonal production, autonomic microangiopathy is implicated in disabetic dysmotility.

Based on studies using the model of experimental obesity it has been suggested that the gastric emptying is accelerated in rats, and this has been related to hyperphagia. Faster rates of gastric emptying were demonstrated in animals after insulin administration (13) and in patients with hypoglycemia (14). Prolongation of gastric emptying has also been reported in patients after vagotomy (15) and hyperglycemia (6, 16, 17). 

Tone and motility of the fundus help mainly in liquid emptying (18) while the distal stomach including the antrum facilitate the propulsion of solid foods (19). With this presumption, early and late stages of gastric emptying should reflect closely the motor functions of the proximal and distal stomach.

The present study was performed to determine the status of gastric emptying of solid foods in streptozotocin (STZ) induced diabetes mellitus and to evaluate the role of the vagus, in the control as well as diabetic groups of animals with regard to gastric motility using the experimental model of vagotomy. 
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METHODS

Animals

Adult female Wistar rats (n =64, body weight = 132.87 ± 8.82 g) were allowed to acclimatize to the laboratory environment and to the housing pattern of four rats to a cage for a period of two weeks prior to the commencement of the experiment. Rat chow (Hindustan lever) and water were provided ad libitum. The animals were assigned to: control  [C] Non-diabetic group (n=32) and Diabetic [D] group (n=32).

Induction of diabetes with STZ

Streptozotocin (STZ, Sigma Chemicals, St. Louis, MO) 1% solution in citrate buffer pH 4.5, was administered at 50 mg/ kg body weight, i.p. (20). Blood samples of all the animals were collected from the orbital sinus using heparinized capillary tubes and the blood-glucose levels were estimated by Nelson-Somogui method. In diabetic animals, the estimation was performed on the 5^th day after the administration of STZ.

Vagotomy

This was performed through a linear midline abdominal incision. The vagi were cut below the level of the diaphragm, near the cardiac sphincter of the stomach. Completeness of vagotomy was tested by stimulating the esophagus with interrupted pulses and observing gastric motility (21). Following vagotomy, the rats were housed singly and were allowed to recover from surgery. Sixteen rats from the control group and an equal number of rats from the diabetic group were subjected to bilateral gastric vagotomy under light ether anesthesia.

Gastric emptying studies

The rats were given 2.0 grams of dry pellets following food deprivation for a period of 6 hours. All the rats consumed the meal within 30 minutes and 120 minutes after the end of the meal. These studies were performed during the day between 14.00 and 16.30 hours.

Collection of gastric contents

Through a linear midline abdominal incision in ether anaesthetized rats, the stomach was carefully dissected out after ligating the lower end of the esophagus and the first part of the duodenum. The stomach was cut above the end of the esophagus and below the first part of the duodenum. The stomach was cut along the greater curvature and the contents were collected in a petri dish, using double distilled water to wash all the contains in to the petri dish. The contents were then transferred into small bottles and dried in an incubator at 40 deg C for 24 hours and then weighed. The contents were weighed thrice for concordancy and the weight of the dry solid food gave the quantity of food remaining in the stomach at the time of sacrifice.

The rats subjected to vagotomy were rendered diabetic (STZ administration) on the tenth post-operative day and sacrificed for collection of gastric contents after blood samples were taken for blood-glucose estimation on the fifth day following STZ administration.

Values of the quantity of blood remaining in the stomach at the end of 30 minute intervals till at the end of 120 minutes were obtained in the various groups of experimental and control rats. Each timed group consisted of four to six animals. From the values, the following parameters of whole stomach emptying (WSE) were derived:- 

(1)   at 30, 60, 90 and 120 minutes (RE 30, 60, 90 and 120).

(2)   Quantity and percentage of food remaining in the stomach at the end of the above mentioned time intervals (R% 30, 60, 90 and 120).

(3)   Rate of WSE between 0-30, 30-60, 60-90 and 90 to 120 minutes intervals by taking in to account the food emptied during these periods (RE 30,60,90 and 120).

(4)   Expected time of 50% gastric emptying (T 50%). 

The body weights of all the animals were monitored weekly, before and after experimental intervention. 

Statistical analysis 

The statistical analysis was done using Students ‘t’ test. P values less than 0.05 were considered significant. 
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RESULTS 

The rate of emptying in the different groups of rats at half hourly intervals is given is Table I. When WSE was calculated in the control [C] rats, is was seen that the emptying of solid foods followed a linear pattern where the rates increased progressively with time (Table I), and there was also a linear decrease in the quantity of food remaining in the stomach with the passage of time (Table III). When the rates of emptying (RE) were derived at 30 minute intervals it was observed that the RE slow down at 60 and 90 minutes, whereas the early and late phases showed a sharp increase (Table II). 

Vagotomy in control animals [CV] markedly reduced WSE. The large quantity of food remaining at the end of 30 and 120 minutes indicated that the removal of parasympathetic effect delayed both, the early and the late phases of gastric emptying. The rate of WSE in STZ induced diabetes in rats was reduced during the early stages as indicated by the food remaining in the stomach at the end of 30 minutes (p<0.001). At the end of 90 minutes there was no significant change, followed once again by prolongation of the emptying time at the end of 120 minutes (p<0.05). Neither RE 60 nor RE 90 showed any significant change from the control values.  

TABLE 1: Gastric emptying results

Table II: Percentage of emptying. (Calculated from the amount of food remaining in and emptied from the stomach at the beginning and end of half hourly intervals)

Table III : Food remaining in the stomach. (Figures in parenthesis indicate the percentage) 

Comparision of the diabetic vagotomised rats [DV] with the diabetic rats with intact vagi [D] showed that the diabetic state did not significantly inhibit WSE in a denervated (vagotomized) stomach except at the end of 120 minutes since the food remaining in the stomach was significantly (p<0.05) more than that remaining in the diabetic rats [D] with intact vagi (Table III). 

On comparing the different parameters in the CV  and the DV groups, it became evident that both these groups exhibited almost similar rates of emptying. It was only towards the end, in the late stages of WSE, that the DV rats group showed a significant (P<0.05) slowing in the rate of emptying (Table II). 

Blood glucose levels were almost five times higher in the diabetic rats as compared with the controls. Both these exhibited almost similar rates of emptying. It was only towards the end, in the late stages of WSE, the DV rats group showed a significant (P<0.05) slowing in the rate of emptying (Table I). 

During the course of the study there was a decrease in the body weight of the diabetic rats though not statistically significant. No animal, however, gained weight after the induction of diabetes. The control animals did gain weight while the vagotomized controls either maintained their body weight or showed a slight decrease. 
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DISCUSSION 

In the present study we investigated the rate of gastric emptying in STZ-induced rat model of diabetes mellitus, by determining the effect of bilateral gastric vagotomy in control as well as in diabetic animals. The data collected and derived in both, control and experimental animals is interpreted in relation to gastric activity. The first record of the food remaining in the stomach was obtained at the end of 30 minutes. Hence, the time lag of the ingested meal is not available. In rats, where the habit of nibbling is observed, the emptying should commence much earlier (22). Food retained in the stomach at the end of the first 30 minutes corresponds roughly to the activity of the proximal stomach. Our technique has the drawback of an inability to directly assess the time required for the redistribution of the meal, and for the meal to move into the distal stomach. It has however, been reported in patients with diabetes mellitus, that there is a poor relationship between solid and liquid gastric emptying and the intragastric meal distribution is frequently abnormal (23). However, the degree of retention at the end (120 minutes) of the study in a group denotes the status of antral and distal gastric activity.

Monitoring of gastric emptying throughout the period of the study in the same animal was not feasible as our method estimated the gastric contents at varying intervals in the different groups. To overcome this shortcoming, derived values for rate of emptying were considered adequate, since all the groups were matched in body weight and the values of food retained in the stomach at different time intervals showed minimum standard error in the animals belonging to a particular group.

Diabetes induced with STZ decreases the WSE, with delay being more prominent in the earlier than in the later phases of gastric emptying. This finding is similar to reports of gastroparesis confirmed by radiological, manometric and other techniques in patients of diabetes mellitus (1,3,24). This delay is possibly due to impaired activity of proximal and distal stomach as supported by earlier studies (4, 19). A similar study in IDDM patients provided evidence that lack of antral myoelectric activity may inhibit emptying of solids. Other factors that can contribute to this, in our study, are insulin lack and hyperglycemia. Insulin stimulates gastric emptying (13) and hyperglycemia alters vagal discharge (25) in rats and inhibits antral tone and facilities pyloric activity in man. (16).

Acute hyperglycemia itself is an important mechanism for the delay of solid gastric emptying in STZ induced diabetic rats (26). It has also been reported in STZ induced diabetic rats that diabetes is associated with an early and specific alteration of the muscarinic control of contraction of antral smooth muscles at a post-synaptic level, associated with an alteration of the GPT-binding proteins coupled to muscarinic receptors (27).

The effect of vagotomy in control rats substantiated the role of the vagus in gastric emptying as observed previously in various animals and human studies (11, 15, 28). Our results demonstrate that the diabetic state did not additionally influence the gastric slowing after vagotomy.  Significant retention in the DV group in the last phase demonstrates the deleterious effect diabetes has on the denervated antrum, thus providing evidence that vagal neuropathy can be ruled out as a causative factor. Indeed, vagal neuropathy is highly unlikely as the duration of diabetes was very short (29).

Sensory and motor denervation of the stomach by vagotomy is often the cause of post-operative hypophagia, lowered body weight and disordered drinking (30,31,32,33). It is also accompanied by changes in pancreatic insulin and glucagons (34) Gastric hypotonia is possibly due to lack of cholinergic neurotransmission, as cholinergic drugs restore the motor activity after vagotomy (1,11). Hyperglycemia after vagotomy may directly influence the activity of the stomach (16,35,36). In another interesting study the role of Cholinergic and peptidergic pathways in the impairment of gastric motility associated with diabetes, at the post-synaptic level in isolated gastric fundus smooth muscle strips was assessed and the findings indicated that the smooth muscle dysfunction evoked by STZ induced diabetes in rats, causing diminished contractions in response to carbachol and galanin is at least partly attributable to increased nitric oxide (NO) synthesis (37). Alterations in signal transduction as evidenced by the expression of GTP-binding proteins at downstream receptors in gastric myocytes, resulting in the impairment gastric motility has been established in STZ induces diabetic rates as well as in spontaneous diabetic (WBN/Kob) rats, although the underlying mechanisms were slightly different (38). This study, however, addresses the mechanisms of impaired gastric motility in experimental diabetes of long standing duration.

Although the WSE was found to increase progressively with time in all the groups, the RE at 60 minutes was the lowest and almost identical in all the groups. This can be explained by the effect of the feedback mechanisms initiated in the intestine. Neural as well as hormonal mechanisms are possibly not changed quantitatively in diabetic rats. Post-prandial release of gastrointestinal (GI) hormones like Cholecystokinin, Gastric Inhibitory Polypeptide, Substance P and Somatostain (36, 39) may suppress the activity of the stomach. A number of reports suggest that the production of the GI hormones may be affected in the diabetes (6,39). Vasoactive intestinal polypeptide (VIP) is an inhibitory non-adrenergic, non-cholinergic transmitter, which mediates the relaxation of sphincters of the gastrointestinal tract. Low tissue level of VIP in the gastroduodenal tract of diabetic rat has been reported and this may contribute in part to the abnormal gut motility (40). In the eventuality of such a situation, the effect will be manifested on WSE. It is therefore difficult to exclude change in the hormonal and local neural mechanisms affecting WSE in our study.

In our earlier work, we have demonstrated the inhibition of gastric acid secretion in STZ induced diabetic rats (41). Accelerated gastric emptying has been documented in hypochlorhydria (42). In our study, hypochlorhydria as a result of STZ induced diabetes is not associated with accelerated WSE.

To summarize, in early STZ-induced diabetes, the daytime emptying of solid food decreases. This is not as a result of autonomic neuropathy as has commonly been attributed to b the cause of delayed gastric emptying in diabetes. The direct toxic effect of STZ on the gastric smooth muscle resulting in decreased WSE can also be ruled out in our study since impaired gastric motility resulting from changes in the profile of GI hormones and not due to the toxic effects of STZ has been established elsewhere, by injecting nicotinamide prior to the administration of STZ (37, 38). There is emerging evidence now, however, that hyperglycemia and mechanisms involving local hormones/factors including nitric oxide (NO) may be responsible for the delayed WSE in early diabetes while neuropathy and alterations in GTP-binding proteins (Gsalpha & Gq/11 alpha) could be the cause of delayed gastric emptying in diabetes of long standing duration.
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