Altered Glucose and Insulin Responses to Brain Medullary Thyrotropin-R
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《内分泌学杂志》
Center for Ulcer Research and Education: Digestive Diseases Research Center, Department of Medicine, Division of Digestive Diseases and Brain Research Institute, University of California, Los Angeles
Department of Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073
Abstract
Insulin secretion is impaired in type 2 diabetes (T2D). The insulin and glucose responses to central autonomic activation induced by excitation of brain medullary TRH receptors were studied in T2D Goto-Kakizaki (GK) rats. Blood glucose levels in normally fed, pentobarbital-anesthetized GK and nondiabetic Wistar rats were 193 and 119 mg/100 ml in males and 214 and 131 mg/100 ml in females. Intracisternal injection (ic) of the stable TRH analog RX 77368 (10 ng) induced significantly higher insulin response in both genders of overnight-fasted GK rats compared with Wistar rats and slightly increased blood glucose in female Wistar rats but significantly decreased it from 193 to 145 mg/100 ml in female GK rats. RX 77368 (50 ng) ic induced markedly greater glucose and relatively weaker insulin responses in male GK rats than Wistar rats. Bilateral vagotomy blocked ic RX 77368-induced insulin secretion, whereas adrenalectomy abolished its hyperglycemic effect. In adrenalectomized male GK but not Wistar rats, ic RX 77368 (50 ng) dramatically increased serum insulin levels by 6.5-fold and decreased blood glucose levels from 154 to 98 mg/100 ml; these changes were prevented by vagotomy. GK rats had higher basal pancreatic insulin II mRNA levels but a lower response to ic RX 77368 (50 ng) compared with Wistar rats. These results indicate that central-vagal activation-induced insulin secretion is susceptible in T2D GK rats. However, the dominant sympathetic-adrenal response to medullary TRH plays a suppressing role on vagal-mediated insulin secretion. This unbalanced vago-sympathetic activation by medullary TRH may contribute to the impaired insulin secretion in T2D.
Introduction
ABNORMAL INSULIN SECRETION and synthesis are key factors in the pathophysiology of type 2 diabetes (T2D) (1). Pancreatic endocrine secretion is regulated by the central nervous system through rich innervation of vagal and sympathetic nerves in the islets (2, 3). Insulin secretion is stimulated by vagal activation and inhibited by sympathetic-adrenal activation (4, 5). Both systems participate in meal-induced insulin secretion but only the vagus-cholinergic component plays a major role in insulin secretion of the cephalic phase and during the early absorption period (6, 7). Maintaining normal glucose tolerance requires the integrity of vagal function (8). Therefore, defects in the autonomic control of pancreatic -cell function might contribute to the relatively insufficient insulin secretion in T2D. Although ample knowledge has been achieved in the cause, prevention, and repair of islet -cell damage and diminished insulin secretion in diabetes at the cellular and molecular levels (9), the altered autonomic regulation of pancreatic insulin secretion in T2D is still poorly understood.
TRH is a neuropeptide synthesized in brain medullary caudal raphe nuclei and the parapyramidal regions. These nuclei send TRH-containing nerve projections to innervate the dorsal vagal complex (DVC), composed of the dorsal motor nucleus of the vagus and the nucleus tractus solitarii, as well as the ventrolateral reticular formation of the medulla and the intermediolateral cell column of the spinal cord (10, 11). It was found in the 1980s that an intracerebroventricular injection of TRH induces hyperglycemia through pathways involving the adrenal gland; however, intracerebroventricular TRH also prevents central and peripheral stimuli-induced hyperglycemia by stimulating insulin release in rats and mice (12, 13, 14). Studies in the last 20 yr have well established that brain medullary TRH plays a physiological role in the autonomic regulation of visceral functions (15). The DVC contains dense TRH-immunoreactive nerve terminals and TRH receptors (16, 17). TRH or its stable analog RX 77368 injected intracisternally (ic) or microinjected into the DVC or TRH endogenously released into the DVC after chemical stimulation of cell bodies in the raphe nuclei increases vagal efferent discharge, induces vagally mediated activation of enteric neurons and increases of gastric secretion and motility (18, 19, 20, 21, 22), and stimulates pancreatic insulin secretion (23). Convincing findings demonstrated that ic TRH or its analog induces centrally initiated vagal and sympathetic activation that mimics physiological or pathophysiological processes (15).
In this study, we tested the hypothesis that autonomic regulation of pancreatic insulin secretion and synthesis are impaired in T2D. We compared blood glucose and pancreatic insulin responses to ic injection of the stable TRH analog, RX 77368, between the nondiabetic Wistar rats and Goto-Kakizaki (GK) rats, the genetically determined nonobese T2D model with impaired insulin response to glucose (24, 25). Surgical approaches were used to determine the mediation of vagal and adrenal-sympathetic pathways in ic TRH analog-induced glucose and insulin changes in the two rat strains and the imbalance of vagal-sympathetic activation in the T2D GK rats.
Materials and Methods
Animals
The GK rats were bred in Animal Facilities of the Veterans Affairs (VA) Greater Los Angeles Area Healthcare System with approved protocol and used at the age of 3 months when body weight was 240–270 g (male) or 180–220 g (female), except in one experiment specified in Results that used 9-month-old female rats. The age- and sex-matched control Wistar rats were purchased from Harlan Laboratory (San Diego, CA) and raised in VA Animal Facilities for 1 wk before the experiments. The body weight of GK rats were about 10–20 g lower than the age-matched Wistar rats, as previously reported (26). The rats were housed under controlled conditions (21–23 C, lights on from 0600–1800 h) with free access to standard rat chow (Prolab Lab Diet; PMI Nutrition International, Brentwood, MO) and tap water. Food, but not water, was removed 16 h before most experiments, except for one group of Wistar and one group of GK rats that were used to obtain basal glucose and insulin levels in normally fed conditions. Animal protocols were approved by the University of California, Los Angeles, Office for the Protection of Research Subjects and VA Greater Los Angeles Area Healthcare System Animal Committee.
Experimental protocol
All experiments were performed between 0900 am and 1500 h. Rats were anesthetized with ip pentobarbital (Abbott Laboratories, North Chicago, IL) (50 mg/kg followed by 20 mg/kg each hour until the end of experiments) to avoid surgery-, ic injection-, and blood samplings-induced stressful influence on glycemic regulation that is usually inevitable in conscious animals. Previous studies have shown that pentobarbital anesthesia has slight to moderate influence on insulin output, glucose production, hepatic insulin resistance, and pancreatic blood flow (27, 28, 29). However, other anesthesia, such as Hypnorm, urethane, or ketamine, can cause strong changes in autonomic activity (30, 31, 32) and insulin resistance (33) or induce hyperglycemia (34, 35, 36). Thus, pentobarbital is relatively less potent in influencing glucose metabolism (34) and has little effect on mean arterial blood pressure, rectal temperature, and brain c-fos expression (32, 37).
A PE-50 cannula was inserted into the external iliac vein for blood sampling. Basal blood samples (0.2 ml/rat) were collected at 30 min after the iv cannulation. In some groups of rats, bilateral cervical vagotomy, bilateral adrenalectomy, both of the surgeries, or the corresponding sham operation was performed immediately after the iv cannulation. In these groups, the basal blood samples were collected after a 60-min postsurgery stabilization period. After the basal blood sampling, each rat was positioned on a stereotaxic instrument (Kopf model 900) and received an ic injection of either physiological saline (vehicle, 10 μl) or RX 77368 (Ferring Pharmaceuticals, Felthan, Middlesex, UK) (10 or 50 ng/10 μl), as performed in our previous studies (22, 38). Blood samples (0.2 ml) were collected at 30, 60, 90, and 120 min after the ic injection. In another two groups of Wistar rats and two groups of GK rats each receiving ic saline or RX 77368 (50 ng), respectively, the pancreas was collected at 120 min after the ic injection for Northern blot analysis of pancreatic insulin II mRNA levels.
Measurement of blood glucose and serum insulin levels
Blood glucose levels were measured by One Touch Ultra Blood Glucose Monitoring System (Lifescan, Milpitas, CA) and serum insulin by rat insulin RIA kit (catalog item RI-13K; Linco Research, St. Charles, MO).
Northern blot analysis of pancreatic insulin II mRNA
Pancreatic total RNA from each sample was extracted with standard RNAzol method using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA). Total RNA (20 μg) was run on agarose gel containing MOPS and formaldehyde (Sigma Chemical Co., St. Louis, MO) and then transferred to a nylon membrane by UV cross-linking. The insulin II DNA probe was prepared from RT (Ambion, Austin, TX)-PCR (QIAGEN, Valencia, CA). The sequence of insulin II primer was designed using Primer Express software (accession J04807). The forward insulin II primer (5'–3') sequence was CCTAAGTGACCAGCTACA; the reverse primer (5'–3') sequence was GTAGTTCTGCAGTTGGTA. The size of the PCR product was 367 bp. DNA fragments from PCR were run on low-melting agarose gel, extracted, and then purified by Wizard PCR Preps DNA purification system (Promega, Madison, WI). Purified PCR product was labeled with [-32P]dCTP (ICN Pharmaceuticals Inc., Bryan, OH) using Random Primer DNA Labeling Systems (New England BioLabs, Inc., Beverly, MA). After labeling, probes were purified using QIAquick Nucleotide Removal Kit (QIAGEN). The hybridization was carried out overnight at 68 C.
Statistical analysis
Data are expressed as mean ± SEM of each experimental group. Statistical comparisons among multiple group mean values were performed using two-way or one-way ANOVA followed by Dunn’s method. Comparisons between group mean values of Wistar and GK rats receiving the same treatment or between RX 77368-treated and saline-treated rats of the same strain were performed using unpaired Student’s t test. Comparisons between mean values before and after ic injection of the same group used paired Student’s t test. All the statistical tests were performed using SigmaStat program. P value < 0.05 was considered statistically significant.
Results
Basal blood glucose and serum insulin levels of anesthetized Wistar and GK rats
In normally fed conditions, Wistar rats were euglycemic, whereas GK rats had remarkable hyperglycemia (Fig. 1). There was no gender difference in glucose levels within the same strain. Serum insulin levels were lower in GK rats compared with Wistar controls and lower in females compared with males of the same strain, although these differences did not reach statistical significance (Fig. 1). Overnight fasting significantly reduced blood glucose levels in Wistar rats, although the levels were still maintained within the physiological range. The high blood glucose levels observed in fed GK rats were normalized in overnight-fasted males but remained significantly higher in overnight-fasted females (Fig. 1). Compared with the normally fed rats, serum insulin levels were lower in all the overnight-fasted rats, which was statistically significant in Wistar males and GK females. However, insulin levels were still higher in fasted male GK than fasted male Wistar rats (Fig. 1). The female GK rats had the lowest insulin levels among all the fasted groups, which were in accordance with their high blood glucose levels (Fig. 1).
Effect of ic TRH analog RX 77368 on blood glucose and serum insulin levels in overnight-fasted male Wistar and GK rats
Saline (10 μl) ic injection did not significantly influence blood glucose and serum insulin levels in overnight-fasted male Wistar and GK rats compared with their basal levels (Fig. 2A). RX 77368 ic at a low dose (10 ng/10 μl) did not change glucose levels but slightly and significantly increased serum insulin levels at 30 min after injection in Wistar rats (Fig. 2B). In contrast, RX 77368 (10 ng) significantly increased glucose levels in GK rats and induced a marked insulin-stimulatory response. The insulin levels in the GK rats reached a significant 3-fold higher peak compared with the basal levels at 30 min that lasted for more than 2 h (Fig. 2B). In rats of both strains injected with a higher dose of RX 77368 (50 ng/10 μl), a significant and potent hyperglycemic response was induced, which was significantly stronger in GK compared with Wistar rats (Fig. 2C). Blood glucose increased from the basal levels of 88 ± 6 and 123 ± 3 mg/100 ml in the Wistar and GK rats to peak levels of 250 ± 29 and 334 ± 26 mg/100 ml at 90 min after the injection, respectively, and remained at significantly high levels until the end of the observation period (120 min after the injection) (Fig. 2C). However, serum insulin levels significantly and strikingly increased 6-fold in Wistar rats but only increased less than 3-fold in GK rats, although it was statistically significant compared with its basal levels (Fig. 2C). The ic 50-ng RX 77368-induced increase of insulin in the GK rats reached similar levels as that induced by ic 10 ng RX 77368 (Fig. 2, B and C).
Effect of ic TRH analog RX 77368 on blood glucose and serum insulin levels in overnight-fasted female Wistar and GK rats
Overnight-fasted female GK rats had higher basal blood glucose and lower serum insulin levels compared with the males (Fig. 1). A low dose of RX 77368 (10 ng) was ic injected in each pentobarbital-anesthetized rat in groups (n = 4 per group) of young female Wistar (2.5 months old; body weight, 237 ± 3 g), young female GK (2.8 months old; body weight, 182 ± 3 g), or old female GK (9 months old; body weight, 224 ± 7 g) rats. RX 77368 (10 ng) slightly but significantly increased blood glucose levels from a basal level of 92 ± 2 to 118 ± 4 mg/100 ml at 120 min after the injection in female Wistar rats but did not significantly influence their serum insulin levels (Fig. 3). In contrast, ic RX 77368 (10 ng) significantly reduced blood glucose from a basal level of 193 ± 13 to 145 ± 9 mg/100 ml (–25%) in young female GK and from 157 ± 11 to 123 ± 3 mg/100 ml (–22%) in old female GK rats (Fig. 3). In accordance with this glucose decrease, serum insulin levels significantly increased to 2.3-fold of the basal levels in the young female GK rats but not in young Wister controls (Fig. 3). The direction and extent of ic RX 77368-induced changes in individual glucose levels at 60 and 120 min after the ic injection were negatively correlated with the individual basal glucose levels in these three groups of rats (Fig. 4). That is, ic RX 77368 (10 ng) slightly increased glucose levels in rats with relatively low basal glucose levels, such as in Wistar rats, but significantly reduced glucose levels in hyperglycemic GK rats (Figs. 3 and 4). The glucose levels converged to the level of 120 mg/100 ml after ic RX 77368 (10 ng) injection in these three groups of rats with significantly different basal glucose levels (Fig. 3).
Effect of ic TRH analog RX 77368 on pancreatic insulin II gene expression in overnight-fasted male Wistar and GK rats
Pancreatic insulin II mRNA levels were significantly higher in the overnight-fasted male GK rats than the Wistar controls (Fig. 5). RX 77368 (50 ng) ic injection significantly increased pancreatic insulin II mRNA levels in Wistar rats but did not further increase it in GK rats measured at 120 min after the ic injection (Fig. 5).
Effect of vagotomy, adrenalectomy, and vagotomy plus adrenalectomy on basal glucose and insulin levels in overnight-fasted male Wistar and GK rats
Basal blood samples were collected 60 min after one of the surgeries in overnight-fasted male rats. Blood glucose levels were influenced by bilateral cervical vagotomy, as shown by a slight but significant decrease in Wistar rats and a significant 26% increase in GK rats (Table 1). Vagotomy also broadened individual variations in serum insulin levels and dulled the significant difference between Wistar and GK rats observed in sham-operated groups (Table 1). In contrast, bilateral adrenalectomy had no effect on basal blood glucose levels in Wistar rats and nonsignificantly increased it in GK rats. Serum insulin levels were significantly increased by 2-fold in both adrenalectomized Wistar and GK rats (Table 1). The influence of vagotomy plus adrenalectomy on basal blood glucose and serum insulin levels was similar to that of adrenalectomy alone (Table 1).
Effect of vagotomy, adrenalectomy, and vagotomy plus adrenalectomy on ic RX 77368-induced changes in glucose and insulin levels in overnight-fasted male Wistar and GK rats
Acute bilateral cervical vagotomy diminished the hyperglycemic effect of ic RX 77368 (50 ng) in both the Wistar and GK rats, although the effect was still significant in both strains (Fig. 6B). Compared with the sham-operated groups, the peak glucose increase at 90 min after ic RX 77368 was reduced from 250 ± 29 to 141 ± 8 mg/100 ml (–44%) in vagotomized Wistar and from 334 ± 26 to 252 ± 25 mg/100 ml (–25%) in vagotomized GK rats (Fig. 6, A and B). The insulin-stimulatory effect of ic RX 77368 (50 ng) was totally prevented by vagotomy (Fig. 6B). Acute bilateral adrenalectomy completely abolished the hyperglycemic effect of ic RX 77368 (50 ng) in the two strains (Fig. 6C). Furthermore, in adrenalectomized GK but not Wistar rats, ic RX 77368 (50 ng) significantly reduced blood glucose levels from a basal level of 154 ± 35 to a nadir of 79 ± 13 mg/100 ml at 90 min after the injection (Fig. 6C). The peak serum insulin response observed at 30 min after ic RX 77368 (50 ng) was a 1.3-fold increase in adrenalectomized Wistar rats, which was not statistically significant compared with the preinjection level. In contrast, a significant and remarkable 6.5-fold increase of peak insulin response was observed in adrenalectomized GK rats (Fig. 6C). The decrease in blood glucose and the increase in insulin levels induced by ic RX 77368 in adrenalectomized GK rats were absent in GK rats that received both bilateral adrenalectomy and cervical vagotomy (Fig. 6D).
Discussion
Results obtained from this study clearly show that the T2D GK rats have altered glucose and insulin responses to a centrally initiated autonomic activation induced by ic TRH analog. These were shown by a susceptible vagal-mediated insulin secretion after a low dose of ic RX 77368 (10 ng) and significantly powerful sympathetic-adrenal-mediated hyperglycemic and insulin-inhibitory responses to an ic high dose of RX 77368 (50 ng).
The GK rat is a polygenic model of nonobese T2D with impaired insulin response to elevated glucose levels (25). In this study, the normally fed GK rats had remarkable hyperglycemia without a matched elevation in serum insulin levels, proving their diabetic status and diminished postprandial insulin secretion. Most of the experiments in this study were performed in overnight-fasted rats to minimize the influence of digestion and postprandial absorption on basal circulating glucose and insulin levels. Previous findings indicate an abnormal vagal-cholinergic regulation of visceral functions in GK rats that contributes to the impaired pancreatic insulin secretion. For instance, vagal-dependent islet blood flow, which is important in glucose-load-induced insulin secretion (39, 40), is diminished in GK rats; this abnormality participates in the progressive deterioration of glucose intolerance (41, 42). Carbachol, which activates muscarinic acetylcholine receptors, fully normalizes insulin secretion responding to 16.7 mmol/liter glucose in GK rats through an effect abolished by atropine (43). The present study further investigated the vagal dysfunction in this T2D rat model by testing glucose and insulin responses to ic injection of a stable TRH analog, which mimics the physiological process of centrally initiated autonomic activation. TRH or RX 77368 exogenously injected into the cisterna magna acts on TRH receptors located on vagal motor neurons in the DVC to elevate vagal efferent discharge (21) that results in vagal-cholinergic-mediated stimulation of gastrointestinal functions (15, 19, 20, 22). TRH or RX 77368 also causes sympathetic-adrenal gland-mediated hyperglycemia by acting on unidentified central sites (44), possibly involving the rostroventrolateral reticular nucleus and the caudoventrolateral reticular nucleus in the brain medulla, where TRH-containing fibers are localized (our unpublished observation), neurons participate in sympathetic regulation of visceral functions (45), and neurons with presumed sympathoexcitatory function are activated by TRH (46).
We have previously reported that acute hyperglycemia induced by iv glucose infusion completely abolishes ic TRH analog-induced gastric acid secretion in nondiabetic rats (47). Based on this finding, we originally hypothesized that the impaired vagal regulation of insulin secretion in GK rats might be the result of an inhibitory influence of its hyperglycemia on medullary TRH action. However, results of the present study show that this is not the case. A low dose of RX 77368 (10 ng) did not significantly influence serum insulin levels in Wistar control rats but remarkably increased it in both the male and female GK rats, indicating that insulin response to central vagal activation induced by medullary TRH is actually more sensitive, rather than dulled, in GK than in Wistar rats. The higher dose of RX 77368 (50 ng) ic injection, on the other hand, induced remarkable hyperglycemia in both strains and a 6-fold increase in serum insulin in Wistar rats. However, this dose (50 ng) did not further increase insulin levels in GK rats compared with the response to 10 ng. In accordance with this relatively lower insulin response, the glucose increase was significantly greater in GK than in Wistar rats. These data suggest that a low dose of RX 77368 (10 ng) induces vagal activation while having less impact on sympathetic-adrenal activation, resulting in a consequent increase in pancreatic insulin secretion. The higher dose of RX 77368 (50 ng), however, activates the vagal and also the sympathetic-adrenal systems, the latter causing hyperglycemia. In supporting this view, the insulin increase after ic RX 77368 was completely prevented by acute bilateral cervical vagotomy, and the hyperglycemia was abolished by adrenalectomy. The greater hyperglycemic response to ic RX 77368 (50 ng) in GK rats indicate that not only the vagus nerve, but also the sympathetic-adrenal system, is more strongly activated by medullary TRH in GK than in Wistar rats.
To further analyze the abnormality in medullary TRH-initiated autonomic regulation of insulin secretion in GK rats, studies with acute surgical blockage of vagal and/or sympathetic-adrenal pathways were performed in overnight-fasted male rats. The surgeries themselves did not influence blood glucose levels in nondiabetic Wistar rats, except bilateral cervical vagotomy, which slightly and significantly reduced glucose. These data indicate that acute surgery in anesthetized rats of the present study had little, if any, stressful influence on glucose levels. In contrast to the Wister rats, blood glucose levels significantly increased in GK rats that underwent vagotomy or vagotomy plus adrenalectomy but not adrenalectomy, indicating a beneficial role of the integrity of the vagus nerve in antagonizing hyperglycemia in GK rats. The serum insulin levels, however, significantly increased by about 2-fold in adrenalectomized and vagotomized plus adrenalectomized, but not the vagotomized, Wistar and GK groups, indicating a sympathetic-adrenal inhibitory tone on basal insulin secretion in both the nondiabetic Wistar rats and the T2D GK rats, which was stronger in the GK rats because these rats had a more remarkable insulin increase after adrenalectomy. As expected, vagotomy completely prevented ic RX 77368-induced hyperinsulinemia and adrenalectomy totally abolished the hyperglycemic effect in both strains. Furthermore, although not affecting glucose levels in adrenalectomized Wistar rats, ic RX 77368 (50 ng) significantly reduced blood glucose in adrenalectomized GK rats, from high diabetic levels to levels the same as in Wistar rats. This glucose-normalizing effect of ic TRH analog in GK rats was achieved by inducing a 6.5-fold increase of serum insulin, which was abolished by vagotomy. Taken together, our data indicate that the dominant sympathetic-adrenal activation by a high dose of ic TRH analog in GK rats plays a suppressing role on the vagal stimulation of pancreatic insulin secretion. The unbalanced central activation of vagal and sympathetic systems may contribute to the impaired insulin secretion in T2D GK rats.
Alteration in the balance of parasympathetic and sympathetic nervous activity, mainly explained by attenuated parasympathetic activity and relative predominance of sympathetic activity, is common in T2D patients. Sustained overactivation of the sympathetic nervous system was attributed to the central effects of hyperinsulinemia and believed to play an important role in the pathological development of T2D, in particular, to contribute to the hypertension and cardiovascular mortality in T2D (48, 49, 50, 51). Our results indicate that the vagal-sympathetic imbalance in T2D could be a result of altered vagal and/or splanchnic outflow responding to the regulation of medullary TRH. The fact that the same dose of RX 77368 (10 ng) ic injection induced different insulin and glucose responses in nondiabetic and T2D rats with different basal glucose levels suggests that modifying medullary TRH action, such as relating the sensitivity of vagal activation responding to medullary TRH with blood glucose levels, is part of the autonomic adaptation for increased demand of insulin secretion in T2D. However, altered medullary TRH action could also significantly impair pancreatic insulin secretion when sympathetic overactivation becomes overt.
The mechanisms of this altered autonomic response to medullary TRH in GK rats are currently unknown. It was reported that peripheral neuropathy could be tested morphologically in 9- or 18-month-old but not in 2-month-old GK rats (26, 52). Most GK rats used in the present study were 3 months old; it is unlikely that peripheral neuropathy had developed seriously enough to be responsible for the altered autonomic response to ic TRH analog. The results showing that vagal-mediated insulin and sympathetic-adrenal-mediated glucose responses to ic TRH analog were actually more sensitive in GK than in Wistar rats also do not favor this possibility. In addition, 9-month-old female GK rats, assumed to have developed peripheral neuropathy (52), displayed the same extent of glucose-decreasing response to ic TRH analog (10 ng) as the 3-month-old female GK rats, indicating that peripheral neuropathy may not be a critical factor responsible for the altered autonomic response in GK rats of the present study. In support of this, central sympathetic hyperactivity was observed in T2D patients without peripheral neuropathy (49). Abnormal gene and/or protein expression of neuropeptides/transmitters and their receptors, such as TRH and its receptor, in medullary autonomic regulatory nuclei and vagal/sympathetic transduction pathways may play a role in the altered autonomic regulation in GK rats because these components might be influenced by the altered metabolism in T2D. Although direct evidence has yet to be obtained, alterations have been observed in the spinal cord and the ventromedial hypothalamic nucleus of GK rats, such as decreased adrenergic receptors, reduced norepinephrine release, and decreased expression of substance P and calcitonin gene-related protein in the spinal cord (26, 53, 54, 55). Additional experiments, such as measuring blood concentration of catecholamines and levels of mRNA and protein of neuropeptides/transmitters and their receptors in the brain medulla and thoracic spinal cord, brain medullary microinjection of TRH or its analog into vagal and sympathetic controlling nuclei, or a direct electrophysiological recording of the hepatic vagal and splanchnic discharges responding to ic TRH analog, will provide more information on the mechanism of unbalanced vagal-sympathetic activation by medullary TRH in GK rats.
In a recently published paper, Dunn (56) emphasized that it is important to keep in mind that the primary abnormality of T2D is the loss of insulin secretion and that a major contributor to insulin resistance is hyperglycemia secondary to insulin deficit. Subjects at risk of developing T2D have -cell dysfunction before they develop glucose intolerance (57). Our present results show that in T2D GK rats, vagal integrity is important for antagonizing hyperglycemia and pancreatic insulin release is sensitively responsive to the central-vagal stimulation induced by medullary TRH receptor activation. Moreover, the insulin-stimulatory action of medullary TRH is glucose-level related. However, the vagal-mediated insulin stimulation can be suppressed by an overactivation of the sympathetic-adrenal system, which is also regulated by medullary TRH. These findings indicate that autonomic response to medullary TRH plays an important role in physiological and pathophysiological regulation of pancreatic endocrine secretion. Increasing vagal activity, decreasing sympathetic-adrenal tone, or correcting the unbalanced autonomic response to central regulation could be potential therapeutic approaches for improving islet -cell functions in T2D patients. A recent clinical observation that the insulin requirement dramatically decreased to less than 50% in a T2D patient who had undergone spinal-sympathetic blockage provides supportive evidence for this possibility (58).
Acknowledgments
We thank Ms. Ai Chen for her technical assistance.
Footnotes
This work was supported by Veterans Affairs Merit Award (to H.Y.) and National Institutes of Health DK-41301 (CURE Center Grant Animal Core).
First Published Online September 22, 2005
Abbreviations: DVC, Dorsal vagal complex; GK rats, Goto-Kakizaki rats; ic, intracisternal; T2D, type 2 diabetes.
Accepted for publication September 7, 2005.
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Department of Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073
Abstract
Insulin secretion is impaired in type 2 diabetes (T2D). The insulin and glucose responses to central autonomic activation induced by excitation of brain medullary TRH receptors were studied in T2D Goto-Kakizaki (GK) rats. Blood glucose levels in normally fed, pentobarbital-anesthetized GK and nondiabetic Wistar rats were 193 and 119 mg/100 ml in males and 214 and 131 mg/100 ml in females. Intracisternal injection (ic) of the stable TRH analog RX 77368 (10 ng) induced significantly higher insulin response in both genders of overnight-fasted GK rats compared with Wistar rats and slightly increased blood glucose in female Wistar rats but significantly decreased it from 193 to 145 mg/100 ml in female GK rats. RX 77368 (50 ng) ic induced markedly greater glucose and relatively weaker insulin responses in male GK rats than Wistar rats. Bilateral vagotomy blocked ic RX 77368-induced insulin secretion, whereas adrenalectomy abolished its hyperglycemic effect. In adrenalectomized male GK but not Wistar rats, ic RX 77368 (50 ng) dramatically increased serum insulin levels by 6.5-fold and decreased blood glucose levels from 154 to 98 mg/100 ml; these changes were prevented by vagotomy. GK rats had higher basal pancreatic insulin II mRNA levels but a lower response to ic RX 77368 (50 ng) compared with Wistar rats. These results indicate that central-vagal activation-induced insulin secretion is susceptible in T2D GK rats. However, the dominant sympathetic-adrenal response to medullary TRH plays a suppressing role on vagal-mediated insulin secretion. This unbalanced vago-sympathetic activation by medullary TRH may contribute to the impaired insulin secretion in T2D.
Introduction
ABNORMAL INSULIN SECRETION and synthesis are key factors in the pathophysiology of type 2 diabetes (T2D) (1). Pancreatic endocrine secretion is regulated by the central nervous system through rich innervation of vagal and sympathetic nerves in the islets (2, 3). Insulin secretion is stimulated by vagal activation and inhibited by sympathetic-adrenal activation (4, 5). Both systems participate in meal-induced insulin secretion but only the vagus-cholinergic component plays a major role in insulin secretion of the cephalic phase and during the early absorption period (6, 7). Maintaining normal glucose tolerance requires the integrity of vagal function (8). Therefore, defects in the autonomic control of pancreatic -cell function might contribute to the relatively insufficient insulin secretion in T2D. Although ample knowledge has been achieved in the cause, prevention, and repair of islet -cell damage and diminished insulin secretion in diabetes at the cellular and molecular levels (9), the altered autonomic regulation of pancreatic insulin secretion in T2D is still poorly understood.
TRH is a neuropeptide synthesized in brain medullary caudal raphe nuclei and the parapyramidal regions. These nuclei send TRH-containing nerve projections to innervate the dorsal vagal complex (DVC), composed of the dorsal motor nucleus of the vagus and the nucleus tractus solitarii, as well as the ventrolateral reticular formation of the medulla and the intermediolateral cell column of the spinal cord (10, 11). It was found in the 1980s that an intracerebroventricular injection of TRH induces hyperglycemia through pathways involving the adrenal gland; however, intracerebroventricular TRH also prevents central and peripheral stimuli-induced hyperglycemia by stimulating insulin release in rats and mice (12, 13, 14). Studies in the last 20 yr have well established that brain medullary TRH plays a physiological role in the autonomic regulation of visceral functions (15). The DVC contains dense TRH-immunoreactive nerve terminals and TRH receptors (16, 17). TRH or its stable analog RX 77368 injected intracisternally (ic) or microinjected into the DVC or TRH endogenously released into the DVC after chemical stimulation of cell bodies in the raphe nuclei increases vagal efferent discharge, induces vagally mediated activation of enteric neurons and increases of gastric secretion and motility (18, 19, 20, 21, 22), and stimulates pancreatic insulin secretion (23). Convincing findings demonstrated that ic TRH or its analog induces centrally initiated vagal and sympathetic activation that mimics physiological or pathophysiological processes (15).
In this study, we tested the hypothesis that autonomic regulation of pancreatic insulin secretion and synthesis are impaired in T2D. We compared blood glucose and pancreatic insulin responses to ic injection of the stable TRH analog, RX 77368, between the nondiabetic Wistar rats and Goto-Kakizaki (GK) rats, the genetically determined nonobese T2D model with impaired insulin response to glucose (24, 25). Surgical approaches were used to determine the mediation of vagal and adrenal-sympathetic pathways in ic TRH analog-induced glucose and insulin changes in the two rat strains and the imbalance of vagal-sympathetic activation in the T2D GK rats.
Materials and Methods
Animals
The GK rats were bred in Animal Facilities of the Veterans Affairs (VA) Greater Los Angeles Area Healthcare System with approved protocol and used at the age of 3 months when body weight was 240–270 g (male) or 180–220 g (female), except in one experiment specified in Results that used 9-month-old female rats. The age- and sex-matched control Wistar rats were purchased from Harlan Laboratory (San Diego, CA) and raised in VA Animal Facilities for 1 wk before the experiments. The body weight of GK rats were about 10–20 g lower than the age-matched Wistar rats, as previously reported (26). The rats were housed under controlled conditions (21–23 C, lights on from 0600–1800 h) with free access to standard rat chow (Prolab Lab Diet; PMI Nutrition International, Brentwood, MO) and tap water. Food, but not water, was removed 16 h before most experiments, except for one group of Wistar and one group of GK rats that were used to obtain basal glucose and insulin levels in normally fed conditions. Animal protocols were approved by the University of California, Los Angeles, Office for the Protection of Research Subjects and VA Greater Los Angeles Area Healthcare System Animal Committee.
Experimental protocol
All experiments were performed between 0900 am and 1500 h. Rats were anesthetized with ip pentobarbital (Abbott Laboratories, North Chicago, IL) (50 mg/kg followed by 20 mg/kg each hour until the end of experiments) to avoid surgery-, ic injection-, and blood samplings-induced stressful influence on glycemic regulation that is usually inevitable in conscious animals. Previous studies have shown that pentobarbital anesthesia has slight to moderate influence on insulin output, glucose production, hepatic insulin resistance, and pancreatic blood flow (27, 28, 29). However, other anesthesia, such as Hypnorm, urethane, or ketamine, can cause strong changes in autonomic activity (30, 31, 32) and insulin resistance (33) or induce hyperglycemia (34, 35, 36). Thus, pentobarbital is relatively less potent in influencing glucose metabolism (34) and has little effect on mean arterial blood pressure, rectal temperature, and brain c-fos expression (32, 37).
A PE-50 cannula was inserted into the external iliac vein for blood sampling. Basal blood samples (0.2 ml/rat) were collected at 30 min after the iv cannulation. In some groups of rats, bilateral cervical vagotomy, bilateral adrenalectomy, both of the surgeries, or the corresponding sham operation was performed immediately after the iv cannulation. In these groups, the basal blood samples were collected after a 60-min postsurgery stabilization period. After the basal blood sampling, each rat was positioned on a stereotaxic instrument (Kopf model 900) and received an ic injection of either physiological saline (vehicle, 10 μl) or RX 77368 (Ferring Pharmaceuticals, Felthan, Middlesex, UK) (10 or 50 ng/10 μl), as performed in our previous studies (22, 38). Blood samples (0.2 ml) were collected at 30, 60, 90, and 120 min after the ic injection. In another two groups of Wistar rats and two groups of GK rats each receiving ic saline or RX 77368 (50 ng), respectively, the pancreas was collected at 120 min after the ic injection for Northern blot analysis of pancreatic insulin II mRNA levels.
Measurement of blood glucose and serum insulin levels
Blood glucose levels were measured by One Touch Ultra Blood Glucose Monitoring System (Lifescan, Milpitas, CA) and serum insulin by rat insulin RIA kit (catalog item RI-13K; Linco Research, St. Charles, MO).
Northern blot analysis of pancreatic insulin II mRNA
Pancreatic total RNA from each sample was extracted with standard RNAzol method using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA). Total RNA (20 μg) was run on agarose gel containing MOPS and formaldehyde (Sigma Chemical Co., St. Louis, MO) and then transferred to a nylon membrane by UV cross-linking. The insulin II DNA probe was prepared from RT (Ambion, Austin, TX)-PCR (QIAGEN, Valencia, CA). The sequence of insulin II primer was designed using Primer Express software (accession J04807). The forward insulin II primer (5'–3') sequence was CCTAAGTGACCAGCTACA; the reverse primer (5'–3') sequence was GTAGTTCTGCAGTTGGTA. The size of the PCR product was 367 bp. DNA fragments from PCR were run on low-melting agarose gel, extracted, and then purified by Wizard PCR Preps DNA purification system (Promega, Madison, WI). Purified PCR product was labeled with [-32P]dCTP (ICN Pharmaceuticals Inc., Bryan, OH) using Random Primer DNA Labeling Systems (New England BioLabs, Inc., Beverly, MA). After labeling, probes were purified using QIAquick Nucleotide Removal Kit (QIAGEN). The hybridization was carried out overnight at 68 C.
Statistical analysis
Data are expressed as mean ± SEM of each experimental group. Statistical comparisons among multiple group mean values were performed using two-way or one-way ANOVA followed by Dunn’s method. Comparisons between group mean values of Wistar and GK rats receiving the same treatment or between RX 77368-treated and saline-treated rats of the same strain were performed using unpaired Student’s t test. Comparisons between mean values before and after ic injection of the same group used paired Student’s t test. All the statistical tests were performed using SigmaStat program. P value < 0.05 was considered statistically significant.
Results
Basal blood glucose and serum insulin levels of anesthetized Wistar and GK rats
In normally fed conditions, Wistar rats were euglycemic, whereas GK rats had remarkable hyperglycemia (Fig. 1). There was no gender difference in glucose levels within the same strain. Serum insulin levels were lower in GK rats compared with Wistar controls and lower in females compared with males of the same strain, although these differences did not reach statistical significance (Fig. 1). Overnight fasting significantly reduced blood glucose levels in Wistar rats, although the levels were still maintained within the physiological range. The high blood glucose levels observed in fed GK rats were normalized in overnight-fasted males but remained significantly higher in overnight-fasted females (Fig. 1). Compared with the normally fed rats, serum insulin levels were lower in all the overnight-fasted rats, which was statistically significant in Wistar males and GK females. However, insulin levels were still higher in fasted male GK than fasted male Wistar rats (Fig. 1). The female GK rats had the lowest insulin levels among all the fasted groups, which were in accordance with their high blood glucose levels (Fig. 1).
Effect of ic TRH analog RX 77368 on blood glucose and serum insulin levels in overnight-fasted male Wistar and GK rats
Saline (10 μl) ic injection did not significantly influence blood glucose and serum insulin levels in overnight-fasted male Wistar and GK rats compared with their basal levels (Fig. 2A). RX 77368 ic at a low dose (10 ng/10 μl) did not change glucose levels but slightly and significantly increased serum insulin levels at 30 min after injection in Wistar rats (Fig. 2B). In contrast, RX 77368 (10 ng) significantly increased glucose levels in GK rats and induced a marked insulin-stimulatory response. The insulin levels in the GK rats reached a significant 3-fold higher peak compared with the basal levels at 30 min that lasted for more than 2 h (Fig. 2B). In rats of both strains injected with a higher dose of RX 77368 (50 ng/10 μl), a significant and potent hyperglycemic response was induced, which was significantly stronger in GK compared with Wistar rats (Fig. 2C). Blood glucose increased from the basal levels of 88 ± 6 and 123 ± 3 mg/100 ml in the Wistar and GK rats to peak levels of 250 ± 29 and 334 ± 26 mg/100 ml at 90 min after the injection, respectively, and remained at significantly high levels until the end of the observation period (120 min after the injection) (Fig. 2C). However, serum insulin levels significantly and strikingly increased 6-fold in Wistar rats but only increased less than 3-fold in GK rats, although it was statistically significant compared with its basal levels (Fig. 2C). The ic 50-ng RX 77368-induced increase of insulin in the GK rats reached similar levels as that induced by ic 10 ng RX 77368 (Fig. 2, B and C).
Effect of ic TRH analog RX 77368 on blood glucose and serum insulin levels in overnight-fasted female Wistar and GK rats
Overnight-fasted female GK rats had higher basal blood glucose and lower serum insulin levels compared with the males (Fig. 1). A low dose of RX 77368 (10 ng) was ic injected in each pentobarbital-anesthetized rat in groups (n = 4 per group) of young female Wistar (2.5 months old; body weight, 237 ± 3 g), young female GK (2.8 months old; body weight, 182 ± 3 g), or old female GK (9 months old; body weight, 224 ± 7 g) rats. RX 77368 (10 ng) slightly but significantly increased blood glucose levels from a basal level of 92 ± 2 to 118 ± 4 mg/100 ml at 120 min after the injection in female Wistar rats but did not significantly influence their serum insulin levels (Fig. 3). In contrast, ic RX 77368 (10 ng) significantly reduced blood glucose from a basal level of 193 ± 13 to 145 ± 9 mg/100 ml (–25%) in young female GK and from 157 ± 11 to 123 ± 3 mg/100 ml (–22%) in old female GK rats (Fig. 3). In accordance with this glucose decrease, serum insulin levels significantly increased to 2.3-fold of the basal levels in the young female GK rats but not in young Wister controls (Fig. 3). The direction and extent of ic RX 77368-induced changes in individual glucose levels at 60 and 120 min after the ic injection were negatively correlated with the individual basal glucose levels in these three groups of rats (Fig. 4). That is, ic RX 77368 (10 ng) slightly increased glucose levels in rats with relatively low basal glucose levels, such as in Wistar rats, but significantly reduced glucose levels in hyperglycemic GK rats (Figs. 3 and 4). The glucose levels converged to the level of 120 mg/100 ml after ic RX 77368 (10 ng) injection in these three groups of rats with significantly different basal glucose levels (Fig. 3).
Effect of ic TRH analog RX 77368 on pancreatic insulin II gene expression in overnight-fasted male Wistar and GK rats
Pancreatic insulin II mRNA levels were significantly higher in the overnight-fasted male GK rats than the Wistar controls (Fig. 5). RX 77368 (50 ng) ic injection significantly increased pancreatic insulin II mRNA levels in Wistar rats but did not further increase it in GK rats measured at 120 min after the ic injection (Fig. 5).
Effect of vagotomy, adrenalectomy, and vagotomy plus adrenalectomy on basal glucose and insulin levels in overnight-fasted male Wistar and GK rats
Basal blood samples were collected 60 min after one of the surgeries in overnight-fasted male rats. Blood glucose levels were influenced by bilateral cervical vagotomy, as shown by a slight but significant decrease in Wistar rats and a significant 26% increase in GK rats (Table 1). Vagotomy also broadened individual variations in serum insulin levels and dulled the significant difference between Wistar and GK rats observed in sham-operated groups (Table 1). In contrast, bilateral adrenalectomy had no effect on basal blood glucose levels in Wistar rats and nonsignificantly increased it in GK rats. Serum insulin levels were significantly increased by 2-fold in both adrenalectomized Wistar and GK rats (Table 1). The influence of vagotomy plus adrenalectomy on basal blood glucose and serum insulin levels was similar to that of adrenalectomy alone (Table 1).
Effect of vagotomy, adrenalectomy, and vagotomy plus adrenalectomy on ic RX 77368-induced changes in glucose and insulin levels in overnight-fasted male Wistar and GK rats
Acute bilateral cervical vagotomy diminished the hyperglycemic effect of ic RX 77368 (50 ng) in both the Wistar and GK rats, although the effect was still significant in both strains (Fig. 6B). Compared with the sham-operated groups, the peak glucose increase at 90 min after ic RX 77368 was reduced from 250 ± 29 to 141 ± 8 mg/100 ml (–44%) in vagotomized Wistar and from 334 ± 26 to 252 ± 25 mg/100 ml (–25%) in vagotomized GK rats (Fig. 6, A and B). The insulin-stimulatory effect of ic RX 77368 (50 ng) was totally prevented by vagotomy (Fig. 6B). Acute bilateral adrenalectomy completely abolished the hyperglycemic effect of ic RX 77368 (50 ng) in the two strains (Fig. 6C). Furthermore, in adrenalectomized GK but not Wistar rats, ic RX 77368 (50 ng) significantly reduced blood glucose levels from a basal level of 154 ± 35 to a nadir of 79 ± 13 mg/100 ml at 90 min after the injection (Fig. 6C). The peak serum insulin response observed at 30 min after ic RX 77368 (50 ng) was a 1.3-fold increase in adrenalectomized Wistar rats, which was not statistically significant compared with the preinjection level. In contrast, a significant and remarkable 6.5-fold increase of peak insulin response was observed in adrenalectomized GK rats (Fig. 6C). The decrease in blood glucose and the increase in insulin levels induced by ic RX 77368 in adrenalectomized GK rats were absent in GK rats that received both bilateral adrenalectomy and cervical vagotomy (Fig. 6D).
Discussion
Results obtained from this study clearly show that the T2D GK rats have altered glucose and insulin responses to a centrally initiated autonomic activation induced by ic TRH analog. These were shown by a susceptible vagal-mediated insulin secretion after a low dose of ic RX 77368 (10 ng) and significantly powerful sympathetic-adrenal-mediated hyperglycemic and insulin-inhibitory responses to an ic high dose of RX 77368 (50 ng).
The GK rat is a polygenic model of nonobese T2D with impaired insulin response to elevated glucose levels (25). In this study, the normally fed GK rats had remarkable hyperglycemia without a matched elevation in serum insulin levels, proving their diabetic status and diminished postprandial insulin secretion. Most of the experiments in this study were performed in overnight-fasted rats to minimize the influence of digestion and postprandial absorption on basal circulating glucose and insulin levels. Previous findings indicate an abnormal vagal-cholinergic regulation of visceral functions in GK rats that contributes to the impaired pancreatic insulin secretion. For instance, vagal-dependent islet blood flow, which is important in glucose-load-induced insulin secretion (39, 40), is diminished in GK rats; this abnormality participates in the progressive deterioration of glucose intolerance (41, 42). Carbachol, which activates muscarinic acetylcholine receptors, fully normalizes insulin secretion responding to 16.7 mmol/liter glucose in GK rats through an effect abolished by atropine (43). The present study further investigated the vagal dysfunction in this T2D rat model by testing glucose and insulin responses to ic injection of a stable TRH analog, which mimics the physiological process of centrally initiated autonomic activation. TRH or RX 77368 exogenously injected into the cisterna magna acts on TRH receptors located on vagal motor neurons in the DVC to elevate vagal efferent discharge (21) that results in vagal-cholinergic-mediated stimulation of gastrointestinal functions (15, 19, 20, 22). TRH or RX 77368 also causes sympathetic-adrenal gland-mediated hyperglycemia by acting on unidentified central sites (44), possibly involving the rostroventrolateral reticular nucleus and the caudoventrolateral reticular nucleus in the brain medulla, where TRH-containing fibers are localized (our unpublished observation), neurons participate in sympathetic regulation of visceral functions (45), and neurons with presumed sympathoexcitatory function are activated by TRH (46).
We have previously reported that acute hyperglycemia induced by iv glucose infusion completely abolishes ic TRH analog-induced gastric acid secretion in nondiabetic rats (47). Based on this finding, we originally hypothesized that the impaired vagal regulation of insulin secretion in GK rats might be the result of an inhibitory influence of its hyperglycemia on medullary TRH action. However, results of the present study show that this is not the case. A low dose of RX 77368 (10 ng) did not significantly influence serum insulin levels in Wistar control rats but remarkably increased it in both the male and female GK rats, indicating that insulin response to central vagal activation induced by medullary TRH is actually more sensitive, rather than dulled, in GK than in Wistar rats. The higher dose of RX 77368 (50 ng) ic injection, on the other hand, induced remarkable hyperglycemia in both strains and a 6-fold increase in serum insulin in Wistar rats. However, this dose (50 ng) did not further increase insulin levels in GK rats compared with the response to 10 ng. In accordance with this relatively lower insulin response, the glucose increase was significantly greater in GK than in Wistar rats. These data suggest that a low dose of RX 77368 (10 ng) induces vagal activation while having less impact on sympathetic-adrenal activation, resulting in a consequent increase in pancreatic insulin secretion. The higher dose of RX 77368 (50 ng), however, activates the vagal and also the sympathetic-adrenal systems, the latter causing hyperglycemia. In supporting this view, the insulin increase after ic RX 77368 was completely prevented by acute bilateral cervical vagotomy, and the hyperglycemia was abolished by adrenalectomy. The greater hyperglycemic response to ic RX 77368 (50 ng) in GK rats indicate that not only the vagus nerve, but also the sympathetic-adrenal system, is more strongly activated by medullary TRH in GK than in Wistar rats.
To further analyze the abnormality in medullary TRH-initiated autonomic regulation of insulin secretion in GK rats, studies with acute surgical blockage of vagal and/or sympathetic-adrenal pathways were performed in overnight-fasted male rats. The surgeries themselves did not influence blood glucose levels in nondiabetic Wistar rats, except bilateral cervical vagotomy, which slightly and significantly reduced glucose. These data indicate that acute surgery in anesthetized rats of the present study had little, if any, stressful influence on glucose levels. In contrast to the Wister rats, blood glucose levels significantly increased in GK rats that underwent vagotomy or vagotomy plus adrenalectomy but not adrenalectomy, indicating a beneficial role of the integrity of the vagus nerve in antagonizing hyperglycemia in GK rats. The serum insulin levels, however, significantly increased by about 2-fold in adrenalectomized and vagotomized plus adrenalectomized, but not the vagotomized, Wistar and GK groups, indicating a sympathetic-adrenal inhibitory tone on basal insulin secretion in both the nondiabetic Wistar rats and the T2D GK rats, which was stronger in the GK rats because these rats had a more remarkable insulin increase after adrenalectomy. As expected, vagotomy completely prevented ic RX 77368-induced hyperinsulinemia and adrenalectomy totally abolished the hyperglycemic effect in both strains. Furthermore, although not affecting glucose levels in adrenalectomized Wistar rats, ic RX 77368 (50 ng) significantly reduced blood glucose in adrenalectomized GK rats, from high diabetic levels to levels the same as in Wistar rats. This glucose-normalizing effect of ic TRH analog in GK rats was achieved by inducing a 6.5-fold increase of serum insulin, which was abolished by vagotomy. Taken together, our data indicate that the dominant sympathetic-adrenal activation by a high dose of ic TRH analog in GK rats plays a suppressing role on the vagal stimulation of pancreatic insulin secretion. The unbalanced central activation of vagal and sympathetic systems may contribute to the impaired insulin secretion in T2D GK rats.
Alteration in the balance of parasympathetic and sympathetic nervous activity, mainly explained by attenuated parasympathetic activity and relative predominance of sympathetic activity, is common in T2D patients. Sustained overactivation of the sympathetic nervous system was attributed to the central effects of hyperinsulinemia and believed to play an important role in the pathological development of T2D, in particular, to contribute to the hypertension and cardiovascular mortality in T2D (48, 49, 50, 51). Our results indicate that the vagal-sympathetic imbalance in T2D could be a result of altered vagal and/or splanchnic outflow responding to the regulation of medullary TRH. The fact that the same dose of RX 77368 (10 ng) ic injection induced different insulin and glucose responses in nondiabetic and T2D rats with different basal glucose levels suggests that modifying medullary TRH action, such as relating the sensitivity of vagal activation responding to medullary TRH with blood glucose levels, is part of the autonomic adaptation for increased demand of insulin secretion in T2D. However, altered medullary TRH action could also significantly impair pancreatic insulin secretion when sympathetic overactivation becomes overt.
The mechanisms of this altered autonomic response to medullary TRH in GK rats are currently unknown. It was reported that peripheral neuropathy could be tested morphologically in 9- or 18-month-old but not in 2-month-old GK rats (26, 52). Most GK rats used in the present study were 3 months old; it is unlikely that peripheral neuropathy had developed seriously enough to be responsible for the altered autonomic response to ic TRH analog. The results showing that vagal-mediated insulin and sympathetic-adrenal-mediated glucose responses to ic TRH analog were actually more sensitive in GK than in Wistar rats also do not favor this possibility. In addition, 9-month-old female GK rats, assumed to have developed peripheral neuropathy (52), displayed the same extent of glucose-decreasing response to ic TRH analog (10 ng) as the 3-month-old female GK rats, indicating that peripheral neuropathy may not be a critical factor responsible for the altered autonomic response in GK rats of the present study. In support of this, central sympathetic hyperactivity was observed in T2D patients without peripheral neuropathy (49). Abnormal gene and/or protein expression of neuropeptides/transmitters and their receptors, such as TRH and its receptor, in medullary autonomic regulatory nuclei and vagal/sympathetic transduction pathways may play a role in the altered autonomic regulation in GK rats because these components might be influenced by the altered metabolism in T2D. Although direct evidence has yet to be obtained, alterations have been observed in the spinal cord and the ventromedial hypothalamic nucleus of GK rats, such as decreased adrenergic receptors, reduced norepinephrine release, and decreased expression of substance P and calcitonin gene-related protein in the spinal cord (26, 53, 54, 55). Additional experiments, such as measuring blood concentration of catecholamines and levels of mRNA and protein of neuropeptides/transmitters and their receptors in the brain medulla and thoracic spinal cord, brain medullary microinjection of TRH or its analog into vagal and sympathetic controlling nuclei, or a direct electrophysiological recording of the hepatic vagal and splanchnic discharges responding to ic TRH analog, will provide more information on the mechanism of unbalanced vagal-sympathetic activation by medullary TRH in GK rats.
In a recently published paper, Dunn (56) emphasized that it is important to keep in mind that the primary abnormality of T2D is the loss of insulin secretion and that a major contributor to insulin resistance is hyperglycemia secondary to insulin deficit. Subjects at risk of developing T2D have -cell dysfunction before they develop glucose intolerance (57). Our present results show that in T2D GK rats, vagal integrity is important for antagonizing hyperglycemia and pancreatic insulin release is sensitively responsive to the central-vagal stimulation induced by medullary TRH receptor activation. Moreover, the insulin-stimulatory action of medullary TRH is glucose-level related. However, the vagal-mediated insulin stimulation can be suppressed by an overactivation of the sympathetic-adrenal system, which is also regulated by medullary TRH. These findings indicate that autonomic response to medullary TRH plays an important role in physiological and pathophysiological regulation of pancreatic endocrine secretion. Increasing vagal activity, decreasing sympathetic-adrenal tone, or correcting the unbalanced autonomic response to central regulation could be potential therapeutic approaches for improving islet -cell functions in T2D patients. A recent clinical observation that the insulin requirement dramatically decreased to less than 50% in a T2D patient who had undergone spinal-sympathetic blockage provides supportive evidence for this possibility (58).
Acknowledgments
We thank Ms. Ai Chen for her technical assistance.
Footnotes
This work was supported by Veterans Affairs Merit Award (to H.Y.) and National Institutes of Health DK-41301 (CURE Center Grant Animal Core).
First Published Online September 22, 2005
Abbreviations: DVC, Dorsal vagal complex; GK rats, Goto-Kakizaki rats; ic, intracisternal; T2D, type 2 diabetes.
Accepted for publication September 7, 2005.
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