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Endocrinology 2005 146:5425-5432 originally published online Sep 22, 2005; , doi: 10.1210/en.2005-0553

Yan Ao, Natalie Toy, Moon K. Song, Vay Liang W. Go and Hong Yang

Diabetic Goto-Kakizaki Rats2Thyrotropin-Releasing Hormone (TRH)-Induced Autonomic Activation in Type

Altered Glucose and Insulin Responses to Brain Medullary

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Altered Glucose and Insulin Responses to BrainMedullary Thyrotropin-Releasing Hormone (TRH)-Induced Autonomic Activation in Type 2 Diabetic Goto-Kakizaki Rats

Yan Ao, Natalie Toy, Moon K. Song, Vay Liang W. Go, and Hong Yang

Center for Ulcer Research and Education: Digestive Diseases Research Center, Department of Medicine, Division of DigestiveDiseases and Brain Research Institute, University of California, Los Angeles, and Department of Veterans Affairs GreaterLos Angeles Healthcare System, Los Angeles, California 90073

Insulin secretion is impaired in type 2 diabetes (T2D). Theinsulin and glucose responses to central autonomic activationinduced by excitation of brain medullary TRH receptors werestudied in T2D Goto-Kakizaki (GK) rats. Blood glucose levelsin normally fed, pentobarbital-anesthetized GK and nondia-betic Wistar rats were 193 and 119 mg/100 ml in males and 214and 131 mg/100 ml in females. Intracisternal injection (ic) ofthe stable TRH analog RX 77368 (10 ng) induced significantlyhigher insulin response in both genders of overnight-fastedGK rats compared with Wistar rats and slightly increasedblood glucose in female Wistar rats but significantly de-creased it from 193 to 145 mg/100 ml in female GK rats. RX77368 (50 ng) ic induced markedly greater glucose and rela-tively weaker insulin responses in male GK rats than Wistarrats. Bilateral vagotomy blocked ic RX 77368-induced insulin

secretion, whereas adrenalectomy abolished its hyperglyce-mic effect. In adrenalectomized male GK but not Wistar rats,ic RX 77368 (50 ng) dramatically increased serum insulin lev-els by 6.5-fold and decreased blood glucose levels from 154 to98 mg/100 ml; these changes were prevented by vagotomy. GKrats had higher basal pancreatic insulin II mRNA levels but alower response to ic RX 77368 (50 ng) compared with Wistarrats. These results indicate that central-vagal activation-in-duced insulin secretion is susceptible in T2D GK rats. How-ever, the dominant sympathetic-adrenal response to medul-lary TRH plays a suppressing role on vagal-mediated insulinsecretion. This unbalanced vago-sympathetic activation bymedullary TRH may contribute to the impaired insulin secre-tion in T2D. (Endocrinology 146: 5425–5432, 2005)

ABNORMAL INSULIN SECRETION and synthesis arekey factors in the pathophysiology of type 2 diabetes

(T2D) (1). Pancreatic endocrine secretion is regulated by thecentral nervous system through rich innervation of vagal andsympathetic nerves in the islets (2, 3). Insulin secretion isstimulated by vagal activation and inhibited by sympathetic-adrenal activation (4, 5). Both systems participate in meal-induced insulin secretion but only the vagus-cholinergiccomponent plays a major role in insulin secretion of thecephalic phase and during the early absorption period (6, 7).Maintaining normal glucose tolerance requires the integrityof vagal function (8). Therefore, defects in the autonomiccontrol of pancreatic �-cell function might contribute to therelatively insufficient insulin secretion in T2D. Although am-ple knowledge has been achieved in the cause, prevention,and repair of islet �-cell damage and diminished insulinsecretion in diabetes at the cellular and molecular levels (9),the altered autonomic regulation of pancreatic insulin secre-tion in T2D is still poorly understood.

TRH is a neuropeptide synthesized in brain medullarycaudal raphe nuclei and the parapyramidal regions. These

nuclei send TRH-containing nerve projections to innervatethe dorsal vagal complex (DVC), composed of the dorsalmotor nucleus of the vagus and the nucleus tractus solitarii,as well as the ventrolateral reticular formation of the medullaand the intermediolateral cell column of the spinal cord (10,11). It was found in the 1980s that an intracerebroventricularinjection of TRH induces hyperglycemia through pathwaysinvolving the adrenal gland; however, intracerebroventric-ular TRH also prevents central and peripheral stimuli-in-duced hyperglycemia by stimulating insulin release in ratsand mice (12–14). Studies in the last 20 yr have well estab-lished that brain medullary TRH plays a physiological rolein the autonomic regulation of visceral functions (15). TheDVC contains dense TRH-immunoreactive nerve terminalsand TRH receptors (16, 17). TRH or its stable analog RX 77368injected intracisternally (ic) or microinjected into the DVC orTRH endogenously released into the DVC after chemicalstimulation of cell bodies in the raphe nuclei increases vagalefferent discharge, induces vagally mediated activation ofenteric neurons and increases of gastric secretion and mo-tility (18 –22), and stimulates pancreatic insulin secretion(23). Convincing findings demonstrated that ic TRH or itsanalog induces centrally initiated vagal and sympatheticactivation that mimics physiological or pathophysiologi-cal processes (15).

In this study, we tested the hypothesis that autonomicregulation of pancreatic insulin secretion and synthesis are

First Published Online September 22, 2005Abbreviations: DVC, Dorsal vagal complex; GK rats, Goto-Kakizaki

rats; ic, intracisternal; T2D, type 2 diabetes.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/05/$15.00/0 Endocrinology 146(12):5425–5432Printed in U.S.A. Copyright © 2005 by The Endocrine Society

doi: 10.1210/en.2005-0553

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impaired in T2D. We compared blood glucose and pancreaticinsulin responses to ic injection of the stable TRH analog, RX77368, between the nondiabetic Wistar rats and Goto-Kak-izaki (GK) rats, the genetically determined nonobese T2Dmodel with impaired insulin response to glucose (24, 25).Surgical approaches were used to determine the mediationof vagal and adrenal-sympathetic pathways in ic TRH ana-log-induced glucose and insulin changes in the two ratstrains and the imbalance of vagal-sympathetic activation inthe T2D GK rats.

Materials and MethodsAnimals

The GK rats were bred in Animal Facilities of the Veterans Affairs(VA) Greater Los Angeles Area Healthcare System with approved pro-tocol and used at the age of 3 months when body weight was 240–270g (male) or 180–220 g (female), except in one experiment specified inResults that used 9-month-old female rats. The age- and sex-matchedcontrol 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 housedunder controlled conditions (21–23 C, lights on from 0600–1800 h) withfree access to standard rat chow (Prolab Lab Diet; PMI Nutrition Inter-national, Brentwood, MO) and tap water. Food, but not water, wasremoved 16 h before most experiments, except for one group of Wistarand one group of GK rats that were used to obtain basal glucose andinsulin levels in normally fed conditions. Animal protocols were ap-proved by the University of California, Los Angeles, Office for theProtection of Research Subjects and VA Greater Los Angeles AreaHealthcare System Animal Committee.

Experimental protocol

All experiments were performed between 0900 am and 1500 h. Ratswere anesthetized with ip pentobarbital (Abbott Laboratories, NorthChicago, IL) (50 mg/kg followed by 20 mg/kg each hour until the endof experiments) to avoid surgery-, ic injection-, and blood samplings-induced stressful influence on glycemic regulation that is usually inev-itable in conscious animals. Previous studies have shown that pento-barbital anesthesia has slight to moderate influence on insulin output,glucose production, hepatic insulin resistance, and pancreatic bloodflow (27–29). However, other anesthesia, such as Hypnorm, urethane, orketamine, can cause strong changes in autonomic activity (30–32) andinsulin resistance (33) or induce hyperglycemia (34–36). Thus, pento-barbital 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 bloodsampling. Basal blood samples (0.2 ml/rat) were collected at 30 min afterthe iv cannulation. In some groups of rats, bilateral cervical vagotomy,bilateral adrenalectomy, both of the surgeries, or the correspondingsham operation was performed immediately after the iv cannulation. Inthese groups, the basal blood samples were collected after a 60-minpostsurgery stabilization period. After the basal blood sampling, eachrat was positioned on a stereotaxic instrument (Kopf model 900) andreceived an ic injection of either physiological saline (vehicle, 10 �l) orRX 77368 (Ferring Pharmaceuticals, Felthan, Middlesex, UK) (10 or 50ng/10 �l), 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 eachreceiving ic saline or RX 77368 (50 ng), respectively, the pancreas wascollected at 120 min after the ic injection for Northern blot analysis ofpancreatic insulin II mRNA levels.

Measurement of blood glucose and serum insulin levels

Blood glucose levels were measured by One Touch Ultra Blood Glu-cose 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 standardRNAzol method using Trizol reagent (Invitrogen Life Technologies,Carlsbad, CA). Total RNA (20 �g) was run on agarose gel containingMOPS and formaldehyde (Sigma Chemical Co., St. Louis, MO) and thentransferred to a nylon membrane by UV cross-linking. The insulin IIDNA probe was prepared from RT (Ambion, Austin, TX)-PCR (QIA-GEN, Valencia, CA). The sequence of insulin II primer was designedusing Primer Express software (accession J04807). The forward insulinII primer (5�–3�) sequence was CCTAAGTGACCAGCTACA; the reverseprimer (5�–3�) sequence was GTAGTTCTGCAGTTGGTA. The size ofthe PCR product was 367 bp. DNA fragments from PCR were run onlow-melting agarose gel, extracted, and then purified by Wizard PCRPreps DNA purification system (Promega, Madison, WI). Purified PCRproduct was labeled with [�-32P]dCTP (ICN Pharmaceuticals Inc.,Bryan, OH) using Random Primer DNA Labeling Systems (New En-gland BioLabs, Inc., Beverly, MA). After labeling, probes were purifiedusing QIAquick Nucleotide Removal Kit (QIAGEN). The hybridizationwas 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 per-formed using two-way or one-way ANOVA followed by Dunn’smethod. Comparisons between group mean values of Wistar and GKrats receiving the same treatment or between RX 77368-treated andsaline-treated rats of the same strain were performed using unpairedStudent’s t test. Comparisons between mean values before and after icinjection of the same group used paired Student’s t test. All the statisticaltests were performed using SigmaStat program. P value � 0.05 wasconsidered statistically significant.

ResultsBasal blood glucose and serum insulin levels ofanesthetized 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 thesame strain. Serum insulin levels were lower in GK ratscompared with Wistar controls and lower in females com-pared with males of the same strain, although these differ-ences did not reach statistical significance (Fig. 1). Overnightfasting significantly reduced blood glucose levels in Wistarrats, although the levels were still maintained within thephysiological range. The high blood glucose levels observedin fed GK rats were normalized in overnight-fasted males butremained significantly higher in overnight-fasted females(Fig. 1). Compared with the normally fed rats, serum insulinlevels were lower in all the overnight-fasted rats, which wasstatistically significant in Wistar males and GK females.However, insulin levels were still higher in fasted male GKthan fasted male Wistar rats (Fig. 1). The female GK rats hadthe lowest insulin levels among all the fasted groups,which were in accordance with their high blood glucoselevels (Fig. 1).

Effect of ic TRH analog RX 77368 on blood glucose andserum insulin levels in overnight-fasted male Wistar andGK rats

Saline (10 �l) ic injection did not significantly influenceblood glucose and serum insulin levels in overnight-fasted

5426 Endocrinology, December 2005, 146(12):5425–5432 Ao et al. • Vagal-Sympathetic Control of Insulin in GK Rats

male Wistar and GK rats compared with their basal levels(Fig. 2A). RX 77368 ic at a low dose (10 ng/10 �l) did notchange 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 increasedglucose levels in GK rats and induced a marked insulin-stimulatory response. The insulin levels in the GK ratsreached a significant 3-fold higher peak compared with thebasal 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 �l), a significant and potent hyperglycemic re-sponse was induced, which was significantly stronger in GKcompared with Wistar rats (Fig. 2C). Blood glucose increasedfrom the basal levels of 88 � 6 and 123 � 3 mg/100 ml in theWistar and GK rats to peak levels of 250 � 29 and 334 � 26mg/100 ml at 90 min after the injection, respectively, andremained at significantly high levels until the end of theobservation period (120 min after the injection) (Fig. 2C).However, serum insulin levels significantly and strikinglyincreased 6-fold in Wistar rats but only increased less than3-fold in GK rats, although it was statistically significantcompared with its basal levels (Fig. 2C). The ic 50-ng RX77368-induced increase of insulin in the GK rats reachedsimilar levels as that induced by ic 10 ng RX 77368 (Fig. 2, Band C).

Effect of ic TRH analog RX 77368 on blood glucose andserum insulin levels in overnight-fasted female Wistar andGK rats

Overnight-fasted female GK rats had higher basal bloodglucose and lower serum insulin levels compared with the

FIG. 1. Basal blood glucose and serum insulin levels of normally fedand overnight-fasted GK and Wistar rats. Each column representsmean � SEM of the number of rats indicated in the bottom of thecolumn. *, P � 0.05 compared with Wistar rats of same gender andfeeding state; #, P � 0.05 compared with normally fed rats of samegender and strain; @, P � 0.05 compared with male rats of the samefeeding state and strain.

FIG. 2. Effect of ic injection of saline or RX 77368 (10 or 50ng) on blood glucose and serum insulin levels in overnight-fasted male Wistar and GK rats. Each point representsmean � SEM of four to six GK (F) or Wistar (E) rats. *, P �0.05 compared with Wistar rats; #, P � 0.05 compared withthe basal value of the group.

Ao et al. • Vagal-Sympathetic Control of Insulin in GK Rats Endocrinology, December 2005, 146(12):5425–5432 5427

males (Fig. 1). A low dose of RX 77368 (10 ng) was ic injectedin each pentobarbital-anesthetized rat in groups (n � 4 pergroup) 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; bodyweight, 224 � 7 g) rats. RX 77368 (10 ng) slightly but sig-nificantly increased blood glucose levels from a basal level of92 � 2 to 118 � 4 mg/100 ml at 120 min after the injectionin female Wistar rats but did not significantly influence theirserum insulin levels (Fig. 3). In contrast, ic RX 77368 (10 ng)significantly reduced blood glucose from a basal level of193 � 13 to 145 � 9 mg/100 ml (�25%) in young female GKand from 157 � 11 to 123 � 3 mg/100 ml (�22%) in oldfemale GK rats (Fig. 3). In accordance with this glucose de-crease, serum insulin levels significantly increased to 2.3-foldof the basal levels in the young female GK rats but not inyoung Wister controls (Fig. 3). The direction and extent of icRX 77368-induced changes in individual glucose levels at 60and 120 min after the ic injection were negatively correlatedwith the individual basal glucose levels in these three groupsof rats (Fig. 4). That is, ic RX 77368 (10 ng) slightly increasedglucose levels in rats with relatively low basal glucose levels,such as in Wistar rats, but significantly reduced glucoselevels in hyperglycemic GK rats (Figs. 3 and 4). The glucoselevels converged to the level of 120 mg/100 ml after ic RX77368 (10 ng) injection in these three groups of rats withsignificantly different basal glucose levels (Fig. 3).

Effect of ic TRH analog RX 77368 on pancreatic insulin IIgene expression in overnight-fasted male Wistar andGK rats

Pancreatic insulin II mRNA levels were significantlyhigher in the overnight-fasted male GK rats than the Wistarcontrols (Fig. 5). RX 77368 (50 ng) ic injection significantlyincreased pancreatic insulin II mRNA levels in Wistar ratsbut did not further increase it in GK rats measured at 120 minafter the ic injection (Fig. 5).

Effect of vagotomy, adrenalectomy, and vagotomy plusadrenalectomy on basal glucose and insulin levels inovernight-fasted male Wistar and GK rats

Basal blood samples were collected 60 min after one of thesurgeries in overnight-fasted male rats. Blood glucose levelswere influenced by bilateral cervical vagotomy, as shown bya slight but significant decrease in Wistar rats and a signif-icant 26% increase in GK rats (Table 1). Vagotomy also broad-ened individual variations in serum insulin levels and dulledthe significant difference between Wistar and GK rats ob-served in sham-operated groups (Table 1). In contrast, bi-lateral adrenalectomy had no effect on basal blood glucoselevels in Wistar rats and nonsignificantly increased it in GKrats. Serum insulin levels were significantly increased by2-fold in both adrenalectomized Wistar and GK rats (Table1). The influence of vagotomy plus adrenalectomy on basal

FIG. 3. Effect of ic injection of RX 77368 (10 ng) on blood glucose andserum insulin levels in overnight-fasted female Wistar and GK rats.Each point represents mean � SEM of number of GK (F, young GK;f, old GK) or Wistar (E) rats indicated in the parentheses. *, P � 0.05compared with Wistar rats; #, P � 0.05 compared with the basal valueof the group.

FIG. 4. Correlations between the change (�) of blood glucose at 60 or120 min after ic RX 77368 (10 ng) and individual basal glucose levelsof overnight-fasted female GK or Wistar rats. R, Correlation coeffi-cient.

FIG. 5. Northern blot analysis of pancreatic insulin II mRNA signalsin saline or RX 77368 (50 ng) ic injected, overnight-fasted male GK orWistar rats. *, P � 0.05 compared with saline-injected Wistar rats.


blood glucose and serum insulin levels was similar to that ofadrenalectomy alone (Table 1).

Effect of vagotomy, adrenalectomy, and vagotomy plusadrenalectomy on ic RX 77368-induced changes in glucoseand insulin levels in overnight-fasted male Wistar andGK rats

Acute bilateral cervical vagotomy diminished the hyper-glycemic effect of ic RX 77368 (50 ng) in both the Wistar andGK rats, although the effect was still significant in bothstrains (Fig. 6B). Compared with the sham-operated groups,the peak glucose increase at 90 min after ic RX 77368 wasreduced from 250 � 29 to 141 � 8 mg/100 ml (�44%) invagotomized Wistar and from 334 � 26 to 252 � 25 mg/100ml (�25%) in vagotomized GK rats (Fig. 6, A and B). Theinsulin-stimulatory effect of ic RX 77368 (50 ng) was totally

prevented by vagotomy (Fig. 6B). Acute bilateral adrenalec-tomy completely abolished the hyperglycemic effect of ic RX77368 (50 ng) in the two strains (Fig. 6C). Furthermore, inadrenalectomized GK but not Wistar rats, ic RX 77368 (50 ng)significantly reduced blood glucose levels from a basal levelof 154 � 35 to a nadir of 79 � 13 mg/100 ml at 90 min afterthe injection (Fig. 6C). The peak serum insulin response ob-served at 30 min after ic RX 77368 (50 ng) was a 1.3-foldincrease in adrenalectomized Wistar rats, which was notstatistically significant compared with the preinjection level.In contrast, a significant and remarkable 6.5-fold increase ofpeak insulin response was observed in adrenalectomized GKrats (Fig. 6C). The decrease in blood glucose and the increasein insulin levels induced by ic RX 77368 in adrenalectomizedGK rats were absent in GK rats that received both bilateraladrenalectomy and cervical vagotomy (Fig. 6D).

TABLE 1. Basal blood glucose and serum insulin levels of overnight-fasted male Wistar and GK rats at 60 min after surgery

SurgeryBlood glucose (mg/100 ml) Serum insulin (ng/ml)

Wistar rats (n) GK rats (n) Wistar rats (n) GK rats (n)

Sham operation 89 � 3 (11) 112 � 6 (12)a 0.45 � 0.06 (11) 0.95 � 0.08 (12)a

Vagotomy (Vx) 74 � 4 (5)b 141 � 3 (4)a,b 1.02 � 0.35 (5) 0.85 � 0.28 (4)Adrenalectomy (Ax) 88 � 3 (7) 154 � 35 (7)a 0.96 � 0.23 (7)b 1.91 � 0.32 (7)a,b

Vx � Ax 86 � 6 (5) 186 � 20 (5)a,b 1.03 � 0.19 (5)b 2.23 � 0.48 (5)a,b

a P � 0.05 compared with Wistar rats.b P � 0.05 compared with sham-operated group of the same strain.

FIG. 6. Effect of ic injection of RX 77368 (50 ng) on bloodglucose and serum insulin levels in overnight-fastedmale Wistar and GK rats that underwent sham opera-tion (A), bilateral cervical vagotomy (B), bilateral adre-nalectomy (C), or vagotomy plus adrenalectomy (D).Each point represents mean � SEM of number of GK (F)or Wistar (E) rats indicated in the parentheses. *, P �0.05 compared with Wistar rats; #, P � 0.05 comparedwith the basal value of the group.


Discussion

Results obtained from this study clearly show that the T2DGK rats have altered glucose and insulin responses to acentrally initiated autonomic activation induced by ic TRHanalog. These were shown by a susceptible vagal-mediatedinsulin secretion after a low dose of ic RX 77368 (10 ng) andsignificantly powerful sympathetic-adrenal-mediated hy-perglycemic and insulin-inhibitory responses to an ic highdose of RX 77368 (50 ng).

The GK rat is a polygenic model of nonobese T2D withimpaired insulin response to elevated glucose levels (25). Inthis study, the normally fed GK rats had remarkable hyper-glycemia without a matched elevation in serum insulin lev-els, proving their diabetic status and diminished postpran-dial insulin secretion. Most of the experiments in this studywere performed in overnight-fasted rats to minimize theinfluence of digestion and postprandial absorption on basalcirculating glucose and insulin levels. Previous findings in-dicate an abnormal vagal-cholinergic regulation of visceralfunctions in GK rats that contributes to the impaired pan-creatic insulin secretion. For instance, vagal-dependent isletblood flow, which is important in glucose-load-induced in-sulin secretion (39, 40), is diminished in GK rats; this abnor-mality participates in the progressive deterioration of glu-cose intolerance (41, 42). Carbachol, which activatesmuscarinic acetylcholine receptors, fully normalizes insulinsecretion responding to 16.7 mmol/liter glucose in GK ratsthrough an effect abolished by atropine (43). The presentstudy further investigated the vagal dysfunction in this T2Drat model by testing glucose and insulin responses to icinjection of a stable TRH analog, which mimics the physio-logical process of centrally initiated autonomic activation.TRH or RX 77368 exogenously injected into the cisternamagna acts on TRH receptors located on vagal motor neu-rons in the DVC to elevate vagal efferent discharge (21) thatresults in vagal-cholinergic-mediated stimulation of gastro-intestinal functions (15, 19, 20, 22). TRH or RX 77368 alsocauses sympathetic-adrenal gland-mediated hyperglycemiaby acting on unidentified central sites (44), possibly involv-ing the rostroventrolateral reticular nucleus and the cau-doventrolateral reticular nucleus in the brain medulla, whereTRH-containing fibers are localized (our unpublished obser-vation), neurons participate in sympathetic regulation of vis-ceral functions (45), and neurons with presumed sympatho-excitatory function are activated by TRH (46).

We have previously reported that acute hyperglycemiainduced by iv glucose infusion completely abolishes ic TRHanalog-induced gastric acid secretion in nondiabetic rats (47).Based on this finding, we originally hypothesized that theimpaired vagal regulation of insulin secretion in GK ratsmight be the result of an inhibitory influence of its hyper-glycemia on medullary TRH action. However, results of thepresent study show that this is not the case. A low dose ofRX 77368 (10 ng) did not significantly influence serum insulinlevels in Wistar control rats but remarkably increased it inboth the male and female GK rats, indicating that insulinresponse to central vagal activation induced by medullaryTRH is actually more sensitive, rather than dulled, in GKthan in Wistar rats. The higher dose of RX 77368 (50 ng) ic

injection, on the other hand, induced remarkable hypergly-cemia in both strains and a 6-fold increase in serum insulinin Wistar rats. However, this dose (50 ng) did not furtherincrease insulin levels in GK rats compared with the responseto 10 ng. In accordance with this relatively lower insulinresponse, the glucose increase was significantly greater inGK than in Wistar rats. These data suggest that a low doseof RX 77368 (10 ng) induces vagal activation while havingless impact on sympathetic-adrenal activation, resulting in aconsequent increase in pancreatic insulin secretion. Thehigher dose of RX 77368 (50 ng), however, activates the vagaland also the sympathetic-adrenal systems, the latter causinghyperglycemia. In supporting this view, the insulin increaseafter ic RX 77368 was completely prevented by acute bilateralcervical vagotomy, and the hyperglycemia was abolished byadrenalectomy. The greater hyperglycemic response to ic RX77368 (50 ng) in GK rats indicate that not only the vagusnerve, but also the sympathetic-adrenal system, is morestrongly activated by medullary TRH in GK than in Wistarrats.

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 sym-pathetic-adrenal pathways were performed in overnight-fasted male rats. The surgeries themselves did not influenceblood glucose levels in nondiabetic Wistar rats, except bi-lateral cervical vagotomy, which slightly and significantlyreduced glucose. These data indicate that acute surgery inanesthetized rats of the present study had little, if any, stress-ful influence on glucose levels. In contrast to the Wister rats,blood glucose levels significantly increased in GK rats thatunderwent vagotomy or vagotomy plus adrenalectomy butnot adrenalectomy, indicating a beneficial role of the integ-rity of the vagus nerve in antagonizing hyperglycemia in GKrats. The serum insulin levels, however, significantly in-creased by about 2-fold in adrenalectomized and vagoto-mized plus adrenalectomized, but not the vagotomized,Wistar and GK groups, indicating a sympathetic-adrenalinhibitory tone on basal insulin secretion in both the non-diabetic Wistar rats and the T2D GK rats, which was strongerin the GK rats because these rats had a more remarkableinsulin increase after adrenalectomy. As expected, vagotomycompletely prevented ic RX 77368-induced hyperinsulin-emia and adrenalectomy totally abolished the hyperglycemiceffect in both strains. Furthermore, although not affectingglucose levels in adrenalectomized Wistar rats, ic RX 77368(50 ng) significantly reduced blood glucose in adrenalecto-mized GK rats, from high diabetic levels to levels the sameas in Wistar rats. This glucose-normalizing effect of ic TRHanalog in GK rats was achieved by inducing a 6.5-fold in-crease of serum insulin, which was abolished by vagotomy.Taken together, our data indicate that the dominant sym-pathetic-adrenal activation by a high dose of ic TRH analogin GK rats plays a suppressing role on the vagal stimulationof pancreatic insulin secretion. The unbalanced central acti-vation of vagal and sympathetic systems may contribute tothe impaired insulin secretion in T2D GK rats.

Alteration in the balance of parasympathetic and sympa-thetic nervous activity, mainly explained by attenuated para-sympathetic activity and relative predominance of sympa-


thetic activity, is common in T2D patients. Sustainedoveractivation of the sympathetic nervous system was at-tributed to the central effects of hyperinsulinemia and be-lieved to play an important role in the pathological devel-opment of T2D, in particular, to contribute to thehypertension and cardiovascular mortality in T2D (48–51).Our results indicate that the vagal-sympathetic imbalance inT2D could be a result of altered vagal and/or splanchnicoutflow responding to the regulation of medullary TRH. Thefact that the same dose of RX 77368 (10 ng) ic injectioninduced different insulin and glucose responses in nondia-betic and T2D rats with different basal glucose levels sug-gests that modifying medullary TRH action, such as relatingthe sensitivity of vagal activation responding to medullaryTRH with blood glucose levels, is part of the autonomicadaptation for increased demand of insulin secretion in T2D.However, altered medullary TRH action could also signifi-cantly impair pancreatic insulin secretion when sympatheticoveractivation becomes overt.

The mechanisms of this altered autonomic response tomedullary TRH in GK rats are currently unknown. It wasreported that peripheral neuropathy could be tested mor-phologically in 9- or 18-month-old but not in 2-month-old GKrats (26, 52). Most GK rats used in the present study were 3months old; it is unlikely that peripheral neuropathy haddeveloped seriously enough to be responsible for the alteredautonomic response to ic TRH analog. The results showingthat vagal-mediated insulin and sympathetic-adrenal-medi-ated glucose responses to ic TRH analog were actually moresensitive in GK than in Wistar rats also do not favor thispossibility. In addition, 9-month-old female GK rats, as-sumed to have developed peripheral neuropathy (52), dis-played the same extent of glucose-decreasing response to icTRH analog (10 ng) as the 3-month-old female GK rats,indicating that peripheral neuropathy may not be a criticalfactor responsible for the altered autonomic response in GKrats of the present study. In support of this, central sympa-thetic hyperactivity was observed in T2D patients withoutperipheral neuropathy (49). Abnormal gene and/or proteinexpression of neuropeptides/transmitters and their recep-tors, such as TRH and its receptor, in medullary autonomicregulatory nuclei and vagal/sympathetic transduction path-ways may play a role in the altered autonomic regulation inGK rats because these components might be influenced bythe altered metabolism in T2D. Although direct evidence hasyet to be obtained, alterations have been observed in thespinal cord and the ventromedial hypothalamic nucleus ofGK rats, such as decreased adrenergic receptors, reducednorepinephrine release, and decreased expression of sub-stance P and calcitonin gene-related protein in the spinal cord(26, 53–55). Additional experiments, such as measuringblood concentration of catecholamines and levels of mRNAand protein of neuropeptides/transmitters and their recep-tors in the brain medulla and thoracic spinal cord, brainmedullary microinjection of TRH or its analog into vagal andsympathetic controlling nuclei, or a direct electrophysiolog-ical recording of the hepatic vagal and splanchnic dischargesresponding to ic TRH analog, will provide more informationon the mechanism of unbalanced vagal-sympathetic activa-tion by medullary TRH in GK rats.

In a recently published paper, Dunn (56) emphasized thatit is important to keep in mind that the primary abnormalityof T2D is the loss of insulin secretion and that a major con-tributor to insulin resistance is hyperglycemia secondary toinsulin deficit. Subjects at risk of developing T2D have �-celldysfunction before they develop glucose intolerance (57).Our present results show that in T2D GK rats, vagal integrityis important for antagonizing hyperglycemia and pancreaticinsulin release is sensitively responsive to the central-vagalstimulation induced by medullary TRH receptor activation.Moreover, the insulin-stimulatory action of medullary TRHis glucose-level related. However, the vagal-mediated insu-lin stimulation can be suppressed by an overactivation of thesympathetic-adrenal system, which is also regulated by med-ullary TRH. These findings indicate that autonomic responseto medullary TRH plays an important role in physiologicaland pathophysiological regulation of pancreatic endocrinesecretion. Increasing vagal activity, decreasing sympathetic-adrenal tone, or correcting the unbalanced autonomic re-sponse to central regulation could be potential therapeuticapproaches for improving islet �-cell functions in T2D pa-tients. A recent clinical observation that the insulin require-ment dramatically decreased to less than 50% in a T2D pa-tient who had undergone spinal-sympathetic blockageprovides supportive evidence for this possibility (58).

Acknowledgments

We thank Ms. Ai Chen for her technical assistance.

Received May 9, 2005. Accepted September 7, 2005.Address all correspondence and requests for reprints to: Hong Yang,

M.D., Ph.D., Center for Ulcer Research and Education: Digestive Dis-eases Research Center, Veterans Affairs Greater Los Angeles HealthcareSystem Building 115, Room 203, 11301 Wilshire Boulevard, Los Angeles,California 90073. E-mail: [email protected].

This work was supported by Veterans Affairs Merit Award (to H.Y.)and National Institutes of Health DK-41301 (CURE Center Grant AnimalCore).

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