3. 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-Kak-
izaki (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 ana-
log-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 pro-
tocol 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 Inter-
national, 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 ap-
proved 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 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 blood
flow (27–29). However, other anesthesia, such as Hypnorm, urethane, or
ketamine, can cause strong changes in autonomic activity (30–32) and
insulin 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 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 l) or
RX 77368 (Ferring Pharmaceuticals, Felthan, Middlesex, UK) (10 or 50
ng/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 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 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 standard
RNAzol method using Trizol reagent (Invitrogen Life Technologies,
Carlsbad, CA). Total RNA (20 g) 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 (QIA-
GEN, Valencia, CA). The sequence of insulin II primer was designed
using Primer Express software (accession J04807). The forward insulin
II primer (5Ј–3Ј) sequence was CCTAAGTGACCAGCTACA; the reverse
primer (5Ј–3Ј) 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 [␣-32
P]dCTP (ICN Pharmaceuticals Inc.,
Bryan, OH) using Random Primer DNA Labeling Systems (New En-
gland 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 Ϯ 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’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 Ͻ 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 com-
pared with males of the same strain, although these differ-
ences 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 l) ic injection did not significantly influence
blood 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
4. male Wistar and GK rats compared with their basal levels
(Fig. 2A). RX 77368 ic at a low dose (10 ng/10 l) 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 l), a significant and potent hyperglycemic re-
sponse was induced, which was significantly stronger in GK
compared with Wistar rats (Fig. 2C). Blood glucose increased
from the basal levels of 88 Ϯ 6 and 123 Ϯ 3 mg/100 ml in the
Wistar and GK rats to peak levels of 250 Ϯ 29 and 334 Ϯ 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
FIG. 1. Basal blood glucose and serum insulin levels of normally fed
and overnight-fasted GK and Wistar rats. Each column represents
mean Ϯ SEM of the number of rats indicated in the bottom of the
column. *, P Ͻ 0.05 compared with Wistar rats of same gender and
feeding state; #, P Ͻ 0.05 compared with normally fed rats of same
gender and strain; @, P Ͻ 0.05 compared with male rats of the same
feeding state and strain.
FIG. 2. Effect of ic injection of saline or RX 77368 (10 or 50
ng) on blood glucose and serum insulin levels in overnight-
fasted male Wistar and GK rats. Each point represents
mean Ϯ SEM of four to six GK (F) or Wistar (E) rats. *, P Ͻ
0.05 compared with Wistar rats; #, P Ͻ 0.05 compared with
the basal value of the group.
Ao et al. • Vagal-Sympathetic Control of Insulin in GK Rats Endocrinology, December 2005, 146(12):5425–5432 5427
5. males (Fig. 1). A low dose of RX 77368 (10 ng) was ic injected
in each pentobarbital-anesthetized rat in groups (n ϭ 4 per
group) of young female Wistar (2.5 months old; body weight,
237 Ϯ 3 g), young female GK (2.8 months old; body weight,
182 Ϯ 3 g), or old female GK (9 months old; body
weight, 224 Ϯ 7 g) rats. RX 77368 (10 ng) slightly but sig-
nificantly increased blood glucose levels from a basal level of
92 Ϯ 2 to 118 Ϯ 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 Ϯ 13 to 145 Ϯ 9 mg/100 ml (Ϫ25%) in young female GK
and from 157 Ϯ 11 to 123 Ϯ 3 mg/100 ml (Ϫ22%) in old
female GK rats (Fig. 3). In accordance with this glucose de-
crease, 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 signif-
icant 26% increase in GK rats (Table 1). Vagotomy also broad-
ened individual variations in serum insulin levels and dulled
the 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 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
FIG. 3. Effect of ic injection of RX 77368 (10 ng) on blood glucose and
serum insulin levels in overnight-fasted female Wistar and GK rats.
Each point represents mean Ϯ SEM of number of GK (F, young GK;
f, old GK) or Wistar (E) rats indicated in the parentheses. *, P Ͻ 0.05
compared with Wistar rats; #, P Ͻ 0.05 compared with the basal value
of the group.
FIG. 4. Correlations between the change (⌬) of blood glucose at 60 or
120 min after ic RX 77368 (10 ng) and individual basal glucose levels
of overnight-fasted female GK or Wistar rats. R, Correlation coeffi-
cient.
FIG. 5. Northern blot analysis of pancreatic insulin II mRNA signals
in saline or RX 77368 (50 ng) ic injected, overnight-fasted male GK or
Wistar rats. *, P Ͻ 0.05 compared with saline-injected Wistar rats.
5428 Endocrinology, December 2005, 146(12):5425–5432 Ao et al. • Vagal-Sympathetic Control of Insulin in GK Rats
6. 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 hyper-
glycemic 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 Ϯ 29 to 141 Ϯ 8 mg/100 ml (Ϫ44%) in
vagotomized Wistar and from 334 Ϯ 26 to 252 Ϯ 25 mg/100
ml (Ϫ25%) 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 adrenalec-
tomy 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 Ϯ 35 to a nadir of 79 Ϯ 13 mg/100 ml at 90 min after
the injection (Fig. 6C). The peak serum insulin response ob-
served 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).
TABLE 1. Basal blood glucose and serum insulin levels of overnight-fasted male Wistar and GK rats at 60 min after surgery
Surgery
Blood glucose (mg/100 ml) Serum insulin (ng/ml)
Wistar rats (n) GK rats (n) Wistar rats (n) GK rats (n)
Sham operation 89 Ϯ 3 (11) 112 Ϯ 6 (12)a
0.45 Ϯ 0.06 (11) 0.95 Ϯ 0.08 (12)a
Vagotomy (Vx) 74 Ϯ 4 (5)b
141 Ϯ 3 (4)a,b
1.02 Ϯ 0.35 (5) 0.85 Ϯ 0.28 (4)
Adrenalectomy (Ax) 88 Ϯ 3 (7) 154 Ϯ 35 (7)a
0.96 Ϯ 0.23 (7)b
1.91 Ϯ 0.32 (7)a,b
Vx ϩ Ax 86 Ϯ 6 (5) 186 Ϯ 20 (5)a,b
1.03 Ϯ 0.19 (5)b
2.23 Ϯ 0.48 (5)a,b
a
P Ͻ 0.05 compared with Wistar rats.
b
P Ͻ 0.05 compared with sham-operated group of the same strain.
FIG. 6. Effect of ic injection of RX 77368 (50 ng) on blood
glucose and serum insulin levels in overnight-fasted
male 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 Ϯ SEM of number of GK (F)
or Wistar (E) rats indicated in the parentheses. *, P Ͻ
0.05 compared with Wistar rats; #, P Ͻ 0.05 compared
with the basal value of the group.
Ao et al. • Vagal-Sympathetic Control of Insulin in GK Rats Endocrinology, December 2005, 146(12):5425–5432 5429
7. 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 hy-
perglycemic 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 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 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 in-
dicate an abnormal vagal-cholinergic regulation of visceral
functions in GK rats that contributes to the impaired pan-
creatic insulin secretion. For instance, vagal-dependent islet
blood 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 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 physio-
logical process of centrally initiated autonomic activation.
TRH or RX 77368 exogenously injected into the cisterna
magna acts on TRH receptors located on vagal motor neu-
rons in the DVC to elevate vagal efferent discharge (21) that
results in vagal-cholinergic-mediated stimulation of gastro-
intestinal functions (15, 19, 20, 22). TRH or RX 77368 also
causes sympathetic-adrenal gland-mediated hyperglycemia
by acting on unidentified central sites (44), possibly involv-
ing the rostroventrolateral reticular nucleus and the cau-
doventrolateral reticular nucleus in the brain medulla, where
TRH-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 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 hyper-
glycemia 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 hypergly-
cemia 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 sym-
pathetic-adrenal pathways were performed in overnight-
fasted male rats. The surgeries themselves did not influence
blood glucose levels in nondiabetic Wistar rats, except bi-
lateral 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, stress-
ful 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 integ-
rity of the vagus nerve in antagonizing hyperglycemia in GK
rats. 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-adrenal
inhibitory tone on basal insulin secretion in both the non-
diabetic 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 hyperinsulin-
emia 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 adrenalecto-
mized 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 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 analog
in GK rats plays a suppressing role on the vagal stimulation
of pancreatic insulin secretion. The unbalanced central acti-
vation of vagal and sympathetic systems may contribute to
the 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-
5430 Endocrinology, December 2005, 146(12):5425–5432 Ao et al. • Vagal-Sympathetic Control of Insulin in GK Rats
8. thetic activity, is common in T2D patients. Sustained
overactivation 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 the
hypertension and cardiovascular mortality in T2D (48–51).
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 nondia-
betic and T2D rats with different basal glucose levels sug-
gests 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 signifi-
cantly 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 mor-
phologically 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-medi-
ated 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, as-
sumed to have developed peripheral neuropathy (52), dis-
played 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 sympa-
thetic hyperactivity was observed in T2D patients without
peripheral neuropathy (49). Abnormal gene and/or protein
expression of neuropeptides/transmitters and their recep-
tors, such as TRH and its receptor, in medullary autonomic
regulatory nuclei and vagal/sympathetic transduction path-
ways 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 sub-
stance P and calcitonin gene-related protein in the spinal cord
(26, 53–55). Additional experiments, such as measuring
blood concentration of catecholamines and levels of mRNA
and protein of neuropeptides/transmitters and their recep-
tors 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 electrophysiolog-
ical recording of the hepatic vagal and splanchnic discharges
responding to ic TRH analog, will provide more information
on the mechanism of unbalanced vagal-sympathetic activa-
tion 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 con-
tributor 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 insu-
lin stimulation can be suppressed by an overactivation of the
sympathetic-adrenal system, which is also regulated by med-
ullary 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 re-
sponse to central regulation could be potential therapeutic
approaches 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 blockage
provides 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 Healthcare
System Building 115, Room 203, 11301 Wilshire Boulevard, Los Angeles,
California 90073. E-mail: hoyang@ucla.edu.
This work was supported by Veterans Affairs Merit Award (to H.Y.)
and National Institutes of Health DK-41301 (CURE Center Grant Animal
Core).
References
1. Rhodes CJ 2005 Type 2 diabetes: a matter of -cell life and death? Science
307:380–384
2. Woods SC, Porte DJ 1974 Neural control of the endocrine pancreas. Physiol
Rev 54:596–619
3. Berthoud HR, Powley TL 1990 Identification of vagal preganglionics that
mediate cephalic phase insulin response. Am J Physiol Regul Integr Comp
Physiol 258:R523–R530
4. Kaneto A, Miki E, Kosaka K 1974 Effects of vagal stimulation on glucagon and
insulin secretion. Endocrinology 95:1005–1010
5. Ahren B 2000 Autonomic regulation of islet hormone secretion: implications
for health and disease. Diabetologia 43:393–410
6. Benthem L, Mundinger TO, Taborsky GJJ 2000 Meal-induced insulin secre-
tion in dogs is mediated by both branches of the autonomic nervous system.
Am J Physiol Endocrinol Metab 278:E603–E610
7. Strubbe JH 1992 Parasympathetic involvement in rapid meal-associated con-
ditioned insulin secretion in the rat. Am J Physiol Regul Integr Comp Physiol
263:R615–R618
8. Trimble ER, Berthoud HR, Siegel EG, Jeanrenaud B, Renold AE 1981 Im-
portance of cholinergic innervation of the pancreas for glucose tolerance in the
rat. Am J Physiol Endocrinol Metab 241:E337–E341
9. Mandrup-Poulsen T 2003 -Cell death and protection. Ann NY Acad Sci
1005:32–42
10. Lynn RB, Kreider MS, Miselis RR 1991 Thyrotropin-releasing hormone-
immunoreactive projections to the dorsal motor nucleus and the nucleus of the
solitary tract of the rat. J Comp Neurol 311:271–288
11. Sasek CA, Wessendorf MW, Helke CJ 1990 Evidence for co-existence of
thyrotropin-releasing hormone, substance P and serotonin in ventral medul-
lary neurons that project to the intermediolateral cell column in the rat. Neu-
roscience 35:105–119
Ao et al. • Vagal-Sympathetic Control of Insulin in GK Rats Endocrinology, December 2005, 146(12):5425–5432 5431
9. 12. Marubashi S, Kunii Y, Tominaga M, Sasaki H 1988 Modulation of plasma
glucose levels by thyrotropin-releasing hormone administered intracerebrov-
entricularly in the rat. Neuroendocrinology 48:640–644
13. Amir S, Harel M, Rivkind AI 1987 Thyrotropin-releasing hormone potently
reverses epinephrine-stimulated hyperglycemia in mice. Brain Res 435:112–
122
14. Amir S, Rivkind AI 1988 Prevention of clonidine-stimulated hyperglycemia
by thyrotropin-releasing hormone. Peptides 9:527–531
15. Tache´ Y, Yang H 1994 Role of medullary TRH in the vagal regulation of gastric
function. In: Wingate DL, Butkd TF, eds. Innervation of the gut: pathophys-
iological implications. Boca Raton, FL: CRC Press; 67–80
16. Rinaman L, Miselis RR, Kreider MS 1989 Ultrastructural localization of
thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex
in rat. Neurosci Lett 104:7–12
17. Manaker S, Rizio G 1989 Autoradiographic localization of thyrotropin-re-
leasing hormone and substance P receptors in the rat dorsal vagal complex.
J Comp Neurol 290:516–526
18. Stephens RL, Tache Y 1989 Intracisternal injection of a TRH analogue stim-
ulates gastric luminal serotonin release in rats. Am J Physiol Gastrointest Liver
Physiol 256:G377–G383
19. Yang H, Ohning GV, Tache´ Y 1993 TRH in dorsal vagal complex mediates acid
response to excitation of raphe pallidus neurons in rats. Am J Physiol Gas-
trointest Liver Physiol 265:G880–G886
20. Garrick T, Prince M, Yang H, Ohning G, Tache´ Y 1994 Raphe pallidus
stimulation increases gastric contractility via TRH projections to the dorsal
vagal complex in rats. Brain Res 636:343–347
21. O-Lee TJ, Wei JY, Tache´ Y 1997 Intracisternal TRH and RX 77368 potently
activate gastric vagal efferent discharge in rats. Peptides 18:213–219
22. Miampamba M, Yang H, Sharkey KA, Tache Y 2001 Intracisternal TRH
analog induces Fos expression in gastric myenteric neurons and glia in con-
scious rats. Am J Physiol Gastrointest Liver Physiol 280:G979–G991
23. Yang H, Tache Y, Ohning G, Go VL 2002 Activation of raphe pallidus neurons
increases insulin through medullary thyrotropin-releasing hormone (TRH)-
vagal pathways. Pancreas 25:301–307
24. Ostenson CG, Khan A, Abdel-Halim SM, Guenifi A, Suzuki K, Goto Y,
Efendic S 1993 Abnormal insulin secretion and glucose metabolism in pan-
creatic islets from the spontaneously diabetic GK rat. Diabetologia 36:3–8
25. Goto Y, Suzuki K, Sasaki M, Ono T, Abe S 1988 GK rat as a model of
nonobese, noninsulin-dependent diabetes. Selective breeding over 35 gener-
ations. In: Renold AE, Renold, eds. Frontiers in diabetes research: lessons from
animal diabetes II. London: John Libbey; 301–303
26. Murakawa Y, Zhang W, Pierson CR, Brismar T, Ostenson CG, Efendic S,
Sima AA 2002 Impaired glucose tolerance and insulinopenia in the GK-rat
causes peripheral neuropathy. Diabetes Metab Res Rev 18:473–483
27. Clark PW, Jenkins AB, Kraegen EW 1990 Pentobarbital reduces basal liver
glucose output and its insulin suppression in rats. Am J Physiol Endocrinol
Metab 258:E701–E707
28. Iwase M, Tashiro K, Uchizono Y, Goto D, Yoshinari M 2001 Pancreatic islet
blood flow in conscious rats during hyperglycemia and hypoglycemia. Am J
Physiol Regul Integr Comp Physiol 280:R1601–R1605
29. Vera ER, Battell ML, Bhanot S, McNeill JH 2002 Effects of age and anesthetic
on plasma glucose and insulin levels and insulin sensitivity in spontaneously
hypertensive and Wistar rats. Can J Physiol Pharmacol 80:962–970
30. Maggi CA, Meli A 1986 Suitability of urethane anesthesia for physiophar-
macological investigations. Part 3: Other systems and conclusions. Experientia
42:531–537
31. Maggi CA, Meli A 1986 Suitability of urethane anesthesia for physiophar-
macological investigations in various systems. Part 1: General considerations.
Experientia 42:109–114
32. Takayama K, Suzuki T, Miura M 1994 The comparison of effects of various
anesthetics on expression of Fos protein in the rat brain. Neurosci Lett 176:
59–62
33. Porter JP 1994 Effect of intrahypothalamic insulin on sympathetic nervous
function in rats drinking a high-sucrose solution. Am J Physiol Regul Integr
Comp Physiol 266:R1463–R1469
34. Johansen O, Vaaler S, Jorde R, Reikeras O 1994 Increased plasma glucose
levels after Hypnorm anaesthesia, but not after Pentobarbital anaesthesia in
rats. Lab Anim 28:244–248
35. Reyes Toso CF, Linares LM, Rodriguez RR 1995 Blood sugar concentrations
during ketamine or pentobarbitone anesthesia in rats with or without ␣- and
-adrenergic blockade. Medicina (B Aires) 55:311–316
36. Tranquilli WJ, Thurmon JC, Neff-Davis CA, Davis LE, Benson GJ, Hoffman
W, Lock TF 1984 Hyperglycemia and hypoinsulinemia during xylazine-ket-
amine anesthesia in Thoroughbred horses. Am J Vet Res 45:11–14
37. Marota JJ, Crosby G, Uhl GR 1992 Selective effects of pentobarbital and
halothane on c-fos and jun-B gene expression in rat brain. Anesthesiology
77:365–371
38. Yang H, Kawakubo K, Tache´ Y 1999 Intracisternal PYY increases gastric
mucosal resistance: role of cholinergic, CGRP, and NO pathways. Am J Physiol
Gastrointest Liver Physiol 277:G555–G562
39. Kiba T 2004 Relationships between the autonomic nervous system and the
pancreas including regulation of regeneration and apoptosis: recent develop-
ments. Pancreas 29:E51–E58
40. Jansson L, Hellerstrom C 1986 Glucose-induced changes in pancreatic islet
blood flow mediated by central nervous system. Am J Physiol Endocrinol
Metab 251:E644–E647
41. Svensson AM, Ostenson CG, Sandler S, Efendic S, Jansson L 1994 Inhibition
of nitric oxide synthase by NG-nitro-l-arginine causes a preferential decrease
in pancreatic islet blood flow in normal rats and spontaneously diabetic GK
rats. Endocrinology 135:849–853
42. Svensson AM, Ostenson CG, Jansson L 2000 Age-induced changes in pan-
creatic islet blood flow: evidence for an impaired regulation in diabetic GK rats.
Am J Physiol Endocrinol Metab 279:E1139–E1144
43. Guenifi A, Simonsson E, Karlsson S, Ahren B, Abdel-Halim SM 2001 Car-
bachol restores insulin release in diabetic GK rat islets by mechanisms largely
involving hydrolysis of diacylglycerol and direct interaction with the exocy-
totic machinery. Pancreas 22:164–171
44. Feldberg W, Pyke D, Stubbs WA 1985 Hyperglycaemia: imitating Claude
Bernard’s piqure with drugs. J Auton Nerv Syst 14:213–228
45. Ishikawa T, Yang H, Tache Y 2001 Microinjection of bombesin into the ven-
trolateral reticular formation inhibits peripherally stimulated gastric acid se-
cretion through spinal pathways in rats. Brain Res 918:1–9
46. Sun MK, Guyenet PG 1989 Effects of vasopressin and other neuropeptides on
rostral medullary sympathoexcitatory neurons ‘in vitro’. Brain Res 492:261–270
47. Doong ML, Yang H 2003 Intravenous glucose infusion decreases intracisternal
thyrotropin-releasing hormone induced vagal stimulation of gastric acid se-
cretion in anesthetized rats. Neurosci Lett 340:49–52
48. Perin PC, Maule S, Quadri R 2001 Sympathetic nervous system, diabetes, and
hypertension. Clin Exp Hypertens 23:45–55
49. Huggett RJ, Scott EM, Gilbey SG, Stoker JB, Mackintosh AF, Mary DA 2003
Impact of type 2 diabetes mellitus on sympathetic neural mechanisms in
hypertension. Circulation 108:3097–3101
50. Lindmark S, Wiklund U, Bjerle P, Eriksson JW 2003 Does the autonomic
nervous system play a role in the development of insulin resistance? A study
on heart rate variability in first-degree relatives of type 2 diabetes patients and
control subjects. Diabet Med 20:399–405
51. Huggett RJ, Scott EM, Gilbey SG, Bannister J, Mackintosh AF, Mary DA 2005
Disparity of autonomic control in type 2 diabetes mellitus. Diabetologia 48:
172–179
52. Wada R, Koyama M, Mizukami H, Odaka H, Ikeda H, Yagihashi S 1999
Effects of long-term treatment with ␣-glucosidase inhibitor on the peripheral
nerve function and structure in Goto-Kakizaki rats: a genetic model for type
2 diabetes. Diabetes Metab Res Rev 15:332–337
53. Yoshida S, Yamashita S, Tokunaga K, Yamane M, Shinohara E, Keno Y,
Nishida M, Kotani K, Shimomura I, Kobayashi H, Nakamura T, Miyagawa
J, Kameda-Takemura K, Odaka H, Ikeda H, Matsuzawa Y 1996 Visceral fat
accumulation and vascular complications associated with VMH lesioning of
spontaneously non-insulin-dependent diabetic GK rat. Int J Obes Relat Metab
Disord 20:909–916
54. Issa BG, Lewis BM, Ham J, Peters JR, Scanlon MF 1998 Glutamate pathways
mediate somatostatin responses to glucose in normal and diabetic rat hypo-
thalamus. J Neuroendocrinol 10:377–381
55. Bitar MS, Pilcher CW 2000 Diabetes attenuates the response of the lumbospi-
nal noradrenergic system to idazoxan. Pharmacol Biochem Behav 67:247–255
56. Dunn PJ 2004 Insulin therapy in type 2 diabetes. NZ Family Physician 31:
395–398
57. Chiasson JL, Rabasa-Lhoret R 2004 Prevention of type 2 diabetes: insulin
resistance and -cell function. Diabetes 53(Suppl 3):S34–S38
58. Kapural L, Hayek SM, Stanton-Hicks M, Mekhail N 2004 Decreased insulin
requirements with spinal cord stimulation in a patient with diabetes. Anesth
Analg 98:745–746
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5432 Endocrinology, December 2005, 146(12):5425–5432 Ao et al. • Vagal-Sympathetic Control of Insulin in GK Rats
