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Endocrinology 148(5):20752084 Copyright 2007 by The Endocrine Society doi: 10.1210/en.2006-1315

Alteration of Glucose Homeostasis in V1a Vasopressin Receptor-Deficient Mice

Toshinori Aoyagi, Jun-ichi Birumachi, Masami Hiroyama, Yoko Fujiwara, Atsushi Sanbe, Junji Yamauchi, and Akito Tanoue
Department of Pharmacology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan
Arginine-vasopressin (AVP) is known to be involved in maintaining glucose homeostasis, and AVP-resistance is observed in poorly controlled non-insulin-dependent diabetes mellitus subjects, resulting in a lowered plasma volume. Recently we reported that V1a vasopressin receptor-deficient (V1aR / ) mice exhibited a decreased circulating blood volume and hypermetabolism of fat accompanied with impaired insulin-signaling. Here we further investigated the roles of the AVP/V1a receptor in regulating glucose homeostasis and plasma volume using V1aR / mice. The plasma glucose levels at the baseline or during a glucose tolerance test were higher in V1aR / than wild-type (WT) mice. Moreover, a hyperinsulinemic-euglycemic clamp revealed that the glucose infusion rate was significantly lower in V1aR / mice than in WT mice and that hepatic glucose production was higher in V1aR / mice than WT mice. In contrast to the increased hepatic glucose production, the liver glycogen content was decreased in the mutant mice. These results indicated that the mutant mice had impaired glucose tolerance. Furthermore, feeding V1aR / mice a high-fat diet accompanied by increased calorie intake resulted in significantly overt obesity in comparison with WT mice. In addition, we found that the circulating plasma volume and aldosterone level were decreased in V1aR / mice, although the plasma AVP level was increased. These results suggested that the effect of AVP on water recruitment was disturbed in V1aR / mice. Thus, we demonstrated that one of the AVP-resistance conditions resulting from deficiency of the V1a receptor leads to decreased plasma volume as well as impaired glucose homeostasis, which can progress to obesity under conditions of increased calorie intake. (Endocrinology 148: 20752084, 2007)

RGININE-VASOPRESSIN (AVP) is a neuropeptide hormone that is involved in diverse functions, including the regulation of osmotic homeostasis, vasoconstriction, and ACTH release. These physiological effects are mediated by three types of AVP receptors, designated as V1a, V1b, and V2 (13). The V1a receptor is widely expressed, whereas the V1b and V2 receptors are predominantly expressed in the anterior pituitary and the kidney, respectively (1 4). The functional role of the V1a receptor is considered to mediate vascular contraction (5), cellular proliferation (6), platelet aggregation (7), and glycogenolysis (1, 8). The V1b receptor stimulates ACTH and insulin release (9, 10). Both V1a and V1b receptors bind to the Gq protein and act through phosphatidylinositol hydrolysis to mobilize intercellular Ca2 (11, 12). The V2 receptor is coupled with the Gs protein and stimulates adenylate cyclase to increase cellular cAMP, which results in the induction of an antidiuretic effect in the kidney (13). There have been several reports indicating the involvement of AVP in regulating the plasma glucose homeostasis. The plasma AVP level was increased in patients with insulinFirst Published Online February 15, 2007 Abbreviations: ANP, Atrial natriuretic peptide; AVP, arginine-vasopressin; BUN, blood urea nitrogen; CNS, central nervous system; GABAergic, secretion of -aminobutyric acid; GTT, glucose tolerance test; HE, hematoxylin-eosin; HF, high fat; IDDM, insulin-dependent diabetes mellitus; NC, normal chow; NIDDM, non-IDDM; V1aR; vasopressin receptor type V1a; WAT, white adipose tissue; WT, wild type. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

dependent diabetes mellitus (IDDM) or non-IDDM (NIDDM) (14, 15), and treating these subjects with insulin or sulfonylurea improved not only the plasma glucose level but also the plasma AVP level (16, 17). In addition, the binding activity of AVP to the liver, in which the V1a receptors were predominantly expressed, decreased in the ob/ob mice and the streptozotocin-induced diabetes mellitus rat, whereas the binding activity to the kidney, in which the V2 receptors were predominantly expressed, was not altered (18, 19). In addition, the antidiuretic or hepatic function of AVP was attenuated in human and rodent subjects with IDDM or NIDDM (20 22). These findings suggest that the altered glucose homeostasis could occasionally result in or from AVP resistance (20 22), which is, in part, mediated via the V1a receptor, although the mechanisms remain to be elucidated. We generated V1a receptor-deficient (V1aR / ) mice and revealed that the insulin-induced Akt phosphorylation was suppressed in adipocytes isolated from V1aR / mice, suggesting that whole-body glucose tolerance was altered in V1aR / mice (23). In addition, we found that the circulating blood volume and blood pressure were decreased in mutant mice (24). By monitoring urine excretion, we found that there was no significant difference between wild-type (WT) and V1aR / mice in urine excretion during water loading in response to stimulation with a V2 receptor-selective agonist, suggesting that the antidiuretic effect through the V2 receptor remains intact (24). The V1aR / mice also showed a decreased level of plasma atrial natriuretic peptide (ANP) (24). The level of plasma ANP, which is a clinically available marker for the diagnosis of an appropriate plasma volume during dialysis, is known to correlate positively with the

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plasma volume (25) and negatively with the body mass index (26, 27). These findings raise the possibility that, in the case of V1a receptor deficiency, altered body fluid homeostasis and the ANP level could affect glucose homeostasis and energy expenditure. In the present study, we analyzed the indicators for glucose homeostasis under a condition with a normal chow (NC) diet or a high-fat (HF) diet. Our results indicate that the abolished AVP function via the V1a receptor could be involved in the development of the metabolic syndrome.
Materials and Methods Animals
The generation of V1aR / mice has been described previously (23). Non-V1aR / mouse littermates (WT) were used as age-matched control subjects for V1aR / mice. The genetic background of WT and V1aR / mice was a mixture of 129sv and C57Back/6J mice. Animals were housed in microisolator cages in a pathogen-free barrier facility. WT and V1aR / mice were housed under a controlled condition at 23 1 C with 50 10% relative humidity on a 12-h light, 12-h dark cycle with ad libitum access to food and water except when the experimental protocol specified otherwise. Animals were used at 4 20 wk of age and fasted for 16 h before the studies. All data presented here were obtained from male mice. All experimentation was performed under approved institutional guidelines.

trifuged at 3000 rpm for 10 min at 4 C, and the supernatants were collected and stored at 80 C. Plasma glucose and BUN were measured by spectrophotometry using a commercially available kit (WAKO, Osaka, Japan). The levels of plasma insulin, ANP, leptin, aldosterone, and AVP were determined using ELISA kits (insulin; Mercodia, Uppsala, Sweden; ANP; Phoenix Pharmaceuticals, Belmont, CA; leptin; Morinaga, Tokyo, Japan; aldosterone; Cayman Chemical, Ann Arbor, MI; AVP; R&D Systems, Minneapolis, MN). The hematocrit value was determined using a heparinized-microcapillary tube (Drummond, Broomall, PA).

Glucose tolerance test (GTT)

Mice were fasted for 16 h before receiving an ip administration of 1.5 g glucose per kilogram body weight in saline (0.9% NaCl). Blood samples were taken from the tail vein at 0, 10, 30, 60, and 120 min in heparinaized microcapillary tubes, and the plasma glucose and insulin levels were determined.

Hyperinsulinemic-euglycemic clamp study

A hyperinsulinemic-euglycemic clamp test was performed using [3-3H] glucose (PerkinElmer Inc., Wellesley, MA) for the estimation of whole glucose appearance and hepatic glucose production as previously described (28 30). Operations were carried out under 50 mg/kg pentobarbital (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan) anesthesia, and a catheter (a stretched PE-10 polyethylene tube connected with a PE-50 polyethylene tube; Becton Dickinson, Franklin Lakes, NJ) filled with heparin solution was inserted in the left internal jugular vein. The catheters were externalized through an incision in the skin flap behind its head, and animals were returned to individual cages after the surgery. After 3 d for complete recovery, mice were fasted overnight, and conscious mice were placed in a restrainer to which they had adapted. Insulin and [3-3H] glucose were infused at a rate of 15 pmol/ kg min and 0.1 Ci/min, respectively, and an infusion rate of 50% glucose was regulated to maintain euglycemia (120 140 mg/dl). A steady-state of clamp was achieved within 90 120 min. Blood samples were taken from the tail tip at 10-min intervals, and the plasma glucose concentration was determined. For the immediate measurement of the plasma glucose level, we used the Ascensia blood glucose meter (Bayer Healthcare, Leverkusen, Germany). For the determination of the plasma [3-3H] glucose concentration, 12 l of plasma samples were deproteinized with 0.3 n Ba(OH)2 and 0.3 n ZnSO4, dried to remove 3H2O, resuspended in water, and counted using a scintillation counter. The clamp whole glucose appearance (micromoles per minute) was calculated as the ratio of the [3-3H] glucose infusion rate (disintegrations per minute per minute) to the specific activity of plasma glucose (disintegrations per minute per micromole) during the final 30 min of the clamp. Hepatic glucose production during the hyperinsulinemic-euglycemic clamp was determined by subtracting the glucose infusion rate from the whole glucose appearance.

Feeding and weighing

Male WT and V1aR / mice were weighed once weekly from weaning at 4 wk of age. The mice were housed individually and allowed to acclimatize for a week. At 5 wk of age, the mice were placed on pellets of a NC diet consisting of 24.9% protein, 51.4% carbohydrate, and 4.6% fat (wt/wt/wt) (CE-2, 346.8 kcal per 100 g; CLEA, Tokyo, Japan) or a HF diet consisting of 25.5% protein, 29.4% carbohydrate, and 32.0% fat (wt/wt/wt) (HFD-32, 507.6 kcal per 100 g; CLEA). The food was weighed daily over a period of 15 wk, and food intake was determined over the time period of 519 wk of age.

Measurement of food intake volume

Daily food consumption in male WT and V1aR / mice at 8 wk of age was measured using a metabolic cage (Shinano, Tokyo, Japan). Mice were housed in the metabolic cage for 3 d to adapt to the new environment, and mice with normal body weight were then used for the measurement of food intake.

Measurements of epididymal-white adipose tissue (WAT) weight and adipocyte diameter

For preparing samples, mice were anesthetized by etherization. Epididymal WAT was excised from 16-h-fasted mice fed the NC or HF diet and then weighed. The WAT to body weight ratios were calculated and expressed as the percentage of body weight. The adipocyte diameter was measured using prepared WAT sections on a microscope.

Measurement of the tissue glycogen content

The total cellular glycogen content was assessed using a glucoamylase method (31). Liver samples were digested in an sodium dodecyl sulfate buffer and centrifuged to obtain the digested supernatant. Glycogen was digested using 1 U/ml glucoamylase (WAKO) in a 0.05 m acetate buffer (pH 5.5) for 30 min at 37 C. After digestion of the glucose from glycogen, sample buffers were neutralized using 0.1 m NaOH, and the glucose concentration was then measured. Glucose released from glycogen was measured by spectrophotometry with a commercially available kit (WAKO) and expressed as micrograms glucose per milligram protein. Undigested samples were used for background subtraction.

Tissue section staining

Paraffin sections of the liver and WAT were stained using hematoxylin-eosin (HE) reagents (Muto Chemicals, Tokyo, Japan) or periodic acid Shiff reagents (Muto Chemicals) to visualize the glycogen granules.

Measurement of plasma parameters

For the measurement of the plasma glucose levels, blood samples (10 l) were immediately mixed with 10 volumes of 0.33 m HClO4 to inhibit the use of glucose before measurement. The mixtures were centrifuged at 3000 rpm for 5 min, the supernatants (10 l) were taken up, and the glucose concentration was determined with an identically treated standard. For the measurement of the levels of blood urea nitrogen (BUN), insulin, ANP, leptin, aldosterone, and AVP, blood samples were cen-

Statistical analysis
All values are expressed as means se. Statistical analysis was performed using the two-way ANOVA. P 0.05 by the unpaired Students t test or unpaired Welchs t test was considered statistically significant.

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Results Plasma parameters (glucose, insulin) and glucose tolerance

Because increased plasma AVP level and decreased binding activity of AVP to the liver have been observed in NIDDM subjects (14 22), we determined the plasma glucose level in male WT and V1aR / mice at 8 wk of age. The glucose level was slightly increased in V1aR / under the fasting condition (82.66 3.88 mg/dl in WT mice, n 31, vs. 103.38 10.17 mg/dl in V1aR / mice, n 20, P 0.0686), and the differences were more remarkable under the feeding condition (135.88 3.74 mg/dl in WT mice, n 30, vs. 153.17 6.88 mg/dl in V1aR / mice, n 21, P 0.0346) (Fig. 1A). After a 16-h fast, the plasma insulin level in V1aR / mice was not significantly different from that in WT mice (613.98 50.88 pg/ml in WT mice, n 27, vs. 558.46 60.26 pg/ml in V1aR / mice, n 25, P 0.482) (Fig. 1C). Because the plasma glucose level was higher in V1aR / mice than WT mice, we then performed a GTT to investigate whether the glucose tolerance was altered in V1aR / mice. After ip loading with 1.5 g/kg body weight of glucose to male mice at 8 wk of age, the plasma glucose level in V1aR / mice was significantly higher than that in WT mice at 10, 30, 60, and 120 min after a glucose injection (Fig. 1B). In a different set of experiments, we performed another GTT with sampling at only 0 and 30 min to minimize the sampling stress for mice. Impaired glucose tolerance in V1aR / mice was also observed (glucose level at 0 min; 80.00 4.02 mg/dl in WT, n 7, vs. 86.8 8.36 mg/dl in V1aR / mice, n 7, P 0.492, glucose level at 30 min; 207.14 17.85 mg/dl in WT mice, n 7, vs. 326.60 26.01 mg/dl in V1aR / mice, n 7, P 0.00595). Thus, the results of all these experiments demonstrate that glucose tolerance is impaired in V1aR / mice. On the other hand, the plasma insulin levels were not different between WT and V1aR / mice (Fig. 1C). These data suggested that the glucose tolerance was altered by the decreased insulin sensitivity in V1aR / mice because the plasma glucose level in V1aR / mice was higher despite the similar plasma insulin level during the GTT. To further investigate the insulin sensitivity in V1aR / mice, we performed the hyperinsulinemic-euglycemic clamp study with conscious WT and V1aR / mice at 8 wk of age.

The plasma glucose levels and whole glucose appearance during the steady-state of clamp were not different between WT and V1aR / mice (clamp plasma glucose, 140.68 4.89 mg/dl in WT mice, n 8, vs. 139.43 3.21 mg/dl in V1aR / mice, n 8; whole glucose appearance, 263.30 30.21 mol/ kg min in WT mice, vs. 278.58 41.41 mol/kg min in V1aR / mice) (Fig. 2A). The glucose infusion rate during the steady-state of clamp was significantly lower in V1aR / mice than WT mice (251.23 32.06 mol/kg min in WT mice vs. 160.89 14.74 mol/kg min in V1aR / mice, P 0.0287), suggesting that V1aR / mice are insulin resistant (Fig. 2B).
Hepatic glucose production and glycogen level in the liver

Because a rodent subject with NIDDM showed increased hepatic glucose production and reciprocally reduced glycogen granules in the liver (32), we evaluated hepatic glucose production in V1aR / mice using the hyperinsulinemiceuglycemic clamp study. The hepatic glucose production was calculated by subtraction of the glucose infusion rate from the whole glucose appearance. V1aR / mice showed significantly higher hepatic glucose production during the steady-state of clamp (12.07 8.28 mol/kg min in WT mice vs. 117.70 32.21 mol/kg min in V1aR / mice, P 0.0132) (Fig. 2C), which suggested that glycogenolysis was increased in V1aR / mice. To confirm the increased glycogenolysis in V1aR / mice, we measured the hepatic glycogen level using several universal methods, such as the amylase method and staining for glycogen granules. The liver glycogen content in V1aR / mice was significantly lower than that in WT mice (232.21 30.78 g/mg protein in WT mice, n 6, vs. 114.05 21.39 g/mg protein in V1aR / mice, n 6, P 0.0117) (Fig. 3A). In addition, the staining level of hepatic glycogen granules was decreased in the liver section of V1aR / mice (Fig. 3B).
Growth curve on the HF diet

Next, we performed a HF diet loading test to confirm the impaired glucose tolerance in V1aR / mice. Growth curves were measured from 4 to 19 wk of age in male WT and V1aR / mice. The mice were fed a NC or HF diet from the age of 5 wk (WT mice, n 14, and V1aR / mice, n 10 on

FIG. 1. Alteration of glucose homeostasis. A, Basal feeding (open bar) or fasting (solid bar) plasma glucose levels in WT (feeding, n 30; fasting, n 31) and V1aR / (feeding, n 21; fasting, n 20) mice. B, Plasma glucose levels during the GTT after a 16-h fast were measured in WT (solid line) and V1aR / (dashed line) mice at 8 wk of age (n 31 for WT mice and n 20 for V1aR / mice). C, Plasma insulin levels during the GTT after a 16-h fast were measured in WT (solid line) and V1aR / (dashed line) mice at 8 wk of age (n 27 for WT mice and n 25 for V1aR / mice). Values are means SE. *, P 0.05, **, P 0.01, ***, P 0.001 vs. WT mice by the unpaired Students t test or unpaired Welchs t test. ###, P 0.001 vs. WT mice by two-way ANOVA.

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FIG. 2. Hyperinsulinemic-euglycemic clamp study. A, Whole glucose appearance during the final 30 min of the clamp in WT (n 8) and V1aR / (n 8) mice. B, Glucose infusion rate during the final 30 min of the clamp in WT and V1aR / mice. C, Hepatic glucose production during the final 30 min of the clamp in WT and V1aR / mice. Values are means SE. *, P 0.05 vs. WT mice by the unpaired Welchs t test.

the NC diet; WT mice, n 14, and V1aR / mice, n 10 on the HF diet). WT mice on the NC diet (Fig. 4, A and B) were significantly heavier than V1aR / mice at 19 wk of age (body weight, 33.53 0.72 g in WT mice vs. 28.38 0.63 g in V1aR / mice, P 3.92 10 5), and the average weight gain (519 wk) was significantly less in V1aR / mice than in WT mice (11.17 0.44 g in WT mice vs. 5.25 0.54 g in V1aR / mice, P 1.97 10 8). In contrast, V1aR / mice on the HF diet were significantly heavier than WT mice at 19 wk of age (body weight, 38.71 2.00 g in WT mice vs. 47.27 1.86 g in V1aR / mice, P 0.00651) (Fig. 4, A and B). Whereas the weight gains resulting from the HF diet were significantly increased in both mice, compared with those resulting from the NC diet, the average weight gain (from 5 to 19 wk) was greater in V1aR / mice than in WT mice (16.25 1.58 g in WT mice vs. 24.01 1.76 g in V1aR / mice, P 0.00376).
WAT weight and plasma leptin level of mice on the HF diet

To investigate which body tissues were responsible for this increased weight, after mice had been fed the HF diet for 15 wk, tissues were collected from a subset of both mice. As shown in Table 1 and Fig. 4C, at 20 wk of age, V1aR / mice showed a remarkable increase in epididymal-WAT depots, compared with WT on HF diets, consistent with the increase in body weight (0.83 0.095 g in WT mice vs. 2.89 0.21 g in V1aR / mice, P 9.44 10 7). The weight of brown adipose tissue, sc WAT, retroperitoneal WAT, and visceral WAT yielded the same results (data not shown). There was no significant difference in the liver, kidney, and heart weight (data not shown). The V1aR / mice on both the NC and HF diets had significantly greater adiposity in the epididymal WAT (measured as the percentage of body weight). In good agreement with the increased adiposity, the adipocyte diameters in epididymal WAT were larger in V1aR / mice than WT mice on the NC or HF diet. Because the plasma leptin level is known to be highly correlated with the adipose tissue mass, we measured the leptin level in these animals on the corresponding diets (33, 34) (Fig. 4D). The plasma leptin level was significantly higher in V1aR / mice than that in WT mice on the NC diet (1.80 0.52 ng/ml in WT mice vs. 4.19 1.06 ng/ml in V1aR / mice, P 0.0334), whereas it tended to be increased in V1aR / mice on the HF diet (10.69 2.53 ng/ml in WT mice vs. 13.70 2.52 ng/ml in V1aR / mice, P 0.271).
Glucose tolerance on the HF diet

effects on glucose homeostasis of feeding WT and V1aR / mice an HF diet. We assessed the basal plasma glucose and insulin levels of mice at 19 wk of age under a fasting or feeding condition with the HF diet. The plasma glucose levels of V1aR / mice were significantly higher than those of WT mice under the fasting or feeding condition (fasting, 88.50 4.92 mg/dl in WT mice, n 14, vs. 112.40 6.39 mg/dl in V1aR / mice, n 10, P 0.00635; feeding, 159.50 6.09 mg/dl in WT mice, n 14, vs. 195.80 10.90 mg/dl in V1aR / mice, n 10, P 0.00505), whereas the fasting insulin levels were not different between WT and V1aR / mice on the HF diet (Fig. 5, A and B). The fasting glucose levels were significantly elevated in both mice on the HF diet, compared with those of mice on the NC diet. Furthermore, the plasma glucose levels of WT or V1aR / mice on the HF diet during the GTT were significantly elevated, compared with those of WT or V1aR / on the NC diet, as assessed by two-way ANOVA (NC in WT mice vs. HF in WT mice, P 0.00297; NC in V1aR / mice vs. HF in V1aR / mice, P 0.00828). These results indicated that the HF diet could affect glucose tolerance (Fig. 5C). In addition, the plasma glucose levels of V1aR / mice on the NC or HF diet during the GTT tended to be elevated, compared with those of WT mice, as assessed by two-way ANOVA (WT mice on the NC diet vs. V1aR / mice on the NC diet, P 0.353; WT mice on the HF diet vs. V1aR / mice on the HF diet, P 0.176). In addition, the plasma glucose level at 2 h after glucose administration was higher in V1aR / than in WT mice on the HF diet (165.43 23.71 mg/dl in WT mice vs. 223.30 25.74 mg/dl in V1aR / mice, P 0.113). These results indicated that HF diet loading could exaggerate glucose intolerance in V1aR / mice more evidently than in WT mice. Moreover, V1aR / mice exhibited a malignant fatty liver after being on the HF diet, whereas a

Because altered glucose tolerance was observed in V1aR / mice on the NC diet, we also investigated the

FIG. 3. Glycogen content in the liver. A, The liver glycogen contents were determined by the amylase method in 8-wk-old WT and V1aR / mice (n 6, each). *, P 0.05 vs. WT mice by the unpaired Students t test. B, Liver sections in WT and V1aR / mice at 8 wk of age were stained with periodic acid Shiff reagents.

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FIG. 4. Effects of a HF diet. A, Body weight changes of mice on the HF or NC diet. Open circles with a solid line, WT mice on the NC diet (n 14); open triangles with a dashed line, V1aR / mice on the NC diet (n 10); closed circles with a solid line, WT mice on the HF diet (n 14); closed triangles with a dashed line, V1aR / mice on the HF diet (n 10). Mice were fed the NC or HF diet from 5 to 20 wk of age. **, P 0.01, ***, P 0.001 WT vs. V1aR / mice on the NC diet at 19 wk of age, WT vs. V1aR / mice on the HF diet at 19 wk of age by the unpaired Students t test; ##, P 0.01 vs. WT by two-way ANOVA; , P 0.001 vs. mice on the NC diet by two-way ANOVA. B, Body weight gain of mice on the NC or HF diet from 5 to 19 wk of age. **, P 0.01, ***, P 0.001 vs. WT mice by the unpaired Students t test; , P 0.01, , P 0.001 vs. mice on the NC diet by Welchs t test. C, Mice were killed at 20 wk of age after 16 h of fasting. Epididymal-WAT sections were stained with the HE reagent, and representative sections are shown with 200 magnification. D, The plasma leptin level was measured in mice at 20 wk of age after 16 h of fasting. *, P 0.05 vs. WT mice by the unpaired Students t test; , P 0.01, , P 0.001 vs. mice on the NC diet by the unpaired Welchs t test.

slightly fatty liver was observed in WT mice (Fig. 5D). These results suggested that the glucose intolerance observed in V1aR / mice was accelerated by the HF diet, leading to hyperglycemia, excessive obesity, and fatty liver.
Feeding behavior in V1aR
/

mice

Calorie intake was also investigated with male mice at 8 wk of age. The daily calorie intake of V1aR / mice was significantly reduced, compared with that of WT mice (14.82 0.98 kcal/d in WT mice, n 16, vs. 11.68 1.15 kcal/d in V1aR / mice, n 12, P 0.0464) (Fig. 6A), whereas the body weights were not different (25.38 0.52 g in WT mice, n 16, vs. 26.07 0.31 g in V1aR / mice, n 12, P 0.2672). The calorie intake ratio of V1aR / / WT on the NC diet was 79%. In addition, we examined the daily calorie intake of mice on the HF diet from 5 to 19 wk (Fig. 6B). The daily calorie intake during the experimental period and daily mean calorie intake for the last 2 months were significantly decreased in V1aR /
TABLE 1. WAT weight and diameter of adipocytes
WT on NC

mice, as assessed by two-way ANOVA (NC in WT mice vs. NC in V1aR / mice, P 0.00114) (Fig. 6B) and the t test (daily mean calorie intake, 13.65 0.15 kcal/d in WT mice on the NC diet, n 14, vs. 10.39 0.15 kcal/d in V1aR / mice on the NC diet, n 10, P 6.00 10 15) (Fig. 6C), respectively. The calorie intake ratio of V1aR / to WT on the NC diet was 76%. When WT mice were fed the HF diet, their daily calorie intake did not differ from that of WT mice on the NC diet (14.18 0.29 kcal/d in WT mice on the HF diet, n 14) (Fig. 6C). The calorie intake ratio of WT on the HF diet to WT on the HF diet was 104%. However, the calorie intake of V1aR / mice on the HF diet was significantly elevated, compared with that of V1aR / mice on the NC diet, as assessed by two-way ANOVA (NC in V1aR / mice vs. HF in V1aR / mice, P 0.0129) and the t test (14.37 0.27 kcal/d in V1aR / mice on the HF diet, n 10, P 3.00 10 17 vs. V1aR / mice on the NC diet) (Fig. 6, B and C). The calorie intake ratio of V1aR / on the HF diet to V1aR / on the NC diet was 138%.

WT on HF

V1aR

on NC

V1aR

on HF

Body weight (g) Epididymal WAT weight (g) Percent of body weight Adipocyte diameter ( m)

29.68 0.30 0.99 48.98

0.89 0.038 0.10 2.63

37.54 0.83 2.14 63.09

1.77 0.095 0.18 3.28

23.93 0.34 1.41 61.87

0.49*** 0.026 0.10** 1.80*

45.51 2.89 6.31 96.49

1.92**, 0.21***, 0.39***, 3.33**,

Mice were killed at 20 wk of age after being on the NC or HF diet for 15 wk. The excised tissues were weighted and stained with HE reagents. The adipocyte diameter with prepared WAT sections was measured on the microscope (n 14 for WT mice on the NC diet, n 14 for WT mice on the HF diet, n 10 for V1aR / mice on the NC diet, and n 10 for V1aR / mice on the HF diet). *, P 0.05, **, P 0.01, ***, P 0.001 vs. WT mice on the corresponding diets by the unpaired Students t test or unpaired Welchs t test. , P 0.05, , P 0.001 vs. mice on the NC diet by the unpaired Students t test or unpaired Welchs t test.

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FIG. 5. Glucose homeostasis and histology in the liver on a HF diet. A, Basal feeding (open bar) or fasting (solid bar) plasma glucose levels in WT (n 14) and V1aR / (n 10) mice at 19 wk of age after being on the NC or HF diet for 14 wk. **, P 0.01 vs. WT mice by the unpaired Students t test; , P 0.05, , P 0.01, , P 0.001 vs. mice on the NC diet by the unpaired Students t test or the unpaired Welchs t test. B, Basal fasting plasma insulin levels in WT (n 14) and V1aR / (n 10) mice at 19 wk of age after being on the NC or HF diet for 15 wk. , P 0.05, , P 0.001 vs. mice on the NC diet by the unpaired Students t test or the unpaired Welchs t test. C, The plasma glucose levels during the GTT after a 16-h fast were measured in WT and V1aR / mice at 19 wk of age. Open circles with a solid line, WT mice on the NC diet (n 14); open triangles with a dashed line, V1aR / mice on the NC diet (n 10); closed circles with a solid line, WT mice on the HF diet (n 14); closed triangles with a dashed line, V1aR / mice on the HF diet (n 10). , P 0.01 vs. mice on the NC diet by two-way ANOVA. D, Pathogenesis of fatty liver stained with HE reagents in 20-wk-old V1aR / mice on the NC or HF diet. Representative sections are shown with 200 magnification.

Plasma parameters for body fluid

Discussion

Because V1aR mice were shown to have a decreased level of circulating blood volume (24), we determined the hematocrit value, BUN level, plasma AVP level, and plasma aldosterone level (Table 2). The hematocrit value, BUN level, and plasma AVP level were significantly increased in V1aR / mice. The plasma ANP level was significantly decreased in V1aR / mice, as described previously (24). The plasma aldosterone level was also significantly decreased in V1aR / mice (Table 2). These results indicated that blockade of the V1a receptor led to a reduced plasma aldosterone level and plasma volume, which could cause decreased circulating blood volume and increased AVP release.

AVP resistance is reported to consist of decreased plasma volume, hypervasopressinemia, and reduced coupling of AVP for the V1a receptor in the liver and kidney and to be accompanied with human or rodent NIDDM, especially under a poorly controlled condition (14 22). These reports suggest that the alteration of AVP signaling could mediate the development of a metabolic syndrome. Recently we reported that the Akt phosphorylation induced by insulin was reduced in primary adipocytes isolated from V1aR / mice, suggesting that whole body glucose tolerance was altered in V1aR / mice (27). Therefore, we investigated the glucose homeostasis in V1aR / mice by assessing the glucose tol-

FIG. 6. Daily food consumption. A, Food consumption of 8-wk-old mice on the NC diet in a metabolic cage (n 16 for WT mice, n 12 for V1aR / mice). *, P 0.05 vs. WT mice by the unpaired Students t test. B, Food consumption of mice on the NC or HF diet. Open circles with a solid line, WT mice on the NC diet (n 14); open triangles with a dashed line, V1aR / mice on the NC diet (n 10); closed circles with a solid line, WT mice on the HF diet (n 14); closed triangles with a dashed line, V1aR / mice on the HF diet (n 10). ##, P 0.01 vs. WT mice; , P 0.05 vs. mice on the NC diet by two-way ANOVA. C, Mean value of food consumption of mice on the NC or HF diet from 11 through 18 wk of age. ***, P 0.001 vs. WT mice by the unpaired Welchs t test; , P 0.001 vs. mice on the NC diet by the unpaired Welchs t test.

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TABLE 2. Parameters for the plasma volume

WT V1aR
/

Hematocrit (%) BUN (mg/dl) AVP (pg/ml) ANP (ng/ml) Aldosterone (ng/ml)

46.27 18.05 45.42 18.29 4.09

1.21 1.44 19.67 1.10 0.14

52.24 23.08 146.50 12.32 3.31

1.65* 1.50* 26.26** 1.74* 0.08**

The hematocrit value (n 6, each), BUN (n 30, each), plasma AVP level (n 8, each), plasma ANP level (n 8, each), and plasma aldosterone level (n 4, each) were measured using the serum obtained from WT and V1aR / mice at 8 wk of age under the feeding condition. *, P 0.05, **, P 0.01 vs. WT mice by the unpaired Students t test or unpaired Welchs t test.

erance and found that 8-wk-old V1aR / mice that were on the NC diet showed hyperglycemia and impaired glucose tolerance, whereas their plasma insulin level was not significantly altered. Thus, the phenotype of V1aR / mice was consistent with clinical data from a NIDDM patient (35), who showed impaired glucose tolerance and intact insulin secretion. Furthermore, insulin resistance observed in V1aR / mice was supported by a decreased glucose infusion rate in the hyperinsulinemic-euglycemic clamp study. In addition, we determined the hepatic glucose production rate using a hyperinsulinemic-euglycemic clamp study and directly estimated the hepatic glycogen content to see whether V1aR / mice exhibited up-regulation in hepatic glucose production, as shown in NIDDM subjects (32). Hepatic glucose production in the steady-state of clamp was increased in V1aR / mice, and, therefore, the glycogen level in the liver of V1aR / mice was reduced by approximately half. These results indicated that the increased hepatic glucose production could contribute in part to developing glucose intolerance in V1aR / mice. In addition, we performed the HF diet loading test to confirm the glucose intolerance in V1aR / mice. V1aR / mice on the HF diet, but not WT mice on the same diet, consequently showed hyperobesity

accompanied with malignant hyperglycemia, glucose intolerance, adipocyte hypertrophy, and hyperleptinemia. The remarkable pathogenesis of fatty liver observed in V1aR / mice on the HF diet also supported the finding of an impaired glucose tolerance condition. If results obtained from the study with GTT were applied to the classification of human diabetes mellitus presently proposed by American Diabetes Association using the data of plasma glucose levels at 0 and 2 h during the GTT (36), abolishing the V1a receptor would lead to a shift to the prediabetes mellitus condition, whereas WT mice would still be classified as being under the normal condition (Fig. 7A). Furthermore, V1aR / mice on the HF diet shifted from a prediabetes mellitus to a diabetes mellitus condition, whereas WT mice on the HF diet had a prediabetes mellitus condition (Fig. 7B). The food intake of V1aR / mice on the NC diet was approximately 80% less than that of WT mice, and the weight gain was less in V1aR / than in WT mice on the NC diet. On the NC diet, hyperglycemia and hyperleptinemia observed in V1aR / mice might lead to hypophagia, consequently resulting in decreased weight gain in V1aR / mice (37). In contrast to V1aR / mice on the NC diet, V1aR / mice on the HF diet had significantly increased calorie intake (ratio of calorie intake, 138%), whereas the calorie intake of WT mice on the HF diet did not differ from that of those on the NC diet (ratio of calorie intake, 104%). Therefore, the calorie intake in V1aR / mice was much more elevated than that in WT mice when the diet was changed from NC to HF. This increased calorie intake in V1aR / mice would accelerate the hypertrophy of adipocytes, consequently resulting in increased weight gain. Moreover, Scarpace et al. (38) reported that the HF diet-treated rats overexpressing leptin cDNA showed leptin resistance, elevated food intake, and increased weight gain, compared with the HF diet-treated rats expressing a control vector. This suggested that leptin resistance accompanied with hyperleptinemia could prefer-

FIG. 7. Alterations in the glucose tolerance of mice on the NC or HF diet and mechanisms for AVP/V1a receptor-mediated glucose tolerance. A, Classification of glucose intolerance using the fasting plasma glucose level and 2-h plasma glucose level after glucose administration in WT (n 31) and V1aR / (n 20) mice at 8 wk of age. ***, P 0.001 vs. WT mice by the unpaired Students t test. B, Classification of glucose intolerance using the fasting plasma glucose level and 2-h plasma glucose level after glucose administration at 19 wk of age; n 14 for WT mice on NC, n 10 for V1aR / mice on NC, n 14 for WT mice on HF, and n 10 for V1aR / mice on HF. **, P 0.01 vs. WT mice by the unpaired Students t test; , P 0.05, , P 0.001 vs. mice on the NC diet by the unpaired Students t test or the unpaired Welchs t test. C, Scheme of mechanisms for AVP/V1a receptor-mediated glucose tolerance.

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entially occur in V1aR / mice on the HF diet. Thus, hyperleptinemia in V1aR / mice fed the HF diet could cause leptin resistance, leading to elevated food intake and, consequently, increased weight gain. AVP plays a crucial role in regulating body fluid retention, which is mediated through the V2 receptor in the kidney (39, 40). Because systematic disruption of the V1a receptor resulted in decreased blood volume and ANP levels without altered V2 receptor function, the functional role of AVP for body fluid recruitment could be mediated through not only the V2 receptor but also the V1a receptor (23). To assess the plasma volume, we examined the hematocrit value, BUN level, plasma AVP level, and plasma aldosterone level in V1aR / mice. The hematocrit value and BUN level were increased, suggesting that the plasma volume was decreased in V1aR / mice. In addition, the plasma AVP level was increased, and the plasma aldosterone level was decreased in V1aR / mice. These findings could reflect insufficient water recruitment and an AVP-resistant condition in V1aR / mice. Because ANP has been known as a mediator for the translocation of extracellular fluid as well as marker for a plasma volume (41), decreased plasma volume may be concomitant with decreased extracellular fluid in V1aR / mice. Taken together, these findings demonstrate that the V1a receptor is involved in maintaining glucose homeostasis as well as body fluid homeostasis and that lack of this receptor could lead to glucose intolerance accompanied with the prediabetes mellitus phenotype despite a lower energy intake. Various sources constituting the mechanisms underlying the blockade of the V1a receptor and glucose intolerance could be used to explain this physiological phenomenon because the V1a receptor is widely distributed and its functional role on glucose homeostasis remains unclear (2, 4). Therefore, we propose the following three mechanisms (Fig. 7C). First, the central AVP/V1a receptor signal in the central nervous system (CNS) regulates hepatic glucose production. AVP is well known to stimulate glycogen degradation through the V1a receptor (8). Therefore, we had expected that the glycogen content would increase. However, the rate of hepatic glucose production was increased, and the glycogen content in the liver of V1aR / mice was decreased. Because the AVP/V1a receptor pathway of glycogen degradation in the liver is abolished in V1aR / mice, other mechanisms could be involved in regulating hepatic glucose production in the liver of V1aR / mice. In addition to the peripheral action in the liver, AVP is known to act within the CNS as a neuromodulator/neurotransmitter regulating an array of CNS-mediated functions (e.g. learning and memory, neuroendocrine reactivity, social behaviors, circadian rhythmicity, thermoregulation, and autonomic function) (4, 42). It has also been reported that AVP stimulates secretion of -aminobutyric acid (GABAergic) neurons in vitro (43) and that GABAergic neurons suppress hepatic glucose production (44). Thus, central blockade of the V1a receptor could attenuate the stimulation of GABAergic neurons, resulting in the up-regulation of hepatic glucose production. Second, AVP can stimulate the insulin-signaling pathway via the V1a receptor. We found that glucose tolerance was altered by insulin resistance in V1aR / mice (Fig. 2B). AVP

has been reported to activate insulin-signaling, such as Akt and p70S6kinase, via the epithelial growth factor receptorphosphatidylinositol 3-kinase pathway (45), suggesting that AVP can modulate glucose homeostasis by affecting insulin signaling. In fact, the phosphorylation of Akt was significantly reduced in the adipocytes of V1aR / mice (27). Thus, a blunted insulin-signaling pathway due to a deficient AVP/ V1a signal could result in reduced glucose uptake in muscles and adipocytes, leading to insulin resistance and impaired glucose tolerance. Third, the AVP/V1a receptor signal may regulate glucose tolerance via body fluid homeostasis. V1aR / mice showed an AVP-resistant condition including hypervasopressinemia and decreased plasma volume. Perraudin et al. (46) reported that AVP directly regulated aldosterone secretion through the V1a receptor, which was expressed in the adrenal cortex. Indeed, the plasma aldosterone level was decreased in V1aR / mice and supported AVP-resistance in mutant mice. Thus, dysfunction of the AVP/V1a receptor-aldosterone system in V1aR / mice could cause a lower response to water recruitment by aldosterone, leading to decreased plasma volume. Consequently, the decreased plasma volume could result in increased blood glucose level as well as increased levels of blood solutes, such as hematocrit and BUN. In addition to the lowered plasma volume, the plasma ANP level was decreased in V1aR / mice. The plasma volume or osmotic level regulates the plasma ANP level, which is well known as an intensive lipolytic factor (24, 25). Thus, the decreased plasma ANP level induced by the lower plasma volume could cause suppressed lipid degradation of adipocytes, resulting in hypertrophy of adipose tissue in V1aR / mice. Although no reports have shown that a decreased plasma volume contributes to the development of diabetes mellitus, it has been reported that a hyperosmolar condition could influence glucose homeostasis (4751). The elevation of plasma osmotic pressure affected glucose tolerance in humans (47, 48). Hyperosmolar stimuli can attenuate the glucose uptake by insulin-stimulated p38MAPK activation and Akt activation in various tissues (49 51). These reports prompted us to consider the relationship between the attenuation of body fluid homeostasis and whole body glucose intolerance. We revealed that V1aR / mice showed increased plasma glucose and BUN levels by 1.2-fold when individually compared with WT mice. These data are a clinically available indicator for the prediction of plasma osmotic levels as well as plasma Na and K concentrations (52). Previously we reported that the plasma Na and K levels were not significantly different in V1aR / and WT mice (23). Therefore, the deficiency of the V1a receptor might lead to an increase in the plasma osmotic level, resulting in the promotion of glucose intolerance in V1aR / mice. In our experiments, V1aR / mice simultaneously showed AVP resistance, impaired glucose tolerance, and increased hepatic glucose production. This suggests that AVP could be involved in maintaining the homeostasis of blood glucose by regulating body fluid, hepatic glucose production, and insulin-signaling, such as Akt (Fig. 7C). Moreover, our data suggest that a selective agonist for the V1a receptor would contribute to the therapeutics for diabetes mellitus and improve impaired glucose tolerance by increas-

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ing the insulin effect, down-regulation of hepatic glucose production, and recruitment of plasma volume. In conclusion, our study with V1aR / mice examined the contributions of AVP to lifestyle-related diseases. We should clarify the mechanisms underlying the regulation of plasma recruitment by the AVP/V1a receptor in future studies.
Acknowledgments
Received September 25, 2006. Accepted February 2, 2007. Address all correspondence and requests for reprints to: Akito Tanoue, M.D., Ph.D., Department of Pharmacology, National Research Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan. E-mail: atanoue@nch.go.jp. This work was supported in part by research grants from the Scientific Fund of the Ministry of Education, Science, and Culture of Japan; the Ministry of Human Health and Welfare of Japan; the Japan Health Science; the NOVARTIS Foundation; and the Takeda Science Foundation. Disclosure Summary: The authors have nothing to disclose.

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