Muscarinic stimulation of pancreatic insulin and glucagon release is abolished in [M.sub.3] muscarinic acetylcholine receptor—deficient mice

Muscarinic stimulation of pancreatic insulin and glucagon release is abolished in [M.sub.3] muscarinic acetylcholine receptor—deficient mice

Alokesh Duttaroy

A key feature of type 2 diabetes is that glucose fails to stimulate adequate release of insulin from pancreatic [beta]-cells (1,2). Characteristically, the [beta]-cell eventually fails to compensate for the gradually developing increase in insulin resistance, resulting in overt hyperglycemia. Sulfonylureas and related compounds stimulate insulin release in the absence of high glucose levels (3). Antidiabetic drugs that can potentiate insulin release in a glucose-dependent fashion would therefore be highly desirable (3).

Glucose-dependent insulin secretion is modulated by several hormones and neurotransmitters, among which acetylcholine plays a prominent role (reviewed in 4-6). Acetylcholine is released from intrapancreatic parasympathetic (vagal) nerve endings during the preabsorptive and, most likely, the absorptive phase of feeding (5,6). The acetylcholine-mediated preabsorptive phase of insulin secretion, although relatively small compared with the total release of insulin after a meal, seems to be of particular importance for maintaining normal glucose tolerance (5,6). A considerable body of evidence also suggests that increased vagal/cholinergic activity may be involved in enhanced insulin secretion in certain animal models of obesity (7-12).

An important feature of the cholinergic regulation of pancreatic insulin release is that acetylcholine stimulates insulin release in a strictly glucose-dependent manner, becoming more and more effective as the plasma glucose concentration increases (5,6). This concept is supported by a large number of in vivo (13-15) and in vitro (16-21) functional studies.

The acetylcholine/vagus effects on pancreatic insulin release are mediated by activation of muscarinic acetylcholine receptors located on the pancreatic [beta]-cells (4-6). Molecular cloning studies have revealed the existence of five molecularly distinct muscarinic receptor subtypes ([M.sub.1]-[M.sub.5]) (22). Receptor localization studies suggest that multiple muscarinic receptors ([M.sub.1], [M.sub.3], [M.sub.4], and [M.sub.5]) are expressed in pancreatic islets/[beta]-cells or [beta]-cell-derived tumor cell lines (23-25). However, the [M.sub.3] muscarinic receptor appears to be the predominant subtype expressed by pancreatic [beta]-cells (4-6,23-25). Interestingly, previous studies suggest that acetylcholine can also stimulate the secretion of glucagon by acting on muscarinic receptors located on pancreatic [alpha]-cells (5,6,26-28).

To better understand the physiological roles of the [M.sub.3] muscarinic receptor, we recently used gene-targeting technology to generate [M.sub.3] muscarinic receptor–deficient mice ([M3.sup.-/]/ mice) (29). In an initial study, we reported that [M3.sup.-/-]/ mice show a pronounced reduction in body weight associated with hypophagia and a significant decrease in serum leptin and insulin levels (29).

To study the potential role of [M.sub.3] muscarinic receptors in [beta]- and [alpha]-cell function, we carried out systematic in vitro insulin and glucagon release studies using isolated pancreatic islets prepared from wild-type (WT), [M3.sup.+/-], and [M3.sup.+/-]/ mice. To examine whether the deficits observed in the in vitro hormone release studies were correlated with changes in blood glucose, insulin, and glucagon levels and altered glucose tolerance, we carried out additional in vivo experiments using WT, [M3.sup.+/-], and [M3.sup.-/-] mice.

Our results demonstrated in an unambiguous fashion that muscarinic stimulation of pancreatic insulin and glucagon release is mediated by the [M.sub.3] muscarinic receptor subtype. These deficits were accompanied by pronounced changes in plasma insulin and glucagon levels in vivo. Our findings highlight the usefulness of gene-targeting technology in shedding light on the metabolic roles of individual members of the muscarinic receptor family.

RESEARCH DESIGN AND METHODS

[M.sub.3] muscarinic receptor-deficient mice were generated as previously described (29). All mice used for the present study (WT, [M3.sup.+/-], and [M3.sup.-/-] mice) were littermates generated by intermating heterozygons [M.sub.3] receptor mutant mice ([M3.sup.+/-] mice; genetic background: 129SvEv/C57BL/6J). Unless indicated otherwise, all experiments were carried out with adult male mice ages 3-7 months at the time of testing.

Mice were housed four to five per cage in a room with a 12-h light/dark cycle (lights on at 6:00 A.M.) and given ad libitum access to food and water. All manipulations were approved by the Animal Care and Use Committee of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.

RT-PCR analysis. Total RNA was extracted from mouse pancreatic islets and total brain using the total RNA isolation kit from Invitrogen. Extracted RNA samples were treated with 4 units of RNAse-free DNase (Ambion) at 37[degrees]C for 1 h to remove residual genomic DNA. The RNA was then reversed transcribed with an oligo-d[T.sub.16] primer and MuLV RT using the GeneAmp RNA PCR kit, as described by the manufacturer (Applied Biosystems). The reverse transcription step was omitted in control samples to test for the presence of contaminating genomic DNA. The reverse-transcribed products were screened for the presence of [M.sub.1]-[M.sub.5] cDNA by PCR, using the GeneAmp RNA PCR kit (Applied Biosystems) and an Eppendorf Mastercycler thermal cycler (40 cycles of 94[degrees]C at 1 min, 56[degrees]C at 2 min, and 72[degrees]C at 3 min). PCRs were carried out in a final volume of 50 [micro]l containing 10 [micro]l of the RT reaction product (corresponding to ~0.5-1 [micro]g RNA), 10 mmol/l Tris-HCl (pH 8.3), 50 mmol/l KCl, 2 mmol/l Mg[Cl.sub.2], 1 mmol/l of each dNTP, 100 ng each of the sense and corresponding antisense primers, and 1.25 units of AmpliTaq DNA polymerase. The identity of the PCR products was confirmed by restriction analysis (data not shown). The RT-PCR products were separated on 1.5% agarose gels.

The sizes of the expected RT-PCR products were [M.sub.1] (497 bp), [M.sub.2] (480 bp), [M.sub.3] (498 bp), [M.sub.4] (474 bp), and [M.sub.5] (485 bp). Subtype-specific primers were designed based on the mouse [M.sub.1]-[M.sub.5] muscarinic receptor sequences (Gen-Bank data accession numbers: [M.sub.1], J04192; Me, AF264049; [M.sub.3], F264050; [M.sub.4], X63473; and [M.sub.5], AF264051). The following primers were used: 1) [M.sub.1]: 5′-TCTGCTCATCAGCTTTGACCG-3′ (forward), 5′-CATCCTCTTCCTCTTCT TCTTTCC-3′ (reverse); 2) [M.sub.2]: 5′-TGTCAGCAATGCCTCCGTTATG-3′ (forward), 5′-GCCTTGCCATTCTGGATCTTG-3′ (reverse); 3) [M.sub.3]: 5′-GGTGTGAT GATYGGTCTGGCTTG-3′ (forward), 5′-GGAAGCAGAGTTTTCCAGGGAG-3′ (reverse); 4) [M.sub.4]: 5′-TCAAGAGCCCTCTGATGAAGCC-3′ (forward), 5′-AGATT GTCCGAGTCACTTTGCG-3′ (reverse); and 5) [M.sub.5]: 5′-GCTGACCTCCAAGGTT CCGATFC-3′ (forward), 5′-CCGTCAGCTTTTACCACCAAT C-3′ (reverse).

Islet isolation. Mice were killed by cervical dislocation, and their abdominal wall was opened. The pancreas was distended by injecting 3-6 ml of dissociation solution (Hanks’ balanced salt solution containing 0.23 mg/ml liberase enzyme; Roche Diagnostics) via the common bile duct. The organ was then removed from the abdomen and incubated with an additional 1.5 ml of dissociation solution for 23 min at 37[degrees]C. Islets of Langerhans were then hand picked from the digested pancreatic tissue using a dissection microscope.

In vitro insulin and glucagon secretion studies. Insulin and glucagon secretion were measured after static incubation of pancreatic islets (23) prepared from WT and [M.sub.3] receptor mutant mice. Islets were preincubated for 60 min in a modified Krebs solution containing (in mmol/l) 5.6 glucose, 120 NaCl, 5 KCl, 1.1 Mg[Cl.sub.2], 2.6 Ca[Cl.sub.2], 25 NaHC[O.sub.3], and 0.5% BSA. The solution was equilibrated with 95% [O.sub.2]/5% C[O.sub.2] and maintained at 37[degrees]C at a pH of 7.4 throughout the experiments. Batches of 10-12 islets were then transferred into tubes containing the same solution supplemented with various amounts of glucose and oxotremorine-M (Oxo-M; Sigma). The tubes were incubated for 1 h in a chamber maintained at 37[degrees]C and equilibrated with 95% [O.sub.2]/5% C[O.sub.2]. The medium was then withdrawn and frozen for the analysis of insulin and glucagon concentrations. The remaining islet insulin and glucagon content was extracted by sonication in acid/ethanol (30). The amount of insulin or glucagon secreted during the incubation period was normalized to the total insulin or glucagon content of the islets in each tube. Insulin concentrations were determined by enzyme-linked immunosorbent assay (ELISA; Alpco). Glucagon concentrations were measured via radioimmunoassay (RIA; Linco).

Oral glucose tolerance test. After an overnight (8-10 h) fast, mice received an oral load of glucose (2 mg/g body wt) via oral gavage. Blood samples were collected via retro-orbital sinus puncture before (t = 0 min) and 15, 30, 60, and 120 min after glucose administration. Blood glucose levels were determined using an automated blood glucose reader (Glucometer Elite Sensor; Bayer).

At the same time points, blood collected via the retro-orbital sinus puncture was transferred into serum separator tubes (0.5 ml, Capojet; Terumo Medical). Serum was obtained by centrifugation (10 min at 4[degrees]C) at 1,200g using an Eppendorf bench top centrifuge. Serum insulin concentrations were determined via ELISA (Alpco) using mouse insulin as a standard.

In a separate experiment, we also determined plasma glucagon levels at defined time points after oral administration of glucose (2 mg/g) to mice that had been fasted for 8-10 h overnight. Plasma glucagon concentrations were measured via RIA (Linco).

Determination of pancreatic insulin content. Mice were killed by cervical dislocation, and whole pancreata were dissected and immediately frozen in liquid nitrogen. Protein extracts were prepared using the acid-ethanol method (26). in brief, pancreata weighing 200-300 mg were homogenized in 1 ml acid-ethanol (95% ethanol and 10.2 N HCl at a ratio of 50:1) by an Ultrasonic Homogenizer (Biologic) for 2 min applying 20 pulses. After an overnight incubation at 4[degrees]C, the extracts were centrifuged at 650g for 30 min at 4[degrees]C. Insulin concentrations in pancreatic extracts were measured via ELISA (Alpco), following the manufacturer’s instructions. The protein content of the pancreatic extracts was determined using the Bio-Rad protein assay kit.

Statistics. Data are expressed as means [+ or -] SE for the indicated number of observations. P values were calculated using one-way ANOVA followed by appropriate post hoc tests (Fisher’s least significant differences method for islet insulin and glucagon measurements and Bouferroni method for all other studies).

RESULTS

RT-PCR analysis of muscarinic receptor expression in mouse pancreatic islets. To examine which muscarinic receptor subtypes are expressed in pancreatic islets of the mouse, we subjected total RNA prepared from WT mouse pancreatic islets to RT-PCR amplification using [M.sub.1]-[M.sub.5] mouse muscarinic receptor-specific primers. Because all five muscarinic receptors are known to be expressed in the brain (31), mouse brain total RNA served as a positive control. As expected, all five muscarinic receptors were found to be expressed in the WT mouse brain (Fig. 1A). In contrast, only [M.sub.1] and [M.sub.3] receptor cDNA could be detected in samples from WT mouse islets (Fig. 1B). Figure 1C shows the absence of [M.sub.3] receptor transcripts in pancreatic islets prepared from [M3.sup.-/-] mice.

[FIGURE 1 OMITTED]

In vitro insulin release studies. To study the potential role of the [M.sub.3] muscarinic receptor subtype in augmenting pancreatic insulin release, we carried out a series of in vitro insulin release studies using isolated islets prepared from WT, [M3.sup.+/-], and [M3.sup.-/-] mice. Oxo-M, a non-subtype-selective muscarinic agonist, was used as a hydrolytically s2table muscarinic stimulant throughout all experiments.

In the absence of Oxo-M, static incubation of pancreatic islets from WT mice with a basal concentration of glucose (5.6 mmol/l) led to the release of only very small amounts of insulin (Fig. 2). The magnitude of this basal secretory response was not significantly affected by the absence of [M.sub.3] receptors or the presence of Oxo-M (0.5 or 20 [micro]mol//) (Fig. 2).

[FIGURE 2 OMITTED]

In contrast, incubation of pancreatic islets from WT and [M.sub.3] receptor mutant mice with a high concentration of glucose (16.7 mmol/l) led to a significant increase (~10- to 15-fold above basal levels determined in the presence of 5.6 mmol/l glucose) in insulin release (Fig. 2). In islets from WT mice (16.7 mmol/l glucose), the addition of Oxo-M (0.5 or 20 [micro]mol/l) led to a pronounced potentiation of glucose-dependent insulin release (Fig. 2). Strikingly, the Oxo-M–mediated potentiation of glucose-dependent insulin release was totally abolished in islets prepared from [M3.sup.-/-] mice and significantly reduced (by ~30-50%) in islets from [M3.sup.+/-] mice (Fig. 2).

Insulin content in the whole pancreas. Total pancreatic insulin content did not differ significantly between 6-month-old WT and [M.sub.3] receptor mutant mice (276 [+ or -] 40 [WT] vs. 228 [+ or -] 15 [[M3.sup.+/-]] vs. 341 [+ or -] 36 [[M3.sup.-/-]] pg insulin/[micro]g pancreatic protein; n = 6). Moreover, mean wet weights of the pancreata (expressed as a percentage of total body weight) did not differ significantly among the three genotypes (0.92 [+ or -] 0.06 [WT] vs. 0.80 [+ or -] 0.05 [[M3.sup.+/-]] vs. 0.95 [+ or -] 0.08% [[M3.sup.-/-]]; n = 6). Preliminary immunohistochemical studies showed that the number and size distribution of pancreatic islets were similar in WT and [M.sub.3] receptor mutant mice (data not shown).

Blood glucose and serum insulin levels. To examine whether [M.sub.3] receptor mutant mice showed altered blood glucose and insulin levels, we continuously monitored these parameters in freely fed WT, [M3.sup.+/-], and [M3.sup.-/-] mice over a 6-month period. As shown in Fig. 3A, [M3.sup.+/-] mice showed blood glucose levels that, at most time points, were not significantly different from the corresponding levels obtained with the WT control mice. In contrast, [M3.sup.-/-] mice showed a significant reduction in blood glucose levels (by ~20-50%) starting at age 3 months (Fig. 3A). This hypoglycemia persisted in [M3.sup.-/-] mice that were > 1 year old (data not shown). The serum insulin levels of 1-month-old WT, [M3.sup.+/-], and [M3.sup.-/-] mice did not differ significantly among the three genotypes (Fig. 3B). In contrast, 2- to 6-month-old [M3.sup.+/-] and [M3.sup.-/-] mice showed significant reductions (by approximately two- to six-fold) in serum insulin levels as compared with their WT littermates (Fig. 3B). As we reported previously (29), [M3.sup.-/-] mice showed pronounced reductions in body weight (by ~25%) throughout the 6-month observation period (Fig. 3C). This difference in body weight persisted throughout the entire life of the [M3.sup.-/-] mice (data not shown). In contrast, [M3.sup.+/-] mice showed body weights that were similar to those of their WT littermates (Fig. 3C).

[FIGURE 3 OMITTED]

Oral glucose tolerance test. To determine whether the lack of [M.sub.3] receptors was associated with changes in glucose tolerance in vivo, WT, [M3.sup.+/-], and [M3.sup.-/-] mice were subjected to an oral glucose tolerance test (OGTT; 2 mg glucose/g body wt). As shown in Fig. 4A, [M3.sup.+/-] mice exhibited a similar temporal pattern of changes in blood glucose levels as their WT littermates. In contrast, [M3.sup.-/-] mice showed a significant reduction in blood glucose levels compared with WT mice (P < 0.05 at 15 min after glucose administration), indicating that [M3.sup.-/-] mice cleared glucose more efficiently from the circulation than the WT control animals.

[FIGURE 4 OMITTED]

In WT mice, the increase in blood glucose levels was accompanied by a significant increase in serum insulin levels that reached peak values 15 min after glucose administration (Fig. 4B). Interestingly, the glucose-induced spike in serum insulin levels observed 15 min after glucose administration was significantly blunted in [M3.sup.+/-] mice (P < 0.05) (Fig. 4B). [M3.sup.+/-] mice also showed a reduction in serum insulin levels at this time point, which, however, failed to reach statistical significance (P = 0.08 vs. WT) (Fig. 4B).

In vitro glucagon release studies. The incubation of isolated pancreatic islets with muscarinic agonists not only facilitates insulin secretion but also stimulates the release of glucagon (5,6,26-28). To assess the potential role of the [M.sub.3] muscarinic receptor subtype in this activity, we carried out in vitro glucagon-release studies using isolated islets prepared from WT, [M3.sup.+/-], and [M3.sup.-/-] mice. In the absence of Oxo-M and in the presence of 16.7 mmol/l glucose, the amount of glucagon released from [M3.sup.+/-] and [M3.sup.-/-] preparations did not differ significantly from the corresponding WT value.

The addition of Oxo-M (20 [micro]mol/l) led to a significant increase (P < 0.05) in glucagon release from WT and [M3.sup.+/-] islets, as compared with the corresponding preparations that were not treated with Oxo-M (Fig. 5). In the presence of Oxo-M, [M3.sup.-/-] islets secreted significantly less (P 0.05) from the amount released by [M3.sup.-/-] islets in the absence of Oxo-M.

[FIGURE 5 OMITTED]

Plasma glucagon levels in vivo. We next measured plasma glucagon levels in freely fed WT, [M3.sup.+/-], and [M3.sup.-/-] mice. Whereas [M3.sup.+/-] mice showed similar plasma glucagon levels as WT mice, [M3.sup.-/-] mice displayed significantly reduced plasma glucagon levels (~30-35% reduction vs. WT mice; P 0.05) for any of the three genotypes (data not shown).

[FIGURE 6 OMITTED]

DISCUSSION

The stimulatory effects of acetylcholine on pancreatic insulin release are known to be mediated by activation of muscarinic receptors (4-6). RT-PCR analysis showed that mouse pancreatic islets express [M.sub.1] and [M.sub.3] muscarinic receptors (Fig. 1). Similar findings were obtained with rat pancreatic islets (23,25). Whereas previous studies (24,25) reported the expression of additional muscarinic receptor subtypes ([M.sub.4] or [M.sub.5]) in islet preparations or in [beta]-cell-derived cell lines, we did not detect [M.sub.2], [M.sub.4], or [M.sub.5] receptor mRNA in mouse pancreatic islets. Because the majority of the cells in rat or mouse islets represent [beta]-cells, it is highly likely that both [M.sub.1] and [M.sub.3] receptors are expressed by the insulin-secreting [beta]-cells.

To better understand the role of the muscarinic cholinergic system in [beta]-cell function, it is essential to identify the muscarinic receptor subtype(s) mediating stimulation of insulin release. Classic pharmacological studies using different “subtype-preferring” muscarinic antagonists have suggested that the [M.sub.3] receptor subtype (previously also referred to as “glandular [M.sub.2] receptor subtype”) (32) plays a key role in the control of insulin secretion (26,27,32). However, the proper interpretation of these experiments is complicated by the limited subtype selectivity of the muscarinic antagonists used in these studies. For example, virtually all antagonists that have a high affinity for [M.sub.3] receptors (e.g., 4-DAMP, derivatives of sila-hexocyclium) also exhibit a high affinity for [M.sub.1] and [M.sub.5], receptors (33,34). Moreover, it is especially difficult to predict the simultaneous involvement of two or more muscarinic receptor subtypes (e.g., [M.sub.1] and [M.sub.3] receptors) in a specific functional response by using the currently available subtype-preferring muscarinic antagonists.

To circumvent these difficulties, we carried out in vitro insulin release studies using pancreatic islets prepared from mutant mice in which the [M.sub.3] muscarinic receptor gene had been inactivated by gene-targeting techniques (29). Consistent with previously published results (16-21), the muscarinic agonist, Oxo-M, had little effect on basal insulin release measured in the presence of a low concentration of glucose (5.6 mmol/l), using isolated islets from WT, [M3.sup.+/-]/, and [M3.sup.-/-] mice (Fig. 2). On the other hand, in the presence of a stimulatory glucose concentration (16.7 mmol/l), the addition of Oxo-M to islets prepared from WT mice resulted in a pronounced potentiation of insulin output (Fig. 2). Strikingly, this insulinotropic activity of Oxo-M was completely abolished in islets prepared from [M3.sup.-/-] mice (Fig. 2). Pancreata from [M3.sup.-/-] mice contained normal amounts of total insulin, and preliminary studies showed that the number and size of pancreatic islets were similar in WT and [M3.sup.-/-] mice (data not shown). These observations demonstrated in an unambiguous fashion that muscarinic receptor-mediated stimulation of pancreatic insulin release is mediated by the [M.sub.3] receptor subtype and that [M.sub.3] or other muscarinic receptor subtypes do not contribute to this response to a significant extent. The potential functional role of islet [M.sub.1] receptors remains to be elucidated.

Interestingly, heterozygous [M.sub.3] receptor mutant mice ([M3.sup.+/-] mice), in which the density of [M.sub.3] receptors is reduced by ~50% (29), also showed significant impairments in muscarinic agonist-mediated potentiation of glucose-dependent insulin release (reduction in maximum secretory responses by ~30-50%). This observation indicated that >50% of the islet [M.sub.3] muscarinic receptors must be occupied to achieve maximum insulinotropic activity, at least in the mouse model. In contrast to many other muscarinic responses, [M.sub.3] muscarinic receptor-mediated augmentation of insulin release is therefore characterized by a very low degree of receptor reserve.

The [M.sub.3] muscarinic receptor, like the [M.sub.1] and [M.sub.5] receptor subtypes, is known to selectively couple to G proteins of the [G.sub.q] family (22,33). Consistent with this finding, muscarinic stimulation of pancreatic [beta]-cells leads to a series of biochemical events that are usually associated with the activation of [G.sub.q]-type G proteins, including the activation of phospholipase C, protein kinase C, and phospholipase A2 (4-6). Stimulation of these signaling cascades eventually results in elevated intracellular calcium levels and an increase in the efficiency of calcium-dependent exocytosis of insulin-containing storage vesicles (4-6). These latter two mechanisms are predicted to be primarily responsible for the insulinotropic effects of [M.sub.3] receptor activation.

Our findings suggest that drugs that can selectively activate [M.sub.3] muscarinic receptors may be of potential therapeutic benefit in the treatment of type 2 diabetes. Such agents appear particularly attractive as they would exert their full insulinotropic effects only in the presence of stimulatory concentrations of glucose, an effect not seen with other antidiabetic drugs such as the commonly used sulfonylureas (3). Unfortunately, muscarinic agonists that can activate [M.sub.3] muscarinic receptors (or other muscarinic receptor subtypes) with a high degree of subtype selectivity are not available at present (22,33). The development of such agents therefore represents an important goal for medicinal chemists.

Consistent with the results of the in vitro insulin release studies, [M3.sup.+/-] and [M3.sup.-/-] mice showed reduced serum insulin levels in vivo (by approximately two- to sixfold, as compared with their WT littermates) (Fig. 3B). Moreover, [M3.sup.-/-] mice displayed a significant decrease in serum insulin levels 15 min after an oral glucose load (2 mg/g) (Fig. 4B). However, the [M3.sup.+/-] and [M3.sup.-/-] mice did not develop hyperglycemia or impaired glucose tolerance in vivo. In fact, [M3.sup.-/-] mice showed significantly reduced blood glucose levels (Fig. 3A) and even displayed a significant increase in glucose tolerance in the OGTT (Fig. 4A), indicative of an increase in insulin sensitivity. Consistent with this observation, [M3.sup.-/-] mice showed a more pronounced and prolonged hypoglycemia response than WT control mice in an insulin tolerance test (29).

We also demonstrated previously that the mass of peripheral fat deposits is significantly reduced in [M3.sup.-/-] mice and that this phenotype was linked to a reduction in food intake (29). It is well known that lean animals (individuals) generally exhibit an increase in insulin sensitivity (35). Muscarinic receptors located in insulin-sensitive tissues, such as liver, skeletal muscle, and fat, do not seem to play a significant role in regulating glucose utilization or other metabolic functions in these tissues (36). It is therefore likely that the increased insulin sensitivity displayed by the [M3.sup.-/-] mice in vivo was primarily due to the reduction in body fat mass (29). This observation may explain why the lack of cholinergic stimulation of insulin release observed with islets from [M3.sup.-/-] mice in vitro did not result in hyperglycemia and/or impaired glucose tolerance in vivo. The increase in insulin sensitivity displayed by the [M3.sup.-/-] mice may represent another major factor contributing to the hypoinsulinemia associated with the lack of [M.sub.3] receptors.

Interestingly, although the [M3.sup.-/-] mice were able to clear glucose from the circulation more efficiently than WT mice (OGTT), glucose clearance was unaltered in [M3.sup.+/-] mice (Fig. 4A). In contrast to [M3.sup.-/-] mice, which showed a significantly reduced body weight, the body weight of [M3.sup.+/-] mice did not differ significantly from that of their WT littermates (Fig. 3C). These findings therefore support the concept that the increased insulin sensitivity displayed by the [M3.sup.-/-] mice is caused primarily by the reduction in body weight/body fat mass (29).

In a recent study (29), we provided evidence that the absence of hypothalamic [M.sub.3] receptors may be responsible, at least partially, for the hypophagia displayed by the [M3.sup.-/-] mice. This hypophagia phenotype may represent a major factor contributing to the reduction in body weight and blood glucose levels associated with the absence of [M.sub.3] receptors (29).

The activation of vagal nepees not only promotes insulin release but also stimulates the release of glucagon from pancreatic [alpha]-cells (5,6,28). In vitro and in vivo experiments suggest that this activity is mediated by muscarinic receptors predicted to be located on pancreatic [alpha]-cells (5,6,26-28). In the present study, we demonstrated that the muscarinic agonist, Oxo-M, failed to stimulate the release of glucagon from isolated islets prepared from [M.sub.3] receptor-deficient mice ([M3.sup.-/-] mice) (Fig. 5), indicating that this response is mediated by the [M.sub.3] receptor subtype in WT animals. In agreement with this finding, previous in vitro and in vivo studies using muscarinic antagonists of limited receptor subtype selectivity have also suggested that the [M.sub.3] subtype plays a key role in muscarinic receptor–mediated glucagon release (26,27).

Consistent with the in vitro glucagon release data, we found that plasma glucagon levels were significantly decreased (by ~30-50%) in both fed and fasted [M3.sup.-/-] mice. Glucagon counteracts the hypoglycemic effects of insulin, primarily by stimulating hepatic glucose production. It is therefore likely that the reduced glucagon plasma levels of the [M3.sup.-/-] mice contribute to the hypoglycemia displayed by these mutant animals.

Because hypoglycemia usually triggers an increase in circulating glucagon levels, it is possible that our findings underestimated the importance of pancreatic [M.sub.3] receptors in mediating glucagon secretion. Moreover, because hypoglycemia normally leads to reduced insulin secretion, the reduced blood glucose levels displayed by the [M3.sup.-/-] mice may also contribute to the hypoinsulinemia associated with the lack of [M.sub.3] receptors.

In conclusion, we have demonstrated that muscarinic stimulation of pancreatic insulin and glucagon release is mediated by the [M.sub.3] muscarinic receptor subtype. Consistent with this observation, [M.sub.3] receptor–deficient mice showed pronounced reductions in plasma insulin and glucagon levels. Given the lack of muscarinic ligands that can selectively block or inhibit specific muscarinic receptor subtypes with a high degree of selectivity, these findings highlight the usefulness of muscarinic receptor mutant mice as a novel tool for dissecting the metabolic roles of the [M.sub.1]- M.sub.5] muscarinic receptors.

ACKNOWLEDGMENTS

This work was supported in part by a grant from the American Diabetes Association (to D.M.).

REFERENCES

(1.) Kahn SE: Clinical review 135: the importance of beta-cell failure in the development and progression of type 2 diabetes. J Clin Endocrinol Metab 86:4047-4058, 2001

(2.) Kahn CR: Banting Lecture: insulin action, diabetogenes, and the cause of type 2 diabetes. Diabetes 43:1066-1084, 1994

(3.) Moller DE: New drug targets for type 2 diabetes and the metabolic syndrome. Nature 414:821-827, 2001

(4.) Satin LS, Kinard TA: Neurotransmitters and their receptors in the islets of Langerhans of the pancreas: what messages do acetylcholine, glutamate, and GABA transmit? Endocrine 8:213-223, 1998

(5.) Ahren B: Autonomic regulation of islet hormone secretion: implications for health and disease. Diabetologia 43:393-410, 2000

(6.) Gilon P, Henquin JC: Mechanisms and physiological significance of the cholinergic control of pancreatic beta-cell function. Eudocr Rev 22:565-604, 2001

(7.) Ahren B, Lundquist I: Modulation of basal insulin secretion in the obese, hyperglycemic mouse. Metabolism 31:172-179, 1982

(8.) Jeanrenaud B: An hypothesis on the aetiology of obesity: dysfunction of the central nervous system as a primary cause. Diabetologia 28:502-513, 1985

(9.) Rohner-Jeanrenaud F, Jeanrenaud B: Involvement of the cholinergic system in insulin and glucagon oversecretion of genetic preobesity. Endocrinology 116:830-834, 1985

(10.) Fukudo S, Virnelli S, Kuhn CM, Cochrane C, Feinglos MN, Surwit RS: Muscarinic stimulation and antagonism and glucoregulation in nondiabetic and obese hyperglycemic mice. Diabetes 38:1433-1438, 1989

(11.) Ahren B, Simonsson E, Scheurink AJ, Mulder H, Myrsen U, Sundler F: Dissociated insulinotropic sensitivity to glucose and carbachol in high-fat diet-induced insulin resistance in C57BL/6J mice. Metabolism 46:97-106, 1997

(12.) Edvell A, Lindstrom P: Vagotomy in young obese hyperglycemic mice: effects on syndrome development and islet proliferation. Am J Physiol 274:E1034-E1039, 1998

(13.) Bloom SR, Edwards AV: Pancreatic endocrine responses to stimulation of the peripheral ends of the vagus nerves in conscious calves. J Physiol 315:31-41, 1981

(14.) Bergman RN, Miller RE: Direct enhancement of insulin secretion by vagal stimulation of the isolated pancreas. Am J Physiol 225:481-486, 1973

(15.) Bloom SR, Edwards AV: Effects of certain metabolites on pancreatic endocrine responses to stimulation of the vagus nerves in conscious calves. J Physiol 362:303-310, 1985

(16.) Honey RN, Weir GC: Acetylcholine stimulates insulin, glucagon, and somatostatin release in the perfused chicken pancreas. Endocrinology 107:1065-1068, 1980

(17.) Holst JJ, Schaffalitzky de Muckadell OB, Fahrenkrug J, Lindkaer S, Nielsen OV, Schwartz TW: Nervous control of pancreatic endocrine secretion in pigs. III. The effect of acetylcholine on the pancreatic secretion of insulin and glucagon. Acta Physiol Scand 111:15-22, 1981

(18.) Garcia MC, Hermans MP, Henquin JC: Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets. Biochem J 254:211-218, 1988

(19.) Persaud SJ, Jones PM, Sugden D, Howell SL: The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans. Biochem J 264:753-758, 1989

(20.) Zawalich WS, Zawalich KC, Rasmussen H: Cholinergic agonists prime the beta-cell to glucose stimulation. Endocrinology 125:2400-2406, 1989

(21.) Verchere CB, Kwok YN, Brown JC: Modulation of acetylcholine-stimulated insulin release by glucose and gastric inhibitory polypeptide. Pharmacology 42:273-282, 1991

(22.) Wess J: Molecular biology of muscarinic acetylcholine receptors. Crit Rev Neurobiol 10:69-99, 1996

(23.) Boschero AC, Szpak-Glasman M, Carneiro EM, Bordin S, Paul I, Rojas E, Atwater I: Oxotremorine-M potentiation of glucose-induced insulin release from rat islets involves [M.sub.3] muscarinic receptors. Am J Physiol 268: E336-E342, 1995

(24.) Tang SH, Sharp GW: Identification of muscarinic receptor subtypes in RINm5F cells by means of polymerase chain reaction, subcloning, and DNA sequencing. Diabetes 46:1419-1423, 1997

(25.) Iismaa TP, Kerr EA, Wilson JR, Carpenter L, Sims N, Biden TJ: Quantitative and functional characterization of muscarinic receptor subtypes in insulin-secreting cell lines and rat pancreatic islets. Diabetes 49:392-398, 2000

(26.) Verspohl EJ, Tacke R, Mutschler E, Lambrecht G: Muscarinic receptor subtypes in rat pancreatic islets: binding and functional studies. Eur J Pharmacol 178:303-311, 1990

(27.) Karlsson S, Ahren BJ: Muscarinic receptor subtypes in carbachol-stimulated insulin and glucagon secretion in the mouse. J Auton Pharmacol 13:439-446, 1993

(28.) Brunicardi FC, Shavelle DM, Andersen DK: Neural regulation of the endocrine pancreas. Int J Pancreatol 18:177-195, 1995

(29.) Yamada M, Miyakawa T, Duttaroy A, Yamanaka A, Moriguchi T, Makita R, Ogawa M, Chou CJ, Xia B, Crawley JN, Felder CC, Den CX, Wess, J: Mice lacking the [M.sub.3] muscarinic aeetylcholine receptor are hypophagic and lean. Nature 410:207-212, 2001

(30.) Davalli AM, Ogawa Y, Scaglia L, Wu YJ, Hollister J, Bolmer-Weir S, Weir GC: Function, mass, and replication of porcine and rat islets transplanted into diabetic nude mice. Diabetes 44:104-111, 1995

(31.) Levey AI: Immunological localization of M1-M5 muscarinic acetylcholine receptors in peripheral tissues and brain. Life Sci 52:441-448, 1993

(32.) Henquin JC, Nenquin M: The muscarinic receptor subtype in mouse pancreatic B-cells. FEBS Lett 236:89-92, 1988

(33.) Caulfield MP, Birdsall NJ: International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 50: 279-290, 1998

(34.) Dorje F, Wess J, Lambreclit G, Tacke R, Mutschler E, Brann MR: Antagonist binding profiles of five cloned human muscarinic receptor subtypes. J Pharmacol Exp Ther 256:727-733, 1991

(35.) Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin D: Central nervous system control of food intake. Nature 404:661-671, 2000

(36.) Hoffman BB, Taylor P: The autonomic and somatic motor nervous systems. In Goodman & Gilman’s The Pharmacological Basis of Therapeutics. 10th ed. Hardman JG, Limbird LE, Eds. New York, McGraw-Hill, 2001, p. 115-153

Alokesh Duttaroy, (1) Charles L. Zimliki, (2) Dinesh Gautam, (1) Yinghong Cui, (1) David Mears, (2) and Jurgen Wess (1)

>From the (1) Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland; and the (2) Department of Anatomy, Physiology and Genetics, Uniformed Services University, Bethesda, Maryland.

Address correspondence and reprint requests to Dr. Jurgen Wess, Chief, Molecular Signaling Section Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8A, Rm. B1A-05, 8 Center Dr., MSC 0810, Bethesda, MD 20892-0810. E-mall: jwess@helLx.nih.gov.

Received for publication 6 January 2004 and accepted in revised form 29 March 2004.

A.D. is currently affiliated with Novartis Institutes for Biomedical Research, Cambridge, Massachusetts.

ELISA, enzyme-linked immunosorbent assay; OGTT, oral glucose tolerance test; Oxo-M; oxolremorine-M; RIA, radioimmunoassay; WT, wild type.

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