Neuropeptides and the Regulation of Islet Function

Neuropeptides and the Regulation of Islet FunctionBo Ahren, Nils Wierup, and Frank Sundler

The pancreatic islets are richly innervated by autonomicnerves. The islet parasympathetic nerves emanate fromintrapancreatic ganglia, which are controlled by pregangli-onic vagal nerves. The islet sympathetic nerves are post-ganglionic with the nerve cell bodies located in gangliaoutside the pancreas. The sensory nerves originate fromdorsal root ganglia near the spinal cord. Inside the islets,nerve terminals run close to the endocrine cells. In addi-tion to the classic neurotransmitters acetylcholine andnorepinephrine, several neuropeptides exist in the isletnerve terminals. These neuropeptides are vasoactive intes-tinal polypeptide, pituitary adenylate cyclase–activatingpolypeptide, gastrin-releasing polypeptide, and cocaine-and amphetamine-regulated transcript in parasympatheticnerves; neuropeptide Y and galanin in the sympatheticnerves; and calcitonin gene–related polypeptide in sensorynerves. Activation of the parasympathetic nerves and ad-ministration of their neurotransmitters stimulate insulinand glucagon secretion, whereas activation of the sympa-thetic nerves and administration of their neurotransmit-ters inhibit insulin but stimulate glucagon secretion. Theautonomic nerves contribute to the cephalic phase of insu-lin secretion, to glucagon secretion during hypoglycemia,to pancreatic polypeptide secretion, and to the inhibitionof insulin secretion, which is seen during stress. In rodentmodels of diabetes, the number of islet autonomic nerves isupregulated. This review focuses on neural regulation ofislet function, with emphasis on the neuropeptides.Diabetes 55 (Suppl. 2):S98–S107, 2006

Since the discovery of nerves in the pancreaticislet by Paul Langerhans in his thesis from 1869,the neural-islet axis has been explored by anumber of neuroanatomists, physiologists, and

endocrinologists (rev. in 1–3). It is currently known thatbranches of the parasympathetic and sympathetic as wellas the sensory nervous system innervate the islets withnerve terminals ending closely to the islet endocrine cells.It is also known that these nerves affect islet hormonesecretion. The classic neurotransmitters in the islet auto-

nomic nerves are acetylcholine and norepinephrine. Dur-ing the last decades, the contribution to neural regulationof islet function also by neuropeptides has been estab-lished (rev. in 4). Several neuropeptides are localized toislet nerve terminals, are released from the pancreaticnerves upon nerve stimulation, and influence islet hor-mone secretion. It is also known that the islet innervationis altered in animal models of type 2 diabetes (5). Theautonomic nervous system has also been suggested to beinvolved in the regulation of islet mass (6). The presentreview highlights the role of neuropeptides in islet function.

PARASYMPATHETIC NERVOUS SYSTEM

Anatomy and effects. The parasympathetic nerves inner-vating the pancreatic islets emanate from the pancreaticganglia, which are innervated by preganglionic parasym-pathetic nerves originating in the dorsal motor nucleus ofthe vagus. Activation of the parasympathetic nerves en-hances insulin and glucagon secretion (1,3). These stimu-latory effects are of physiological relevance under at leastthree conditions: 1) for the cephalic phase of insulinsecretion during meal ingestion, 2) for the glucagon re-sponse to hypoglycemia, and 3) for pancreatic polypeptide(PP) secretion.Parasympathetic nerves and cephalic phase of insulinsecretion. The cephalic phase of insulin secretion leadsto the rapid and early increase in insulin levels during thefirst minutes after food ingestion (7). This is due toactivation of olfactory-gustatory sensory receptors in as-sociation with psychological stimuli, which activatescentral parasympathetic nerves that stimulate insulin se-cretion. The importance of the cephalic phase of insulinsecretion was recently examined by an approach to blockthe autonomic ganglia with the ganglionic blocker, tri-metophane, in healthy subjects (7). In this approach,trimetophane was infused intravenously to inhibit theautonomic ganglia. A meal was served during the tri-metophane infusion, and the trimetophane infusion wasstopped 15 min later. It was first demonstrated thatcirculating insulin increased within the first 10 min aftermeal ingestion, which is before any rise in circulatingglucose was observed. Of more importance in this context,however, was the second finding that this 10-min insulinresponse to meal ingestion was reduced by �75% bytrimetophane (Fig. 1). This demonstrates that a neurallymediated cephalic phase of meal-related insulin secretionexists in humans. This reduction was accompanied byimpairment of glucose elimination, even though the inhib-ited insulin response was limited to the first 15 min. Hence,the rapid and early insulin secretion contributes substan-tially to the glucose tolerance after meal ingestion. Thismay in turn be ascribed to the inhibition of hepatic glucoseproduction by insulin.Parasympathetic nerves and stimulation of glucagonsecretion during hypoglycemia. The parasympatheticnerves might also be of physiological importance for thestimulation of glucagon secretion during hypoglycemia

From the Department of Clinical Sciences, Lund University, Lund, Sweden,and the Department of Experimental Medical Sciences, Lund University,Lund, Sweden.

Address correspondence and reprint requests to Dr. Bo Ahren, Departmentof Clinical Sciences, B11 BMC, SE-221 84 Lund, Sweden. E-mail: [email protected].

Received for publication 18 March 2006 and accepted in revised form 25May 2006.

This article is based on a presentation at a symposium. The symposium andthe publication of this article were made possible by an unrestricted educa-tional grant from Servier.

CART, cocaine- and amphetamine-regulated transcript; CGRP, calcitoningene–related polypeptide; GRP, gastrin-releasing polypeptide; GSIS, glucose-stimulated insulin secretion; NPY, neuropeptide Y; PACAP, pituitary adenylatecyclase–activating polypeptide; PP, pancreatic polypeptide; TH, tyrosinehydroxylase; VIP, vasoactive intestinal polypeptide.

DOI: 10.2337/db06-S013© 2006 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked “advertisement” in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

S98 DIABETES, VOL. 55, SUPPLEMENT 2, DECEMBER 2006

(8). Glucagon secretion during counterregulation might bemediated by low glucose, low islet insulin, and highepinephrine, which all are consequences of hypoglycemiaand all stimulate glucagon secretion. A possible contribu-tion by the islet nerves was examined using the trimeto-phane protocol to inhibit autonomic ganglia in thepresence of insulin-induced hypoglycemia in healthy sub-jects (9). It was found that the glucagon response tohypoglycemia (�2.5 mmol/l glucose) was markedly re-duced by trimetophane (Fig. 2). Hence, a significant pro-portion of the glucagon response to hypoglycemia de-pends on autonomic nerves.Parasympathetic nerves and PP secretion. The isletparasympathetic nerves seem of importance for PP secre-tion. This is evident from findings that vagus nerve stimu-lation increases PP secretion (10,11) and that the PPresponse to hypoglycemia is reduced by autonomic gan-glionic blockade, as demonstrated in humans (9). In fact,the coupling of PP secretion to parasympathetic activity

has suggested that plasma PP levels may be used as amarker or index of parasympathetic activity (12). How-ever, the physiological relevance of the PP response toparasympathetic activation is not known.Islet parasympathetic neurotransmitters. It is wellknown that acetylcholine stimulates insulin secretionthrough a direct action on the islet �-cells (3). In addition,several neuropeptides are localized to islet parasympa-thetic nerve terminals and therefore potentially contributeto the islet effects of parasympathetic activation. Theseneuropeptides are vasoactive intestinal polypeptide (VIP),pituitary adenylate cyclase–activating polypeptide (PACAP),gastrin-releasing polypeptide (GRP), neuropeptide Y (NPY),and cocaine- and amphetamine-regulated transcript (CART)peptide (2–4,13–15). Figure 3 illustrates immunofluorescenceof rodent pancreatic tissue showing the close proximity ofthese nerves with the pancreatic islets by showing VIP as anexample: nerves co-harboring VIP and the cholinergicmarker vesicular acetylcholine transporter (VAchT) enter theislets to terminate in close proximity to the islet endocrinecells (Fig. 3A–C). Figure 3 also shows nerves harboring GRP(Fig. 3G) and CART (Fig. 3H) in islets. Figure 3I–J shows thata great proportion of the CART-containing fibers are also VIPimmunoreactive. Similar findings have previously been re-ported for PACAP (16) and, in the pig, for GRP (17) and,recently, for CART peptide (14,15). Therefore, VIP, PACAP,GRP, and CART are parasympathetic co-transmitter neu-ropeptides in autonomic nerve endings in the islets.Effects of parasympathetic co-transmitters on insulinand glucagon secretion in mice. VIP, PACAP, and GRPstimulate insulin and glucagon secretion when adminis-tered both in vivo in several species, including humans,and in vitro in isolated islets or the perfused pancreas(3,4,16–19). Here we report that VIP, PACAP, and GRP allpotentiate glucose-stimulated insulin secretion (GSIS) andarginine-stimulated glucagon secretion, both when exam-ined in isolated mouse islets (Figs. 4A and 5A) and whenintravenously administered to mice together with glucose(Fig. 4C) or arginine (Fig. 5C). Increased insulin secretionhas also been documented in transgenic mice overexpress-ing the VIP gene (20) or the PACAP gene (21) in the islet

FIG. 1. Serum insulin and plasma glucose levels in six healthy womensubjected to a 30-min intravenous infusion of the ganglionic antagonisttrimetophane or saline (from �15 to �15 min). At time 0, a standard-ized mixed meal was served. Small insert in upper panel shows theinsulin levels between 0 and 10 min in detail. Data are means � SE.Asterisks indicate probability level of random difference between thetwo protocols, as revealed by ANOVA: *P < 0.05, **P < 0.01. Repro-duced with permission from the American Diabetes Association (7).

FIG. 2. Plasma glucagon in healthy postmenopausal women before andduring insulin and glucose infusions producing hypoglycemia of �2.5mmol/l during intravenous infusion of the ganglionic antagonist tri-metophane (E) or saline (F). Data are means � SE. Adapted withpermission from the American Diabetes Association (9).

B. AHREN, N. WIERUP, AND F. SUNDLER

DIABETES, VOL. 55, SUPPLEMENT 2, DECEMBER 2006 S99

�-cells. The molecular basis of their effects is still far fromfully understood, although it is established that VIP andPACAP signal through cAMP.

The importance of VIP and PACAP for islet physiologyhas been explored in model experiments using specificreceptor antagonists and peptide ligand or receptor knock-out mice. A study using PACAP�/� mice showed impairedGSIS in these mice (22). Also VIP�/� mice have beengenerated (23), but insulin secretion in these animalsremains to be examined. Another strategy to explore thephysiology of VIP and PACAP is to inhibit the activity orexpression of their receptors. The two neuropeptidesactivate both VPAC1 and VPAC2 receptors and PACAP inaddition to PAC1 receptors. Of these receptors, VPAC2 andPAC1 are expressed in islet cells (16). By disrupting thePAC1 receptor gene in mice, expression of truncated PAC1receptors, which do not bind PACAP, evolves (24).PAC1R�/� mice display a marked reduction in the insulin

response to both oral and intravenous glucose, showingthat PACAP is of importance for a normal GSIS. Further-more, the insulin response to intravenous administrationof 2-deoxy-glucose (2-DG), is reduced in PAC1R�/� mice(Fig. 6). 2-deoxy-glucose competes with glucose for phos-phorylation, which results in neuroglycopenia. This in turnactivates the autonomic nerves, which results in a stimu-lation of insulin secretion after 2 min (25). Other studieshave shown that PAC1 receptor antagonists pharmacolog-ically inhibit the insulin response to oral glucose in mice(26) and to vagal nerve activation in the pig pancreas (27).A recent study compared the reduction in insulin secretionafter gastric glucose versus intravenous glucose inPAC1R�/� mice by matching the glucose levels underthese two conditions. The reduction was more markedafter gastric glucose, again suggesting that PACAP contrib-utes to the insulin response to oral glucose (28). Theimpairment of the insulin response in these mice suggests

FIG. 3. Immunofluorescence micrographs of rodent islets. A–C: Mouse islet double stained for VIP (A) and vesicular acetylcholine transporter(B), merged in C. Note the high degree of colocalization, illustrating that VIP immunoreactive fibers are cholinergic. D–F: Part of rat islet doublestained for NPY (D) and TH (E), merged in F. A great proportion of the NPY immunoreactive fibers are adrenergic. G: Bombesin/GRPimmunoreactive fiber in a mouse islet. H: CGRP immunoreactive fiber in a mouse islet. I–K: Part of rat islet double stained for VIP (I) and CART(J), merged in K. A great proportion of the CART-containing fibers are also VIP immunoreactive. Note that CART immunoreactivity is also seenin a �-cell (J). In brief, pancreatic tissue from normal C57BL/6J and normal Sprague-Dawley rats were fixed overnight in Stefanini’s solution and,after being frozen on dry ice, were cut in a cryostat, mounted on slides, and incubated with a guinea pig anti-VIP antibody (code 8701, dilution1:1,280; EuroDiagnostica, Malmo, Sweden), rabbit anti-TH (code P401010-0, dilution 1:320; Pel-Freeze Biologicals, Rogers, AR), rabbit anti–COOH-terminal flanking peptide of NPY (dilution 1:1,280; Genosys Biotech, Pampisford, U.K.), rabbit anti-CART (code 12/D, dilution 1:1,280;Cocalico, Reamstown, PA), guinea pig anti-CGRP (code M8513, dilution 1:640; EuroDiagnostica), and anti-bombesin/GRP (code 81051, dilution1:600; EuroDiagnostica). Sections were incubated for 1 h at room temperature with a secondary antibody, coupled with fluorescein isothiocyanateor Texas red with specificity for IgG (dilution 1:100 and 1:400, respectively; DAKO, Copenhagen, Denmark).

NEUROPEPTIDES AND ISLET FUNCTION


that PACAP may contribute to the cephalic phase ofinsulin secretion. In contrast, mice with VPAC2 genedeletion have a normal glucose tolerance during an oralglucose tolerance test in association with a reduced insu-lin response (29). This suggests increased insulin sensitiv-ity in VPAC2

�/� mice with an appropriate downregulationof the insulin response to maintain normal glucose toler-ance, which indirectly would support normal �-cell func-tion. A recent study has also demonstrated that PAC1R�/�

mice display impaired glucagon secretion during hypogly-cemia (30). Thus, PACAP may contribute to the parasym-pathetic involvement in the glucagon response tocounterregulation, in addition to its potential physiological

importance in regulating insulin secretion after 1) glucosestimulation, 2) vagal nerve activation, and 3) meal intake.

Because VIP and PACAP both strongly stimulate insulinsecretion, they may be of potential interest in the treat-ment of type 2 diabetes. PACAP has been shown to reducethe hyperglycemia in rodent diabetes (high fat–fed miceand GK rats) (31). However, one problem is that PACAPstimulates glucagon secretion and the peptide has potentvasoactive effects, which would limit its usefulness in treat-ment. Instead, specific activation of VPAC2 receptors wouldbe advantageous. Recently, a specific VPAC2 receptor agonistwas described (32): it augments GSIS in both rodent andhuman islets and potentiates insulin secretion and glucose

FIG. 4. Effects of islet neurotransmitters on insulin secretion in vivo and in vitro in NMRI mice. A and B: Insulin secretion fromovernight-incubated collagenase-isolated islets after incubation for 60 min in a medium containing various glucose concentrations with orwithout addition of islet cholinergic co-transmitters (Fig. 4A) or islet adrenergic co-transmitters (Fig. 4B). Synthetic porcine VIP (100 nmol/l),ovine PACAP-38 (100 nmol/l), porcine GRP (100 nmol/l), norepinephrine (1 �mol/l), porcine NPY (100 nmol/l), or rat galanin (100 nmol/l) wereused. There were 24–32 incubations at each data point; means are shown. C and D: Plasma insulin levels after intravenous administration ofglucose (1 g/kg) alone or together with synthetic porcine VIP (1.5 nmol/kg), ovine PACAP-38 (1.3 nmol/kg), porcine GRP (4 nmol/kg),norepinephrine (320 nmol/kg), porcine NPY (1.1 nmol/kg), or rat galanin (2 nmol/kg) in anesthetized mice. There were 16–20 animals in eachgroup. Means are shown. For the in vitro studies, pancreatic islets from normal NMRI mice were isolated by collagenase digestion and handpickedunder microscope. After overnight incubation in RPMI medium, the islets were preincubated in HEPES balanced salt solution (HBSS) containing125 mmol/l NaCl, 5.9 mmol/l KCl, 1.28 mmol/l CaCl2, 1.2 mmol/l MgCl2, 25 mmol/l HEPES (pH 7.4), 3.3 mmol/l glucose, and 0.1% fatty acid–freebovine albumin (Boehringer Mannheim, Mannheim, Germany) for 60 min. Islets in groups of three were then incubated in 200 �l HBSS for 60 minat 37°C, with varying concentrations of glucose alone or with addition of the peptides (all peptides from Peninsula Laboratories Europe,Merseyside, U.K.). For the in vivo studies, plasma insulin levels were determined after intravenous administration of glucose (1 g/kg) alone ortogether with the peptides in NMRI mice anesthetized with 0.5 mg/mouse fluanison, 0.02 mg/mouse fentanyl (Hypnorm; Janssen, Beerse,Belgium), and 0.25 mg/mouse midazolam (Dormicum; Hoffman-LaRoche, Basel, Switzerland). Samples (75 �l) were taken from the retrobulbarintraorbital capillary plexus before and at various time points after the intravenous injections. Plasma was separated and analyzed for insulinwith radioimmunoassay (Linco Research, St. Charles, MO).



disposal in rats, thereby offering a novel target for treatmentof type 2 diabetes based on islet neuropeptides.

The islet parasympathetic nerve endings also harborGRP. GRP is released from the pancreas during parasym-pathetic nerve stimulation and stimulates insulin andglucagon secretion (3,4,17,19). It has been shown that theGRP receptor is expressed in islets (33), suggesting adirect action of the neuropeptide on islet cells. A study inGRP receptor gene–deficient mice has shown that theinsulin response to oral glucose is impaired (34), whichwould support a role in the meal-related insulin response.It was also shown that insulin secretion in response toendogenous nerve activation in mice (by 2-deoxyglucose)is impaired in these mice, suggesting that GRP contributesto neurally mediated islet hormone secretion. These find-ings suggest that islet neuronal GRP, like VIP and PACAP,is of physiological importance.

It was recently also demonstrated that CART, an an-orexigenic peptide that is highly expressed in the brain(rev. in 35), is also a neuropeptide of the rat and mousepancreas (14,15, rev. in 36). Figure 3H–J shows the loca-tion of CART-containing nerves within an islet. Colocaliza-tion with VIP demonstrates the parasympathetic identityof the majority of the CART fibers. Recent studies haveshown that CART affects islet hormone secretion (37).CART inhibits GSIS from isolated rat islets. On the otherhand, CART potentiates GSIS augmented by glucagon-likepeptide 1. Although there is hitherto no CART receptoridentified, recent data suggest that CART exerts the poten-

tiating effect on glucagon-like peptide 1–mediated GSIS viaincreased cAMP and the protein kinase A–dependentpathway (37). Furthermore, the potential impact of CARTof islet function was demonstrated by using CART�/� mice(14). These mice displayed blunted GSIS both in vivo andin vitro, together with impaired glucose elimination.

SYMPATHETIC NERVOUS SYSTEM

Anatomy and function. The islet sympathetic nerves arepostganglionic, with their nerve cell bodies mainly locatedin the celiac ganglion or in the paravertebral sympatheticganglia. Electrical stimulation of the sympathetic nervesinhibits insulin secretion and stimulates glucagon secre-tion (1–4). This is of physiological importance duringstress and physical exercise to elicit a hyperglycemicresponse by increasing hepatic glucose delivery. An exper-imental tool for the study of function of the sympatheticnerves is administration of 6-hydroxydopamine to rodents.6-hydroxydopamine is taken up by way of vesicular mono-amine transporter localized to nerve endings of sympa-thetic neurons and selectively destroys sympathetic nerveterminals. As a consequence, islet nerves staining for thesympathetic marker tyrosine hydroxylase (TH) are absentin mice at 48 h after administration of 6-hydroxydopamine(38). This is associated with augmented GSIS (39,40),reduced insulin gene expression, and increased �-cellmass (38). These findings therefore suggest that the sym-

FIG. 5. Effects of islet neurotransmitters on glucagon secretion in vivo and in vitro in NMRI mice. A and B: Glucagon secretion from overnightincubated collagenase-isolated islets after incubation for 60 min in a medium containing glucose at 5 mmol/l and arginine at 20 mmol/l with orwithout addition of islet cholinergic co-transmitters (Fig. 5A) or islet adrenergic co-transmitters (Fig. 5B). Synthetic porcine VIP (100 nmol/l),ovine PACAP-38 (100 nmol/l), porcine GRP (100 nmol/l), norepinephrine (1 �mol/l), porcine NPY (100 nmol/l), or rat galanin (100 nmol/l) wereused. There were 16 incubations at each data point; means are shown. C and D: Plasma glucagon levels after intravenous administration ofarginine (0.25 g/kg) alone or together with synthetic porcine VIP (1.5 nmol/kg), ovine PACAP-38 (1.3 nmol/kg), porcine GRP (4 nmol/kg),norepinephrine (320 nmol/kg), porcine NPY (1.1 nmol/kg), or rat galanin (2 nmol/kg) in anesthetized mice. There were eight animals in eachgroup. Means are shown. Symbols are same as in Fig. 4. Glucagon was determined with radioimmunoassay (Linco Research).



pathetic nerves are of importance both for insulin secre-tion, insulin gene expression, and islet �-cell mass.Islet sympathetic neurotransmitters. The classic sym-pathetic neurotransmitter is norepinephrine, which inhib-its GSIS and stimulates glucagon secretion (3,39).However, combined �- and �-adrenoceptor blockade doesnot prevent sympathetic nerve activation from inhibitinginsulin secretion (41). This suggests that neurotransmit-ters other than norepinephrine contribute to some sympa-thetic islet effects. Neuropeptides localized to isletsympathetic nerve terminals are NPY and galanin (1–4).They are both released from the pancreas during sympa-thetic nerve activation (13,39) and, like sympathetic nervestimulation, inhibit insulin secretion and stimulate gluca-gon secretion (3,4). Figure 3D–E illustrates that NPYnerves are localized to islets and the colocalization withTH illustrates the sympathetic nature of the NPY-contain-ing nerve endings. It should be emphasized, however, thatNPY is localized also to nerve endings that do not harborTH. Also, galanin is localized to TH-containing nerveendings in the islets in a number of species (2,42). BothNPY and galanin are released from the pancreas duringsympathetic nerve activation (13,43) and, like sympatheticnerve stimulation, inhibit insulin secretion and stimulateglucagon secretion (3,4). Figures 4B and D and 5B and Dshow that norepinephrine, NPY, and galanin all inhibitGSIS and augment arginine-stimulated glucagon secretionin isolated mouse islets and in vivo in mice. Because NPYand galanin thus mimic the effects of sympathetic nervestimulation, they might contribute to sympathetic isleteffects.

In the dog, galanin has been suggested to be of majorimportance in contributing to the inhibitory influence ofsympathetic nerve activation on insulin secretion (43).Also in mice, galanin seems to be of physiological impactbecause immunoneutralization of galanin prevents theinhibition of insulin secretion, which is seen during astress model (swimming) in mice (44). To study thephysiological impact of galanin on islet function in moredetail, mice with a loss-of-function mutation in the galaningene have been examined (45). The inhibition of insulinsecretion that is seen after administration of 2-deoxyglu-

cose (and that reflects activation of sympathetic nerves)was impaired in galanin�/� mice. This further supports acontribution by galanin of the response to sympatheticactivation.

SENSORY NERVES

The islets are innervated by sensory nerves that harborcalcitonin gene–related polypeptide (CGRP) (1–4). Thefibers leave the pancreas along the sympathetic fibers withthe splanchnic nerves and reach the spinal cord. Figure 3Hillustrates an islet fiber harboring CGRP. The relevance ofthese nerves for islet function is far from understood.These sensory nerves may be targeted by the use of thetoxin capsaicin, which causes degeneration of small un-myelinated C-fibers. Capsaicin is an agonist for the tran-sient receptor potential vanilloid receptor (TRPV1);acutely, it activates the receptors, which leads to a releaseof the neurotransmitters. After the acute effect, however,capsaicin leads to loss of unmyelinated sensory fibers inconjunction with a substantial number of CGRP nerves(46). If capsaicin is given to neonatal rodents, the sensorydeactivation is permanent, whereas if given to adults, thedeactivation is transient. One effect of capsaicin-inducedsensory deactivation is increased insulin secretion (47).This would suggest that sensory nerve activation in isletsinhibits insulin secretion.Islet sensory neurotransmitters. CGRP nerves are scat-tered through the pancreas but with particular densityaround small blood vessels and islets (48). Furthermore,exogenous administration of CGRP inhibits GSIS (48).This suggests that sensory nerves inhibit insulin secretion;however, the physiological importance of this remainselusive. It should be mentioned in this context that CARTis also present in CGRP-containing fibers in rat and mousepancreas (14,15). The biological significance of CART inthese fibers needs further investigation.Sensory deafferentation and treatment of diabetes.Since sensory nerves apparently inhibit insulin secretion,possibly through CGRP, and since sensory deafferentationby capsaicin increases insulin secretion, it has been pro-posed that sensory deactivation might be a novel target fortreatment of type 2 diabetes. This hypothesis was substan-tiated in a recent study in obese Zucker rats (49). Thisnovel potential neurally based therapeutic approach hasbeen further explored by using the toxin resiniferatoxin.This is a vanilloid that has the same mechanism in causingdeactivation/degeneration of C-fibers (and A�-fibers) ascapsaicin but is less toxic. By administering resinifera-toxin to obese Zucker rats (50) and to Zucker diabetic fatty(ZDF) rats (51), improved glycemia has been observed,together with improved insulin secretion, as demonstratedin ZDF rats.

ISLET NERVES AND DIABETES

Several studies have indicated altered islet neurohormonalinfluences in models of insulin resistance and type 2diabetes, and therefore it has been speculated whetherislet nerve dysfunction may contribute to the developmentof type 2 diabetes. A study in high fat–fed rats, which is amodel of glucose intolerance and type 2 diabetes, dis-closed an increased islet innervation (52). Furthermore,insulin resistance in high fat–fed mice is accompanied byaugmented insulin secretion after cholinergic activation asa sign of cholinergic hypersensitivity (53) and as a signthat the hyperinsulinemia in ob/ob mice is highly sensitive

FIG. 6. Plasma insulin response to intravenous administration of2-deoxyglucose (2-DG; 0.5 g/kg) in anesthetized PAC1R�/� mice (E) ortheir wild-type counterparts (F). Asterisks indicate probability levelof random difference between the groups: *P < 0.05, **P < 0.01, ***P <0.001.



to atropine (54). Indeed, cholinergic activation by carba-chol normalizes insulin secretion in high fat–fed mice (55).It was therefore of interest that reduced islet innervationwas evident in a model of type 2 diabetes, the Chinesehamster (5).

We present here immunocytochemical data of isletsfrom two rodent models of diabetes. The models are theGoto-Kakizaki (GK) rats and the db/db mice. The GK ratmodel is one of the best-described models of type 2diabetes, as recently was reviewed (56). Its basis is a �-cell

FIG. 7. Immunofluorescence micrographs of GK rat islets illustrating that the numbers of both parasympathetic and sympathetic nerves aregreatly increased in the islets. A–C: Double staining for VIP (A) and vesicular acetylcholine transporter (B), merged in C. Note also the highdegree of colocalization (exemplified by arrow heads). D–F: Double staining for NPY (D) and TH (E), merged in F. NPY is largely colocalized withTH (exemplified by arrow heads); note that a substantial population of the NPY immunoreactive varicosities are devoid of TH, presumablyrepresenting parasympathetic fibers.

FIG. 8. Immunofluorescence micrographs of db/db mouse islets illustrating that both parasympathetic and sympathetic nerves are greatlyincreased in the islets. A–C: Double staining for VIP (A) and vesicular acetylcholine transporter (B), merged in C. Note the high degree ofcolocalization (exemplified by arrow heads). D: Staining for NPY. E: Staining for TH. F: Staining for CART.



defect, which occurs through changes in several indepen-dent genes, leading to impaired insulin secretion in com-bination with metabolic impairment reducing �-cellneogenesis and a potential acquired loss of �-cell differ-entiation due to glucotoxicity. The db/db mouse is charac-terized by a leptin receptor deficiency, resulting in insulinresistance and impaired islet function (57). Figures 7 and8 show that in both these two rodent models of diabetes,the islet innervation is upregulated. This emphasizes thatislet innervation is of importance for the islet dysfunctionin diabetes. We also found that certain neuropeptides, e.g.,VIP and CART (Table 1) (37), are expressed in isletendocrine cells in the diabetic rodents. This is similar toour previous observation of marked expression of NPY inislet �-cells in dexamethasone-induced insulin resistancein rats (58). We propose that augmented endocrine cellexpression of neurotransmitters in the islets is a sign ofislet adaptation for normalization of glucose homeostasis.We suggest the term “neuro-islet plasticity” for this phe-nomenon; a thorough examination of which needs to beundertaken now.

A novel potential effect of parasympathetic nerves,which may be of pathophysiological relevance for type 2diabetes, is stimulation of �-cell mass. Regulation of �-cellmass has recently come into focus because of studiessuggesting that �-cell mass is reduced in type 2 diabetes(6,59). In adult life, �-cell growth and renewal is mainlyregulated by replication and differentiation from isletprecursor cells within islets and within the duct epithelium(60). An important mechanism for increase in �-cell massmay therefore be the increased demand created by insulinresistance; therefore, islets are enlarged in, for instance,the pre-diabetic state and obesity. On the other hand, inovert type 2 diabetes, �-cell mass seems to be reduced,which may be ascribed to exaggerated apoptosis triggeredby, for instance, glucotoxicity or lipotoxicity. However,neural effects may also contribute to these perturbations.Thus, it has been demonstrated that vagotomy attenuatesthe islet hyperplasia in ob/ob mice, the mechanism ofwhich needs to be further explored (61). From a therapeu-tic point of view, the potential of the incretin hormoneglucagon-like peptide 1 to augment islet �-cell mass, pre-sumably by both increasing neogenesis and inhibitingapoptosis, has been extensively discussed (60). However,islet neuropeptides may also be of interest in this context;for example, exogenous administration of PACAP hasbeen shown to maintain �-cell mass in an experimentaldiabetes model in mice, which is due to increased �-cellapoptosis (�-cell overexpression of calmodulin [62]).

CONCLUSIONS AND OUTSTANDING ISSUES

The pancreatic islets are innervated by the autonomicnervous system, which affect islet function through bothclassic neurotransmitters and neuropeptides. Table 1 sum-marizes the neuropeptides, which are of greatest im-portance in this context. The islet nerves seem ofphysiological relevance for 1) the insulin secretion duringthe cephalic phase of meal ingestion, 2) the glucagonresponse to hypoglycemia, 3) the PP release, and 4) theislet stress response. The islet nerves may also be ofsignificance for the regulation of islet mass. Novel poten-tial targets for treatment of diabetes may evolve fromstudies of islet effects of autonomic nerves and neuropep-tides. Of most interest at the moment is stimulation ofinsulin secretion by 1) cholinergic agonism, 2) VPAC2receptor agonists, 3) PACAP, 4) CART in combinationwith glucagon-like peptide 1, and 5) sensory nerve deac-tivation. However, much remains to be established regard-ing the potential role for the autonomic nerves in relationto islet function. Complex issues relate to mechanisms ofactivation of the autonomic nerves and regulation ofrelease of the neurotransmitters in relation to each otherand the possibility of synergistic action of the neurotrans-mitters. Other issues relate to the involvement of neu-ropeptides in islet physiology in humans as well as topotential importance of the autonomic nerves for the isletcompensation to insulin resistance, to diabetes develop-ment, and as targets for treatment of diabetes. Therefore,although almost 140 years have passed since the firstdescription of islet nerves by Paul Langerhans, much efforthas yet to be paid to understand the role for these nervesin islet physiology and pathophysiology.

ACKNOWLEDGMENTS

These studies have been supported by the Swedish ResearchCouncil (grants 6834 and 4499), Swedish Diabetes Associa-tion, Albert Påhlson Foundation, The Royal PhysiographicSociety, Novo Nordisk Foundation, Tore Nilsson, Åke Wibergand Gyllenstiernska-Krapperup Foundations, Region Skåne,and the Faculty of Medicine, Lund University.

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TABLE 1Neuropeptides that are localized to islet autonomic nerves: their primary and main localization in normal rodent islets, in GK rats,and in ob/ob mice and their effect on insulin and glucagon secretion

NeuropeptideLocalization Effects

Normal rodents GK rat db/db mouse Insulin secretion Glucagon secretion

VIP PS nerves Innervation 1/�-Cells Innervation 1/�-Cells Stimulation StimulationPACAP PS nerves NT NT Stimulation StimulationGRP PS nerves NT NT Stimulation StimulationNPY S nerves Innervation 1 Innervation 1 Inhibition StimulationGalanin S nerves NT NT Inhibition Stimulation

CGRPSensory nerves/

D-cells Innervation 1 Unchanged Inhibition StimulationCART PS nerves Innervation 1/�-Cells Innervation 1/�-Cells Stimulation Inhibition

NT, not tested.



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Neuropeptides and the Regulation of Islet Function