Phosphoproteome reveals molecular mechanisms of aberrant rhythm in neurotransmitter-mediated islet hormone secretion in diabetic mice.

BACKGROUND: Acetylcholine (ACh) and norepinephrine (NE) are representative neurotransmitters of parasympathetic and sympathetic nerves, respectively, that antagonize each other to coregulate internal body functions. This also includes the control of different kinds of hormone secretion from pancreatic islets. However, the molecular mechanisms have not been fully elucidated, and whether innervation in islets is abnormal in diabetes mellitus also remains unclear. METHODS AND RESULTS: Immunofluorescence colocalization and islet perfusion were performed and the results demonstrated that ACh/NE and their receptors were highly expressed in islet and rapidly regulated different hormones secretion. Phosphorylation is considered an important posttranslational modification in islet innervation and it was identified by quantitative proteomic and phosphoproteomic analyses in this study. The phosphorylated islet proteins were found involved in many biological and pathological processes, such as synaptic signalling transduction, calcium channel opening and insulin signalling pathway. Then, the kinases were predicted by motif analysis and further screened and verified by kinase-specific siRNAs in different islet cell lines (αTC1-6, Min6 and TGP52). After functional verification, Ksr2 and Pkacb were considered the key kinases of ACh and NE in insulin secretion, and Cadps, Mlxipl and Pdcd4 were the substrates of these kinases measured by immunofluorescence co-staining. Then, the decreased expression of receptors, kinases and substrates of ACh and NE were found in diabetic mice and the aberrant rhythm in insulin secretion could be improved by combined interventions on key receptors (M3 (pilocarpine) or α2a (guanfacine)) and kinases (Ksr2 or Pkacb). CONCLUSIONS: Abnormal innervation was closely associated with the degree of islet dysfunction in diabetic mice and the aberrant rhythm in insulin secretion could be ameliorated significantly after intervention with key receptors and kinases in the early stage of diabetes mellitus, which may provide a promising therapeutic strategy for diabetes mellitus in the future.

Autonomic control of pancreatic beta cells: What is known on the regulation of insulin secretion and beta-cell proliferation in rodents and humans.

Insulin secretion and pancreatic beta-cell proliferation are tightly regulated by several signals such as hormones, nutrients, and neurotransmitters. However, the autonomic control of beta cells is not fully understood. In this review, we describe mechanisms involved in insulin secretion as well as metabolic and mitogenic actions on its target tissues. Since pancreatic islets are physically connected to the brain by nerves, parasympathetic and sympathetic neurotransmitters can directly potentiate or repress insulin secretion and beta-cell proliferation. Finally, we highlight the role of the autonomic nervous system in metabolic diseases such as diabetes and obesity.

The Transcriptome and Epigenome Reveal Novel Changes in Transcription Regulation During Pancreatic Rat Islet Maturation.

Islet function is critical for normal glucose homeostasis. Unlike adult ß cells, fetal and neonatal islets are more proliferative and have decreased insulin secretion in response to stimuli. However, the underlying mechanisms governing functional maturity of islets have not been completely elucidated. Pancreatic islets comprise different cell types. The microenvironment of islets and interactions between these cell types are critical for ß-cell development and maturation. Thus, the study of intact islets is optimal to identify novel molecular mechanisms controlling islet functional development. Transcriptomes and genome-wide histone landscapes of H3K4me3, H3K27me3, and H3K27Ac from intact islets isolated from 2- and 10-week-old Sprague-Dawley rats were integrated to elucidate genes and pathways modulating islet development, as well as the contribution of epigenetic regulation. A total of 4489 differentially expressed genes were identified; 2289 and 2200 of them were up- and down-regulated in 10-week islets, respectively. Ingenuity Pathway Analysis revealed critical pathways regulating functional maturation of islets, including nutrient sensing, neuronal function, immune function, cell replication, and extracellular matrix. Furthermore, we identified significant changes in enrichment of H3K4me3, H3K27me3, and H3K27Ac marks, which correlated with expression changes of genes critical for islet function. These histone marks were enriched at critical transcription factor-binding motifs, such as Hoxa9, C/EBP-ß, Gata1, Foxo1, E2f1, E2f3, and Mafb. In addition, our chromatin immunoprecipitation sequencing data revealed multiple potential bivalent genes whose poised states changed with maturation. Collectively, our current study identified critical novel pathways for mature islet function and suggested a role for histone modifications in regulating islet development and maturation.

Heterogeneity and neurovascular integration of intraportally transplanted islets revealed by 3-D mouse liver histology.

Intraportal islet transplantation has been clinically applied for treatment of unstable type 1 diabetes. However, in the liver, systematic assessment of the dispersed islet grafts and the graft-hepatic integration remains difficult, even in animal models. This is due to the lack of global and in-depth analyses of the transplanted islets and their microenvironment. Here, we apply three-dimensional (3-D) mouse liver histology to investigate the islet graft microstructure, vasculature, and innervation. Streptozotocin-induced diabetic mice were used in syngeneic intraportal islet transplantation to achieve euglycemia. Optically cleared livers were prepared to enable 3-D morphological and quantitative analyses of the engrafted islets. 3-D image data reveal the clot- and plaque-like islet grafts in the liver: the former are derived from islet emboli and associated with ischemia, whereas the latter (minority) resemble the plaques on the walls of portal vessels (e.g., at the bifurcation) with mild, if any, perigraft tissue damage. Three weeks after transplantation, both types of grafts are revascularized, yet significantly more lymphatics are associated with the plaque- than clot-like grafts. Regarding the islet reinnervation, both types of grafts connect to the periportal nerve plexus and develop peri- and intragraft innervation. Specifically, the sympathetic axons and varicosities contact the α-cells, highlighting the graft-host neural integration. We present the heterogeneity of the intraportally transplanted islets and the graft-host neurovascular integration in mice. Our work provides the technical and morphological foundation for future high-definitional 3-D tissue and cellular analyses of human islet grafts in the liver.NEW & NOTEWORTHY Modern 3-D histology identifies the clot- and plaque-like islet grafts in the mouse liver, which otherwise cannot be distinguished with the standard microtome-based histology. The two types of grafts are similar in blood microvessel density and sympathetic reinnervation. Their differences, however, are their locations, severity of associated liver injury, and access to lymphatic vessels. Our work provides the technical and morphological foundation for future high-definitional 3-D tissue/cellular analyses of human islet grafts in the liver.

Islet sympathetic innervation and islet neuropathology in patients with type 1 diabetes.

Dysregulation of glucagon secretion in type 1 diabetes (T1D) involves hypersecretion during postprandial states, but insufficient secretion during hypoglycemia. The sympathetic nervous system regulates glucagon secretion. To investigate islet sympathetic innervation in T1D, sympathetic tyrosine hydroxylase (TH) axons were analyzed in control non-diabetic organ donors, non-diabetic islet autoantibody-positive individuals (AAb), and age-matched persons with T1D. Islet TH axon numbers and density were significantly decreased in AAb compared to T1D with no significant differences observed in exocrine TH axon volume or lengths between groups. TH axons were in close approximation to islet α-cells in T1D individuals with long-standing diabetes. Islet RNA-sequencing and qRT-PCR analyses identified significant alterations in noradrenalin degradation, α-adrenergic signaling, cardiac ß-adrenergic signaling, catecholamine biosynthesis, and additional neuropathology pathways. The close approximation of TH axons at islet α-cells supports a model for sympathetic efferent neurons directly regulating glucagon secretion. Sympathetic islet innervation and intrinsic adrenergic signaling pathways could be novel targets for improving glucagon secretion in T1D.

Brain control of blood glucose levels: implications for the pathogenesis of type 2 diabetes.

Despite a rapidly growing literature, the role played by the brain in both normal glucose homeostasis and in type 2 diabetes pathogenesis remains poorly understood. In this review, we introduce a framework for understanding the brain's essential role in these processes based on evidence that the brain, like the pancreas, is equipped to sense and respond to changes in the circulating glucose level. Further, we review evidence that glucose sensing by the brain plays a fundamental role in establishing the defended level of blood glucose, and that defects in this control system contribute to type 2 diabetes pathogenesis. We also consider the possibility that the close association between obesity and type 2 diabetes arises from a shared defect in the highly integrated neurocircuitry governing energy homeostasis and glucose homeostasis. Thus, whereas obesity is characterised by an increase in the defended level of the body's fuel stores (e.g. adipose mass), type 2 diabetes is characterised by an increase in the defended level of the body's available fuel (e.g. circulating glucose), with the underlying pathogenesis in each case involving impaired sensing of (or responsiveness to) relevant humoral negative feedback signals. This perspective is strengthened by growing preclinical evidence that in type 2 diabetes the defended level of blood glucose can be restored to normal by therapies that restore the brain's ability to properly sense the circulating glucose level. Graphical abstract.

Loss of sympathetic innervation to islets of Langerhans in canine diabetes and pancreatitis is not associated with insulitis.

Canine diabetes mellitus (DM) affects 0.6% of the canine population and yet, its etiology is poorly understood. Most affected dogs are diagnosed as adults and are insulin-dependent. We compared pan-leukocyte and sympathetic innervation markers in pancreatic islets of adult dogs with spontaneous DM (sDM), spontaneous pancreatitis (sPanc), both (sDMPanc), toxin-induced DM (iDM) and controls. We found evidence of decreased islet sympathetic innervation but no significant infiltration of islets with leukocytes in all disease groups. We show that loss of sympathetic innervation is ongoing in canine DM and does not necessarily precede it. We further found selective loss of islet-associated beta cells in dogs with sDM and sDMPanc, suggesting that collateral damage from inflammation in the exocrine pancreas is not a likely cause of DM in these dogs. The cause of this selective loss of beta cells needs to be further elucidated but overall, our findings are not supportive of an autoimmune process as a cause of sDM in adult dogs. The loss of sympathetic innervation in sPanc in dogs that do not suffer from DM links the disease in the exocrine pancreas to a pathological process in the endocrine pancreas, suggesting pancreatitis might be a potential precursor to DM.

CNS control of the endocrine pancreas.

Increasing evidence suggests that, although pancreatic islets can function autonomously to detect and respond to changes in the circulating glucose level, the brain cooperates with the islet to maintain glycaemic control. Here, we review the role of the central and autonomic nervous systems in the control of the endocrine pancreas, including mechanisms whereby the brain senses circulating blood glucose levels. We also examine whether dysfunction in these systems might contribute to complications of type 1 diabetes and the pathogenesis of type 2 diabetes. Graphical abstract.

Expression of Microtubule Associated Protein Tau in Mouse Pancreatic Islets Is Restricted to Autonomic Nerve Fibers.

BACKGROUND: Evidence from clinical studies and basic research has shown a strong correlation between Alzheimer's disease (AD) and type 2 diabetes. Tau, a neuronal microtubule-associated protein, is hyperphosphorylated and aggregated into neurofibrillary tangles in the AD brain. However, the expression of tau in pancreas is under debate. OBJECTIVE: We determined the expression of tau in mouse pancreas. METHODS: We used western blots, immunoprecipitation, and immunohistochemical staining to analyze pancreatic expression of tau in mice. RESULTS: We found that neither total tau nor phosphorylated tau was detectable in the mouse pancreas by western blots. Immunostaining with pan tau antibodies R134d and Tau-5 revealed bright and dense varicosities in the pancreatic islets and the exocrine pancreas. These varicosities were immunoreactive to synapsin 1, a presynaptic marker which can outline autonomic nerve profiles in pancreas, exhibiting complete colocalization with tau. Importantly, endocrine cells in islets did not exhibit specific immunoreactivity to any of pan tau antibodies tested, nor did the exocrine cells. CONCLUSION: In the mouse pancreas, we found that tau is exclusively expressed in autonomic nerve fibers, but there is no detectable expression in endocrine cells in the islet.

Cholinergic-pathway-weakness-associated pancreatic islet dysfunction: a low-protein-diet imprint effect on weaned rat offspring.

Currently, metabolic disorders are one of the major health problems worldwide, which have been shown to be related to perinatal nutritional insults, and the autonomic nervous system and endocrine pancreas are pivotal targets of the malprogramming of metabolic function. We aimed to assess glucose-insulin homeostasis and the involvement of cholinergic responsiveness (vagus nerve activity and insulinotropic muscarinic response) in pancreatic islet capacity to secrete insulin in weaned rat offspring whose mothers were undernourished in the first 2 weeks of the suckling phase. At delivery, dams were fed a low-protein (4% protein, LP group) or a normal-protein diet (20.5% protein, NP group) during the first 2 weeks of the suckling period. Litter size was adjusted to six pups per mother, and rats were weaned at 21 days old. Weaned LP rats presented a lean phenotype (P < 0.01); hypoglycaemia, hypoinsulinaemia and hypoleptinaemia (P < 0.05); and normal corticosteronaemia (P > 0.05). In addition, milk insulin levels in mothers of the LP rats were twofold higher than those of mothers of the NP rats (P < 0.001). Regarding glucose-insulin homeostasis, weaned LP rats were glucose-intolerant (P < 0.01) and displayed impaired pancreatic islet insulinotropic function (P < 0.05). The M3 subtype of the muscarinic acetylcholine receptor (M3mAChR) from weaned LP rats was less responsive, and the superior vagus nerve electrical activity was reduced by 30% (P < 0.01). A low-protein diet in the suckling period malprogrammes the vagus nerve to low tonus and impairs muscarinic response in the pancreatic ß-cells of weaned rats, which are imprinted to secrete inadequate insulin amounts from an early age.

Human pancreatic afferent and efferent nerves: mapping and 3-D illustration of exocrine, endocrine, and adipose innervation.

The pancreas consists of both the exocrine (acini and ducts) and endocrine (islets) compartments to participate in and regulate the body's digestive and metabolic activities. These activities are subjected to neural modulation, but characterization of the human pancreatic afferent and efferent nerves remains difficult because of the lack of three-dimensional (3-D) image data. Here we prepare transparent human donor pancreases for 3-D histology to reveal the pancreatic microstructure, vasculature, and innervation in a global and integrated fashion. The pancreatic neural network consists of the substance P (SP)-positive sensory (afferent) nerves, the vesicular acetylcholine transporter (VAChT)-positive parasympathetic (efferent) nerves, and the tyrosine hydroxylase (TH)-positive sympathetic (efferent) nerves. The SP+ afferent nerves were found residing along the basal domain of the interlobular ducts. The VAChT+ and TH+ efferent nerves were identified at the peri-acinar and perivascular spaces, which follow the blood vessels to the islets. In the intrapancreatic ganglia, the SP+ (scattered minority, ~7%) and VAChT+ neurons co-localize, suggesting a local afferent-efferent interaction. Compared with the mouse pancreas, the human pancreas differs in 1) the lack of SP+ afferent nerves in the islet, 2) the lower ganglionic density, and 3) the obvious presence of VAChT+ and TH+ nerves around the intralobular adipocytes. The latter implicates the neural influence on the pancreatic steatosis. Overall, our 3-D image data reveal the human pancreatic afferent and efferent innervation patterns and provide the anatomical foundation for future high-definition analyses of neural remodeling in human pancreatic diseases.NEW & NOTEWORTHY Modern three-dimensional (3-D) histology with multiplex optical signals identifies the afferent and efferent innervation patterns of human pancreas, which otherwise cannot be defined with standard histology. Our 3-D image data reveal the unexpected association of sensory and parasympathetic nerves/neurons in the intrapancreatic ganglia and identify the sympathetic and parasympathetic nerve contacts with the infiltrated adipocytes. The multiplex approach offers a new way to characterize the human pancreas in remodeling (e.g., fatty infiltration and duct lesion progression).

A new mode of pancreatic islet innervation revealed by live imaging in zebrafish.

Pancreatic islets are innervated by autonomic and sensory nerves that influence their function. Analyzing the innervation process should provide insight into the nerve-endocrine interactions and their roles in development and disease. Here, using in vivo time-lapse imaging and genetic analyses in zebrafish, we determined the events leading to islet innervation. Comparable neural density in the absence of vasculature indicates that it is dispensable for early pancreatic innervation. Neural crest cells are in close contact with endocrine cells early in development. We find these cells give rise to neurons that extend axons toward the islet as they surprisingly migrate away. Specific ablation of these neurons partly prevents other neurons from migrating away from the islet resulting in diminished innervation. Thus, our studies establish the zebrafish as a model to interrogate mechanisms of organ innervation, and reveal a novel mode of innervation whereby neurons establish connections with their targets before migrating away.

Autonomic nervous system and pancreatic islet blood flow.

Vascularization and innervation of the islet of Langerhans are highly interconnected and are critical for intercellular and intertissular communication. They are both involved in the control of islet blood flow which has been shown to have an important role in the control of endocine secretion. Both parameters are disturbed during the course of metabolic pathologies and particularly diabetes. A better understanding of these mechanisms has and will greatly benefit from the rapidly-emerging technologies particularly in vivo imaging enabling to study both anatomy and functions of the islet.

Signals in the pancreatic islet microenvironment influence ß-cell proliferation.

The progressive loss of pancreatic ß-cell mass that occurs in both type 1 and type 2 diabetes is a primary factor driving efforts to identify strategies for effectively increasing, enhancing or restoring ß-cell mass. While factors that seem to influence ß-cell proliferation in specific contexts have been described, reliable stimulation of human ß-cell proliferation has remained a challenge. Importantly, ß-cells exist in the context of a complex, integrated pancreatic islet microenvironment where they interact with other endocrine cells, vascular endothelial cells, extracellular matrix, neuronal projections and islet macrophages. This review highlights different components of the pancreatic microenvironment, and reviews what is known about how signaling that occurs between ß-cells and these other components influences ß-cell proliferation. Future efforts to further define the role of the pancreatic islet microenvironment on ß-cell proliferation may lead to the development of successful approaches to increase or restore ß-cell mass in diabetes.

The Different Faces of the Pancreatic Islet.

Type 1 diabetes (T1D) patients who receive pancreatic islet transplant experience significant improvement in their quality-of-life. This comes primarily through improved control of blood sugar levels, restored awareness of hypoglycemia, and prevention of serious and potentially life-threatening diabetes-associated complications, such as kidney failure, heart and vascular disease, stroke, nerve damage, and blindness. Therefore, beta cell replacement through transplantation of isolated islets is an important option in the treatment of T1D. However, lasting success of this promising therapy depends on durable survival and efficacy of the transplanted islets, which are directly influenced by the islet isolation procedures. Thus, isolating pancreatic islets with consistent and reliable quality is critical in the clinical application of islet transplantation.Quality of isolated islets is important in pre-clinical studies as well, as efforts to advance and improve clinical outcomes of islet transplant therapy have relied heavily on animal models ranging from rodents, to pigs, to nonhuman primates. As a result, pancreatic islets have been isolated from these and other species and used in a variety of in vitro or in vivo applications for this and other research purposes. Protocols for islet isolation have been somewhat similar across species, especially, in mammals. However, given the increasing evidence about the distinct structural and functional features of human and mouse islets, using similar methods of islet isolation may contribute to inconsistencies in the islet quality, immunogenicity, and experimental outcomes. This may also contribute to the discrepancies commonly observed between pre-clinical findings and clinical outcomes. Therefore, it is prudent to consider the particular features of pancreatic islets from different species when optimizing islet isolation protocols.In this chapter, we explore the structural and functional features of pancreatic islets from mice, pigs, nonhuman primates, and humans because of their prevalent use in nonclinical, preclinical, and clinical applications.

The Brain-to-Pancreatic Islet Neuronal Map Reveals Differential Glucose Regulation From Distinct Hypothalamic Regions.

The brain influences glucose homeostasis, partly by supplemental control over insulin and glucagon secretion. Without this central regulation, diabetes and its complications can ensue. Yet, the neuronal network linking to pancreatic islets has never been fully mapped. Here, we refine this map using pseudorabies virus (PRV) retrograde tracing, indicating that the pancreatic islets are innervated by efferent circuits that emanate from the hypothalamus. We found that the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMN), and lateral hypothalamic area (LHA) significantly overlap PRV and the physiological glucose-sensing enzyme glucokinase. Then, experimentally lowering glucose sensing, specifically in the ARC, resulted in glucose intolerance due to deficient insulin secretion and no significant effect in the VMN, but in the LHA it resulted in a lowering of the glucose threshold that improved glucose tolerance and/or improved insulin sensitivity, with an exaggerated counter-regulatory response for glucagon secretion. No significant effect on insulin sensitivity or metabolic homeostasis was noted. Thus, these data reveal novel direct neuronal effects on pancreatic islets and also render a functional validation of the brain-to-islet neuronal map. They also demonstrate that distinct regions of the hypothalamus differentially control insulin and glucagon secretion, potentially in partnership to help maintain glucose homeostasis and guard against hypoglycemia.

Leptin Controls Parasympathetic Wiring of the Pancreas during Embryonic Life.

The autonomic nervous system plays a critical role in glucose metabolism through both its sympathetic and parasympathetic branches, but the mechanisms that underlie the development of the autonomic innervation of the pancreas remain poorly understood. Here, we report that cholinergic innervation of pancreatic islets develops during mid-gestation under the influence of leptin. Leptin-deficient mice display a greater cholinergic innervation of pancreatic islets beginning in embryonic life, and this increase persists into adulthood. Remarkably, a single intracerebroventricular injection of leptin in embryos caused a permanent reduction in parasympathetic innervation of pancreatic ß cells and long-term impairments in glucose homeostasis. These developmental effects of leptin involve a direct inhibitory effect on the outgrowth of preganglionic axons from the hindbrain. These studies reveal an unanticipated regulatory role of leptin on the parasympathetic nervous system during embryonic development and may have important implications for our understanding of the early mechanisms that contribute to diabetes.

3-D imaging of islets in obesity: formation of the islet-duct complex and neurovascular remodeling in young hyperphagic mice.

BACKGROUND: Obesity and insulin resistance lead to islet hyperplasia. However, how the islet remodeling influences the pancreatic environment and the associated neurovascular networks is largely unknown. The lack of information is primarily due to the difficulty of global visualization of the hyperplasic islet (>200 µm) and the neurovascular environment with high definition. METHODS: We modulated the pancreatic optical property to achieve 3-dimensional (3-D) whole-islet histology and to integrate transmitted light microscopy (which provides the ground-truth tissue information) with confocal fluorescence imaging. The new optical and imaging conditions were used to globally examine the hyperplastic islets of the young (2 months) obese db/db and ob/ob mice, which otherwise cannot be easily portrayed by the standard microtome-based histology. The voxel-based islet micrographs were digitally processed for stereo projection and qualitative and quantitative analyses of the islet tissue networks. RESULTS: Paired staining and imaging of the pancreatic islets, ducts and neurovascular networks reveal the unexpected formation of the 'neuro-insular-ductal complex' in the young obese mice. The complex consists of the peri- and/or intra-islet ducts and prominent peri-ductal sympathetic nerves; the latter contributes to a marked increase in islet sympathetic innervation. In vascular characterization, we identify a decreased perivascular density of the ob/ob islet pericytes, which adapt to ensheathing the dilated microvessels with hypertrophic processes. CONCLUSIONS: Modulation of pancreatic optical property enables 3-D panoramic examination of islets in the young hyperphagic mice to reveal the formation of the islet-duct complex and neurovascular remodeling. On the basis of the morphological proximity of the remodeled tissue networks, we propose a reactive islet microenvironment consisting of the endocrine cells, ductal epithelium and neurovascular tissues in response to the metabolic challenge that is experienced early in life.

3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration.

The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

Short-term diabetic hyperglycemia suppresses celiac ganglia neurotransmission, thereby impairing sympathetically mediated glucagon responses.

Short-term hyperglycemia suppresses superior cervical ganglia neurotransmission. If this ganglionic dysfunction also occurs in the islet sympathetic pathway, sympathetically mediated glucagon responses could be impaired. Our objectives were 1) to test for a suppressive effect of 7 days of streptozotocin (STZ) diabetes on celiac ganglia (CG) activation and on neurotransmitter and glucagon responses to preganglionic nerve stimulation, 2) to isolate the defect in the islet sympathetic pathway to the CG itself, and 3) to test for a protective effect of the WLD(S) mutation. We injected saline or nicotine in nondiabetic and STZ-diabetic rats and measured fos mRNA levels in whole CG. We electrically stimulated the preganglionic or postganglionic nerve trunk of the CG in nondiabetic and STZ-diabetic rats and measured portal venous norepinephrine and glucagon responses. We repeated the nicotine and preganglionic nerve stimulation studies in nondiabetic and STZ-diabetic WLD(S) rats. In STZ-diabetic rats, the CG fos response to nicotine was suppressed, and the norepinephrine and glucagon responses to preganglionic nerve stimulation were impaired. In contrast, the norepinephrine and glucagon responses to postganglionic nerve stimulation were normal. The CG fos response to nicotine, and the norepinephrine and glucagon responses to preganglionic nerve stimulation, were normal in STZ-diabetic WLD(S) rats. In conclusion, short-term hyperglycemia's suppressive effect on nicotinic acetylcholine receptors of the CG impairs sympathetically mediated glucagon responses. WLD(S) rats are protected from this dysfunction. The implication is that this CG dysfunction may contribute to the impaired glucagon response to insulin-induced hypoglycemia seen early in type 1 diabetes.