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GC-globulin (GC), or vitamin D-binding protein, is a multifunctional protein involved in the transport of circulating vitamin 25(OH)D and fatty acids, as well as actin scavenging. In the pancreatic islets, the gene encoding GC, GC/Gc, is highly localized to glucagon-secreting α-cells. Despite this, the role of GC in α-cell function is poorly understood. We previously showed that GC is essential for α-cell morphology, electrical activity, and glucagon secretion. We now show that loss of GC exacerbates α-cell failure during metabolic stress. High-fat diet-fed GC-/- mice have basal hyperglucagonemia, which is associated with decreased α-cell size, impaired glucagon secretion and Ca2+ fluxes, and changes in glucose-dependent F-actin remodelling. Impairments in glucagon secretion can be rescued using exogenous GC to replenish α-cell GC levels, increase glucagon granule area, and restore the F-actin cytoskeleton. Lastly, GC levels decrease in α-cells of donors with type 2 diabetes, which is associated with changes in α-cell mass, morphology, and glucagon expression. Together, these data demonstrate an important role for GC in α-cell adaptation to metabolic stress.

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The importance of pancreatic endocrine cell activity modulation by autonomic innervation has been debated. To investigate this question, we established an in vivo imaging model that also allows chronic and acute neuromodulation with genetic and optogenetic tools. Using the GCaMP6s biosensor together with endocrine cell fluorescent reporters, we imaged calcium dynamics simultaneously in multiple pancreatic islet cell types in live animals in control states and upon changes in innervation. We find that by 4 days post fertilization in zebrafish, a stage when islet architecture is reminiscent of that in adult rodents, prominent activity coupling between beta cells is present in basal glucose conditions. Furthermore, we show that both chronic and acute loss of nerve activity result in diminished beta-beta and alpha-beta activity coupling. Pancreatic nerves are in contact with all islet cell types, but predominantly with beta and delta cells. Surprisingly, a subset of delta cells with detectable peri-islet neural activity coupling had significantly higher homotypic coupling with other delta cells suggesting that some delta cells receive innervation that coordinates their output. Overall, these data show that innervation plays a vital role in the maintenance of homotypic and heterotypic cellular connectivity in pancreatic islets, a process critical for islet function.

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In diabetes, glucagon secretion from pancreatic α cells is dysregulated. The underlying mechanisms, and whether dysfunction occurs uniformly among cells, remain unclear. We examined α cells from human donors and mice using electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses α cell exocytosis by reducing P/Q-type Ca2+ channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat feeding of mice, α cells shift toward a "ß cell-like" electrophysiological profile in concert with indications of impaired identity. In human α cells we identified links between cell membrane properties and cell surface signaling receptors, mitochondrial respiratory chain complex assembly, and cell maturation. Cell-type classification using machine learning of electrophysiology data demonstrated a heterogenous loss of "electrophysiologic identity" in α cells from donors with type 2 diabetes. Indeed, a subset of α cells with impaired exocytosis is defined by an enrichment in progenitor and lineage markers and upregulation of an immature transcriptomic phenotype, suggesting important links between α cell maturation state and dysfunction.

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The pancreatic islets contain beta-cells and alpha-cells, which are responsible for secreting two principal gluco-regulatory hormones; insulin and glucagon, respectively. However, they also contain delta-cells, a relatively sparse cell type that secretes somatostatin (SST). These cells have a complex morphology allowing them to establish an extensive communication network throughout the islet, despite their scarcity. Delta-cells are electrically excitable cells, and SST secretion is released in a glucose- and KATP-dependent manner. SST hyperpolarises the alpha-cell membrane and suppresses exocytosis. In this way, islet SST potently inhibits glucagon release. Recent studies investigating the activity of delta-cells have revealed they are electrically coupled to beta-cells via gap junctions, suggesting the delta-cell is more than just a paracrine inhibitor. In this Review, we summarize delta-cell morphology, function, and the role of SST signalling for regulating islet hormonal output. A distinguishing feature of this Review is that we attempt to use the discovery of this gap junction pathway, together with what is already known about delta-cells, to reframe the role of these cells in both health and disease. In particular, we argue that the discovery of gap junction communication between delta-cells and beta-cells provides new insights into the contribution of delta-cells to the islet hormonal defects observed in both type 1 and type 2 diabetes. This reappraisal of the delta-cell is important as it may offer novel insights into how the physiology of this cell can be utilised to restore islet function in diabetes.

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Insulin-induced hypoglycemia is a major treatment barrier in type-1 diabetes (T1D). Accordingly, it is important that we understand the mechanisms regulating the circulating levels of glucagon. Varying glucose over the range of concentrations that occur physiologically between the fed and fuel-deprived states (8 to 4 mM) has no significant effect on glucagon secretion in the perfused mouse pancreas or in isolated mouse islets (in vitro), and yet associates with dramatic increases in plasma glucagon. The identity of the systemic factor(s) that elevates circulating glucagon remains unknown. Here, we show that arginine-vasopressin (AVP), secreted from the posterior pituitary, stimulates glucagon secretion. Alpha-cells express high levels of the vasopressin 1b receptor (V1bR) gene (Avpr1b). Activation of AVP neurons in vivo increased circulating copeptin (the C-terminal segment of the AVP precursor peptide) and increased blood glucose; effects blocked by pharmacological antagonism of either the glucagon receptor or V1bR. AVP also mediates the stimulatory effects of hypoglycemia produced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We show that the A1/C1 neurons of the medulla oblongata drive AVP neuron activation in response to insulin-induced hypoglycemia. AVP injection increased cytoplasmic Ca2+ in alpha-cells (implanted into the anterior chamber of the eye) and glucagon release. Hypoglycemia also increases circulating levels of AVP/copeptin in humans and this hormone stimulates glucagon secretion from human islets. In patients with T1D, hypoglycemia failed to increase both copeptin and glucagon. These findings suggest that AVP is a physiological systemic regulator of glucagon secretion and that this mechanism becomes impaired in T1D.

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Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in ß cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.

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OBJECTIVES: Elevated plasma glucagon is an early symptom of diabetes, occurring in subjects with impaired glucose regulation. Here, we explored alpha-cell function in female mice fed a high-fat diet (HFD). METHODS: Female mice expressing the Ca2+ indicator GCaMP3 specifically in alpha-cells were fed a high-fat or control (CTL) diet. We then conducted in vivo phenotyping of these mice, as well as experiments on isolated (ex vivo) islets and in the in situ perfused pancreas. RESULTS: In HFD-fed mice, fed plasma glucagon levels were increased and glucagon secretion from isolated islets and in the perfused mouse pancreas was also elevated. In mice fed a CTL diet, increasing glucose reduced intracellular Ca2+ ([Ca2+]i) oscillation frequency and amplitude. This effect was also observed in HFD mice; however, both the frequency and amplitude of the [Ca2+]i oscillations were higher than those in CTL alpha-cells. Given that alpha-cells are under strong paracrine control from neighbouring somatostatin-secreting delta-cells, we hypothesised that this elevation of alpha-cell output was due to a lack of somatostatin (SST) secretion. Indeed, SST secretion in isolated islets from HFD-fed mice was reduced but exogenous SST also failed to suppress glucagon secretion and [Ca2+]i activity from HFD alpha-cells, in contrast to observations in CTL mice. CONCLUSIONS: These findings suggest that reduced delta-cell function, combined with intrinsic changes in alpha-cells including sensitivity to somatostatin, accounts for the hyperglucagonaemia in mice fed a HFD.

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OBJECTIVE: Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the ß-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates ß-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the ß-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation. METHODS: We used isolated islets from mice deleted selectively in the ß-cell for Stard10 (ßStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein. RESULTS: ßStard10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3' position. Lipidomic analysis of ßStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in ßStard10KO islets. CONCLUSION: Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing.

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Pancreatic islets are complex micro-organs consisting of at least three different cell types: glucagon-secreting α-, insulin-producing ß- and somatostatin-releasing δ-cells1. Somatostatin is a powerful paracrine inhibitor of insulin and glucagon secretion2. In diabetes, increased somatostatinergic signalling leads to defective counter-regulatory glucagon secretion3. This increases the risk of severe hypoglycaemia, a dangerous complication of insulin therapy4. The regulation of somatostatin secretion involves both intrinsic and paracrine mechanisms5 but their relative contributions and whether they interact remains unclear. Here we show that dapagliflozin-sensitive glucose- and insulin-dependent sodium uptake stimulates somatostatin secretion by elevating the cytoplasmic Na+ concentration ([Na+]i) and promoting intracellular Ca2+-induced Ca2+ release (CICR). This mechanism also becomes activated when [Na+]i is elevated following the inhibition of the plasmalemmal Na+-K+ pump by reductions of the extracellular K+ concentration emulating those produced by exogenous insulin in vivo 6. Islets from some donors with type-2 diabetes hypersecrete somatostatin, leading to suppression of glucagon secretion that can be alleviated by a somatostatin receptor antagonist. Our data highlight the role of Na+ as an intracellular second messenger, illustrate the significance of the intraislet paracrine network and provide a mechanistic framework for pharmacological correction of the hormone secretion defects associated with diabetes that selectively target the δ-cells.

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Hypoglycaemia (low plasma glucose) is a serious and potentially fatal complication of insulin-treated diabetes. In healthy individuals, hypoglycaemia triggers glucagon secretion, which restores normal plasma glucose levels by stimulation of hepatic glucose production. This counterregulatory mechanism is impaired in diabetes. Here we show in mice that therapeutic concentrations of insulin inhibit glucagon secretion by an indirect (paracrine) mechanism mediated by stimulation of intra-islet somatostatin release. Insulin's capacity to inhibit glucagon secretion is lost following genetic ablation of insulin receptors in the somatostatin-secreting δ-cells, when insulin-induced somatostatin secretion is suppressed by dapagliflozin (an inhibitor of sodium-glucose co-tranporter-2; SGLT2) or when the action of secreted somatostatin is prevented by somatostatin receptor (SSTR) antagonists. Administration of these compounds in vivo antagonises insulin's hypoglycaemic effect. We extend these data to isolated human islets. We propose that SSTR or SGLT2 antagonists should be considered as adjuncts to insulin in diabetes therapy.

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Pancreatic ß-cells form highly connected networks within isolated islets. Whether this behaviour pertains to the situation in vivo, after innervation and during continuous perfusion with blood, is unclear. In the present study, we used the recombinant Ca2+ sensor GCaMP6 to assess glucose-regulated connectivity in living zebrafish Danio rerio, and in murine or human islets transplanted into the anterior eye chamber. In each setting, Ca2+ waves emanated from temporally defined leader ß-cells, and three-dimensional connectivity across the islet increased with glucose stimulation. Photoablation of zebrafish leader cells disrupted pan-islet signalling, identifying these as likely pacemakers. Correspondingly, in engrafted mouse islets, connectivity was sustained during prolonged glucose exposure, and super-connected 'hub' cells were identified. Granger causality analysis revealed a controlling role for temporally defined leaders, and transcriptomic analyses revealed a discrete hub cell fingerprint. We thus define a population of regulatory ß-cells within coordinated islet networks in vivo. This population may drive Ca2+ dynamics and pulsatile insulin secretion.

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Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type 2 diabetes mellitus (T2DM). The incretin hormone glucagon-like peptide-1 (GLP-1) is released from the gut and is used in T2DM therapy. Uniquely, it both stimulates insulin and inhibits glucagon secretion and thereby lowers plasma glucose levels. In this study, we have investigated the action of GLP-1 on glucagon release from human pancreatic islets. Immunocytochemistry revealed that only <0.5% of the α-cells possess detectable GLP-1R immunoreactivity. Despite this, GLP-1 inhibited glucagon secretion by 50-70%. This was due to a direct effect on α-cells, rather than paracrine signaling, because the inhibition was not reversed by the insulin receptor antagonist S961 or the somatostatin receptor-2 antagonist CYN154806. The inhibitory effect of GLP-1 on glucagon secretion was prevented by the PKA-inhibitor Rp-cAMPS and mimicked by the adenylate cyclase activator forskolin. Electrophysiological measurements revealed that GLP-1 decreased action potential height and depolarized interspike membrane potential. Mathematical modeling suggests both effects could result from inhibition of P/Q-type Ca2+ channels. In agreement with this, GLP-1 and ω-agatoxin (a blocker of P/Q-type channels) inhibited glucagon secretion in islets depolarized by 70 mmol/L [K+ ]o , and these effects were not additive. Intracellular application of cAMP inhibited depolarization-evoked exocytosis in individual α-cells by a PKA-dependent (Rp-cAMPS-sensitive) mechanism. We propose that inhibition of glucagon secretion by GLP-1 involves activation of the few GLP-1 receptors present in the α-cell membrane. The resulting small elevation of cAMP leads to PKA-dependent inhibition of P/Q-type Ca2+ channels and suppression of glucagon exocytosis.

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Islet ß-cells are responsible for secreting all circulating insulin in response to rising plasma glucose concentrations. These cells are a phenotypically diverse population that express great functional heterogeneity. In mice, certain ß-cells (termed 'hubs') have been shown to be crucial for dictating the islet response to high glucose, with inhibition of these hub cells abolishing the coordinated Ca2+ oscillations necessary for driving insulin secretion. These ß-cell hubs were found to be highly metabolic and susceptible to pro-inflammatory and glucolipotoxic insults. In this study, we explored the importance of hub cells in human by constructing mathematical models of Ca2+ activity in human islets. Our simulations revealed that hubs dictate the coordinated Ca2+ response in both mouse and human islets; silencing a small proportion of hubs abolished whole-islet Ca2+ activity. We also observed that if hubs are assumed to be preferentially gap junction coupled, then the simulations better adhere to the available experimental data. Our simulations of 16 size-matched mouse and human islet architectures revealed that there are species differences in the role of hubs; Ca2+ activity in human islets was more vulnerable to hub inhibition than mouse islets. These simulation results not only substantiate the existence of ß-cell hubs, but also suggest that hubs may be favorably coupled in the electrical and metabolic network of the islet, and that targeted destruction of these cells would greatly impair human islet function.

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Glucagon, the principal hyperglycemic hormone, is secreted from pancreatic islet α cells as part of the counter-regulatory response to hypoglycemia. Hence, secretory output from α cells is under high demand in conditions of low glucose supply. Many tissues oxidize fat as an alternate energy substrate. Here, we show that glucagon secretion in low glucose conditions is maintained by fatty acid metabolism in both mouse and human islets, and that inhibiting this metabolic pathway profoundly decreases glucagon output by depolarizing α cell membrane potential and decreasing action potential amplitude. We demonstrate, by using experimental and computational approaches, that this is not mediated by the KATP channel, but instead due to reduced operation of the Na+-K+ pump. These data suggest that counter-regulatory secretion of glucagon is driven by fatty acid metabolism, and that the Na+-K+ pump is an important ATP-dependent regulator of α cell function.

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The α-, ß- and δ-cells of the pancreatic islet exhibit different electrophysiological features. We used a large dataset of whole-cell patch-clamp recordings from cells in intact mouse islets (N = 288 recordings) to investigate whether it is possible to reliably identify cell type (α, ß or δ) based on their electrophysiological characteristics. We quantified 15 electrophysiological variables in each recorded cell. Individually, none of the variables could reliably distinguish the cell types. We therefore constructed a logistic regression model that included all quantified variables, to determine whether they could together identify cell type. The model identified cell type with 94% accuracy. This model was applied to a dataset of cells recorded from hyperglycaemic ßV59M mice; it correctly identified cell type in all cells and was able to distinguish cells that co-expressed insulin and glucagon. Based on this revised functional identification, we were able to improve conductance-based models of the electrical activity in α-cells and generate a model of δ-cell electrical activity. These new models could faithfully emulate α- and δ-cell electrical activity recorded experimentally.

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Animal and human data indicate pathological afferent signaling emanating from the carotid body that drives sympathetically mediated elevations in blood pressure in conditions of hypertension. This first-in-man, proof-of-principle study tested the safety and feasibility of unilateral carotid body resection in 15 patients with drug-resistant hypertension. The procedure proved to be safe and feasible. Overall, no change in blood pressure was found. However, 8 patients showed significant reductions in ambulatory blood pressure coinciding with decreases in sympathetic activity. The carotid body may be a novel target for treating an identifiable subpopulation of humans with hypertension.

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Type 2 diabetes involves a ménage à trois of impaired glucose regulation of pancreatic hormone release: in addition to impaired glucose-induced insulin secretion, the release of the hyperglycaemic hormone glucagon becomes dysregulated; these last-mentioned defects exacerbate the metabolic consequences of hypoinsulinaemia and are compounded further by hypersecretion of somatostatin (which inhibits both insulin and glucagon secretion). Glucagon secretion has been proposed to be regulated by either intrinsic or paracrine mechanisms, but their relative significance and the conditions under which they operate are debated. Importantly, the paracrine and intrinsic modes of regulation are not mutually exclusive; they could operate in parallel to control glucagon secretion. Here we have applied mathematical modelling of α-cell electrical activity as a novel means of dissecting the processes that underlie metabolic regulation of glucagon secretion. Our analyses indicate that basal hypersecretion of somatostatin and/or increased activity of somatostatin receptors may explain the loss of adequate counter-regulation under hypoglycaemic conditions, as well as the physiologically inappropriate stimulation of glucagon secretion during hyperglycaemia seen in diabetic patients. We therefore advocate studying the interaction of the paracrine and intrinsic mechanisms; unifying these processes may give a more complete picture of the regulation of glucagon secretion from α-cells than studying the individual parts.

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KEY POINTS: Sympathetic activity exhibits respiratory modulation that is amplified in hypertensive rats. Respiratory modulated sympathetic activity produces greater changes in vascular resistance than tonic stimulation of the same stimulus magnitude in normotensive but not hypertensive rats. Mathematical modelling demonstrates that respiratory modulated sympathetic activity may fail to produce greater vascular resistance changes in hypertensive rats because the system is saturated as a consequence of a dysfunctional noradrenaline reuptake mechanism. Respiratory modulated sympathetic activity is an efficient mechanism to raise vascular resistance promptly, corroborating its involvement in the ontogenesis of hypertension. ABSTRACT: Sympathetic nerve activity (SNA) exhibits respiratory modulation. This component of SNA is important - being recruited under cardiorespiratory reflex conditions and elevated in the spontaneously hypertensive (SH) rat - and yet the exact influence of this modulation on vascular tone is not understood, even in normotensive conditions. We constructed a mathematical model of the sympathetic innervation of an arteriole, and used it to test the hypothesis that respiratory modulation of SNA preferentially increases vasoconstriction compared to a frequency-matched tonic pattern. Simulations supported the hypothesis, where respiratory modulated increases in vasoconstriction were mediated by a noradrenergic mechanism. These predictions were tested in vivo in adult Wistar rats. Stimulation of the sympathetic chain (L3) with respiratory modulated bursting patterns, revealed that bursting increases vascular resistance (VR) more than tonic stimulation (57.8 ± 3.3% vs. 44.8 ± 4.2%; P < 0.001; n = 8). The onset of the VR response was also quicker for bursting stimulation (rise time constant = 1.98 ± 0.09 s vs. 2.35 ± 0.20 s; P < 0.01). In adult SH rats (n = 8), the VR response to bursting (44.6 ± 3.9%) was not different to tonic (37.4 ± 3.5%; P = 0.57). Using both mathematical modelling and in vivo techniques, we have shown that VR depends critically on respiratory modulation and revealed that this pattern dependency in Wistar rats is due to a noradrenergic mechanism. This respiratory component may therefore contribute to the ontogenesis of hypertension in the pre-hypertensive SH rat - raising VR and driving vascular remodelling. Why adult SH rats do not exhibit a pattern-dependent response is not known, but further modelling revealed that this may be due to dysfunctional noradrenaline reuptake.

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This paper explores the influence of burst properties of the sympathetic nervous system on arterial contractility. Specifically, a mathematical model is constructed of the pathway from action potential generation in a sympathetic postganglionic neurone to contraction of an arterial smooth muscle cell. The differential equation model is a synthesis of models of the individual physiological processes, and is shown to be consistent with physiological data. The model is found to be unresponsive to tonic (regular) stimulation at typical frequencies recorded in sympathetic efferents. However, when stimulated at the same average frequency, but with repetitive respiratory-modulated burst patterns, it produces marked contractions. Moreover, the contractile force produced is found to be highly dependent on the number of spikes in each burst. In particular, when the model is driven by preganglionic spike trains recorded from wild-type and spontaneously hypertensive rats (which have increased spiking during each burst) the contractile force was found to be 10-fold greater in the hypertensive case. An explanation is provided in terms of the summative increased release of noradrenaline. Furthermore, the results suggest the marked effect that hypertensive spike trains had on smooth muscle cell tone can provide a significant contribution to the pathology of hypertension.

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Hypertension is associated with pathologically increased sympathetic drive to the vasculature. This has been attributed to increased excitatory drive to sympathetic preganglionic neurons (SPN) from brainstem cardiovascular control centers. However, there is also evidence supporting increased intrinsic excitability of SPN. To test this hypothesis, we made whole cell recordings of muscle vasoconstrictor-like (MVClike) SPN in the working-heart brainstem preparation of spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats. The MVClike SPN have a higher spontaneous firing frequency in the SH rat (3.85 ± 0.4 vs. 2.44 ± 0.4 Hz in WKY; P = 0.011) with greater respiratory modulation of their activity. The action potentials of SH SPN had smaller, shorter afterhyperpolarizations (AHPs) and showed diminished transient rectification indicating suppression of an A-type potassium conductance (IA). We developed mathematical models of the SPN to establish if changes in their intrinsic properties in SH rats could account for their altered firing. Reduction of the maximal conductance density of IA by 15-30% changed the excitability and output of the model from the WKY to a SH profile, with increased firing frequency, amplified respiratory modulation, and smaller AHPs. This change in output is predominantly a consequence of altered synaptic integration. Consistent with these in silico predictions, we found that intrathecal 4-aminopyridine (4-AP) increased sympathetic nerve activity, elevated perfusion pressure, and augmented Traube-Hering waves. Our findings indicate that IA acts as a powerful filter on incoming synaptic drive to SPN and that its diminution in the SH rat is potentially sufficient to account for the increased sympathetic output underlying hypertension.

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