Cannabinoid receptor type 1 activation causes a water diuresis by inducing an acute central diabetes insipidus in mice.

Cannabis and synthetic cannabinoid consumption are increasing worldwide. Cannabis contains numerous phytocannabinoids that act on the G protein-coupled cannabinoid receptor type 1 (CB1R) and cannabinoid receptor type 2 expressed throughout the body, including the kidney. Essentially every organ, including the kidney, produces endocannabinoids, which are endogenous ligands to these receptors. Cannabinoids acutely increase urine output in rodents and humans, thus potentially influencing total body water and electrolyte homeostasis. As the kidney collecting duct (CD) regulates total body water, acid/base, and electrolyte balance through specific functions of principal cells (PCs) and intercalated cells (ICs), we examined the cell-specific immunolocalization of CB1R in the mouse CD. Antibodies against either the C-terminus or N-terminus of CB1R consistently labeled aquaporin 2 (AQP2)-negative cells in the cortical and medullary CD and thus presumably ICs. Given the well-established role of ICs in urinary acidification, we used a clearance approach in mice that were acid loaded with 280 mM NH4Cl for 7 days and nonacid-loaded mice treated with the cannabinoid receptor agonist WIN55,212-2 (WIN) or a vehicle control. Although WIN had no effect on urinary acidification, these WIN-treated mice had less apical + subapical AQP2 expression in PCs compared with controls and developed acute diabetes insipidus associated with the excretion of large volumes of dilute urine. Mice maximally concentrated their urine when WIN and 1-desamino-8-d-arginine vasopressin [desmopressin (DDAVP)] were coadministered, consistent with central rather than nephrogenic diabetes insipidus. Although ICs express CB1R, the physiological role of CB1R in this cell type remains to be determined.NEW & NOTEWORTHY The CB1R agonist WIN55,212-2 induces central diabetes insipidus in mice. This research integrates existing knowledge regarding the diuretic effects of cannabinoids and the influence of CB1R on vasopressin secretion while adding new mechanistic insights about total body water homeostasis. Our findings provide a deeper understanding about the potential clinical impact of cannabinoids on human physiology and may help identify targets for novel therapeutics to treat water and electrolyte disorders such as hyponatremia and volume overload.

Diabetes Insipidus: New Concepts for Diagnosis.

Diabetes insipidus (DI), be it from central or from nephrogenic origin, has to be differentiated from primary polydipsia. This differentiation is crucial since wrong treatment can have dangerous consequences. For decades, the "gold standard" for differential diagnosis has been the standard water deprivation test. However, this test has several limitations leading to an overall limited diagnostic accuracy. In addition, the test has a long duration of 17 h and is cumbersome for patients. Also clinical signs and symptoms and MRI characteristics overlap between patients with DI and primary polydipsia. Direct measurement of arginine vasopressin (AVP) upon osmotic stimulation was first shown to overcome these limitations, but failed to enter clinical practice mainly due to technical limitations of the AVP assay. Copeptin is secreted in equimolar ratio to AVP, mirroring AVP concentrations in the circulation. We have shown that copeptin, without prior fluid deprivation, identifies patients with nephrogenic DI. For the more difficult differentiation between central DI and primary polydipsia, a copeptin level of 4.9 pmol/L stimulated with hypertonic saline infusion differentiates between these 2 entities with a high diagnostic accuracy and is superior to the water deprivation test. However, it is important to note that close and regular sodium monitoring every 30 min during the hypertonic saline test is a prerequisite, which is not possible in all hospitals. Furthermore, side effects are common. Therefore, a nonosmotic stimulation test would be advantageous. Arginine significantly stimulates copeptin and therefore is a novel, so far unknown stimulus of this peptide. Consequently, infusion of arginine with subsequent copeptin measurement was shown to be an even simpler and better tolerated test, but head to head comparison is still lacking.

A novel mechanism of autophagy-associated cell death of vasopressin neurons in familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI), characterized by delayed-onset progressive polyuria and loss of arginine vasopressin (AVP) neuron, is an autosomal dominant disorder caused by AVP gene mutations. We previously generated a knock-in mouse model for FNDI, which recapitulated the phenotype of human FNDI. To address the mechanisms underlying AVP neuron loss, we subjected FNDI mice to intermittent water deprivation, which accelerated the phenotype and induced AVP neuron loss within a relative short period. Electron microscopic analyses revealed that aggregates were confined to a sub-compartment of the endoplasmic reticulum (ER), ER-associated compartment (ERAC), in AVP neurons of FNDI mice under normal conditions. In contrast, aggregates scattered throughout the dilated ER lumen, and phagophores, autophagosome precursors, emerged and surrounded the ER containing scattered aggregates in FNDI mice subjected to water deprivation for 4 weeks, suggesting that failure of ERAC formation leads to autophagy induction for degradation of aggregates. Furthermore, the cytoplasm was entirely occupied with large vacuoles in AVP neurons of FNDI mice subjected to water deprivation for 12 weeks, at which stage 30-40% of AVP neurons were lost. Our data demonstrated that although autophagy should primarily be a protective mechanism, continuous autophagy leads to gradual loss of organelles including ER, resulting in autophagy-associated cell death of AVP neurons in FNDI mice.

Hemorrhagic fever with renal syndrome accompanied by panhypopituitarism and central diabetes insipidus: a case report.

Central diabetes insipidus (DI) was detected in a patient with hemorrhagic fever with renal syndrome (HFRS) who had been molecularly and serologically diagnosed with Hantaan virus infection. We recommend that clinicians differentiate central DI in HFRS patients with a persistent diuretic phase even when pituitary MRI findings are normal.

The Novel Ser18del AVP Variant Causes Inherited Neurohypophyseal Diabetes Insipidus by Mechanisms Shared with Other Signal Peptide Variants.

BACKGROUND/AIM: Variability in the severity and age at onset of autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) may be associated with certain types of variants in the arginine vasopressin (AVP) gene. In this study, we aimed to describe a large family with an apparent predominant female occurrence of polyuria and polydipsia and to determine the underlying cause. METHODS: The family members reported their family demography and symptoms. Two subjects were diagnosed by fluid deprivation and dDAVP challenge tests. Eight subjects were tested genetically. The identified variant along with 3 previously identified variants in the AVP gene were investigated by heterologous expression in a human neuronal cell line (SH-SY5Y). RESULTS: Both subjects investigated clinically had a partial neurohypophyseal diabetes insipidus phenotype. A g.276_278delTCC variant in the AVP gene causing a Ser18del deletion in the signal peptide (SP) of the AVP preprohormone was perfectly co-segregating with the disease. When expressed in SH-SY5Y cells, the Ser18del variant along with 3 other SP variants (g.227G>A, Ser17Phe, and Ala19Thr) resulted in reduced AVP mRNA, impaired AVP secretion, and partial AVP prohormone degradation and retention in the endoplasmic reticulum. Impaired SP cleavage was demonstrated directly in cells expressing the Ser18del, g.227G>A, and Ala19Thr variants, using state-of-the-art mass spectrometry. CONCLUSION: Variants affecting the SP of the AVP preprohormone cause adFNDI with variable phenotypes by a mechanism that may involve impaired SP cleavage combined with effects at the mRNA, protein, and cellular level.

Electron Microscopic Study of the Inner Medulla in Rat Kidneys under Conditions of Vasopressin Treatment Combined with Prostaglandin Synthesis Blockade.

Ultrastructural changes in cells of the renal inner medulla involved in the realization of the antidiuretic effect of vasopressin under conditions of prostaglandin synthesis blockade were studied in the kidneys of Wistar rats and endogenous vasopressin-deficient homozygous Brattleboro rats. The results indicated uniform trend to an increase in the number of clathrincoated vesicles under conditions of hormone treatment combined with prostaglandin synthesis blockade in animals with different neurohypophyseal status. These changes reflected translocation of aquaporins and an increase in the permeability of the collecting tubular epithelium for water. Brattleboro rats, but not Wistar rats, exhibited ultrastructural signs of synthesis activation in the epithelium and widening of the intercellular gaps, which could indicate more intense paracellular water transport.

Central diabetes insipidus associated with impaired renal aquaporin-1 expression in mice lacking liver X receptor ß.

The present study demonstrates a key role for the oxysterol receptor liver X receptor ß (LXRß) in the etiology of diabetes insipidus (DI). Given free access to water, LXRß(-/-) but not LXRα(-/-) mice exhibited polyuria (abnormal daily excretion of highly diluted urine) and polydipsia (increased water intake), both features of diabetes insipidus. LXRß(-/-) mice responded to 24-h dehydration with a decreased urine volume and increased urine osmolality. To determine whether the DI was of central or nephrogenic origin, we examined the responsiveness of the kidney to arginine vasopressin (AVP). An i.p. injection of AVP to LXRß(-/-) mice revealed a partial kidney response: There was no effect on urine volume, but there was a significant increase of urine osmolality, suggesting that DI may be caused by a defect in central production of AVP. In the brain of WT mice LXRß was expressed in the nuclei of magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus. In LXRß(-/-) mice the expression of AVP was markedly decreased in the magnocellular neurons as well as in urine collected over a 24-h period. The persistent high urine volume after AVP administration was traced to a reduction in aquaporin-1 expression in the kidney of LXRß(-/-) mice. The LXR agonist (GW3965) in WT mice elicited an increase in urine osmolality, suggesting that LXRß is a key receptor in controlling water balance with targets in both the brain and kidney, and it could be a therapeutic target in disorders of water balance.

Endoplasmic reticulum stress in vasopressin neurons of familial diabetes insipidus model mice: aggregate formation and mRNA poly(A) tail shortening.

The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone, which binds to newly synthesized secretory and transmembrane proteins to facilitate protein folding. BiP mRNA is expressed in the arginine vasopressin (AVP) neurons in the supraoptic nucleus of wild-type mice even in basal conditions, and the expression levels increase in response to dehydration. These data suggest that AVP neurons are subjected to ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is caused by mutations in the gene locus of AVP. The mutant proteins could accumulate in the ER and possibly increase ER stress in the AVP neurons. We bred mice possessing a mutation causing FNDI, which manifested progressive polyuria, as do the patients with FNDI. Electron microscopic analyses demonstrated that aggregates accumulated in the ER of AVP neurons in FNDI mice. Despite polyuria, which could potentially induce dehydration, AVP mRNA expression was decreased in the supraoptic nucleus, and the AVP mRNA poly(A) tail length was shortened in FNDI mice compared with wild-type mice. Incubation of hypothalamic explants of wild-type mice with ER stressors caused shortening of the poly(A) tail length of AVP mRNA, accompanied by decreases in the expression. These data revealed a mechanism by which ER stress decreases poly(A) tail length of AVP mRNA, and this reduces the load of unfolded proteins that form the aggregates in ER of the AVP neurons in FNDI mice.

Simplified drug efficacy evaluation system for vasopressin neurodegenerative disease using mouse disease-specific induced pluripotent stem cells.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.

Reversible impairment of the processing of proopiomelanocortin into ACTH in pituitary enlargement suspected of lymphocytic hypophysitis.

We describe a 64-year-old woman with a cystic pituitary mass presenting with central diabetes insipidus. Brain magnetic resonance imaging (MRI) with enhancement showed enlargement of the pituitary gland with cystic portions and thickening of the pituitary stalk with homogeneous enhancement. Combined anterior pituitary stimulation test and insulin-induced hypoglycemic test confirmed the diagnosis of panhypopituitarism, including adrenocortical insufficiency due to pituitary and hypothalamic dysfunction by stalk compression. Interestingly, the response of serum cortisol to CRH was low and delayed, in contrast to the marked increase in plasma ACTH. Molecular analysis of her plasma ACTH by Sephadex G75 gel exclusion chromatography coupled with radioimmunoassay (RIA) indicated a peak for high molecular weight ACTH, i.e., proACTH, in addition to that for 1-39 ACTH. Three years later, enlargement of the pituitary gland with cystic portions and thickening of the pituitary stalk disappeared completely, followed by the decrease in plasma proACTH level. By the results of endocrinological study and the change of pituitary MRI findings, lymphocytic hypophysitis was suggested. Synthesis of immature ACTH is generally thought to be due to impaired processing of the precursor proopiomelanocortin (POMC) through activation of prohormone convertase (PC)-1 by CRH. It is possible that the immature ACTH in this case was produced by impaired processing of the precursor POMC due to decreased CRH, dysfunction of corticotrophs in the anterior pituitary by compression of the normal pituitary, or antibodies targeting hypothalamic and/or pituitary cells. This report suggested that impaired processing of POMC may unusually play a role in adrenocortical insufficiency exhibited in lymphocytic hypophysitis.

Dominant pro-vasopressin mutants that cause diabetes insipidus form disulfide-linked fibrillar aggregates in the endoplasmic reticulum.

Autosomal dominant neurohypophyseal diabetes insipidus results from mutations in the precursor protein of the antidiuretic hormone arginine vasopressin. Mutant prohormone is retained in the endoplasmic reticulum of vasopressinergic neurons and causes their progressive degeneration by an unknown mechanism. Here, we show that several dominant pro-vasopressin mutants form disulfide-linked homo-oligomers and develop large aggregations visible by immunofluorescence and immunogold electron microscopy, both in a fibroblast and a neuronal cell line. Double-labeling showed the pro-vasopressin aggregates to colocalize with the chaperone calreticulin, indicating that they originated from the endoplasmic reticulum. The aggregates revealed a remarkable fibrillar substructure. Bacterially expressed and purified mutant pro-vasopressin spontaneously formed fibrils under oxidizing conditions. Mutagenesis experiments showed that the presence of cysteines, but no specific single cysteine, is essential for disulfide oligomerization and aggregation in vivo. Our findings assign autosomal dominant diabetes insipidus to the group of neurodegenerative diseases associated with the formation of fibrillar protein aggregates.

Short-term stimulation of the thiazide-sensitive Na+-Cl- cotransporter by vasopressin involves phosphorylation and membrane translocation.

Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na+-Cl- cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11beta-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V(2) receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT.

Amyloid-like aggregation of provasopressin.

The antidiuretic hormone vasopressin is synthesized as a longer precursor protein. After folding in the endoplasmic reticulum (ER), provasopressin is transported through the secretory pathway, forms secretory granules in the trans-Golgi network (TGN), is processed, and finally secreted into the circulation. Mutations in provasopressin cause autosomal dominant diabetes insipidus. They prevent native protein folding and cause fibrillar, amyloid-like aggregation in the ER, which eventually results in cell death. Secretory granules of peptide hormones were proposed to constitute functional amyloids and thus might be the cause of amyloid formation of misfolded mutant protein in the ER. Indeed, the same two segments in the precursor-vasopressin and a C-terminal glycopeptide-were found to be responsible for pathological aggregation in the ER and physiological aggregation in granule formation in the TGN. Furthermore, even wild-type provasopressin tends to aggregate in the ER, but is controlled by ER-associated degradation. When essential components thereof, Sel1L or Hrd1, were inactivated, wild-type provasopressin accumulated as fibrillar aggregates in vasopressinergic neurons in mice, causing diabetes insipidus. Evolution of amyloidogenic sequences for granule formation thus made provasopressin dependent on ER quality control mechanisms. These principles may similarly apply to other peptide hormones.

Role of protein aggregation and degradation in autosomal dominant neurohypophyseal diabetes insipidus.

This review focuses on the cellular and molecular aspects underlying familial neurohypophyseal diabetes insipidus (DI), a rare disorder that is usually transmitted in an autosomal-dominant fashion. The disease, manifesting in infancy or early childhood and gradually progressing in severity, is caused by fully penetrant heterozygous mutations in the gene encoding prepro-vasopressin-neurophysin II, the precursor of the antidiuretic hormone arginine vasopressin (AVP). Post mortem studies in affected adults have shown cell degeneration in vasopressinergic hypothalamic nuclei. Studies in cells expressing pathogenic mutants and knock-in rodent models have shown that the mutant precursors are folding incompetent and fail to exit the endoplasmic reticulum (ER), as occurs normally with proteins that have entered the regulated secretory pathway. A portion of these mutants is eliminated via ER-associated degradation (ERAD) by proteasomes after retrotranslocation to the cytosol. Another portion forms large disulfide-linked fibrillar aggregates within the ER, in which wild-type precursor is trapped. Aggregation capacity is independently conferred by two domains of the prohormone, namely the AVP moiety and the C-terminal glycopeptide (copeptin). The same domains are also required for packaging into dense-core secretory granules and regulated secretion, suggesting a disturbed balance between the physiological self-aggregation at the trans-Golgi network and avoiding premature aggregate formation at the ER in the disease. The critical role of ERAD in maintaining physiological water balance has been underscored by experiments in mice expressing wild-type AVP but lacking critical components of the ERAD machinery. These animals also develop DI and show amyloid-like aggregates in the ER lumen. Thus, the capacity of the ERAD is exceeded in autosomal dominant DI, which can be viewed as a neurodegenerative disorder associated with the formation of amyloid ER aggregates. While DI symptoms develop prior to detectable cell death in transgenic DI mice, the eventual loss of vasopressinergic neurons is accompanied by autophagy, but the mechanism leading to cell degeneration in autosomal dominant neurohypophyseal DI still remains unknown.

Autosomal dominant neurohypophyseal diabetes insipidus in two families. Molecular analysis of the vasopressin-neurophysin II gene and functional studies of three missense mutations.

BACKGROUND: Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease with symptoms of polydipsia, polyuria and dehydration caused by arginine vasopressin deficiency. Disease onset is within infancy or adolescence. A variety of disease-causing mutations of the arginine vasopressin neurophysin II gene (AVP) on chromosome 20p13 have been described. METHODS: Two Polish families with adFNDI were screened for mutations. Processing of wild-type (WT) and mutant AVP was monitored using immunocytochemical methods in stably transfected Neuro2A cells. AVP secretion into the cell culture supernatant was investigated with an enzyme immunoassay. RESULTS: In the first family a heterozygous p.G96D mutation was identified. Some patients additionally carried a novel heterozygous mutation p.A159T. The second family presented with a heterozygous mutation p.C98G. Confocal laser microscopy unveiled accumulation of p.G96D and p.C98G prohormones in the cellular bodies, whereas WT and p.A159T prohormones and/or processed products were located in the tips of cellular processes. Reduced levels of AVP in supernatant culture medium of p.G96D and p.C98G transfected cells in comparison to p.A159T and WT cells were found. CONCLUSIONS: We conclude that the p.G96D and p.C98G mutations cause adFNDI in the two reported families. The sequence variant p.A159T does not seem to have disease-causing effects.

Management of central diabetes insipidus with oral desmopressin in a patient with ectrodactyly and cleft lip/palate (ECP) syndrome.

We present a female infant with facial abnormalities such as bilateral cleft lip and palate, ectrodactyly and central diabetes insipidus. She had a history of recurrent hypernatremic attacks and she was treated successfully with oral desmopressin. As an alternative to the nasal route, long-term management was achieved using oral route and she had a favorable growth and development during infancy.

Disorders of water and salt metabolism associated with pituitary disease.

Disorders of water and sodium homeostasis are very common problems encountered in clinical medicine. Disorders of water metabolism are divided into hyperosmolar and hypoosmolar states, with hyperosmolar disorders characterized by a deficit of body water in relation to body solute and hypoosmolar disorders characterized by an excess of body water in relation to total body solute. This article briefly reviews the physiology of hyperosmolar and hypoosmolar syndromes, then focuses on a discussion of the pathophysiology, evaluation, and treatment of specific pre- and postoperative disorders of water metabolism in patients with pituitary lesions.

Functional ectopic neural lobe increases GAP-43 expression via PI3K/AKT pathways to alleviate central diabetes insipidus after pituitary stalk lesion in rats.

Central diabetes insipidus can occur after hypothalamic-hypophyseal tract injury. This injury is linked with a deficit in circulating vasopressin and oxytocin, which are produced in the supraoptic nuclei and the hypothalamic paraventricular nuclei. Previous studies indicate that an ectopic neural lobe forms after pituitary stalk lesion in rats, and while the relationship between an ectopic neural lobe and CDI outcomes is unclear, the underlying mechanisms are also unknown. Here, we report that two different CDI characteristics are shown in rats that underwent pituitary stalk electric lesion and are defined by two different groups classified as the recovery group and the no-recovery group. Rats showed an enlarged functional ectopic neural lobe at the lesion site with a low CDI index. Moreover, growth associated protein-43, p-PI3K and p-AKT were up-regulated in the unmyelinated fibers of the ectopic neural lobe. Our findings suggest that the enlarged structure formed a functional ectopic neural lobe after the pituitary stalk lesion, and its regeneration might influence the CDI outcome. This regeneration might be due to an increase in GAP-43 expression through the PI3K/AKT pathway.

Chemical chaperone 4-phenylbutylate reduces mutant protein accumulation in the endoplasmic reticulum of arginine vasopressin neurons in a mouse model for familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI), characterized by progressive polyuria and loss of arginine vasopressin (AVP) neurons, is an autosomal dominant disorder caused by AVP gene mutations. Our previous studies with FNDI model mice demonstrated that mutant proteins accumulated in the endoplasmic reticulum (ER) of AVP neurons. Here, we examined therapeutic effects of the chemical chaperone 4-phenylbutylate (4-PBA) in FNDI mice. Treatment with 4-PBA reduced mutant protein accumulation in the ER of FNDI mice and increased AVP release, leading to reduced urine volumes. Furthermore, AVP neuron loss under salt loading was attenuated by 4-PBA treatment. These data suggest that 4-PBA ameliorated mutant protein accumulation in the ER of AVP neurons and thereby prevented FNDI phenotype progression.

Anterior and posterior pituitary function testing with simultaneous insulin tolerance test and a novel copeptin assay.

CONTEXT: Posterior pituitary function in patients with suspected diabetes insipidus is usually assessed by a water deprivation test. Alternatively, a nonosmotic stimulus such as hypoglycemia may be used to stimulate vasopressin [arginine vasopressin (AVP)] secretion. Plasma AVP measurement may aid in the diagnosis and, especially, differential diagnosis of diabetes insipidus and polydipsia. However, AVP measurement is cumbersome. Copeptin, the stable C-terminal glycopeptide of the AVP prohormone, is stoichiometrically secreted from the posterior pituitary. OBJECTIVE: The aim was to study the value of copeptin levels in the diagnosis of diabetes insipidus during insulin-induced hypoglycemia. PATIENTS AND METHODS: A total of 38 patients were studied during insulin-induced hypoglycemia as part of a combined pituitary function test for possible anterior pituitary disease. There were 29 patients who had normal posterior pituitary function, and nine had central diabetes insipidus. Blood sampling was done before and 30, 45, and 90 min after iv insulin injection. Copeptin was measured with a new sandwich immunoassay. RESULTS: Patients with intact posterior pituitary function had basal copeptin levels of 3.7 +/- 1.5 pm, with a maximal increase to 11.1 +/- 4.6 pm 45 min after insulin injection. Copeptin levels in patients with diabetes insipidus were 2.4 +/- 0.5 pm before insulin injection, with a maximum increase to 3.7 +/- 0.7 pm. Both basal and stimulated copeptin levels were lower in patients with diabetes insipidus as compared with patients with intact posterior pituitary function. A stimulated copeptin level 45 min after insulin injection of less than 4.75 pm had an optimal diagnostic accuracy to detect diabetes insipidus. CONCLUSION: Copeptin measurement may be used to assess posterior together with anterior pituitary function during insulin-induced hypoglycemia.