A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk.

The angiotensin AT1 receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT1 receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT1 receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET2 technique to monitor the effect of angiotensin II on the interaction between Rluc8 tagged insulin receptor and GFP2 tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin receptor and IRS4 interactions whereas Gαs- and Gαi-coupled 7TM receptors had no effect. Furthermore, we used a panel of kinase inhibitors to show that angiotensin II engages different pathways when regulating insulin receptor interactions with IRS1 and IRS4. Angiotensin II inhibited the interaction between insulin receptor and IRS1 through activation of ERK1/2, while the interaction between insulin receptor and IRS4 was partially inhibited through protein kinase C dependent mechanisms. We conclude that the crosstalk between angiotensin AT1 receptor and insulin receptor signaling shows a high degree of specificity, and involves Gαq protein, and activation of distinct kinases. Thus, the BRET2 technique can be used as a platform for studying molecular mechanisms of crosstalk between insulin receptor and 7TM receptors.

The Volume Activated Potassium Channel KCNK5 is Up-Regulated in Activated Human T Cells, but Volume Regulation is Impaired.

BACKGROUND/AIMS: The potential role of the two-pore domain potassium channel KCNK5 (also known as TASK-2 and K(2P)5.1) in activated T cell physiology has only recently been described. So far KCNK5 has been described to be up-regulated in T cells in multiple sclerosis patients and to be implicated in the volume regulatory mechanism regulatory volume decrease (RVD) in T cells. METHODS: We investigated the time-dependent expression pattern of KCNK5 in CD3/CD28 activated human T cells using qPCR and Western blotting and its role in RVD using a Coulter Counter. RESULTS: KCNK5 is highly up-regulated in CD3/CD28 activated T cells both at mRNA (after 24 h) and protein level (72 and 144 h), but despite this up-regulation the RVD response is inhibited. Furthermore, the swelling-activated Cl- permeability in activated T cells is strongly decreased, and the RVD inhibition is predominantly due to the decreased Cl- permeability. CONCLUSION: The up-regulated KCNK5 in activated human T cells does not play a volume regulatory role, due to decreased Cl- permeability. We speculate that the KCNK5 up-regulation might play a role in hyperpolarization of the cell membrane leading to increased Ca2+ influx and proliferation of T cells.

The Effect of Cataract Surgery on Circadian Photoentrainment: A Randomized Trial of Blue-Blocking versus Neutral Intraocular Lenses.

PURPOSE: Cataract decreases blue light transmission. Because of the selective blue light sensitivity of the retinal ganglion cells governing circadian photoentrainment, cataract may interfere with normal sleep-wake regulation and cause sleep disturbances. The purpose was to investigate the effect of cataract surgery on circadian photoentrainment and to determine any difference between blue-blocking and neutral intraocular lenses (IOLs). DESIGN: The study was a single-center, investigator-driven, double-masked, block-randomized clinical trial. PARTICIPANTS: One eye in 76 patients with bilateral age-related cataract eligible for cataract surgery was included. METHODS: Intervention was cataract surgery by phacoemulsification. Patients were randomized to receive a blue-blocking or neutral IOL. MAIN OUTCOME MEASURES: Primary outcome was activation of intrinsic photosensitive ganglion cells using post-illumination pupil response (PIPR) to blue light from 10 to 30 seconds after light exposure as a surrogate measure. Secondary outcomes were circadian rhythm analysis using actigraphy and 24-hour salivary melatonin measurements. Finally, objective and subjective sleep quality were determined by actigraphy and the Pittsburgh Sleep Quality Index. RESULTS: The blue light PIPR increased 2 days (17%) and 3 weeks (24%) after surgery (P < 0.001). The majority of circadian and sleep-specific actigraphy parameters did not change after surgery. A forward shift of the circadian rhythm by 22 minutes (P = 0.004) for actigraphy and a tendency toward an earlier melatonin onset (P = 0.095) were found. Peak salivary melatonin concentration increased after surgery (P = 0.037). No difference was detected between blue-blocking and neutral IOLs, whereas low preoperative blue light transmission was inversely associated with an increase in PIPR (P = 0.021) and sleep efficiency (P = 0.048). CONCLUSIONS: Cataract surgery increases photoreception by the photosensitive retinal ganglion cells. Because of inconsistency between the significant findings and the many parameters that were unchanged, we can conclude that cataract surgery does not adversely affect the circadian rhythm or sleep. Longer follow-up time and fellow eye surgery may reveal the significance of the subtle changes observed. We found no difference between blue-blocking and neutral IOLs, and, because of the minor effect of surgery in itself, an effect of IOL type seems highly unlikely.

Reduced CSF hypocretin-1 levels are associated with cluster headache.

BACKGROUND: Cluster headache (CH) is a debilitating disorder characterized by unilateral, severe pain attacks with accompanying autonomic symptoms, often waking the patient from sleep. As it exhibits strong chronobiological traits and genetic studies have suggested a link with the hypocretin (HCRT) system, the objective of this study was to investigate HCRT-1 in CH patients. METHODS: Cerebrospinal fluid HCRT-1 concentration was measured in 12 chronic and 14 episodic CH patients during an active bout, and in 27 healthy controls. The patients were well characterized and clinical features compared to the HCRT concentration. RESULTS: We found significantly lower HCRT levels both in chronic (p = 0.0221) and episodic CH (p = 0.0005) patients compared with controls. No significant relationship was found with other clinical features. CONCLUSIONS: This is the first report of significantly reduced HCRT concentrations in CH patients. We speculate that decreased HCRT may reflect insufficient antinociceptive activity of the hypothalamus. The mechanism of the antinociceptive effect of HCRT is not known and requires further investigation. This study supports the hypothesis of a connection between arousal regulation and CH.

KCNK5 is functionally down-regulated upon long-term hypotonicity in Ehrlich ascites tumor cells.

BACKGROUND/AIMS: Regulatory volume decrease (RVD) in response to acute cell swelling is well described and KCNK5 (also known as TASK-2 or K2P5.1) has been shown to be the volume sensitive K(+) channel in Ehrlich cells. Very little is, on the other hand, known about the effects of long-term hypotonicity on expression and function of KCNK5, thus we have investigated the effect of long-term hypotonicity (24h - 48h) on KCNK5 in Ehrlich cells on the mRNA, protein and physiological levels. METHODS: Physiological effects of long-term hypotonicity were measured using patch-clamp and Coulter counter techniques. Expression patterns of KCNK5 on mRNA and protein levels were established using real-time qPCR and western blotting respectively. RESULTS: The maximum swelling-activated current through KCNK5 was significantly decreased upon 48h of hypotonicity and likewise the RVD response was significantly impaired after both 24 and 48h of hypotonic stimulation. No significant differences in the KCNK5 mRNA expression patterns between control and stimulated cells were observed, but a significant decrease in the KCNK5 protein level 48h after stimulation was found. CONCLUSION: The data suggest that the strong physiological impairment of KCNK5 in Ehrlich cells after long-term hypotonic stimulation is predominantly due to down-regulation of the KCNK5 protein synthesis.

A BRET assay for monitoring insulin receptor interactions and ligand pharmacology.

The insulin receptor (IR) belongs to the receptor tyrosine kinase super family and plays an important role in glucose homeostasis. The receptor interacts with several large docking proteins that mediate signaling from the receptor, including the insulin receptor substrate (IRS) family and Src homology-2-containing proteins (Src). Here, we applied the bioluminescence resonance energy transfer 2 (BRET2) technique to study the IR signaling pathways. The interaction between the IR and the substrates IRS1, IRS4 and Shc was examined in response to ligands with different signaling properties. The association between IR and the interacting partners could successfully be monitored when co-expressing green fluorescent protein 2 (GFP2) tagged substrates with Renilla reniformis luciferase 8 (Rluc8) tagged IR. Through additional optimization steps, we developed a stable and flexible BRET2 assay for monitoring the interactions between the IR and its substrates. Furthermore, the insulin analogue X10 was characterized in the BRET2 assay and was found to be 10 times more potent with respect to IRS1, IRS4 and Shc recruitment compared to human insulin. This study demonstrates that the BRET2 technique can be applied to study IR signaling pathways, and that this assay can be used as a platform for screening and characterization of IR ligands.

Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists.

Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists" with intrinsic "functional selectivity" that simultaneously blocks Galpha(q) protein activity and activates G protein-independent pathways of the AT(1)R confer important perspectives in treatment of cardiovascular diseases. In this study, we performed a global quantitative phosphoproteomics analysis of the AT(1)R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT(1)R agonist angiotensin II and the biased agonist [Sar(1),Ile(4),Ile(8)]angiotensin II (SII angiotensin II), which only activates the Galpha(q) protein-independent signaling. We quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analogue SII angiotensin II. 36% of the AT(1)R-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by Galpha(q) protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Galpha(q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation of the signaling properties of biased agonists to other receptors in the future.

Rapid eye movement sleep behaviour disorder in patients with narcolepsy is associated with hypocretin-1 deficiency.

Rapid eye movement sleep behaviour disorder is characterized by dream-enacting behaviour and impaired motor inhibition during rapid eye movement sleep. Rapid eye movement sleep behaviour disorder is commonly associated with neurodegenerative disorders, but also reported in narcolepsy with cataplexy. Most narcolepsy with cataplexy patients lack the sleep-wake, and rapid eye movement sleep, motor-regulating hypocretin neurons in the lateral hypothalamus. In contrast, rapid eye movement sleep behaviour disorder and hypocretin deficiency are rare in narcolepsy without cataplexy. We hypothesized that rapid eye movement sleep behaviour disorder coexists with cataplexy in narcolepsy due to hypocretin deficiency. In our study, rapid eye movement sleep behaviour disorder was diagnosed by the International Classification of Sleep Disorders (2nd edition) criteria in 63 narcolepsy patients with or without cataplexy. Main outcome measures were: rapid eye movement sleep behaviour disorder symptoms; short and long muscle activations per hour rapid eye movement and non-rapid eye movement sleep; and periodic and non-periodic limb movements per hour rapid eye movement and non-rapid eye movement sleep. Outcome variables were analysed in relation to cataplexy and hypocretin deficiency with uni- and multivariate logistic/linear regression models, controlling for possible rapid eye movement sleep behaviour disorder biasing factors (age, gender, disease duration, previous anti-cataplexy medication). Only hypocretin deficiency independently predicted rapid eye movement sleep behaviour disorder symptoms (relative risk = 3.69, P = 0.03), long muscle activations per hour rapid eye movement sleep (ln-coefficient = 0.81, P < 0.01), and short muscle activations per hour rapid eye movement sleep (ln-coefficient = 1.01, P < 0.01). Likewise, periodic limb movements per hour rapid eye movement and non-rapid eye movement sleep were only associated with hypocretin deficiency (P < 0.01). A significant association between hypocretin deficiency and cataplexy was confirmed (P < 0.01). In a sub-analysis, hypocretin deficiency suggested the association of periodic limb movements and rapid eye movement sleep behaviour disorder outcomes (symptoms, non-periodic short and long muscle activity) in rapid eye movement sleep. Our results support the hypothesis that hypocretin deficiency is independently associated with rapid eye movement sleep behaviour disorder in narcolepsy. Thus, hypocretin deficiency is linked to the two major disturbances of rapid eye movement sleep motor regulation in narcolepsy: rapid eye movement sleep behaviour disorder and cataplexy. Hypocretin deficiency is also significantly associated with periodic limb movements in rapid eye movement and non-rapid eye movement sleep, and provides a possible pathophysiological link between rapid eye movement sleep behaviour disorder and periodic limb movements in narcolepsy. The study supports the hypothesis that an impaired hypocretin system causes a general instability of motor regulation during wakefulness, rapid eye movement and non-rapid eye movement sleep in human narcolepsy.

Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation.

The swelling-activated K(+) currents (I(K,vol)) in Ehrlich ascites tumor cells (EATC) has been reported to be through the two-pore domain (K(2p)), TWIK-related acid-sensitive K(+) channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by I(K,vol) indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower cell volume. Swelling-activated K(+) efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K(+) efflux 1.3 times. To exclude K(+) efflux via a KCl cotransporter, cellular Cl(-) was substituted with NO(3)(-). Also under these conditions K(+) efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volume-sensitive K(+) channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the swelling-induced phosphorylation and activation of TASK-2 in EATC.

Validation of the ICSD-2 criteria for CSF hypocretin-1 measurements in the diagnosis of narcolepsy in the Danish population.

STUDY OBJECTIVES: The International Classification of Sleep Disorders (ICSD-2) criteria for low CSF hypocretin-1 levels (CSF hcrt-1) still need validation as a diagnostic tool for narcolepsy in different populations because inter-assay variability and different definitions of hypocretin deficiency complicate direct comparisons of study results. DESIGN AND PARTICIPANTS: Interviews, polysomnography, multiple sleep latency test, HLA-typing, and CSF hcrt-1 measurements in Danish patients with narcolepsy with cataplexy (NC) and narcolepsy without cataplexy (NwC), CSF hcrt-1 measurements in other hypersomnias, neurological and normal controls. Comparisons of hypocretin deficiency and frequency of HLA-DQB1*0602-positivity in the Danish and eligible NC and NwC populations (included via MEDLINE search), by (re)calculation of study results using the ICSD-2 criterion for low CSF hcrt-1 (< 30% of normal mean). MEASUREMENTS AND RESULTS: In Danes, low CSF hcrt-1 was present in 40/46 NC, 3/14 NwC and 0/106 controls (P < 0.0001). Thirty-nine of 41 NC and 4/13 NwC patients were HLA-DQB1*0602-positive (P < 0.01). Hypocretin-deficient NC patients had higher frequency of cataplexy, shorter mean sleep latency, more sleep onset REM periods (P < 0.05) and more awakenings (NS) than did NC patients with normal CSF hcrt-1. Across populations, low CSF hcrt-1 and HLA-DQB1*0602-positivity characterized the majority of NC (80% to 100%, P = 0.53; 80% to 100%, P = 0.11) but a minority of NwC patients (11% to 29%, P = 0.75; 29% to 89%, P = 0.043). CONCLUSION: The study provides evidence that hypocretin deficiency causes a more severe NC phenotype. The ICSD-2 criterion for low CSF hcrt-1 (< 30% of normal mean) is valid for diagnosing NC, but not NwC. HLA-typing should precede CSF hcrt-1 measurements because hypocretin deficiency is rare in HLA-DQB1*0602-negative patients.

Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling.

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.

Variation within the PPARG gene is associated with residual beta-cell function and glycemic control in children and adolescents during the first year of clinical type 1 diabetes.

CONTEXT: Conflicting evidence exists as to whether the Pro12Ala single nucleotide polymorphism of the type 2 diabetes susceptibility gene peroxisome proliferator-activated receptor gamma (PPARG) also confers risk for type 1 diabetes (T1D). OBJECTIVE: The objective of this study was to investigate the PPARG gene in relation to residual beta-cell function and glycemic control in newly diagnosed T1D. DESIGN: Prospective, non-interventional, 12-month follow-up study, conducted in 18 centers in 15 countries. PATIENTS: Two hundred and fifty-seven children and adolescents (aged <16 yr) with newly diagnosed T1D. MAIN OUTCOME MEASURES: Beta-cell function was determined as 90-min meal-stimulated C-peptide (Boost test) 1, 6, and 12 months after diagnosis. Hemoglobin A1c (HbA1c) and daily insulin dose (IU/kg/d) were recorded at 1, 3, 6, 9, and 12 months after diagnosis. Haplotypes within PPARG were estimated by SNPHap program. Statistical analyses were performed in a repeated measurements model. RESULTS: Five haplotypes within PPARG were generated (h1, 68.4%; h2, 16.3%; h3, 8.3%; h4, 3.5%; and hx, 3.5%). Compared with the most frequent h1 haplotype, the haplotypes h3 and h4 of the PPARG associated with residual beta-cell function during the first year of clinical disease: h3 with a 27% lower C-peptide (p = 0.02) and h4 with a 39% lower C-peptide (p = 0.01). Haplotype h4 also associated with a 0.86% (absolute) higher HbA1c, after adjustment for the insulin dose (p = 0.02). CONCLUSION: Variation in the PPARG locus may influence disease progression during the first year after the presentation of T1D.

[Sar1, Ile4, Ile8]-angiotensin II Potentiates Insulin Receptor Signalling and Glycogen Synthesis in Hepatocytes.

The angiotensin II type I receptor (AT1R) is involved in the regulation of cardiovascular function. Excessive activation of AT1R by angiotensin II (Ang II) leads to cardiovascular disease and may be involved in the development of insulin resistance and diabetes. Functionally selective Ang II analogues, such as the [Sar1, Ile4, Ile8]-angiotensin II (SII Ang II) analogue, that only activate a subset of signalling networks have been demonstrated to have beneficial effects on cardiovascular function in certain settings, including lowering blood pressure and increasing cardiac performance. Here, we studied the effect of SII Ang II on insulin receptor (IR) signalling and glucose metabolism in primary rat hepatocytes. We show that long-term pre-treatment of hepatocytes with SII Ang II increased insulin-stimulated glycogen synthesis, while Ang II and the AT1R antagonist losartan had no effect. Insulin-stimulated suppression of hepatic glucose output was not affected by Ang II or SII Ang II. It is well known that insulin regulates glycogen synthesis and glucose output through Akt-mediated phosphorylation of glycogen synthase kinase α/ß (GSK3α/ß) and forkhead box protein O1 (FOXO1), respectively. In line with this, we show that SII Ang II potentiated insulin-stimulated phosphorylation of Akt and GSK3α/ß, but not FOXO1. Furthermore, we demonstrate that the effect of SII Ang II on insulin-stimulated signalling and glycogen synthesis was dependent on Src and Gαq, as inhibitors of these proteins abolished the potentiating effect of SII Ang II. Thus, our results demonstrate that SII Ang II may have a positive effect on IR signalling and glucose metabolism in hepatocytes.

Functional brown adipose tissue and sympathetic activity after cold exposure in humans with type 1 narcolepsy.

Study Objectives: To investigate the activity of brown adipose tissue (BAT) in patients with type 1 narcolepsy during cold exposure using two separate scans of sympathetic and metabolic activity of BAT to evaluate whether orexin deficiency leads to altered nonshivering thermoregulation in narcolepsy. Methods: Seven patients with type 1 narcolepsy and seven healthy controls underwent two consecutive scans after 2 hr cold exposure: 123I-meta-iodo-benzyl-guanidine (123I-MIBG) single photon emission computed tomography and18F-2-deoxy-glucose (18F-FDG) positron emission tomography and computed tomography to visualize sympathetic innervation and metabolic activity of BAT, respectively. Plasma levels of eight hormones regulating BAT activity were measured before and after 2 hr in the cold. Results: 18F-FDG-uptake and uptake of 123I-MIBG in BAT after 2 hr cold exposure were observed in all individuals, but the activity of BAT was not significantly different between patients with type 1 narcolepsy and healthy controls (p > 0.05). Plasma levels of GLP-1 were higher in patients with type 1 narcolepsy compared with controls (p < 0.05), but not altered by cold adaptation in patients and controls (p > 0.05). FGF21 concentrations decreased after 2 hr cold exposure in both patients with type 1 narcolepsy and healthy participants (p < 0.05). Conclusions: Sympathetic and metabolic activity of BAT was observed after cold exposure in patients with type 1 narcolepsy. Increased GLP-1 in narcolepsy may suggest autonomic dysfunction with metabolic changes. We conclude that BAT is functional after cold exposure in spite of the loss of orexinergic neurons in narcolepsy.

Co-localisation of the Kir6.2/SUR1 channel complex with glucagon-like peptide-1 and glucose-dependent insulinotrophic polypeptide expression in human ileal cells and implications for glycaemic control in new onset type 1 diabetes.

OBJECTIVE: The ATP-dependent K+-channel (K(ATP)) is critical for glucose sensing and normal glucagon and insulin secretion from pancreatic endocrine alpha- and beta-cells. Gastrointestinal endocrine L- and K-cells are also glucose-sensing cells secreting glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotrophic polypeptide (GIP) respectively. The aims of this study were to 1) investigate the expression and co-localisation of the K(ATP) channel subunits, Kir6.2 and SUR1, in human L- and K-cells and 2) investigate if a common hyperactive variant of the Kir6.2 subunit, Glu23Lys, exerts a functional impact on glucose-sensing tissues in vivo that may affect the overall glycaemic control in children with new-onset type 1 diabetes. DESIGN AND METHODS: Western blot and immunohistochemical analyses were performed for expression and co-localisation studies. Meal-stimulated C-peptide test was carried out in 257 children at 1, 6 and 12 months after diagnosis. Genotyping for the Glu23Lys variant was by PCR-restriction fragment length polymorphism. RESULTS: Kir6.2 and SUR1 co-localise with GLP-1 in L-cells and with GIP in K-cells in human ileum tissue. Children with type 1 diabetes carrying the hyperactive Glu23Lys variant had higher HbA1C at diagnosis (coefficient = 0.61%, P = 0.02) and 1 month after initial insulin therapy (coefficient = 0.30%, P = 0.05), but later disappeared. However, when adjusting HbA1C for the given dose of exogenous insulin, the dose-adjusted HbA1C remained higher throughout the 12 month study period (coefficient = 0.42%, P = 0.03). CONCLUSIONS: Kir6.2 and SUR1 co-localise in the gastrointestinal endocrine L- and K-cells. The hyperactive Glu23Lys variant of the K(ATP) channel subunit Kir6.2 may cause defective glucose sensing in several tissues and impaired glycaemic control in children with type 1 diabetes.

Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells.

We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increased activation of both wild-type Rsk proteins could be achieved by coexpression of a constitutively active (CA) mitogen-activated protein kinase (MAPK) kinase, MEK1-DD, which is known to cause differentiation of PC12 cells even in the absence of NGF. Rsk1 and Rsk2 mutated in the PDK1-binding site were not activated by either NGF or MEK1-DD. Expression of constitutively active Rsk1 or Rsk2 in PC12 cells resulted in highly active proteins whose levels of activity did not change either with NGF treatment or after coexpression with MEK1-DD. Rsk2-CA expression had no detectable effect on the cells. However, expression of Rsk1-CA led to differentiation of PC12 cells even in the absence of NGF, as evidenced by neurite outgrowth. Differentiation was not observed with a nonactive Rsk1-CA that was mutated in the PDK1-binding site. Expression of Rsk1-CA did not lead to activation of the endogenous MAPK pathway, indicating that Rsk1 is sufficient to induce neurite outgrowth and is the only target of MAPK required for this effect. Collectively, our data demonstrate a key role for Rsk1 in the differentiation process of PC12 cells.

The angiotensin type 1 receptor activates extracellular signal-regulated kinases 1 and 2 by G protein-dependent and -independent pathways in cardiac myocytes and langendorff-perfused hearts.

The angiotensin II (AngII) type 1 receptor (AT(1)R) has been shown to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) through G proteins or G protein-independently through beta-arrestin2 in cellular expression systems. As activation mechanisms may greatly influence the biological effects of ERK1/2 activity, differential activation of the AT(1)R in its native cellular context could have important biological and pharmacological implications. To examine if AT(1)R activates ERK1/2 by G protein-independent mechanisms in the heart, we used the [Sar(1), Ile(4), Ile(8)]-AngII ([SII] AngII) analogue in native preparations of cardiac myocytes and beating hearts. We found that [SII] AngII does not activate G(q)-coupling, yet stimulates the beta-arrestin2-dependent ERK1/2. The G(q)-activated pool of ERK1/2 rapidly translocates to the nucleus, while the beta-arrestin2-scaffolded pool remains in the cytosol. Similar biased agonism was achieved in Langendorff-perfused hearts, where both agonists elicit ERK1/2 phosphorylation, but [SII] AngII induces neither inotropic nor chronotropic effects.

Differential extracellular signal-regulated kinases 1 and 2 activation by the angiotensin type 1 receptor supports distinct phenotypes of cardiac myocytes.

The angiotensin II (AngII) type 1 receptor (AT(1)R) is a seven-transmembrane receptor well established to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) by discrete G protein-dependent and beta-arrestin2-dependent pathways. The biological importance of this, however, remains obscure. Application of the modified analogue [Sar(1), Ile(4), Ile(8)]-AngII ([SII] AngII) allowed us to dissect the two pathways of ERK1/2 activation in native cardiac myocytes. Although cytosol-retained, the beta-arrestin2-bound pool of ERK1/2 represents an active signalling component that phosphorylates p90 Ribosomal S6 Kinase, a ubiquitous and versatile mediator of ERK1/2 signal transduction. Moreover, the beta-arrestin2-dependent ERK1/2 signal supports intact proliferation of cardiac myocytes. In contrast to G(q)-activated ERK1/2, and in keeping with its failure to translocate to the nucleus, the beta-arrestin2-scaffolded pool of ERK1/2 does not phosphorylate the transcription factor Elk-1, induces no increased transcription of the immediate-early gene c-Fos, and does not entail myocyte hypertrophy. These results clearly demonstrate the biological significance of differential signalling by the AT(1)R. The opportunity to separate desirable cardiac myocyte division from detrimental hypertrophy holds promise that novel pharmacological approaches will allow targeting of pathway-specific actions.

Normal Morning Melanin-Concentrating Hormone Levels and No Association with Rapid Eye Movement or Non-Rapid Eye Movement Sleep Parameters in Narcolepsy Type 1 and Type 2.

STUDY OBJECTIVES: Other than hypocretin-1 (HCRT-1) deficiency in narcolepsy type 1 (NT1), the neurochemical imbalance of NT1 and narcolepsy type 2 (NT2) with normal HCRT-1 levels is largely unknown. The neuropeptide melanin-concentrating hormone (MCH) is mainly secreted during sleep and is involved in rapid eye movement (REM) and non-rapid eye movement (NREM) sleep regulation. Hypocretin neurons reciprocally interact with MCH neurons. We hypothesized that altered MCH secretion contributes to the symptoms and sleep abnormalities of narcolepsy and that this is reflected in morning cerebrospinal fluid (CSF) MCH levels, in contrast to previously reported normal evening/afternoon levels. METHODS: Lumbar CSF and plasma were collected from 07:00 to 10:00 from 57 patients with narcolepsy (subtypes: 47 NT1; 10 NT2) diagnosed according to International Classification of Sleep Disorders, Third Edition (ICSD-3) and 20 healthy controls. HCRT-1 and MCH levels were quantified by radioimmunoassay and correlated with clinical symptoms, polysomnography (PSG), and Multiple Sleep Latency Test (MSLT) parameters. RESULTS: CSF and plasma MCH levels were not significantly different between narcolepsy patients regardless of ICSD-3 subtype, HCRT-1 levels, or compared to controls. CSF MCH and HCRT-1 levels were not significantly correlated. Multivariate regression models of CSF MCH levels, age, sex, and body mass index predicting clinical, PSG, and MSLT parameters did not reveal any significant associations to CSF MCH levels. CONCLUSIONS: Our study shows that MCH levels in CSF collected in the morning are normal in narcolepsy and not associated with the clinical symptoms, REM sleep abnormalities, nor number of muscle movements during REM or NREM sleep of the patients. We conclude that morning lumbar CSF MCH measurement is not an informative diagnostic marker for narcolepsy.

Cerebrospinal Fluid Biomarkers of Neurodegeneration Are Decreased or Normal in Narcolepsy.

Objectives: To investigate whether cerebrospinal fluid (CSF) biomarkers of neurodegeneration are altered in narcolepsy in order to evaluate whether the hypocretin deficiency and abnormal sleep-wake pattern in narcolepsy leads to neurodegeneration. Methods: Twenty-one patients with central hypersomnia (10 type 1 narcolepsy, 5 type 2 narcolepsy, and 6 idiopathic hypersomnia cases), aged 33 years on average and with a disease duration of 2-29 years, and 12 healthy controls underwent CSF analyses of the levels of ß-amyloid, total tau protein (T-tau), phosphorylated tau protein (P-tau181), α-synuclein, neurofilament light chain (NF-L), and chitinase 3-like protein-1 (CHI3L1). Results: Levels of ß-amyloid were lower in patients with type 1 narcolepsy (375.4 ± 143.5 pg/mL) and type 2 narcolepsy (455.9 ± 65.0 pg/mL) compared to controls (697.9 ± 167.3 pg/mL, p < .05). Furthermore, in patients with type 1 narcolepsy, levels of T-tau (79.0 ± 27.5 pg/mL) and P-tau181 (19.1 ± 4.3 pg/mL) were lower than in controls (162.2 ± 49.9 pg/mL and 33.8 ± 9.2 pg/mL, p < .05). Levels of α-synuclein, NF-L, and CHI3L1 in CSF from narcolepsy patients were similar to those of healthy individuals. Conclusion: Six CSF biomarkers of neurodegeneration were decreased or normal in narcolepsy indicating that taupathy, synucleinopathy, and immunopathy are not prevalent in narcolepsy patients with a disease duration of 2-29 years. Lower CSF levels of ß-amyloid, T-tau protein, and P-tau181 in narcolepsy may indicate that hypocretin deficiency and an abnormal sleep-wake pattern alter the turnover of these proteins in the central nervous system.