Traditional Japanese medicine Kamikihito ameliorates sucrose preference, chronic inflammation and obesity induced by a high fat diet in middle-aged mice.

The high prevalence of obesity has become a pressing global public health problem and there exists a strong association between increased BMI and mortality at a BMI of 25 kg/m2 or higher. The prevalence of obesity is higher among middle-aged adults than among younger groups and the combination of aging and obesity exacerbate systemic inflammation. Increased inflammatory cytokines such as interleukin 6 and tumor necrosis factor alpha (TNFα) are hallmarks of obesity, and promote the secretion of hepatic C-reactive protein (CRP) which further induces systematic inflammation. The neuropeptide oxytocin has been shown to have anti-obesity and anti-inflammation effects, and also suppress sweet-tasting carbohydrate consumption in mammals. Previously, we have shown that the Japanese herbal medicine Kamikihito (KKT), which is used to treat neuropsychological stress disorders in Japan, functions as an oxytocin receptors agonist. In the present study, we further investigated the effect of KKT on body weight (BW), food intake, inflammation, and sweet preferences in middle-aged obese mice. KKT oral administration for 12 days decreased the expression of pro-inflammatory cytokines in the liver, and the plasma CRP and TNFα levels in obese mice. The effect of KKT administration was found to be different between male and female mice. In the absence of sucrose, KKT administration decreased food intake only in male mice. However, while having access to a 30% sucrose solution, both BW and food intake was decreased by KKT administration in male and female mice; but sucrose intake was decreased in female mice alone. In addition, KKT administration decreased sucrose intake in oxytocin deficient lean mice, but not in the WT lean mice. The present study demonstrates that KKT ameliorates chronic inflammation, which is strongly associated with aging and obesity, and decreases food intake in male mice as well as sucrose intake in female mice; in an oxytocin receptor dependent manner.

Systemic Co-Administration of Low-Dose Oxytocin and Glucagon-Like Peptide 1 Additively Decreases Food Intake and Body Weight.

INTRODUCTION: GLP-1 receptor agonists are the number one drug prescribed for the treatment of obesity and type 2 diabetes. These drugs are not, however, without side effects, and in an effort to maximize therapeutic effect while minimizing adverse effects, gut hormone co-agonists received considerable attention as new drug targets in the fight against obesity. Numerous previous reports identified the neuropeptide oxytocin (OXT) as a promising anti-obesity drug. The aims of this study were to evaluate OXT as a possible co-agonist for GLP-1 and examine the effects of its co-administration on food intake (FI) and body weight (BW) in mice. METHODS: FI and c-Fos levels were measured in the feeding centers of the brain in response to an intraperitoneal injection of saline, OXT, GLP-1, or OXT/GLP-1. The action potential frequency and cytosolic Ca2+ ([Ca2+]i) in response to OXT, GLP-1, or OXT/GLP-1 were measured in ex vivo paraventricular nucleus (PVN) neuronal cultures. Finally, FI and BW changes were compared in diet-induced obese mice treated with saline, OXT, GLP-1, or OXT/GLP-1 for 13 days. RESULTS: Single injection of OXT/GLP-1 additively decreased FI and increased c-Fos expression specifically in the PVN and supraoptic nucleus. Seventy percent of GLP-1 receptor-positive neurons in the PVN also expressed OXT receptors, and OXT/GLP-1 co-administration dramatically increased firing and [Ca2+]i in the PVN OXT neurons. The chronic OXT/GLP-1 co-administration decreased BW without changing FI. CONCLUSION: Chronic OXT/GLP-1 co-administration decreases BW, possibly via the activation of PVN OXT neurons. OXT might be a promising candidate as an incretin co-agonist in obesity treatment.

NMDA Receptor Antagonists Increase the Release of GLP-1 From Gut Endocrine Cells.

Type 2 diabetes mellitus (T2DM) remains one of the most pressing health issues facing modern society. Several antidiabetic drugs are currently in clinical use to treat hyperglycaemia, but there is a need for new treatments that effectively restore pancreatic islet function in patients. Recent studies reported that both murine and human pancreatic islets exhibit enhanced insulin release and ß-cell viability in response to N-methyl-D-aspartate (NMDA) receptor antagonists. Furthermore, oral administration of dextromethorphan, an over-the-counter NMDA receptor antagonist, to diabetic patients in a small clinical trial showed improved glucose tolerance and increased insulin release. However, the effects of NMDA receptor antagonists on the secretion of the incretin hormone GLP-1 was not tested, and nothing is known regarding how NMDA receptor antagonists may alter the secretion of gut hormones. This study demonstrates for the first time that, similar to ß-cells, the NMDA receptor antagonist MK-801 increases the release of GLP-1 from a murine L-cell enteroendocrine model cell line, GLUTag cells. Furthermore, we report the 3' mRNA expression profiling of GLUTag cells, with a specific focus on glutamate-activated receptors. We conclude that if NMDA receptor antagonists are to be pursued as an alternative, orally administered treatment for T2DM, it is essential that the effects of these drugs on the release of gut hormones, and specifically the incretin hormones, are fully investigated.

Acetyl-CoA-carboxylase 1 (ACC1) plays a critical role in glucagon secretion.

Dysregulated glucagon secretion from pancreatic alpha-cells is a key feature of type-1 and type-2 diabetes (T1D and T2D), yet our mechanistic understanding of alpha-cell function is underdeveloped relative to insulin-secreting beta-cells. Here we show that the enzyme acetyl-CoA-carboxylase 1 (ACC1), which couples glucose metabolism to lipogenesis, plays a key role in the regulation of glucagon secretion. Pharmacological inhibition of ACC1 in mouse islets or αTC9 cells impaired glucagon secretion at low glucose (1 mmol/l). Likewise, deletion of ACC1 in alpha-cells in mice reduced glucagon secretion at low glucose in isolated islets, and in response to fasting or insulin-induced hypoglycaemia in vivo. Electrophysiological recordings identified impaired KATP channel activity and P/Q- and L-type calcium currents in alpha-cells lacking ACC1, explaining the loss of glucose-sensing. ACC-dependent alterations in S-acylation of the KATP channel subunit, Kir6.2, were identified by acyl-biotin exchange assays. Histological analysis identified that loss of ACC1 caused a reduction in alpha-cell area of the pancreas, glucagon content and individual alpha-cell size, further impairing secretory capacity. Loss of ACC1 also reduced the release of glucagon-like peptide 1 (GLP-1) in primary gastrointestinal crypts. Together, these data reveal a role for the ACC1-coupled pathway in proglucagon-expressing nutrient-responsive endocrine cell function and systemic glucose homeostasis.

Abcc5 Knockout Mice Have Lower Fat Mass and Increased Levels of Circulating GLP-1.

OBJECTIVE: A previous genome-wide association study linked overexpression of an ATP-binding cassette transporter, ABCC5, in humans with a susceptibility to developing type 2 diabetes with age. Specifically, ABCC5 gene overexpression was shown to be strongly associated with increased visceral fat mass and reduced peripheral insulin sensitivity. Currently, the role of ABCC5 in diabetes and obesity is unknown. This study reports the metabolic phenotyping of a global Abcc5 knockout mouse. METHODS: A global Abcc5-/- mouse was generated by CRISPR/Cas9. Fat mass was determined by weekly EchoMRI and fat pads were dissected and weighed at week 18. Glucose homeostasis was ascertained by an oral glucose tolerance test, intraperitoneal glucose tolerance test, and intraperitoneal insulin tolerance test. Energy expenditure and locomotor activity were measured using PhenoMaster cages. Glucagon-like peptide 1 (GLP-1) levels in plasma, primary gut cell cultures, and GLUTag cells were determined by enzyme-linked immunosorbent assay. RESULTS: Abcc5-/- mice had decreased fat mass and increased plasma levels of GLP-1, and they were more insulin sensitive and more active. Recombinant overexpression of ABCC5 protein in GLUTag cells decreased GLP-1 release. CONCLUSIONS: ABCC5 protein expression levels are inversely related to fat mass and appear to play a role in the regulation of GLP-1 secretion from enteroendocrine cells.

A Ketone Ester Drink Lowers Human Ghrelin and Appetite.

OBJECTIVE: The ketones d-ß-hydroxybutyrate (BHB) and acetoacetate are elevated during prolonged fasting or during a "ketogenic" diet. Although weight loss on a ketogenic diet may be associated with decreased appetite and altered gut hormone levels, it is unknown whether such changes are caused by elevated blood ketones. This study investigated the effects of an exogenous ketone ester (KE) on appetite. METHODS: Following an overnight fast, subjects with normal weight (n = 15) consumed 1.9 kcal/kg of KE, or isocaloric dextrose (DEXT), in drinks matched for volume, taste, tonicity, and color. Blood samples were analyzed for BHB, glucose, insulin, ghrelin, glucagon-like peptide 1 (GLP-1), and peptide tyrosine tyrosine (PYY), and a three-measure visual analogue scale was used to measure hunger, fullness, and desire to eat. RESULTS: KE consumption increased blood BHB levels from 0.2 to 3.3 mM after 60 minutes. DEXT consumption increased plasma glucose levels between 30 and 60 minutes. Postprandial plasma insulin, ghrelin, GLP-1, and PYY levels were significantly lower 2 to 4 hours after KE consumption, compared with DEXT consumption. Temporally related to the observed suppression of ghrelin, reported hunger and desire to eat were also significantly suppressed 1.5 hours after consumption of KE, compared with consumption of DEXT. CONCLUSIONS: Increased blood ketone levels may directly suppress appetite, as KE drinks lowered plasma ghrelin levels, perceived hunger, and desire to eat.

Molecular action of sulphonylureas on KATP channels: a real partnership between drugs and nucleotides.

Sulphonylureas stimulate insulin secretion from pancreatic ß-cells primarily by closing ATP-sensitive K(+) channels in the ß-cell plasma membrane. The mechanism of channel inhibition by these drugs is unusually complex. As direct inhibitors of channel activity, sulphonylureas act only as partial antagonists at therapeutic concentrations. However, they also exert an additional indirect inhibitory effect via modulation of nucleotide-dependent channel gating. In this review, we summarize current knowledge and recent advances in our understanding of the molecular mechanism of action of these drugs.

Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: a mechanistic study.

Sulfonylureas, which stimulate insulin secretion from pancreatic ß-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K(+) (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on ß-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on ß-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.

Molecular mechanism of sulphonylurea block of K(ATP) channels carrying mutations that impair ATP inhibition and cause neonatal diabetes.

Sulphonylurea drugs are the therapy of choice for treating neonatal diabetes (ND) caused by mutations in the ATP-sensitive K(+) channel (KATP channel). We investigated the interactions between MgATP, MgADP, and the sulphonylurea gliclazide with KATP channels expressed in Xenopus oocytes. In the absence of MgATP, gliclazide block was similar for wild-type channels and those carrying the Kir6.2 ND mutations R210C, G334D, I296L, and V59M. Gliclazide abolished the stimulatory effect of MgATP on all channels. Conversely, high MgATP concentrations reduced the gliclazide concentration, producing a half-maximal block of G334D and R201C channels and suggesting a mutual antagonism between nucleotide and gliclazide binding. The maximal extent of high-affinity gliclazide block of wild-type channels was increased by MgATP, but this effect was smaller for ND channels; channels that were least sensitive to ATP inhibition showed the smallest increase in sulphonylurea block. Consequently, G334D and I296L channels were not fully blocked, even at physiological MgATP concentrations (1 mmol/L). Glibenclamide block was also reduced in ß-cells expressing Kir6.2-V59M channels. These data help to explain why patients with some mutations (e.g., G334D, I296L) are insensitive to sulphonylurea therapy, why higher drug concentrations are needed to treat ND than type 2 diabetes, and why patients with severe ND mutations are less prone to drug-induced hypoglycemia.

Tetrameric structure of SUR2B revealed by electron microscopy of oriented single particles.

The ATP-sensitive potassium (K(ATP)) channel is a hetero-octameric complex that links cell metabolism to membrane electrical activity in many cells, thereby controlling physiological functions such as insulin release, muscle contraction and neuronal activity. It consists of four pore-forming Kir6.2 and four regulatory sulfonylurea receptor (SUR) subunits. SUR2B serves as the regulatory subunit in smooth muscle and some neurones. An integrative approach, combining electron microscopy and homology modelling, has been used to obtain information on the structure of this large (megadalton) membrane protein complex. Single-particle electron microscopy of purified SUR2B tethered to a lipid monolayer revealed that it assembles as a tetramer of four SUR2B subunits surrounding a central hole. In the absence of an X-ray structure, a homology model for SUR2B based on the X-ray structure of the related ABC transporter Sav1866 was used to fit the experimental images. The model indicates that the central hole can readily accommodate the transmembrane domains of the Kir tetramer, suggests a location for the first transmembrane domains of SUR2B (which are absent in Sav1866) and suggests the relative orientation of the SUR and Kir6.2 subunits.

A universally conserved residue in the SUR1 subunit of the KATP channel is essential for translating nucleotide binding at SUR1 into channel opening.

The sulphonylurea receptor (SUR1) subunit of the ATP-sensitive potassium (KATP) channel is a member of the ATP-binding cassette (ABC) protein family. Binding of MgADP to nucleotide-binding domain 2 (NBD2) is critical for channel activation.We identified a residue in NBD2 (G1401) that is fully conserved among ABC proteins and whose functional importance is unknown. Homology modelling places G1401 on the outer surface of the protein, distant from the nucleotide-binding site. The ATPase activity of purified SUR1-NBD2-G1410R (bound to maltose-binding protein) was slightly inhibited when compared to the wild-type protein, but its inhibition by MgADP was unchanged, indicating that MgADP binding is not altered. However, MgADP activation of channel activity was abolished. This implies that the G1401R mutation impairs the mechanism by which MgADP binding to NBD2 is translated into opening of the KATP channel pore. The location of G1401 would be consistent with interaction of this residue with the pore-forming Kir6.2 subunit. Channel activity in the presence of MgATP reflects the balance between the stimulatory (at SUR1) and inhibitory (at Kir6.2) effects of nucleotides. Mutant channels were 2.5-fold less sensitive to MgATP inhibition and not activated by MgATP. This suggests that ATP block of the channel is reduced by the SUR1 mutation. Interestingly, this effect was dependent on the functional integrity of the NBDs. These results therefore suggest that SUR1 modulates both nucleotide inhibition and activation of the KATP channel.

MKP-1 knockout does not prevent glucocorticoid-induced bone disease in mice.

Glucocorticoid-induced osteoporosis (GCOP) is predominantly caused by inhibition of bone formation, resulting from a decrease in osteoblast numbers. Employing mouse (MBA-15.4) and human (MG-63) osteoblast cell lines, we previously found that the glucocorticoid (GC) dexamethasone (Dex) inhibits cellular proliferation as well as activation of the MAPK/ERK signaling pathway, essential for mitogenesis in these cells, and that both these effects could be reversed by the protein tyrosine phosphatase (PTP) inhibitor vanadate. In a rat model of GCOP, the GC-induced changes in bone formation, mass, and strength could be prevented by vanadate cotreatment, suggesting that the GC effects on bone were mediated by one or more PTPs. Employing phosphatase inhibitors, qRT-PCR, Western blotting, and overexpression/knockdown experiments, we concluded that MKP-1 was upregulated by Dex, that this correlated with the dephosphorylation of ERK, and that it largely mediated the in vitro effects of GCs on bone. To confirm the pivotal role of MKP-1 in vivo, we investigated the effects of the GC methylprednisolone on the quantitative bone histology of wild-type (WT) and MKP-1 homozygous knockout (MKP-1(-/-)) mice. In WT mice, static bone histology revealed that GC administration for 28 days decreased osteoid surfaces, volumes, and osteoblast numbers. Dynamic histology, following time-spaced tetracycline labeling, confirmed a significant GC-induced reduction in osteoblast appositional rate and bone formation rate. However, identical results were obtained in MKP-1 knockout mice, suggesting that in these animals upregulation of MKP-1 by GCs cannot be regarded as the sole mediator of the GC effects on bone.

Disease progression and search for monogenic diabetes among children with new onset type 1 diabetes negative for ICA, GAD- and IA-2 Antibodies.

BACKGROUND: To investigate disease progression the first 12 months after diagnosis in children with type 1 diabetes negative (AAB negative) for pancreatic autoantibodies [islet cell autoantibodies(ICA), glutamic acid decarboxylase antibodies (GADA) and insulinoma-associated antigen-2 antibodies (IA-2A)]. Furthermore the study aimed at determining whether mutations in KCNJ11, ABCC8, HNF1A, HNF4A or INS are common in AAB negative diabetes. MATERIALS AND METHODS: In 261 newly diagnosed children with type 1 diabetes, we measured residual ß-cell function, ICA, GADA, and IA-2A at 1, 6 and 12 months after diagnosis. The genes KCNJ11, ABCC8, HNF1A, HNF4A and INS were sequenced in subjects AAB negative at diagnosis. We expressed recombinant K-ATP channels in Xenopus oocytes to analyse the functional effects of an ABCC8 mutation. RESULTS: Twenty-four patients (9.1%) tested AAB negative after one month. Patients, who were AAB-negative throughout the 12-month period, had higher residual ß-cell function (P = 0.002), lower blood glucose (P = 0.004), received less insulin (P = 0.05) and had lower HbA1c (P = 0.02) 12 months after diagnosis. One patient had a heterozygous mutation leading to the substitution of arginine at residue 1530 of SUR1 (ABCC8) by cysteine. Functional analyses of recombinant K-ATP channels showed that R1530C markedly reduced the sensitivity of the K-ATP channel to inhibition by MgATP. Morover, the channel was highly sensitive to sulphonylureas. However, there was no effect of sulfonylurea treatment after four weeks on 1.0-1.2 mg/kg/24 h glibenclamide. CONCLUSION: GAD, IA-2A, and ICA negative children with new onset type 1 diabetes have slower disease progression as assessed by residual beta-cell function and improved glycemic control 12 months after diagnosis. One out of 24 had a mutation in ABCC8, suggesting that screening of ABCC8 should be considered in patients with AAB negative type 1 diabetes.

Activation of the K(ATP) channel by Mg-nucleotide interaction with SUR1.

The mechanism of adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel activation by Mg-nucleotides was studied using a mutation (G334D) in the Kir6.2 subunit of the channel that renders K(ATP) channels insensitive to nucleotide inhibition and has no apparent effect on their gating. K(ATP) channels carrying this mutation (Kir6.2-G334D/SUR1 channels) were activated by MgATP and MgADP with an EC(50) of 112 and 8 µM, respectively. This activation was largely suppressed by mutation of the Walker A lysines in the nucleotide-binding domains of SUR1: the remaining small (∼10%), slowly developing component of MgATP activation was fully inhibited by the lipid kinase inhibitor LY294002. The EC(50) for activation of Kir6.2-G334D/SUR1 currents by MgADP was lower than that for MgATP, and the time course of activation was faster. The poorly hydrolyzable analogue MgATPγS also activated Kir6.2-G334D/SUR1. AMPPCP both failed to activate Kir6.2-G334D/SUR1 and to prevent its activation by MgATP. Maximal stimulatory concentrations of MgATP (10 mM) and MgADP (1 mM) exerted identical effects on the single-channel kinetics: they dramatically elevated the open probability (P(O) > 0.8), increased the mean open time and the mean burst duration, reduced the frequency and number of interburst closed states, and eliminated the short burst states. By comparing our results with those obtained for wild-type K(ATP) channels, we conclude that the MgADP sensitivity of the wild-type K(ATP) channel can be described quantitatively by a combination of inhibition at Kir6.2 (measured for wild-type channels in the absence of Mg(2+)) and activation via SUR1 (determined for Kir6.2-G334D/SUR1 channels). However, this is not the case for the effects of MgATP.

The ATPase activities of sulfonylurea receptor 2A and sulfonylurea receptor 2B are influenced by the C-terminal 42 amino acids.

Unusually among ATP-binding cassette proteins, the sulfonylurea receptor (SUR) acts as a channel regulator. ATP-sensitive potassium channels are octameric complexes composed of four pore-forming Kir6.2 subunits and four regulatory SUR subunits. Two different genes encode SUR1 (ABCC8) and SUR2 (ABCC9), with the latter being differentially spliced to give SUR2A and SUR2B, which differ only in their C-terminal 42 amino acids. ATP-sensitive potassium channels containing these different SUR2 isoforms are differentially modulated by MgATP, with Kir6.2/SUR2B being activated more than Kir6.2/SUR2A. We show here that purified SUR2B has a lower ATPase activity and a 10-fold lower K(m) for MgATP than SUR2A. Similarly, the isolated nucleotide-binding domain (NBD) 2 of SUR2B was less active than that of SUR2A. We further found that the NBDs of SUR2B interact, and that the activity of full-length SUR cannot be predicted from that of either the isolated NBDs or NBD mixtures. Notably, deletion of the last 42 amino acids from NBD2 of SUR2 resulted in ATPase activity resembling that of NBD2 of SUR2A rather than that of NBD2 of SUR2B: this might indicate that these amino acids are responsible for the lower ATPase activity of SUR2B and the isolated NBD2 of SUR2B. We suggest that the lower ATPase activity of SUR2B may result in enhanced duration of the MgADP-bound state, leading to channel activation.

Asymmetric switching in a homodimeric ABC transporter: a simulation study.

ABC transporters are a large family of membrane proteins involved in a variety of cellular processes, including multidrug and tumor resistance and ion channel regulation. Advances in the structural and functional understanding of ABC transporters have revealed that hydrolysis at the two canonical nucleotide-binding sites (NBSs) is co-operative and non-simultaneous. A conserved core architecture of bacterial and eukaryotic ABC exporters has been established, as exemplified by the crystal structure of the homodimeric multidrug exporter Sav1866. Currently, it is unclear how sequential ATP hydrolysis arises in a symmetric homodimeric transporter, since it implies at least transient asymmetry at the NBSs. We show by molecular dynamics simulation that the initially symmetric structure of Sav1866 readily undergoes asymmetric transitions at its NBSs in a pre-hydrolytic nucleotide configuration. MgATP-binding residues and a network of charged residues at the dimer interface are shown to form a sequence of putative molecular switches that allow ATP hydrolysis only at one NBS. We extend our findings to eukaryotic ABC exporters which often consist of two non-identical half-transporters, frequently with degeneracy substitutions at one of their two NBSs. Interestingly, many residues involved in asymmetric conformational switching in Sav1866 are substituted in degenerate eukaryotic NBS. This finding strengthens recent suggestions that the interplay of a consensus and a degenerate NBS in eukaroytic ABC proteins pre-determines the sequence of hydrolysis at the two NBSs.

Review. SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator.

SUR1 is an ATP-binding cassette (ABC) transporter with a novel function. In contrast to other ABC proteins, it serves as the regulatory subunit of an ion channel. The ATP-sensitive (KATP) channel is an octameric complex of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits, and it links cell metabolism to electrical activity in many cell types. ATPase activity at the nucleotide-binding domains of SUR results in an increase in KATP channel open probability. Conversely, ATP binding to Kir6.2 closes the channel. Metabolic regulation is achieved by the balance between these two opposing effects. Precisely how SUR1 talks to Kir6.2 remains unclear, but recent studies have identified some residues and domains that are involved in both physical and functional interactions between the two proteins. The importance of these interactions is exemplified by the fact that impaired regulation of Kir6.2 by SUR1 results in human disease, with loss-of-function SUR1 mutations causing congenital hyperinsulinism and gain-of-function SUR1 mutations leading to neonatal diabetes. This paper reviews recent data on the regulation of Kir6.2 by SUR1 and considers the molecular mechanisms by which SUR1 mutations produce disease.

Analysing steroid modulation of BK(Ca) channels reconstituted into planar lipid bilayers.

A number of recent studies have described the activation of BK(Ca) channels by steroid hormones such as estrogen. The proposed mechanisms are diverse and include both the direct interaction with the ion channel subunits and the stimulation via receptor activation and cell signalling pathways. To investigate the activation of BK(Ca) channels by estrogen we devised a cell-free system by incorporating recombinant channels of known subunit composition into artificial bilayers and recorded single channel currents. This chapter describes the methods used to prepare purified membrane fractions from cultured cells and the construction of artificial phospholipids bilayers for the incorporation and recording of ion channels.

A mutation (R826W) in nucleotide-binding domain 1 of ABCC8 reduces ATPase activity and causes transient neonatal diabetes.

Activating mutations in the pore-forming Kir6.2 (KCNJ11) and regulatory sulphonylurea receptor SUR1 (ABCC8) subunits of the K(ATP) channel are a common cause of transient neonatal diabetes mellitus (TNDM). We identified a new TNDM mutation (R826W) in the first nucleotide-binding domain (NBD1) of SUR1. The mutation was found in a region that heterodimerizes with NBD2 to form catalytic site 2. Functional analysis showed that this mutation decreases MgATP hydrolysis by purified maltose-binding protein MBP-NBD1 fusion proteins. Inhibition of ATP hydrolysis by MgADP or BeF was not changed. The results indicate that the ATPase cycle lingers in the post-hydrolytic MgADP.P(i)-bound state, which is associated with channel activation. The extent of MgADP-dependent activation of K(ATP) channel activity was unaffected by the R826W mutation, but the time course of deactivation was slowed. Channel inhibition by MgATP was reduced, leading to an increase in resting whole-cell currents. In pancreatic beta cells, this would lead to less insulin secretion and thereby diabetes.