A Kir6 .2 mutation causing severe functional effects in vitro produces neonatal diabetes without the expected neurological complications Aims/hypothesis Heterozygous activating mutations in the pancreatic ATP-sensitive K + channel cause permanent neonatal diabetes mellitus -LRB- PNDM -RRB- . This results from a decrease in the ability of ATP to close the channel , which thereby suppresses insulin secretion . PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy . We identified a heterozygous mutation -LRB- L164P -RRB- in the pore-forming -LRB- Kir6 .2 -RRB- subunit of the channel in three unrelated patients and examined its functional effects . Introduction ATP-sensitive potassium -LRB- KATP -RRB- channels link cellular metabolism to membrane electrical activity by regulating K + fluxes across the plasma membrane -LSB- 1 -RSB- . They are found in multiple tissues but are of particular importance in regulating insulin secretion from pancreatic beta cells -LSB- 1 , 2 -RSB- . At substimulatory glucose concentrations , KATP channels are open so that the membrane potential is hyperpolarised and Ca2 + influx and insulin secretion are prevented -LSB- 3 -RSB- . Glucose metabolism enhances ATP production , resulting in closure of KATP channels , stimulation of electrical activity , opening of voltage-gated Ca2 + channels and exocytosis of insulin granules . KATP channels are hetero-octamers of Kir6.x and sulfonylurea receptor -LRB- SUR -RRB- subunits -LSB- 4 -- 6 -RSB- . Four inwardly rectifying subunits -LRB- Kir6 .2 in pancreatic beta cells -RRB- form the pore of the channel and four auxiliary SUR subunits -LRB- SUR1 in pancreatic beta cells -RRB- associate with the tetrameric pore and regulate its gating -LSB- 7 -- 9 -RSB- . Binding and/or hydrolysis of Mg-nucleotides by the intracellular nucleotide-binding domains -LRB- NBDs -RRB- of SUR produces channel opening -LSB- 10 -- 13 -RSB- . It is believed that reciprocal changes in the intracellular concentrations of ATP and MgADP are involved in the metabolic regulation of KATP channels . Over the last 4 years , many different missense mutations in the genes encoding Kir6 .2 -LRB- KCNJ11 -RRB- and SUR1 -LRB- ABCC8 -RRB- have been shown to cause permanent neonatal diabetes mellitus -LRB- PNDM -RRB- . This is a rare disorder characterised by high blood glucose levels that manifests within the first 6 months of life . A subgroup of mutations were associated with a more severe clinical profile characterised by Delayed development of motor , intellectual and social skills , muscle weakness , Epilepsy , facial dysmorphism and Neonatal Diabetes -LRB- DEND syndrome -RRB- -LSB- 14 -- 16 -RSB- . Mutations in Kir6 .2 have also been found to cause a remitting relapsing form of neonatal diabetes that resembles transient neonatal diabetes mellitus -LSB- 17 , 18 -RSB- . All Kir6 .2 mutations analysed to date were heterozygous and most were de novo mutations -LSB- 14 , 19 -- 21 -RSB- . In most cases , the diabetes they caused could be successfully treated with sulfonylureas -LSB- 14 , 20 , 22 -RSB- , which directly close KATP channels by binding to the SUR1 subunit of the channel -LSB- 23 -RSB- . All PNDM mutations result in a reduced KATP channel sensitivity to inhibition by MgATP in vitro -LSB- 15 , 24 -RSB- . This is expected to cause an increased KATP current amplitude and reduced insulin secretion . Studies to date suggest that the severity of the clinical phenotype reflects the extent of the reduction of the channel ATP sensitivity . Thus , mutations that produce a small increase in KATP current in the presence of physiological concentrations of MgATP -LRB- 1 -- 5 mmol/l -RRB- lead to PNDM , whereas mutations that cause a larger increase in KATP current give rise to DEND syndrome -LSB- 15 -RSB- . In this paper , we identify a KCNJ11 mutation -LRB- L164P -RRB- that causes neonatal diabetes without obvious neurological complications . In functional studies , we show that the L164P mutation produces a large increase in the resting whole-cell current and a marked reduction in KATP channel sensitivity to inhibition by ATP . These effects are a secondary consequence of an increase in the channel open probability -LRB- Po -RRB- produced by the mutation . Surprisingly , other mutations that produce a similar increase in Po cause DEND syndrome . The L164P mutant channel was also far less blocked by the sulfonylurea tolbutamide , which explains why the patients were unable to transfer to glibenclamide therapy . Methods Participants Informed consent was obtained from all individuals investigated -LRB- or from their parents if they were children -RRB- . Molecular genetic analysis Genomic DNA was extracted from peripheral lymphocytes using standard procedures . The KCNJ11 gene was amplified and sequenced as described -LSB- 25 -RSB- . Other family members were also tested for the novel mutation . Family relationships were confirmed using a combination of six microsatellites on chromosome 11 : D11S902 , D11S419 , D11S1397 , D11S1901 , D11S921 and D11S1888 . Oocyte preparation Female Xenopus laevis were anaesthetised with ethyl 3-aminobenzoate methanesulfonate salt -LRB- MS222 ; 2 g/l added to the water -RRB- . One ovary was removed via a mini-laparotomy , the incision sutured and the animal allowed to recover . Subsequently , animals were operated on for a second time , but under terminal anaesthesia . Immature stage V -- VI oocytes were incubated for 60 min with 1 mg/ml collagenase -LRB- Type V ; Sigma , Poole , UK -RRB- and manually defolliculated . All procedures were carried out in accordance with UK Home Office Legislations and the University of Oxford ethical guidelines . Oocytes were coinjected with ∼ 0.8 ng wild-type or mutant Kir6 .2 mRNA and ∼ 4 ng mRNA encoding SUR . The final injection volume was 50 nl per oocyte . Isolated oocytes were maintained in Barth 's solution and studied 1 -- 4 days after injection . Electrophysiology Wild-type or mutant Kir6 .2 -LRB- GenBank D50581 -RRB- were coexpressed with SUR1 in Xenopus oocytes -LRB- GenBank L40624 -RRB- as described -LSB- 26 -RSB- . Because all the patients were heterozygous for the L164P mutation , their pancreatic beta cells will contain a mixture of wild-type and mutant Kir6 .2 subunits . To simulate this heterozygosity , we coinjected Xenopus oocytes with a 1:1 mixture of mutant and wild-type Kir6 .2 together with SUR1 mRNA . This is expected to give rise to a mixed population of channels composed of homomeric wild-type channels , homomeric mutant channels and heteromeric channels containing between one and three mutant subunits -LSB- 21 -RSB- . We refer to this global channel population as heterozygous channels.Whole-cell currents were recorded using a two-electrode voltage clamp in response to voltage steps of ± 20 mV from a holding potential of − 10 mV , filtered at 1 kHz and digitised at 4 kHz . Oocytes were perfused with a solution containing -LRB- in mmol/l -RRB- : 90 KCl , 1 MgCl2 , 1.8 CaCl2 and 5 HEPES -LRB- pH 7.4 with KOH -RRB- . Metabolic inhibition was produced by 3 mmol/l sodium azide.Macroscopic currents were recorded from giant inside-out patches using an EPC10 amplifier -LRB- List Medical Electronics , Darmstadt , Germany -RRB- controlled with Pulse v8 .74 software -LRB- Heka Electronik , Lambrecht , Germany -RRB- . Macroscopic currents were elicited by 3 s voltage ramps from − 110 to +100 mV -LRB- holding potential 0 mV -RRB- , or recorded at a constant potential of − 60 mV . They were filtered at 0.5 kHz and digitised at 1 kHz . The pipette solution contained -LRB- mmol/l -RRB- : 140 KCl , 1.2 MgCl2 , 2.6 CaCl2 , 10 HEPES -LRB- pH 7.4 with KOH -RRB- . The internal -LRB- bath -RRB- solution contained -LRB- mmol/l -RRB- : 107 KCl , 1 K2SO4 , 2 MgCl2 , 10 EGTA , 10 HEPES -LRB- pH 7.2 with KOH -RRB- and Mg-nucleotides as indicated . Experiments were conducted at 20 -- 22 °C . Solutions were changed using a local perfusion system consisting of tubes of 200 μm diameter into which the tip of the patch pipette was inserted.Nucleotide concentration -- inhibition curves were fit with the Hill equation : where G is the KATP conductance in the presence of ATP , Gc is the KATP conductance in the absence of the nucleotide , -LSB- ATP -RSB- is the ATP concentration , IC50 is the nucleotide concentration at which inhibition is half maximal and h is the slope factor -LRB- Hill coefficient -RRB- . Single-channel currents were measured at − 60 mV , filtered at 5 kHz and digitised at 20 kHz . Unitary amplitude and Po were measured from the Gaussian fit to all-points amplitude histograms of tracts of current of 30 -- 90 s duration.Data were analysed with in-house routines developed in the IgorPro platform -LRB- Wavematrics , Portland , OR , USA -RRB- . Data are given as means ± SEM in the text and in the figures . Statistical significance was evaluated using a two-tailed Student t test and p < 0.05 taken to indicate a significant difference . Results Patient characteristics and genetics Three unrelated probands with permanent neonatal diabetes were shown to be heterozygous for the KCNJ11 gene mutation L164P -LRB- c. 491T > C , p.Leu164Pro -RRB- . All were female and had unaffected parents of different ethnic origin . Two patients have been reported previously -LSB- 27 , 28 -RSB- . Mutation testing and microsatellite analysis of DNA from both parents and the child for two of the cases confirmed that the mutation had arisen de novo -LRB- the parents of the third case were not available for testing -RRB- . None of the patients had any neurological complications or obvious developmental delay.The first patient is currently 8 years old . She was born in Singapore from Sri Lankan parents , at 38 weeks of gestation with a birthweight of 2.6 kg . She developed diabetes at 30 weeks of age . When she came to Australia at the age of 6.5 years , her HbA1c level was elevated -LRB- 7.4 % -RRB- and her diabetes required insulin treatment -LRB- 0.45 U kg − 1 day − 1 -RRB- . Glibenclamide treatment -LRB- 1.0 mg kg − 1 day − 1 -RRB- was trialled for 2 months -LRB- at 7 years of age -RRB- but did not produce a decrease in her insulin requirement . She continues to require insulin -LRB- up to 0.7 U kg − 1 day − 1 -RRB- with an HbA1c ranging from 7.2 to 8.2 % . She has normal developmental milestones , her neurological examination is normal and she has no evidence of diabetic complications.The second patient , born in Australia of Afghan origin , is currently 2.7 years old . She was born at term following an uneventful pregnancy , with a birthweight of 2.7 kg . She presented at 8 weeks in diabetic ketoacidosis and was subsequently treated -LRB- at 2.5 months for 4 weeks , then at 5 months for 3 months -RRB- with glibenclamide -LRB- up to 1.1 mg kg − 1 day − 1 , regimens of two times per day or three times per day -RRB- , but had no reduction in insulin requirement -LRB- 0.4 U kg − 1 day − 1 -RRB- -LSB- 28 -RSB- . Her mean HbA1c since diagnosis has been 7.6 % -LRB- range 7.2 -- 8.7 % -RRB- . Assessment at 5 months by a neurologist was entirely normal , with a normal EEG . Subsequently , she has had normal developmental milestones and her neurological examinations have been normal at all follow up visits every 3 months . Diabetic complications have not been assessed due to the young age of the patient.The third patient is from Slovakia -LSB- 27 -RSB- . She was born at term with a birthweight of 2.6 kg , and was diagnosed with diabetes at 5 weeks of age . She is currently 20 years old and is treated with insulin -LRB- 1.24 U kg − 1 day − 1 -RRB- . Glibenclamide transfer was not attempted because the patient also suffers from hepatitis C . She has poor glycaemic control , with an HbA1c level of 15.2 % , and various diabetic complications including retinopathy and nephropathy . Psychomotoric development in childhood was normal . A neurological examination suggested the presence of diabetic motoric polyneuropathy in the lower extremities but there were no further neurological findings . Although her IQ was not tested , she successfully completed a specialised business school training suggesting normal mental development . Functional analysis : effects on whole-cell KATP currents We examined the functional effects of the L164P mutation by heterologous expression in Xenopus oocytes . When wild-type Kir6 .2 / SUR1 channels are expressed in oocytes they are normally closed because they are inhibited by resting intracellular ATP concentrations -LRB- -LSB- ATP -RSB- i -RRB- . They can be opened , however , by azide -LRB- 3 mmol/l ; Fig. 1 -RRB- , a metabolic inhibitor that lowers -LSB- ATP -RSB- i -LSB- 29 -RSB- . Heterozygous L164P -LRB- hetL164P -RRB- currents were about 18-fold larger at rest than wild-type currents , but increased twofold on metabolic inhibition indicating that the channel is only partially closed at resting ATP levels -LRB- Fig. 1 -RRB- . In contrast , homomeric L164P -LRB- homL164P -RRB- channels displayed a much greater resting current and were little affected by metabolic inhibition -LRB- Fig. 1 -RRB- . Fig. 1Whole-cell KATP current . Mean steady state whole-cell currents evoked by voltage steps from − 10 to − 30 mV before -LRB- control , white bars -RRB- and after application of 3 mmol/l azide -LRB- grey bars -RRB- and in the presence of 3 mmol/l azide plus 0.5 mmol/l tolbutamide -LRB- black bars -RRB- . The number of oocytes was five to seven in each case . G , KATP conductance ; Gc , KATP conductance expressed relative to the conductance in the absence of the nucleotide . WT , wild-type ; hetL164P and homL164P channels as indicated The sulfonylurea tolbutamide -LRB- 0.5 mmol/l -RRB- blocked whole-cell KATP currents by 98 % but had no effect on homL164P currents -LRB- 4 % block -RRB- . HetL164P channels were blocked by only 54 % -LRB- Fig. 1 -RRB- . Functional analysis : effects on KATP channel ATP sensitivity The increase in resting whole-cell KATP currents suggests that the L164P mutation may reduce the channel ATP sensitivity , as found for other PNDM mutations -LSB- 21 -RSB- . We first measured the ATP sensitivity of wild-type and mutant KATP currents in the absence of Mg2 + , to isolate the effects of ATP on Kir6 .2 -LRB- in the absence of Mg2 + , ATP does not interact with SUR1 -LSB- 11 -RSB- -RRB- . Both homL164P and hetL164P channels had severely impaired ATP sensitivity . HomL164P channels were not blocked at all by ATP , even at concentrations as high as 10 mmol/l . The concentration -- inhibition curve for hetL164P showed a striking shift to higher ATP concentrations , with an IC50 of about 100 μmol/l , and a marked pedestal of unblocked current at very high ATP concentrations . The data were best fitted by assuming that in the heterozygous state about 20 % of channels are never closed by ATP -LRB- Fig. 2a , b and Table 1 -RRB- . Fig. 2ATP-inhibition of L164P channels is less that that of wild-type channels . a , c Currents recorded in inside-out patches excised from Xenopus oocytes expressing hetKir6.2-L164P / SUR1 -LRB- hetL164P -RRB- or homKir6.2-L164P / SUR1 -LRB- homL164P -RRB- channels , as indicated , in response to 3 s voltage ramps from − 110 to +100 mV . ATP -LRB- 10 mmol/l -RRB- was applied as indicated by the horizontal bars in the absence -LRB- a -RRB- or presence -LRB- c -RRB- of 2 mmol/l Mg2 + . b , d , mean relationship between -LSB- ATP -RSB- and KATP conductance -LRB- G -RRB- , expressed relative to the conductance in the absence of the nucleotide -LRB- Gc -RRB- , for wild-type -LRB- white circles , n = 9 -RRB- , hetL164P -LRB- white/black circles , n = 6 -RRB- or homL164P -LRB- black circles , n = 4 -RRB- channels . Experiments were carried out in the absence -LRB- b -RRB- or presence -LRB- d -RRB- of 2 mmol/l Mg2 + . The continuous lines through the black circles were drawn by eye . The smooth curves are the best fit to the Hill equation with IC50 of 11 μmol/l -LRB- wild-type -RRB- and 100 μmol/l -LRB- hetL164P -RRB- -LRB- b , 0 mmol/l Mg2 + -RRB- or IC50 of 16 μmol/l -LRB- wild-type -RRB- and 122 μmol/l -LRB- hetL164P -RRB- -LRB- d , 2 mmol/l Mg2 + -RRB- Table 1ATP sensitivity of wild-type and mutant channels IC50 -LRB- Mg-free -RRB- h -LRB- Mg-free -RRB- IC50 -LRB- 2 mmol/l Mg2 + -RRB- h -LRB- 2 mmol/l Mg2 + -RRB- A -LRB- 2 mmol/l Mg2 + -RRB- % Imax -LRB- 3 mmol/l MgATP -RRB- Wild-type9.6 ± 1.61.30 ± 0.0815.8 ± 3.00.99 ± 0.05 n.a. 0.01 ± 0.01 hetL164P100 ± 8 * 1.10 ± 0.05118 ± 29 * 1.16 ± 0.340.29 ± 0.07 * 33.6 ± 3.4 * homL164Pn.a.n.a.n.a.n.a.n.a .98 ± 1 * Values are means ± SEM . The number of patches was four to nine in each case.A The fraction of unblocked current used to fit the ATP concentration -- inhibition relationship ; h Hill coefficient ; IC50 , ATP concentration -LRB- μmol/l -RRB- producing half-maximal inhibition ; % Imax the per cent unblocked current in the presence of 3 mmol/l MgATP ; n.a. not applicable -LRB- as no block -RRB- . * p < 0.05 vs wild-type Molecular mechanism of the reduced ATP sensitivity Mutations that reduce the ATP sensitivity of the KATP channel can act in several ways . They may prevent ATP binding directly . They may impair the mechanism by which nucleotide binding is coupled to channel gating . They may also stabilise the intrinsic open state of the channel -LRB- i.e. that in the absence of ATP -RRB- , which shifts the gating equilibrium in the presence of ATP towards channel opening and thus indirectly reduces the channel ATP sensitivity -LRB- e.g. -LSB- 15 , 30 , 31 -RSB- -RRB- . In a structural model of Kir6 .2 -LSB- 32 -RSB- , L164P lies within the channel pore , at a considerable distance from the ATP-binding site -LRB- Fig. 3 -RRB- . It is not predicted to interact directly with ATP . We therefore examined whether the L164P mutation alters intrinsic gating . Experiments were carried out in the absence of ATP , where intrinsic gating can be assessed . The L164P mutation had no effect on single-channel current amplitude -LRB- Table 2 -RRB- . However , the intrinsic Po was markedly increased , being 0.86 -LRB- n = 6 -RRB- for homL164P compared with 0.4 -LRB- n = 4 -RRB- for wild-type channels -LRB- Fig. 4 and Table 2 -RRB- . This suggests that , at least in part , the L164P mutation alters channel ATP sensitivity indirectly , via an increase in Po . Fig. 3Homology model of Kir6 .2 -LSB- 32 -RSB- . For clarity , only two subunits are shown . ATP -LRB- yellow -RRB- is shown in its binding site . Residue L164 is shown in redFig . 4The L164P mutation enhances single-channel activity . Representative single KATP channel currents recorded at − 60 mV in inside-out patches from oocytes expressing wild-type or homL164P channels , as indicated . Currents were recorded in the absence of Mg2 + and nucleotidesTable 2Single-channel parameters for wild-type and homL164P channels Poi -LRB- pA -RRB- Wild-type0.39 ± 0.054.0 ± 0.1 homL164P0 .86 ± 0.01 * 4.1 ± 0.1 Mean ± SEM values of intrinsic open probability -LRB- Po -RRB- and single-channel current -LRB- i -RRB- measured at − 60 mVThe number of patches was five to six in each case * p < 0.05 vs wild-type Effects on KATP channel ATP sensitivity in the presence of Mg2 + Previous studies have shown that Kir6 .2 mutations associated with neonatal diabetes may not only decrease the sensitivity of Kir6 .2 to ATP , but can also enhance channel activation by Mg-nucleotides -LSB- 24 -RSB- . We therefore explored the effect of the L164P mutation on the ATP sensitivity in the presence of 2 mmol/l Mg2 + . Surprisingly , there was no difference in the effect of ATP in the presence and absence of Mg2 + on homL164P channels -LRB- Fig. 2c , d -RRB- . This contrasts with what has been observed for mutations in the ATP-binding site that completely abolished ATP inhibition in the absence of Mg2 + , such as R50P and G334D : channels containing these mutations were activated by MgATP -LSB- 33 , 34 -RSB- . The IC50 for ATP inhibition of hetL164P channels was also not significantly affected by Mg2 + , although the pedestal of unblocked current at high ATP concentrations was increased by about 50 % -LRB- Table 1 -RRB- . At 3 mmol/l MgATP , a concentration within the physiological range , the unblocked current was 34 % , substantially greater than that found for wild-type channels -LRB- < 1 % ; Table 1 -RRB- . Functional analysis : effects on MgADP sensitivity The lack of MgATP activation of hetL164P channels could be due to reduced functional coupling between SUR1 and Kir6.2-L164P , or to reduced MgATP binding/hydrolysis at the NBDs of SUR1 . To explore the former possibility , we measured the ability of MgADP to activate hetL164P channels preblocked by 100 μmol/l MgATP . It was necessary to preblock the channels as the mutant channel has an open probability close to maximal and further activation is therefore not possible . Figure 5 shows that 30 μmol/l MgADP activates wild-type currents by 3.7 ± 1.8 fold -LRB- n = 3 -RRB- and that 100 μmol/l MgADP did not produce any further increase -LRB- 3.7 ± 1.2-fold , n = 3 -RRB- . In contrast , hetL164P channels were activated less : 1.4 ± 0.1 fold -LRB- n = 3 -RRB- by 30 μmol/l MgADP and 1.5 ± 0.1 fold -LRB- n = 4 -RRB- by 100 μmol/l MgADP . Fig. 5Sensitivity to MgADP of wild-type and hetL164P channels . a Representative currents -LRB- I -RRB- recorded at − 60 mV from inside-out excised membrane patches from Xenopus oocytes expressing wild-type or hetL164P channels , as indicated . Patches were exposed to 100 μmol/l ADP in the continuous presence of 100 μmol/l ATP : 2 mmol/l Mg2 + was present throughout . b Mean current in the presence of 100 μmol/l MgATP or 30 or 100 μmol/l MgADP plus 100 μmol/l MgATP , normalised to the current in the absence of nucleotides for wild-type -LRB- grey bars -RRB- and hetL164P -LRB- black bars -RRB- channels . Bars indicate means ± SEM . The number of patches was three to four in each case Despite the fact that 100 μmol/l MgATP blocked mutant channels less , the lower extent of activation of hetL164P channels by 30 μmol/l MgADP is not due to the fact that channel activity is already maximal , as the amplitude is still significantly less than that in control solution -LRB- Fig. 5 -RRB- . Discussion We describe the clinical and functional effects of a KCNJ11 mutation , L164P , associated with permanent neonatal diabetes . This mutation causes a marked reduction in KATP channel inhibition by ATP primarily by stabilising the open state of the channel . This leads to an increase in the whole-cell KATP current , and in beta cells is expected to result in a reduction in insulin secretion . Surprisingly , unlike other mutations that reduced the channel ATP sensitivity by a similar amount , no motor or mental developmental delay was associated with the L164P mutation . Structural considerations and molecular basis for reduced ATP sensitivity In a homology model of Kir6 .2 -LSB- 32 -RSB- , L164 lies partway along the permeation pathway , 35 Å away from the ATP-binding site -LRB- Fig. 3 -RRB- . It is therefore unlikely that it acts by reducing ATP binding directly . The side-chains of L164 point into the pore , forming a hydrophobic girdle that is narrow enough to prevent the passage of water and hydrated K + ions -LSB- 35 -RSB- , which suggests that L164 may form a hydrophobic gate within the pore . Functional studies support this idea . Following mutation of L164 to cysteine , cadmium ions were able to block the KATP channel with high affinity -LSB- 36 , 37 -RSB- , suggesting that the four cysteines -LRB- one on each subunit -RRB- come together close enough to form a high-affinity binding site for Cd2 + . These data indicate that the side-chain of L164 must face into the pore , and that the pore is very narrow at this position . Substitution of a proline for L164 is expected to produce a kink in the α-helix -LSB- 38 -RSB- and disrupt the hydrophobic gate . It is therefore not surprising that the L164P mutation caused a dramatic effect on the Po . Mutation of L164 to cysteine , alanine , valine , threonine or glycine also produces a very large increase in Po -LSB- 30 , 36 , 37 , 39 -RSB- , consistent with the importance of this residue in channel gating . The fact that the L164P mutation enhanced Po can explain , in part , the reduced ATP sensitivity of the channel . It is also possible that the mutation may have effects additional to stabilisation of the channel open state . Effect of Mg2 + on Kir6.2-L164P / SUR1 channel ATP sensitivity In contrast to all PNDM mutations studied to date -LSB- 24 -RSB- , MgATP was unable to enhance the activity of either homL164P or hetL164P channels . The lack of MgATP activation of homL164P channels may be due to the fact that Po is already very high and thus there is no scope for further activation . However , little MgATP activation was also observed for hetL164P channels . This is in marked contrast to other mutations that caused enhanced Po -LRB- I296L , -LSB- 16 -RSB- , V59G -LSB- 15 -RSB- -RRB- where MgATP activated both homomeric and heterozygous channels . It is therefore possible that the L164P mutation reduces the efficacy of coupling between SUR1 and Kir6 .2 , and thereby decreases the ability of MgATP to stimulate channel activity . In support of this idea , MgADP activation of hetL164P channels was also reduced . This may explain the inability of Mg2 + to reduce ATP inhibition of hetL164P channels , since it is well established that MgATP must be hydrolysed to MgADP to stimulate channel activity.The reason for the lack of Mg-nucleotide activation of hetL164P channels is unclear . Because L164 lies within the pore , far away from the NBDs of SUR1 , the effect must be mediated allosterically . It is possible that this is mediated by an interaction between the backbone of L164 itself , or transmembrane domain 2 -LRB- within which L164 lies -RRB- , and the transmembrane domains of SUR1 . In the absence of an atomic resolution structure of the KATP channel complex , however , this can not be definitely determined.HomL164P channels were not blocked by tolbutamide , as expected because of their high Po -LSB- 40 -RSB- . The lower efficacy of tolbutamide on hetL164P channels -LRB- about 50 % block -RRB- may reflect the enhanced Po of channels within the heterozygous population containing mutant subunits . It could also reflect impaired coupling between SUR1 and Kir6 .2 , such as that found for MgADP . Clinical implications Previous studies of KCNJ11 mutations have suggested that there is a good correlation between the percentage of current that remains unblocked in the presence of 3 mmol/l MgATP and the clinical phenotype . Namely , currents that are > 30 % of maximal are associated with DEND syndrome , and those that lie between 5 -- 10 % of maximal with neonatal diabetes alone , compared with a current of < 1 % for wild-type channels -LRB- Fig. 6 -RRB- . The L164P mutation does not conform to this simple relationship between the functional effects of the mutation and the clinical phenotype . The magnitude of the hetL164P current in 3 mmol/l MgATP was 36 % , yet none of the patients had extra-pancreatic symptoms . Fig. 6Mean KATP current -LRB- expressed as a % of maximum -RRB- measured in the presence of 3 mmol/l MgATP from inside-out patches expressing heterozygous mutant channels as indicated . The dashed line indicates the maximal current amplitude normally associated with PNDM . iDEND , intermediate DEND syndrome -LRB- i.e. neonatal diabetes with developmental delay -LSB- 21 -RSB- -RRB- . Data for wild-type -LRB- WT -RRB- and Kir6.2-R201H channels are from -LSB- 14 -RSB- , for Y330C and F333I from -LSB- 26 -RSB- , for Q52R , V59G and R201C from -LSB- 15 -RSB- , for V59M from -LSB- 24 -RSB- , for I296L from -LSB- 16 -RSB- and for G334D from -LSB- 34 -RSB- The reason for this anomaly is not clear , particularly as the resting whole-cell current was also very large . Previous studies have also indicated that such large whole-cell currents are invariably associated with more severe clinical symptoms . The lack of responsiveness of the patient to sulfonylureas is consistent with the very large increase in Po and the resting whole-cell current . It is therefore likely that hetL164P channels expressed in Xenopus oocytes are a reasonable model for the pancreatic beta cell KATP channels of the patients . We are therefore forced to conclude that compensatory mechanisms must ameliorate the extra-pancreatic effects of this mutation . The fact that the patients had very different ethnic origins suggests the compensatory mechanism is unlikely to reflect a shared genetic background : however , it does appear to be specific to the L164P mutation , which is unique -LRB- to date -RRB- in having a marked functional effect in vitro despite causing neonatal diabetes without neurological complications.Importantly , a maximally effective concentration of tolbutamide only blocked hetL164P channels by 50 % . All patients to date whose channels are blocked by < 65 % have not been able to transfer from insulin treatment to sulfonylurea therapy . This suggested that our patients would be unable to transfer to sulfonylurea therapy , as indeed was found to be the case . This is likely to be due to the fact that L164P destabilises the long closed state of the channel , to which sulfonylureas preferentially bind , and which is rarely entered in channels with enhanced Po . Our results further suggest that not all patients with permanent neonatal diabetes will necessarily respond to sulfonylurea treatment , and indicates that knowledge of the functional effect of the mutation is helpful for predicting the drug response in patients .

O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O


The effect of ambient temperature on gross-efficiency in cycling Time-trial performance deteriorates in the heat . This might potentially be the result of a temperature-induced decrease in gross-efficiency -LRB- GE -RRB- . The effect of high ambient temperature on GE during cycling will be studied , with the intent of determining if a heat-induced change in GE could account for the performance decrements in time trial exercise found in literature . Ten well-trained male cyclists performed 20-min cycle ergometer exercise at 60 % -LRB- power output at which VO2max was attained -RRB- in a thermo-neutral climate -LRB- N -RRB- of 15.6 ± 0.3 °C , 20.0 ± 10.3 % RH and a hot climate -LRB- H -RRB- of 35.5 ± 0.5 °C , 15.5 ± 3.2 % RH . GE was calculated based on VO2 and RER . Skin temperature -LRB- Tsk -RRB- , rectal temperature -LRB- Tre -RRB- and muscle temperature -LRB- Tm -RRB- -LRB- only in H -RRB- were measured . GE was 0.9 % lower in H compared to N -LRB- 19.6 ± 1.1 % vs. 20.5 ± 1.4 % -RRB- -LRB- P < 0.05 -RRB- . Tsk -LRB- 33.4 ± 0.6 °C vs. 27.7 ± 0.7 °C -RRB- and Tre -LRB- 37.4 ± 0.6 °C vs. 37.0 ± 0.6 °C -RRB- were significantly higher in H. Tm was 38.7 ± 1.1 °C in H. GE was lower in heat . Tm was not high enough to make mitochondrial leakage a likely explanation for the observed reduced GE . Neither was the increased Tre . Increased skin blood flow might have had a stealing effect on muscular blood flow , and thus impacted GE . Cycling model simulations showed , that the decrease in GE could account for half of the performance decrement . GE decreased in heat to a degree that could explain at least part of the well-established performance decrements in the heat . Introduction Performance decrements are widely observed during exercise in the heat compared to thermo-neutral circumstances -LRB- Febbraio et al. 1996 ; Gonzalez Alonso et al. 1999 ; Parkin et al. 1999 ; Romer et al. 2003 ; Tatterson et al. 2000 ; Tucker et al. 2004 -RRB- . It has been shown that fatigue at exhaustion is related to factors associated with thermoregulation and hyperthermia -LRB- Febbraio et al. 1996 ; Nielsen et al. 1990 ; Parkin et al. 1999 -RRB- . Time trial performance and fatigue -LRB- evidenced by decrements in power output over the race -RRB- and finish time have received much less attention in the literature . Accordingly , the present study will focus on the effect of a hot ambient temperature on thermal and cardio respiratory strain during the exercise that might contribute to the well-established decrease in power output . Tatterson et al. -LRB- 2000 -RRB- measured time trial performance on a 30 min self-paced cycling time trial in 32 °C , 60 % RH versus 23 °C , 60 % RH . They observed that power output was reduced in the heat by 6.5 % -LRB- 345 ± 9 W vs. 323 ± 8 W -RRB- . Tucker et al. -LRB- 2004 -RRB- compared 20 km time trials in 35 and 15 °C and found a comparable reduction in average power output in the heat of about 6.3 % -LRB- 255 ± 47 W vs. 272 ± 45 W -RRB- . This change in average power output led to a difference in final time on a 20 km time trial of about 48 s -LRB- 29.6 ± 1.9 min vs. 28.8 ± 1.8 min -RRB- , which equals about 2.8 % . Possible causes for this deterioration in performance are associated with elevated body temperature -LRB- Tatterson et al. 2000 -RRB- and anticipatory changes in pacing strategy to avoid hyperthermia -LRB- Tucker et al. 2004 -RRB- . Although most research has revolved around neuromuscular function , central drive and fatigue at the point of exhaustion , a plausible explanation for the deterioration in time trial performance in heat may also be found in a temperature-induced change in gross-efficiency -LRB- GE -RRB- . GE is an important variable in cycling performance -LRB- Lucia et al. 2002 ; Moseley et al. 2004 -RRB- and a linear relationship between body temperature and GE has been observed -LRB- Daanen et al. 2006 -RRB- . A possible explanation for the decreased GE in the heat could be mitochondrial leakage . A temperature induced metabolic disruption caused by non-specific proton-leakage across the inner mitochondrial membrane has been shown to occur at high muscle temperatures -LRB- Brooks et al. 1971 ; Willis and Jackman 1994 -RRB- , resulting in a decrease in ADP :O ratio . Further , a heat induced skin vasodilatation could occur in heat . To prevent a resulting decrease in blood flow to exercising and respiratory muscles -LRB- Nielsen et al. 1990 ; Romer et al. 2003 ; Rowell et al. 1966 -RRB- a higher cardiac output must exist to continue supplying the muscles with the same blood flow , but still sending extra blood to the skin for cooling . In the present study we sought to determine the effect of heat on GE . GE can be assessed accurately during sub-maximal exercise at intensities as high as 60 − 80 % VO2max by calculating the ratio between mechanical and metabolic -LRB- mainly dependent on aerobic energy metabolism -RRB- power output , as has been done in Daanen et al. -LRB- 2006 -RRB- , Foster et al. -LRB- 2003 -RRB- , Hettinga et al. -LRB- 2006 -RRB- , Lucia et al. -LRB- 2002 -RRB- , Moseley et al. -LRB- 2001 , 2004 -RRB- . The presence of a possible effect of ambient temperature on GE can serve as input for an energy flow model -LRB- De Koning et al. 1999 -RRB- to quantify the impact of this effect on time trial performance . Materials and methods Subjects Ten healthy , non-smoking , well-trained male subjects , familiar with cycling exercise at the club-level , participated in this study . They were informed of the nature of the experiment and provided written informed consent . Subject characteristics are presented in Table 1 . The study was approved by the Medical Ethical Committee of the University Medical Centre Utrecht -LRB- The Netherlands -RRB- . Table 1Subject characteristicsMean value ± SDAge -LRB- years -RRB- 23.5 ± 4.4 Height -LRB- cm -RRB- 179.7 ± 9.1 Body mass -LRB- kg -RRB- 72.5 ± 7.2 VO2max -LRB- l min − 1 -RRB- 4.78 ± 0.41 -LRB- W -RRB- 354 ± 29Values are mean ± SD Experimental design Incremental test The subjects first performed an incremental bicycle test to determine at which power output VO2max was attained The incremental test was performed in the heat , under similar conditions as the constant intensity bouts , to make sure that would not be overestimated in the hot condition . This incremental test was used solely to determine the relative intensities for the constant intensity cycling bouts . The test began at a power output -LRB- PO -RRB- of 150 W , after which PO was increased with 15 W every minute . Exercise was performed on an electronically braked cycle ergometer -LRB- Lode Excalibur , Lode NV , Groningen , The Netherlands -RRB- until exhaustion or until pedal frequency dropped below 80 rpm . Oxygen consumption -LRB- VO2 -RRB- was measured breath by breath , using open circuit spirometry -LRB- Oxycon Pro-Delta , Jaeger , Hoechberg , Germany -RRB- . The gas analyzer was calibrated using a Jaeger 2 l syringe , room air and a calibration gas mixture . Heart rate -LRB- HR -RRB- was monitored using radiotelemetry -LRB- Polar Electro , Kempele , Finland -RRB- . Constant intensity exercise bouts On separate days , subjects performed a constant-intensity exercise bout at 60 % of in a thermo-neutral climate -LRB- N -RRB- and in a hot , dry climate -LRB- H -RRB- . Temperature in the thermo-neutral climate was 15.6 ± 0.3 °C , relative humidity -LRB- RH -RRB- was 20.0 ± 10.3 % . In the hot , dry climate , the temperature was 35.5 ± 0.5 °C and RH was 15.5 ± 3.2 % . These temperatures were equivalent to the temperatures in the studies of Tatterson et al. -LRB- 2000 -RRB- and Tucker et al. -LRB- 2004 -RRB- . All tests were performed in a climate-controlled room with a continuous airflow of 0.2 m s − 1 . RH was set low to increase the evaporative capacity of the environment . Further , all trials in the H-climate were performed with a simulated wind velocity of 1.72 m s − 1 -LRB- 6.2 km/h -RRB- to further increase evaporative heat loss , which is the most important form of heat loss in cycling in hot conditions -LRB- Saunders et al. 2005 -RRB- . Prior to the experiments , the subjects were asked to refrain from vigorous exercise for at least 48 h . They were also asked not to consume coffee , alcohol or drugs after 10 p.m. the day before the exercise and not to eat for two hours prior to the experiments . Subjects drank water ad libitum before the experiment . Before the constant intensity bout in the H-condition , the subject stabilized for 50 min in the 35 °C , 20 % RH room . For the comfort of the subjects and to prevent them from shivering , the stabilizing period was reduced to 35 min for the N-condition in the 15 °C , 20 % RH room . During the test , oxygen consumption -LRB- VO2 -RRB- , respiratory exchange ratio -LRB- RER -RRB- and ventilation -LRB- VE -RRB- were measured breath by breath . Power output -LRB- PO -RRB- and heart rate were registered continuously . After stabilization , a 5-min warm-up was performed at 100 W with a pedal frequency of ∼ 100 rpm . After 1 min of rest , the constant intensity bout was started -LRB- ∼ 100 rpm -RRB- . The subjects cycled for 20 min at a constant power output of 60 % of the power output at which maximal VO2 was attained at the incremental test in the H condition . Directly after the end of exercise , blood lactate concentration -LRB- BLC -RRB- was measured -LRB- Lactate Pro , Arkray , Kyoto , Japan -RRB- . Rectal temperature -LRB- Tre -RRB- was monitored every 5 s during the entire test using a thermistor temperature probe -LRB- YSI 701 , Yellow Springs Instrument , Dayton , USA -RRB- inserted about ten centimeters in the rectum . Skin temperature -LRB- Tsk -RRB- was measured every 5 s at 14 different skin loci , conform ISO 9886 , using thermocouples -LRB- YSI 709B , Yellow Springs Instrument , Dayton , USA -RRB- . Data were recorded with a Data Translation acquisition board -LRB- DT2821 , Viewdac , Keithley Instruments , Cleveland , USA -RRB- . Further , muscle temperature -LRB- Tm -RRB- was measured at a minimum depth of two centimeters and recorded every 5 s . Since this measurement was invasive , measurements were restricted to the constant intensity bout in H -LRB- n = 6 -RRB- . Tm was measured with a sterile thermal thermocouple-probe -LRB- type MAC08170A275SM , Ellab A/S , Rodovre , Denmark -RRB- in the right vastus lateralis muscle , inserted by a physician . One hour before inserting the temperature probe , a lidocaine plaster was attached to the skin , as a local anaesthetic . Calculating gross-efficiency -LRB- GE -RRB- Metabolic power -LRB- Pmet -RRB- was calculated by multiplying oxygen consumption with the oxygen equivalent : Pmet -LRB- W -RRB- = VO2 × -LSB- -LRB- 4,940 × RER + 16,040 -RRB- / 60 -RSB- according to Garby and Astrup -LRB- 1987 -RRB- , assuming that respiratory exchange ratio -LRB- RER -RRB- equaled respiratory quotient -LRB- RQ -RRB- at sub-maximal intensities . Further , we assumed that respiratory exchange ratios in excess of 1.00 were attributable to buffering of lactate by bicarbonate . Ratios in excess of 1.00 were thus treated as if they equaled 1.00 . The measured mechanical power output -LRB- PO -RRB- divided by the calculated Pmet defined GE . GE was calculated from 90 s after the start of exercise until the end of exercise . Energy flow model The energy flow model as described by De Koning et al. -LRB- 1999 -RRB- was used to calculate the effect of changes in GE on performance , by simulating a 20 km time trial as is studied in literature -LRB- Tatterson et al. 2000 ; Tucker et al. 2004 -RRB- . This model is based on power equations and has been reasonably successful in predicting performance in cyclic events as cycling -LRB- De Koning et al. 1999 -RRB- and speed skating -LRB- De Koning et al. 2005 -RRB- . The energy flow model , also referred to as power balance model , relates to power production and power dissipation : Pprod = Plost + dE/dt , where Pprod is total power that can be produced , Plost power that is used to overcome frictional losses , and dE/dt rate of change of kinetic energy of the mass centre of the body . These terms can be calculated as described in De Koning et al. -LRB- 1999 -RRB- , and the influence of changing one single variable , in this case GE , can be predicted . Statistics Paired Student t test 's were performed to test if data were significantly different between the H-condition and N-condition , P values less than 0.05 were accepted as statistically significant . Results Power output at 60 % was 211.5 ± 18.6 W. All subjects were able to complete the trials in both H and N. VO2 was significantly higher in H than in N , resulting in GE being significantly lower in H compared to N. Further , HR and VE were higher in H compared to N. For RER and BLC , no main effect of temperature was found . Mean values are shown in Table 2 . Table 2Gross-efficiency -LRB- GE -RRB- , VO2 , respiratory exchange ratio -LRB- RER -RRB- , respiratory minute volume -LRB- VE -RRB- , heart rate -LRB- HR -RRB- and blood lactate concentration -LRB- BLC -RRB- at 60 % in the thermo-neutral -LRB- N -RRB- and in the hot dry -LRB- H -RRB- climate60 % NHGE -LRB- % -RRB- 20.5 ± 1.419.6 ± 1.1 * VO2 -LRB- ml min − 1 -RRB- 3,002.8 ± 290.13,126.5 ± 268.3 * RER0 .89 ± 0.030.90 ± 0.01 VE -LRB- l min − 1 -RRB- 76.9 ± 7.782.0 ± 9.4 * HR -LRB- bpm -RRB- 145.1 ± 6.5155.4 ± 12.0 * BLC -LRB- mmol l − 1 -RRB- 2.6 ± 1.03.9 ± 2.1 Values are mean ± SD * Significant differences with N -LRB- P < 0.05 -RRB- Mean GE over time is plotted for both conditions in Fig. 1 . The difference in GE between conditions was 0.9 % over the entire trial . From minute 5 -- 8 , the N − H difference was 0.6 % ± 0.7 % , from minute 15 -- 18 , the difference was 1.1 ± 1.3 % , both significant . No significant change in GE within trials was observed comparing GE over the first half -LRB- min 5 -- 8 -RRB- with GE over the second half -LRB- min 15 -- 18 -RRB- . Fig. 1Gross-efficiency -LRB- GE -RRB- plotted over time at 60 % in the thermo-neutral -LRB- N -RRB- climate -LRB- dashed line -RRB- and in the hot dry -LRB- H -RRB- climate -LRB- solid line -RRB- ± SD -LRB- dotted lines -RRB- Tsk and Tre were significantly higher in H. Rectal and skin temperature changes combined , the difference in body heat content between N and H amounts to ∼ 138.5 ± 35.1 kJ , calculated assuming that the specific heat of the body tissue was 3.4 kJ kg − 1 °C − 1 . Maximal muscle temperature -LRB- Tmmax -RRB- in H was 38.7 ± 1.1 °C . Mean temperature values are shown in Table 3 . Figure 2 shows Tre , Tsk and Tm plotted over time for both conditions . Figure 3 shows the correlation between Tre and GE for both conditions . R2 in N was 0.04 , R2 in H was 0.36 . Table 3Rectal temperature -LRB- Tre -RRB- , skin temperature -LRB- Tsk -RRB- and maximal muscle temperature -LRB- Tmmax -RRB- at 60 % in the thermo-neutral -LRB- N -RRB- and in the hot dry -LRB- H -RRB- climate60 % NHTre -LRB- °C -RRB- 37.03 ± 0.5837.35 ± 0.63 * Tsk -LRB- °C -RRB- 27.74 ± 0.7133.39 ± 0.57 * Tmmax -LRB- °C -RRB- 38.7 ± 1.1 -LRB- n = 6 -RRB- Values are mean ± SD * Significant differences with N -LRB- P < 0.05 -RRB- Fig. 2Top panel Rectal temperature -LRB- Tre -RRB- plotted over time at 60 % in the thermo-neutral -LRB- N -RRB- climate -LRB- dashed line -RRB- and in the hot dry -LRB- H -RRB- climate -LRB- solid line -RRB- ± SD -LRB- dotted lines -RRB- . Center panel Skin temperature -LRB- Tsk -RRB- plotted over time at 60 % in the thermo-neutral -LRB- N -RRB- climate -LRB- dashed line -RRB- and in the hot dry -LRB- H -RRB- climate -LRB- solid line -RRB- ± SD -LRB- dotted lines -RRB- . Bottom panel Muscle temperature -LRB- Tm -RRB- plotted over time at 60 % in the hot dry -LRB- H -RRB- climate -LRB- solid line -RRB- ± SD -LRB- dotted lines -RRB- Fig. 3Top panel Correlation between gross-efficiency -LRB- GE -RRB- and rectal temperature -LRB- Tre -RRB- for the thermo-neutral -LRB- N -RRB- climate . Bottom panel Correlation between gross-efficiency -LRB- GE -RRB- and rectal temperature -LRB- Tre -RRB- for the hot , dry -LRB- H -RRB- climate To quantify the effect of the decrease in GE of 0.9 % , a simulation with an energy flow model -LRB- De Koning et al. 1999 -RRB- was performed . This simulation showed that a change in GE of 0.9 % would result in a difference in final time of 25.6 s over 20 km . Thus , about half of the decrement in performance reported in the literature -LRB- a difference in final time of 48 s over 20 km , Tucker et al. 2004 -RRB- could be accounted for by the measured decrease in GE . To explain the entire deterioration in time trial performance in the heat , the mean decrease in GE had to be 2 % . Discussion GE during sub-maximal cycling exercise in the heat was significantly reduced compared to exercise in N by 0.9 % . Values for GE corresponded to values found in literature , between 17 and 22 % -LRB- Foster et al. 2003 ; Moseley et al. 2001 -RRB- . It has to be kept in mind that in calculating GE at 60 % anaerobic contribution was assumed to be negligible . But it might have been influenced by the different climates . Gonzalez-Alonso et al. -LRB- 1999 -RRB- reported that exercise in a hot environment results in a higher anaerobic contribution and found an increase in carbohydrate utilization and lactate accumulation during exercise . Though not significant , BLC in the present study was higher in H . According to di Prampero et al. -LRB- 1999 -RRB- , an increase of BLC of 1 mmol l − 1 of blood is equivalent to an O2 consumption of 3 ml kg − 1 body weight . Differences in BLC in the present study between climates -LRB- 2.6 mmol l − 1 in N vs. 3.9 mmol l − 1 in H -RRB- thus correspond with a difference in O2 consumption equivalent of ∼ 14.1 ml min − 1 . Accordingly , differences in GE between climates might be even larger then reported in the present study . If one adds this ` anaerobic O2 equivalent ' to the VO2 , then GE decreases about ∼ 0.19 % in N compared to ∼ 0.27 % in H , which means that the difference in GE between N and H increases with ∼ 0.07 % . Mean Tre was about 0.3 °C higher in H compared to N. Mean Tsk was about 5.7 °C higher in H than in N. Comparable differences of 0.2 -- 0.4 °C in Tre -LRB- Tatterson et al. 2000 ; Tucker et al. 2004 -RRB- and ∼ 6.0 °C in Tsk -LRB- Tatterson et al. 2000 -RRB- were reported during cycling bouts of ∼ 30 min in climates comparable to the present study . Although the difference in rectal temperature between H and N was significant , it was not a large difference compared to literature . The measured absolute values for Tre were somewhat low , presumably due to the relatively short period of exercise in our protocol and the use of a fan to optimize losing heat to the environment , as was done in the studies of Tatterson et al. -LRB- 2000 -RRB- and Tucker et al. -LRB- 2004 -RRB- . The fan was used to simulate competitive circumstances , and although wind velocity in this study was not as high as the cycling speed , combined with the relatively low RH , the effect would have been considerable -LRB- Saunders et al. 2005 -RRB- . The differences in ambient temperature between H and N represent a considerable difference in thermal stress . Thermal strain is mainly visible in the increased skin temperature , but although the difference of 0.3 °C in core temperature is small , it is significant . Rectal and skin temperature changes combined , the difference in body heat content between N and H amounts to 138.5 ± 35.1 kJ , which is considerable . Further , the differences between climates have been shown to be large enough to evoke a difference in GE of 0.9 % , the main purpose of the present study . A possible explanation for the lower GE in the heat may be the higher core temperature . Daanen et al. -LRB- 2006 -RRB- found a strong linear relationship between body temperature , mainly determined by Tre , and GE at 60 % in 30 °C . Their study showed that our difference in Tre of 0.3 °C could account for a reduction in GE of ∼ 0.2 % , and thus was not large enough to explain the entire reduction in GE of 0.9 % . In the study of Daanen et al. -LRB- 2006 -RRB- , the skin temperature however was almost unchanged , while in the present study skin temperature in the H condition was about 5.6 °C higher , indicating an increased blood flow to the skin . The present study did not find a strong correlation in heat -LRB- r2 = of 0.36 -RRB- between Tre and GE . Further , remarkably , in the N-condition , no correlation between Tre and GE was found at all , even though Tre reached values which are also observed in the H-condition , only later in time -LRB- see Fig. 3 -RRB- . Apparently , Tre does not seem to be the main cause of the reduced GE . This is supported by the observation that even though Tre rose significantly during the time trial with about 1 °C , GE did not increase significantly comparing the first half with the second half within the trials . Two other explanations for the reduced GE in heat can be given . Firstly , an explanation for the temperature-induced reduction in GE might be a metabolic disruption that is known to occur at elevated muscle temperatures -LRB- Brooks et al. 1971 ; Willis and Jackman 1994 -RRB- . Though Ferguson et al. -LRB- 2006 -RRB- found no effect of increasing muscle temperature on energy turnover in dynamic exercise in a range of 34.2 -- 38.3 °C , ADP :O ratio has been shown to decrease at higher muscle temperatures -LRB- Brooks et al. 1971 ; Willis and Jackman 1994 -RRB- . During heavy exercise in the heat , non-specific proton leakage across the inner mitochondrial membrane is increased , resulting in a decrease in the efficiency of oxidative phosphorylation and increasing the resting metabolic rate -LRB- Willis and Jackman 1994 -RRB- . This will result in a reduction in GE . Willis and Jackman -LRB- 1994 -RRB- found a decrease of 10 -- 20 % in ADP :O ratio at muscle temperatures of 40 °C and higher compared to 37 °C . They found that this decrease resulted in a 400 -- 800 ml min − 1 increase in VO2 . In the present study , a smaller but significant increase in VO2 of 124 ml min − 1 was found . Mean Tmmax in heat was 38.7 °C , lower than the 40 °C as found in Willis and Jackman -LRB- 1994 -RRB- , but higher than 34.2 -- 38.3 °C , the Tm range in which Ferguson et al. -LRB- 2006 -RRB- found no effect of increasing Tm on energy turnover in dynamic exercise . Since Tm was not measured in both climates , it can not be confirmed if muscle temperature was significantly different . Tsk and Tre were significantly different but did not approach 40 °C in either condition . It seems that , as suggested by Brooks -LRB- 1971 -RRB- , the core of the body functions as a heat sink for the skeletal musculature helping to maintain Tm below the point where significant reductions in the ADP :O ratio occur . Another potential cause for the decrease of GE in a hot ambient temperature was the larger vasodilatation of the skin to lose heat , as was shown by the significantly higher Tsk in H compared to N . The resulting decreased blood flow to exercising and respiratory muscles may be compensated by increasing cardiac output -LRB- Nielsen et al. 1990 ; Rowell et al. 1966 -RRB- , since a significant increase in heart rate was found in heat . The extra VO2 in the H condition is at least partially attributable to the extra myocardial VO2 , since a higher cardiac output has to exist to continue supplying the muscles with the same blood flow , but have to send extra blood to the skin for cooling . Additionally , ventilation was increased , which may also lead to a reduction in GE . Assuming that the mechanical work per breath is 80 -- 125 J -LRB- Milic-Amili et al. 2001 -RRB- , it can be estimated that the higher VE in this study can account for maximally 10 % of the increase in VO2 . Lastly , it has to be noticed , that GE decreases if the proportion of energy expenditure that is used to maintain homeostasis is increased . Thus , the lower GE in H could have been solely due to an increase in resting metabolic rate , while net efficiency remained unchanged . This would be consistent with the hypothesis that muscle temperature in the hot conditions was not high enough to make mitochondrial leakage a likely explanation for the observed reduced GE . Unfortunately , resting metabolic rate has not been measured . To determine the potential importance of the measured decrease in GE on time trial performance , a 20-km time trial , which was also studied by Tucker et al. -LRB- 2004 -RRB- , was modeled by the use of the energy flow model -LRB- De Koning et al. 1999 -RRB- . A time trial of this distance can be seen as a mainly aerobic exercise bout . Tucker et al. -LRB- 2004 -RRB- found a difference in final time of 48 s between exercise in 35 °C compared to 15 °C . Using the energy flow model of De Koning et al. -LRB- 1999 -RRB- , it was calculated that the measured difference in GE of 0.9 % would lead to a difference in final time between H and N of 25.6 s . This explains about half of the 48 s found by Tucker et al. -LRB- 2004 -RRB- . For the entire reduction in final time of 48 s , a decrease in GE of 2 % would be necessary , which was not found . It can be concluded that about half of the decrease in time trial performance can be accounted for by the reduction in GE . It has to be noted that in this simulation , it is assumed that the difference in GE between H and N at higher intensities will not change . Although it has been shown that GE increases with exercise intensity , since the relative share of resting metabolism diminishes at higher sub-maximal intensities -LRB- Mosely and Jeukendrup 2001 -RRB- , this effect seems to be equal for both conditions and will at most have only a minor effect on the difference between conditions . Conclusion GE was lower in the heat . Tm was not high enough to make mitochondrial leakage a likely explanation for the observed reduced GE . Neither was the increased Tre . The extra VO2 in the H condition seems to be at least partially attributable to the extra myocardial VO2 , since a higher cardiac output has to exist to continue supplying the muscles with the same blood flow , but have to send extra blood to the skin for cooling and thus impacted GE . Based on our findings under the current circumstances , it can be concluded that the temperature-induced change in GE could account for about half of the well-established performance decrements found during time trial exercise in the heat .

O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O B O O O B O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O