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Aims/hypothesis Impaired awareness of hypoglycaemia (IAH) in type 1 diabetes increases the risk of severe hypoglycaemia sixfold and can be resistant to intervention. We explored the impact of IAH on central responses to hypoglycaemia to investigate the mechanisms underlying barriers to therapeutic intervention. Methods We conducted [ O]water positron emission tomography studies of regional brain perfusion during euglycaemia (target 5 mmol/l), hypoglycaemia (achieved level, 2.4 mmol/l) and recovery (target 5 mmol/l) in 17 men with type 1 diabetes: eight with IAH, and nine with intact hypoglycaemia awareness (HA). Results Hypoglycaemia with HA was associated with increased activation in brain regions including the thalamus, insula, globus pallidus (GP), anterior cingulate cortex (ACC), orbital cortex, dorsolateral frontal (DLF) cortex, angular gyrus and amygdala; deactivation occurred in the temporal and parahippocampal regions. IAH was associated with reduced catecholamine and symptom responses to hypoglycaemia vs HA (incremental AUC: autonomic scores, 26.2 ± 35.5 vs 422.7 ± 237.1; neuroglycopenic scores, 34.8 ± 88.8 vs 478.9 ± 311.1; both p < 0.002). There were subtle differences (p < 0.005, k ≥ 50 voxels) in brain activation at hypoglycaemia, including early differences in the right central operculum, bilateral medial orbital (MO) cortex, and left posterior DLF cortex, with additional differences in the ACC, right GP and post- and pre-central gyri in established hypoglycaemia, and lack of deactivation in temporal regions in established hypoglycaemia. Conclusions/interpretation Differences in activation in the post- and pre-central gyri may be expected in people with reduced subjective responses to hypoglycaemia. Alterations in the activity of regions involved in the drive to eat (operculum), emotional salience (MO cortex), aversion (GP) and recall (temporal) suggest differences in the perceived importance and urgency of responses to hypoglycaemia in IAH compared with HA, which may be key to the persistence of the condition. . . . . Keywords Counterregulation Hypoglycaemia Impaired awareness of hypoglycaemia Neuroimaging Positron emission tomography Type 1 diabetes Electronic supplementary material The online version of this article Abbreviations (https://doi.org/10.1007/s00125-018-4622-2) contains peer-reviewed but ACC Anterior cingulate cortex unedited supplementary material, which is available to authorised users. CT Computerised tomography DLF Dorsolateral frontal * Stephanie A. Amiel fMRI Functional MRI email@example.com GP Globus pallidus Division of Imaging Sciences and Biomedical Engineering, King’s HA Hypoglycaemia awareness College London, London, UK IAH Impaired awareness of hypoglycaemia Diabetes Research Group, King’s College London, King’sCollege iAUC Incremental AUC Hospital Campus, Weston Education Centre, 10 Cutcombe Road, k Cluster size London SE5 9RJ, UK MO Medial orbital Institute of Diabetes and Obesity, King’s Health Partners, PET Positron emission tomography London, UK rCBF Regional cerebral blood flow Singapore General Hospital, Singapore, Republic of Singapore SPM Statistical Parametric Mapping 2 School of Life Sciences, University of Nottingham, Nottingham, UK Diabetologia (2018) 61:1676–1687 1677 Introduction extensive activation, including the dorsal-medial thalamus, globus pallidus (GP) and anterior cingulate cortex (ACC), For people with diabetes, the main defence against severe has been described in healthy people at 3 mmol/l and consid- hypoglycaemia, in which blood glucose falls too low to sus- ered to be associated with autonomic stress responses . tain cognitive function , is subjective awareness of minor Using [ O]water positron emission tomography (PET) scans episodes. Impaired awareness of hypoglycaemia (IAH) is as- repeated during induction of, maintenance of and recovery sociated with delayed and diminished counterregulatory re- from hypoglycaemia at 2.8 mmol/l, we described activation sponses to hypoglycaemia . It is reported in 25–40% of of the thalamus and ACC, with progressive involvement of people with longstanding type 1 diabetes mellitus [3, 4], and pathways involved in feeding behaviour and reward, symp- 10% with insulin-treated type 2 diabetes , increasing risk of tom perception and aversion . severe hypoglycaemia 6- and 17-fold respectively [3–5]. Less is known about the impact of diabetes and The brain triggers counterregulatory neuroendocrine re- hypoglycaemia awareness (HA) status. Hormone responses sponses to hypoglycaemia, symptom perception, and coordi- to hypoglycaemia are diminished in type 1 diabetes , but nation of endogenous and behavioural protective responses. the importance for symptoms experienced is unclear . Animal studies  have hypothesised that IAH, which is in- Thalamic activation has been described as enhanced in a mod- ducible by exposure to plasma glucose below 3 mmol/l [7, 8], el of IAH  but reduced in IAH itself . A recent study is associated with increased brain capacity for glucose uptake. compared a single measurement of global and regional cere- However, early studies of enhanced global brain glucose up- bral blood flow (rCBF) after 45 min at 2.8 mmol/l in people take in humans  are not compatible with the clinical picture with type 1 diabetes with and without IAH using arterial spin- of IAH, where deterioration of cognitive function precedes the labelling MRI. This reported a global increase in cerebral onset of diminished and asymptomatic counterregulatory re- blood flow only in IAH, and failure of an enhanced regional sponses . Early neuroimaging studies failed to confirm a thalamic response , but there are no reports of IAH- global increase in glucose uptake in IAH . specific differences in other regional responses. Functional neuroimaging investigates the brain’sresponse Understanding the central pathophysiology of IAH is key to a challenge by measuring regional changes in glucose up- to its management. Although IAH can be restored by avoiding take and metabolism or perfusion as markers of neuronal ac- hypoglycaemia [2, 20], this can be difficult to achieve, with tivation. Activation of the hypothalamus, important in glucose 8% of adults with type 1 diabetes showing low concern about sensing, has been described with modest decrements of blood hypoglycaemia despite being at high risk . Cognitions glucose (to 4.3 mmol/l) in healthy volunteers . More around hypoglycaemia may create barriers to its avoidance 1678 Diabetologia (2018) 61:1676–1687 . We therefore extended the use of repeated [ O]water (over 15–20 min) for 30 min. The insulin was then stopped, PET scans to measure changes in regional brain perfusion and the participant withdrawn from the scanner. He was then during sequential euglycaemia, hypoglycaemia and recovery given lunch, with his usual subcutaneous fast-acting insulin in men with type 1 diabetes with and without IAH. pre-meal dose and, if a morning dose of basal insulin had been omitted, a reduced basal insulin dose to provide cover until the evening dose. The glucose infusion was reduced and plasma Methods glucose monitored until concentrations were spontaneously maintained. The cannulae were then removed. Participants Participants were advised about monitoring and dosing to minimise risk of hypoglycaemia over the next 24 h. Right-handed men with type 1 diabetes aged between 20 and 50 years, with HbA levels less than 86 mmol/mol (<10%), Scanning protocol 1c were recruited into two groups, based on their awareness of hypoglycaemia as defined by history and 8-item Clarke score This was as previously reported . In brief, the brain was . HA was defined as good awareness of occasional localised in the PET view field using a planar CT scout. A hypoglycaemia, no severe hypoglycaemia in the past year low-dose CT scan was acquired to correct attenuation in sub- and a Clarke score ≤3. IAH was defined by history, including sequent PET scans. Head position was checked, and 3 min [ experience of severe hypoglycaemia, and Clarke score ≥4. O]water PET scans were made at 10 min intervals. For each The protocol was approved by the Ethics Committee of scan, 350 MBq [ O]water in 10 ml sterile water was manu- King’s College Hospital London and the Administration of ally injected intravenously over 10 s. Three scans were ac- Radioactive Substances Committee (ARSCAC, Health quired during euglycaemia: one during the fall in glucose, five Protection Agency, Didcot, Oxfordshire, UK). All participants during the hypoglycaemic phase and three during recovery. gave written informed consent. Scans were acquired in 3D mode, reconstructed to a single static frame using the 3D FORE algorithm , and 2D- Protocol filtered back-projection with scatter correction and CT-based correction of attenuation. To minimise movement artefacts, Scans were performed at the PET Imaging Centre, St Thomas’ each participant’s CT scan was realigned to each PET scan Hospital, London, using a GE Discovery ST PET/ using the rigid-body registration algorithm in the Statistical computerised tomography (CT) scanner (GE Medical Parametric Mapping 2 program (SPM2; www.fil.ion.ucl.ac. Systems, Milwaukee, WI, USA) with a 15.8 cm axial field uk/spm, accessed 15 March 2013). The realigned CT was of view. Participants were admitted the evening before the used to correct the attenuation in the PET reconstruction. study. Two intravenous cannulae were inserted for infusion of soluble insulin (Actrapid; NovoNordisk, Copenhagen, Assessment of physiological responses Denmark) in a 4% (vol./vol.) saline (154 mmol/l NaCl) solu- tion of autologous blood; blood was sampled hourly. After scanning, arterial blood was taken to measure Participants fasted after their evening meal, sips of water being counterregulatory hormones. The participant was asked ver- allowed, and omitted their evening background insulin dose. bally torate (from 1to7) 13 hypoglycaemia-associated symp- To achieve normoglycaemia without hypoglycaemia, plasma toms (see below) . glucose was maintained between 4 and 7 mmol/l overnight by adjusting the insulin infusion. Biochemical analyses In the morning, the left radial artery was cannulated. After at least 20 min, each participant rested supine on the scanner Blood was kept on ice, spun, separated and flash-frozen on trolley, in a headrest with a forehead positioning strap. Once in dry ice until storage. A volume of 1 ml was added to 30% (wt/ the scanner, the head position was checked using gantry la- vol.) polyethylene glycol for free insulin radioimmunoassay sers. A new primed-continuous infusion of soluble insulin was (Diagnostic Systems Laboratories, London, UK). Blood −1 −1 started, at a maintenance rate 1.5 mU kg min .Fourmi- (3 ml) for catecholamines was taken into heparinised tubes nutes later, an infusion of 10% (wt/vol.) glucose (Baxter containing 15 μl sodium metabisulphite; plasma was separat- Healthcare, Thetford, Norfolk, UK) was started and was ad- ed at the time of study, stored at −80°C and analysed by justed using 5 min arterial plasma glucose readings (YSI 2300 radioimmunoassay . Stat Plus; Yellow Springs Instruments, Yellow Springs, OH, USA). The target plasma glucose level was 5 mmol/l for Symptom scores Scores for sweating, shakiness, anxiety, 40 min, with a reduction over 20 min to 2.6 mmol/l, mainte- warmth, palpitation and tingling were summed as autonomic. nance at 2.6 mmol/l for 45 min, and restoration to 5 mmol/l Scores for dizziness, irritability, difficulty in speaking, Diabetologia (2018) 61:1676–1687 1679 confusion, lack of energy, drowsiness and poor concentration For analysis, scans were grouped as: euglycaemia/baseline were totalled as neuroglycopenic. (scans 1–3), early hypoglycaemia (scans 4–6), established hypoglycaemia (scans 7–9) and recovery (scans 10–12). Statistical analyses of non-imaging data Two-way repeated measures ANOVA was used to investigate the main effects of stage of hypoglycaemia (early, late or re- Age, BMI, HbA and duration of diabetes were com- covery vs baseline), group and interactions . 1c pared between groups using independent sample t tests, Regions of significant effects were calculated using a and Clarke scores by Mann–Whitney U testing. Hormonal voxel-level p value <0.001 and cluster sizes (k) ≥100 and symptom responses to hypoglycaemia and impact of voxels; these were recalculated at p <0.005, k ≥ 50 voxels awareness status were examined by calculating the main to examine for smaller differences where more significant and interaction effects of scan and group on each variable differences had been excluded. t values <2 (or <−2) indi- using a repeated measures linear mixed model (first-order cate significance with >95% confidence. Clusters were autoregressive covariance structure). To assess the effect localised using the Tziortzi atlas . of hypoglycaemia, baseline values for each variable were calculated by averaging data from scans 1–3. A summary statistic of response to the total hypoglycaemic period was Results calculated as the incremental AUC (iAUC) of the re- sponse in scans 4–9. The effect of hypoglycaemia was Participants tested using a one-sample t test of the iAUC if the mixed model revealed significant scan or interaction effects. To Of the 17 men with type 1 diabetes recruited, nine had assess differences between the two groups, the baseline intact HA. These nine individuals were aged 37.6 ± andiAUCmeasureswereusedintwo-sample t tests if 9.3 years and had a diabetes duration of 14.3 ± 12.3 years, the mixed model revealed significant group or interaction BMI, 23.4 ± 3.4 kg/m and HbA , 58.5 ± 12.4 mmol/mol 1c effects. Analyses were performed using SPSS software (7.5 ± 1.3%) (all means ± SD). They reported good aware- version24(www-01.ibm.com/software/uk/analytics/ ness of hypoglycaemia, with a median Clarke score of 2 spss/). (range 1–3) and no severe hypoglycaemia in the past year. The other eight men (means ± SD: age, 36.4 ± 7.8 years; diabetes duration, 27.1 ± 11.9 years; BMI, 26.6 ± 1.4 kg/ Neuroimaging analysis m ;HbA , 57.7 ± 9.3 mmol/mol [7.3 ± 0.8%]) had IAH, 1c with a median Clarke score of 5.5 (range 4–6); all had a The analytic program was chosen to allow interrogation of history of severe hypoglycaemia. The IAH and HA the data without preconceptions of the brain regions that groups did not differ in terms of age (p= 0.79), duration might respond to hypoglycaemia in people with type 1 of diabetes (p= 0.07) or HbA (p= 0.89) but had slight- 1c diabetes or respond differently between HA and IAH. ly a greater BMI (p= 0.02). By design, Clarke scores SPM2 was used to preprocess and analyse the PET data. were significantly different between groups (p <0.001). Image processing and analysis of regional perfusion were performed automatically and identically in each partici- Plasma insulin, glucose, symptoms pant or group, removing the need for blinding. PET im- and counterregulatory hormones ages were transformed into standard anatomical space conforming to the standard Montreal Neurological During the studies, steady-state free plasma insulin was Institute (MNI) space using the [ O]water PET template (mean ± SD) 476.4 ± 138.48 and 487.9 ± 225.3 pmol/l supplied with SPM2, masked to exclude the scalp and for the HA and IAH group, respectively (p= 0.89). smoothed using a Gaussian kernel (full width at half max- The hypoglycaemia achieved (Fig. 1a) did not differ imum [FWHM] = 6 mm). between groups (2.4 ± 0.1 and 2.4 ± 0.1 mmol/l, respec- Within SPM2, each image was spatially normalised to its tively; p= 0.7); there were no significant differences in whole-brain mean using SPM2, which performs an initial af- starting glucose level, or glucose concentration during fine registration followed by a basis function method non- recovery. Mean glucose levels were: scan 4, 3.4 ± 0.2 linear registration  and calculates regional perfusion rela- vs 3.5 ± 0.4; scan 5, 2.7 ± 0.2 vs 2.7 ± 0.3; and scan 6 tive to the whole-brain mean. SPM2 calculates the mean im- 2.2 ± 0.3 vs 2.3 ± 0.2 (all p > 0.05). Hormone concentra- age intensity across the whole brain, without segmentation, tions did not differ between the groups during using its default threshold method (mean of the overall mean × euglycaemia. Adrenaline (epinephrine) and noradrenaline 0.8), and uses integrated activity over time (here 3 min), (norepinephrine) (Fig. 1b, c) showed significant main which is proportional to rCBF . effects of scan number (see also electronic 1680 Diabetologia (2018) 61:1676–1687 Symptom responses Significant main effects for scan, group and interaction were revealed in autonomic and neuroglycopenic total scores (Fig. 1d, e; ESM Table 1). The HA group showed significant re- sponses (iAUC) for both scores (422.7 ± 237.1, p= 0.001 and 478.9 ± 311.1, p= 0.002, respectively). The IAH group showed a small response for autonomic scores (26.2 ± 35.5; p= 0.038), significantly lower than the response with HA (p= 0.002), and no significant neuroglycopenic symptom re- sponse (34.8 ± 88.8, p= 0.15 from baseline, p= 0.002 vs HA -20 0 20 40 60 80 100 120 140 group). Baseline autonomic scores were slightly higher for Time from scan 1 (min) HA than IAH (p= 0.024), with a mean difference in score b c of 1.5, which was small in comparison with the mean increase of 10.5 seen during hypoglycaemia. Neuroimaging data Regional brain responses to acute hypoglycaemia in type 1 diabetes with preserved HA The response to early 12 3 4 5 6 7 8 9 101112 12 34 5 6 7 8 9 101112 Scan number Scan number hypoglycaemia across HA participants (Fig. 2a, ESM Table 2) included a regional increase in perfusion (compared d e with baseline) in the thalamic pulvinar bilaterally, bilateral dor- 30 40 solateral frontal (DLF) cortices, right insular cortex and ACC; 20 a decrease in perfusion was seen in the left inferior temporal gyrus. As hypoglycaemia progressed (Fig. 2b, ESM Table 3), 10 the activation area became more extensive, with additional activation (vs baseline) in the following: posterior, middle 0 0 123456789 101112 1 2 3 4 5 6 7 8 9 101112 and anterior thalamus and GP; bilateral insula and frontal oper- Scan number Scan number cula; frontal cortex including the DLF cortex bilaterally and Fig. 1 Plasma glucose, catecholamine and symptom responses. Blue lateral orbital cortex; ACC; and precuneus and right angular lines, HA; red lines, IAH. (a) Plasma glucose levels (mean ± SD). Grey bars represent scan time points. iAUC was not significantly different gyrus/supramarginal gyrus/superior temporal gyrus. Perfusion between groups. (b, c) Mean ± SD at each scan for (b) plasma adrenaline was reduced in established hypoglycaemia bilaterally in the (iAUC, p= 0.007 between groups) and (c) plasma noradrenaline (signif- parahippocampal and posterior parietal cortex, inferior tempo- icant response to hypoglycaemia in HA only (p < 0.009); no significant ral gyri and parts of the cerebellum including the vermis. difference in iAUC between groups. (d, e) Box plots showing median (circles), upper and lower quartiles (box), range (vertical lines) and out- During recovery (Fig. 2c, ESM Table 4), regions including liers (crosses) for (d) autonomic symptom scores and (e) neuroglycopenic the ACC, GP, right insula and left precuneus showed activa- symptom scores. For both (d)and (e), difference between groups, tion; there was persisting deactivation in the inferior temporal p= 0.002 and posterior parietal gyri, and new activation of the amygdala. supplementary material [ESM] Table 1), with a signifi- Impact of awareness of hypoglycaemia on regional brain re- cant interaction effect of scan and group for adrenaline sponses to hypoglycaemia Regional brain responses to levels. Post hoc tests of effect of hypoglycaemia using iAUC hypoglycaemia in IAH involved various regions similar to revealed significant responses in HA for adrenaline (p < 0.001) those showing responses in the HA group (Fig. 2,ESM and noradrenaline (p= 0.009). The adrenaline response with Tables 5–7), with some differences that were explored in a IAH was significant (p= 0.003) but significantly lower than formal comparison. Directly comparing responses between for HA (p for comparison = 0.007). There was a significant groups identified clusters showing significantly different noradrenaline response only in the HA group (p= 0.009). The hypoglycaemia responses between the IAH and HA groups iAUC for cortisol did not reach significance in either group, at the second statistical threshold voxel level (p <0.005, k ≥ 50 with a trend towards significance for HA (p= 0.075; see voxels) (Fig. 3; Table 1). In early hypoglycaemia (Fig. 3a–c), ESM Table 1). The iAUC for growth hormone was significant five clusters were identified. One in the left lingual gyrus in both groups (HA, p= 0.035; IAH, p= 0.006; see ESM showed deactivation for IAH and activation for HA, and four—the right central operculum, medial orbital (MO) cortex Table 1). Autonomic score Adrenaline (nmol/l) Plasma glucose (mmol/l) Neuroglycopenic score Noradrenaline (nmol/l) Diabetologia (2018) 61:1676–1687 1681 Fig. 2 SPM2 results showing a 9.9 significant (voxel level p <0.001, HA k ≥ 100 voxels) regional changes 3.1 in brain perfusion compared with baseline euglycaemia during (a) early hypoglycaemia, (b) -3.1 IAH established hypoglycaemia and (c) recovery from hypoglycaemia -7.1 measured with repeated b 9.9 [ O]water PETscans in men with HA IAH and without (HA). For 3.1 analysis, scans were grouped as euglycaemia (scans 1–3), early hypoglycaemia (scans 4–6), -3.1 IAH established hypoglycaemia (scans -7.1 7–9) and recovery (scans 10–12). 9.9 Significant increases in rCBF are shown in red-yellow, and HA decreases are shown in blue- 3.1 white. t values are shown in the right-hand scale. Scans were -3.1 overlaid onto an MRI scan in IAH Montreal Neurological Institute -7.1 standard space (greyscale) bilaterally and left posterior DLF cortex (two clusters)— hypoglycaemia between participants with preserved (HA) or showed activation in IAH vs deactivation in HA. impaired (IAH) awareness of hypoglycaemia were accompa- As hypoglycaemia continued (Fig. 3d–f), eight clusters nied by subtle differences in brain responses. These occurred showed significantly different changes in activation between not only in regions associated with the generation and subjec- groups. Five showed limited or absent responses for IAH but tive awareness of stress responses, but also in regions associ- activation for HA: the right GP, ACC, dorsal cingulate cortex, ated with executive control, reward, memory and emotional right pre- and bilateral post-central gyri, and left precuneus. salience. Two further clusters, in the right MO and left parietal cortices Defective hormonal responses to hypoglycaemia in IAH (parietal lobule and angular gyrus), showed activation in IAH are well recognised [2, 8, 16]. They are inducible by exposure but no change or deactivation in HA. A cluster including the to hypoglycaemia [7, 8] and restored by avoiding it [2, 20]. left posterior middle temporal gyrus, angular gyrus and occip- The IAH group showed the expected diminution of symptom- ital pole showed no change in IAH but deactivation in HA. atic and catecholamine responses to our hypoglycaemic Fourteen clusters showed significantly different activation be- challenge. tween groups during the recovery period (Fig. 3g–i). IAH showed deactivation or minimal response compared with acti- Responses in men with type 1 diabetes and intact vation in the HA group in the following: right fusiform cortex; awareness a midbrain cluster, extending to the right amygdala; right su- perior temporal gyrus/insula; left pre- and post-central gyri; Our neuroimaging data in HA participants are largely consis- and a cluster including right white matter, anterior temporal tent with studies in people without diabetes and extend earlier pole and amygdala. The IAH group failed to show the deacti- observations, particularly by describing the evolution of re- vation seen with HA in the bilateral middle and left inferior sponses during development of and recovery from temporal gyri. However, there was activation in IAH compared hypoglycaemia. Our ‘early’ scans were made as arterialised with deactivation in HA in the left posterior medial frontal plasma glucose level was falling, and include data collected at cortex, left posterior DLF cortex extending to pre-central gy- glucose values between 3.5 and 2.2 mmol/l; the ‘established’ rus, and left angular gyrus/occipital pole/parietal lobule/ data were collected at 2.4 mmol/l. Thalamic activation seen in supramarginal gyrus/left posterior superior temporal gyrus. both early and established hypoglycaemia is consistent with data from individuals without diabetes, and with the role of the thalamus in relaying sensory signals to cortical areas . Discussion Novel findings include insula activation, seen in all three phases of hypoglycaemia and not previously described in di- In this neuroimaging study of men with type 1 diabetes, dif- abetes. Using similar techniques, we previously described in- ferences in symptomatic and hormonal responses to sular activation in established hypoglycaemia only in people Recovery Established Early 1682 Diabetologia (2018) 61:1676–1687 4.0 3.1 -3.1 -5.0 5 5 0 0 -5 -5 -10 -10 LG MO pDLF CO pDLF L R L d 4.0 3.1 -3.1 -5.0 e f 10 10 5 5 -5 -5 -10 -10 GP aCC dCC PoCG PrCG MO pmTG Par R L R L L g 4.0 3.1 -3.1 -5.0 h i 10 10 0 0 -5 -5 -10 -10 aTP TOF MidB sTG PoCG PoCG mTG iTG pmTG SMG AG MF pDLF pDLF R R R L L L L R L L L L L HA > IAH HA < IAH Fig. 3 SPM2 results for comparisons of HA and IAH responses to response is more positive for HA than IAH in early (b), established (e) hypoglycaemia (see Table 1). (a, d, g) Images showing regions with and recovered (h) hypoglycaemia. (c, f, i) Data for clusters where re- significantly different responses between HA and IAH to early (a), sponse is more negative for HA than IAH in early (c), established (f) established (d) and recovered (g) hypoglycaemia (change from baseline). and recovered (i) hypoglycaemia. Black bars, HA; white bars, IAH. a, Changes were detected using SPM2 statistical thresholds set at voxelwise anterior; AG, angular gyrus; CC, cingulate cortex; CO, central opercu- p <0.005 and k ≥ 50 voxels. No clusters were seen with stricter thresholds lum; d, dorsal; i, inferior; , left; LG, lingual gyrus; m, medial; MF, medial (k ≥ 100 voxels, p < 0.001). Yellow clusters show regions where response frontal cortex; MidB, midbrain; p, posterior; Par, parahippocampal gyrus; is significantly more positive for HA than IAH; blue clusters show re- PoCG, postcentral gyrus; PrCG, precentral gyrus; , right; s, superior; gions where response is more negative in HA than IAH. t values are SMG, supramarginal gyrus; TG, temporal gyrus; TOF, temporal occipital shown in the right-hand scale. Associated bar charts show the mean and fusiform gyrus; TP, temporal gyrus. Where no laterality is noted ( or ), L R SE of group responses for whole clusters; all between-group differences the cluster was represented bilaterally. Where there are two data sets for were significant, as defined above. (b, e, h) Data for clusters where one brain region, there were two clusters in that region without diabetes ; we speculate that earlier and more per- sensitivity to changes in plasma glucose or prior experience sistent activation in type 1 diabetes relates to heightened of more fluctuating glucose concentrations. Wiegers et al did Recovery response (%) Established response (%) Early response (%) Recovery response (%) Established response (%) Early response (%) Diabetologia (2018) 61:1676–1687 1683 Table 1 Regions of the brain with different activation responses to hypoglycaemia in men with type 1 diabetes with HA vs those with IAH Period vs baseline, Group (region name) k (voxels) t (peak voxel) p(peak voxel) p(uncorrected Atlas coordinates for Subcluster size response direction cluster) location in the brain xy z Early HA > IAH Lingual gyrus L 54 3.57 0.00024 0.060 0 −74 −631 Lingual gyrus R 23 HA < IAH Post dorsolateral 79 −3.47 0.00034 0.026 −48 22 12 79 frontal cortex L Post dorsolateral 54 −4.40 0.00001 0.060 −32 26 46 54 frontal cortex L Insular cortex R 55 −3.53 0.00027 0.058 44 −210 4 Precentral gyrus R 3 Central operculum cortex R 44 Cerebral white matter R 4 Medial orbital cortex L 70 −4.17 0.00003 0.035 4 62 −14 11 Medial orbital cortex R 59 Established HA > IAH Precentral gyrus L 128 3.84 0.00009 0.007 −6 −38 66 9 Postcentral gyrus L 95 Cerebral white matter R 30 Precentral gyrus R 89 4.01 0.00005 0.020 20 −28 66 56 Postcentral gyrus R 33 Dorsal ant cingulate cortex 42 Cerebral white matter R 8 Ant cingulate cortex 68 4.03 0.00004 0.037 −236 6 68 HA < IAH Parietal lobule L 109 −4.75 <0.00001 0.011 −34 −58 40 54 Supramarginal gyrus L 2 Angular gyrus L 53 Middle temp gyrus post L 171 −4.18 0.00002 0.002 −54 −62 16 76 Angular gyrus L 63 Occipital pole L 32 Medial orbital cortex L 133 −3.76 0.00012 0.006 4 60 −12 6 Medial orbital cortex R 127 Recovery HA > IAH Outside atlas 127 3.53 0.00027 0.007 40 −40 −16 4 Inf temp gyrus post R 1 Cerebral white matter R 7 Postcentral gyrus L 58 Outside atlas 111 3.79 0.00011 0.010 10 −8 −16 23 Cerebral white matter R 17 Hippocampus R 9 Amygdala R 22 Midbrain 30 Precentral gyrus L 119 3.84 0.00009 0.008 −38 −24 62 25 Postcentral gyrus L 94 Insular cortex R 78 3.42 0.0004 0.027 42 −22 0 11 Sup temp gyrus ant R 52 Sup temp gyrus post R 7 Cerebral white matter R 8 Outside atlas 79 3.81 0.0001 0.026 32 2 −20 3 1684 Diabetologia (2018) 61:1676–1687 Table 1 (continued) Period vs baseline, Group (region name) k (voxels) t (peak voxel) p(peak voxel) p(uncorrected Atlas coordinates for Subcluster size response direction cluster) location in the brain xy z Insular cortex R 10 Ant temp pole R 21 Parahippocampal 1 ambiens gyrus ant R Cerebral white matter R 27 Amygdala R 17 HA < IAH Middle temp gyrus ant L 97 −5.02 <0.00001 0.015 −64 −14 −14 32 Middle temp gyrus post L 56 Inf temp gyrus ant L 3 Inf temp gyrus post L 6 Sup temp gyrus post L 138 −4.56 <0.00001 0.005 −58 −48 16 43 Middle temp gyrus post L 1 Supramarginal gyrus L 68 Angular gyrus L 23 Cerebral white matter L 3 Ant dorsolateral 78 −4.42 <0.00001 0.027 −10 50 34 10 frontal cortex L Anterior medial 6 frontal cortex L Postero-medial 62 frontal cortex L Middle temp gyrus post L 90 −4.01 0.00005 0.019 −56 −34 −22 22 Inf temp gyrus post L 68 Parietal lobule L 685 −3.96 0.00006 <0.001 −38 −58 54 88 Supramarginal gyrus L 38 Angular gyrus L 425 Occipital pole L 134 Middle temp gyrus post R 53 −3.89 0.00007 0.062 60 −46 2 53 Precentral gyrus L 95 −3.57 0.00024 0.016 −40 2 52 32 Post dorsolateral 63 frontal cortex L Post dorsolateral 97 −3.46 0.00035 0.015 −36 18 50 97 frontal cortex L Region names derive from the intersection of regions identified by SPM using the Tziortzi atlas . t values are calculated from a linear contrast for every voxel, the peak t value being the voxel with highest t value in the cluster p(peak voxel) is the p value that corresponds to the peak t value within each cluster, calculated with 154 degrees of freedom. p(uncorrected cluster) is the p value calculated using SPM2, reflecting the significance of cluster size of regions that meet the criteria for significance (i.e. the likelihood that a cluster of that size being found based on Gaussian field theory and functional image smoothness) Regions that did not meet the criteria for significance Ant, anterior; Inf, inferior; L left; Post, posterior; R right; Sup, superior; Temp, temporal not find insular activation in people without diabetes and de- regional responses and may be better at detecting differences scribed reduced insular perfusion using functional MRI between groups in studies of similar size . (fMRI) in seven HA individuals with type 1 diabetes . Activation of the GP in established hypoglycaemia and Comparing direction of signal change across studies using recovery, and the ACC in all three phases, is consistent with different technologies is complex; however, [ O]water PET some reports involving individuals without diabetes at similar is less susceptible to low signal to noise ratios and movement glucose concentrations [13, 14] but has not been described in than fMRI. It also allows more quantitative measurement of type 1 diabetes. The GP and ACC, involved in reward, might Diabetologia (2018) 61:1676–1687 1685 be reacting to the hypoglycaemic stress responses. Activation is clinically associated with reduced motivation to avoid of orbital cortex is likewise consistent with non-diabetic re- hypoglycaemia [21, 22], with reduced incentive to treat sponses to comparable hypoglycaemia [13, 14, 18] but not hypoglycaemia as important . previously observed in type 1 diabetes . The orbital cortex Parts of the dorsal and posterior DLF cortex responded encodes stimulus value or salience , and the lateral orbital from early hypoglycaemia through to recovery with activation cortex forms a ‘salience’ network with the ACC . responses in IAH compared with deactivation in HA. Activation of the DLF cortex in response to hypoglycaemia Hypoglycaemia is associated with impaired inhibitory control; has not previously been described [12–14, 18, 19]. It has a role perhaps the deactivation in HA represents conscious attempts in working memory—the short-term recall and processing of to maintain inhibitory control of behaviour during information necessary for complex task performance, includ- hypoglycaemia. ing learning and reasoning —and is involved in inhibitory The ACC is involved in decision-making and conflict res- control [35, 36]. Changing activity during hypoglycaemia is olution between options, and is key in monitoring perfor- consistent with clinically observed changes in cognition and mance, evaluating actions and detecting events that require behavioural disinhibition during hypoglycaemia, and recall behavioural modification and re-evaluation . Lack of after it. The report of activation of the precuneus and angular ACC activation only in IAH fits with views of IAH as a gyrus during established hypoglycaemia and recovery is also habituation response. A similar lack of activation in IAH in novel [12–14, 19]. The precuneus is part of the ‘default mode the somatosensory post-central (somatosensory) and pre- network’, showing reduced activity compared with the resting central (motor) gyri, persisting in recovery, may reflect re- state when undertaking tasks ; activation may reflect less- duced somatic sensations (e.g. warmth, shakiness) and motor er ability to perform tasks during hypoglycaemia. The angular responses (e.g. tremor) experienced by the IAH group in gyrus, linked to the DLF cortex  and showing parallel hypoglycaemia. responses, plays a role in regulating shift of attention to more Minimal responses in IAH, vs deactivation in HA, in the left salient stimuli . The amygdala encodes the predicted bio- posterior middle temporal gyrus during established logical relevance of a stimulus ; its activation in recovery hypoglycaemia, and the bilateral posterior middle and left infe- may be a key determinant of responses to subsequent rior temporal gyri in recovery, are also consistent with different hypoglycaemic events. memory formation during hypoglycaemia and recovery [40, Deactivation of the inferior temporal gyri in all three 41]. The same is true of differences in the left parietal lobule/ phases, of parietal regions during established hypoglycaemia angular gyrus in established hypoglycaemia, with activation in and recovery, and of parahippocampal regions during IAH but minimal response in HA, and in recovery in the left established hypoglycaemia, described in some studies of indi- angular gyrus and supramarginal gyrus, with activation in IAH and deactivation in HA. The lateral parietal cortex shows func- viduals without diabetes but not previously in type 1 diabetes, provide a neurological correlate of failure to form memory tional connectivity with the hippocampal formation and is as- during hypoglycaemia: temporal gyri for semantic or concep- sociated with recollection of experiences [41, 45]. tual memory , and parahippocampal gyrus and lateral pa- In recovery, in addition to persisting differential responses rietal cortex for episodic memory . in somatosensory and memory networks, we found activation in IAH and deactivation in HA in part of the medial frontal Impact of IAH cortex; this was in a cluster corresponding to regions of the dorsal-medial prefrontal cortex identified as having a role in The subtle differences in hypoglycaemia responses in IAH are self-referential mental activity, such as making judgements potentially important. In early hypoglycaemia, deactivation about unpleasantness/pleasantness . It may also have a seen in parts of the central operculum, MO cortex and poste- role in episodic or experiential memory . This may pro- rior and lateral DLF cortex in HA was replaced by activation. vide a correlate for individuals with type 1 diabetes with IAH Operculum activation changes in response to food cues, mod- and HA forming differently valenced memories of the experi- ulated by feeding state and degree of liking the food : ence of hypoglycaemia. However, the medial frontal cortex, differences between IAH and HA may relate to differences along with the lateral parietal regions discussed above, is also in the drive to eat to treat. These were paralleled by different a component of the default mode network , and these responses in the MO cortex, encoding stimulus value and differences may represent hypoglycaemia being a different salience , in early and established hypoglycaemia; this ‘task’ for the brain in IAH than HA. may underlie differences in perceived importance of Our participants were matched well for age, diabetes duration hypoglycaemia, including lack of aversion. Lack of activation and diabetes control but imperfectly for BMI. Obesity alters brain of the GP, with its role in memory of unpleasant experiences responses to food and food cues, including the responses of some or aversion , is also consistent with not finding frontal regions described as different in their response to hypoglycaemia here . However, none of our participants hypoglycaemia unpleasant. These are key findings, as IAH 1686 Diabetologia (2018) 61:1676–1687 was obese, so it is unlikely our observed differences in response and behaviours unhelpful to future hypoglycaemia avoidance; to hypoglycaemia were related to this. The recruitment of only further research is required into how best to address these in men facilitates research involving radio-isotopes and importantly clinical practice. reduces variability of responses due to sex differences in Acknowledgements We are grateful to the following: research nurses A. counterregulation . The clinical picture of IAH is not sex- Pernet and B. Wilson (King’s College London and King’s College specific so our data interpretation probably also applies to wom- Hospital NHS Foundation Trust), who assisted in the care of the partici- en; however, adaptation to antecedent hypoglycaemia may vary pants and supported the clinical studies and sample-handling; M. by sex, at least in individuals without diabetes , and studies in O’Doherty and the radiologists, radiographers and chemists of the King’s College London PET Imaging Centre for their support; L. Reed, women would be of interest. Right-handedness was chosen as then of the Institute of Psychiatry, King’s College London, for significant many brain functions are lateralised. intellectual input into the interpretation of our neuroimaging data; D. The strengths of our study include pre-study determination Forster, Nottingham, and J. Jones London, for the biochemical analyses; of awareness status on clinical grounds, so individuals defined and finally our volunteers. as having IAH were representative of those with clinically Data availability The primary data are medical imaging data for which problematic hypoglycaemia. In addition, the analysis did not there are no publicly available repositories. The authors are able to pro- require a preconception of brain regions that might respond vide data in response to email requests. differently to hypoglycaemia by awareness status. Although Funding This work was funded by a research grant from the JDRF less powerful than a region of interest analysis, in which data International. are compared between groups only in prespecified brain re- gions, this enabled us to identify areas not traditionally asso- Duality of interest The authors declare that there is no duality of interest ciated with stress responses. That differences between the two associated with this manuscript. groups (the effect of awareness status on rCBF responses) Contribution statement SAA, JTD and PKM conceived and designed were identified at lower thresholds than those used to find the study. MMT and PC acquired the data; JTD, PKM, SAA, KFH, IM differences within groups (the effect of hypoglycaemia) is and PC analysed and/or interpreted the data. SAA, JTD and KFH drafted statistically explicable: within-group comparison is always the article. All authors revised it critically for important intellectual con- tent and gave final approval of the version to be published. SAA is the more powerful than between-group comparison, where differ- guarantor of this work. ences between participants come into play. It is also biologi- cally plausible as hypoglycaemia is a large stress stimulus Open Access This article is distributed under the terms of the Creative whereas differences between HA and IAH are probably an Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, order of magnitude less. It is, however, possible that other distribution, and reproduction in any medium, provided you give appro- brain regions responding differently were missed. priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Conclusion References In conclusion, we used [ O]water PET to describe the evolution 1. International Hypoglycaemia Study Group (2017) Glucose concen- of the brain’s responses to hypoglycaemia over time in men with trations of less than 3.0 mmol/l (54 mg/dl) should be reported in type 1 diabetes and found differences related to HA status. 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