Hypothalamic Nutrient Sensing

Nutrient sensing neurons are unique in coupling changes in the concentration of nutrients to changes in neuronal activity. These neurons typically exist in regions of the brain where the blood brain barrier is fenestrated, such as the arcuate nucleus of the hypothalamus. Glucose and leucine are nutrients known to be sensed by neurons in this brain region, but the mechanisms by which they are sensed, and cells that sense them require further study. Using calcium imaging of adult neuron cultures from the mouse mediobasal hypothalamus, I demonstrated that leucine bidirectionally regulates neuronal activity in a neurochemically heterogeneous population of neurons, including AgRP/NPY and POMC neurons. Using pharmacological tools, I demonstrated, unexpectedly, that this acute sensing is independent of mTOR and leucine metabolism, known pathways involved in leucine sensing in vivo. Leucine sensing is LAT1 independent. The response principally relies on calcium entry into the cell across the plasma membrane, but IP3 sensitive calcium stores play a role in neurons inhibited by leucine. Using phosphoTRAP and single cell RNA sequencing, I aimed to identify a molecular marker for leucine sensing cells to allow their manipulation in vivo. PhosphoTRAP, and subsequent pharmacological studies identified a T Type calcium channel may be a marker for leucine sensing cells. AgRP neurons are essential for feeding, and also play roles in controlling glucose homeostasis. Using chemogenetics to selectively activate these neurons, I demonstrated, in contrast to a similar, recently published study, that blood glucose concentrations did not rise upon activation of these neurons. A subpopulation of AgRP neurons express glucokinase, and some AgRP neurons are glucose inhibited, but the role of glucokinase in these neurons has not been characterised. Our lab generated an AgRP neuron specific glucokinase knock out mouse line. Preliminary results suggest 18 – 25 week old female AgRP glucokinase knock out mice may have altered glucose tolerance, but conclusions can only be drawn once further mice have been phenotyped, and the success of the glucokinase knock out from AgRP neurons has been confirmed.University of Cambridge Clinical Schoo

Nicotinic α4 Receptor-Mediated Cholinergic Influences on Food Intake and Activity Patterns in Hypothalamic Circuits.

Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine's (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4β2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHβE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHβE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHβE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4β2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4β2 nAChR-mediated transmission.This work was supported by the Royal Society and the European Union (Latin America/European Liason, LAEL).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/1371/journal.pone.013332

Rehabilitation versus surgical reconstruction for non-acute anterior cruciate ligament injury (ACL SNNAP): a pragmatic randomised controlled trial

BackgroundAnterior cruciate ligament (ACL) rupture is a common debilitating injury that can cause instability of the knee. We aimed to investigate the best management strategy between reconstructive surgery and non-surgical treatment for patients with a non-acute ACL injury and persistent symptoms of instability.MethodsWe did a pragmatic, multicentre, superiority, randomised controlled trial in 29 secondary care National Health Service orthopaedic units in the UK. Patients with symptomatic knee problems (instability) consistent with an ACL injury were eligible. We excluded patients with meniscal pathology with characteristics that indicate immediate surgery. Patients were randomly assigned (1:1) by computer to either surgery (reconstruction) or rehabilitation (physiotherapy but with subsequent reconstruction permitted if instability persisted after treatment), stratified by site and baseline Knee Injury and Osteoarthritis Outcome Score—4 domain version (KOOS4). This management design represented normal practice. The primary outcome was KOOS4 at 18 months after randomisation. The principal analyses were intention-to-treat based, with KOOS4 results analysed using linear regression. This trial is registered with ISRCTN, ISRCTN10110685, and ClinicalTrials.gov, NCT02980367.FindingsBetween Feb 1, 2017, and April 12, 2020, we recruited 316 patients. 156 (49%) participants were randomly assigned to the surgical reconstruction group and 160 (51%) to the rehabilitation group. Mean KOOS4 at 18 months was 73·0 (SD 18·3) in the surgical group and 64·6 (21·6) in the rehabilitation group. The adjusted mean difference was 7·9 (95% CI 2·5–13·2; p=0·0053) in favour of surgical management. 65 (41%) of 160 patients allocated to rehabilitation underwent subsequent surgery according to protocol within 18 months. 43 (28%) of 156 patients allocated to surgery did not receive their allocated treatment. We found no differences between groups in the proportion of intervention-related complications.InterpretationSurgical reconstruction as a management strategy for patients with non-acute ACL injury with persistent symptoms of instability was clinically superior and more cost-effective in comparison with rehabilitation management

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Nicotinic α4 receptor-mediated cholinergic influences on food intake and activity patterns in hypothalamic circuits

Abstract: Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine's (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4β2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHβE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHβE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHβE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4β2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4β2 nAChR-mediated transmission

The effects of systemic DHβE injections on HO, MCH, A13-TH and A12-TH activity.

<p>In animals that showed greater food intake compared to controls, DHβE injections at 2 mg/kg increased cFos expression in laterally located HO neurons (A1-2) (Saline and DHβE, n = 6) and decreased cFos expression in total MCH neurons (A3) (Saline, n = 4; DHβE, n = 5). There was no difference between 2 mg/kg DHβE and Saline groups in TH A13 (Saline and DHβE, n = 6) and A12 cFos expression (Saline and DHβE, n = 4) (B1-2). Abbreviations: HO, hypocretin/orexin; MCH, melanin concentrating hormone; TH, tyrosine hydroxylase; f, fornix; DMH, dorsomedial hypothalamus; LH, lateral hypothalamus; PeF, perifornical region; PeFLH, perifornical region of the hypothalamus; Arc, arcuate nucleus. Scale bar: A, 300 μm; B, 100 μm.</p

The effects of systemic DHβE injection on food intake and PVH activity.

<p>A1. Schematic demonstrating the experimental setup. A2. Animals injected with 2 mg/kg DHβE i.p. consumed more food than saline injected animals (saline, n = 7; 1 mg/kg, n = 7; 2 mg/kg, n = 6). Total cFos (B1) (Saline and DHβE, n = 5) and percent of PVH oxytocin-expressing neurons (B2) (Saline and DHβE, n = 4) were not significantly different between DHβE and Control conditions (B3). Abbreviations: PVH, paraventricular nucleus of the hypothalamus; OXY, oxytocin 3V, third ventricle. Scale bar: 100 μm.</p

Hypothalamic TH and oxytocin neurons expressing the α4 nicotinic subunit.

<p>A. 50.1% of TH-containing neurons in the A13 were colocalized with α4 (A1,2) while 50.7% of A12 TH-containing neurons in the arcuate nucleus showed α4 nAChR co-expression (A1,A2) (α4-TH, n = 3). There was no difference in colocalization across the different arcuate subdivisions analyzed (A2). B. Within the PVH, 41.3% of oxytocin neurons were also immunolabeled for α4 (α4-oxy, n = 4). Abbreviations: TH, tyrosine hydroxylase; Oxy, oxytocin; PVH, paraventricular nucleus of the hypothalamus; Arc, Arcuate nucleus; 3V, third ventricle. Scale bar: 200 μm.</p

The effects of DHβE infusions into the lateral hypothalamus on food intake.

<p>A. Scheme demonstrating the experimental setup. Intra-cerebral cannulae were confined to the lateral hypothalamic region (B2) an example of which is shown in B1. Cumulative measurements of food intake showed that DHβE-infused animals consumed more food 2 hours post ad libitum food access following a 12 hour fast (C) (saline, n = 8; 20 μg DHβE, n = 7). Abbreviations: mt, mammillothalamic tract; f, fornix.</p

Example immunolabeling for α4 (A), oxytocin in the PVH (B), tyrosine-hydroxylase in the A13 (C1) and arcuate nucleus (C2), and HO (D1) and MCH (D2) immunoreactivity throughout the hypothalamus.

<p>Abbreviations: OXY, oxytocin; PVH, paraventricular nucleus of the hypothalamus; TH, tyrosine hydroxylase; HO, Hypocretin/Orexin; MCH, Melanin Concentrating Hormone; 3V, third ventricle; mt, mammillothalamic tract; f, fornix. Scale bar: A, B 100 μm; C-E 300 μm.</p