Physiology - Theses
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ItemChronic ephedrine administration decreases brown adipose tissue activity in a randomised controlled human trial: implications for obesity( 2018)Aim Activation of Brown Adipose Tissue (BAT) may have therapeutic potential to combat obesity. Acute treatment of mice with sympathomimetic drugs activates BAT thermogenesis, and chronic treatment increases BAT thermogenic capacity. It has previously been demonstrated that human BAT is acutely responsive to oral administration of the sympathomimetic ephedrine. This study aimed to determine whether chronic treatment with ephedrine could mimic adaptive thermogenesis in humans. Methods Twenty-three healthy young men were recruited via general advertisement from Melbourne, Australia to participate in a randomised, placebo-controlled, parallel group trial. Recruited individuals were unmedicated, non-smokers, physically inactive and had no prior history of either cardiovascular disease, insulin resistance or diabetes. They were allocated to either a placebo (n=11; 22±2 years, 23±2 kg/m2) or 1.5 mg/kg/day ephedrine (active group; n=12, age 23±1 years, BMI 24±1 kg/m2) treatment group for twenty-eight days. Body composition was measured before and after the intervention by dual energy x-ray absorptiometry. BAT activity, measured before and after the twenty-eight day intervention period, via 18F-fluorodeoxyglucose positron emission tomography computed-tomography (18F-FDG PET/CT) in response to a single dose of 2.5mg/kg ephedrine, was the primary outcome measure. Results After twenty-eight days of treatment, the active treatment lost significantly more total body fat (placebo 1.1± 0.3 kg, ephedrine -0.9 ± 0.5kg; p<0.01) and visceral adipose tissue (placebo 6.4 ± 19.1g, ephedrine -134 ± 43g; p<0.01), with no change in lean mass or bone mineral content, compared with the placebo group. In response to acute ephedrine, BAT activity (change in mean standardised uptake value: placebo -3 ± 7 %, ephedrine -22 ± 6%) and the increase in systolic blood pressure were significantly reduced (p<0.05) in the active group compared with placebo. Conclusion Chronic ephedrine treatment reduced body fat content, however, it was independent of an increase in BAT activity. Rather, chronic ephedrine treatment suppressed BAT glucose disposal, suggesting that chronic ephedrine treatment decreased, rather than increased BAT activity.
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ItemInvestigating cortical oscillations, coherence and seizure susceptibility in a mouse model of autism( 2018)Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder diagnosed by difficulties in social communication, repetitive behaviour and/or restricted interests. A high proportion of ASD patients also experience seizures and abnormal brain activity as recorded via electroencephalography, however the underlying biological mechanisms of increased seizure susceptibility in ASD are unknown. Neuroligin-3 (NL3) is a neuronal adhesion protein involved in regulating synaptic structure and function. A rare point mutation at position 451 of the Neuroligin-3 amino acid sequence converts an arginine to a cysteine residue is associated with ASD and reduces NL3 protein levels by 90%. This NL3 R451C mutation was identified in two Swedish brothers with ASD, one of whom was diagnosed with comorbid epilepsy. The mutation is replicated in NL3R451C mice that exhibit reduced preference for social interactions, increased repetitive behaviours, and increased performance in memory and some motor testing. NL3R451C mice also display region-specific differences in inhibitory and excitatory neurotransmission in brain slices increased dendritic complexity, and reduced number of PV-inhibitory interneurons, all of which could contribute to changes in seizure susceptibility and oscillatory activity. Several animal models of ASD show spontaneous seizures and increased susceptibility to experimentally induced seizures, however whether NL3R451C mice have altered seizure susceptibility is unknown. It is well established that brain neuronal activity generates electrical rhythms that underlie cognitive phenomena in humans and may be altered in ASD. Gamma oscillations in the 30-100Hz range are frequently increased with demanding cognitive load and are thought to orchestrate spatially disparate cortical regions. Several clinical studies have pointed to alterations in gamma oscillations and in the coherence of oscillatory activity measured between two disparate cortical regions in ASD patients. To determine whether gamma oscillations are altered in NL3R451C mice, the glutamatergic NMDA receptor antagonist ketamine (20mg/kg; a potent inducer of gamma oscillations) was administered to adult male and female NL3R451C and WT mice and oscillatory activity recorded via electrocorticography (EcOG). PV-interneurons have been experimentally isolated to reveal they are the major drivers of gamma rhythms following ketamine administration. Baseline oscillatory activity from 6 and 10 week old male mice was examined as well as interhemispheric coherence in male and female NL3 mutants and wild type littermates. Gamma oscillatory power in male wild type and NL3R451C mice was comparable following administration of saline and was enhanced to a similar degree following ketamine (20mg/kg) administration. Non-gamma oscillations were also unchanged by ketamine administration. Interhemispheric coherence, however, was significantly higher in 6- week old male NL3R451C compared to control mice for both beta and gamma oscillation ranges. In 10-week old male mutants, there was a significant effect detected for theta, alpha and beta oscillations. Because female mice carry two copies of the X-linked NL3 gene it was hypothesised that any oscillatory activity effects may be exacerbated by the R451C mutation. Gamma band oscillations following administration of ketamine (20 mg/kg) were statistically not significant when WT, heterozygous and homozygous females were compared. Additionally, no statistically significant differences were found for interhemispheric coherence in females. To gain further understanding of the role of the NL3 R451C mutation in epilepsy, seizure susceptibility was investigated in NL3R451C male mice using pentalenetetrazole (PTZ) at both low (20 and 30 mg/kg) and high (50mg/kg) doses. Administration of high dose PTZ is associated with behavioural changes culminating to convulsive seizures. The Racine behavioural scale was used to quantify behavioural seizure severity over time in response to high dose PTZ. In mice, low dose PTZ results spike-wave discharges (SWDs) and a loss of responsiveness resembling absence seizures in patients. Electroencephalography (EcOG) recordings of neuronal activity at the cortical surface was utilised to detect SWDs following administration of low dose PTZ over a 30 minute period. NL3R451C mice showed a strong trend for shorter SWD duration although EcOG analyses of SWD frequency and duration showed similar susceptibility in both mutants and WT littermates to low dose PTZ-induced seizures. Following administration of high-dose PTZ (50mg/kg), NL3R451C mutants were slower to progress to severe seizure scores and overall progressed to milder seizures on the racine scale over the 30-minute testing period. The results of the present study show reduced seizure susceptibility and increased coherence in male NL3R451C mice. These changes might reflect underlying structural and neurochemical alterations present in NL3R451C mice. The reported increase in cortical inhibitory neurotransmission in NL3R451C mice may underlie the resistance to PTZ-induced seizures identified in this study. Similarities in gamma oscillations may indicate similar functioning of NMDA receptors and the PV-interneurons that are strongly implicated in the generation of gamma rhythms. Higher interhemispheric coherence in male NL3R451C mice could stem from differences in cognitive, behavioural and cortical changes present in this model. Further research is needed to clarify relationships between behaviour and oscillatory activity in NL3R451C mice.
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ItemEffects of compression garments on recovery from intermittent exercise( 2013)Purpose: To examine the effects of wearing compression garments for 24 h post-exercise on the biochemical, physical and perceived recovery of highly-trained athletes. Method: Eight field hockey players completed a match-simulation exercise protocol on two occasions separated by 4 wks after which lower-limb compression garments (CG) or loose pants (CON) were worn for 24 h. Blood was collected pre-exercise and 1, 24 and 48 h post-exercise for IL-6, IL-1β, TNF-α, CRP and CK. Blood lactate was monitored throughout exercise and for 30 min after. A 5 counter-movement jump (5CMJ) and squat jump (SJ) were performed and perceived soreness rated at pre-exercise and 1, 24 and 48 h post-exercise. Perceived recovery was assessed post-exercise using a questionnaire related to exercise readiness. Repeated measures ANOVA was used to assess changes in blood, perceptual and physical responses to recovery. Results: CK and CRP were significantly elevated 24 h post-exercise in both conditions (p<0.05). No significant differences were observed for TNF- α , IL1-β, IL-6 between treatments (p>0.05). Power and force production in the 5CMJ was reduced and perceived soreness was highest at 1 h post-exercise (p<0.05). Perceived recovery was lowest at 1 h post exercise in both conditions (p<0.01), whilst overall, perceived recovery was greater when CG were worn (p<0.005). Conclusion: None of the blood or physical markers of recovery indicate any benefit of wearing compression garments post-exercise. However, muscle soreness and perceived recovery indicators suggest a psychological benefit may exist.
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ItemEffect of fasting on adipose tissue glycogen levels in the obese Zucker rat( 2012)Current scientific investigations have shown that obesity is associated with several alterations affecting not only the metabolic state of adipose tissue, since it also interferes with functional outcomes in target organs including liver and skeletal muscle. Metabolic alterations linked with obesity in the adipose tissue include early development of insulin resistance, impaired glucose transport and increased adipogenesis. In this regards, glucose uptake is the rate limiting step for glucose metabolism in adipose tissue. Insulin-mediated glucose uptake provides the cells not only with glucose as an energy substrate towards glycolysis, but also participates in de novo synthesis of fatty acids, fatty acid re-esterification, and a small amount of the incoming glucose is stored as glycogen. Glycogen is the mechanism by which cells store and mobilize glucose to meet their energetic and synthetic demands. During feeding, high blood glucose and plasma insulin command glycogen synthesis. Conversely, glycogen degradation during fasting requires sequential inactivation of glycogen synthase and activation of glycogen phosphorylase – the glycogen-metabolising enzymes – both actions orchestrated by the upstream kinase PKA. Little is known about the regulatory mechanisms controlling glycogen metabolism in the adipose tissue and how it is affected by pathological states related to obesity. The aim of the project was to examine adipose glycogen metabolism and the effects of fasting on glycogen-metabolising enzymes in order to determine whether adipose glycogen metabolism is altered as consequence of the obesity-induced insulin resistance. It was hypothesised that obesity imposes a blunted response to fasting that is reflected by impaired glycogen mobilization. Data obtained in this study showed that lean Zucker rats activated the lipolytic pathway in response to fasting, and NEFA were released into circulation. In contrast, fed and fasted plasma NEFA and glycerol levels were elevated several fold in obese rats, although with a reduced response to fasting. Considering that lipolysis occurs in parallel with glycogen degradation, we then tested the hypothesis that the blunted fasting response was associated with altered glycogen metabolism. This study showed for the first time that glycogen is not mobilized by 24-h fasting in the adipose tissue of obese Zucker rats. It could be initially attributed to an impaired degradation due to the lack of GP activation, although the finding of an impaired GS inactivation could similarly contribute to higher glycogen content following fasting. Additionally, obesity induced a differential effect on the protein content of GS and GP; increasing GS whereas reducing GP proteins expression. Following fasting, protein contents of both enzymes were reduced which could be interpreted as an attempt to counterbalance the unaltered glycogen content during fasting. Accordingly, it can be argued that obesity in Zucker rats may be associated with a higher content of glycogen after 24-h of fasting mainly caused by obesity-induced dysregulation upon GS and GP protein expression, with a lack of GP activation and defective inactivation of GS. These latter findings led us questioning about the involvement of upstream steps into the activation and inactivation of glycogen-metabolising enzymes. Therefore we next examined the activity of the upstream kinase PKA. The PKA activity was increased by fasting in lean rats. However, PKA activity was reduced in obese fasted rats, which was associated with the observed high adipose tissue glycogen levels found among fasted animals. In order to confirm this finding, we tested the activation of CREB, the downstream effector of PKA signalling. CREB phosphorylation was increased 3-fold by fasting in the lean group in parallel with increase in PKA activity. Interestingly, a similar level of phosphorylation was found in obese fed rats when compared with lean fed rats, which implies basal activation of this transcription factor. This over-activated CREB could be explained considering the role of insulin in the activation of CREB. On the other hand, after 24-h fasting, the pCREB/totalCREB ratio was significantly reduced in the obese rats which means that CREB is less active during fasting in obese Zucker rats. Taking into consideration the repressive effect exerted by CREB on the expression of GLUT4, we analysed GLUT4 content in the adipose tissue. As anticipated, fasting reduced GLUT4 protein content in the adipose tissue of lean animals. In the obese rats, GLUT4 protein level was markedly reduced to one third of lean fed content, whereas fasting did not modify GLUT4 protein content in this group, which led us to consider that GLUT4 protein content in the obese was decreased secondary to obesity and that fasting did not induce any further repressive effect within the time course of 24-h of food deprivation. In sum, these novel findings have shed light on how obesity may alter adipose tissue glycogen metabolism impairing its degradation during fasting. Obesity impairs cAMP-PKA signalling leading to dysregulation of glycogen-metabolising enzymes. Additionally, the obesity-induced repressive effect on the protein contents of GLUT4 and GP in association with increased protein level of GS might be related to alterations in carbohydrate metabolism in the adipose tissue. Further analyses are required in order to investigate other potential mechanisms involved in the regulation of glycogen metabolism, impaired cAMP-PKA signalling and GLUT4 expression with obesity.