Medicine (Austin & Northern Health)

In recent years, obesity has reached pandemic proportions and leading risk factor for the development of cardiovascular diseases. Obese individuals have been shown to have 3.5 times greater risk of developing hypertension than individuals with normal body weight. According to the Framingham Study, up to 75% newly-diagnosed cases of hypertension may be attributable to obesity. While sympathoactivation had been established as one of the main contributors to obesity-related hypertension, recent evidence suggests that attenuated sympathoinhibitory mechanisms may be just as important. Disruption to the delicate balance of sympathoinhibitory and sympathoexcitatory mechanisms may cause a disturbance to vasoconstrictor and vasodilator mechanisms, thus impacting cardiovascular homeostasis. The hormone leptin has received considerable attention in the aetiology of obesity-related hypertension. This peptide is primarily derived from adipose tissue and acts centrally to reduce appetite and increase energy expenditure. However obesity is associated with leptin resistance as many obese individuals have abnormally elevated circulating levels of leptin (hyperleptinaemia). This phenomenon is known as selective leptin resistance, in which the beneficial metabolic effects of leptin on energy expenditure are abolished but the sympathoexcitatory effects are sustained. This prolonged sympathoactivation is thought to contribute to the development of hypertension in obesity. Leptin is not only found in adipose tissue but also the stomach. This gastric-derived leptin is involved in the short-term regulation of food intake and satiety signalling, unlike adipose leptin that is involved on long-term regulatory mechanisms via transcriptional changes occurring in forebrain regions. The gut peptide, cholecystokinin (CCK), is involved in regulating satiety and many other digestive processes. Studies have shown that feeding and CCK administration induce the release of gastric leptin. Our laboratory was the first to demonstrate that the gut peptides, CCK and leptin, play a role in cardiovascular control via a centrally-mediated tri-synaptic reflex. CCK and gastric leptin have an interactive relationship in cardiovascular regulation, and the effects of gastric leptin are due to CCK release within the gut. CCK elicits a centrally-mediated bimodal sympathetic reflex dependent on the activation of CCK1 receptors located on subdiaphragmatic vagal afferents. Our laboratory has found that CCK inhibits a subpopulation of presympathetic vasomotor neurons in the rostral ventrolateral medulla (RVLM) that are critical to blood pressure regulation. Furthermore, this subset of neurons is proposed to modulate sympathetic vasomotor outflow specifically to the gastrointestinal and renal vascular beds, inhibition of which results in withdrawal of sympathetic vasomotor tone to promote vasodilation. The gastrointestinal and renal circulations receive up to 50% of total blood volume postprandially. Since the satiety effects of gastric leptin and CCK are affected in obesity, we hypothesised that disruption to the sympathoinhibitory and vasodilator effects of gut hormones may impact on cardiovascular homeostasis, thus contributing to hypertension in obesity. Specifically, this thesis has focussed on the cardiovascular role of gastrointestinal hormones in the aetiology of obesity-related hypertension. In order to address this hypothesis, we first established a diet induced obesity (DIO) model using polygenic out-bred male Sprague-Dawley rats fed a moderately high-fat diet (MHFD; 32% kcal from fat). After a 13-15 week feeding period, MHFD fed animals were segregated according to weight gain, with the upper tertile weight-gainers being assigned to the obesity-prone (OP) group and the lower tertile to the obesity-resistant (OR) group. Control animals were fed a low-fat diet (LFD; 9% kcal from fat). Initial weights of the animals were not significantly different between the groups. OP animals typically developed elevated resting arterial pressure (AP), increased weight gain, adiposity index and plasma leptin levels when compared with OR or control animals. However the plasma lipids, insulin and glucose were not significantly different between OP, OR or control animals, indicating that this model is independent of the metabolic syndrome. Using this DIO model, we examined the effects of CCK (2 µg/kg) and leptin (15 µg/kg) administered close to the coeliac artery and within the gastrointestinal circulation (termed "close arterial") on splanchnic sympathetic nerve discharge (SND), in artificially ventilated, isoflurane-anaesthetised animals. To determine whether any effects were attributable to obesity or the high-fat diet, animals were analysed according to weight gain (OP versus OR) or diet (all MHFD animals (including middle tertile) versus LFD animals). When analysed according to weight gain, the splanchnic sympathoinhibitory responses to CCK and leptin were significant attenuated or reversed respectively, in both the OP and OR animals when compared with control animals (P < 0.05). The splanchnic SND responses to these peptides were not significantly different between OP or OR animals (P > 0.05). Collectively, MHFD animals had significantly lower levels of CCK when compared to the LFD animals (P < 0.05) and this corresponded with attenuated or reversed splanchnic SND responses to CCK (P < 0.05) and leptin (P < 0.001), respectively. Since plasma CCK was only lower in MHFD animals, this suggested that diet alters the release of this peptide. The sympathoinhibitory effects of gastric leptin have been proposed to be due to CCK release. Therefore, in light of the attenuated sympathoinhibitory effects of CCK in MHFD rats, it is not surprising that the modest effects of close arterial leptin were reversed, possibly due to the unmasking of sympathoexcitatory mechanisms. These findings demonstrated that a high-fat diet is associated with blunted/reversed splanchnic sympathoinhibitory responses to CCK and gastric leptin, possibly impacting on sympathetic vasomotor mechanisms involved in circulatory control. Renal sympathetic nerve activity (SNA) has been implicated in the development of hypertension in obesity. Furthermore, hypertension has been associated with increased vascular resistance and impaired vasodilator mechanisms. To test whether renal SND and regional vasodilator responses to CCK were affected in obesity we recorded renal SNA and examined vasodilator responses in renal and superior mesenteric arteries using Doppler flowmetry techniques. We found that CCK (0.1 - 8 µg/kg) inhibited renal SND and increased renal vascular conductance (VC) in control and OR animals, however these responses were significantly attenuated in OP rats (P < 0.05 for all). While there was a tendency for the OP rats to have reduced mesenteric VC, this did not reach statistical significance. Resting AP was directly correlated with weight gain and was inversely correlated with CCK-induced vasodilatation in both the renal and mesenteric arteries (P < 0.05 for all). Animals with higher resting AP tended to have vasoconstrictor rather than vasodilator responses. These results suggest that in obese rats, disruption of CCK-induced sympathoinhibitory signals evoked by CCK reduces vasodilation in the splanchnic or renal regions. Our laboratory has proposed that the cardiovascular effects of gastric leptin are dependent on the release of CCK. We have shown that rats on a MHFD have lower circulating levels of CCK, suggesting that in these animals the release of this peptide is compromised. Since the splanchnic sympathoinhibitory effects of gastric leptin are abolished/reversed in MHFD animals, we therefore sought to investigate whether the renal sympathoinhibitory and regional vasodilator effects of gastric leptin are also altered. Close arterial leptin (15 µg/kg) inhibited renal SND in control animals, however this response was abolished in OP and OR rats (P < 0.01 for both). Renal VC was increased in control animals in response to leptin, but this response was significantly blunted only in the OP rats (P < 0.05). However the vasodilator response in the superior mesenteric artery was not significantly different between OP, OR or control rats (P > 0.05 for all). In agreement with our previous findings, renal sympathoinhibitory responses of gastric leptin were affected by diet. However the vasodilator response of this peptide in the renal vascular bed was only affected in OP animals, suggesting these responses are altered as a result of obesity. While we established that the typical sympathoinhibitory and vasodilatory effects of CCK and gastric leptin are attenuated or reversed in obese hypertensive animals, we did not identify the mechanisms for these changes. In the following study we sought to investigate whether these changes are due to aberrant central or peripheral mechanisms. The renal and splanchnic sympathoinhibitory effects of CCK are dependent on the activation of CCK1 receptors situated on subdiaphragmatic vagal afferents, and subsequent reflex inhibition of a subpopulation of CCK-sensitive presympathetic vasomotor neurons in the RVLM. We used extracellular single unit recording to examine whether the sensitivity of presympathetic vasomotor RVLM neurons to gut peptides is altered in obese hypertensive animals. Typically presympathetic vasomotor neurons inhibited by CCK are fast-firing and fast-conducting, and these were typical characteristics of neurons examined in this study. CCK induced inhibitory responses in RVLM neurons of OR or control animals but these responses were abolished or were excitatory in OP animals (P < 0.05 for all). RVLM neuronal responses to CCK correlated with resting AP and weight gain; animals with lower AP and weight gain tended to have inhibitory responses to CCK, whereas those with higher AP and weight gain had reduced inhibitory or excitatory responses. A further aim was to examine the barosensitivity of RVLM neurons in the different groups of animals. Barosensitivity of the RVLM neurons was significantly attenuated in OP animals (P < 0.05), suggesting altered baroreflex gain. These results suggest that in obese hypertensive animals there is a generalised blunted responsiveness of RVLM neurons to inhibitory inputs. Our second aim was to examine whether subdiaphragmatic vagal responses to CCK and CCK1 receptor mRNA expression on the nodose ganglia are altered in obese hypertensive animals. We examined subdiaphragmatic vagal nerve discharge (SVND) responses to close arterial CCK (0.1 - 4 µg/kg) administration, and evaluated CCK1 receptor mRNA expression using quantitative PCR, respectively. Although CCK evoked a dose-dependent increase in SVND, this was not significantly different between OP, OR or control rats (P > 0.05 for all). Furthermore, there were no differences in CCK1 receptor mRNA expression in the nodose ganglia between the three groups (P > 0.05 for all), suggesting that vagal afferent responses to CCK are intact in obesity. Both the baroreflex and CCK-induced reflex are dependent on the activation of neurons in the nucleus of the solitary tract (NTS) and caudal ventrolateral medulla (CVLM). To investigate whether altered sympathoinhibitory effects in obesity result from aberrant signalling in key cardiovascular medullary centres, we examined whether obese hypertensive rats have reduced Fos-like immunoreactivity (Fos-IR) in response to CCK, using immunohistochemical techniques. Intraperitoneal injection of CCK (8 µg/kg) induced significantly less Fos-IR neurons in the NTS of OP animals when compared to control rats only (P < 0.01). There were significantly fewer Fos-IR neurons in the CVLM neurons of OP animals in response to CCK, when compared to both control or OR rats (P < 0.05). Overall, this study suggests that blunted sympathoinhibitory and vasodilator responses in obesity-related hypertension are due aberrant central but not peripheral signalling mechanisms. In summary, the results of these studies have demonstrated that the sympathoinhibitory and regional vasodilator effects of gut peptides are blunted or abolished in obesity-related hypertension, and that these changes may result from aberrant central signalling mechanisms. Our findings provide evidence of impaired sympathoinhibitory mechanisms from the gut in the development of hypertension in obesity. These studies specifically demonstrate that renal and splanchnic sympathoinhibitory and regional vasodilator effects of CCK and gastric leptin are attenuated or reversed in obese hypertensive animals. Blunted vasodilator responses to CCK were directly correlated with resting AP, weight gain and adiposity index. The typical inhibitory RVLM neuronal responses induced by CCK were blunted or were instead sympathoexcitatory in obese hypertensive animals and RVLM neuronal responses to CCK were directly correlated with resting AP and weight gain. These animals also had attenuated barosensitivity of RVLM neurons, indicative of altered baroreflex gain. Furthermore, in obese hypertensive rats the number of Fos-IR neurons was significantly reduced in key cardiovascular medullary centres involved in sympathoinhibitory reflexes. Nevertheless, subdiaphragmatic vagal transmission and CCK1 receptor mRNA expression in the nodose ganglia were not significantly different between the obese animals and their normotensive counterparts, suggesting that vagal afferent transmission remains intact in obese animals. Collectively, these studies support the notion that central brainstem processing of sympathoinhibitory reflexes is compromised in obese hypertensive animals, at least with respect to the CCK-induced sympathoinhibitory reflex. Altered cardiovascular responses of gut peptides, may have serious implications on postprandial cardiovascular homeostasis and lead to increased vascular resistance in the renal and gastrointestinal beds. In obesity, these changes may disrupt sympathoinhibitory mechanisms and lead to increased sympathoexcitatory mechanisms and subsequently increased vascular resistance in the gut and kidney, contributing to the aetiology of hypertension. The implications of these studies are important in the development of novel targeted therapeutic interventions to better combat and treat hypertension in obesity.

As rates of obesity continue to rise, obesity related hypertension (ORH) is becoming an increasing health burden on a global scale. Multiple mechanisms underlie the development of hypertension in obesity but important common end-pathways are vasoconstriction and volume expansion due to enhanced tubular reabsorption of sodium. The mechanisms that lead to obesity-related sodium retention at a tubular level are largely unknown. The effects of high fat diet (HFD) induced obesity on distal tubular sodium transporters were studied at different time points. At 2.5 weeks the key sodium-retaining change identified was increased expression of NCC (the target for thiazide diuretics). In contrast, at 14 weeks, NCC expression had returned to baseline but the activities of NKCC2 (the target for frusemide) and ENaC (the target for amiloride) were increased. The increase in NKCC2 activity was shown to be due to increased activating-phosphorylation. There was a marked increase at S126-NKCC2, which identifies a new role for this site in the regulation of blood pressure. A smaller phosphorylation change at T96/T101 due to increased activity of SPAK/OSR1 was also noted in the cortex. This suggests that different mechanisms underlie the establishment and maintenance of sodium retention in obesity. The energy-sensing kinase AMPK has been implicated as a mediator of several adverse consequences of obesity. AMPK has previously been found to phosphorylate S126-NKCC2 in vitro. AMPK activity was reduced in the renal cortex and in cells of the thick ascending limb after 14 weeks of HFD, making it unlikely AMPK was responsible for phosphorylation change at S126-NKCC2 noted at this time. However, in vitro studies employing activation of AMPK and the WNK/SPAK/OSR1 pathway in wild type and AMPK-null murine embryonic fibroblasts have revealed a role for AMPK in negatively regulating activating-phosphorylation of SPAK/OSR1. This suggests obesity-related AMPK suppression is responsible for the enhanced T96/T101-NKCC2 found in the renal cortex after 14 weeks of HFD through activation of SPAK/OSR1. The widely expressed basolateral sodium potassium 2 chloride co-transporter NKCC1 is closely related to NKCC2 and shares many structural and functional properties. A role for NKCC1 in regulating blood pressure through effects on vascular tone has previously been identified. A role for AMPK in regulating the function of NKCC1 was studied. AMPK was found to negatively regulate NKCC1 activity through inhibiting activating-phosphorylation at T212/217. This was due to reduced activity of SPAK/OSR1; phosphatase activity and surface expression were unchanged by activation of AMPK. In summary, the current study has revealed that different tubular mechanisms are involved in the establishment and maintenance of sodium retention in obesity. Increased phosphorylation of S126-NKCC2 has been identified as a hitherto unrecognized mediator of enhanced sodium reabsorption in ORH. Furthermore, for the first time, obesity-related suppression of AMPK has been linked to activation of SPAK/OSR1. Through downstream effects on NKCC2 and NKCC1, respectively, this links AMPK suppression with sodium retention and vasoconstriction, the key mechanisms that drive the development of ORH.

Medicine (Austin & Northern Health) - Theses

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