Sympathetic activation is a hallmark in obesity. While uncomplicated obesity is accompanied by an increase in muscle sympathetic nerve activity (MSNA; Lambert et al., ^2010b), sympathetic activation is more pronounced in obese subjects with central fat accumulation (Alvarez et al., ²⁰⁰²; Grassi et al., ²⁰⁰⁴), hypertension (Grassi et al., ²⁰⁰⁰; Lambert et al., ²⁰⁰⁷), obstructive sleep apnoea (Grassi et al., ^2005b), and when associated with the metabolic syndrome (MS; Grassi et al., ^2005a). Such sympathoexcitation is most likely involved in the generation and aggravation of the hemodynamic and metabolic profile of obese individuals as it has been shown to favor blood pressure (BP) elevation (Grassi et al., ²⁰⁰⁴), reduced insulin sensitivity and increased serum triglycerides (Landsberg, ²⁰⁰¹) and to be associated with left ventricular hypertrophy (Schlaich et al., ²⁰⁰³) and dysfunction (Grassi et al., ²⁰⁰⁹). Reinforcing the possible deleterious impact of sympathetic overactivity in obesity, we recently demonstrated a direct relationship between sympathetic drive and the degree of kidney, cardiac, and endothelial dysfunction in a population of young overweight individuals prior to the development of any elevation in BP (Lambert et al., ^2010b). Life style modification is the first line treatment for the management of the MS. Hence, we have demonstrated that diet-induced calorie restriction not only improved all components of the MS but also significantly lowered BP (Straznicky et al., ²⁰⁰⁵), improved renal function (Straznicky et al., ^2011b), and that these improvements were underpinned by a marked decrease in multi-unit MSNA activity. As such sympathoinhibition is an important goal in the therapeutic approach in the MS (Grassi, ²⁰⁰⁶; Mancia et al., ²⁰⁰⁷). Direct recording of multi-unit MSNA provides an estimation of overall sympathetic activity but does not indicate how the activation is brought about nor the mechanism by which changes in sympathetic nerve firing can occur. Indeed, different mechanisms, including recruitment of active fibers, increased probability or rate of firing of active fibers, or increased discharge (multiple firing) within a sympathetic burst can be at play (Macefield et al., ¹⁹⁹⁴). We recently demonstrated that in subjects with obesity, recruitment of additional fibers was the central mechanism by which sympathoexcitation occurs because the firing rate of individual fiber was normal and incidence of multiple firing was rare (Lambert et al., ²⁰⁰⁷). In hypertensive subjects with the MS, we also observed that the incidence of multiple firing was linked with anxiety proneness (Lambert et al., ^2010a). Therefore, recording the firing pattern of single vasoconstrictor neurons provides additional information about the mechanisms underlying the ultimate degree of sympathetic outflow. We therefore sought to determine whether the pattern of sympathoexcitation of single-unit MSNA was altered following diet-induced weight loss in subjects with the MS.

Subjects and data were drawn from our previous investigations examining the role of diet on sympathetic nervous activity in subjects with the MS (Straznicky et al., ²⁰⁰⁵, ^2011a).

The clinical trial was registered at ClinicalTrials.gov trial NCT00163943. Only patients with successful single-unit MSNA measurements obtained on two occasions (before and after the diet) were included (n = 14). These subjects comprised nine men and five postmenopausal women, aged 49–70 years (mean = 57 ± 2 years), who fulfilled Adult Treatment Panel III criteria for the MS (Third Report of the National Cholesterol Education Program, ²⁰⁰²). Participants had central obesity (waist circumference ≥102 cm in men and ≥88 cm in women) and two or more MS parameters. All were non-smokers, sedentary, defined as physical exercise two or less times per week for <20 min/session with a stable body weight (<1 kg) in the previous 6 months. Exclusion criteria included type 2 diabetes (fasting glucose ≥7 mmol/l), a history of secondary hypertension or cardiovascular, cerebrovascular, renal, liver, or thyroid disease, and use of drugs known to affect measured parameters. Participants treated for hypertension were studied after medications had been discontinued for at least 6 weeks. Screening investigations comprised physical examination, medical and dietary histories, 12-lead electrocardiogram, blood biochemistry, and lipid analyses. Supine BP was measured on three occasions 1 week apart as the average of five readings after a 5-min rest (Dinamap, Model 1846SX; Critikon, Tampa, FL, USA). The third of these measurements was defined as baseline BP. The study was approved by the Alfred Hospital Ethics Committee, and all subjects gave written informed consent.

We examined the effects of diet-induced weight loss on the autonomic nervous system in middle aged individuals with the MS by combining multi-unit and single-unit MSNA recordings with the assessment of cardiac and sympathetic baroreflex function. Our results indicate that in response to weight loss, along with favorable hemodynamic and metabolic changes, autonomic function is greatly improved with changes in the firing pattern of active vasoconstrictive fibers contributing to the reduction of sympathetic outflow.

The presence of sympathoexcitation in patients with the MS has previously been described (Grassi et al., ^2005a; Straznicky et al., ²⁰⁰⁵). Importantly, sympathetic activation can result from a combination of changes in the behavior of the firing activity of individual vasoconstrictor fibers including either a change in the number of active fibers (recruitment) or/and changes in the pattern of firing such as increased firing rate and probability and increased discharge during a sympathetic burst (multiple spikes; Macefield et al., ¹⁹⁹⁴). In conditions associated with elevated sympathetic outflow such as in heart failure (Macefield et al., ¹⁹⁹⁹; Murai et al., ²⁰⁰⁹), essential hypertension (Greenwood et al., ¹⁹⁹⁹; Lambert et al., ²⁰⁰⁷), and obstructive sleep apnoea (Elam et al., ²⁰⁰²), it has been shown that changes in the firing pattern contribute to the degree of sympathoexcitation. We however previously demonstrated that this is not the case in obesity where the mechanism by which sympathetic activation occurs predominantly involves recruitment of active fibers as the firing pattern of individual vasoconstrictor fibers is similar to that of non-obese individuals (Lambert et al., ²⁰⁰⁷). The characteristics of the firing pattern of sMSNA of the MS subjects in the present study were comparable to those of our previously published obese subjects (Lambert et al., ²⁰⁰⁷) indicating that sympathoexcitation in our MS participants seems to involve an increase in the recruitment of vasoconstrictor fibers rather than abnormalities in the firing pattern. Surprisingly, the present study indicates that despite a normal firing pattern, the decrease in sympathetic activity associated with weight loss involves alteration in the firing pattern of sympathetic nerves including a decrease in firing rate, probability of firing as well as a decrease in the incidence of multiple firing within a sympathetic burst. Whether changes in the firing pattern alone are sufficient to induce the observed decrease in multi-unit MSNA is not known. Given that recruitment of active fibers is the main mechanism by which sympathetic activation occurs it is likely that a decrease in the recruitment of active fibers would also contribute to the reduction in sympathetic nervous activation. Previous studies have demonstrated that changes in the firing pattern was critical in the sympathoexcitation associated with acute physiological stressors in healthy individuals (Macefield and Wallin, ¹⁹⁹⁹; Murai et al., ²⁰⁰⁶) as well as in patients with chronic heart failure (Murai et al., ²⁰⁰⁹). For example Murai et al. (²⁰⁰⁹) observed that in response to handgrip exercise, heart failure patients displayed an aberrant firing pattern of individual vasoconstrictor fiber and that this most likely contributed to the exaggerated sympathoexcitation occurring during exercise. Our study extends these observations by indicating that the firing pattern of single vasoconstrictor fibers may be crucial in the adjustment of chronic sympathoinhibition associated with weight loss.

As expected, decreased sympathetic nervous activity induced by body weight loss was also associated with a number of hemodynamic, metabolic, and reflex changes suggesting that the changes in sympathetic outflow are likely to involve numerous mechanisms. Grassi et al. (¹⁹⁹⁵) observed that in obese normotensive subjects the baroreceptor modulation of heart rate and MSNA was blunted and suggested a baroreflex impairment as a possible cause of the obesity-related sympathetic activation. In the present study we found that the decrease in sympathetic outflow was accompanied by a marked improvement of both cardiac and sympathetic baroreflex function which is in line of previous finding in obese normotensive subjects (Grassi et al., ¹⁹⁹⁸).This suggests that the changes in sympathetic activity associated with body weight modifications may have a reflex origin and that the improvement of the baroreflex control of the cardiovascular system may lead to reduced sympathetic outflow. Single-unit vasoconstrictor neurons are known to be strongly influenced by baroreceptor stimuli within the physiological range (Macefield et al., ¹⁹⁹⁴) hence, a change in baroreflex function is likely to affect their firing properties.

The improvement in the metabolic profile of our participants was significant and is likely to have played an important role in the reduction of sympathetic outflow. In particular, plasma insulin concentration and insulin resistance (HOMA) were reduced. We have previously observed that sympathetic nervous activity, as assessed from measures of whole body noradrenaline spillover and MSNA, is strongly correlated to insulin resistance in MS patients (Straznicky et al., ²⁰⁰⁵). Furthermore, reflex and metabolic mechanisms may reinforce each other in the sympatho-inhibitory effect because insulin may lead to sympathetic activation through an impairment of the baroreceptor function (Rowe et al., ¹⁹⁸¹). Sympathetic activation in turn can induce insulin resistance and hyperinsulinemia (Lembo et al., ¹⁹⁹⁴). An alternative mechanism may involve changes in plasma leptin concentration which was reduced following weight loss. There exist reports indicating that leptin causes sympathoexcitation (Rahmouni, ²⁰¹⁰). Interestingly, in this study we noted a small but significant reduction in anxiety state and trait in our participants following the weight loss intervention. Previously we demonstrated that in hypertensive patients with the MS, state and trait anxiety scores correlated with the firing rate, firing probability, and incidence of multiple firing of individual vasoconstrictor fibers but not with multi-unit MSNA indicating that a more disturbed firing pattern was associated with anxiety proneness (Lambert et al., ^2010a). Our results of a simultaneous change in anxiety and firing pattern are suggestive of a potential causal effect.

The decrease in the firing probability and rate and incidence of multiple spikes associated with weight loss may have important physiological and clinical relevance. Previous reports have demonstrated that irregularity of the sympathetic firing pattern may influence effector organ responses. Indeed, irregular electrical stimulation produces faster and more pronounced vasoconstrictor responses than continuous stimulation at a regular frequency in cat skeletal muscle (Andersson et al., ¹⁹⁸³), in small arteries and veins from rats (Nilsson et al., ¹⁹⁸⁵), and in rabbit hindquarters (Ando et al., ¹⁹⁹³). Dibona and Sawin (¹⁹⁹⁹) observed that the pattern of renal sympathetic nerve stimulation in rats significantly influenced the rapidity, magnitude, and selectivity of the renal vascular and tubular responses. Furthermore we recently demonstrated that a more disturbed firing pattern of vasoconstrictor fibers to the skeletal muscle was associated with higher rates of noradrenaline spillover from the heart (Lambert et al., ²⁰¹¹), a finding suggesting that a more disturbed firing pattern may be associated with increased cardiovascular risk. Indeed, a direct association between left ventricular hypertrophy and the firing rate of sympathetic fibers has been shown in hypertensive individuals (Burns et al., ²⁰⁰⁷). Taken together, this observation suggests that a decrease in the firing rate and probability, and a reduction in incidence of multiple spikes of individual vasoconstrictor fiber may confer reduced damage to the cardiovascular and renal systems.

There are a number of limitations that we must acknowledge. The absence of a weight stable control group means that we may not have fully accounted for the effects of time and familiarization on study parameters. However sympathetic nerve recording is highly reproducible over a long period of time (Fagius and Wallin, ¹⁹⁹³) and we previously found no time effect in sympathetic outflow in subjects with the MS (Straznicky et al., ^2011a) hence the changes in sympathetic parameters are likely to be driven by the dietary restriction. The small number of subjects in our study was dictated by the technical difficulties of obtaining single-unit recordings and did not allow us to uncover possible associations between changes in single-unit parameters and other physiological characteristics. Furthermore, we cannot conclude as to whether the changes in sympathetic nerve firing properties that we observed are due to the weight loss or the negative energy balance. Weight loss maintenance was indeed recently shown to be associated with a rebound in mMSNA (Straznicky et al., ^2011a).

In conclusion, sympathoinhibition associated with weight loss in patients with the MS involves marked changes in the firing pattern of active vasoconstrictive fibers and this may due to improved reflex mechanisms and metabolic profile.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We acknowledge the contribution of Ms. Ania Schlaich in analyzing the data. Source(s) of funding: This study was supported by grants from the National Health and Research Council of Australia (NHMRC), the National Heart Foundation of Australia, and in part by the Victorian Government’s Operational Infrastructure Support Program. Dr. E. Lambert, Ass. Prof. Schlaich, Prof. Esler, and Dr. G. Lambert are supported by career fellowships from the National Health and Medical research council of Australia.