OBJECTIVE: To determine whether plasma leptin and insulin concentrations are related to adiposity-associated elevations in muscle sympathetic nerve activity (MSNA) with age in healthy adult humans.
DESIGN: Cross-sectional investigation of young and older adult men.
SUBJECTS: Thirty healthy adult men, 16 young (25±1 y, mean±s.e.) and 14 older (61±1 y).
MEASUREMENTS/RESULTS: The older men had higher (P<0.05) levels of body mass, BMI, total fat mass and truncal fat mass (dual energy X-ray absorptiometry) than the young men. MSNA burst frequency (microneurography) was ∼75% higher in the older men (P<0.001). Plasma leptin concentrations were ∼150% higher (P<0.01), whereas plasma insulin concentrations were ∼70% higher (P<0.05) in the older subjects. MSNA was related to both total (r=0.51, P<0.01) and truncal (r=0.56, P<0.01) fat mass. Plasma leptin concentrations were related to total and truncal fat mass (both r=0.83, P<0.001), and to MSNA (r=0.49, P<0.01). Plasma insulin concentrations were related to MSNA (r=0.38, P<0.05). We used partial correlation analyses to assess whether leptin and/or insulin are potential contributors to the relation between body fat and MSNA.
Adjusting for the effects of plasma leptin, but not insulin, concentrations eliminated the significant relations between MSNA and total and truncal fat mass.
CONCLUSION: Our results: (1) demonstrate a positive relation between MSNA and plasma leptin concentrations in young and older healthy men; and (2) support the concept that circulating leptin concentrations may act as a humoral signal contributing to adiposity-associated elevations in MSNA with age in adult humans.
The authors1 and others2,3,4 have demonstrated that tonic (basal) skeletal muscle sympathetic nerve activity (MSNA) increases with age even in healthy adult humans. Recently, we found that this elevation in MSNA with age is related in part to increases in whole-body and central (ie abdominal or trunk) body fatness.5,6 However, the mechanism underlying the association between elevations in adiposity and MSNA with age is unknown.
Circulating levels of leptin are one possible physiological signal linking adiposity with age-related increases in MSNA. Leptin stimulates central sympathetic outflow to the hindlimb in experimental animals,7,8 and plasma concentrations of leptin correlate positively with MSNA in younger adult humans.9 Moreover, plasma leptin levels are strongly related to total fat mass10,11,12 and in some cases abdominal or truncal fat mass13,14 in humans. Elevations in plasma leptin concentrations with age have been observed in some,11,13,15 but not all10,12 men or women studied to date. However, the exact nature of the inter-relations among MSNA, adiposity and plasma leptin concentrations with age are unknown.
Circulating concentrations of insulin represent another possible humoral signal linking body fat and MSNA with advancing age. In young adult humans, a significant association between fasting insulin concentrations and MSNA has been observed,16 although not in all cases.17,18 Fasting insulin levels are positively related to adiposity,19,20,21 and tend to increase with age, even in healthy adults.22 However, the possible role of plasma insulin concentrations in the age-associated elevations in basal MSNA has not been determined.
The experimental aim of the present investigation was to determine the relations among fasting plasma leptin and insulin concentrations, total and truncal fat mass, and MSNA with age in healthy adult humans. We hypothesized that circulating leptin and insulin concentrations may act as humoral signals contributing to the observed relation between MSNA and adiposity with age. If so, we expected to find that these putative signals would correlate with MSNA, and that accounting for their respective influences would abolish or at least weaken the relations between MSNA and total and truncal adiposity.
Thirty healthy sedentary adult males, 16 young (25±1 y, mean±s.e.) and 14 older (60±1 y) were studied. All subjects were non-diabetic, normotensive (systolic/diastolic blood pressure<140/90 mmHg), free of known cardiovascular and metabolic disease, and otherwise healthy as assessed by medical history. Older subjects were further evaluated with electrocardiograms during rest and maximal exercise. Subjects were non-smokers and were not taking any regular medications. All subjects had thyroid hormone levels within the normal range. The nature, purpose and risks of the study were explained to each subject before written informed consent was obtained. The experimental protocol was approved by the Human Research Committee at the University of Colorado at Boulder.
All measurements were made in the morning after a 12 h fast. Subjects were studied under quiet resting conditions in the semi-recumbent position. Arterial blood pressure was measured by auscultation over the brachial artery according to American Heart Association guidelines.23
Multiunit recordings of MSNA were obtained from the right peroneal nerve at the fibular head using the microneurographic method previously described by our laboratory.1,24 The neural activity was amplified, filtered (bandwidth=700–2000 Hz), full-wave rectified, and integrated (time constant=100 ms) to obtain a mean voltage neurogram determined to be acceptable according to criteria previously described.1,25 After stable baseline activity was established, recordings of MSNA were obtained over a subsequent 20 min period.
Total body mass and composition and trunk region adiposity were determined as described previously by our laboratory.26 Body mass was measured on a physician's scale. Whole-body composition was measured using dual-energy X-ray absorptiometry (DXA-IQ; Lunar Radiation Corp., Madison, WI, software version 4.1). Truncal body fat was determined from extended analysis of the LUNAR software. DXA-determined truncal fat correlates strongly (r=0.90) with computed tomographic (CT) measurements of central body fat.27,28
Antecubital venous blood samples were obtained at 1, 10 and 20 min for subsequent determinations of plasma leptin (assay kit, LINCO Research Inc., St Charles, MO), insulin (assay kit, American Laboratory Products Company, Windham, NH) and glucose concentrations (Beckman glucose analyzer 2, Beckman Instruments Inc., Fullerton, CA) using standardized biochemical approaches.
Data analysis and statistics
Bursts of MSNA were identified by visual inspection. For consistency, the same investigator analyzed all neurograms. MSNA was calculated as the mean burst frequency (bursts/min) over the final 10 min of the 20 min recording period. The reliability of these measurements has been established previously by our laboratory.24 The average of the values obtained from the three blood samples was used to determine fasting plasma concentrations of leptin, insulin and glucose.
Group differences in selected subject characteristics were determined by unpaired t-tests. Correlation coefficients were obtained by Pearson product–moment correlations to determine the simple relations between variables of interest. Multiple regression analysis was used to determine the relations between MSNA and measures of adiposity before and after partialling out the effects of humoral factors. The level of statistical significance was set at P<0.05. Data are expressed as mean±s.e.
Selected subject characteristics are shown in Table 1. There were no age-group differences in resting arterial blood pressure. The older subjects had a higher body mass, body mass index (BMI), total fat mass, and truncal fat mass than the young subjects (all P<0.05). MSNA burst frequency was ∼75% higher in the older men (P<0.01). Fasting plasma concentrations of leptin, insulin and glucose are shown in Table 2. Plasma leptin concentrations were ∼150% higher (P<0.01) and plasma insulin concentrations were ∼70% higher (P<0.05) in the older subjects. Fasting plasma glucose concentrations tended to be higher in the older men (P=0.06). The age-related difference in leptin levels was explained by differences in adiposity because removing the influence of fat mass eliminated this difference (P=0.99).
Simple relations among MSNA, adiposity and humoral factors
In the pooled population MSNA was positively related to both total fat mass (r=0.51, P<0.01) and truncal fat mass (r=0.56, P<0.001). Fasting plasma concentrations of leptin were related to both total fat mass (r=0.83, P<0.001) and truncal fat mass (r=0.83, P<0.001), and to MSNA (r=0.49, P<0.01) (Figure 1A). Fasting plasma concentrations of insulin also were significantly related to total (r=0.59, P<0.001) and truncal (r=0.67, P<0.001) fat mass, and to MSNA (r=0.38, P<0.05) (Figure 1B). Plasma glucose concentrations were not significantly related to total (r=0.37, P=0.06) and truncal (r=0.38, P=0.06) fat mass, and to MSNA (r=0.38, P=0.06) (Figure 1C).
Effects of plasma concentrations of leptin and insulin on the MSNA-adiposity relations
Removing the influence of plasma leptin concentrations using partial correlation analysis eliminated the significant relations between MSNA and both total fat mass (r=0.22, P=0.26) and truncal fat mass (r=0.31, P=0.10). In contrast, accounting for the influence of plasma insulin concentrations did not affect the significant relations between MSNA and adiposity (all remained P<0.01).
The primary new findings from the present study are: (1) tonic levels of MSNA are related to fasting plasma leptin and insulin concentrations in healthy young and older adult males; and (2) plasma leptin, but not insulin, concentrations appear to contribute to the MSNA–adiposity relation with age in these men. Thus, our findings provide initial experimental support for the hypothesis that circulating levels of leptin may serve as an important physiological signal linking age-associated increases in adiposity to elevations in MSNA.
Our conclusion that plasma leptin concentrations may act as a humoral signal coupling MSNA and adiposity with age is based on at least three lines of evidence. First, mean levels of both MSNA and leptin were higher in the older men. Second, among the individual subjects, univariate correlational analysis revealed significant positive relations between plasma leptin concentrations and: (a) MSNA (r=0.49); and (b) total and truncal fat mass (both r=0.83). Third and most importantly, partialling out the effect of plasma leptin concentrations eliminated the significant relations between MSNA and both total and truncal fat mass.
Our finding of a significant positive relation between plasma leptin concentrations and MSNA with increasing age in men is consistent with the recent observations of Snitker et al.9 They reported a correlation of r=0.44 between MSNA and plasma leptin concentrations in a group of healthy men with an average age of 29 y. Based on this correlation, these investigators concluded that circulating levels of leptin may be the physiological mechanism explaining the previously observed relation between MSNA and adiposity in younger adults.16,18,24 The present findings extend those of Snitker and colleagues in at least two ways. First, we have shown that the relation between plasma leptin concentrations and MSNA is maintained in middle-aged and older adult males. Second, we have demonstrated that accounting for the influence of plasma leptin concentrations abolishes the significant relations between MSNA and total and truncal fat mass in this population. The latter is a new and critical line of support for the working hypothesis that plasma leptin levels may play an important role in the tight physiological coupling between MSNA and adiposity. This provides an initial rationale for pursuing this possibility experimentally (eg by chronic interventional manipulation of leptin and body fat levels in older individuals to determine a resultant change in basal MSNA).
It should be noted that all of the present measurements were made in the fasting state. Diurnal fluctuations in plasma leptin levels, and sympathoexcitatory responses evoked by other stimuli may alter this relation. However, in the present context we are attempting to identify a possible mechanism for the previously observed correlation between age-associated increases in adiposity and basal (fasting) MSNA. Thus, the relations of MSNA and leptin to adiposity are key, and because adiposity does not vary acutely, the interpretation of these data should not be affected by the fasting state or diurnal variations in leptin.
We also considered the possibility that plasma insulin concentrations could be the physiological link between increases in adiposity and MSNA with age. We found that, although statistically significant, the relation between MSNA and fasting plasma insulin concentrations in our pooled population was not as strong (r=0.38) as that observed for leptin. This observation is in agreement with a previous study;16 however, two other investigations found no significant relation between MSNA and fasting insulin levels in healthy adults.17,18 More importantly, in the present study partialling out the influence of plasma insulin concentrations did not affect the significant relations between MSNA and adiposity with age. Taken together, these findings indicate that MSNA and plasma insulin concentrations are, at most, weakly related, and that it is unlikely that circulating fasting insulin levels serve as a physiologically significant signal through which tonic MSNA and adiposity are linked.
We wish to emphasize that acute hyperinsulinemia, evoked experimentally (eg insulin infusion) or in response to energy intake, is clearly associated with a temporary increase in MSNA.29,30,31,32 At such high circulating plasma concentrations insulin may indeed serve as a physiological mediator of sympathoexcitation via a direct stimulatory effect on the CNS7 and/or an indirect arterial baroreflex-mediated response to peripheral vasodilation.33 In any case, the conclusions for the present study should be confined to the relation between basal MSNA and fasting plasma insulin concentrations.
It should be noted that the relation of fasting plasma glucose concentration to MSNA and to total and truncal fat mass did not reach statistical significance in the present investigation. In an earlier study, Spraul et al 34 failed to observe a significant association between MSNA and plasma glucose levels in younger adults. Considered together, these findings suggest that fasting plasma glucose concentrations in the normal euglycemic range is unlikely to be an important mechanism explaining the associations between tonic MSNA and adiposity with age. This conclusion recognizes that both acute hypoglycemia35 and hyperglycemia34 produce temporary and potentially marked elevations in MSNA in humans.
In the present study, we found that fasting plasma leptin concentrations were ∼150% higher in older compared with young healthy adult males. This agrees with the findings of some previous reports of higher plasma leptin concentrations in older adults.11,13,15 The present results support the idea that the increase in plasma leptin concentration observed with advancing age is associated with elevations in adiposity because adjusting for total or truncal fat mass via ANCOVA eliminated the significant age-group differences in leptin. Some previous studies have not found age-related increases in fasting leptin concentrations in men and/or women.10,12 The lack of consistent findings may be due to differences in age or the level of adiposity of the subjects, or the statistical technique used to adjust for the effects of adiposity. Regardless, our results support the idea that age-associated elevations in fasting leptin concentrations are secondary to increases in total and central adiposity.
There are two potentially important limitations associated with the present study that should be considered. First, our conclusions are based solely on data from correlational analyses, which cannot determine cause and effect. Thus, further work must be done to identify whether leptin acts as a signal stimulating MSNA as adiposity increases with age. Secondly, because of the cross-sectional study design employed, it is possible that genetic or other constitutional factors independent of age or adiposity influenced our results.
In conclusion, the results of the present study demonstrate a positive relation between MSNA and plasma leptin concentrations in young and older healthy men. Moreover, removing the influence of plasma leptin concentrations abolished the significant relations between MSNA and fat mass. These findings provide experimental support for the concept that circulating leptin levels may act as a physiologically important humoral signal underlying adiposity-associated elevations in MSNA with age in healthy adult humans. Further studies are needed, however, to demonstrate that these events are causally linked.
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The authors would like to thank Mary Jo Reiling and Sonya Craig for their technical assistance, and the Colorado Clinical Nutrition Research Unit (DK48520) for the plasma leptin analyses. This study was supported by NIH awards AG39966, AG06537, Institutional Training grant DK07658 (MBM), AG05705 and K01 AG00828 (PPJ).
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Monroe, M., Van Pelt, R., Schiller, B. et al. Relation of leptin and insulin to adiposity-associated elevations in sympathetic activity with age in humans. Int J Obes 24, 1183–1187 (2000). https://doi.org/10.1038/sj.ijo.0801364
- muscle sympathetic nerve activity
- abdominal body fat
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