Subjects

Of the 63 subjects, the 23 females were, on average, slightly older than males (mean age ± std: female 50.3 ± 7.8 years, male 45.9 ± 9.1 year), although the difference was not significant (P > 0.05, independent samples t test). Body mass index also did not differ significantly (female 23.9 ± 5.0 kg/m2, male 25,2 ± 2.8 kg/m2; group difference P = 0.2, independent samples t test). Seven females reported being left handed, three ambidextrous, and 13 right handed. Five males reported being left handed, two ambidextrous, and 33 right handed. The difference was not significant (P = 0.13, chi-square).

Physiology

Heart rate showed significant sex, time, and sex × time effects (all P ≤ 0.001), with relative increases in females and males during the grip period (model statistics: chi-square = 2722, −2 res log-likelihood = 162,589; Fig. 2a). Visual inspection of the trends over the protocol confirms similar responses for each of the four challenges (Fig. 2b). As in other healthy populations, the absolute heart rates were higher in females than males (Fig. 2b). Four females and three males showed artifact in the mean saturation signal at times during the series and were excluded from the SaO 2 analysis. The remaining 37 males showed a decrease in oxygen saturation (SaO 2 ) from 5 s into the challenge, whereas the 19 females showed no significant change in SaO 2 (Fig. 2c). The response patterns differed between the two groups from 9 to 14 s into the challenge. However, the plot of SaO 2 over the protocol (Fig. 2d) suggests that the two groups started at a similar level, but the males decreased further during the recovery from the first challenge and then remained at a lower level throughout the remainder of the challenges.

Fig. 2 Heart rate (HR) and SaO 2 changes during a series of four static handgrip exercise challenges, averaged for female and male groups. HR is from all 63 subjects, and SaO 2 is from 58 subjects with artifact-free mean saturation data. a HR % change relative to baseline and c SaO 2 , averaged over four challenges (mean ± SE). Time points of significant within-group responses are indicated by blue xs (males) and red circles (females), and significant between-group differences in red-yellow asterisks, based on P < 0.05 with repeated measures ANOVA (RMANOVA). b Absolute heart rate and d SaO 2 over series of four static handgrip exercise challenges, averaged separately over females and males with SE shaded Full size image

fMRI responses: sex differences

The static handgrip exercise challenge elicited significant fMRI signal responses that differed from baseline in all insular gyri in both sexes (Fig. 3). For both males and females, neural responses were present during the challenge and recovery periods (blue X’s and red O’s in Fig. 3). Female and male responses differed in all gyri except the left ASG (Table 1; Fig. 3). The responses generally showed an increase, peaking between 5 and 6 s into the challenge, followed by a decrease in the response to a nadir between 12 and 16 s into the challenge and a second peak 4–10 s into the recovery period. While patterns included increases and decreases relative to baseline, the magnitude of female responses was consistently higher than in males during the static handgrip exercise challenge in the gyri where female and male responses differed, as shown by the higher group averages (that is, the solid female lines higher than dashed male lines in Fig. 3). Group differences were present from 4 s into the challenge, shortly after the first static handgrip exercise (P < 0.05, RMANOVA red-yellow stars in Fig. 3). In the left gyri, the female response initially increased faster than the male response, but the peak response did not differ; however, the male response declined faster and farther than the female response, except in the PSG, where the female trough approached the response from the males and then diverged briefly at the onset of the recovery. In the PSG, the female response briefly dipped lower than the male response 28 s into the recovery period. Females also exhibited lower responses at 38 and 44 s into recovery in the left ALG. In the right gyri, females exhibited a higher response from 2–4 s into the challenge until the end of the challenge, or 2 s into recovery, with the exception of the time of the peak response in the right ALG and PLG where the male response approached the female response. In the right PSG, the secondary peak was higher in females at 6 s into the recovery.

Fig. 3 Mean fMRI insula responses over four static handgrip exercise challenges, averaged for female and male groups. All left and right gyri response patterns are shown. Time points of significant within-group responses and between group differences are indicated above the x-axis and below the graphs (RMANOVA P < 0.05; Table 1) Full size image

Table 1 Female vs. male model fit. Overall model chi-square (ChiSq) was always significant (P < 0.0001) Full size table

Additional analyses are shown Additional file 1 (age-related models) and Additional file 2 (right-handed only models). These files illustrate the significance comparisons via color-coded cells. Inclusion of age-affected finings only in the left MSG, with a change from non-significant to significant of sex in two models (2 and 3 in the “Additional analyses: age effects and handedness” section). Inclusion of age by time interactions affected only the left PSG, with a change from significant to non-significant of time and time by sex interaction in two models (3 and 4 in the “Additional analyses: age effects and handedness” section). Inclusion of age by sex interactions affected all right gyri, with a change from significant to non-significant of sex in two models (4 and 5 in the “Additional analyses: age effects and handedness” section). Analysis of right-handed only subjects resulted in mostly similar findings. Considering the original model (1 in the “Additional analyses: age effects and handedness” section), a change from non-significant to significant in the effect of sex appeared in the left MSG, with the other 29 model effects being unchanged. The remaining right-handed models showed 14 of 200 effects with significance changes (full details in Additional file 2).

fMRI responses: anterior-posterior organization

In the right insular gyral responses relative to the right PLG, both female and male responses in the anterior gyri (ASG, MSG, and PSG) showed a higher response from 2 to 4 s into the challenge until 4–6 s into the recovery period (Fig. 4). In the left gyri (relative to the left PLG), in females, MSG > ALG and PSG > ALG from 6 s through the remainder of the challenge period. The left PSG response remained higher than the PLG response until 6 s into the recovery period. In the left gyri in males, ASG > ALG from 2 s into the challenge through the challenge, except at 12 s into the challenge. MSG > ALG and PSG > ALG from 2 s into the challenge through to the end of the challenge (MSG) and 2 s into the recovery period (PSG), except at 4 s into the challenge when the PLG response peaks. The anterior gyri showed mostly similar patterns of response (Table 2).

Fig. 4 Anterior-to-posterior organization of insula fMRI responses over four static handgrip exercise challenges, illustrated by time trends relative to pattern in posterior-most gyrus (PLG). Females in top and males in bottom. Time points of between-gyrus differences are indicated by symbols above the x-axis and below the graphs (RMANOVA P < 0.05; Table 2) Full size image

Table 2 Anterior-posterior model fit. Overall model chi-square (ChiSq) was always significant (P < 0.0001) Full size table

Additional analyses are shown in Additional file 3 (age-related models) and Additional file 4 (right-handed only models). These files illustrate the significant comparisons via color-coded cells. Inclusion of age by gyrus interactions affected finings in females and males in the right side only, with a change from significant to non-significant of sex in two models (4 and 5 in the “Additional analyses: age effects and handedness” section). Analysis of right-handed only subjects resulted in similar findings to all subjects across 80 effects assessed (full details in Additional file 4).

fMRI responses: left-right organization

Lateralization of activity during the challenge was evident in the ASG for males but only sporadically for females (Fig. 5). In males, the right response was less than the left. The lateralization response in males differed from females, which was similar at the onset of the challenge, but the right side dipped less than the left side from 8 to 12 s. The males show a significantly more left-dominant response in the anterior ASG and MSG gyri but not the more posterior regions (Table 3).

Fig. 5 Lateralization of insula fMRI responses averaged over four static handgrip exercise challenges, illustrated by right − left time trends, such that a higher signal indicates a greater right-sided response. Time points of between-hemisphere differences in females (red circles) and males (blue xs) are indicated, as well as time points of group differences (red-yellow asterisks; RMANOVA P < 0.05; Table 3) Full size image

Table 3 Female vs. male in laterality (right minus left) model fit Full size table

Additional analyses are shown in Additional file 5 (age-related models) and Additional file 6 (right-handed only models). These files illustrate the significant comparisons via color-coded cells. Age did not alter effects in the PSG. Inclusion of age by time interactions affected finings in all other gyri, with a change from significant to non-significant of time in two models (3 and 4 in the “Additional analyses: age effects and handedness” section), and in the MSG changes from significant to non-significant of sex by time interactions. Inclusion of age by sex interactions affected findings in the ASG, with changes from significant to non-significant in the effects of sex in two models (4 and 5 in the “Additional analyses: age effects and handedness” section). Analysis of right-handed only subjects resulted in similar findings to all subjects across all but four of the 100 effects assessed (full details in Additional file 6). In the PSG, the time by sex interactions in the original model and in the model with age effect (2 in the “Additional analyses: age effects and handedness” section) changed from non-significant to significant. In the ALG, the time effect shifted from non-significant to significant in the models with age by time interactions (3 and 4 in the “Additional analyses: age effects and handedness” section).

Interpretation: overview of key findings

Females and males show similarities in fMRI signal responses during and after the short static handgrip exercise challenge, with a peak early in the challenge period, followed by a return to or below baseline, and an increase to another peak upon release, then by a gradual return to baseline. However, the magnitude of signal change differed by sex in all but the left anterior-most gyrus (ASG), with males showing lower fMRI signals in all other regions during and just after the challenge. Females showed lower heart rate increases during the challenge, but males dropped SaO 2 levels, which remained low throughout the protocol. The right insula showed an anterior dominance in both sexes, although with no distinction between the three short gyri. The left showed similar patterns, except that in females, the left ASG showed a lower response than the other short gyri. The left showed higher responses in all gyri in the males but only in the posterior gyri (ALG, PLG) in females. In fact, the two anterior-most gyri (ASG, MSG) showed greater right-sided responses in females. Thus, as with the Valsalva [23], the static handgrip exercise elicits generally similar responses in females and males, but selected areas, especially the ASG, show opposite patterns.

Cardiovascular responses

Heart rate increases to static handgrip exercise in females were lower than males. An earlier finding in a younger sample also showed slightly higher heart rate increases (but not significantly so) in males vs. females during a 2-min static handgrip exercise at 40% of maximum grip strength, even though in contrast with our study, both groups started at equivalent resting heart rate levels [43]. In a smaller and younger sample (7 men and 6 women, mean age 25–26), a 2-min static handgrip exercise at 30% of maximum elicited a substantially greater heart rate increase in males than females [44]. Unlike females, males also greatly increased muscle sympathetic nerve activity (MSNA), although this activity occurred later in the challenge. An earlier study found no sex difference in heart rate responses to a 30% static handgrip exercise [45], but only one measure at 60 s was used, and that analysis would be less sensitive that the time-trend comparisons used in more recent studies. The combined evidence suggests that females, on average, have a reduced change in heart rate to the static handgrip exercise. Since heart rate is a significant component of cardiac output increase to a pressor challenge, females presumably have a lower need or resort to other vascular and heart control mechanisms to accommodate day-to-day perfusion challenges.

The SaO 2 sex differences showed an enduring effect of prior static handgrip exercise challenges across the four task (~6 min) protocol. The RMANOVA performed on the averaged challenges was therefore confounded by the lack of return to baseline in such a way as to increase false negatives, but even so, SaO 2 showed significant declines during the challenge. The whole-protocol plot shows SaO 2 in both males and females declining around the first task and remaining low. Dips are visible in the males during the three subsequent grip periods, reflected also in the RMANOVA outcomes. However, intriguingly, the SaO 2 declines from before the initial task period, about 20–30 s into the baseline, suggesting an anticipation effect. The decline is over 0.5%, which for the fMRI BOLD signal is a substantial change; typical fMRI activations are measured as signal changes around 1%. However, the BOLD effect as a response to neuronal activation is relatively independent of baseline state [46]; therefore, while blood SaO 2 could affect the resting level of the fMRI signal, the activation should be similar.

The reason the males showed an overall lower SaO 2 is unclear. One possibility is that males were holding their breath because they were trying harder, a phenomenon observed with other tasks [47]. Tasks deemed “masculine” such as strengthening are associated with greater effort by males [48].

Insular function

The insula is involved in regulation of autonomic actions but also has an integrative role for body sensations, and during the static handgrip exercise, both of these functions will be represented in the fMRI signals. However, the sensory and interoceptive responses are principally located in the posterior insula [49–51], whereas the predominant responses to the static handgrip exercise were in the more anterior short gyri. Other functions associated with the insula such as pain and mood are unlikely to be represented during this short static handgrip exercise challenge.

While the anterior insula is active during many tasks involving sympathetic activation, the signal increases here show that the activity in the structure may also increase with suppression of parasympathetic action, which is accompanied by cardiovascular changes, including rising heart rate, blood pressure, and cardiac output [52]. Presumably, sympathetic outflow was not substantially increased during this brief static handgrip exercise [26], leaving, we speculate, a process dependent on suppression of parasympathetic activity. Previous neuroimaging studies on static handgrip exercise over a longer challenge period also show there is no direct relationship between MSNA and insular activation [53].

Lateralization

The left-sided dominance in all gyri in males likely reflects a combination of parasympathetic responses associated with vagal withdrawal and contralateral representation of the right-hand sensory-motor signals. That is, parasympathetic withdrawal could involve an active process in the insula perhaps reflecting an increase in inhibition [54]. Additionally, right-handed sensori-motor representation is in left cortical brain regions, which include the insula [55]. Thus, in males, any right-sided sympathetic dominance was likely masked by left-sided representation of the hand. The concept that sympathetic action is dominated by the right side of the brain is well supported by human and animal data [56–59], as well as by the Valsalva study conducted during the same series of experiments as the present static handgrip exercise challenge [23]. An interesting complementary experiment would be a left static handgrip exercise, during which we predict a larger right-sided insular response.

In contrast, while females showed equivalent left-sided dominance in the posterior long gyri, the two anterior-most gyri showed right-sided dominance (and the mid-region, MSG, showed no lateralization). Thus, females and males showed a different response pattern organization. Considering a simple model, the findings could reflect a greater sympathetic-related activation on the right, less sensory-motor-related activation on the left, or less parasympathetic activity in the left. Since the distribution of autonomic functions is more anterior [49], the pattern is consistent with greater or lower parasympathetic-related activation in females than males. That is, if we assume that representation of limb sensations is similar in males and females across the whole insula and is primarily localized in posterior regions, the similar female-male responses in the posterior long gyri suggest no difference in the contralateral sensorimotor activation. The female static handgrip exercise pattern of higher right ASG signals is opposite to the Valsalva-induced pattern of lower responses in this region in females [23].

Anterior autonomic dominance

On the right side, the three anterior short gyri showed larger responses than the two posterior long gyri, highlighting a dominant role over the posterior regions during increased cardiovascular activity, a pattern consistent with other challenges [22]. Unlike the Valsalva maneuver, the anterior, mid, and posterior right short gyri showed similar response patterns, and the anterior and posterior right long gyri were also very similar. Thus, the anterior-most ASG may have distinct functions only when strong sympathetic activation is occurring, which is not the case with the present short static handgrip exercise.

The males showed a similar pattern on both left and right, with the anterior short gyri following similar time courses and the ALG signal being close to the PLG. However, females showed only the MSG and PSG with similar increases. The left ASG patterns in females were lower than the other two short gyri for most of the grip period and remained close to those of the long gyri. Thus, the left ASG patterns in females were inconsistent with those of other short gyri in males and females on the left and right. This distinction is in contrast to the Valsalva, where the right ASG showed unique patterns of response. The present findings reinforce the difference in organization in the anterior-most gyrus of the insula, specifically in females.

Age and handedness influences

Age differed slightly between the sexes, and age modestly influenced the findings, including some sex by age-related variation that may relate to menopausal status in females. The models with sex × age (4 and 5 in the “Additional analyses: age effects and handedness” section) altered the time effect in a consistent manner across multiple analyses: of the 19 original models, nine showed a significant group effect (5 between-sex, 3 between gyri, 1 between-sex laterality); the group effect reflects differences in fMRI signal magnitude averaged over the entire period including baseline and challenge. All of these time effects changed from significant to non-significant with the inclusion of age × sex, showing that consistent differences in average magnitude of signal responses are accounted for with inclusion of this interaction. The lack of significant intensity differences over the entire period is consistent with the normalization of the signal to a percent change from baseline (the standard fMRI approach). Only other sex-difference models significantly affected with inclusion of age factors: (1) the left MSG showed a change from non-significant to significant of the effect of sex with age as a variable (models 2 and 3 in the “Additional analyses: age effects and handedness” section) and (2) the left PSG showed a change in time and sex × time from significant to non-significant in the models with age × time (models 3 and 4 in the “Additional analyses: age effects and handedness” section). These two changes were modest, suggesting that the influences are minor.

The inclusion of only right-handed subjects did not substantially alter the pattern of results. Handedness can influence autonomic function [60] but not necessarily to a static handgrip exercise [61, 62].

Clinical implications for patients with insular injury

Insular lesions or stroke compromises autonomic regulation [63, 64]. The findings here suggest that unilateral injury may result in dysregulation varying according to the stimulus and differ by sex. Assuming that greater fMRI activation represents a more active subregion role, a lesion or stroke in the right anterior-most insula may affect sympathetic regulation in males more than females. Similarly, a right-sided anterior insular insult could affect parasympathetic regulation in females more than males. Right-sided insular stroke leads to autonomic imbalance, but such effects have not been separated by sex [65]. Other regulatory actions such as heart rhythm and blood glucose control are also affected in people with insular stroke. Right-sided stroke is strongly associated with cardiac arrhythmias, yet there is a dearth of sex-specific data [66]. Similarly, right-sided stroke is associated with hyperglycemia [67], and sex-based metabolic issues are a particular concern. The data suggest there may be substantial sex differences in clinical consequences of insular damage.

Future studies

The findings raise new research questions. One broad question is whether activity in the right anterior insula is closely related to sympathetic activation. A simple extension of existing experiments or a secondary analysis of longer paradigm data could address the question: does the right insula respond once sympathetic activation to the static handgrip exercise occurs (30–60 s into the challenge)? In such a longer paradigm that leads to sympathetic activation, does the insular right side show an increase? Since MSNA studies show activity from approximately 1 min into a static handgrip exercise, an fMRI analysis could look at that time period, as opposed to immediately after onset. Another question is whether the lateral representation of the hand performing the grip is strongly represented in the results. Comparison with a left static handgrip exercise, and passive motion or very low grip strength fMRI changes could allow the sensory-motor effects to be separated.

The clinical consequences of lateralized insular injury are now well established, yet the sex-specific patterns remain to be comprehensively characterized. Many of the existing datasets could be analyzed on a sex-specific basis. The findings do indicate that new projects should collect sex and hormonal information such as menopausal status.

Limitations

The sample likely included pre- and post-menopausal women, a factor that would only indirectly be reflected in the additional models with age by sex interactions (4 and 5 in the “Additional analyses: age effects and handedness” section). We did not measure hormone status in females, which likely contributed to variability, since menopausal status and stage of the menstrual cycle influence many aspects of autonomic function [68, 69]. One study in younger people did show a slight reduction in diastolic and to a lesser extent systolic pressure in women in mid luteal vs. early follicular phase (estrogen is lower during the early follicular phase), but the effect size was many times smaller than the male female difference regardless of phase [43]. Heart rate did not show such phase-related differences in that study, suggesting hormonal status is unlikely to have been a main driver of the present findings.

Variability in individual anatomy could easily have led to variability in true separation of gyri [39, 70], but the fMRI signal itself is only sensitive to within a few millimeters, so a finer anatomical distinction would be unlikely to make a noticeable difference in the findings.