Keeping weight off: Mindfulness-Based Stress Reduction alters amygdala functional connectivity during weight loss maintenance in a randomized control trial

Obesity is associated with significant comorbidities and financial costs. While behavioral interventions produce clinically meaningful weight loss, weight loss maintenance is challenging. The objective was to improve understanding of the neural and psychological mechanisms modified by mindfulness that may predict clinical outcomes. Individuals who intentionally recently lost weight were randomized to Mindfulness-Based Stress Reduction (MBSR) or a control healthy living course. Anthropometric and psychological factors were measured at baseline, 8 weeks and 6 months. Functional connectivity (FC) analysis was performed at baseline and 8 weeks to examine FC changes between regions of interest selected a priori, and independent components identified by independent component analysis. The association of pre-post FC changes with 6-month weight and psychometric outcomes was then analyzed. Significant group x time interaction was found for FC between the amygdala and ventromedial prefrontal cortex, such that FC increased in the MBSR group and decreased in controls. Non-significant changes in weight were observed at 6 months, where the mindfulness group maintained their weight while the controls showed a weight increase of 3.4% in BMI. Change in FC at 8-weeks between ventromedial prefrontal cortex and several ROIs was associated with change in depression symptoms but not weight at 6 months. This pilot study provides preliminary evidence of neural mechanisms that may be involved in MBSRs impact on weight loss maintenance that may be useful for designing future clinical trials and mechanistic studies.

, and by 3-5 years they have 103 frequently returned to baseline weight (Franz et al., 2007). Thus, it is important to 104 develop treatment strategies that address factors associated with weight regain. 105 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint Although findings are mixed (Elfhag & Rossner, 2005), there is compelling evidence 106 from weight loss maintenance studies that psychological factors, particularly stress 107 and depression, are significant predictors (Brantley et  interventions, in particular MBSR, for reducing aversive symptoms such as 122 depression and anxiety is well-established (Goyal et al., 2014). Given that these 123 symptoms increase the risk of unhealthy behaviors, MBSR may support 124 maintenance of weight loss following successful initiation of health behavior change 125 (Fulwiler et al., 2016). 126 127 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. importantly, the change in behavior and treatment response over the course of 150 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Because fMRI allows for the observation of brain activation in real time via blood 168 oxygenation level dependent (BOLD) imaging, it provides the opportunity to analyze 169 the FC of various brain regions in an actively processing or RS brain (Biswal, Zerrin 170 Yetkin, Haughton, & Hyde, 1995). This refers to the concept that regions of the brain 171 that are functionally interconnected will co-activate in synchrony or predictable 172 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint cadence, and thus temporal analysis of fMRI data can be used to determine which 173 brain regions are functionally linked (van den Heuvel & Hulshoff Pol, 2010). 174 Furthermore, higher-level FC analysis can establish functionally linked brain 175 networks, or "independent components," (ICs) of interconnected brain regions. This preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint Stice, 2011) (Fig.1). Our second aim was to investigate the association of RS change 196 post-intervention with 6-month outcomes for psychological and anthropometric 197 factors. We hypothesized that increased RS connectivity would be associated with 198 improvement in depressive symptoms and inversely related to decreases in weight 199 and waist circumference. In the current study, we present findings for our primary 200 outcomes: 1) Change in RS fMRI signal following the 8-week MBSR intervention; 2) 201 The association of RS fMRI changes with changes in weight (BMI) and depression 202 symptoms at 6 months follow up. Exploratory analyses of other factors such as self-203 reported change in mindfulness, meditation practice time, and other psychological 204 symptoms and health behaviors will be presented in future reports. 205 206 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint 12 207 208 209 210 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint minimum 5% weight loss in the past year; 3 that were currently enrolled in a weight 242 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint loss study, 8 that had a past weight loss surgery, 2 MRI exclusion, 7 who engaged in 243 current meditation or yoga practice, 11 who had participated in an MBSR class 244 previously, and 8 that had diabetes. Forty-two of those screened and found eligible 245 declined to participate when told of the time commitment required for the 8-week 246 classes, and 18 individuals could not be scheduled. Ninety-one participants passed 247 all exclusion criteria and were invited to attend the initial visit. Of these, 59 attended 248 the baseline visit and 57 were randomized. 249

250
Of the 57 subjects randomized, 5 were either unable to complete initial MRI, or 251 dropped out during the 8-week intervention. 52 returned for the 8-week follow-up 252 visit, and all 52 completed the 6-month follow-up visit (91% retention). 83% of 253 participants completed 5 or more sessions, and 73% completed 6 or more. 254 255

Randomization 256
Study participants, who were blind to the hypotheses, 57 were randomized to either 257 the MBSR or the healthy living course (HLC) based on a permuted blocks 258 randomization scheme (Fulwiler et al., 2016). Sample sizes consisted of 29 in the 259 MBSR group and 28 in the HLC group. Randomization and investigators blinding 260 was implemented using sealed envelopes and unique identification numbers by the 261 study coordinator. In addition to the participants who were aware of which 262 intervention they were receiving but not the study hypotheses, only the study 263 coordinator among the research team was non-blinded to participant assignment 264 until the study conclusion. MBSR teachers at the Center for Mindfulness were not 265 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint aware of who in their classes were study participants. HLC teachers were aware that 266 classes were being offered as part of the research study but were blinded to study 267 hypotheses. day-long retreat), small-group format, and process (didactic teaching and group 287 discussion). The MSBR course was taught by certified teachers from the UMass 288 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. paper. In addition, fMRIs were conducted at baseline and at the first follow up 299 assessment. Due to claustrophobia and other contraindications, 5 subjects were not 300 included in the MRI analysis as they did not undergo MRI, bringing the analysis to 301 51 subjects (MBSR N= 28, HLC N=23). All MRI data were acquired on a GE 3 Tesla 302 scanner with a 12-channel coil. High resolution structural images were acquired 303 using a using a magnetization-prepared rapid acquisition with gradient echo 304 (MPRAGE) pulse sequence with the following parameters: TR/TE¼ 2.1 s/2.25 ms, 305 slices¼ 128, matrix¼ 256 256, flip angle¼ 12, resolution¼ 1.0 1.0 1.33 mm. 306 Gradient echo echo-planar images sensitive to BOLD contrast were acquired using 307 the following parameters: TR/ TE¼ 2.0 s/30 ms, flip angle¼ 90, slices¼ 34, voxel 308 size¼ 3.5mm isotropic. During the 6:04 minute RS fMRI scan participants were 309 asked to remain awake with their eyes open FMRI and was carried out as described 310 in our previous manuscript (Fulwiler et al., 2016). We used region of interest-based 311 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. Gaussian-weighted least-squares straight line fit with a high pass cutoff = 150 s. 325 fMRI data were registered to MNI152 standard space is done in two steps. First, the 326 fMRI data were aligned to the high-resolution structural image using 6 degree-of-327 freedom (DOF) affine transformation. Second, the structural scan was aligned with 328 MNI152 standard brain using non-linear registration. Finally, transformation of the 329 functional results into MNI space was done following concatenation of the two 330 alignments into a single matrix. All spatially normalized fMRI data were re-sampled 331 to 2mm 3 resolution. 332 333 334 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  (Beckmann 337 and Smith, 2004)] was used to perform an ICA of the fMRI data from all subjects and 338 sessions (pre-and post-intervention). Pre-processed functional data was masked 339 with MNI152 brain image to ensure exclusion of non-brain data, before inputting 340 them into MELODIC. MELODIC decomposes the spatially normalized fMRI data from 341 all subjects (e.g. concatenated into a single data matrix) into a set of ICs, with each IC 342 being a distributed set of brain regions with a temporal trace that describes the 343 evolution of that particular spatial pattern over time. For group ICA, each IC is comprised of a spatial map and a corresponding time 354 course and represents a spatio-temporal pattern of activity that is common to the 355 entire set of participants. To estimate the spatial patterns and time courses for each 356 subject (which capture the between-subject variability), dual regression (Nickerson 357 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint et al., 2017) was applied as follows. Before conducting dual regression, the melodic 358 maps were thresholded to ± Z score of 2.3 (cluster significance P < .05). Each IC was 359 then normalized to maximum value 1 by dividing each component by its maximum Z 360 score to account for differences in the scale of the spatial maps. In the first stage of 361 dual regression, the group ICA maps were regressed onto each subject's fMRI data preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Change in resting state connectivity following the 8-week interventions 396
The connectivity between each of the 4 ROIs and each of the five ICs of interest 397 (namely the estimated marginal means values) were analyzed individually for a 398 group x time interaction using repeated measures ANOVA. A third category of 399 hemisphere was added to the interaction to assess lateralization. A group x time 400 interaction was found between the amygdala and vmPFC (Fig.3)  analyses revealed that this interaction was driven by a significant group difference 405 at 8 weeks (p=0.010) rather than the non-significant baseline differences (p>0.6). 406 No other significant group x time interaction was found for any other ROI x 407 component of interest, with or without an effect of hemisphere. Additionally, there 408 were no additional significant effects found after removal of interaction effects. 409 410 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint There were no statistically significant changes in the measured anthropometric 421 metrics (weight, BMI, waist circumference) and psychological variables (depression, 422 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint stress, internal disinhibition) from baseline to 6-month follow up. However, post-423 hoc analysis revealed that the mean weight of MBSR participants remained about 424 the same (losing 0.7 lbs, or 0.4% of body weight and 0.1kg/m 2 , or 0.5% of BMI, and 425 3.2cm, or 3.5% of total waist circumference), while increasing in the HLC group 426 (gaining 5.9 lbs or 3.2%, BMI by 1.0kg/m 2 or 3.4%, and waist circumference by 427 1.2cm or 1.2%). No statistically significant differences between the groups were 428 observed in psychological or eating measures. 429 430 Our second primary aim was to investigate the association of FC change with 431 changes in psychological and anthropometric outcomes (Fulwiler et al., 2016). A 432 paired t-test of the association between connectivity change at 8 weeks and weight 433 and depression at 6 months did not reveal between-group differences. However, 434 using a single all-subject analysis combining both groups and standard linear 435 regression, a significant correlation was found for decrease in FC at 8-weeks 436 (between vmPFC and a cluster of structural regions that contained the precuneus, 437 inferior frontal gyrus, and parietal operculum), and increased CESD score change (in 438 either direction) at 6 months (p = 0.02). Other significant correlations were found 439 between increased 8-week FC change in basal ganglia and the cluster consisting of 440 hippocampus, nucleus accumbens, and OFC and increased 6-month CESD change (p 441 = 0.032); and increased 8-week FC change in the salience network and occipital and 442 inferior temporal gyrus and increased CESD change (p = 0.02). Conversely, lower 443 connectivity between ventromedial prefrontal cortex and the cluster containing 444 precuneus, inferior frontal gyrus, and parietal operculum was associated with 445 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint The goal of the present study was to identify neural processes modified by MBSR 451 that may be associated with weight loss maintenance and related emotional 452 symptoms. We also examined changes in RS following MBSR vs. a rigorous control 453 condition and, in an exploratory analysis, examined the association of these changes 454 with depression symptoms and weight stability at 6-month follow-up. We 455 confirmed our hypothesis that change in functional connectivity would be greater in 456 the MBSR group compared to the comparison group. Specifically, this significant 457 effect for FC changes was for amygdala-vmPFC connectivity, in agreement with 458 previous studies. Secondly, our exploratory analysis of the association between FC 459 change at 8 weeks and clinical outcomes found that FC change is associated with 460 change in depression symptoms at 6-month follow-up. 461 462

Neural targets of mindfulness and relevance to weight loss studies 463
Our findings are consistent with studies in other populations which show that 464 mindfulness training leads to changes in medial prefrontal-amygdala connectivity 465 critically implicated in the regulation of emotion. In one study, a mindfulness 466 strategy for regulating emotional response was associated with regulation of 467 amygdala activation by medial prefrontal cortex, in contrast to emotion suppression 468 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint involved connectivity with dorsolateral prefrontal cortex (Murakami et al., 2015). 469 In other studies, MBSR training led to decreased amygdala reactivity accompanied 470 by increased amygdala-vmPFC functional coupling (Kral et al., 2018) and 471 improvement in anxiety symptoms that were correlated with increased amygdala-472 vmPFC coupling (Hölzel et al., 2013). Considering these reports of increased 473 functional coupling, our finding of increased RS connectivity following MBSR 474 strengthens the evidence for the importance and specificity of the amygdala-vmPFC 475 connection as a marker of MBSR treatment progress, efficacy, and outcomes. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint control, and decreased activity of the medial PFC predicts weight gain in obese 515 individuals (Kishinevsky et al., 2012). Thus, for an experimental medicine approach 516 to refining mindfulness interventions for emotion regulation and behavior change 517 research (Onken et al., 2014), neuroimaging markers such as connectivity between 518 amygdala and medial PFC may prove useful for optimizing the intervention and 519 identifying subpopulations most likely to respond (see Gabrieli et al. (Gabrieli,520 Ghosh, & Whitfield-Gabrieli, 2015) for a review of the neuromarker approach). As 521 obesity is the cumulative result of a range of specific eating behavior traits (Carnell,522 Gibson, Benson, Ochner, & Geliebter, 2012), understanding neural targets specific to 523 different subgroups will be essential. For example, we have reported preliminary 524 evidence that MBSR leads to reduced emotional eating especially for those with high 525 emotional eating scores at baseline (Levoy, Lazaridou, Brewer, & Fulwiler, 2017). 526 There may be specific neural targets for emotional eaters vs. other subtypes of 527 overeaters. 528 529

Association of connectivity changes with clinical outcomes 530
Our second primary aim was to examine whether pre-post change in FC would 531 predict 6-month change in weight and depression symptoms. A correlation with 532 weight was not found. On the other hand, we report for the first time that FC change 533 is associated with change in depression symptoms at 6-month follow-up. This 534 finding was not specific to MBSR as our sample size was too small to detect 535 differences between groups. Given this sample size issue, despite non-significant 536 changes over time in our collected anthropometric and psychologic measures, this 537 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint lack of statistical significance does not necessarily preclude real group differences in 538 these anthropometric and psychologic measures. The finding included several ROI's 539 but did not include amygdala-vmPFC connectivity. However, future studies may examine its association with clinical outcomes in 555 specific subpopulations. In contrast, vmPFC connectivity with other areas such as a 556 cluster that included the IFG was correlated with depression symptoms. The IFG 557 plays a critical role in attentional control and various forms of response inhibition, 558 including emotional inhibition (Aron, Robbins, & Poldrack, 2004), and disruption of 559 IFG FC by negative emotion is believed to be responsible for the adverse effect of 560 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Study strengths and limitations 565
This study had a number of strengths. The two groups were well-matched in terms 566 of baseline demographic, psychometric and anthropometric characteristics, with no 567 statistically significant baseline differences. The comparison condition was well-568 matched to the experimental condition on course length, style, and number of 569 sessions, employing both a small-group format that included didactics and group 570 discussion. In addition, we employed a 6-month follow-up for testing the effect of 571 interventions on clinical outcomes. 572 573 A significant limitation is that recruitment goals were not achieved resulting in the 574 trial being underpowered. Our primary outcomes were powered on a sample size of 575 80 participants, but only 52 participants completed the study. Thus, while the 576 sample size allowed us to detect a significant group difference in functional 577 connectivity change in pre-selected regions of interest in the MBSR group as 578 predicted, between group changes in clinical outcomes were not significant and we 579 were unable to confirm the hypothesis that functional connectivity changes would 580 predict clinical outcomes in the MBSR group. This limitation is not uncommon in 581 trials involving mindfulness-based interventions, in part because the intervention 582 demands a large investment by participants. After expressing initial enthusiasm for 583 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint the study, many potential recruits declined to participate because of the time 584 requirement for the interventions. 585 586 Another limitation of the study is the largely white and female demographics of the 587 sample, especially in light of evidence that race, sex, and socioeconomic status may 588 strongly affect weight loss outcomes (Ball & Crawford, 2005 . In 590 addition, although baseline differences between groups were not significant, the 591 magnitude of differences in some variables may have been important and should be 592 adjusted for in future studies with a larger sample. Our initial hypotheses were 593 exploratory, so FC between several regions of interest and independent components 594 were tested. Thus, a limitation of this study is the use of multiple testing which may 595 inflate false positives. Furthermore, this study did not address the continued 596 longitudinal follow-up of FC throughout weight loss maintenance, and whether long-597 term success in weight loss maintenance is correlated to connectivity markers. In 598 addition, the study did not establish a quantitative approach to using FC as a 599 biomarker. A more in-depth approach correlating clinical measures to FC would be 600 needed to establish amygdala-PFC connectivity as a measure with clinical utility. 601 Finally, without data on participants' practice of mindfulness between sessions, we 602 do not know the actual "dose" of mindfulness received. 603 604 605 606 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint Conclusions 607 608 The present study provides evidence that mindfulness training in people who have 609 lost weight and intend to keep it off leads to increased RS FC in a neural circuit 610 involved in emotion regulation. These findings are consistent with previous 611 research suggesting that short-term training in mindfulness meditation alters the 612 neural circuitry of emotion regulation, specifically connectivity between the 613 amygdala and vmPFC. While connectivity change in this circuit was not associated 614 with later clinical outcomes, preliminary evidence suggests an association between 615 other vmPFC connectivity changes and depression symptoms which may be 616 relevant to weight loss maintenance. 617 618 619 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this this version posted November 17, 2020. ; https://doi.org/10.1101/2020.11.16.20226290 doi: medRxiv preprint 1 780 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.