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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Obesity (Silver Spring). 2020 Mar 5;28(5):851–856. doi: 10.1002/oby.22758

Weight loss after obesity is associated with increased food motivation and faster weight regain

Bridget A Matikainen-Ankney 1, Mohamed A Ali 1, Nanami L Miyazaki 1, Sydney A Fry 1, Julia A Licholai 1, Alexxai V Kravitz 1,2,3
PMCID: PMC7180106  NIHMSID: NIHMS1549960  PMID: 32133782

Abstract

Objective

While changes in diet often result in short-term weight loss, weight loss is not typically maintained. It remains unclear why long-term weight loss is so difficult. We hypothesized obesity produces persistent changes in behavior that bias animals towards weight re-gain after weight loss.

Method

Mice were induced to gain weight with high-fat diet for 6 weeks, and then induced to lose this weight with low-fat diet for 7 subsequent weeks. A control group was maintained on the low-fat diet for all 13 weeks. Activity was measured continuously with home-cage activity monitors for the entire experiment. Motivation for sweetened food pellets was tested following weight loss. A separate group of mice were re-exposed to high-fat diet following 2, 4, or 8 weeks of withdrawal, to assess their rate of weight re-gain.

Results

Activity levels decreased as animals gained weight, and partially recovered following weight loss. Motivation for sucrose pellets was persistently heightened after weight loss. Consistent with these behavioral changes, mice also re-gained weight at a faster rate when re-exposed to a high-fat diet after a period of weight loss.

Conclusion

Weight loss after obesity was associated with increased motivation for palatable food, and an increased rate of weight re-gain.

Keywords: Obesity, weight loss, physical activity, motivation

Introduction

Two-thirds of Americans are overweight or obese 1. While weight loss is a common goal of obesity treatments, many patients are unsuccessful in this pursuit. This has led to the proposal that obesity is a chronic, progressive disease 2. Changes in diet or exercise frequently cause short-term weight loss (< 2 years) which is unfortunately followed by weight regain in subsequent years 3,4. It remains unclear why weight loss is so difficult to maintain. Here, we tested the hypothesis that behavioral changes that persist after weight loss bias animals towards weight re-gain, and may explain why weight loss is so difficult to maintain. We tested this hypothesis by quantifying activity levels, food motivation, and weight regain in mice after a period of obesity and weight loss.

Methods

Animals/diets

56 male and 35 female C57Bl6 mice were housed in a 12-hour light/dark cycle with ad lib access to food and water. Mice were provided laboratory chow diet (5001 Rodent Diet) or a 60% HFD (D12492, Research Diets). For weight gain calculations, the rate of gain over four weeks after diet reintroduction was compared. PR and open-field experiments were conducted during light-period (1pm to 5pm). All procedures were approved by the NIDDK Animal Care and Use Committee.

Continuous activity monitoring

Mice were singly housed and their activity was recorded continuously with passive infrared detector-based monitors 5. Data was binned hourly for analysis. One device was dropped because data did not record.

Open field

Open field activity was measured in 30-minute tests conducted in 20 cm diameter buckets. Ethovision software (Noldus XT) was used for tracking.

Operant behaviour

Operant behavior was tested with the Feeding Experimentation Device, version 3 (FED3, https://hackaday.io/project/106885-feeding-experimentation-device-3-fed3) 6. A nosepoke on the active port of FED3 yielded a combined tone and light conditioned stimulus, as well as 20 mg sucrose pellet (TestDiet, chocolate flavored sucrose pellet). Nosepokes on the inactive port were logged but had no consequence. Acquisition was conducted in a FR schedule wherein one poke yielded one pellet. For progressive ratio (PR) sessions, a Richardson Roberts 7 schedule was followed wherein the number of pokes to obtain a reward increased after every completed trial; i.e. the number of responses required follows: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, etc. PR sessions were 2.5 hours, mice completed two separate sessions 48 hours apart, and results were averaged from both sessions for each mouse.

Blood measures

Trunk blood from sacrificed animals was collected at room temperature, spun at 10,000 rpm for 6 minutes, and serum was frozen until assayed. FFA (Roche Diagnostics Gmbh, Mannheim, Germany), TG (Pointe Scientific Inc., Canton, MI), and cholesterol (Thermo Scientific, Middletown, VA) were measured by colorimetric assays. Serum glucose was measured by Glucometer Contour. Insulin (Millipore, St. Charles, MO) was measured by radioimmunoassay. Leptin (R&D Systems, Minneapolis, MN) was measured by ELISA.

Calorie measurement

Food consumption was measured every 2–3 days from singly housed mice, by weighing food removed from rodent feeding dishes (rodent café, OYS America). Daily calorie intake was calculated for laboratory chow and HFD periods based on the caloric density of these diets (3.1kCal/g for laboratory chow, 5.2kCal/g for HFD). The control group for this experiment was re-analyzed from previously published work 8. The weight-loss group was run at the same time as the control group, but this data was not previously published.

Statistics and visualization

Mann-Whitney U tests and 1-way ANOVA were run using Python (SciPy), 2-way ANOVAs and mixed effects models were run with GraphPad Prism (v 8.0). Data was processed and plotted with custom python scripts available at https://osf.io/kw4v6/.

Results and Discussion

A home-cage based activity monitor5 was used to continuously record physical activity levels of two groups of male mice over 13 weeks: one group received high fat diet (HFD) for 6 weeks followed by low-fat rodent chow for 7 weeks (weight loss, n=9), while the other received low-fat laboratory chow for all 13 weeks (control, n=10) (Fig 1A). Continuous recordings allowed for an assessment of animal activity across all 13 weeks of the experiment. Mice gained weight while maintained on HFD, which was then lost when switched back to chow (Fig 1B,C). Dark cycle activity levels of the weight loss group were significantly reduced during the HFD phase, as previously reported 8. After weight-loss, activity levels were no longer statistically distinct from the control group, consistent with a recovery of activity after weight loss. However, this recovery appeared to be only partial (reaching ~85% of the control activity levels, Fig 1D,E). As our study was powered to detect >20% reductions in activity, we could not draw firmer statistical conclusions on whether these animals exhibited a persistent reduction in activity following weight loss. Future work powered to detect more subtle changes will be needed to test this. It is worth noting that such persistent decreases in activity following weight loss have been reported in humans9–12, non-human primates13, and dogs 14,15. Finally, we examined physical activity across the circadian period and found the obesity-induced reduction in activity was strongest in the first 6 hours of the dark cycle (Fig 1F,G).

Fig 1. Obesity-linked changes in physical activity.

Fig 1.

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(a) Schematic of weight loss (orange) and control (grey) diet groups. (b) Average weight over time for both groups. (c) Weights at weeks 0, 6, and 12. Significant interaction between group and week (F (2, 36) = 190.6, p<0.0001), with a significant difference between groups at week 6 (p<0.0001); no significant difference between groups at baseline or week 12 (p=0.980, 0.118, respectively). (d) Average dark cycle activity normalized to control over the experiment. (e) Activity levels at baseline, week 6, and week 13. There was a significant interaction between group and week (F (2, 31) = 4.350, p=0.0216). At 6 weeks there was a significant reduction in average activity in weight loss group relative to control (p=0.0006). Between-groups comparison showed no significant difference at baseline or week 13 (p=0.983, 0.583, respectively). (f) Circadian activity plots during baseline, weight gain (weeks 1–6), and weight loss (weeks 7–13), in seconds of PIR sensor activation per min. X-axis indicates time from the start of dark cycle. (g) Early dark cycle activity (sec moved per min), significant interaction between group and time (F (2, 32) = 5.811, p=0.0070). During weeks 1–6 there was a significant decrease in weight loss vs control (p=0.0064, no significant difference between groups for baseline or weeks 7–13 (p=0.971, 0.280, respectively). Correlations between weight gain (h) and loss (i) vs average activity for the two phases of the experiment. There was no significant relationship between weight gain (p=0.835, r=−0.082) or loss (p=0.955, r=0.022) and activity. In (c) 2-way ANOVA with repeated measures was conducted. In (e) and (g), linear models with mixed effects were used to compare means. Sidak’s post hoc tests were used for between group comparisons. P-values on panels (c), (e), and (g) indicate interactions. Pearson regressions were run in (h) and (i). Error bars indicate S.E.M.

Although decreases in physical activity often accompany weight gain, it remains unclear whether activity levels are causal to weight gain or loss. We therefore looked for evidence of a causal relationship by correlating activity levels with weight changes in individual mice. Surprisingly, there was no evidence of a relationship between weight gain and activity levels during that period (Fig 1H), indicating that less active mice were not more prone to gaining weight on HFD. Activity levels were also not correlated with the amount of weight loss when HFD was removed (Fig 1I), further supporting that activity levels are not a strong driver of weight loss in mice. To obtain orthogonal measures of activity for this analysis, mice were removed from their cages for a video-taped open field test at three timepoints in this experiment: weeks 0, 6 and 13. Activity levels in the open field were also not correlated with weight gain or loss (p=0.221, r=−0.425 for weight gain at week 6; p=0.300, r=0.365 for weight loss at week 13). We conclude that variance in activity levels does not relate to the propensity of individual mice to gain or lose weight.

Withdrawal from palatable food can increase preference 16, lever pressing 17, and cravings 18 for such foods. Therefore, we hypothesized that mice may have increased motivation for palatable food following weight loss. To test this, after 6 weeks of withdrawal from HFD and associated weight loss, weight-loss and control mice were trained on an operant nose-poking task using a home-cage-based device, FED36. Successful nose pokes in the active port (Fig 2A) yielded a 20mg chocolate flavored sucrose pellet. Mice of both groups learned the task in a single 24 hour fixed-ratio one (FR1) session, with no significant differences in learning rate or performance on the FR1 session (Fig 2B,C). In a subsequent progressive ratio (PR) task (Fig 2D,E), weight loss mice exhibited a significant increase in total responses (Fig 2E), and a trend towards an increase in pellets earned (Fig 2F), consistent with a heightened reinforcing efficacy of palatable food following weight loss 7. We conclude that obesity induces lasting changes in brain systems that drive motivation, as observed at a behavioral level in the mice in this study.

Fig 2. Weight loss increases drive for palatable food in formerly obese mice.

Fig 2.

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(a) Home-cage operant behavior device. (b) Average active nosepokes of weight loss and control groups in a 24-hour fixed-ratio acquisition period. (c) Box/scatter plots showing total active nosepokes during acquisition. (d) Average active nosepokes over PR task. Box/scatter plots showing total active nosepokes (e) and pellets earned (f) during PR. (g) There was no significant change in FFA, TG, cholesterol, insulin, leptin, or glucose. (h) Calories consumed per day across diet paradigm, quantified in (i), showing average calories consumed increased in weight-loss group only during ad lib HFD period (days 30–90). Significant interaction between group and time, F (2, 28) = 37.38, p = 0.0001. Posthoc test between groups show a significant difference between groups for days 60–90 p=0.0001. There was no significant difference between groups for days 0–29 p=0.999, or days 120–150, p=0.6538. Two-tailed Mann Whitney U test was conducted in (c), one-tailed Mann Whitney U conducted in (e) and (f). Student t-tests were run in (g). 2-way ANOVA with repeated measures and Sidak’s post hoc tests were run in (i). ****p<=0.0001

To test if the increased motivation for sucrose pellets was due to persistent changes in circulating energy stores or hunger signaling molecules, serum samples were analyzed from both groups at the conclusion of the experiment. Free-fatty acids (FFA), triglycerides (TG), cholesterol, insulin, and leptin were not different between the weight loss and control groups, although there was a trend towards an increase in blood glucose in the weight loss group (Fig 2G). We also tested whether the increase in motivation for sweetened food related to a persistent increase in ad lib food intake following weight loss. In a separate cohort of mice, we measured food intake for 8 weeks, directly following 8 weeks of either ad lib HFD (weight loss group) or chow (control group, Fig 2H). We did not detect any difference in ad lib food intake following weight-loss (Fig 2I). This suggests that the enhanced motivation for food in these mice may be specific for palatable foods such as sucrose pellets.

Based on their increased motivation for palatable food, we predicted that previously-obese mice would re-gain weight at a faster rate when re-exposed to HFD. To test this prediction, a new cohort of mice (n=48, equal number of males and females) were exposed to HFD for 4 weeks and then restricted to chow for two (2 wk weight loss, n=16), four (4 wk weight loss, n=16), or eight (8 wk weight loss, n=16) weeks before being re-exposed to ad lib HFD. Two control groups were given access to chow (ad lib chow, n=8) or HFD (ad lib HFD, n=15) for the duration of the experiment (Fig 3A). Supporting our prediction, mice from 2- and 4- week weight loss groups regained weight significantly faster than their initial rates of weight gain upon reintroduction to HFD (Fig 3B,C). Interestingly, the 8 wk weight loss group did not regain weight significantly faster than their initial weight gain period (Fig 3C), suggesting that a faster rate of weight gain following weight loss may be a transient phenomenon. There were no sex differences in these results, with both male and female mice in 2- and 4-wk weight loss groups exhibiting significantly increased rates of weight gain (data not shown).

Fig 3. Formerly obese mice regain weight at a significantly higher rate upon re-exposure to HFD.

Fig 3.

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(a) Diet paradigms for five separate groups. Weight loss groups (2-, 4-, and 8 wk weight loss) were removed from HFD at week 4 and put on chow for 2, 4, or 8 weeks, respectively, before being switched back to HFD. (b) Average weights over time. (c) Within-mouse comparisons of weight gain rates between baseline (weeks 0–4) and HFD reintroduction 2 (pink, left), 4 (purple, middle), or 8 (blue, right) weeks after withdrawal. Grey lines show ad lib chow mice at time-matched control periods. 2-way ANOVAs: (2 wk: interaction F (1, 22) = 4.354, p=0.049; posthoc Sidak’s test within group, weight loss p=0.001, control p=0.932; 4 wk: interaction F(1,22)=9.228, p=0.006; posthoc Sidak’s test within group, weight loss p=0.0012, control p=0.621; 8 wk: interaction F(1,43)=2.293, p=0.137; posthoc Sidak’s test within group, weight loss p=0.588, control p=0.418). Pearson regression correlations between rate of weight re-gain and (d) initial weight gain rate (p=0.495, r=−0.101) or (e) weight loss (p=0.001, r=0.461) for all weight loss mice. **p<=0.01, ***p<=0.001

We next asked whether the rates of weight re-gain would correlate with the rate of initial weight gain or the rate of weight loss during the HFD withdrawal period. We observed no significant relationship between initial weight gain and weight re-gain (Fig 3D), suggesting that the mechanisms that govern weight re-gain may differ from those that govern the rate of initial weight gain. Indeed, some animals that gained weight slowly in the first 4 wk exposure period gained weight at a fast rate when re-exposed to HFD (Figure 3C). In contrast, weight re-gain rates were positively correlated with the amount of weight lost during HFD withdrawal (Fig 3E). This implies that mice who lost the most weight may be most vulnerable for weight regain when re-exposed to ad lib HFD.

Here we examined why mice experience pressures to re-gain weight following weight loss. As physical activity levels recovered after weight loss, and were not correlated with weight gain or loss, we believe that activity is unlikely to be a dominant driver of weight re-gain after weight loss. Instead, we believe that persistent changes in food motivation may be responsible for the increased rate of weight regain. This is in contrast with other interpretations that changes in physical activity can be a dominant factor in weight gain and loss 19. In addition, metabolic changes after obesity can contribute to increased rate of weight re-gain 20,21. Rather than arguing for one single mechanism, we believe it likely that multiple mechanisms underlie weight re-gain after weight loss, and that changes in motivation to obtain palatable food is one such mechanism. Our findings support the notion that obesity is a chronic, progressive disease that can be sustained by persistent changes in behavior. Understanding the biological underpinnings of these changes will be critical for informing lasting interventions to treat obesity and reduce the health burden of the obesity epidemic.

Study Importance Questions.

  1. What is already known about this subject?
    1. Though long-term maintenance of weight loss is one of the largest clinical challenges facing obesity treatment, it remains unclear why weight loss is so difficult to maintain
    2. Data from humans and rodents suggests that weight loss after obesity is associated with persistent behavioral changes including decreases in physical activity
  2. What are the new findings in your manuscript?
    1. Weight loss after obesity is associated with sustained increases in motivation for sucrose in mice
    2. Previously obese mice re-gained weight at a higher rate when re-exposed to high fat diet, suggesting the persistent increase in food motivation may contribute to chronic obesity
  3. How might your results change the direction of research or the focus of clinical practice?
    1. Our data supports that chronic obesity is sustained by persistent changes in motivated behavior. This suggests that the study of the neurobiology of obesity should be expanded to include an analysis of how neural circuits underlying motivated behaviors are affected in a lasting way as a result of weight gain and loss. Understanding these changes will be critical for informing therapies to treat obesity.
    2. In the interest of supporting open-science and enhancing reproducibility, data and analysis scripts in this manuscript are available for download (https://osf.io/kw4v6/).

Acknowledgements

We thank the NIDDK Mouse Metabolism Core for assistance with serum assays, and the NIDDK animal care staff, especially Ms. Remona Davis, for enabling the home-cage based assays.

Funding: Research funded by the National Institutes of Health Intramural Research Program at NIDDK, and the NARSAD Young Investigator award (AVK).

Footnotes

Disclosure: The authors declare no conflict of interest.

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