Obesity has substantially increased in prevalence in modern times and is associated with both chronic health conditions (e.g., type 2 diabetes, cardiovascular disease, cancer) and increased mortality rate (( 1 , 2 )). Although intentional weight loss improves the acute risk for noncommunicable diseases and biomarkers of health (( 3 , 4 )), the effect of weight loss on longevity as assessed by observational studies remains disputed (( 5 - 8 )). This may be particularly so given the difficulty in maintaining achieved weight loss for extended durations of time, resulting in subsequent unintentional weight regain. This pattern is often repeated as a recurring loss‐regain cycle referred to as weight cycling or “yo‐yo dieting” (( 9 , 10 )). Relationships between weight change and mortality may be further obfuscated by attempts to assess whether the weight loss was intentional or unintentional, such as that resulting from occult disease (( 11 , 12 )). Even the expression of intention for weight loss alone may not be sufficient to remove this confounding (( 13 )). These difficulties with weight loss and uncertainties concerning benefits for longevity have left some questioning whether weight loss is even worth the effort (( 9 )). To more directly study the associations between weight change and mortality, we used an established murine model to perform a randomized controlled feeding study with more than 900 individually housed mice to assess whether repeated bouts of weight loss by calorie (energy) restriction (CR) and weight regain by ad libitum feeding (weight cycling) in overweight or obese rodents would alter survival relative to maintained obesity ( ad libitum feeding) or weight reduction (sustained CR).

Methods

Animals C57BL/6J male and female mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) at 6 weeks of age and were acclimated to the specific‐pathogen‐free facility for 2 weeks. All mice were singly housed for the duration of the study in standard, ventilated mouse cages within a Thoren Rack Mobile Housing System (Thoren Caging Systems, Inc., Hazleton, Pennsylvania) as described ((14, 15)). Animal rooms were maintained at 20°C to 22°C on a 12‐hour light‐dark cycle from 6:00 am to 6:00 pm. Animal health was checked daily, and moribund animals were euthanized according to the study protocol. All study protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Some data from this study have been described elsewhere ((15)).

Study design Beginning at 8 weeks of age, the C57BL/6J male and female mice were provided free access to a high‐fat diet (45% kcal fat and 20% protein based on D12451 [calorie: 4.73 kcal/g]; Research Diets, New Brunswick, New Jersey) (Supporting Information Table S1) to determine ad libitum intake. At 10 months of age, the mice were weighed, and the heaviest two‐thirds of the mice were subsequently randomized by quartile of body weight within each sex into diet groups (continuing with the high‐fat diet feeding until death). In order to assess the impact of weight loss and cycling from a state of excess body weight, the other one‐third of mice, with the lowest body weight, were removed from the study, as they were naturally resistant to diet‐induced obesity. The four diet groups were as follows: the Ever Obese (EO) group, which continued ad libitum feeding; the Obese Weight Loss (OWL) group, in which energy intake was restricted by nearly 30% of EO intake (with the vitamin and mineral mix supplemented in the high‐fat diet when restriction was > 20%; Research Diets D11022101) (Supporting Information Table S1); the Obese Weight Loss Moderate (OWLM) group, in which energy intake was restricted by nearly 20% of EO intake; and the Weight Cyclers (WC) group, in which energy restriction was enforced by dietary restriction followed by subsequent periods of ad libitum refeeding. The OWL group was designed to reduce the body weight of the mice to a weight comparable with that of C57BL/6J male and female mice (n = 15/sex) fed a low‐fat diet (10% kcal fat) at our facility (Supporting Information Table S1 and Table S10). This low‐fat‐fed (sex‐specific) body weight was also used as a target mean body weight for the WC group during periods of energy restriction (which was applied through a gradual, stepped restriction ~10% intervals). Periods of weight loss (~3‐month periods) and regain (~4‐month periods) were repeated as sequential intervals for the remaining life of the WC group following randomization. The OWLM group was designed to reduce the body weight of the mice to approximately the midpoint of the EO and OWL groups. Because of the size of the study, the total sample was divided and performed in two waves, and the two cohorts of animals were separated by approximately one calendar year. Experimental wave 1 included both male and female mice, whereas wave 2 contained only male mice because of a higher than expected incidence of ulcerative dermatitis early in life for the females in experimental wave 1. Additionally, the vitamin A levels in the high‐fat diet for experimental wave 2 were reduced to the levels recommended by the National Research Council (1995), with the same high‐fat diet formulation (Research Diets number D11112301) (Supporting Information Table S1), as vitamin A had been implicated in ulcerative dermatitis development ((16)). Following randomization, weekly food intake was measured for animals provided ad libitum access (e.g., EO group) along with weekly body weights for all animals. For animals receiving a daily allotment of a restricted amount of food (OWL and OWLM groups), fresh allotments were provided approximately 1 to 2 hours before lights off, and any food remaining after 24 hours was recorded and discarded before the next day’s provisions were given.

Life‐span For the C57BL/6J mice, observed life‐span (days) was recorded as the age when the animals died naturally or were euthanized owing to morbidity. Natural death or moribund status termination was recorded to the nearest day. Causes of death were categorized as (1) euthanized for ulcerative dermatitis or similar skin lesions (n = 290), (2) euthanized for other reasons, such as disability to eat or drink (n = 42), (3) found dead (n = 211), and (4) died from technical complications related to procedures (e.g., anesthesia, physical cage injury, veterinary treatment [n = 9]). Sex and experimental waves of each individual were adjusted when comparing life‐span among treatment assignments.

Body composition From 47 weeks of age, body composition (fat and fat‐free mass) was determined in vivo in all groups by quantitative magnetic resonance (EchoMRI 3‐in‐1, V2.1; Echo Medical Systems, Houston, Texas) at peak and trough points of the WC group (~3‐4 month intervals). The specific week of measurement differed between male and female mice to allow the time needed to reach the targeted weight or to stagger measurements to accommodate the number of animals.

Dissection and tissue collection Following the diet group randomization and initial weight loss phase, wherein mean body weight of OWL and WC was similar to low‐fat‐fed reference animals (~13 months of age), subsamples of mice from each of the various diet groups (n = 5/sex/group) were euthanized by decapitation for blood and tissue collection (e.g., adipose depots, skeletal muscle, liver, heart), with an additional sample of 5/sex from the WC group following refeeding and weight regain. Tissues were weighed to the nearest 0.01 g and either fixed or quickly frozen in screw‐capped microtubes in liquid nitrogen and stored at −80°C until use. Again, at approximately 23 months of age (following the ~50% mortality point), random subsamples of mice remaining alive from all groups were collected as described, with an additional 5/sex in the WC group. All dissections were performed with wave 1 animals during the light phase and by the same individuals, who were blinded to group assignment.

Fat cell size and number Samples of inguinal and gonadal white adipose tissue were collected from the subgroups (n = 5/sex/group) at the specified time points above. Cell size and number were measured by using osmium fixation technique as described previously, with ~100 mg of right inguinal and gonadal adipose tissue weighed to the nearest 0.01 g ((17, 18)).

Serum analytes Total blood was collected from the trunk by decapitation and kept on ice for at least 15 minutes. Samples were centrifuged for 10 minutes at 3,000g at 4°C, with the serum layer carefully collected. Serum glucose was measured by using an Analox GM7 analyzer (Analox Instruments, Lunenburg, Massachusetts), serum insulin by using Millipore Sensitive Rat Insulin Radioimmunoassay (RIA) (Millipore, Billerica, Massachusetts), serum adiponectin and leptin with Millipore enzyme‐linked immunosorbent assay (ELISA) kits, and proinflammatory profiles with the V‐PLEX Panel 1 kit (Meso Scale Diagnostics, Rockville, Maryland).

Gene expression Quantitative real‐time polymerase chain reaction (PCR) was used to determine gene expression as previously described ((19)). Briefly, total RNA was isolated using RNeasy mini‐columns (Qiagen, Inc., Valencia, California) and reverse‐transcribed into single‐stranded complementary DNA (cDNA) using random hexamers and M‐MLV Reverse Transcriptase (Invitrogen, Carlsbad, California). Quantitative amplification of cDNA of interest by PCR was carried out using gene‐specific primers and iQ SYBR Green Supermix (Bio‐Rad Laboratories, Hercules, California). To prevent the amplification of any contaminating genomic DNA, the forward and reverse PCR primers were derived from two different exons that are separated by at least a 1‐kilobase intron. The size of the amplicon was confirmed by agarose gel electrophoresis. Cyclophilin A mRNA level was used to normalize total RNA input. The difference in PCR cycle numbers at the specified fluorescence thresholds (within the linear amplification range) for the gene of interest and cyclophilin A (delta Ct) was used to calculate the mRNA level of the gene of interest. All quantitative real‐time PCRs were performed in triplicate, and the arithmetic mean of the triplicate was used in subsequent calculations.

Statistical analysis Power estimates were performed using the primary outcome of survival for male and female C57BL/6J mice from published survival data. Calculated samples sizes were estimated to provide ≥ 80% to reject the null hypothesis of no difference in survival for each group pairwise comparison with no intentional weight loss (EO group). Statistical analyses were performed using SAS software (version 9.4 for Windows, SAS Institute, Cary, North Carolina). The mean life‐spans of the three CR groups were compared with the mean life‐span of the EO group using survival analysis by running the Cox proportional hazard regression model. Hazard ratios were estimated between each CR group and the EO group. Primary survival analyses were performed for all‐cause mortality (as an intent‐to‐treat model with no censoring) as well as with accommodations by censoring for type of death (e.g., technical complications, technical complications and euthanized for reasons other than ulcerative dermatitis, found dead) (Supporting Information Table S7). Our study was designed to last until all the animals died, permitting us to also run generalized linear models to compare the mean life‐spans of the CR groups with the mean life‐span of the EO group.

Mediation analysis In investigating the effect on life‐span of groups created on the basis of CR, we also hypothesized that there could be a clinical variable acting as a mediator. Food efficiency, percent body fat, and fat to lean ratio were tested for mediation effect. Given known concerns with the use of ratios, additional sensitivity analyses were performed (data not shown) using covariates in the analyses as previously described ((20)). Because the WC group animals were subjected to increase and decrease their body weights, the mediation analysis was done only between specific time points, during which the animals in the WC group were in either the weight gain (week 57 to week 73) or the weight loss (week 73 to week 85) phase. Sobel’s test was performed to test the significance of mediation effect in predicting life‐span ((21)). The differences in levels of serum cytokines and mRNA expression among the EO and CR groups were tested by using analysis of covariance (ANCOVA) (adjusting for sex) within the inguinal and gonadal adipose depots at four time points.