IMPORTANCE Time-restricted eating has increased in popularity among people trying to lose weight. However, the efficacy and safety have not been explored in a large, prospective randomized trial.

OBJECTIVE To compare the effects of weight loss in people who are overweight and obese prescribed a time-restricted eating protocol versus standard three meals per day.

DESIGN, SETTING, AND PARTICIPANTS The Time-Restricted Eating (TREAT) study was a 12-week randomized controlled trial of men and women aged 18-64 with a body mass index of 27-43 kg/m2. The study was conducted on a custom mobile study app on the Eureka Research Platform, and participants received a Bluetooth scale for daily use. Participants lived anywhere in the United States, with a subset of participants (n=50) living within 60 miles of San Francisco who underwent comprehensive, in-person metabolic testing.

INTERVENTION Subjects were randomized to one of two eating plans and received daily reminders about their eating windows through the study app. The consistent meal timing (CMT) group was instructed to eat three structured meals per day. The time-restricted eating (TRE) group was instructed to eat ad libitum from 12:00 pm until 8:00 pm and completely abstain from caloric intake from 8:00 pm until 12:00 pm the following day (16h fast:8h eat). Specific recommendations for caloric intake or macronutrient content were not prescribed to either group, and participants were not advised to make any changes to their physical activity.

MAIN OUTCOMES AND MEASURES The primary outcome was weight loss as measured at home on the Bluetooth-connected scale. Secondary outcomes were weight loss in the in-person cohort and changes in fat mass, lean mass, fasting insulin, fasting glucose, HbA1C, total energy expenditure, and resting energy expenditure. Bonferroni-corrected alphas and confidence intervals were used to correct for multiple comparisons for secondary outcomes. All other outcomes were considered exploratory and were not corrected for multiple outcomes.

RESULTS 116 subjects (mean [SD] age, 46.5 [10.5] years), participated in the study. There was a significant decrease in weight in the TRE (-0.94kg, 95% CI, -1.68, -0.20, p=0.013), but no significant change in the CMT group (-0.68kg, 95% CI, -1.41, 0.05, p=0.07) or between groups (-0.26kg, 95% CI, -1.30, 0.78, p=0.63). In the in-person cohort, (n=25 TRE, n=25 CMT), there was a significant within-group decrease in weight in the TRE group (-1.70kg, 95% CI, -2.56, -0.83, p<0.001), but no significant changes in any of the other secondary outcomes. There was a significant within-group decrease in lean mass in the TRE group (-1.10kg, 95% CI, -1.73, -0.48, p<0.001). There was also a significant difference in appendicular lean mass index between groups (-0.162kg/m2, 95% CI, -0.274, -0.050, p=0.005).

CONCLUSIONS AND RELEVANCE Prescribing TRE to people who are overweight and obese led to modest weight loss, but there was no difference in weight loss when compared to those randomized to CMT. Moreover, the majority of weight lost in people randomized to TRE came from loss of lean mass rather than fat mass. The mechanism(s) underlying this phenomenon is unknown, and future studies will be aimed at exploring the relationship between fasting and lean body mass.

Human obesity results from the interaction between a genetic predisposition and deleterious environmental factors and is considered to be a global epidemic associated with mortality. Therefore, research to understand the pathogenic mechanisms underlying obesity is of great importance and is required in order to develop of rational and effective treatments. This thesis work includes three separate research projects with the common intention of exploring the molecular gentics of the central control of energy homeostasis.

The major central regulator of long-term regulation of body weight in humans is the leptin-melanocortin axis is centered in the hypothalamus. This pathway senses perifpheral energy stores and signal saitiety, and it's disruption leads to the most severe forms of human obesity. Melanocortin-4 receptor plays a key role in transmitting the anorexigenic signal of the adipocyte secreted hormone leptin. We systematically and comparatively evaluated the functional consequences of rare mutations found in both MC4R and MC3R in a large cohort of severely obese North American adults and lean controls. We demonstrated that naturally occurring MC4R mutations but not the MC3R mutations are strongly associated with human obesity.

Both common variants with small effects and rare variants with larger individual effects have been shown to contribute to the genetic predisposition to obesity. We ialso nvestigated whether common genetic variants in and around MC4R are associated with adiposity in a population-based cohort, Health ABC. We found that the strongest association with adiposity was with a SNPsin the 3' non-coding DNA of MC4R. A conserved region near this associated nsSNPs was tested for possible enhancer activity.

Using mice heterozygote for Rab23 nonsense mutations we explore the novel mechanistic link between syndromic and non-syndromic human forms of obesity by exploring the hypothesis that Rab23 deficiency causes obesity through functional impairment of the first order hypothalamic neurons involved in leptin-mediated regulation of energy homeostasis, eventually through disruption of leptin signaling at the level of the POMC neurons.

Human obesity is a multi-factorial trait regulated by environmental and genetic factors and is considered to be a global epidemic associated with mortality. Therefore, understanding the molecular pathways controlling the energy homeostasis is of great importance in order to develop pharmaceutical agents to treat obesity. This thesis work includes four separate research projects with the common intention of exploring the central control of energy homeostasis using three distinct research approaches. Leptin-melanocortin axis is the major central regulator of energy homeostasis where MC4R and POMC play key roles in transmitting the anorexigenic signal of the adipocyte secreted hormone leptin. In a genetic linkage study, we demonstrated that naturally occurring MC4R mutations but not the POMC or MC3R mutations are strongly associated with human obesity. Secondly, in an attempt to determine an alternative signaling pathway to leptin, the inhibition of Gi protein signaling in the POMC neurons decreased the energy expenditure in a transgenic mouse model. Finally, we scrutinized the molecular mechanism underlying the physiologically relevant constitutive activity of MC4R. The data supports the evolutionary role of the N-terminal domain in the activity regulation of the G protein-coupled receptors. Overall, our findings provide essential pieces to the bigger puzzle of the melanocortin system, and contribute to the biomedical research in the pursuit of understanding the regulation of energy homeostasis in humans.

The melanocortin 4 Receptor (MC4R) is a central component of the leptin-melanocortin pathway in the hypothalamus. MC4R plays a critical role in the long-term regulation of energy homeostasis and mutations in MC4R are the most common cause of monogenic obesity. However, the precise molecular and cellular mechanisms underlying the maintenance of energy balance within MC4R expressing neurons are unknown. The expression of MC4R in neurons from the Paraventricular Nucleus of the hypothalamus (PVN) has been shown to be both necessary and sufficient to regulate food intake and body weight, and we discovered that MC4R localizes at the primary cilium of those neurons. Primary cilia are unique microtubule-based organelles that protrude from the cell membrane to sense and relay extracellular signals. In Chapter one, we first mapped the expression of MC4R in the brain, to then determine the extent of MC4R localization at the primary cilia in some of the identified regions. We discovered that, in the PVN, MC4R localization at the primary cilium is dynamic and depends on age and physiological status relayed by MC4R ligands levels. Together, these results shed light on the complex regulation of MC4R bioavailability at the primary cilium. In Chapter two, we found that cilia were required specifically on MC4R-expressing neurons for the control of energy homeostasis. Moreover, these cilia were critical for pharmacological activators of MC4R to exert an anorexigenic effect. Using a combination of mouse genetic approaches, we demonstrated that MC4R signals via cilia of PVN neurons to control food intake and body weight. In Chapter three, we demonstrate that the Melanocortin Receptor Associated Protein 2 (MRAP2) is critical for the ciliary localization and weight-regulating function of MC4R. These findings demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggests that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.

Mutations in the melanocortin 4 receptor (MC4R) are the leading cause of monogenic obesity. MC4R is a key player in the leptin melanocortin pathway, which regulates long term energy homeostasis. Mutations in anorexigenic components of this pathway leads to monogenic obesity. However, the method in which this pathway encodes, and stores body weight set point, thus energy homeostasis, is unknown.In chapter 1, we will characterize and validate two different knock-in mouse models that will allow for studying MC4R localization and specific manipulation of MC4R neurons. In chapter 2, we will show how dynamic localization of MC4R encodes information on peripheral energy stores by integrating information from upstream components of the leptin melanocortin pathway.

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