Is lack of sleep making me fat? The story of leptin, circadian rhythms, and appetite A presentation by Tyler Bradley

Sleep deprivation may do more than just make you tired all day! Getting enough sleep is important For normal functioning It could also be affecting obesity Let’s find out how

Big characters in this story The circadian clock The peripheral clock Leptin The leptin loop components White adipose tissues (fat!) Appetite Energy expenditure The circadian clock- Controls the leptin-mediated endocrine feedback loop The central (circadian) clock plays a dominant role in the homeostasis of leptin signaling The peripheral clock directly regulates leptin transcription in adipose tissue Energy homeostasis in mammals is maintained by the interactions of peripheral signals with the CNS Leptin neuroendocrine feedback loop is a prime example

Wait, sleep deprivation is making me fat!?!? Not necessarily, but it is a factor Obesity has many other contributing factors Excessive food intake Sedentary lifestyle Genetic components Sleep is important for energy balance Sleep duration REM sleep Chronic sleep deprivation Obesity and being overweight are associate with severe co-morbidities including: Type 2 diabetes, cardiovascular disease, and cancer

Leptin knockout mouse Normal mouse

The circadian rhythm The circadian “clock” Allows mammals to coordinate their behavior with daily changes in light and darkness Usually in 24 hours intervals called diurnal oscillations Present in sleeping and feeding behaviors But what controls this circadian rhythm?

The suprachiasmatic nucleus (SCN) Referred to as the primary circadian clock in mammals Located in the hypothalamus Directly affects Sleep Alertness Physical activity Hormone levels Wait another clock?

The peripheral clock Other peripheral oscillators Found in Adrenal gland Esophagus Lung Liver Pancreas Spleen Thymus Skin Directly regulates leptin transcription in adipose tissue

Leptin Hormone made by adipose cells Referred to as the “satiety hormone” Regulates energy balance Inhibiting hunger Promotes energy expenditure Acts primarily on receptors in the arcuate nucleus (ARC) ARC found in the hypothalamus Leptin induces the opposite effects of ghrelin (The hunger hormone) Leptin is primarily synthesized in the adipocytes of white adipose tissue

Leptin Circulates the blood in free form and bound to proteins This means it can be measured Blood leptin levels are higher between midnight and early morning Perhaps suppressing appetite and energy drive during “normal” sleeping hours

The leptin endocrine feedback loop 3 basic components White adipose tissue (WAT) Serum leptin levels Leptin responsive neurons in various hypothalamic centers The arcuate nucleus (ARC) Activation of leptin responsive receptors in the ARC suppresses food intake and stimulates energy expenditure Implies that the body is in an energy stable state It has been shown that the basal level of serum leptin is proportional to body fat content

Leptin in the ARC Targets the leptin receptor in the ARC(LEPR- B) NPY/AgRP and POMC neurons are the direct targets of leptin NPY/AgRP neurons release an orexigenic neuropeptide Y (NPY) and AgRP (antagonist of melanocortin receptors) The POMC neurons secrete α-MSH α-MSH activates central melanocortin signaling ↓ food intake and ↑ energy expenditure The direct targets of leptin in the ARC are NPY/AgRP neurons and POMC neurons NPY- Neuropeptide Y- A potent hunger promoter secreted by cells in the gut and hypothalamus AgRP- Agouti-related protein- An appetite stimulating hormone POMC- Pro-opiomelanocortin- Cleaved into α-MSH and other peptide hormones α-MSH- Alpha melanocyte stimulating hormone α-MSH from the ARC plays an important role in the regulation of appetite

Leptin in the ARC ↑ in plasma leptin ↓ in plasma leptin Inhibits NPY/AgRP neurons Activates Janus kinase 2-Signal Transducer and Activator of Transcription 3 (JAK2-STAT3) in POMC neurons Leads to STAT3-activated POMC transcription α-MSH synthesized and secreted ↓ in plasma leptin Activates NPY/AgRP neurons Inhibits POMC neurons Let’s take a look at what happens when plasma leptin either increases or decreases Increased plasma leptin- Decreased food intake and increased energy expenditure Decreased plasma leptin- Increased energy storage by stimulating food intake and suppressing energy expenditure

White adipose tissue (WAT) Primary energy store in mammals Obese individuals usually have more WAT Site of leptin synthesis by adipocytes Expression of leptin in adipose tissues is directly regulated by clock genes Independent of food cues ↑ fat mass ↑ circulating leptin levels Chronic circadian dysfunction desensitizes leptin receptors in ARC Results in leptin resistance Similar to insulin resistance in Type 2 Diabetics Differs from brown adipose tissue

Chapter 1- Circadian dysfunction induces leptin resistance in mice Nicole M. Kettner, Sara A. Mayo, Jack Hua, Choogon Lee, David D. Moore, Loning Fu

New characters in this chapter Transcription factors in WAT CLOCK/BMAL1 Cryptochromes 1 & 2 (Cry 1 & 2)- Repressor NPAS2/BMAL1 Period 1-3 (Per 1-3) Repressor Transcriptional activator in WAT CCAAT-enhancer-binding protein alpha (C/EBPα) CLOCK/BMAL1- Circadian Locomotor Output Cycles Kaput- Plays a central role in the transcription of the circadian pacemaker NPAS2/BMAL1- Neuronal PAS domain-containing protein 2- C/EBPα- CCAAT-enhancer-binding protein alpha- The most potent transcriptional activator for leptin CLOCK/BMAL1- In adipose the heterodimer directly control leptin expression by regulating activity of C/EBPα

Leptin resistance as a focal point Mice with lacking either leptin (ob/ob) or LEPR-B (db/db) displayed severe obesity Administration of leptin ↓ food intake and body weight in (ob/ob) But not in (db/db) Most obese humans display ↓ responsiveness to0 ↑ plasma leptin and administration of leptin Leptin resistance contributes to obesity Serum leptin levels display diurnal variation in both humans and rodents External food cues have been though to be the main factor controlling diurnal leptin oscillation However, recent studies have shown that circadian disruptions can abolish diurnal oscillation of plasma leptin regardless of external food cues

ARC Leptin receptor

Methods All mice were C57BL/6J Wild type (WT) Lacking BMAL1 (Bmal1 -/-) Lacking Per 1 & 2 (Per 1 -/-; Per 2 -/-) Lacking Cry 1 & 2 (Cry 1 -/-; Cry 2 -/-) Both WT mice and mutant mice were ran through a series of experiments

The role of the clock in energy homeostasis WT mice and mutants were treated with either normal light/dark cycles or jet-lagged 8 hour light phase advance (Monday) 8 hour phase delay (Thursday) Jet lag→ Altered body weight of WT and Per Altered body composition of all types, except Bmal1 Insensitive to chronic disruption of light cues Takeaway- Jet lag→ significantly ↑ body weight and fat composition in WT

Figure 1 A) Body weights of male mice kept in 24 hr LD cycles and fed with regular chow (Ctrl) from 4 to 40 weeks of age (B) Ratios of gonadal fat mass versus body weight (BW) of control male mice at 7 and 20 weeks of age (±SEM). (C) Total fat composition of control male mice from 4 to 40 weeks of age (±SEM). (D) Body weight of chronically jet-lagged (CJ) male mice from 4 to 40 weeks of age (E) Fat (left) and lean body mass (LBM) (right) compositions of male jet-lagged mice from 4 to 40 weeks of age (±SEM). (F) Fat composition in control and jet-lagged female (F) and male (M) mice from 4 to 40 weeks of age (±SEM).

The role of the clock in energy expenditure WT and mutant mice, equal numbers of normal and jet-lagged were monitored using CLAMS Measured food intake, physical activity, and respiration exchanging rate (RER) Takeaway- Disruptions of circadian homeostasis can have differential impacts on energy balance WT (normal and jet-lagged) has similar total daily food intake, physical activity, and RER Per and Cry mutants ate same amount of food daily, but in arrhythmic profiles Per were obesity prone Normal- Low physical activity and RER Jet-lag- Decreased body weight due to ↑ physical activity and RER in light phase Cry mutant body weight did not change, they had abnormally high RER profiles WT had robust VO2 and VCO2, Per had low peak VO2 and VCO2, Cry had drastically high VO2 and VCO2 Chronic jet lag significantly changed energy expenditure in WT (similar to control Per), in jet lagged Per (Similar to control Cry) The distinct body weight of WT, Per, Cry under control and jet lag can be explained by their unique energy expenditure profiles and Their responses to disruptions of environmental light cues, not daily food intake or energy expenditure

Figure 2 Circadian Dysfunction Disrupts Energy Homeostasis (A and B) The average of total daily food intake (A) FI in light or dark phase (B) of control and jet-lagged WT, Cry1−/−;Cry2−/−, and Per1−/−;Per2−/− mice on days 2 and 3 of CLAMS study (±SEM). (C and D) The average of total daily activity (C) activity in light or dark phase (D) to dark phase of control and jet-lagged mice on days 2 and 3 of CLAMS study (±SEM). (E and F) Average daily respiration exchanging rates (RER) (E) and RER in light or dark phase (F) in control and jet-lagged mice on days 2 and 3 of CLAMS study (±SEM). (G and H) The circadian profiles of oxygen consumption (VO2) (G) and carbon dioxide production (VCO2) (H) in control and jet-lagged mice over 3 days of CLAMS analysis. (I and J) Average rates of VO2 (I) and VCO2 (J) produced by control and jet-lagged mice in light and dark phases on days 2 and 3 of CLAMS study (±SEM).

The role of the clock in diurnal plasma leptin Explored the leptin-mediated neuroendocrine pathway Best characterized pathway for body weight WT in 24 hour DD cycles maintained circadian rhythm of plasma leptin WT with acute jet lag resisted changes from circadian rhythm of plasma leptin WT with chronic jet lag showed high and arrhythmic plasma leptin levels Takeaway- Diurnal oscillation of plasma leptin is controlled by circadian clock controls Plasma leptin displays a robust circadian rhythm coupled with diurnal profiles of food intake, physical activity, and energy expenditure in control WT Ablation of Per & Cry genes abolished diurnal oscillation of plasmas leptin Although Per mutants still had a dampened circadian rhythm of food and activity

(A–D) Serum levels of Leptin in WT, Cry1−/−;Cry2−/−, and Per1−/−;Per2−/− mice in 24 hr LD cycles (A), in WT and Per1−/−;Per2−/− mice in 24 hr DD cycles (B) and during 22 hr fasting (C), and in acutely jet-lagged (AJ) WT mice and chronically jet-lagged WT, Cry1−/−;Cry2−/−, and Per1−/−;Per2−/− mice (D) (±SEM). (E and F) The leptin mRNA (E) and protein (F) expression follow a robust circadian rhythm in both LD (top) and DD (bottom) cycles in WAT of control WT mice, but not in WAT from Per mutants or jet-lagged WT mice. (G and H) Northern blots show disruption of adipose clock and circadian expression of leptin mRNA in Per(G) and Cry mutant (H) mice. (I) A summary of three independent northern blots on leptin mRNA expression in mouse WAT in 24 hr LD cycles (±SEM). (J) Western blotting on PER2, BMAL1, CRY1, Leptin (LEP), and β-Actin (β-ACT) in WAT of control and jet-lagged WT (left) and Cry mutants (right). (K) A summary of three independent western blots on Leptin expression in mouse WAT in 24 hr LD cycles (±SEM). (L and M) Western blotting shows lack of BMAL1 expression in nuclear extracts (L) and dampened and arrhythmic Leptin expression (M) in WAT of Ap2Cre;Bmal1fl/fl mice. ZT, zeitgeber time, with light on at ZT0 and off at ZT12; CT, circadian time, with CT0 as the beginning of a subjective day and CT12 as the beginning of a subjective night.

Leptin expression in adipose tissue Control and jet-lagged WT, Per, Cry, and BMAL1 were examined for diurnal food intake, activity, energy expenditure, and plasma leptin All 4 types in the control group had peak plasma leptin levels at mid-active phase All 4 types in the jet-lag group showed a disruption of the endogenous adipose clock and circadian expression of leptin mRNA &/or protein Attempted to find whether the clock controls leptin expression in adipose to drive serum leptin levels Leptin mRNA and protein expression followed a robust circadian rhythm in 24 hr. LD and DD cycles Peaked at mid-active phase Luciferase reporter to assess transcriptional activity NPAS2 is not expressed in adipose- Thus Cry mutants

(A) Circadian heterodimers marginally stimulate a leptin promoter-driven luciferase (lep2762-Luc) reporter at a low concentration but inhibit the reporter activity at higher concentrations independent of CRY1. Numbers indicate the amounts of reporter and expression vector DNA (ng) used in each transfection (±SEM;∗stimulation, #suppression). (B) The C/EBPα binding sites (green) overlap with consensus E boxes (red boxes) in both human and mouse leptin promoters. (C) The strategy for generating mutant leptin promoter-driven luciferase reporters with mutated bases shown in blue. (D) The heterodimer potentiates C/EBPα-mediated lep450-Luc reporter activation at a low concentration but inhibits C/EBPα activity at higher concentrations. Mutation of E1 and E2 abolishes the inhibitory effect of heterodimer. Mutation of E3 abolishes the stimulatory effect of heterodimer (±SEM; ∗stimulation,

Control of leptin expression by the SCN Circadian control of central melanocortin signaling Expression of phosphorylated STAT3 and POMC were arrhythmic and dampened in jet- lagged WT and in Per mutants Expressed high levels of plasma leptin Cry mutants had the reverse ↑ pSTAT3-POMC & ↓ plasma leptin levels Takeaway- Chronic circadian disruption leads to leptin resistance in the CNS of WT mice The differential disruption of leptin-mediated central melanocortin signaling drives the body weight And composition phenotypes of all mouse models studied Injection of leptin into Jet-lagged WT and Per was attempted Both lost weight but with both either leptin or saline injection So weight loss was related to the stress of injection Not exogenous leptin

How is leptin synthesized in adipose tissue? CLOCK/BMAL1 heterodimer binds to leptin promoter C/EBPα is stimulated, and mediates leptin transcription This occurs in the early sleep phase CLOCK/BMAL1 suppresses C/EBPα in late sleep phase

Conclusions Circadian homeostasis of the leptin-mediated neuroendocrine feedback loop is a major mechanism for controlling long term energy balance Leptin transcription in adipose drives the rhythm of plasma leptin levels The SCN clock potentiates the response of leptin receptors in ARC neurons ↑ fat mass ↑ plasma leptin Chronic circadian dysfunction downregulates leptin receptors leading to leptin resistance Jet-lagged WT, Per, Cry & adipose specific Bmal1 knockout all showed a similar disruption Of the fat clock and inhibition of BMAL1 expression, which is correlated with the Suppression of leptin expression in adipose regardless of food intake profiles Suggesting that disruption of clock leads to decreased leptin expression In the adipose, CLOCK/BMAL1 directly controls circadian leptin transcription that drives the rhythm of serum leptin In the CNS, the SCN clock potentiates the response of LEPR-B expressing ARC neurons to circulating leptin To maintain balance of food intake and energy expenditure Increased fat mass elevates circulating Leptin, however chronic circadian dysfunction desensitizes the LEPR-B neurons to increased leptin signaling resulting in resistance

Leptin feedback loop α -MSH Arcuate nucleus

Christy Olson, Nancy Hamilton, Virend Somers Chapter 2-Percentage of REM sleep is associated with overnight change in leptin Christy Olson, Nancy Hamilton, Virend Somers

REM sleep as a focal point Rapid Eye Movement sleep Bouts of REM sleep are shorter initially and become progressively longer as sleep goes on Sleep deprivation=Less time spent in REM sleep Studies show that shorter sleep times are correlated with a higher BMI Could lack of REM sleep be the culprit, as opposed to overall sleep loss?

Methods 58 volunteers in healthy condition were selected (15 women and 43 men) Mean participant age was 35.6 years of age All participants were healthy with no acute or chronic medical conditions Sleep duration and % of REM sleep were obtained using an overnight polysomnography Body fat % was measured along with BMI Blood samples were taken before sleep and upon awakening for leptin levels 5 year larger study in Minnesota examining genetic and cardiovascular factors related to sleep Individuals with sleep apnea were excluded for this study Following the evening blood draw participants watched tv, read, or did quiet activities until bed

Polysomnography A test used to diagnose sleep disorders Polysomnography records: Brain waves the Oxygen level in your blood Heart rate Breathing Eye and leg movements

REM sleep and leptin Sex did not produce a significant change in leptin levels A statistically significant relationship between REM sleep and overnight change in leptin was shown Older age was correlated with change in leptin levels (Body fat did not show a significant change) Spending a greater portion of the night in REM sleep predicted a greater overnight change in leptin A one minute decrease in total REM sleep was associated with a .02 decrease in morning leptin

Non-REM sleep and leptin A separate model was run to determine the relationship between leptin levels and NREM sleep Stage 1, stage 2, and slow-wave sleep were analyzed No significant relationship was found between NREM sleep and leptin levels

Conclusions A higher percentage of REM sleep is related to a greater reduction in leptin during sleep Greater ↓ in overnight leptin may reflect greater amplitude of the diurnal leptin profile Due to ↑ REM sleep Greater amplitude of diurnal leptin may contribute to the suppression of appetite Takeaway- REM sleep is associated with changes in leptin levels Reduction in REM sleep may cause a lower amplitude of diurnal leptin Consistently elevated levels of leptin can lead to leptin resistance, a characteristic of obesity 24 hour leptin profiles are a better way to assess with consistency than morning leptin levels Possible mechanisms linking REM sleep and change in leptin during sleep may include: Orexin and Melanin-stimulating hormone- Thought to play a role in food intake and sleep It was not possible to take individual sleep-wake habits into account Or meal patterns (caloric timing)

Diurnal leptin amplitude

Chapter 3- Chronic sleep fragmentation induces hypothalamic endoplasmic reticulum stress Fahed Hakim, Yang Wang, Alba Carreras, Camila Hirotsu, Jing Zhang, Eduard Peris, and David Gozal

Endoplasmic reticular stress and sleep fragmentation as a focal point The ER is a key player in responding to cellular stress Chronic stress conditions disrupt ER homeostasis and lead to accumulation of mis/unfolded proteins in the ER lumen The cell responds to stress by initiating UPR signaling Signal cascade- (IRE1α), (PERK), & (ATF6) Protein refolding or degradation follows Under chronic ER stress, protein folding cannot be compensated for and UPR induces autophagy Obesity is associated with activation of cell stress signaling pathways and inflammatory pathways To cope, ER links to UPR (unfolded protein response) GRP78, a binding immunoglobulin protein initiates UPR cascade UPR has 3 ER-localized protein sensors IRE1α, ATF6, and PERK PERK prevents general protein synthesis through translation repression

Dephosphorylation of JAK2 is important in terminating leptin signal transduction Can be induced by PTP1B (protein-tyrosine phosphatase 1B)- Negative regulator of leptin signal transduction pathway in hypothalamus PTP1B is elevated in high fat fed rats Deficiency of PTP1b results in resistance to diet-induced obesity caused by leptin hypersensitivity

Methods Male C57BL/6J mice were housed in a 12 hour light/dark cycle Sleep fragmentation was induced in some mice during the light period EEG recorded waves during wake (W), slow wave sleep (SWS), and REM sleep Food intake and body weight were monitored Tauroursodeoxycholic acid (TUDCA) was used in some to improve ER function 2 minute interval of sweeper to induce sleep fragmentation Food and water were readily available Mice were euthanized after and their hypothalami were examined

TUDCA Tauroursodeoxycholic acid Bile acid derivative Works on unfolded proteins to prevent apoptosis Programmed death of a cell Chemical chaperone used to stabilize protein conformation, improve ER folding capacity, and facilitate the trafficking of mutant proteins TUDCA was used to abrogate SF induction of ER stress

Results Sleep Fragmentation (SF) induces hyperphagic behaviors and ↑ daily food intake SF induces ER stress in the hypothalamus Activates the UPR SF ↓ leptin receptor signaling in hypothalamus Restored by TUDCA treatment C/EBPα was identified as an ER stress induced transcription factor that is involved with UPR signaling Mice without C/EBPα (CHOP) were used to further assess the role of Sleep fragmentation on UPR activation TUDCA injection attenuated the early increases in ER stress induced by SF

Figures Sleep fragmentation (SF) exposed mice exhibit increased food intake behaviors and accelerated weight gain. (A) Mice exposed to sleep fragmentation (n = 24) developed increased food intake starting within the first 2–4 days from initiation of SF. The hyperphagic behaviors were sustained throughout the duration of disrupted sleep (P < 0.01). (B) Body weight gain differences between the two groups emerged after 4 w of SF (n = 24) and sustained thereafter (P < 0.02). SC, sleep control.

Sleep fragmentation-exposed mice develop hyperleptinemia over time and attenuation of leptin receptor (ObR) signaling in the hypothalamus. (A) Leptin levels were measured in plasma drawn from fasting mice, and when compared to mice undergoing sleep control (SC) showed increasing levels starting after 1-w exposure to sleep fragmentation (SF), and further increasing at 21 days of SF exposure (B) Leptin receptor (ObR) expression showed a nonstatistically significant trend toward increases in expression (C) p-STAT3/STAT3 in SF decreased over time when compared with SC (P < 0.03), suggesting reduced ObR receptor signaling.  (D) SF mice showed low levels of p-STAT3 expression after leptin injection when compared to baseline (saline injection) or to SC conditions. SF-induced ObR resistance was abrogated when mice were treated with the chemical chaperone TUDCA. PBS, phosphate buffered saline; STAT3, signal transducer and activator of transcription 3

Conclusions Chronic SF caused ↑ food intake and ↓ leptin receptor signaling in the hypothalamus Appears to be maintained by ER stress Activation of UPR cascade Exogenous TUDCA, a protein chaperone, nullified hyperphagic behavior in SF mice ER stress and activation of the UPR response in the hypothalamus are important in energy homeostasis Chronic SF in mice induces an increased weight gain and an increase in visceral and cutaneous fat (Weng et al.) Assessed by MRI Studies show that in acute SF there are adaptive mechanisms that are unregulated and protect neurons from injury Sustained ER stress peaks after several days Activation of UPR response and also leptin resistance Leptin resistance evident by high leptin levels and reduced STAT3 phosphorylation Potential work in PTP1B signaling pathway ER stress and UPR relate sleep integrity not just duration to leptin SF induces ER stress, activates UPR, downregulates leptin receptor signaling in hypothalamus, Most likely through PTP1B pathway

The big picture- Leptin resistance SCN controls diurnal leptin levels ↑ fat mass→ ↑ leptin levels ↑ leptin levels → leptin resistance at ARC receptors ↓ affinity for leptin→ ↓ energy expenditure and ↑ food intake SCN controls diurnal leptin levels Obese individuals displays arrthymic diurnal leptin levels Chronic sleep deprivation lead to increased body weight and fat composition This could be attributed to leptin resistance at receptors in the ARC

REM sleep is important ↑ % REM sleep→ ↓ in leptin during sleep Greater amplitude of diurnal leptin profile ↓ REM sleep→ ↑ leptin levels→ resistance to leptin

Chronic SF induces ER stress and leptin resistance SF ↑ ER stress UPR pathways in hypothalamus activated ↓ LEPR-B signaling ↑ in PTP1B → ↓ Leptin sensitivity throughout body

So what does that mean? Sleep deprivation is bad for you! Obesity can lead to multiple forms of resistances Resistance to insulin Resistances to leptin Resistance to leptin can alter your feeding habits And how the food you eat is stored

Thanks for listening! Any questions/comments?