Leptin Intro/Signaling. ATeamP: Angelo, Anthony, Charlie, Gabby, Joseph

Transcription

1 Leptin Intro/Signaling ATeamP: Angelo, Anthony, Charlie, Gabby, Joseph

2 Overview Intro to Leptin Definition & Sources Physiology Bound vs. Free Receptors Signaling JAK/STAT MAPK PI3K ACC Experimental findings

3 What is leptin? Hormone primarily synthesized and secreted by white adipose tissue Not the only source White is more for energy storage Brown adipose tissue: contains mitochondria Produced by adipocytes in proportion to fat size stores to signal repletion of body energy stores -> hypothalamus Correlates closely with both BMI and total body fat Plays important roles in the regulation of food intake, energy expenditure, metabolism, neuroendocrine functions More recent discovery 1994; opened up whole new perspective to study role of adipocyte-derived factors in energy balance homeostasis

4 Bound vs. Free Leptin In humans, the majority of circulating leptin is bound to serum macromolecules These may modulate ligand bioactivity and bioavailability to target tissues In lean people with small adipose tissue mass, majority of leptin is bound In obese people, the proportion of free leptin is increased Fasting Free leptin may have more rapid turnover due to proteolytic cleavage or increased clearance Decrease in free leptin concentrations has been observed More pronounced in lean volunteers compared with obese subjects No change observed in bound leptin in either group

5 Physiology Highly pleiotropic Capable of stimulating variety of biological responses in a wide spectrum of cell types Placenta, ovary, skeletal muscles, pituitary gland, and lymphoid tissue Both in CNS and the periphery Leptin levels fluctuate according to changes in caloric intake Increase Overfed and obese states Insulin, glucocorticoids, and pro-inflammatory cytokines Decrease Starvation Catecholamines Secreted in pulsatile fashion; circadian rhythm Sexual Dimorphism

6 Physiology Cont d High expression in hypothalamus - ARC and VMH Leptin targets POMC/CART and AgRP/NPY neurons re: Anorexigenic pathway from insulin signaling pathway presentation POMC/CART, AgRP/NPY energy metabolism, food intake and body weight Reduces expression of MCH + orexins in LHA

7 Signaling Bears structural and functional similarity to cytokine family Like cytokines, there are a variety of different receptor types This helps explain the pleiotropic effects of leptin Internalization/degradation cycles control levels of receptor Clathrin-coating mechanism of cytokine receptors 5-25% at cell surface

8 Leptin Receptors Universal distribution of OB-R (aka LEP-R) Acts via transmembrane receptors Performs signal transduction Produced in several alternatively spliced forms: OB-Ra, OB-Rb, OB-Rc, OB-Rd, OB-Re, and OB-Rf OB-Rb = long full-length isoform OB-R isoform deletion in neurons -> obesity in mice Implications in db/db and fa/fa mice reveal role High OB-Rb levels in hypothalamus

9

10 Phosphorylation theme Phosphorylation is a common trend in signal transduction

11 JAK/STAT JAK2 activation through binding to box1 motif Leads to OB-R phosphorylation + homodimerization Phosphorylated receptor leads to binding of STAT3 STAT3 itself is phosphorylated and dimerized Nuclear localization of STAT3 modulates gene expression Stimulating pomc and inhibiting agrp Expression of SOCS3 exhibits negative feedback on JAK2

12 MAPK

13 MAPK signal transduction Maximal activation occurs with ObRb Activation of c-fos and egr-1: Promotes cell proliferation and differentiation Activation of phospholipase A: Leads to caspase 9 induced apoptosis for anti-cancer properties Activation of p42/44 pathway: Cell signaling (NO synthase) ex. Insulin secretion Stress + heat shock + cytokine binding + more = p38 pathway JNK stimulation TNFalpha production In vascular smooth muscle, can induce hypertrophy

14 PI3K

15 Leptin & PI3K Functions similarly to insulin induced PI3K pathway Both leptin and insulin can influence the PI3K pathway Anorexigenic pathway Critical for signaling leptin to the hypothalamus Induces akt for more downstream effects like GLUT-4 recruitment and PDE3B to inhibit insulin secretion Can induce hyperpolarization Can stimulate or inhibit pkc for insulin secretion

16 AMPK 5 -AMP-activated Protein Kinase Stimulates fatty acid oxidation in skeletal muscle Responds to decreases in ATP Begins response in ~15min Opposite of ACC which is used to buildup energy Leptin inhibits ACC Disinhibits CPT1 to allow mitochondrial fuel transfer

17 Experiments: Overview Zhang & Scarpace performed several studies detailing the role of leptin resistance on obesity in mice, focusing on these factors Age-related leptin resistance Variations in caloric intake Physiological responses of leptin Leptin-induced leptin resistance Compounds with caloric intake

18 Hypothalamic Leptin Signaling: The OB Gene The experiments detail the OB gene that codes rat leptin. - Involves multiple pathways: JAK/STAT, PI3K - The OB ribosome initiates these pathways

19 Experiment 3A: Leptin Resistance and STAT-3 Phosphorylation Details the increase/decrease of STAT3 and serum leptin with a small dose of leptin to the hypothalamus. - Both STAT3 and serum leptin increased and decreased nearly parallel to each other - Appears to peak for an hour and then gradually decrease and stabilize as time moves on

20 Experiment 3: Age-Related Leptin Resistance Similarly to humans, body weight and the amount of adipose tissue in rats increase with age. Energy expenditure and food responses in adult rats are weaker than young rats, so they are considered more leptin resistant. Procedure: observe changes in P-STAT3 activity in both young (~6 months) and adult (~24 months) rats, dependent on the dose of leptin injected - Both young and adult mice developed similar peaks around the 10 ng mark - STAT3 phosphorylation was 41% higher in the young mice than in adults

21 Experiment 3: Age-Related Leptin Resistance (cont.) - More P-STAT3 activity was found in the young rats than the adult in the control group - This discrepancy is elevated after the leptin injection - There appear to be less leptin receptors in adult (obese) rats - Revealed diminished leptin signaling in the adult rats -> adult onset obesity

22 Experiment 4: Leptin Signaling and Caloric Intake Diet and caloric intake also affect leptin resistance. Procedure: compares groups of mice assigned to assigned to either chow-fed or high-fat feeding, with some delegated to caloric restriction. They were then given a 2 ug injection of leptin and studied the effects - large discrepancy in P-STAT3 between chow-fed & DIO even without leptin injection - The leptin injection provided a large increase for chow-fed rats but the effects were blunted for the DIO rats - Caloric restriction developed a large increase in leptin activity for both diet variations - Caloric restriction reduces serum leptin, which is increased by DIO, so this offsets the changes - More Ob gene activity during caloric restriction in both cases, despite there being less during DIO

23 Experiment 5- Connection leptin signals & physiological responses Procedure: removal of doxycycline in water -> removal leptin transgene expression Leptin then applied directly to hypothalamus -> reversibly regulate leptin transgene Interesting Findings: When turned on (doxycycline) -> increased Hypothalamic STAT3 phosphy Less food consumption Body weight loss

24 Experiment 5- Connection leptin signals & physiological responses What happens when leptin receptors are activated then deactivated for periods of time? Continuous 2 months vs 1on/1off Months Continuous: Rats had transgene expression applied for two consecutive months 1:1 Months: Rats had transgene active for 1 months & inactive for the next month Reduced hypothalamic leptin receptors Significant elevation in the leptin receptor expression

25 Experiment 5- Connection leptin signals & physiological responses What does this table tell us? Adiposity - increase was in rats where the transgene was active then inactive, while continuous activation led to no change in weight P-STAT3 - increment in group with active gene, while on/off group stayed relatively normal Meaning there is some kind of effect present in the physical and physiological aspect of the rats

26 Experiment 6- Leptin-Induced Leptin Resistance Based on the previous findings, does this mean rats can build a leptin transgene receptor tolerance over time? (especifically the raav gene)

27 Experiment 6- Leptin-Induced Leptin Resistance Experiment: Two different group of rats were compared for more than 300 days:: Lean/Young rats treated with raav-leptin vs Controlled Group

28 Experiment 6- Leptin-Induced Leptin Resistance Findings: Food Intake -The amount eaten by each group differed from about day 4 to 140 BUT gradually reached the same level Body Mass - raav treated rats mass was significantly lower than controlled group from days 9-300, but gradually kept incrementing until almost evening Oxygen Consumption - raav treated rats had elevated levels of O consumption BUT as food intake became restricted by day 223, oxygen consumption levelled with controlled group

29 Experiment 6- Leptin-Induced Leptin Resistance Conclusion: YES!! There is a leptin tolerance build up in the hypothalamus present In young and lean rats

30 Experiment 7- Leptin Resistance lead to diet-induced obesity Based on the previous findings, raav treated rats initially differed in their body weight but over time began regaining it until reaching the same weight as controlled group BUT what will happen if these group of rats where exposed to a constant high calorie diet?

31 Experiment 7- Leptin Resistance lead to diet-induced obesity Controlled Group: Consumed 43% more calories Gained 175% more weight Gained 60% more internal fat raav-leptin Treated Group: Consumed 36% more than the controlled group (an additional 36% from the 43% found) Weight gain was almost double of that of controlled group Gained 26% more in internal fat

32 Experiment 7- Leptin Resistance lead to diet-induced obesity Conclusion:

33

34 Works Cited Fruhbeck, G. Intracellular signalling pathways activated by leptin. Biochemical Society, 393, 2006, Zhang, Y., Scarpace, P. J. The role of leptin in leptin resistance and obesity, Physiology & Behavior, 88, 2006, Park, H-K., Ahima, R. S., Leptin Signaling, F1000Prime Reports, 6, 73, 2014, 1-8.

35 Make sure to hit: -What is Leptin? Definition -Structure: -What things produce leptin? -White Adipose + Others in small amounts -Discern white & brown adipose -Brown has mitochondria, White is energy storage (so it secretes leptin) -Bound leptin v. free leptin -less free leptin in fasting -More bound in individuals -Leptin similar to cytokines -Biochem stuffs -Leptin and its correlation to fat