(December 7, 2022) Webinar: Low-Dose Lithium Supplementation Promotes Adipose Tissue Browning and Sarco(endo)plasmic Reticulum Ca2+ ATPase Uncoupling in Muscle
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Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) uncoupling in skeletal muscle and mitochondrial uncoupling via uncoupling protein 1 (UCP1) in brown/beige adipose tissue are two primary mechanisms implicated in energy expenditure. Here, the effects of glycogen synthase kinase 3 (GSK3) inhibition via lithium chloride (LiCl) treatment on SERCA uncoupling in skeletal muscle and UCP1 expression in adipose were investigated. C2C12 and 3T3-L1 cells treated with LiCl had increased SERCA uncoupling and UCP1 protein levels, respectively, ultimately raising cellular respiration; however, this was only observed when LiCl treatment occurred throughout differentiation. In vivo, LiCl treatment (10 mg/kg/day) increased food intake in chow-fed and high-fat diet (HFD, 60% kcal) fed male mice without increasing body mass – a result attributed to elevated daily energy expenditure. In soleus muscle, the lab determined LiCl treatment promoted SERCA uncoupling via increased expression of SERCA uncouplers, sarcolipin and/or neuronatin, under chow and HFD-fed conditions. They attribute these effects to the GSK3 inhibition observed with LiCl treatment as partial muscle specific GSK3 knockdown produced similar effects. In adipose, LiCl treatment inhibited GSK3 in inguinal WAT (iWAT) but not in brown adipose tissue under chow-fed conditions, which in turn led to an increase in UCP1 in iWAT and a beiging-like effect with a multilocular phenotype. The beiging-like effect was not observed, and increase in UCP1 when mice were fed a HFD, as LiCl could not overcome the ensuing overactivation of GSK3. Nonetheless, the study establishes novel regulatory links between GSK3 and SERCA uncoupling in muscle and GSK3 and UCP1 and beiging in iWAT.
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(December 7, 2022) Webinar: Low-Dose Lithium Supplementation Promotes Adipose Tissue Browning and Sarco(endo)plasmic Reticulum Ca2+ ATPase Uncoupling in Muscle
- 1. Copyright © 2010 Pearson Education, Inc.
- 2. Copyright © 2010 Pearson Education, Inc. About me
- 3. Copyright © 2010 Pearson Education, Inc. Supervisor: Dr. Val Fajardo
- 4. Copyright © 2010 Pearson Education, Inc. Read Here!
- 5. Copyright © 2010 Pearson Education, Inc. Obesity As of 2015, 1/3 of the global population was reported overweight or obese Energy Expenditure Obesity Type 2 Diabetes Alzheimer’s Disease Cardiovascular disease Hypertension
- 6. Copyright © 2010 Pearson Education, Inc. Obesity and Energy Imbalance
- 7. Copyright © 2010 Pearson Education, Inc. Lowell, 2000 Adaptive Thermogenesis in Total Energy Expenditure 10-15%
- 8. Copyright © 2010 Pearson Education, Inc. What is Adaptive Thermogenesis? 2 locations for adaptive thermogenesis: Location #1: o Adipose • Brown & Beige Adipose Tissue • Uncoupling protein 1 : UCP-1 White Adipose tissue (WAT) •Large lipid droplets •Few mitochondria Brown Adipose tissue (BAT) •Small lipid droplets •Abundance of mitochondria •Metabolically active : UCP1 Thermogenesis
- 9. Copyright © 2010 Pearson Education, Inc. Brondani et al., 2012 UCP1 in Brown Adipose UCP1 dissipates proton gradient releasing energy as Heat
- 10. Copyright © 2010 Pearson Education, Inc. What is Adaptive Thermogenesis? 2 locations for adaptive thermogenesis: Location 2: o Skeletal Muscle • Shivering thermogenesis • Non shivering thermogenesis : SERCA
- 11. Copyright © 2010 Pearson Education, Inc. SERCA in skeletal muscle • Responsible for Muscle Relaxation • Active reuptake of Ca2+ into the SR lumen • Maintain intracellular Ca2+ homeostasis Contributes to 40- 50% of resting metabolic rate & 25% of daily energy use
- 12. Copyright © 2010 Pearson Education, Inc. SERCA Coupling Ratio • Under Optimal Conditions 2 Ca2+ transported per 1 ATP hydrolyzed • In vivo SERCA Ca2+ transport is less efficient • Lowered coupling ratio Braun et., al 2020 Presence of Uncouplers Sarcolipin (SLN) Neuronatin (NNAT)
- 13. Copyright © 2010 Pearson Education, Inc. Braun et al., 2020
- 14. Copyright © 2010 Pearson Education, Inc. Bal et al. 2012 Sarcolipin (SLN)
- 15. Copyright © 2010 Pearson Education, Inc. Editors choice : Braun et al., 2021 Neuronatin (NNAT) Braun et al., 2021
- 16. Copyright © 2010 Pearson Education, Inc. Millership et al., 2012 Neuronatin (NNAT)
- 17. Copyright © 2010 Pearson Education, Inc. How can we regulate the expression of SLN and NNAT …
- 18. Copyright © 2010 Pearson Education, Inc. GSK3 • Constitutively active • GSK3α & GSK3β • Implicated in numerous disease states • Inactivation of GSK3 • Possible Target?
- 19. Copyright © 2010 Pearson Education, Inc. o Phosphorylation Ser21 (GSK3⍺) or Ser9 (GSK3β) Beurel et al. 2015 GSK3 Inactivation
- 20. Copyright © 2010 Pearson Education, Inc. • Recently shown to be a negative regulator thermogenesis in brown adipocytes • GSK3 overexpression increased body mass in male mice Modified from Markussen et al. 2018 Pearce et al. 2004 Does GSK3 negatively regulate muscle thermogenesis via SERCA uncoupling? GSK3 in Adipose thermogenesis
- 21. Copyright © 2010 Pearson Education, Inc. oGSK3 opposes Calcineurin action Rotter,2018 GSK3 and Calcineurin
- 22. Copyright © 2010 Pearson Education, Inc. 22 Energy Expenditure Possible Mechanism in muscle?
- 23. Copyright © 2010 Pearson Education, Inc. Lithium •Known GSK3 inhibitor •Traditionally used as treatment for Bipolar Disorder
- 24. Copyright © 2010 Pearson Education, Inc. Low-dose lithium Low doses High doses Li 10mg/kg/day Toxic effects outside of therapeutic range (0.5-1.0mM) Hamstra et al. 2019
- 25. Copyright © 2010 Pearson Education, Inc. Low dose Lithium GSK3 Thermogenesis ??
- 26. Copyright © 2010 Pearson Education, Inc. Audience Poll
- 27. Copyright © 2010 Pearson Education, Inc. JAX C2C12 myotubes 3T3-L1 adipocytes 0.5mM 10 mg/kg/day
- 28. Copyright © 2010 Pearson Education, Inc. Methods cellular models: Created with BioRender.com
- 29. Copyright © 2010 Pearson Education, Inc. C2C12 Cells LiCl treatment inhibits GSK3, increases cellular respiration, and promotes SERCA uncoupling in C2C12 myocytes.
- 30. Copyright © 2010 Pearson Education, Inc. 3T3-L1 Cells LiCl treatment inhibits GSK3 and increases UCP1 and cellular respiration in 3T3-L1 adipocytes
- 31. Copyright © 2010 Pearson Education, Inc. Methods animal models:
- 32. Copyright © 2010 Pearson Education, Inc. In vivo measures chow fed LiCl supplementation does not alter body mass despite increases in food intake due increases in daily energy expenditure in male chow-fed mice
- 33. Copyright © 2010 Pearson Education, Inc. Ex vivo Soleus Muscle: chow fed LiCl supplementation inhibits GSK3 and promotes SERCA uncoupling in soleus muscles from male chow-fed mice
- 34. Copyright © 2010 Pearson Education, Inc. Ex vivo Adipose: chow fed LiCl supplementation inhibits GSK3 and promotes a beige-like phenotype in iWAT from mice fed a chow diet. iWAT BAT
- 35. Copyright © 2010 Pearson Education, Inc. Is Thermogenesis activated under High Fat Feeding?
- 36. Copyright © 2010 Pearson Education, Inc. High fat diet food intake measures LiCl supplementation does not alter body mass despite increases in food intake in male high-fat diet (HFD)–fed mice.
- 37. Copyright © 2010 Pearson Education, Inc. High Fat Diet ex vivo Soleus Muscle LiCl supplementation inhibits GSK3 and promotes SERCA uncoupling in soleus muscles from male C57BL/6J high-fat diet (HFD)–fed mice.
- 38. Copyright © 2010 Pearson Education, Inc. High Fat Diet ex vivo Adipose LiCl supplementation did not inhibit GSK3 in iWAT or BAT from mice fed a high-fat diet (HFD)
- 39. Copyright © 2010 Pearson Education, Inc. Is there a connection between GSK3 and uncoupling proteins ?
- 40. Copyright © 2010 Pearson Education, Inc. GSK3KD Soleus Muscle Muscle-specific partial GSK3 knockdown (GSK3mKD) promotes SERCA uncoupling in soleus muscles from chow-fed male mice.
- 41. Copyright © 2010 Pearson Education, Inc. Low doses of lithium inhibits GSK3, increases expression of NNAT & SLN and SERCA uncoupling in skeletal muscle and promotes a beiging effect in white adipose tissue Potential new target for offsetting Obesity
- 42. Copyright © 2010 Pearson Education, Inc. Future Directions using the Small Animal DXA Examine the effects of GSK3 inhibition on muscle and adipose thermogenesis in FEMALE mice changes in body composition relative to energy expenditure
- 43. Copyright © 2010 Pearson Education, Inc. THANK YOU Supervisor Dr. Fajardo The Fajardo Research Team Jessica Braun Ryan Baranowski Brianna Hockey Sophie Hamstra Emily Copeland Luc Wasilewicz Bianca Marcella Michael Barfoot Mia S. Geromella1,2,‡ , Chantal R. Ryan3,‡ , Jessica L. Braun1,2 , Michael S. Finch3 , Lucas A. Maddalena4 , Olivia Bagshaw4 , Briana L. Hockey1,2, Fereshteh Moradi4 , Rachel K. Fenech3 , Jisook Ryoo1,2, Daniel M. Marko3 , Roopan Dhaliwal3 , Jake Sweezey- Munroe3 , Sophie I. Hamstra1,2 , Georgina Gardner4 , Sebastian Silvera1,2, Rene Vandenboom1,2, Brian D. Roy1,2 , Jeffrey A. Stuart4 , Rebecca E. K. MacPherson3,* ,§ , and Val A. Fajardo1,2,* ,§ From the 1 Department of Kinesiology, 2 Centre for Bone and Muscle Health, 3 Department of Health Sciences, and 4 Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada JBC paper contributors
- 44. Copyright © 2010 Pearson Education, Inc. Q&A Session WWW.SCINTICA.COM INFO@SCINTICA.COM Please enter your questions in the Q&A section. Thank You!
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- So starting off with a big picture issue which is obesity Obesity and overweightness have become a growing health concern across the globe. 2015 approximately one third of the global population has been reported as overweight or obese. Obesity is a multifactorial disease where individuals with obesity have an increased risk of developing comorbidities including type 2 diabetes mellitus, alzheimers disease, cardiovascular disease, hypertension, and other related disorders so strategies that are in place in attempt to combat obesity aim to mitigate the energy imbalance behind its pathology.
- Obesity is often a result of a chronic imbalance between caloric intake and energy expenditure where caloric intake supersedes total energy expended. This results in increased adiposity and fat mass. CLICK, Cellular pathways that increase energy expenditure have become of particular interest in the effort to combat obesity in order to maintain a balance between caloric intake and energy expended. Exploiting cellular pathways contributing to energy expenditure would allow a Shift in the balance between caloric intake and energy expenditure, where energy expenditure can be further increased to outweigh caloric intake..,.. overall resulting in reduced adiposity CLICK one of these pathways is adaptive thermogenesis
- Total energy expenditure can be best described as the amount of energy we use in a day. Taken together with energy intake or food this can determine our metabolism. Total energy expenditure can be broken down into Energy expended by performing work, physical activity, obligatory energy and adaptive thermogenesis. Adaptive thermogenesis, or the regulated production of heat, is the cellular process in which during prolonged cold exposure or caloric excess, energy expenditure and heat production is increased resulting in a greater combustion of metabolic substrates including fats and carbs CLICK It is estimated that adaptive thermogenesis represents 10-15% of our total energy expenditure therefore targeting this process has potential to increase overall energy use in the context of offsetting the imbalance of energy intake vs energy out seen in obesity
- there are 2 sites for adaptive thermogenesis in the body CLICK The first site of adaptive thermogenesis is within the adipose tissue, THERMOGENESIS occurs specifically the brown and beige adipose tissue tbrough uncoupling protein 1 There are three types of adipose tissue, CLICK white with large lipid drops and little mitochondria, this is a typical storage fat , brown adipose CLICK on the other hand has numerous small droplets, not really for storing fat, plenty of mitochondria and a protein known as UCP1 that facilitates thermogenesis and makes brown fat a highly metabolically active tissue. We also see this intermediate phenotype where white adipose adopts a brown like phenotype through inductions from the environment like metabolic stressors such as cold. Beiging of white adipose tissue has become of interest in the context of increasing energy expenditure due to the amount of white adipose or storage fat we as humans possess with the potential of converting it to a more thermogenic site.
- The mitochondria found in the brown adipose cells are responsible for generating energy in the form of ATP through the electron transport chain and oxidative phosphorylation. Oxidative phosphorylation utilizes different metabolic fuels and results in the release of protons into the intermembrane space which are pumped back through the ATP synthase to generate ATP. In the presence of UCP1, CLICK these protons can enter back through UCP1 into the matrix and the energy from doing so is released as HEAT. MORE UCP1 more fuel or energy will be required to input into the electron transport chain to maintain the mitochondrial gradient and ATP synthesis CLICK …….. https://www.frontiersin.org/articles/10.3389/fendo.2020.00498/full
- The second site of adaptive thermogenesis is CLICK within the skeletal muscle which can increase energy expenditure first through shivering thermogenesis Shivering thermogenesis occurs through repeated contractions of the muscle, with every contraction there is heat generated in response Once shivering thermogenesis ceases,CLICK non shivering thermogenesis begins skeletal muscle non shivering thermogenesis which is mediated by the sarcoendoplasmic reticulum calcium atpase or SERCA.
- in the skeletal muscle, The SERCA pumps play a critical role in the excitation contraction coupling cycle by initiating muscle relaxation. SERCA facilitates muscle relaxation through the active reuptake of calcium from the cytosol back into the SR lumen. Following muscle contraction cytosolic calcium levels are high CLICK which allows for binding of 2 Calcium ions to SERCA CLICK. Calcium binding and atp hydrolysis initiates release of calcium into the SR CLICK. Since SERCA requires ATP utilization for mediating muscle relaxation and maintaining intracellular calcium levels, CLICK it is thought that SERCA contributes a significant amount of energy to resting metabolic rate as well as overall metabolism
- Under optimal conditions SERCA transports 2 calcium into the SR per 1 ATP hydrolyzed known as optimal coupling ratio . In vivo, SERCA transport is much less efficient lowering the coupling ratio. Click Think of it Like a fuel inefficient car requiring more gas per kilometers travelled, CLICK an inefficient SERCA pump with a lowered coupling ratio will require more energy to regulate calcium levels in our muscles. One of the factors contributing to this is CLICK The presence of SERCA uncouplers CLICK such as SLN and nnat can interact with the serca pump making SERCA less efficient at pumping calcium. CLICK
- SARCOLIPIN and Neuronatin are able to interact with the SERCA pumps and alter the accessibility to the calcium binding sites. CLICK When calcium trys to bind it in the presence of SLN or NNAT, it will “slip” out even though ATP hydrolysis has occurred. This in turn will LOWER SERCAs coupling ratio, meaningthere less calcium transport into the SR per ATP hydrolyzed in the presence of these uncouplers.
- Sarcolipin is a small uncoupling protein that is known to lower SERCA efficiency thereby increasing the energetic cost of calcium pumping. It has been shown that mice lacking this SLN protein are more susceptible to diet induced obesity and show greater weight gain than their wild type counterparts. Conversely, CLICK mice overexpressing SLN can attenuate weight gain when placed on a high fat diet. CLICK Further, daily energy expenditure in SLN null mice is lowered than WT suggesting SLN may play a critical role in energy expenditure weight gain through SERCA uncoupling.
- Like SLN, Neuronatin has been recently identified as a SERCA uncoupling protein in muscle by one of my fellow lab mates, JESSICA BRAUN where we can see reductions in calcium uptake SERCAs coupling ratio is reduced when increasing the amount of neuronatin relative to SERCA. This suggests that in the presence of NNAT SERCA calcium transport efficiency is reduced. NNAT is expressed in a wider variety of muscle types including soleus EDL RG and WG as well as in other tissues in comparison to SLN which is found primarily in slow oxidative fibers making it a potentially even more attractive target to increase whole body energy expenditure.
- Click Further, it has been previously shown that NNAT null mice have lower daily energy expenditure than their WT counterparts. These NNATko mice also have increased weight gain when place on a high fat diet CLICK also have recently obtained this same NNATKO colony in our lab which we did some DXA scans on just last week based off the first round of scans it looks like the KO mice have greater body weights and potentially fat mass than WT counterparts although this is speculative for now.
- We sought to examine potential upstream regulators of SERCA uncoupling in muscle in order to increase the expression of these uncouplers and therefore increase energy expenditure
- One of these potential regulators is GSK3 GSK3 is a constituitively active kinase which exists in 2 isoforms: GSK3a and GSK3b and is most well known for its role in regulating glycogen metabolism . GSK3 has been implicated in numerous disease states in a number of tissues in the body including in heart failure, bipolar disorder, cancer, diabetes etc. and now GSK3 has become of particular interest as a target in obesity. CLICK GSK3 may have a negative role in obesity and CLICK therefore its inactivation is of particular interest as a possible molecular target Gsk3 as a thermogenic regulator in brown (study) and in white adipocytes (unpublished becca) Gsk3 can also potentially have a role in skeletal thermogenesis by regulating calcineurin Calcineurin increases pgc1 , pgc1 can increase sarcolipin Li inhibits gsk3 , gsk3 inhibits calcineurin , inhibiting gsk3 removes inhibition on calcineurin which can increase sln expression li in theory could attenuate diet induced obesity and insulin resistance in part because of sln induction Why looking at nnat? Well if gsk3 is a regulator of thermogenesis why not look at nnat ? Even if we don’t really know the mechanism involved
- Inactivation of GSK3 through phosphorylation of ser21 (gsk3a) or ser9 in gsk3beta prevents substrate recognition by blocking the binding pocket and blocking its substrate recognition. GSK3 needs to bind primed or phosphorylated substrates, therefore when GSK3 itself is phosphorylated on these sites it recognizes itself as a pseudosubstrate blocking any activity. SO… GSK3 may have a negative role in obesity and diabetes therefore INACTIVATING GSK3 as a potential upstream regulator of serca uncoupling and adipose based thermogenesis presents as a unique target to increase energy expenditure Gsk3 as a thermogenic regulator in brown (study) and in white adipocytes (unpublished becca) Gsk3 can also potentially have a role in skeletal thermogenesis by regulating calcineurin Calcineurin increases pgc1 , pgc1 can increase sarcolipin Li inhibits gsk3 , gsk3 inhibits calcineurin , inhibiting gsk3 removes inhibition on calcineurin which can increase sln expression li in theory could attenuate diet induced obesity and insulin resistance in part because of sln induction Why looking at nnat? Well if gsk3 is a regulator of thermogenesis why not look at nnat ? Even if we don’t really know the mechanism involved
- GSK3 has been shown to negatively regulate the thermogenic program in brown adipocytes where it inhibits the expression of genes that are involved in adipose based thermogenesis On the other hand, CLICK reduction in GSK3 activity activates thermogenesis through increased oxygen consumption, which is a measure of energy use in brown adipocytes suggesting inactivation of GSK3 may be favourable in promoting thermogenesis in brown adipose cells CLICK It has also been demonstrated that overexpression of GSK3 in male mice resulted in increased body mass and fat mass further providing evidence for its potential role in obesity and the obese phenotype. Click While we can see the potential of GSK3's involvement in adipose thermogenesis to our knowledge it has not been determined if GSK3 is also a negative regulator of muscle thermogenesis via SERCA uncoupling. MAPK, p38 ( involved in signal cascade that activate b adregnergic stimulation such as fgf1)
- So how is it possible that GSK3 influences SERCA uncoupling? GSK3 is known to oppose calcineurin signalling. Calcineurin is a phosphtase while GSK3 is a kinase enzyme. While calcineurin promotes dephosphorylation of the transcription factor nfat permitting its nuclear entry in the cell, GSK3 promotes cytosolic accumulation of NFAT through its phosphorylation. CLICK Calcineurin and NFAT signalling has been implicated in the expression of oxidative genes including the uncoupler SLN. Active GSK3 therefore has the potential to reduce muscle based nonshivering thermogenesis by lowering SLN expression.
- WE PROPOSE that Active GSK3 therefore has the potential to reduce muscle based nonshivering thermogenesis by CLICK lowering SLN expression and CLICK SERCA uncoupling. which would lead to a reduction in energy expenditure CLICK Therefore the inhibition of GSK3 CLICK , should result in an CLICK increase in SLN expression with a subsequent CLiCK increase in SERCA uncoupling and therefore increased energy expenditure. To explore the effects of gsk3 inhibition on BOTH SERCA uncoupling and adipose thermogenesis we sought to inhibit GSK3 by using NEXT SLIDE *****
- Lithium which is a well known GSK3 inhibitor and is commonly used for the treatment of bipolar disorder. CLICK Lithium can inhibit GSK3 through phosphorylation of GSK3 on its inhibitory serine sites therefore inactivating it. CLICK Our lab has recently demonstrated that low doses of lithium are able to inhibit GSK3.
- To avoid the toxic effects associated with lithium treatment, it must be used within a narrow therapeutic range of 0.5-1 millimolar serum concentration. We have shown CLICK that lithium at a low dose of 10mg/kg/day results in CLICK a serum concentration of 0.02mM which is well below the recommended therapeutic dose.
- We wanted to examine the relationship between low dose lithium , GSK3, and thermogenesis change the plus T
- To this extent we examined the effect of low dose lithium treatment on GSK3 inhibition on adipose based and muscle thermogenesis first using a cellular model of muscle and adipose followed by an in vivo MALE mouse model and ex vivo analysis of muscle and adipose tissue
- For both muscle and adipocytes Cells were cultured with a control and treated group which received 0.5mM of LiCl CLICK In our C2C12 muscle cells, we performed respiration experiments to quantify cellular energy expenditure. We then measured Calcium uptake using an INDO-1 based assay to quantify ca uptake into the SR. a spectrofluorometric assay was to quantify SERCA ATP hydrolysis in both groups. Western blot analysis will determine SERCA1a/SERCA2a content, extent of Ser9 phosphorylation GSK3B, along with RYR, PGC1a, SLN, content in both groups. CLICK In our adipocytes, similar experiments of cellular respiration were performed and western blotting was used to identify markers of adipose based thermogenesis incuding UCP1 content
- First examining our results inC2C12 cells : Looking at figure a: We showed significant increased in inhibitory phosphorylation of GSK3 content in the lithium treated cells suggesting that lithium is inhibiting GSK3 at this dose. We also noted an increase in PGC1a content, a mitochondrial biogenesis marker with lithium treatment. CLICK CLICK we did not see any changes in other mitochondrial markers between control and lithium groups CLICK CLICK In cells we look at respiration or oxygen consumption as a measure of energy expenditure, and we do see that with lithium treatment energy expenditure was significantly increased. CLICK CLICK By inhibiting calcium release from the SR we can determine SERCA’s contribution to oxygen consumption and When looking at SERCAs contribution to energy expenditure we saw SERCA contributed a higher percentage to basal respiration in the lithium treated cells suggesting its role in increasing this energy use. CLICK CLICK Here this was met with a significant reduction in calcium uptake but no change in SERCA activity leading to significant reductions inSERCA coupling ratio which may have contributed to the increase in respiration/energy expenditure seen with litihium treatment . CLICK CLICK This increased inhibition was not however accompanied by an increase in our uncoupling proteins SLN and NNAT but rather an increase in RYR , a calcium leak protein that can increase futile calcium cycling CLICK . --- so in muscle cells GSK3 inhibition does promote increased energy expenditure through SERCA due to a lowered coupling ratio
- Now examining our results from adipocytes: We again showed that LiCl treatment increased inhibitory phosphorylation of GSK3β with 0.5 mM LiCl treatment CLICK CLICK B, when examining resting cellular respiration rates in LiCl-treated cells had increase oxygen consumption aka increased energy expenditure. CLICK CLICK C, UCP1 and PGC-1α protein content were increased in lithium treated adipocytes, suggesting a potential connection to the increase in oxygen consumption click click MITOCHONDRIAL FOOTPRINTS WERE UNCHANGED BETWEEN GROUPS CLIC CLICK Overall, in adipose we see similar inhibition of GSK3 using lithium as in skeletal muscle, along with increases in cellular respiration perhaps due to increased in UCP1
- Now we can look at some of our in vivo data : Here CLICK We took Body composition measures of lean mass, fat mass and overall body weights using the SCINTICA small animal DXA were also able to take measures of daily energy expenditure using these CLICK promethion metabolic cages which can monitor activity, oxygen consumption, food and water intake, TOGETHER with the DXA we were able to normalize these energy expenditure measures to lean and fat mass. Following the study duration soleus muscles and adipose tissue were collected and ex vivo measures similar to those described in the cellular experiments were performed in muscle with the addition of histology in adipose tissue.
- Here are some of the in vivo energy results: Under a normal chow diet, 12 weeks of LiCl supplementation did not alter body mass; however, LiCl treatment appeared to increase food consumption with a significant difference in cumulative food intake by week 12 compared to the controls. (A+B). CLICK CLICK Furthermore, using the small animal DXA, body composition analysis of fat mass showed no significant differences between LiCl and control, similarly we saw no notable changes in lean mass or muscle mass between groups (Fig. 3, D and E). That being said, The apparent increase in food consumption without an increase in body mass or percent of body fat therefore suggests an increase in energy expenditure with LiCl supplementation, CLICK CLICK WHICH WE DID OBSERVE across light, dark and daily periods when normalizing energy expenditure to both lean and fat mass in the lithium supplemented group CLICK CLICK Importantly, these changes in energy expenditure could not be explained by any differences in cage ambulation CLICK CLICK suggesting some sort of cellular mechanism was increasing energy expenditure.
- to investigate this increase in energy expenditure seen in vivo we performed these ex vivo measures on the soleus muscle CLICK Under a normal chow diet, western blot analysis showed a significant increase in inhibitory phosphor- ylation on GSK3β in soleus muscles from LiCl-treated mice versus control (Fig. 5A). Confirming that this dose did result in gsk3 inhibition CLICK CLICK When measuring rates of Ca2+ uptake and SERCA activity separately, we did not find any significant effect of LiCl treatment; however, LiCl treatment resulted in a significant reduction in the apparent coupling ratio, particu- larly at 1000 nM [Ca2+]free (Fig. 5, B–D). CLICK CLICK The promotion of SERCA uncoupling was associated with a significant increase in the uncoupler NNAT but not SLN CLICK Overall this does shed light on the potential underlying cellular mechanisms that are contributing to the increase in energy expenditure we saw in vivo.
- Taking a look at the ex vivo adipose measures lithium significantly increased GSK3 serine9 phosphorylation in iWAT however, this was not observed in BAT CLICK CLICK Additionally, Lithium treatment resulted in significant elevations of UCP1 and several other mitochondria-related proteins including PGC-1α in iWAT (FIGURE B) but not BAT (FIGURE C) CLICK CLICK Our histology revealed that GSK3 inhibition associated with LiCl supplementation leads to a significant “beiging” effect on iWAT, taking on a multilocular-like phenotype seen typical in BAT CLICK Overall it appears that in adipose GSK3 inhibition turns on the thermogenic program in White adipose tissue allowing for a beige phenotype to be seen.
- Under chow conditions we did see thermogenesis activated in the muscle and adipose , now we want to look at HFD a condition that would benefit from thermogenesis or increased energy expenditure.
- We then examined similar measures in mice fed a High fat diet (a condition that would benefit from thermogenesis) No differences in body mass observed between HFD and HFD+Li although we saw that HFD and lithum fed mice had the increased food consumption : similar to the previous slide we assumed that there would be increases in energy expenditure to account for this at the cellular level
- Sure enough when we examined the soleus muscles we saw that there was increased inhibitory phosphorylation of GSK3 along with reductions in coupling ratio and increased presence of serca uncoupling proteins
- In contrast with chow-fed mice, LiCl supplementation did not lead to any alterations in iWAT or BAT in HFD-fed mice There were no changes in gsk3 phosphorylation between groups, This suggests that LiCl supplementation could not overcome the overactivation of GSK3 in iWAT observed with high fat feeding. CLICK CLICK measuring protein content in iWAT, there were significant reductions found in several mitochondrial- Related proteins with no change in UCP1 in both HFD and HFD-Li groups compared with chow-fed mice, In BAT only increases in CS were detected. Histological analysis did not reveal any beiging effect with lithium supplementation. CLICK CLICK Overall, Under HFD conditions in adipose, lithium could not inhibit GSK3 and induce thermogenesis in iWAT or BAT.
- We also were aware that lithium could have effects outside of GSK3 inhibition that could have been activating thermogenesis. So to establish whether there was a true connection between GSK3 and uncoupling proteins we generated a muscle specific knockdown model of GSK3. CLICK to thank for that is briana hockey in our lab who put a lot of work into this colony
- Lastly, our lab has generated a GSK3 KD model where essentially the GSK3 gene is less expressed To test our findings a step further where gsk3 inhibition is lowering serca coupling ratio we examined these measures of coupling ratio in the soleus of GSK3KD mice CLICK we did examine reductions in GSK3b in our KD model which was not supsrising click click we found that gsk3KDdid not actually have any changes in calcium uptake or serca activity although when measures as apparent coupling ratio CLICK CLICK Simliar to our inhibitory findings with lithium have lowered SERCA apparent coupling ratio and we attributed this CLICK CLICK to increased expression of both SLN and NNAT uncoupling proteins
- We know that the hormone estrogen has a number of metabolic implications, and right now many studies examining some of these measures of thermogenesis have been done solely in male mice, in my phd I will be evaluating the effects of gsk3 inhibition of muscle and adipose thermogenesis in female mice to expand on this current knowledge gap. Utilizing the scintica small animal dxa for these studies will have provide us with critical information on in vivo measures of energy expenditure and body composition changes in this female model.