Low-Dose Lithium Supplementation Influences GSK3ß Activity in a Brain Region Specific Manner in C57BL6 Male Mice.

BACKGROUND: Lithium, a commonly used treatment for bipolar disorder, has been shown to have neuroprotective effects for other conditions including Alzheimer's disease via the inhibition of the enzyme glycogen synthase kinase-3 (GSK3). However, dose-dependent adverse effects of lithium are well-documented, highlighting the need to determine if low doses of lithium can reliably reduce GSK3 activity. OBJECTIVE: The purpose of this study was to evaluate the effects of a low-dose lithium supplementation on GSK3 activity in the brain of an early, diet-induced Alzheimer's disease model. METHODS: Male C57BL/6J mice were divided into either a 6-week or 12-week study. In the 6-week study, mice were fed a chow diet or a chow diet with lithium-supplemented drinking water (10 mg/kg/day) for 6 weeks. Alternatively, in the 12-week study, mice were fed a chow diet, a high-fat diet (HFD), or a HFD with lithium-supplemented drinking water for 12 weeks. Prefrontal cortex and hippocampal tissues were collected for analysis. RESULTS: Results demonstrated reduced GSK3 activity in the prefrontal cortex as early as 6 weeks of lithium supplementation, in the absence of inhibitory phosphorylation changes. Further, lithium supplementation in an obese model reduced prefrontal cortex GSK3 activity as well as improved insulin sensitivity. CONCLUSION: Collectively, these data provide evidence for low-dose lithium supplementation to inhibit GSK3 activity in the brain. Moreover, these results indicate that GSK3 activity can be inhibited despite any changes in phosphorylation. These findings contribute to an overall greater understanding of low-dose lithium's ability to influence GSK3 activity in the brain and its potential as an Alzheimer's disease prophylactic.

Beyond its Psychiatric Use: The Benefits of Low-dose Lithium Supplementation.

Lithium is most well-known for its mood-stabilizing effects in the treatment of bipolar disorder. Due to its narrow therapeutic window (0.5-1.2 mM serum concentration), there is a stigma associated with lithium treatment and the adverse effects that can occur at therapeutic doses. However, several studies have indicated that doses of lithium under the predetermined therapeutic dose used in bipolar disorder treatment may have beneficial effects not only in the brain but across the body. Currently, literature shows that low-dose lithium (≤0.5 mM) may be beneficial for cardiovascular, musculoskeletal, metabolic, and cognitive function, as well as inflammatory and antioxidant processes of the aging body. There is also some evidence of low-dose lithium exerting a similar and sometimes synergistic effect on these systems. This review summarizes these findings with a focus on low-dose lithium's potential benefits on the aging process and age-related diseases of these systems, such as cardiovascular disease, osteoporosis, sarcopenia, obesity and type 2 diabetes, Alzheimer's disease, and the chronic low-grade inflammatory state known as inflammaging. Although lithium's actions have been widely studied in the brain, the study of the potential benefits of lithium, particularly at a low dose, is still relatively novel. Therefore, this review aims to provide possible mechanistic insights for future research in this field.

Low-dose lithium supplementation promotes adipose tissue browning and sarco(endo)plasmic reticulum Ca2+ ATPase uncoupling in muscle.

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 mechanisms implicated in energy expenditure. Here, we investigated 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. 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 diet 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, we determined that LiCl treatment promoted SERCA uncoupling via increased expression of SERCA uncouplers, sarcolipin and/or neuronatin, under chow-fed and HFD-fed conditions. We 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 white adipose tissue (iWAT) but not in brown adipose tissue under chow-fed conditions, which led to an increase in UCP1 in iWAT and a beiging-like effect with a multilocular phenotype. We did not observe this beiging-like effect and increase in UCP1 in mice fed a HFD, as LiCl could not overcome the ensuing overactivation of GSK3. Nonetheless, our study establishes novel regulatory links between GSK3 and SERCA uncoupling in muscle and GSK3 and UCP1 and beiging in iWAT.

Sarco(endo)plasmic reticulum Ca2+-ATPase function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice.

The DBA/2J (D2) mdx mouse is a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. Both D2 and C57 mdx mice exhibited signs of impaired Ca2+ uptake in the gastrocnemius, diaphragm, and left ventricle; however, the level of impairment was more severe in D2 mdx mice. Reductions in maximal SERCA activity were also more prominent in the D2 mdx gastrocnemius and diaphragm when compared to those from C57 mdx mice; however, there were no differences detected in the left ventricle. Across all muscles, D2 mdx mice had the highest levels of oxidative stress as indicated by protein nitrosylation and/or nitration. In conclusion, our study shows that SERCA function is more impaired in young D2 mdx mice compared with age-matched C57 mdx mice.

Heterozygous SOD2 deletion selectively impairs SERCA function in the soleus of female mice.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) restores intracellular Ca2+ ([Ca2+ ]i ) to resting levels after muscle contraction, ultimately eliciting relaxation. SERCA pumps are highly susceptible to tyrosine (T)-nitration, impairing their ability to take up Ca2+ resulting in reduced muscle function and increased [Ca2+ ]i and cellular damage. The mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), converts superoxide radicals into less reactive H2 O2 . Heterozygous deletion of SOD2 (Sod2+/- ) in mice increases mitochondrial oxidative stress; however, the consequences of reduced SOD2 expression in skeletal and cardiac muscle, specifically the effect on SERCA pumps, has yet to be investigated. We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from 6 to 7 month-old wild-type (WT) and Sod2+/- female C57BL/6J mice. Ca2+ -dependent SERCA activity assays were performed to assess SERCA function. Western blotting was conducted to examine the protein content of SERCA, phospholamban, and sarcolipin; and immunoprecipitation experiments were done to assess SERCA2a- and SERCA1a-specific T-nitration. Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus or LV but reduced SERCA2a in the EDL compared with WT, though this was not statistically significant. Soleus muscles from Sod2+/- mice showed a significant reduction in SERCA's apparent affinity for Ca2+ when compared to WT, corresponding with significantly elevated SERCA2a T-nitration in the soleus. No effect was seen in the EDL or the LV. This is the first study to investigate the effects of SOD2 deficiency on muscle SERCA function and shows that it selectively impairs SERCA function in the soleus.

Characterizing SERCA Function in Murine Skeletal Muscles after 35-37 Days of Spaceflight.

It is well established that microgravity exposure causes significant muscle weakness and atrophy via muscle unloading. On Earth, muscle unloading leads to a disproportionate loss in muscle force and size with the loss in muscle force occurring at a faster rate. Although the exact mechanisms are unknown, a role for Ca2+ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump actively brings cytosolic Ca2+ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca2+ ([Ca2+]i). SERCA dysfunction contributes to elevations in [Ca2+]i, leading to cellular damage, and may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content, and reactive oxygen/nitrogen species (RONS) protein adduction in murine skeletal muscle after 35-37 days of spaceflight. In male and female soleus muscles, spaceflight led to drastic impairments in Ca2+ uptake despite significant increases in SERCA1a protein content. We attribute this impairment to an increase in RONS production and elevated total protein tyrosine (T) nitration and cysteine (S) nitrosylation. Contrarily, in the tibialis anterior (TA), we observed an enhancement in Ca2+ uptake, which we attribute to a shift towards a faster muscle fiber type (i.e., increased myosin heavy chain IIb and SERCA1a) without elevated total protein T-nitration and S-nitrosylation. Thus, spaceflight affects SERCA function differently between the soleus and TA.

Neuronatin regulates whole-body metabolism: is thermogenesis involved?

Neuronatin (NNAT) was originally discovered in 1995 and labeled as a brain developmental gene due to its abundant expression in developing brains. Over the past 25 years, researchers have uncovered NNAT in other tissues; notably, the hypothalamus, pancreatic ß-cells, and adipocytes. Recent evidence in these tissues indicates that NNAT plays a significant role in metabolism whereby it regulates food intake, insulin secretion, and adipocyte differentiation. Furthermore, genetic deletion of Nnat in mice lowers whole-body energy expenditure and increases susceptibility to diet-induced obesity and glucose intolerance; however, the underlying cellular mechanisms remain unknown. Based on its sequence homology with phospholamban, NNAT has a purported role in regulating the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump. However, NNAT also shares sequence homology with sarcolipin, which has the unique property of uncoupling the SERCA pump, increasing whole-body energy expenditure and thus promoting adaptive thermogenesis in states of caloric excess or cold exposure. Thus, in this article, we discuss the accumulating evidence suggestive of NNAT's role in whole-body metabolic regulation, while highlighting its potential to mediate adaptive thermogenesis via SERCA uncoupling.

The role of phospholamban and GSK3 in regulating rodent cardiac SERCA function.

Cardiac contractile function is largely mediated by the regulation of Ca2+ cycling throughout the lifespan. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is paramount to cardiac Ca2+ regulation, and it is well established that SERCA dysfunction pathologically contributes to cardiomyopathy and heart failure. Phospholamban (PLN) is a well-known inhibitor of the SERCA pump and its regulation of SERCA2a-the predominant cardiac SERCA isoform-contributes significantly to proper cardiac function. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase involved in several metabolic pathways, and we and others have shown that it regulates SERCA function. In this mini-review, we highlight the underlying mechanisms behind GSK3's regulation of SERCA function specifically discussing changes in SERCA2a and PLN expression and its potential protection against oxidative stress. Ultimately, these recent findings that we discuss could have clinical implications in the treatment and prevention of cardiomyopathies and heart failure.

GSK3 inhibition with low dose lithium supplementation augments murine muscle fatigue resistance and specific force production.

Calcineurin is a Ca2+ -dependent serine/threonine phosphatase that dephosphorylates nuclear factor of activated T cells (NFAT), allowing for NFAT entry into the nucleus. In skeletal muscle, calcineurin signaling and NFAT activation increases the expression of proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) and slow myosin heavy chain (MHC) I ultimately promoting fatigue resistance. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that antagonizes calcineurin by re-phosphorylating NFAT preventing its entry into the nucleus. Here, we tested whether GSK3 inhibition in vivo with low dose lithium chloride (LiCl) supplementation (10 mg kg-1  day-1 for 6 weeks) in male C57BL/6J mice would enhance muscle fatigue resistance in soleus and extensor digitorum longus (EDL) muscles by activating NFAT and augmenting PGC-1α and MHC I expression. LiCl treatment inhibited GSK3 by elevating Ser9 phosphorylation in soleus (+1.8-fold, p = .007) and EDL (+1.3-fold p = .04) muscles. This was associated with a significant reduction in NFAT phosphorylation (-50%, p = .04) and a significant increase in PGC-1α (+1.5-fold, p = .05) in the soleus but not the EDL. MHC isoform analyses in the soleus also revealed a 1.2-fold increase in MHC I (p = .04) with no change in MHC IIa. In turn, a significant enhancement in soleus muscle fatigue (p = .04), but not EDL (p = .26) was found with LiCl supplementation. Lastly, LiCl enhanced specific force production in both soleus (p < .0001) and EDL (p = .002) muscles. Altogether, our findings show the skleletal muscle contractile benefits of LiCl-mediated GSK3 inhibition in mice.

Low-dose lithium feeding increases the SERCA2a-to-phospholamban ratio, improving SERCA function in murine left ventricles.

NEW FINDINGS: What is the central question of this study? Inhibition of glycogen synthase kinase-3 (GSK3) has been shown to improve cardiac SERCA2a function. Lithium can inhibit GSK3, but therapeutic doses used in treating bipolar disorder can have toxic effects. It has not been determined whether subtherapeutic doses of lithium can improve cardiac SERCA function. What is the main finding and its importance? Using left ventricles from wild-type mice, we found that subtherapeutic lithium feeding for 6 weeks decreased GSK3 activity and increased cardiac SERCA function compared with control-fed mice. These findings warrant the investigation of low-dose lithium feeding in preclinical models of cardiomyopathy and heart failure to determine the therapeutic benefit of GSK3 inhibition. ABSTRACT: The sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) pump is responsible for regulating calcium (Ca2+ ) within myocytes, with SERCA2a being the dominant isoform in cardiomyocytes. Its inhibitor, phospholamban (PLN), acts by decreasing the affinity of SERCA for Ca2+ . Changes in the SERCA2a:PLN ratio can cause Ca2+ dysregulation often seen in patients with dilated cardiomyopathy and heart failure. The enzyme glycogen synthase kinase-3 (GSK3) is known to downregulate SERCA function by decreasing the SERCA2a:PLN ratio. In this study, we sought to determine whether feeding mice low-dose lithium, a natural GSK3 inhibitor, would improve left ventricular SERCA function by altering the SERCA2a:PLN ratio. To this end, male wild-type C57BL/6J mice were fed low-dose lithium via drinking water (10 mg kg-1  day-1 LiCl for 6 weeks) and left ventricles were harvested. GSK3 activity was significantly reduced in LiCl-fed versus control-fed mice. The apparent affinity of SERCA for Ca2+ was also increased (pCa50 ; control, 6.09 ± 0.03 versus LiCl, 6.26 ± 0.04, P < 0.0001) along with a 2.0-fold increase in SERCA2a:PLN ratio in LiCl-fed versus control-fed mice. These findings suggest that low-dose lithium supplementation can improve SERCA function by increasing the SERCA2a:PLN ratio. Future studies in murine preclinical models will determine whether GSK3 inhibition via low-dose lithium could be a potential therapeutic strategy for dilated cardiomyopathy and heart failure.

A Low-Therapeutic Dose of Lithium Inhibits GSK3 and Enhances Myoblast Fusion in C2C12 Cells.

Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/ß-catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3α) or Ser9 (GSK3ß) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5-1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3ß (ser9) and GSK3α (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2-2.5 fold, p < 0.05), a result associated with an increase in total ß-catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (-86%, p < 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p < 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p < 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p < 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditions.

SERCA2a tyrosine nitration coincides with impairments in maximal SERCA activity in left ventricles from tafazzin-deficient mice.

The sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) is imperative for normal cardiac function regulating both muscle relaxation and contractility. SERCA2a is the predominant isoform in cardiac muscles and is inhibited by phospholamban (PLN). Under conditions of oxidative stress, SERCA2a may also be impaired by tyrosine nitration. Tafazzin (Taz) is a mitochondrial-specific transacylase that regulates mature cardiolipin (CL) formation, and its absence leads to mitochondrial dysfunction and excessive production of reactive oxygen/nitrogen species (ROS/RNS). In the present study, we examined SERCA function, SERCA2a tyrosine nitration, and PLN expression/phosphorylation in left ventricles (LV) obtained from young (3-5 months) and old (10-12 months) wild-type (WT) and Taz knockdown (TazKD ) male mice. These mice are a mouse model for Barth syndrome, which is characterized by mitochondrial dysfunction, excessive ROS/RNS production, and dilated cardiomyopathy (DCM). Here, we show that maximal SERCA activity was impaired in both young and old TazKD LV, a result that correlated with elevated SERCA2a tyrosine nitration. In addition PLN protein was decreased, and its phosphorylation was increased in TazKD LV compared with control, which suggests that PLN may not contribute to the impairments in SERCA function. These changes in expression and phosphorylation of PLN may be an adaptive response aimed to improve SERCA function in TazKD mice. Nonetheless, we demonstrate for the first time that SERCA function is impaired in LVs obtained from young and old TazKD mice likely due to elevated ROS/RNS production. Future studies should determine whether improving SERCA function can improve cardiac contractility and pathology in TazKD mice.