Follistatin (often shortened to FST) was discovered decades ago as a protein in ovarian fluid that shuts down a fertility hormone. Since then, scientists have learned its bigger job: it grabs onto and neutralizes a family of signaling proteins called the TGF-beta superfamily, which includes myostatin and activin, the molecules that put the brakes on muscle growth and drive things like tissue scarring and tumor growth. Because blocking myostatin sounds like a shortcut to more muscle, follistatin has become popular in bodybuilding and research-chemical circles, sold as injectable 'Follistatin 344.' The real research base, however, is almost entirely mice, rats, and cell dishes.
How strong is the evidence?
Of the 40 papers reviewed, none are human clinical trials of follistatin given as a treatment. Most are animal studies (mice and rats), lab/cell studies, or reviews explaining the biology. A handful of human studies exist, but they only measure people's own natural follistatin levels in blood (as a marker of disease or fitness) - they don't test giving people follistatin. One human trial gave people a supplement (astaxanthin) and exercise, then measured how their natural follistatin levels changed - it did not administer follistatin itself. Because no human dosing or safety trial exists, everything below about benefits is drawn from animal and lab research, not proof in people.
Uses
What people use it for
Building or protecting muscle mass (animal research)
Animal / labResearchers have raised follistatin levels in mice and rats to try to grow muscle or stop it from wasting away, by blocking myostatin and activin, the natural molecules that cap muscle size. This has only been tested in animals so far.
Fighting cancer-related muscle wasting, or cachexia (animal research)
Animal / labIn mice with ovarian or lung tumors, delivering the follistatin gene as mRNA blocked a tumor-driving hormone called activin A, helped preserve muscle and body weight, and improved how well the mice tolerated chemotherapy.
Boosting calorie-burning fat and metabolism (animal research)
Animal / labMice engineered to make extra follistatin developed more active 'brown fat,' the type of fat that burns calories as heat instead of storing them, hinting at a possible future role in weight and metabolic health.
Bodybuilding and physique use ('Follistatin 344,' unregulated)
AnecdotalBecause of the muscle-growth rationale, unregulated injectable products labeled 'Follistatin 344' are sold online to bodybuilders. There is no human dosing, effectiveness, or safety data behind this use, it is not approved by any drug regulator, and it is banned in competitive sport.
Potential benefits
What it may help with
May help build and protect muscle
Animal / labFollistatin blocks myostatin and activin, the natural 'off switches' for muscle growth. In obese mice, removing the body's own follistatin from muscle-support cells made muscle loss worse, showing follistatin normally helps protect muscle. In rats, delivering follistatin protein after nerve injury increased the number of muscle stem cells (satellite cells), though its effect on actual muscle strength was mixed - and in one part of the study it made things slightly worse.
May help prevent muscle wasting caused by cancer (cachexia)
Animal / labIn mice with ovarian or lung cancer, follistatin delivered as mRNA blocked activin A (a hormone that drives tumor growth and muscle wasting), preserved muscle and fat mass, improved food intake and body weight, and made chemotherapy work better.
May activate calorie-burning 'brown fat'
Animal / labMice given extra follistatin developed more brown and 'beige' fat activity, the kind that burns energy as heat, which researchers think could eventually be useful for obesity and metabolic disease.
May support blood vessel health in a lung condition called pulmonary hypertension
Animal / labIn cell and mouse studies, follistatin blocked activin A's role in narrowing and stiffening lung blood vessels, and worked better than an existing blood-pressure drug (bosentan) at reversing vessel remodeling. This supports why newer activin-blocking drugs are being explored for this disease.
Studies:41924877
What to watch for
Side effects & risks
- Moderate
May worsen nerve pain
In mice with nerve injury, follistatin acted directly on pain-sensing nerve cells and made nerve pain (neuropathic pain) worse; blocking the body's own follistatin reduced pain instead. This suggests raising follistatin could aggravate chronic nerve pain conditions.
- Serious
Possible link to cancer growth and treatment resistance
Follistatin and its relative FSTL3 are elevated in several cancers (ovarian cancer, leukemia, and other solid tumors) and are linked to more aggressive disease, immune evasion, and resistance to cancer therapy in lab and animal models.
- Moderate
May disrupt blood sugar control
In mouse models of insulin resistance, follistatin made by the liver raised blood sugar and blocked insulin's effects on fat tissue; reducing it improved blood sugar control. In people, higher blood follistatin is associated with fattier, more damaged livers and metabolic disease, though this is observational, not proof that follistatin causes the problem.
- Moderate
Affects fertility hormones and pregnancy biology
Follistatin's original discovery was as a blocker of FSH, a hormone essential for ovulation and fertility. Its levels shift naturally and dramatically during pregnancy and labor, meaning artificially changing follistatin could plausibly disrupt reproductive hormone balance.
- Serious
Black-market injectable products are unregulated and often mislabeled
A lab analysis of 17 black-market products sold as 'Follistatin 344' found only 9 actually contained follistatin; others contained different, unlisted growth-promoting peptides instead. There's no quality control on these products.
Dosing
Dosing — what studies used
There is no established human dose for follistatin, because it has never been tested in a human clinical trial as a treatment. Every study that actually gave follistatin protein, follistatin gene therapy, or follistatin mRNA was done in mice or rats. The single human study in this research file only measured how people's own natural follistatin levels changed with 12 weeks of exercise and an astaxanthin supplement - it did not give anyone follistatin. Any dose or protocol you might see for 'Follistatin 344' online, including from black-market sellers, is not backed by clinical research and should be treated as unverified, not as a real medical protocol.
Muscle recovery after nerve reinnervation (rat study)
Animal studyRecombinant follistatin protein (FS-288 isoform); exact dose not reported in the available abstract
Continuous delivery · Studied after 3 and 6 months of nerve denervation · Subcutaneous, via implanted osmotic pump
Effects were mixed: smaller muscles and less force at 3 months, only a trend toward more force at 6 months, alongside more muscle stem cells. This was in rats, not people.
Raising circulating follistatin via gene therapy (mouse study)
Animal studySingle dose of an AAV viral vector carrying the FST315 gene, raising blood follistatin about 5-fold
One-time injection · Effects followed for up to 50 days · Intramuscular
Used to test whether raising follistatin could calm testicular inflammation in mice; it partially reduced but did not prevent the condition.
Blocking cancer-related muscle wasting (mouse studies)
Animal studyFollistatin-encoding mRNA packaged in lipid nanoparticles; specific mRNA dose not detailed in the available abstracts
Repeated dosing over the course of tumor treatment (exact schedule not detailed) · Length of the cancer study, alongside chemotherapy in one experiment · Injection (into the abdomen or targeted to lung tumors)
An experimental gene-therapy delivery method tested only in mice with cancer, not a human protocol.
No follistatin product is FDA-approved for any use. The World Anti-Doping Agency (WADA) bans follistatin in competitive sport under its S4 category. Lab testing of black-market 'Follistatin 344' vials found inconsistent contents, including some that contained no follistatin at all or were mixed with other unapproved peptides.
These figures describe what researchers used in studies. They are not a recommendation or a prescription.
Mechanism
How it works
Follistatin is a protein your body already makes. Its job is to grab onto and switch off a group of signaling proteins called the TGF-beta superfamily - most importantly myostatin and activin, which act like brakes that limit how big your muscles can grow, and it also blocks a fertility hormone called FSH. By soaking up these molecules, follistatin can, in theory, release the brakes on muscle growth, tell fat cells to burn more energy, and calm certain inflammation and scarring signals. The catch is that this same braking system touches many organs - the liver, ovaries, immune system, nerves, and even tumors - so turning follistatin up doesn't just build muscle in isolation; animal studies show it can also raise blood sugar, worsen nerve pain, or feed cancer growth depending on where and how it acts.
Who should avoid it
- Anyone who is pregnant or breastfeeding - follistatin naturally shifts hormone balance during pregnancy and labor, and artificially changing it is unstudied and risky.
- Anyone with a current or past cancer diagnosis - follistatin and related proteins are linked to tumor growth and treatment resistance in several cancers.
- Anyone with chronic nerve pain or neuropathy - animal data suggest follistatin can make nerve pain worse.
- Anyone considering black-market 'Follistatin 344' injections - these are unregulated, unapproved, banned in sport, and have been found to contain the wrong ingredients.
Interactions to know
- Not studied in humans, so no confirmed drug interactions exist.
- Because it works on the same activin/myostatin pathway as newer prescription drugs like sotatercept (used for pulmonary hypertension), combining follistatin with activin-pathway drugs could plausibly add up or clash - this has not been tested.
- Theoretical interaction with fertility treatments or hormone therapy, given follistatin's role in blocking FSH.
The papers that matter most
Key studies
In mice, follistatin made by the liver drove insulin resistance and high blood sugar; reducing it fixed glucose control, and in people who had bariatric surgery, falling follistatin tracked with improving blood sugar.
Inactivating hepatic follistatin alleviates hyperglycemia.
Removing the body's own follistatin from muscle-support cells made muscle loss and weakness worse in obese mice, showing follistatin normally helps protect muscle during metabolic stress.
Deletion of fibro-adipogenic progenitors-specific follistatin impairs muscle function and accelerates skeletal muscle atrophy in obese mice.
Delivering the follistatin gene as mRNA in mice with ovarian cancer preserved muscle, improved response to chemotherapy, and extended survival - an early but promising anti-wasting strategy.
Nanoparticle-Based Follistatin Messenger RNA Therapy for Reprogramming Metastatic Ovarian Cancer and Ameliorating Cancer-Associated Cachexia.
Giving rats follistatin protein after nerve injury boosted muscle stem cell counts but had mixed, sometimes negative, effects on actual muscle size and strength.
Follistatin Protein Enhances Satellite Cell Counts in Reinnervated Muscle.
Follistatin directly increased pain-nerve excitability in mice, worsening neuropathic pain after nerve injury - a clear caution sign, not just a benefit story.
Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons.
Testing of 17 black-market 'Follistatin 344' products found only 9 actually contained follistatin; it is banned by WADA and sold with no quality control.
Detection of black market follistatin 344.
Bottom line
Follistatin has a genuinely interesting biological story - it blocks the natural brakes on muscle growth and can activate calorie-burning fat in animals - but it has never been tested as a treatment in a single human being. Combined with real signs it could worsen nerve pain, disrupt blood sugar, or feed certain cancers, plus unregulated and often mislabeled black-market products, there's no responsible way to use it today outside of a research setting.
Research papers
Studies we have on file for Follistatin. Tap a title to open it on PubMed. Labels like “animal” or “human trial” are rough guides.
40 papers
Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons.
Neuropathic pain is a debilitating chronic condition that lacks effective treatment. The role of cytokine- and chemokine-mediated neuroinflammation in its pathogenesis has been well documented. Follistatin (FST) is a secreted protein known to antagonize the biological activity of cytokines in the transforming growth factor-β (TGF-β) superfamily. The involvement of FST in neuropathic pain and the underlying mechanism remain largely unknown. Here, we report that FST was up-regulated in A-fiber sensory neurons after spinal nerve ligation (SNL) in mice. Inhibition or deletion of FST alleviated neuropathic pain and reduced the nociceptive neuron hyperexcitability induced by SNL. Conversely, intrathecal or intraplantar injection of recombinant FST, or overexpression of FST in the dorsal root ganglion (DRG) neurons, induced pain hypersensitivity. Furthermore, exogenous FST increased neuronal excitability in nociceptive neurons. The biolayer interferometry (BLI) assay and coimmunoprecipitation (co-IP) demonstrated direct binding of FST to the insulin-like growth factor-1 receptor (IGF1R), and IGF1R inhibition reduced FST-induced activation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT), as well as neuronal hyperexcitability. Further co-IP analysis revealed that the N-terminal domain of FST exhibits the highest affinity for IGF1R, and blocking this interaction with a peptide derived from FST attenuated Nav1.7-mediated neuronal hyperexcitability and neuropathic pain after SNL. In addition, FST enhanced neuronal excitability in human DRG neurons through IGF1R. Collectively, our findings suggest that FST, released from A-fiber neurons, enhances Nav1.7-mediated hyperexcitability of nociceptive neurons by binding to IGF1R, making it a potential target for neuropathic pain treatment.
AAV-Sparcl1 promotes hair cell regeneration by increasing supporting cell plasticity.
Sensorineural hearing deficiency caused by hair cell damage represents a prevalent sensory deficit disorder. In mammals, age-related reduction in plasticity of inner ear supporting cells (recognized as hair cell precursors) compromises their trans-differentiation capacity, resulting in impaired spontaneous hair cell regeneration post-injury. Therapeutic reprogramming of supporting cells to functionally replace damaged hair cells has emerged as a promising strategy for sensorineural hearing loss treatment. In this study, we demonstrate that the secretory protein Sparcl1 enhances supporting cell reprogramming and hair cell regeneration in both in vitro and in vivo models. Through the adeno-associated virus (AAV)-mediated overexpression system, we successfully achieved in vivo expansion of inner ear organoids accompanied by hair cell differentiation. RNA-seq analysis revealed that Sparcl1 overexpression stimulates supporting cell proliferation via follistatin (Fst) activation and extracellular matrix (ECM) remodeling. Notably, both AAV-ie-Sparcl1 delivery and recombinant Sparcl1 protein administration effectively induced supporting cell differentiation into hair cells in vivo. Collectively, our findings establish Sparcl1 as a potent positive regulator of hair cell regeneration and elucidate mechanisms by which secretory proteins regulate supporting cell plasticity.
FSTL3 is a biomarker of poor prognosis and associated with immunotherapy resistance in ovarian cancer.
High-grade serous ovarian carcinoma (HGSOC) is associated with high mortality rates due to late-stage diagnosis and limited treatment options. We investigated the role of FSTL3 in ovarian cancer progression both as a prognostic biomarker and as a potential therapeutic target.We measured levels of follistatin (FST) and follistatin-like 3 (FSTL3) in 96 ovarian cancer patient ascites samples and found that FSTL3 overexpression was more predominant than FST and associated with poorer survival outcomes. Mice implanted with an HGSOC syngeneic cell line bearing common alterations in ovarian cancer (KRASG12 V, P53R172H, CCNE1oe, AKT2oe) had increasing levels of FST and FSTL3 in serum during tumor growth. Further alteration of this model to generate a knockout of FST (KPCA.FSTKO) and an overexpression of human FSTL3 (KPCA.FSTKO_hFSTL3) revealed that FSTL3 expression was associated with a more fibrotic tumor microenvironment, correlating with an increased abundance of cancer-associated myofibroblasts (myCAFs), and cancer cells with a more mesenchymal phenotype. Tumors overexpressing FSTL3 also had significantly less immunocyte infiltration, reduced intratumoral T-cell abundance, and increased CD8+ T cell exhaustion. FSTL3 overexpression completely abrogated tumor response to PPC treatment (Prexasertib combined with PD-1 and CTLA-4 blockade) compared to controls, suggesting that FSTL3 may be involved in immunotherapy resistance. In conclusion, this study suggests a role for FSTL3 as a prognostic marker and as therapeutic target in HGSOC, where it may play a role in promoting a mesenchymal tumor phenotype, maintaining an immunosuppressive tumor microenvironment, and driving immunotherapy resistance.
Follistatin and follistatin-like 3 in metabolic disorders.
Follistatin (FST) is a glycoprotein which main role is antagonizing activity of transforming growth factor β superfamily members. Folistatin-related proteins such as follistatin-like 3 (FSTL3) also reveal these properties. The exact function of them has still not been established, but it can be bound to the pathogenesis of metabolic disorders. So far, there were performed a few studies about their role in type 2 diabetes, obesity or gestational diabetes and even less in type 1 diabetes. The outcomes are contradictory and do not allow to draw exact conclusions. In this article we summarize the available information about connections between follistatin, as well as follistatin-like 3, and metabolic disorders. We also emphasize the strong need of performing further research to explain their exact role, especially in the pathogenesis of diabetes and obesity.
Sarcopenia--The search for emerging biomarkers.
Sarcopenia, an age-related decline in skeletal muscle mass and function, dramatically affects the life quality of elder people. In view of increasing life expectancy, sarcopenia renders a heavy burden on the health care system. However, although there is a consensus that sarcopenia is a multifactorial syndrome, its etiology, underlying mechanisms, and even definition remain poorly delineated, thus, preventing development of a precise treatment strategy. The main aim of our review is to critically analyze potential sarcopenia biomarkers in light of the molecular mechanisms of their involvement in sarcopenia pathogenesis. Normal muscle mass and function maintenance are proposed to be dependent on the dynamic balance between the positive regulators of muscle growth such as bone morphogenetic proteins (BMPs), brain-derived neurotrophic factor (BDNF), follistatin (FST) and irisin, and negative regulators including TGFβ, myostatin, activins A and B, and growth and differentiation factor-15 (GDF-15). We hypothesize that the shift in this balance to muscle growth inhibitors, along with increased expression of the C- terminal agrin fragment (CAF) associated with age-dependent neuromuscular junction (NMJ) dysfunction, as well as skeletal muscle-specific troponin T (sTnT), a key component of contractile machinery, is a main mechanism underlying sarcopenia pathogenesis. Thus, this review proposes and emphasizes that these molecules are the emerging sarcopenia biomarkers.
Deletion of fibro-adipogenic progenitors-specific follistatin impairs muscle function and accelerates skeletal muscle atrophy in obese mice.
Follistatin is a potent regulator of various TGF-β superfamily members, including myostatin (MSTN) and activin A. Previous studies have shown that follistatin is crucial in enhancing myogenesis during acute muscle injury. The mechanism by which fibro-adipogenic progenitors (FAPs)-specific follistatin influences muscle homeostasis in obese mice remains unknown. Therefore, we investigated the physiological role of follistatin in PDGFRα-positive FAPs in the regulation of muscle homeostasis and exercise in obese mice. A PDGFRα-specific follistatin knockout (follistatin KO) mouse model was generated using PDGFRα-GFP-CreERT2 (PDGFRα-GCE) and follistatinflox/flox mice. These mice were fed a 60% high-fat diet (HFD) for 20 weeks, followed by a series of analyses, including exercise tolerance test, grip strength test, glucose and insulin tolerance assays, gene expression analysis, histology, western blotting, and immunohistochemistry. We showed that follistatin KO mice had reduced expression of Fst in skeletal muscle and white adipose tissue. We also showed that follistatin KO mice exhibited decreased exercise performance and altered skeletal homeostasis during obesity. Deletion of follistatin in FAPs activated the MSTN: Activin A/SMADs signaling pathways, which negatively impacted muscle homeostasis. Furthermore, follistatin KO mice showed reduced muscle mass, increased muscle degradation, and atrophic myofibers. Mitochondrial biogenesis, oxidative phosphorylation, and fatty acid oxidation were also altered in the skeletal muscles of follistatin KO mice. Follistatin plays a protective role in mice by maintaining the metabolic health of skeletal muscles; it restores muscle function during HFD challenge, thereby reducing diet-induced obesity-related complications.
Fructose and follistatin potentiate acute MASLD during complete hepatic insulin resistance.
MASLD (metabolic-associated steatotic liver disease) and MASH (steatohepatitis) are closely associated with hepatic IR (insulin resistance) and T2D. Regardless, insulin-stimulated hepatic lipogenesis is considered essential for MASLD development, as mouse models of complete hepatic IR become diabetic without MASLD when fed high-fat diets. Challenging this notion, we found that male LDKO mice lacking hepatic insulin receptor substrates acutely developed MASLD if fed a fructose-enriched "MASH diet" (GAN) or high-fructose diet. Fructose potentiated hepatic re-esterification of abundant circulating fatty acids in LDKO mice, evidenced by excess 13C incorporation into the glycerol backbone-but not fatty acid chains-of hepatic triacylglyceride after gavage with [U13C]fructose. Suppressing adipose lipolysis in LDKO mice by inactivating hepatic Fst (Follistatin) prevented acute MASLD, whereas over-expressing Fst in wild-type mouse liver accelerated GAN-promoted MASLD/MASH. Compatibly, higher serum FST levels among Tübingen Diabetes Family Study participants clustered with increased adipose IR and greater hepatic triacylglyceride accumulation.
Circulating hormones in biopsy-proven steatotic liver disease and steatohepatitis: A Multicenter Observational Study.
The role of metabolic/inflammatory hormonal systems in metabolic dysfunction associated steatotic liver disease (MASLD) remains to be fully elucidated. To report the levels of the novel total and H-specific growth differentiation factor-15 (GDF-15) and other established hormonal systems and to describe hormonal patterns in controls and patients with MASLD and its stages. This is a multicenter study from two Gastroenterology-Hepatology Departments (Greece and Australia) and one Bariatric-Metabolic Surgery Department (Italy). Overall, n = 455 serum samples of patients with biopsy-proven MASLD (n = 374) and Controls (n = 81) were recruited. We report for the first time that total and H-specific GDF-15 levels are higher in MASLD, at-risk metabolic dysfunction associated steatohepatitis (MASH), and severe fibrosis than in Controls. In addition, follistatin-like-3 (FSTL-3), free insulin-like growth factor-1 (IGF-1), leptin, and insulin levels were higher in MASLD patients than in Controls, while adiponectin levels were lower in MASLD subjects than in Controls. Activin-A, follistatin (FST), FSTL-3, and insulin levels significantly increased in severe fibrosis compared to no/mild fibrosis, while free IGF-1 decreased. In addition, adiponectin levels were lower in subjects without fibrosis vs. any fibrosis. Moreover, GDF-15 presented a strong positive association for the likelihood of having MASLD and at-risk MASH, while in adjusted analyses, FST and adiponectin showed inverse associations. Two different patterns of at-risk MASH were revealed through unsupervised analysis (total variation explained=54%). The most frequent pattern met in our sample (34.3%) was characterized by higher levels of total and H-specific GDF-15, follistatins, and activins, as well as low adiponectin levels. The second pattern revealed was characterized by high levels of free IGF-1, insulin, and leptin, with low levels of activin-A and adiponectin. Similar patterns were also generated in the case of overall MASLD. Total and H-specific GDF-15 levels increase as MASLD severity progresses. FSTL-3, free IGF-1, leptin, and insulin are also higher, whereas adiponectin and activin-A levels are lower in the MASLD group than in Controls. Hormonal systems, including GDF-15, may not only be involved in the pathophysiology but could also prove useful for the diagnostic workup of MASLD and its stages and may potentially be of therapeutic value.
MiR-9-1 controls osteoblastic regulation of lymphopoiesis.
The highly conserved MicroRNA-9 (miR-9) family consists of three members. We discovered that miR-9-1 deletion reduced mature miR-9 expression, causing 43% of the mice to display smaller size and postweaning lethality. MiR-9-1-deficient mice with growth defects experienced severe lymphopenia, but other blood cells were unaffected. The lymphopenia wasn't due to defects in hematopoietic progenitors, as mutant bone marrow (BM) cells underwent normal lymphopoiesis after transplantation into wild-type recipients. Additionally, miR-9-1-deficient mice exhibited impaired osteoblastic bone formation, as mutant mesenchymal stem cells (MSCs) failed to differentiate into osteoblastic cells (OBs). RNA sequencing revealed reduced expression of master transcription factors for osteoblastic differentiation, Runt-related transcription factor 2 (Runx2) and Osterix (Osx), and genes related to collagen formation, extracellular matrix organization, and cell adhesion, in miR-9-1-deficient MSCs. Follistatin (Fst), an antagonist of bone morphogenetic proteins (BMPs), was found to be a direct target of miR-9-1. Its deficiency led to the up-regulation of Fst, inhibiting BMP signaling in MSCs, and reducing IL-7 and IGF-1. Thus, miR-9-1 controls osteoblastic regulation of lymphopoiesis by targeting the Fst/BMP/Smad signaling axis.
Inactivating hepatic follistatin alleviates hyperglycemia.
Unsuppressed hepatic glucose production (HGP) contributes substantially to glucose intolerance and diabetes, which can be modeled by the genetic inactivation of hepatic insulin receptor substrate 1 (Irs1) and Irs2 (LDKO mice). We previously showed that glucose intolerance in LDKO mice is resolved by hepatic inactivation of the transcription factor FoxO1 (that is, LTKO mice)-even though the liver remains insensitive to insulin. Here, we report that insulin sensitivity in the white adipose tissue of LDKO mice is also impaired but is restored in LTKO mice in conjunction with normal suppression of HGP by insulin. To establish the mechanism by which white adipose tissue insulin signaling and HGP was regulated by hepatic FoxO1, we identified putative hepatokines-including excess follistatin (Fst)-that were dysregulated in LDKO mice but normalized in LTKO mice. Knockdown of hepatic Fst in the LDKO mouse liver restored glucose tolerance, white adipose tissue insulin signaling and the suppression of HGP by insulin; however, the expression of Fst in the liver of healthy LTKO mice had the opposite effect. Of potential clinical significance, knockdown of Fst also improved glucose tolerance in high-fat-fed obese mice, and the level of serum Fst was reduced in parallel with glycated hemoglobin in obese individuals with diabetes who underwent therapeutic gastric bypass surgery. We conclude that Fst is a pathological hepatokine that might be targeted for diabetes therapy during hepatic insulin resistance.
Activin/follistatin and atherosclerosis--a review.
Activin-A, a member of the TGF-beta superfamily, has a variety of important biological functions. Concerning Møs, we demonstrated that MSR which has a key role in disposing of modified LDL is downregulated by activin-A. This leads to a decrease in binding, cell association, and degradation of Ac-LDL, resulting in the inhibition of foam cell formation. Follistatin, presumably by blocking the effect of intrinsic activin-A, upregulates MSR expression, thereby promoting Ac-LDL disposal and foam cell formation. Because both activin-A and MSR are induced during Mø differentiation, these results suggest that MSR expression is suppressed by simultaneous production of activin-A in an autocrine manner. In addition to Møs, activin-A and follistatin exert influences on SMCs and ECs. Examination of in vivo expression of activin-A and follistatin revealed that they are present in various atherosclerotic lesions, including human coronary arteries, suggesting that they are locally produced. Activin-A and follistatin are produced by Møs, SMCs, and ECs in vitro. Thus, the activin-A/follistatin system plays an important role in the development of atherosclerosis.
Regulation of ovarian function by the TGF-beta superfamily and follistatin.
The role of follistatin as an activin-binding protein has dominated the study of this molecule for the last 10 years. However, there is emerging evidence that follistatin has a role in modulating the biology of other members of the transforming growth factor beta (TGF-beta) superfamily. This review summarizes the current concepts encompassing follistatin biochemistry as well as molecules with which it is functionally associated. Moreover, the importance of the two follistatin isoforms (follistatin-288 and follistatin-315) is discussed with particular emphasis on the regulation of the ovary. In addition to activin, this review discusses the functions of other members of the TGF-beta superfamily, for example growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15), BMP-6, BMP-4 and BMP-7, in the ovary, and the potential interactions between follistatin and these growth factors. The complex network of TGF-beta superfamily growth factor members involved in the modulation of ovarian function and the interactions of follistatin with these proteins is highlighted.
The emerging role of follistatin under stresses and its implications in diseases.
Follistatin (FST), a single-chain glycosylated protein, is expressed in various tissues. The essential biological function of FST is binding and neutralizing transforming growth factor β (TGF-β) superfamily, including activin, myostatin, and bone morphogenetic protein (BMP). Emerging evidence indicates that FST also serves as a stress responsive protein, which plays a protective role under a variety of stresses. In most cases, FST performs the protective function through its neutralization of TGF-β superfamily. However, under certain circumstances, FST translocates into the nucleus to maintain cellular homeostasis independent of its extracellular antagonism activity. This review provides integrated insight into the most recent advances in understanding the role of FST under various stresses, and the clinical implications corresponding to these findings and discusses the mechanisms to be further studied.
Follistatin: a multifunctional regulatory protein.
Follistatin was first described in 1987 as a follicle-stimulating hormone inhibiting substance present in ovarian follicular fluid. We now know that this effect of follistatin is only one of its many properties in a number of reproductive and nonreproductive systems. A majority of these functions are facilitated through the affinity of follistatin for activin, where activin's effects are neutralized through its binding to follistatin. As such, the interplay between follistatin and activin represents a powerful regulatory mechanism that impinges on a variety of cellular processes within the body. In this review we focus on the biochemical characteristics of follistatin and its interaction with activin and discuss the emerging role of these proteins as potent tissue regulators in the gonad, pituitary gland, pregnancy membranes, vasculature, and liver. Consideration is also given to the larger family of proteins that contain follistatin-like modules, in particular with regard to their functional and structural implications.
Circulating follistatin in relation to energy metabolism.
Recently, substantial evidence has emerged that the liver contributes significantly to the circulating levels of follistatin and that circulating follistatin is tightly regulated by the glucagon-to-insulin ratio. Both observations are based on investigations of healthy subjects. These novel findings challenge the present view of circulating follistatin in human physiology, being that circulating follistatin is a result of spill-over from para/autocrine actions in various tissues and cells. Follistatin as a liver-derived protein under the regulation of glucagon-to-insulin ratio suggests a relation to energy metabolism. In this narrative review, we attempt to reconcile the existing findings on circulating follistatin with the novel concept that circulating follistatin is a liver-derived molecule regulated by the glucagon-to-insulin ratio. The picture emerging is that conditions associated with elevated levels of circulating follistatin have a metabolic denominator with decreased insulin sensitivity and/or hyperglucagoneimia.
Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia.
Internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3/ITD) occurs in about 30% of acute myeloid leukemia (AML) and is associated with poor response to conventional treatment and adverse outcome. Here, we reported that human FLT3/ITD expression led to axis duplication and dorsalization in about 50% of zebrafish embryos. The morphologic phenotype was accompanied by ectopic expression of a morphogen follistatin (fst) during early embryonic development. Increase in fst expression also occurred in adult FLT3/ITD-transgenic zebrafish, Flt3/ITD knock-in mice, and human FLT3/ITD AML cells. Overexpression of human FST317 and FST344 isoforms enhanced clonogenicity and leukemia engraftment in xenotransplantation model via RET, IL2RA, and CCL5 upregulation. Specific targeting of FST by shRNA, CRISPR/Cas9, or antisense oligo inhibited leukemic growth in vitro and in vivo. Importantly, serum FST positively correlated with leukemia engraftment in FLT3/ITD AML patient-derived xenograft mice and leukemia blast percentage in primary AML patients. In FLT3/ITD AML patients treated with FLT3 inhibitor quizartinib, serum FST levels correlated with clinical response. These observations supported FST as a novel therapeutic target and biomarker in FLT3/ITD AML.
Novel Roles of Follistatin/Myostatin in Transforming Growth Factor-β Signaling and Adipose Browning: Potential for Therapeutic Intervention in Obesity Related Metabolic Disorders.
Obesity is a global health problem and a major risk factor for several metabolic conditions including dyslipidemia, diabetes, insulin resistance and cardiovascular diseases. Obesity develops from chronic imbalance between energy intake and energy expenditure. Stimulation of cellular energy burning process has the potential to dissipate excess calories in the form of heat via the activation of uncoupling protein-1 (UCP1) in white and brown adipose tissues. Recent studies have shown that activation of transforming growth factor-β (TGF-β) signaling pathway significantly contributes to the development of obesity, and blockade or inhibition is reported to protect from obesity by promoting white adipose browning and increasing mitochondrial biogenesis. Identification of novel compounds that activate beige/brown adipose characteristics to burn surplus calories and reduce excess storage of fat are actively sought in the fight against obesity. In this review, we present recent developments in our understanding of key modulators of TGF-β signaling pathways including follistatin (FST) and myostatin (MST) in regulating adipose browning and brown adipose mass and activity. While MST is a key ligand for TGF-β family, FST can bind and regulate biological activity of several TGF-β superfamily members including activins, bone morphogenic proteins (BMP) and inhibins. Here, we review the literature supporting the critical roles for FST, MST and other proteins in modulating TGF-β signaling to influence beige and brown adipose characteristics. We further review the potential therapeutic utility of FST for the treatment of obesity and related metabolic disorders.
Activin A-Endothelin-1 Axis Governs Pulmonary Vascular Remodeling: Mechanistic Basis for Emerging Therapies in PAH.
Pulmonary arterial hypertension remains a life-threatening disease despite advances in vasodilator therapy. Vascular remodeling, partly driven by pulmonary artery endothelial cell dysfunction, is accompanied by vasoactive mediators imbalance such as ET-1 (endothelin-1). Although endothelin receptor antagonists alleviate vasoconstriction, they incompletely address the remodeling process. We previously reported how endothelial-derived activin A promotes vascular remodeling, leading to the clinical development of the activin signaling inhibitor sotatercept, which improves outcomes when added to endothelin receptor antagonists. As both activin A and ET-1 originate from endothelial cells and promote remodeling, we investigated whether activin A regulates ET-1 production and activity in pulmonary arterial hypertension. In vitro, we used pulmonary artery endothelial cell models of activin A overabundance alone or cocultured with pulmonary artery smooth muscle cells. Cells were treated with either the activin A inhibitor FST (follistatin), the endothelin receptor antagonist bosentan, the FST/bosentan combination, or vehicle for analysis. In vivo, we exposed wild-type or endothelial-specific INHBA (inhibin β-A)-overexpressing mice (VEcadherin-INHBA-Transgenic/VEcad-INHBA-Tg) to chronic hypoxia pulmonary hypertension model, with the addition of FST, bosentan, FST and bosentan, or vehicle treatments after the first week of hypoxia exposure. Activin A upregulated ET-1 expression via canonical SMAD2/3 (small mother against decapentaplegic family member 2/3) signaling in pulmonary artery endothelial cells. This induction, as well as ET-1-driven downstream effects-including reduced eNOS (endothelial NO synthase), pulmonary artery smooth muscle cell phenotypic switching, oxidative stress, and endothelial-to-mesenchymal transition-was reversed by FST alone or in combination with bosentan. In vivo, FST-based therapy achieved greater hemodynamic, right ventricular remodeling, and vascular structural normalization in wild-type and VEcad-INHBA-Tg mice than bosentan alone, accompanied by stronger ET-1 suppression. We identified ET-1 as a downstream effector of activin A in pulmonary arterial hypertension development, supporting activin A blockade as a strategy to inhibit ET-1-mediated vasoconstriction and remodeling. This mechanistic link provides a rationale for the rapid clinical benefits observed with sotatercept and suggests its potential role earlier in the pulmonary arterial hypertension treatment paradigm.
Follistatin Targets Distinct Pathways To Promote Brown Adipocyte Characteristics in Brown and White Adipose Tissues.
We previously demonstrated that Fst expression is highest in brown adipose tissue (BAT) and skeletal muscle, but is also present at substantial levels in epididymal and subcutaneous white adipose tissues (WATs). Fst promotes mouse brown preadipocyte differentiation and promotes browning during differentiation of mouse embryonic fibroblasts. Fst-transgenic (Fst-Tg) mice show substantial increases in circulating Fst levels and increased brown adipose mass. BAT of Fst-Tg mice had increased expression of brown adipose-associated markers including uncoupling protein 1 (UCP1), PRDM16, PGC-1α, and Glut4. WATs from Fst-Tg mice show upregulation of brown/beige adipose markers and significantly increased levels of phosphorylated p38 MAPK/ERK1/2 proteins compared with the wild-type (WT) mice. Pharmacological inhibition of pp38 MAPK/pERK1/2 pathway of recombinant mouse Fst (rFst) treated differentiating 3T3-L1 cells led to significant blockade of Fst-induced UCP1 protein expression. On the other hand, BAT from Fst-Tg mice or differentiating mouse BAT cells treated with rFst show dramatic increase in Myf5 protein levels as well as upregulation of Zic1 and Lhx8 gene expression. Myf5 levels were significantly downregulated in Fst knock-out embryos and small inhibitory RNA-mediated inhibition of Myf5 led to significant inhibition of UCP1, Lhx8, and Zic1 gene expression and significant blockade of Fst-induced induction of UCP1 protein expression in mouse BAT cells. Both interscapular BAT and WAT tissues from Fst-Tg mice display enhanced response to CL316,243 treatment and decreased expression of pSmad3 compared with the WT mice. Therefore, our results indicate that Fst promotes brown adipocyte characteristics in both WAT and BAT depots in vivo through distinct mechanisms.
Activin and follistatin in female reproduction.
Activin and follistatin were initially identified in the follicular fluid based on their effects on pituitary FSH secretion in the mid-1980s. It is now evident that activin, follistatin and activin receptors are widely expressed in many tissues where they function as autocrine/paracrine regulators of a variety of physiological processes including reproduction. The major function of follistatin is to bind to activin with high affinity and block activin binding to its receptors. Total activin A and follistatin are also found in the maternal circulation throughout pregnancy. Activin A levels are increased in abnormal pregnancies such as pre-eclampsia, fetal growth restriction and gestational hypertension. The placenta, vascular endothelial cells and activated peripheral mononuclear cells (PBMC) may all contribute to the raised levels of activin A in pre-eclampsia with unaltered follistatin in pre-eclamptic placenta, PBMCs or vascular endothelial cells suggesting the availability of 'free' activin A that could be biologically active in these cells.
Nanoparticle-Based Follistatin Messenger RNA Therapy for Reprogramming Metastatic Ovarian Cancer and Ameliorating Cancer-Associated Cachexia.
This study presents the first messenger RNA (mRNA) therapy for metastatic ovarian cancer and cachexia-induced muscle wasting based on lipid nanoparticles that deliver follistatin (FST) mRNA predominantly to cancer clusters following intraperitoneal administration. The secreted FST protein, endogenously synthesized from delivered mRNA, efficiently reduces elevated activin A levels associated with aggressive ovarian cancer and associated cachexia. By altering the cancer cell phenotype, mRNA treatment prevents malignant ascites, delays cancer progression, induces the formation of solid tumors, and preserves muscle mass in cancer-bearing mice by inhibiting negative regulators of muscle mass. Finally, mRNA therapy provides synergistic effects in combination with cisplatin, increasing the survival of mice and counteracting muscle atrophy induced by chemotherapy and cancer-associated cachexia. The treated mice develop few nonadherent tumors that are easily resected from the peritoneum. Clinically, this nanomedicine-based mRNA therapy can facilitate complete cytoreduction, target resistance, improve resilience during aggressive chemotherapy, and improve survival in advanced ovarian cancer.
Endogenous targeting lipid nanoparticles for systemic mRNA delivery to lung cancer tumors.
Systemic delivery of mRNA therapeutics to lung cancer tumors remains challenging despite recent advances in organ-targeting lipid nanoparticles (LNPs). This study introduces novel LNPs, comprising commercially available DC-cholesterol (a cationic derivative of cholesterol) and 113-O12B ionizable lipid (DC_113), which provide selective mRNA translation in healthy lungs while achieving preferential protein expression in pulmonary tumors compared to adjacent lung tissue in an orthotopic cancer model. Analysis of LNPs with diverse ionizable lipids revealed that DC_113 has an apparent pKa above 7.9, the threshold required for efficient interaction with plasma vitronectin (Vtn) and subsequent targeting of Vtn receptors expressed in lung tissue and upregulated in tumors. DC_113 loaded with follistatin (FST)-coded mRNA preferentially delivered therapeutic cargo to tumors, resulting in significant FST expression and subsequent inhibition of Activin A, a key driver of cancer progression and cachexia. This cancer-preferential mRNA delivery achieved approximately 2.5-fold greater reduction in tumor burden than liver-tropic LNPs producing systemic FST. Furthermore, it efficiently mitigated cancer cachexia by improving food intake, maintaining body weight, and preserving muscle and adipose tissues without adverse effects. These findings present a novel strategy for endogenous targeting of LNPs to lung tumors that can enhance the potential of mRNA-based interventions for pulmonary cancer.
Follistatin controls the number of murine teeth by limiting TGF-β signaling.
Supernumerary teeth are common developmental anomalies of dentition. However, the factors and mechanisms driving their formation remain largely unknown. Here, we report that conditional knockout of Fst, encoding an antagonist for the transforming growth factor β (TGF-β) signaling pathway, in both oral epithelium and mesenchyme of mice (Fst CKO ) led to supernumerary upper incisor teeth, arising from the lingual dental epithelium of the native teeth and preceded by an enlarged and split lingual cervical loop. Fst-deficiency greatly activated TGF-β signaling in developing maxillary incisor teeth, associated with increased epithelium cell proliferation. Moreover, Fst CKO teeth exhibited increased expression of Tbx1, Sp6, and Sox2, which were identified as direct targets of TGF-β/SMAD2 signaling. Finally, we show that upregulation of Tbx1 in response to Fst-deficiency was largely responsible for the formation of extra teeth in Fst CKO mice. Taken together, our investigation indicates a novel role for Fst in controlling murine tooth number by restricting TGF-β signaling.
The Reign of Follistatin in Tumors and Their Microenvironment: Implications for Drug Resistance.
Follistatin (FST) is a potent neutralizer of the transforming growth factor-β superfamily and is associated with normal cellular programs and various hallmarks of cancer, such as proliferation, migration, angiogenesis, and immune evasion. The aberrant expression of FST by solid tumors is a well-documented observation, yet how FST influences tumor progression and therapy response remains unclear. The recent surge in omics data has revealed new insights into the molecular foundation underpinning tumor heterogeneity and its microenvironment, offering novel precision medicine-based opportunities to combat cancer. In this review, we discuss these recent FST-centric studies, thereby offering an updated perspective on the protean role of FST isoforms in shaping the complex cellular ecosystem of tumors and in mediating drug resistance.
Regulation of brown adipocyte metabolism by myostatin/follistatin signaling.
Obesity develops from perturbations of cellular bioenergetics, when energy uptake exceeds energy expenditure, and represents a major risk factor for the development of type 2 diabetes, dyslipidemia, cardiovascular disease, cancer, and other conditions. Brown adipose tissue (BAT) has long been known to dissipate energy as heat and contribute to energy expenditure, but its presence and physiological role in adult human physiology has been questioned for years. Recent demonstrations of metabolically active brown fat depots in adult humans have revolutionized current therapeutic approaches for obesity-related diseases. The balance between white adipose tissue (WAT) and BAT affects the systemic energy balance and is widely believed to be the key determinant in the development of obesity and related metabolic diseases. Members of the transforming growth factor-beta (TGF-β) superfamily play an important role in regulating overall energy homeostasis by modulation of brown adipocyte characteristics. Inactivation of TGF-β/Smad3/myostatin (Mst) signaling promotes browning of white adipocytes, increases mitochondrial biogenesis and protects mice from diet-induced obesity, suggesting the need for development of a novel class of TGF-β/Mst antagonists for the treatment of obesity and related metabolic diseases. We recently described an important role of follistatin (Fst), a soluble glycoprotein that is known to bind and antagonize Mst actions, during brown fat differentiation and the regulation of cellular metabolism. Here we highlight various investigations performed using different in vitro and in vivo models to support the contention that targeting TGF-β/Mst signaling enhances brown adipocyte functions and regulates energy balance, reducing insulin resistance, and curbing the development of obesity and diabetes.
Clinical and Therapeutic Implications of Follistatin in Solid Tumours.
Follistatin (FST), as a single-chain glycosylated protein, has two major isoforms, FST288 and FST315. The FST315 isoform is the predominant form whilst the FST288 variant accounts for less than 5% of the encoded mRNA. FST is differentially expressed in human tissues and aberrant expression has been observed in a variety of solid tumours, including gonadal, gastric and lung cancer, hepatocellular carcinoma, basal cell carcinoma and melanoma. Based on the current evidence, FST is an antagonist of transforming growth factor beta family members, such as activin and bone morphogenetic proteins (BMPs). FST plays a role in tumourigenesis, metastasis and angiogenesis of solid tumours through its interaction with activin and BMPs, thus resulting in pathophysiological function. In terms of diagnosis, prognosis and therapy, FST has shown strong promise. Through a better understanding of its biological functions, potential clinical applications may yet emerge.
Follistatin (FST) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development.
Follistatin (FST), a member of the transforming growth factor-β (TGF-β) superfamily, has been identified as an inhibitor of follicle-stimulating hormone. Previous studies showed that it plays an important role in animal reproduction. Therefore, this study aims to investigate its effect on the maturation of buffalo oocytes in vitro, and the underlying mechanism of FST affecting oocyte maturation was also explored in buffalo cumulus cells. Results showed that FST was enriched in the ovary and expressed at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. The FST expression level was up-regulated in MII buffalo oocytes compared with the GV stage (p < .05). To study the effects of FST on buffalo oocytes' maturation and early embryonic development, we added the pcD3.1 skeleton vector and PCD3.1-EGFP-FST vector into the maturation fluid of buffalo oocytes, respectively. It was demonstrated that FST promoted the in vitro maturation rate of buffalo oocytes and the blastocyst rate of embryos cultured in vitro (p < .05). By interfering with FST expression, we discovered that FST in cumulus cells plays a crucial role in oocyte maturation. Interference with the FST expression during the buffalo oocyte maturation did not affect the first polar body rate of buffalo oocyte (p > .05). In contrast, the location of mitochondria in oocytes was abnormal, and the cumulus expansion area was reduced (p < .05). After parthenogenetic activation, the cleavage and blastocyst rates of the FST-interfered group were reduced (p < .05). Furthermore, RT-qPCR was performed to investigate further the underlying mechanism by which FST enhances oocyte maturation. We found that overexpression of FST could up-regulate the expression level of apoptosis suppressor gene Bcl-2 and TGF-β/SMAD pathway-related genes TGF-β, SMAD2, and SMAD3 (p < .05). In contrast, the expression levels of SMAD4 and pro-apoptotic gene BAX were significantly decreased (p < .05). The FST gene could affect buffalo oocyte maturation by regulating the oocyte mitochondria integrity, the cumulus expansion, cumulus cell apoptosis, and the expression levels of TGF-β/SMAD pathway-related genes.
Spermatogenesis does not require the local production of follistatin.
It has been proposed that follistatin can modulate the actions of activins and/or other members of the transforming growth factor-beta superfamily of proteins on testicular function, since mice overexpressing follistatin showed spermatogenic disruption. However, since mice with targeted disruption of the follistatin gene die soon after birth, it is not feasible to determine the effect of the absence of follistatin on testicular function using this model. To further understand the role of follistatin on the development and maintenance of spermatogenesis, fetal testes, collected by Caesarean section at day 18 of gestation from follistatin null mice, were transplanted to the external ear of castrated recombination activating gene 1 immunocompromised male mice. The testicular grafts were then analysed 7-8 weeks after transplantation and showed that full spermatogenesis developed in both the testes of wild-type and follistatin null mice. This study indicates that, if follistatin is required to modulate spermatogenic development, it is not supplied by local testicular production but by circulating follistatin from the host mouse.
Induction of experimental autoimmune orchitis in mice: responses to elevated circulating levels of the activin-binding protein, follistatin.
Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in the regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild-to-severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood-testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels were not sufficient to prevent EAO development.
PACAP, an autocrine/paracrine regulator of gonadotrophs.
Hypothalamic-hypophysiotropic peptides are the proximate regulators of pituitary cells, but they cannot fully account for the complex functioning of these cells. Accordingly, awareness is growing that an array of peptides produced in the pituitary exert paracrine/autocrine functions. One such peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), was originally identified as a hypothalamic activator of cAMP production in pituitary cells. Gonadotrophs and folliculostellate cells are the main source of pituitary PACAP, and each pituitary cell type expresses a PACAP receptor. PACAP increases alpha-subunit (Cga) and Lhb mRNAs, and it stimulates the transcription of follistatin (Fst) that, in turn, restrains activin signaling to repress Fshb and gonadotropin-releasing hormone-receptor (Gnrhr) expression as well as other activin-responsive genes. The PACAP (Adcyap1) promoter is activated by cAMP, and pituitary cells may communicate by a feed-forward, cAMP-dependent mechanism to maintain a high level of PACAP in the fetal pituitary. At birth, pituitary PACAP declines and pituitary follistatin levels decrease, which together with increased gonadotropin-releasing hormone secretion allow Gnrhr and Fshb to increase and facilitate activation of the newborn gonads. Changes in Adcyap1 expression levels in the adult pituitary may contribute to the selective rise in follicle-stimulating hormone (FSH) from age 20-30 days to the midcycle surge and to the secondary increase in FSH that occurs before estrus. These results provide further support for the notion that PACAP is a key player in reproduction through its actions as a pituitary autocrine/paracrine hormone.
Activin A and follistatin are dynamically regulated during human pregnancy.
Activin A (beta A-beta A) and activin B (beta B-beta B) are related dimeric proteins that regulate numerous cellular activities. Activin activity is bioneutralized by follistatin, a specific and high-affinity binding protein. Recently, our group developed specific and sensitive enzyme-linked immunosorbent activin assays that do not detect either activin isoform when bound to follistatin, therefore, the assays are specific for biologically relevant ligands. Activin A is measurable in the serum of pregnant women (cross-sectional sample collection), while activin B is not detected in maternal serum. However, activin B is measurable in amniotic fluid and cord blood sera. The purpose of this study was to measure serum activin A, activin B, and follistatin prospectively in longitudinally collected samples during pregnancy. This study design offered observations of relative changes in serum hormone concentration with each person serving as an internal reference. Serum samples were collected bimonthly from seven pregnant women beginning within the second month of gestation, and up to, but not including, the onset of labor. Six of the seven women had normal labor and delivery. One patient required pitocin (an oxytocin agonist) for induction of labor which led to delivery. Activin A, activin B, total follistatin, free follistatin, human chorionic gonadotropin, estradiol, progesterone, FSH, and LH were measured in maternal serum samples using specific assays. Serum activin A levels increased in the final month of pregnancy in the six patients who delivered following normal labor (< 0.78 ng/ml (first trimester) to 1-6 ng/ml (term)). Activin B was not detected in any serum sample (< 0.78 pg/ml). Total serum follistatin (free follistatin, follistatin-activin, and follistatin-inhibin) increased 10- to 45-fold in the final month of pregnancy in four of the women undergoing normal labor (10 ng/ml (first trimester) to 100-450 ng/ml (final month)). Total follistatin was high and variable in two women throughout pregnancy. Total follistatin returned to basal serum concentration in three of the patients during the last 2 weeks of pregnancy. Free follistatin was detected throughout pregnancy (range < 2-35 ng/ml). Free follistatin represented a small percentage of the total follistatin throughout the time of pregnancy and did not rise coincident with the rise in total follistatin. Serum activin A and activin B were not detected during the entire course of pregnancy in the one patient who did not have normal labor and total follistatin did not rise in the last trimester of pregnancy. Gonadotropin and steroid hormones were measured in all patients and were within normative ranges for human pregnancy (inclusive of the non-laboring patient). The results suggest that immunodetectable activin A is present in the third trimester of pregnant women who have normal onset labor. The total follistatin assay results suggest that follistatin-activin (or -inhibin) complexes are upregulated during the third trimester of pregnancy. Importantly, activin A production exceeds the binding capacity of circulating follistatin. Because binding protein free activin A is biologically active we conclude that the activin A detected in late pregnancy is biologically relevant. The findings are consistent with our hypothesis that activin A is an endocrine factor during the last trimester of human pregnancy and may be involved in normal labor.
Follistatin serum concentrations during full-term labour in women--significant differences between spontaneous and induced labour.
Follistatin has been isolated from human placenta and has been identified in human foetal membranes and fluids. Serum follistatin levels in women rise during pregnancy particularly near term. In this study, we examined the effect of induction and stage of labour on maternal plasma concentrations of follistatin. Women who gave birth after a normal pregnancy were retrospectively divided into three groups: those who went in labour spontaneously (n = 33), needed induction by amniotomy and IV oxytocin (n = 18) or underwent planned caesarean section (n = 10). Serum was collected at 38-40 weeks of gestation, periodically through labour with a vaginal examination and once within 36 h postpartum and assayed for oestradiol, progesterone, prolactin and C-reactive protein. Follistatin was measured using a rabbit antiserum (#204) raised against purified 35 kDa bovine follistatin. Human recombinant follistatin was used as both standard and tracer. Concentrations of follistatin at 38-40 weeks of gestation were significantly different between groups. Those who had a spontaneous labour had concentrations higher than those who were induced. Similarly, those who were induced had concentrations higher than those who underwent a caesarean. In the spontaneous group, follistatin rose during labour, peaking at 57.9 +/- 5.48 ng/ml at > 3 cm of cervical dilation, and after delivery follistatin decreased to 26.16 +/- 3.4 ng/ml at 24 h post-delivery. In induced patients follistatin continued increasing to peak following delivery at 26.9 +/- 3.0 ng/ml and decreased at > 3 h post-delivery. Follistatin concentrations in caesarean section patients at 24 h post-surgery (18.53 +/- 3.74 ng/ml) were not different from that before the surgery and were comparable with the other two groups. Follistatin is clearly implicated in the onset of labour; however, further studies with a larger cohort of women are necessary to determine the nature of its role.
The testis is not the major source of circulating follistatin in the ram.
The aims of this study were to determine the plasma concentrations of follistatin in rams and to assess if the testis contributes to circulating follistatin and if there is uptake or production of follistatin by the head in rams. Catheters were inserted in the carotid artery, jugular vein and spermatic vein of intact rams during the non-breeding season (experiment 1; n = 5) and breeding season (experiment 2; n = 4). In experiment 1, blood samples were collected from 5 rams every 10 min for 4 h, commencing 20-60 min after surgery. After 2 h of sampling 1 microgram gonadotrophin-releasing hormone (GnRH) was injected intravenously. In experiment 2, blood samples were collected from 4 of the rams used in experiment 1 by venipuncture 30 and 15 min before surgery and every 15 min throughout surgery. Commencing 1 h after surgery, matched samples were taken from each of the vessels every 10 min for 4 h (1-4 h after surgery), then every hour for 20 h (4-24 h after surgery) and then every 10 min for 4 h (24-28 h after surgery). In both experiments, follistatin secretion was non-pulsatile and there were no significant differences between the concentrations of follistatin in any of the vessels. There was a significant (P < 0.05) increase in the concentrations of follistatin in each of the vessels throughout the 4 h of 10-min sampling in both experiments. In experiment 2 plasma concentrations of follistatin in the jugular vein were significantly (P < 0.05) lower before surgery than at other stages of the experiment. During the non-breeding season (experiment 1) the concentrations of follistatin in all vessels were about 2-fold higher (P < 0.001) than during the breeding season (experiment 2). Concentrations of follistatin were measured in the testicular tissue of the ram, bull, monkey and rat and were found to be 13.6, 2.1, 2.5, 0.8 ng/g testis respectively. In experiment 3, blood samples were collected every 15 min for 4 h from castrated rams (n = 6) in the absence of treatment with testosterone propionate (TP) and after 7 days of treatment with a physiological dose of TP during the breeding and non-breeding seasons. There was no effect of stage of breeding season or TP on the plasma concentrations of follistatin and these concentrations in the castrated rams were similar to the concentrations in the intact rams in experiment 2. In experiment 4, the function of Leydig cells was stimulated by administration of human chorionic gonadotrophin but this had no effect on plasma concentrations of follistatin. These experiments show that the concentrations of follistatin in the plasma of rams are measurable, that the testis is not the major contributor to circulating follistatin and that there is no significant uptake or production of follistatin by the head in rams. It appears that the contribution of the testis to circulating follistatin may vary with the stage of the breeding season, being greater during the non-breeding season than the breeding season. The gonadotrophins and testosterone do not appear to have a direct effect on the secretion of follistatin in rams. The increase in concentrations of circulating follistatin during surgery and more frequent blood sampling suggest a stress-related effect on the production of follistatin.
Exploring the Impact of Astaxanthin Supplementation in Conjunction with a 12-Week CrossFit Training Regimen on Selected Adipo-Myokines Levels in Obese Males.
Obesity is associated with an exacerbated metabolic condition that is mediated through impairing balance in the secretion of some adipo-myokines. Therefore, the objective of the present study was to explore the impact of astaxanthin supplementation in conjunction with a 12-week CrossFit training regimen on some selected adipo-myokines, insulin insensitivity, and serum lipid levels in obese males. This study is a randomized control trial design; 60 obese males were randomly divided into four groups of 15, including the control group (CG), supplement group (SG), training group (TG), and combined training and supplement group (TSG). The participants were subjected to 12 weeks of astaxanthin (AST) supplementation [20 mg/d capsule, once/d] or CrossFit training or a combination of both interventions. The training regimen comprised 36 sessions of CrossFit, each lasting 60 min, conducted three times per week. The metabolic indices, body composition, anthropometrical, cardio-respiratory, and also some plasma adipo-myokine factors, including decorin (DCN), activin A, myostatin (MST), transforming growth factor (TGF)-β1, and follistatin (FST), were examined 12 and 72 h before the initiation of the main interventional protocols, and then 72 h after the final session of the training protocol. There was no significant difference in the baseline data between the groups (p > 0.05). There were significant interactions between group x time for DCN (η2 = 0.82), activin A (η2 = 0.50), FST (η2 = 0.92), MST (η2 = 0.75), and TGFB-1 (η2 = 0.67) (p < 0.001 for all the variables). Significantly changes showed for DCN in TSG compared to TG and SG and also TG compared to SG (p = 0.0001); for activin A in SG compared to TG (p = 0.01) and TSG (p = 0.002); for FST in SG compared to TG and TSG (p = 0.0001), also in TSG compared to TG (p = 0.0001); for MST in SG, TG, and TSG compared to CG (p = 0.0001) and also in TSG compared to SG (p = 0.0001) and TG (p = 0.001); for TGFB-1 in SG, TG, and TSG compared to CG (p = 0.0001) and also TSG compared to SG (p = 0.0001) and TG (p = 0.001). The 12-week CrossFit training concurrent with AST supplementation reduced anthropometric and metabolic factors and also serum lipid levels while producing positive changes in body composition and cardiovascular factors. Increased FST and DCN and reduced activin A, MST, and TGF-β1 were other affirmative responses to both interventions.
Follistatin Protein Enhances Satellite Cell Counts in Reinnervated Muscle.
Background Muscle recovery following peripheral nerve repair is sup-optimal. Follistatin (FST), a potent muscle stimulant, enhances muscle size and satellite cell counts following reinnervation when administered as recombinant FST DNA via viral vectors. Local administration of recombinant FST protein, if effective, would be more clinically translatable but has yet to be investigated following muscle reinnervation. Objective  The aim of this study is to assess the effect of direct delivery of recombinant FST protein on muscle recovery following muscle reinnervation. Materials and Methods  In total, 72 Sprague-Dawley rats underwent temporary (3 or 6 months) denervation or sham denervation. After reinnervation, rats received FST protein (isoform FS-288) or sham treatment via a subcutaneous osmotic pump delivery system. Outcome measures included muscle force, muscle histomorphology, and FST protein quantification. Results  Follistatin treatment resulted in smaller muscles after 3 months denervation ( p  = 0.019) and reduced force after 3 months sham denervation ( p  < 0.001). Conversely, after 6 months of denervation, FST treatment trended toward increased force output ( p  = 0.066). Follistatin increased satellite cell counts after denervation ( p  < 0.001) but reduced satellite cell counts after sham denervation ( p  = 0.037). Conclusion  Follistatin had mixed effects on muscle weight and force. Direct FST protein delivery enhanced satellite cell counts following reinnervation. The positive effect on the satellite cell population is intriguing and warrants further investigation.
Detection of black market follistatin 344.
Follistatin, a myostatin-inhibiting protein, is prohibited according to chapter S4 of the "WADA 2019 List of Prohibited Substances and Methods". While currently no approved pharmaceutical formulations of follistatin are available, follistatin can be bought on the black market. Most of the products are labeled "follistatin 344" (FS344), a few "follistatin 315". A study on FS344 black market products was performed and an electrophoretic detection method for serum and urine developed. While only nine of the 17 tested products actually contained follistatin, in some of the others growth promoting peptides were found (e.g. MGF, GHRP-2). Surprisingly, all nine products contained His-tagged FS344 and a high degree of its oligomers. The detection method is based on immunomagnetic purification followed by SDS-PAGE and Western blotting with a monoclonal anti-His antibody. Alternatively, a monoclonal anti-follistatin antibody can be used. For immunoprecipitation (IP), a polyclonal anti-follistatin antibody is applied. An evaluation of suitable antibodies for IP and immunoblotting is also presented. Furthermore, practically all currently available follistatin standards were investigated. The detection limit of the method for black market FS344 in urine is ca 0.1 ng/mL for 10 mL. For a sample volume of 100 μL, an LOD of 5 ng/mL could be achieved for serum. Due to the presence of His-tags an unambiguous differentiation from endogenous follistatin is possible.
Activin A and follistatin during the oestrous cycle and early pregnancy in ewes.
The activin pathway has been postulated to be involved in regulation of multiple reproductive processes important for survival of the conceptus. These processes include luteinisation of the follicular cells and thus function of the corpus luteum, early embryo development and uterine function including implantation of the conceptus. Therefore, the aim of the current study was to determine whether the concentrations of activin A and follistatin (FST), an activin-binding protein, differed between ewes with a lifetime history of enhanced or reduced embryonic survival (ES). The mRNAs encoding FST and activin A (inhibin beta A subunit; INHBA) were present in the uterus and abundant in the uterine luminal or glandular epithelia by day 18 of gestation. A peak of activin A was observed in the systemic circulation around the time of oestrus, and activin A concentrations were elevated in animals with reduced ES during the oestrous cycle and early gestation. Concentrations of activin A in uterine fluid were approximately twofold greater on day 16 of gestation in ewes with reduced ES compared to those with enhanced ES. No consistent differences in FST were observed between these groups. Treatment of luteinising ovine granulosa cells with activin A in vitro suppressed progesterone secretion providing evidence of a potential pathway whereby increased concentrations of activin A may decrease ES.
Comparison of bioactivities, binding properties and intrafollicular levels of bovine follistatins.
Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, and 41  kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin and heparan sulphate proteoglycan (HSP) binding properties and biopotencies in the neutralisation of activin A action in vitro revealed that all five isoforms bound activin A, but they did so with different affinities. Only the 31  kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency, and the 35 and 41  kDa isoforms were the least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analysed bFF from dominant follicles (DFs) and subordinate follicles (SF) collected at strategic times during a synchronised oestrous cycle. Total FST, activin A and activin AB were measured by immunoassay, whereas individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with oestrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin A (r=-0.34), activin AB (r=-0.80) and 'total' FST (r=-0.70) levels. Follicle diameter was positively correlated with the abundance of the 41  kDa isoform (r=0.59) but negatively correlated with the abundance of the 33 and 31  kDa isoforms (r=-0.56 and r=-0.41 respectively). Both follicle statuses (DF and SF) and cycle stage affected total FST, activin A and activin B levels, whereas follicle status, but not cycle stage, affected the abundance of the 41, 37, 33 and 31  kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms, which differ in their ability to bind and neutralise activins and to associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may play an important negative role, either directly or via the inhibition of the positive effects of activins, on follicle growth and function during follicular waves.
Evidence for the existence of a local activin follistatin negative feedback loop in the goldfish pituitary and its regulation by activin and gonadal steroids.
Activin is an important regulator of gonadotropin expression and production in the vertebrate pituitary, and its activity is fine-tuned by its binding protein follistatin. In the present study, a full-length cDNA for follistatin was cloned in the goldfish, which shows 74% amino acid sequence identity with that of mammals. Recombinant goldfish follistatin expressed in the Chinese hamster ovary cells significantly blocked activin-induced F5-5 cell differentiation. Goldfish follistatin is expressed in a wide range of tissues including the brain, pituitary, ovary, and testis. The expression of follistatin mRNA in the pituitary is regulated by both activin and gonadal steroids in vitro. Treatment with goldfish activin B for 48 h significantly up-regulated follistatin expression in cultured pituitary cells, suggesting a closed activin-follistatin feedback loop in the pituitary. In agreement with this, both human and goldfish follistatin down-regulated the expression of follistatin itself, probably due to the neutralization of endogenous activin. Examination of FSHbeta and LHbeta expression in the same samples supports the role of activin and follistatin in the differential regulation of FSH and LH as demonstrated previously. Since the expression level of activin beta(B) in the pituitary is rather stable both in vitro and in vivo, it is conceivable that follistatin may play a pivotal regulatory role in the intra-pituitary activin system. Both estradiol and testosterone up-regulated follistatin expression in vitro, suggesting a mediating role for follistatin in steroid feedback on pituitary hormones. These results provide clues to the potential physiological roles of activin-follistatin system in the regulation of gonadotropin biosynthesis in teleosts.
Site-specific mutagenesis of human follistatin.
Follistatin is a monomeric protein originally discovered in ovarian follicular fluid as a suppressor of pituitary follicle-stimulating hormone (FSH) secretion, and later identified as a binding protein for activin. To explore the role of the Asn-linked carbohydrate chains on the follistatin molecule in regard to the inhibition of FSH secretion and activin binding ability, site-specific mutations were introduced at either or both of the two potential Asn-linked glycosylation sites of human follistatin with 315 amino acids (hFS-315). The three types of follistatin mutants were expressed individually in Chinese hamster ovary cells. When tested for their ability to inhibit FSH secretion and to bind activin, each mutant was found to have a similar property as the non-mutated recombinant hFS-315, suggesting that glycosylation of the follistatin molecule has no effect in these functions. However, a two amino acid insertion in between the second and the third amino acid residues in hFS-315 caused the resulting compound to lose completely its inhibitory activity on FSH secretion from the pituitary as well as its binding ability to activin. This finding suggests that the amino-terminal region of the follistatin molecule is critical for both of these functions.
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