Defining the In Vivo Role of mTORC1 in Thyrocytes by Studying the TSC2 Conditional Knockout Mouse Model.

Background: The thyroid gland is susceptible to abnormal epithelial cell growth, often resulting in thyroid dysfunction. The serine-threonine protein kinase mechanistic target of rapamycin (mTOR) regulates cellular metabolism, proliferation, and growth through two different protein complexes, mTORC1 and mTORC2. The PI3K-Akt-mTORC1 pathway's overactivity is well associated with heightened aggressiveness in thyroid cancer, but recent studies indicate the involvement of mTORC2 as well. Methods: To elucidate mTORC1's role in thyrocytes, we developed a novel mouse model with mTORC1 gain of function in thyrocytes by deleting tuberous sclerosis complex 2 (TSC2), an intracellular inhibitor of mTORC1. Results: The resulting TPO-TSC2KO mice exhibited a 70-80% reduction in TSC2 levels, leading to a sixfold increase in mTORC1 activity. Thyroid glands of both male and female TPO-TSC2KO mice displayed rapid enlargement and continued growth throughout life, with larger follicles and increased colloid and epithelium areas. We observed elevated thyrocyte proliferation as indicated by Ki67 staining and elevated cyclin D3 expression in the TPO-TSC2KO mice. mTORC1 activation resulted in a progressive downregulation of key genes involved in thyroid hormone biosynthesis, including thyroglobulin (Tg), thyroid peroxidase (Tpo), and sodium-iodide symporter (Nis), while Tff1, Pax8, and Mct8 mRNA levels remained unaffected. NIS protein expression was also diminished in TPO-TSC2KO mice. Treatment with the mTORC1 inhibitor rapamycin prevented thyroid mass expansion and restored the gene expression alterations in TPO-TSC2KO mice. Although total thyroxine (T4), total triiodothyronine (T3), and TSH plasma levels were normal at 2 months of age, a slight decrease in T4 and an increase in TSH levels were observed at 6 and 12 months of age while T3 remained similar in TPO-TSC2KO compared with littermate control mice. Conclusions: Our thyrocyte-specific mouse model reveals that mTORC1 activation inhibits thyroid hormone (TH) biosynthesis, suppresses thyrocyte gene expression, and promotes growth and proliferation.

Maternal exposure to tributyltin alters the breast milk, hormonal profile, and thyroid morphology of dams and induces sex-specific changes in neonate rat offspring.

Tributyltin (TBT) is the chemical substance commonly used worldwide to prevent biofouling of vessels. Due to its ability to bioaccumulate and biomagnify, even after being banned, significant concentrations of TBT can be detected in sediment, affecting marine and human life. Although studies have shown that direct exposure to TBT alters physiological parameters in mammals, the relationship between exposure to TBT during pregnancy and lactation, considered critical windows for metabolic programming, has not been fully elucidated. Our hypothesis is that offspring whose mothers were exposed to TBT during critical stages of development may exhibit dysfunctions in endocrine-metabolic parameters. We used pregnant Wistar rats that were divided into groups and received the following treatments from gestational day 7 until the end of lactation by intragastric gavage: vehicle (ethanol 0.01%; Control), low TBT dose (100 ng/kg of body weight (bw)/day; TBT100ng) and high TBT dose (1000 ng/kg bw/day; TBT1000ng). Dams and offspring at birth and weaning (21 days old) were studied. Maternal exposure to TBT promoted dose-dependent changes in dams. The findings for adiposity, milk composition and lipid profile were more pronounced in TBT100 ng dam; however, thyroid morphology was altered in TBT1000 ng dam. Female offspring were differentially affected by the dose of exposure. At birth, females in the TBT100ng group had low body weight, lower naso-anal length (NAL), and higher plasma T4, and at weaning, females in the TBT100ng group had lower insulin and leptin levels. Females in the TBT1000ng group had lower NAL at birth and lower leptinemia and weight of white adipose tissue at weaning. Male offspring from TBT groups showed high T3 at birth, without biometric alterations at birth or weaning. Despite these findings, both sexes exhibited dose-dependent morphological changes in the thyroid gland. Thus, maternal exposure to TBT constitutes an important route of contamination for both dams and offspring.

Neonatal nicotine exposure affects adult rat hepatic pathways involved in endoplasmic reticulum stress and macroautophagy in a sex-dependent manner.

Nonalcoholic fatty liver disease (NAFLD) involves changes in hepatic pathways, as lipogenesis, oxidative stress, endoplasmic reticulum (ER) stress, and macroautophagy. Maternal nicotine exposure exclusively during lactation leads to fatty liver (steatosis) only in the adult male offspring, not in females. Therefore, our hypothesis is that neonatal exposure to nicotine sex-dependently affects the signaling pathways involved in hepatic homeostasis of the offspring, explaining the hepatic lipid accumulation phenotype only in males. For this, between postnatal days 2 and 16, Wistar rat dams were implanted with osmotic minipumps, which released nicotine (NIC; 6 mg/Kg/day) or vehicle. The livers of offspring were evaluated at postnatal day 180. Only the male offspring that had been exposed to nicotine neonatally showed increased protein expression of markers of unfolded protein response (UPR), highlighting the presence of ER stress, as well as disruption of the activation of the macroautophagy repair pathway. These animals also had increased expression of diacylglycerol O-acyltransferase 1 and 4-hydroxynonenal, suggesting increased triglyceride esterification and oxidative stress. These parameters were not altered in the female offspring that had been neonatally exposed to nicotine, however they exhibited increased phospho adenosine monophosphate-activated protein kinase pAMPK expression, possibly as a protective mechanism. Thus, the disturbance in the hepatic homeostasis by UPR, macroautophagy, and oxidative stress modifications seem to be the molecular mechanisms underlying the liver steatosis in the adult male offspring of the nicotine-programming model. This highlights the importance of maternal smoking cessation during breastfeeding to decrease the risk of NAFLD development, especially in males.

Disruption of neuromedin B receptor improves mitochondrial oxidative phosphorylation capacity in gastrocnemius muscle of female mice.

Neuromedin B (NB), a bombesin-like peptide, exerts its specific actions by binding to the neuromedin B receptor (NBR), a G protein-coupled receptor. Female NBR-knockout (NBR-KO) mice exhibit resistance to diet-induced obesity, without hyperphagia, suggesting possible increase in energy expenditure. Skeletal muscle (SM) is crucial for whole body energy homeostasis, however, the presence of NB-NBR signaling and its effects in SM are unknown. Here, we show that male and female wild type express Nmbr and Nmb mRNA in SM, with higher levels in females. Female NBR-KO gastrocnemius showed increased Myh7 mRNA level, which characterizes type I fibers (oxidative profile). Their permeabilized gastrocnemius fibers, studied by high-resolution respirometry, exhibited higher consumption of O2 coupled to ATP synthesis and unaltered uncoupled respiration. NBR-KO gastrocnemius had higher protein levels of ATP-synthase and Nduf9 mRNA, corresponding to mitochondrial complex I subunit. NBR-KO gastrocnemius exhibited slight increase in mitochondria number, increased thickness of Z line at electron microscopy, and unaltered mitochondrial dynamics markers. Therefore, in the females' gastrocnemius, a predominantly glycolytic SM, the NBR absence promotes changes that favor mitochondrial oxidative phosphorylation capacity. In addition, in L6 myocytes, NB treatment (5 µg/mL/16 h) promoted lower O2 consumption coupled to ATP synthesis, suggesting direct action at SM cells. Altogether, the study reinforces the hypothesis that inhibition of NB-NBR signaling enhances the capacity for oxidative phosphorylation of white SM, encouraging future studies to elucidate their contribution on other types of SM and whole body energy expenditure, which may lead to a new target to drug development for obesity treatment.NEW & NOTEWORTHY This study describes neuromedin B (NB) and NB receptor as new regulators of skeletal muscle mitochondrial function. The white skeletal muscle mitochondrial oxidative phosphorylation capacity was increased by NB receptor genetic disruption in female mice. These findings may contribute to the resistance to diet-induced obesity, previously found in these mice, which requires future studies. Thus, investigations are necessary to clarify if blockade of NB receptor may be an approach to develop drugs to combat obesity.

COVID-19 and thyroid function: What do we know so far?

Coronavirus disease 2019 (COVID-19) was characterized as a pandemic in March, 2020 by the World Health Organization. COVID-19 is a respiratory syndrome that can progress to acute respiratory distress syndrome, multiorgan dysfunction, and eventually death. Despite being considered a respiratory disease, it is known that other organs and systems can be affected in COVID-19, including the thyroid gland. Thyroid gland, as well as hypothalamus and pituitary, which regulate the functioning of most endocrine glands, express angiotensin-converting enzyme 2 (ACE2), the main protein that functions as a receptor to which SARS-CoV-2 binds to enter host cells. In addition, thyroid gland is extremely sensitive to changes in body homeostasis and metabolism. Immune system cells are targets for thyroid hormones and T3 and T4 modulate specific immune responses, including cell-mediated immunity, natural killer cell activity, the antiviral action of interferon (IFN) and proliferation of T- and B-lymphocytes. However, studies show that patients with controlled hypothyroidism and hyperthyroidism do not have a higher prevalence of COVID-19, nor do they have a worse prognosis when infected with the virus. On the other hand, retrospective observational studies, prospective studies, and case reports published in the last two years reported abnormal thyroid function related to acute SARS-CoV-2 infection or even several weeks after its resolution. Indeed, a variety of thyroid disorders have been documented in COVID-19 patients, including non-thyroidal illness syndrome (NTIS), subacute thyroiditis and thyrotoxicosis. In addition, thyroid disease has already been reported as a consequence of the administration of vaccines against SARS-CoV-2. Overall, the data revealed that abnormal thyroid function may occur during and in the convalescence post-COVID condition phase. Although the cellular and molecular mechanisms are not completely understood, the evidence suggests that the "cytokine storm" is an important mediator in this context. Thus, future studies are needed to better investigate the pathophysiology of thyroid dysfunction induced by COVID-19 at both molecular and clinical levels.

Metformin ameliorates body mass gain and early metabolic changes in ovariectomized rats.

Estradiol has been used to prevent metabolic diseases, bone loss and menopausal symptoms, even though it might raise the risk of cancer. Metformin is usually prescribed for type 2 diabetes mellitus and lowers food intake and body mass while improving insulin resistance and the lipid profile. Ovariectomized rats show increased body mass, insulin resistance and changes in the lipid profile. Thus, the aim of this work was to evaluate whether metformin could prevent the early metabolic dysfunction that occurs early after ovariectomy. Female Wistar rats were divided into the following groups: SHAM-operated (SHAM), ovariectomized (OVX), ovariectomized + estradiol (OVX + E2) and ovariectomized + metformin (OVX + M). Treatment with metformin diminished approximately 50% of the mass gain observed in ovariectomized animals and reduced both the serum and hepatic triglyceride levels. The hepatic levels of phosphorylated AMP-activated protein kinase (pAMPK) decreased after OVX, and the expression of the inactive form of hepatic acetyl-CoA carboxylase (ACC) was also reduced. Metformin was able to increase the levels of pAMPK in the liver of OVX animals, sustaining the balance between the inactive and total forms of ACC. Estradiol effects were similar to those of metformin but with different proportions. Our results suggest that metformin ameliorates the early alterations of metabolic parameters and rescues hepatic AMPK phosphorylation and ACC inactivation observed in ovariectomized rats.

Sexual dimorphism of liver endoplasmic reticulum stress susceptibility in prepubertal rats and the effect of sex steroid supplementation.

NEW FINDINGS: What is the central question of this study? Is there sexual dimorphism in the occurrence of hepatic endoplasmic reticulum stress? What is the main finding and its importance? The transition from prepubertal to the adult age is associated with an increase in the unfolded protein response markers in the liver of male rats, which is probably due to an increase in serum testosterone levels. ABSTRACT: Male rodents present a higher predisposition to obesity and insulin resistance than females. These disorders have been associated with endoplasmic reticulum (ER) stress. To investigate a possible sexual dimorphism in the hepatic occurrence of ER stress, we evaluated the expression of ER stress markers in the livers of male and female rats in two phases of sexual development. In the first experimental model, male and female prepubertal and adult Wistar rats were used. Adult males presented higher body mass and greater mass of the adipose tissue and liver than adult females. Prepubertal animals presented no differences in these parameters between males and females. Despite this finding, the hepatic expression levels of Bip, Ire1α and Xbp1s mRNA were lower in prepubertal males than in females, while in adult animals, they did not differ between sexes. In the second experimental model, we anticipated the sexually mature phase by daily injections of testosterone propionate for 10 days in prepubertal males or by daily injections of oestradiol benzoate for 7 days in prepubertal females. Oestradiol administration in prepubertal females did not change any of the parameters evaluated. Testosterone administration to prepubertal males led to a higher body mass and greater expression of Bip, Ire1α, Atf4 and Xbp1s in the liver. These findings suggest that the increased ER stress predisposition observed in males during puberty is due to an increase in testosterone levels, indicating that ER stress is sexually dimorphic before puberty due to the lack of testosterone in males.