RNA-seq reveals that anti-obesity irisin and triiodothyronine (T3) hormones differentially affect the purinergic signaling transcriptomics in differentiated human adipocytes.

The anti-obesity thyroid hormone, triiodothyronine (T3), and irisin, an exercise- and/or cold-induced myokine, stimulate thermogenesis and energy consumption while decreasing lipid accumulation. The involvement of ATP signaling in adipocyte cell function and obesity has attracted increasing attention, but the crosstalk between the purinergic signaling cascade and anti-obesity hormones lacks experimental evidence. In this study, we investigated the effects of T3 and irisin in the transcriptomics of membrane-bound purinoceptors, ectonucleotidase enzymes and nucleoside transporters participating in the purinergic signaling in cultured human adipocytes. The RNA-seq analysis revealed that differentiated adipocytes express high amounts of ADORA1, P2RY11, P2RY12, and P2RX6 gene transcripts, along with abundant levels of transcriptional products encoding to purine metabolizing enzymes (ENPP2, ENPP1, NT5E, ADA and ADK) and transporters (SLC29A1, SCL29A2). The transcriptomics of purinergic signaling markers changed in parallel to the upsurge of "browning" adipocyte markers, like UCP1 and P2RX5, after treatment with T3 and irisin. Upregulation of ADORA1, ADORA2A and P2RX4 gene transcription was obtained with irisin, whereas T3 preferentially upregulated NT5E, SLC29A2 and P2RY11 genes. Irisin was more powerful than T3 towards inhibition of the leptin gene transcription, the SCL29A1 gene encoding for the ENT1 transporter, the E-NPP2 (autotaxin) gene, and genes that encode for two ADP-sensitive P2Y receptors, P2RY1 and P2RY12. These findings indicate that anti-obesity irisin and T3 hormones differentially affect the purinergic signaling transcriptomics, which might point towards new directions for the treatment of obesity and related metabolic disorders that are worth to be pursued in future functional studies.

Effects of Triiodothyronine on Human Osteoblast-Like Cells: Novel Insights From a Global Transcriptome Analysis.

Background: Thyroid hormones play a significant role in bone development and maintenance, with triiodothyronine (T3) particularly being an important modulator of osteoblast differentiation, proliferation, and maintenance. However, details of the biological processes (BPs) and molecular pathways affected by T3 in osteoblasts remain unclear. Methods: To address this issue, primary cultures of human adipose-derived mesenchymal stem cells were subjected to our previously established osteoinduction protocol, and the resultant osteoblast-like cells were treated with 1 nm or 10 nm T3 for 72 h. RNA sequencing (RNA-Seq) was performed using the Illumina platform, and differentially expressed genes (DEGs) were identified from the raw data using Kallisto and DESeq2. Enrichment analysis of DEGs was performed against the Gene Ontology Consortium database for BP terms using the R package clusterProfiler and protein network analysis by STRING. Results: Approximately 16,300 genes were analyzed by RNA-Seq, with 343 DEGs regulated in the 1 nm T3 group and 467 upregulated in the 10 nm T3 group. Several independent BP terms related to bone metabolism were significantly enriched, with a number of genes shared among them (FGFR2, WNT5A, WNT3, ROR2, VEGFA, FBLN1, S1PR1, PRKCZ, TGFB3, and OSR1 for 1nM T3; and FZD1, SMAD6, NOG, NEO1, and ENG for 10 nm T3). An osteoblast-related search in the literature regarding this set of genes suggests that both T3 doses are unfavorable for osteoblast development, mainly hindering BMP and canonical and non-canonical WNT signaling. Conclusions: Therefore, this study provides new directions toward the elucidation of the mechanisms of T3 action on osteoblast metabolism, with potential future implications for the treatment of endocrine-related bone pathologies.

Pitfalls and challenges of the purinergic signaling cascade in obesity.

Obesity is a worldwide health problem which have reached pandemic proportions, now also including low and middle-income countries. Excessive or abnormal fat deposition in the abdomen especially in the visceral compartment is tightly associated with a high metabolic risk for arterial hypertension, type II diabetes, cardiovascular diseases, musculoskeletal disorders (especially articular degeneration) and some cancers. Contrariwise, accumulation of fat in the subcutaneous compartment has been associated with a neutral metabolic impact, favoring a lower risk of insulin resistance. Obesity results more often from an avoidable imbalance between food consumption and energy expenditure. There are several recommended strategies for dealing with obesity, including pharmacological therapies, but their success remains incomplete and may not compensate the associated adverse effects. Purinergic signaling operated by ATP and its metabolite, adenosine, has attracted increasing attention in obesity. The extracellular levels of purines often reflect the energy status of a given cell population. Adenine nucleotides and nucleosides fine tuning control adipogenesis and mature adipocytes function via the activation of P2 and P1 purinoceptors, respectively. These features make the purinergic signaling cascade a putative target for therapeutic intervention in obesity and related metabolic syndromes. There are, however, gaps in our knowledge regarding the role of purines in adipocyte precursors differentiation and mature adipocytes functions, as well as their impact among distinct adipose tissue deposits (e.g. white vs. brown, visceral vs. subcutaneous), which warrants further investigations before translation to clinical trials can be made.

Irisin modulates genes associated with severe coronavirus disease (COVID-19) outcome in human subcutaneous adipocytes cell culture.

Obesity patients are more susceptible to develop COVID-19 severe outcome due to the role of angiotensin-converting enzyme 2 (ACE2) in the viral infection. ACE2 is regulated in the human cells by different genes associated with increased (TLR3, HAT1, HDAC2, KDM5B, SIRT1, RAB1A, FURIN and ADAM10) or decreased (TRIB3) virus replication. RNA-seq data revealed 14857 genes expressed in human subcutaneous adipocytes, including genes mentioned above. Irisin treatment increased by 3-fold the levels of TRIB3 transcript and decreased the levels of other genes. The decrease in FURIN and ADAM10 expression enriched diverse biological processes, including extracellular structure organization. Our results, in human subcutaneous adipocytes cell culture, indicate a positive effect of irisin on the expression of multiple genes related to viral infection by SARS-CoV-2; furthermore, translatable for other tissues and organs targeted by the novel coronavirus and present, thus, promising approaches for the treatment of COVID-19 infection as therapeutic strategy to decrease ACE2 regulatory genes.

The roles of triiodothyronine and irisin in improving the lipid profile and directing the browning of human adipose subcutaneous cells.

Triiodothyronine (T3) and irisin (I) can modulate metabolic status, increase heat production, and promote differentiation of white adipose tissue (WAT) into brown adipose tissue (BAT). Herein, human subcutaneous white adipocytes were treated with 10 nM T3 or 20 nM I for 24 h to evaluate intracellular lipid accumulation, triglyceride, and glycerol levels, oxidative stress, DNA damage, and protein levels of uncoupling protein 1 (UCP1), adiponectin, leptin, peroxisome proliferator-activated receptor gamma (PPARγ), and fibronectin type III domain-containing protein 5 (FNDC5). T3 and irisin improved UCP1 production, lipid profile, oxidative stress, and DNA damage. T3 elevated adiponectin and leptin levels with a concomitant decrease in PPARy and FNDC5 levels. However, irisin did not alter adipokine, PPARy, and FNDC5 levels. The results indicate that T3 may be used to increase leptin and adiponectin levels to improve insulin sensitivity, and irisin may be used to prevent obesity or maintain weight due to its impact on the lipid profile without altering adipokine levels.

Triiodothyronine activated extranuclear pathways upregulate adiponectin and leptin in murine adipocytes.

Adiponectin and leptin, important for metabolic regulation, are synthesized and secreted by adipose tissue and are influenced by triiodothyronine (T3) that activates the MAPK/ERK and integrin αVß3 pathways, modulating gene expression. Adipocytes were treated with T3 (10 nM), for 1 h, in the absence or presence of PD98059 (PD) and tetraiodothyroacetic acid (Tetrac), which are pathways inhibitors. The cells were incubated with Adipo Red/Oil Red O reagents, and intracellular lipid accumulation [glycerol and triacylglycerol (TAG)], MTT, 8-hydroxideoxyguanosine (8-OH-dG), and mRNA and protein expression were assessed. T3 increased leptin mRNA and protein expression, and, in contrast, there was a decrease in the Tetrac + T3 group. Adiponectin mRNA expression was not altered by T3, though it had increased its protein expression, which was terminated by inhibitors PD + T3 and Tetrac + T3. However, T3 did not alter PPARγ protein expression, lipid accumulation, TAG, glycerol, and DNA damage, but PD + T3 and Tetrac + T3 reduced these parameters. T3 activated the MAPK/ERK pathway on adipocytes to modulate the adiponectin protein expression and integrin αvß3 to alter the leptin gene expression.

Systemic arterial hypertension leads to decreased semen quality and alterations in the testicular microcirculation in rats.

Arterial hypertension is a cardiovascular disease that leads to important systemic alterations and drastically impairs normal organ function over time. Hypertension affects around 700 million men of reproductive age and hypertensive men present increased risk for reproductive disorders, such as erectile dysfunction. However, the link between arterial hypertension and male reproductive disorders is associative at best. Moreover, many studies have reported associations between decreased male fertility and/or semen quality and alterations to general male health. In this study we aim to investigate the effect of systemic high blood pressure in sperm quality, sperm functional characteristics and testicular physiology in a rat model. Hypertensive rats presented altered testicular morphology - mainly vascular alterations and impaired testicular vasomotion. Hypertensive rats also presented decrease in sperm concentration, DNA integrity and increased percentages of sperm with dysfunctional mitochondria, intracellular superoxide anion activity and abnormal morphology. This study provides mechanistic insights by which arterial hypertension affects the testes, evidencing the testes as another target organ for hypertension as well as its impact on sperm quality.

The importance of estrogen for bone protection in experimental hyperthyroidism in human osteoblasts.

Triiodothyronine (T3) and estrogen (E2) play important roles in the bone remodeling process and signaling of receptor activator of the nuclear factor-kappa ß (RANKL) and osteoprotegerin (OPG) expressed by osteoblasts. However, little is known of the molecular action of these hormones in conditions of hyperthyroidism and associated E2 in human cells. AIMS: This study evaluated the effects of the physiological concentration of E2 (10 nM), alone or in association with physiological (1 nM) and supraphysiological (10 nM) concentrations of T3, on RANKL and OPG gene expression in human osteoblasts. MAIN METHODS: Alkaline phosphatase and osteocalcin assays were performed to verify the presence of mature osteoblasts. After mimicking the experimental hyperthyroidism in osteoblasts untreated or treated with E2, RANKL and OPG gene expression was analyzed by real-time PCR and protein expression by western Blot and ELISA. Alizarin Red staining analyzed the amount of bone matrix after hormonal treatments. KEY FINDINGS: E2 enhanced the gene expression of OPG when associated with 1 nM and 10 nM T3. E2 was able to restore the bone matrix after an initial decrease using 1 nM and 10 nM T3. The protective effect of E2 on the RANKL and OPG signaling pathway was demonstrated. E2 restored the bone matrix induced by experimental hyperthyroidism. SIGNIFICANCE: The data highlight the importance of E2 to maintain OPG expression and osteoblast activity against possible loss of bone mass, especially in conditions where T3 is in excess.

Disruptive Effect of Organotin on Thyroid Gland Function Might Contribute to Hypothyroidism.

A considerable increase in endocrine abnormalities has been reported over the last few decades worldwide. A growing exposure to endocrine-disrupting chemicals (EDCs) can be one of the causes of endocrine disorders in populations, and these disorders are not only restricted to the metabolic hormone system but can also cause abnormal functions. Thyroid hormone (TH) disruption is defined as an abnormal change in TH production, transport, function, or metabolism, which results in some degree of impairment in body homeostasis. Many EDCs, including organotin compounds (OTCs), are environmental contaminants that are commonly found in antifouling paints used on ships and in several other industrial procedures. OTCs are obesogenic and can disrupt TH metabolism; however, abnormalities in thyroid function resulting from OTC exposure are less well understood. OTCs, one of the most prevalent EDCs that are encountered on a daily basis, modulate the thyroid axis. In most toxicology studies, it has been reported that OTCs might contribute to hypothyroidism.

Adiponectin and Serine/Threonine Kinase Akt Modulation by Triiodothyronine and/or LY294002 in 3T3-L1 Adipocytes.

Adipose tissue (AT), an endocrine organ that modulates several physiological functions by synthesizing and releasing adipokines such as adiponectin, is a metabolic target of triiodothyronine (T3). T3 and adiponectin play important roles in controlling normal metabolic functions such as stimulation of fatty acid oxidation and increase in thermogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway is important for the differentiation of preadipocytes into adipocytes and can be activated by T3 for the transcription of specific genes, such as adiponectin. We examined the role of PI3K in adiponectin modulation by T3 action in murine adipocytes (3T3-L1). The 3T3-L1 adipocytes were treated with 1000 nM T3 for 1 h in the presence or absence of 50 µM LY294002 (LY), a PI3K inhibitor. Then, we assessed the expression of adiponectin and the phosphorylated serine/threonine kinase Akt (pAkt), a PI3K signaling protein, in the adipocytes. Adiponectin and pAKT levels were higher in the T3-adipocyte cells, whereas in the LY group adiponectin was elevated and pAKT was decreased compared to the control (C). PI3K pathway inhibition for 1 h and posterior treatment with T3, in LY + T3, reduced the adiponectin level and increased pAKT levels compared to those in LY. T3 stimulated adiponectin levels by PI3K pathway activation and T3 can compensate alteration in the PI3K pathway, because with inhibition of the pathway it is able to maintain the basal levels of adiponectin and pAKT.

The importance of estrogen for bone protection in experimental hyperthyroidism in human osteoblasts

Triiodothyronine (T3) and estrogen (E2) play important roles in the bone remodeling process and signaling of receptor activator of the nuclear factor-kappa ß (RANKL) and osteoprotegerin (OPG) expressed by osteoblasts. However, little is known of the molecular action of these hormones in conditions of hyperthyroidism and associated E2 in human cells. AIMS: This study evaluated the effects of the physiological concentration of E2 (10?nM), alone or in association with physiological (1nM) and supraphysiological (10nM) concentrations of T3, on RANKL and OPG gene expression in human osteoblasts. MAIN METHODS: Alkaline phosphatase and osteocalcin assays were performed to verify the presence of mature osteoblasts. After mimicking the experimental hyperthyroidism in osteoblasts untreated or treated with E2, RANKL and OPG gene expression was analyzed by real-time PCR and protein expression by western Blot and ELISA. Alizarin Red staining analyzed the amount of bone matrix after hormonal treatments. KEY FINDINGS: E2 enhanced the gene expression of OPG when associated with 1nM and 10nMT3. E2 was able to restore the bone matrix after an initial decrease using 1nM and 10nMT3. The protective effect of E2 on the RANKL and OPG signaling pathway was demonstrated. E2 restored the bone matrix induced by experimental hyperthyroidism. SIGNIFICANCE: The data highlight the importance of E2 to maintain OPG expression and osteoblast activity against possible loss of bone mass, especially in conditions where T3 is in excess.

The importance of estrogen for bone protection in experimental hyperthyroidism in human osteoblasts

Triiodothyronine (T3) and estrogen (E2) play important roles in the bone remodeling process and signaling of receptor activator of the nuclear factor-kappa ß (RANKL) and osteoprotegerin (OPG) expressed by osteoblasts. However, little is known of the molecular action of these hormones in conditions of hyperthyroidism and associated E2 in human cells. AIMS: This study evaluated the effects of the physiological concentration of E2 (10?nM), alone or in association with physiological (1nM) and supraphysiological (10nM) concentrations of T3, on RANKL and OPG gene expression in human osteoblasts. MAIN METHODS: Alkaline phosphatase and osteocalcin assays were performed to verify the presence of mature osteoblasts. After mimicking the experimental hyperthyroidism in osteoblasts untreated or treated with E2, RANKL and OPG gene expression was analyzed by real-time PCR and protein expression by western Blot and ELISA. Alizarin Red staining analyzed the amount of bone matrix after hormonal treatments. KEY FINDINGS: E2 enhanced the gene expression of OPG when associated with 1nM and 10nMT3. E2 was able to restore the bone matrix after an initial decrease using 1nM and 10nMT3. The protective effect of E2 on the RANKL and OPG signaling pathway was demonstrated. E2 restored the bone matrix induced by experimental hyperthyroidism. SIGNIFICANCE: The data highlight the importance of E2 to maintain OPG expression and osteoblast activity against possible loss of bone mass, especially in conditions where T3 is in excess.