Tetra methyl bisphenol F: another potential obesogen.

BACKGROUND/OBJECTIVES: Obesity and its associated metabolic diseases are increasing globally. Sedentary lifestyle, high caloric diet, and genetic predisposition are known to contribute to the onset of obesity. It is increasingly recognized that exposure to environmental chemicals such as Bisphenol A (BPA) may also play a significant role. BPA has been correlated with an array of adverse health effects, including obesity and metabolic disorders. Due to public concern, manufacturers are replacing BPA with structural analogues for which there is limited toxicological data. The objective of this study was to assess the effects of these BPA analogues on adipogenesis. METHODS: The adipogenic effects of Tetra Methyl Bisphenol F (TMBPF), Bisphenol F (BPF), Bisphenol AP (BPAP), and fluorine-9-bisphenol (BHPF) were evaluated in murine 3T3-L1 cells. The cells were treated with BPA and its analogues at concentrations from 0.01 µM to 20 µM, throughout differentiation, in the absence of Dexamethasone (Dex). Lipid accumulation, mRNA and protein levels of adipogenic markers was assessed. RESULTS: We found that TMBPF, BPF and BPA increased 3T3-L1 lipid accumulation and the expression levels of adipogenic markers lipoprotein lipase (Lpl), fatty acid binding protein 4 (Fabp4) and perilipin (Plin) (1-20 µM; p < 0.05), whereas BHPF and BPAP had no effect in this model. Further, TMBPF induced adipogenesis to a greater extent than all the other chemicals including BPA (1-20 µM; p < 0.05). The effect mediated by TMBPF on expression levels of Fabp4, but not Plin, is likely mediated via peroxisome proliferator-activated receptor (PPAR) γ activation. CONCLUSIONS: Of the BPA analogues tested, BPF was most similar to BPA in its effects, while TMBPF was most adipogenic. In addition, TMBPF is likely a PPARγ agonist, it is likely an obesogenic chemical and may be a metabolic disruptor.

Regulators of thymic stromal lymphopoietin production by human adipocytes.

Thymic stromal lymphopoietin (TSLP) is a cytokine that is known to play a role in inflammatory conditions, especially asthma and atopic dermatitis. It is also recognized to be expressed in human adipose tissue. TSLP production from human adipocytes is stimulated by thyroid-stimulating hormone (TSH). This study aimed to identify TSH-dependent signaling routes that regulate TSLP, to determine if TSLP production is stimulated by other cytokines (IL-1ß and TNF-α), and to examine if TSLP production depends on the adipose depot. Human abdominal differentiated adipocytes were stimulated with TSH, IL-1ß, or TNF-α. Activation of cell signaling kinases was measured by phospho-immunoblot analysis, and TSLP in medium was assessed by ELISA. TSLP responses from abdominal subcutaneous and omental adipocytes were compared. TSH-stimulated TSLP secretion from subcutaneous adipocytes was enhanced by IBMX (raises cAMP levels) and was blocked by UO126 (inhibitor of MEK1/2-ERK1/2). TSLP secretion was stimulated by IL-1ß and by TNF-α. SC-514 (inhibitor of IKKß/NF-κB) only reduced the former. There was no effect of SB203580 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) on the stimulation by TSH, IL-1ß or TNF-α. Interferon-γ inhibited TSLP responses to TSH, IL-1ß, and TNF-α; IL-4 only blocked the response to TNFα. Intra-abdominal omental adipocytes also release TSLP in response to TSH, IL-1ß, and TNF-α. We conclude TSLP is produced by human differentiated adipocytes derived from subcutaneous or omental depots in response to a variety of agonists. Further studies will be needed to understand what role it may play in adipose biology.

mTORC1 activates SREBP-2 by suppressing cholesterol trafficking to lysosomes in mammalian cells.

mTORC1 is known to activate sterol regulatory element-binding proteins (SREBPs) including SREBP-2, a master regulator of cholesterol synthesis. Through incompletely understood mechanisms, activated mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi where SREBP-2 is cleaved to translocate to the nucleus and activate gene expression for cholesterol synthesis. Low ER cholesterol is a well-established trigger for SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing cholesterol delivery to the ER. We report here that mTORC1 activation is accompanied by low ER cholesterol and an increase of SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with a rise in ER cholesterol and a decrease in SERBP-2 activity. This rise in ER cholesterol is of lysosomal origin: blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from increasing and, consequently, SREBP-2 is activated without mTORC1 activation. Furthermore, when mTORC1 activity is low, cholesterol is delivered to lysosomes through two membrane trafficking pathways: autophagy and rerouting of endosomes to lysosomes. Indeed, with dual blockade of both pathways by Atg5-/- and dominant-negative rab5, ER cholesterol fails to increase when mTORC1 activity is low, and SREBP-2 is activated. Conversely, overexpressing constitutively active Atg7, which forces autophagy and raises ER cholesterol even when mTORC1 activity is high, suppresses SREBP-2 activation. We conclude that mTORC1 actively suppresses autophagy and maintains endosomal recycling, thereby preventing endosomes and autophagosomes from reaching lysosomes. This results in a reduction of cholesterol in the ER and activation of SREBP-2.

Dechlorane Plus increases adipogenesis in 3T3-L1 and human primary preadipocytes independent of peroxisome proliferator-activated receptor γ transcriptional activity.

BACKGROUND/OBJECTIVES: Polybrominated diphenyl ethers (PBDEs) are chemicals that were added to consumer products to reduce flammability but were deemed toxic and bioaccumulative and were phased out of commerce. Flame retardants (FRs) such as Dechlorane Plus (DP) were introduced as replacements. DP is being produced in high volumes and is detected in the environment, human milk, and human serum. Although human exposure to DP is evident, little is known about its potential effects on human health. We and others have shown that some FRs are potential obesogens, i.e., promote adipogenesis. However, the effects of DP on adipogenesis are not known. METHODS: Murine 3T3-L1 and human primary subcutaneous (Sc) and omental (Om) preadipocytes were differentiated in the presence of DP (0.001-10 µM) and adipogenic effects were measured. Further, the ability of DP to activate the adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) was also assessed. RESULTS: We show that treatment of murine preadipocytes with DP significantly (p < 0.05) increased lipid accumulation (2.5-fold) and the mRNA expression of adipogenic markers: fatty acid binding protein 4 (Fabp4), lipoprotein lipase (Lpl), perilipin (Plin), adipsin, and adiponectin. DP also significantly (p < 0.05) increased the protein levels of selected mature adipocyte markers. We further show using luciferase reporter assays that DP increased PPARγ transcriptional activity by threefold (p < 0.05). When the PPARγ agonist was replaced by DP in the human preadipocyte differentiation cocktail, DP significantly (p < 0.05) increased the mRNA levels of adipogenic markers, PPARγ, FABP4, and PLIN in human Sc as well as Om cultures. Finally, PPARγ antagonist studies revealed that DP-mediated upregulation of adipogenic markers Fabp4 and Lpl did not occur via PPARγ activation. CONCLUSION: The current study shows that DP can induce adipogenesis of murine and human preadipocytes. We show that, although DP can directly activate PPARγ, its adipogenic effects may be mediated via other pathways.

Effect of hemodialysis on extracellular vesicles and circulating submicron particles.

BACKGROUND: Although hemodialysis is a highly effective treatment for diffusive clearance of low molecular weight uremic toxins, its effect on circulating extracellular vesicles and submicron particles is less clear. The purpose of this study was to examine the impact of hemodialysis on circulating levels of submicron particles. METHODS: Plasma samples from patients were collected immediately before and after the mid-week hemodialysis session. Total submicron particles were assessed by nanoparticle tracking analysis and levels of endothelial (CD144+), platelet (CD41+), leukocyte (CD45+), and total (Annexin V+) membrane microparticles (MPs) were assessed by flow cytometry. RESULTS: Total submicron particle number was significantly lower post-dialysis with reductions in particles < 40 nm, 40-100 nm, and 100-1000 nm in size. Circulating annexin V+ MPs, platelet MPs, leukocyte MPs, and endothelial MPs were all reduced following dialysis. Assessment of protein markers suggested that extracellular vesicles were not present in the dialysate, but rather adsorbed to the dialysis membrane. CONCLUSIONS: In summary, hemodialysis is associated with reductions in circulating submicron particles including membrane MPs. Accordingly, there may be significant interdialytic variation in circulating submicron particles. Investigators interested in measuring extracellular vesicles in patients undergoing hemodialysis should therefore carefully consider the timing of biosampling.

Thyroid-Stimulating Hormone-Stimulated Human Adipocytes Express Thymic Stromal Lymphopoietin.

When recombinant human (rh) thyroid-stimulating hormone (TSH) is administered to thyroid cancer survivors, an acute extra-thyroidal effect raises pro-inflammatory cytokines and activates platelets. Thymic stromal lymphopoietin (TSLP) is a cytokine recently implicated in platelet activation. Our aim was to measure platelet microparticle levels after rhTSH stimulation in vivo, and to investigate TSLP expression in TSH-stimulated human adipocytes in culture. Blood samples for total and platelet microparticle analysis were obtained from thyroid cancer survivors before (day 1) and after rhTSH administration (day 5). Adipocytes, differentiated from stromal preadipocytes isolated from adipose tissue from surgical patients, were stimulated with TSH. TSLP mRNA expression, protein expression, and protein release into the adipocyte medium were measured. The level of platelet microparticles in thyroid cancer patients rose 5-fold after rhTSH stimulation. TSH upregulated TSLP mRNA expression in adipocytes in culture through a pathway that was inhibited by 66% by H89, a protein kinase A inhibitor. TSLP protein expression rose in response to TSH, and TSH-stimulated TSLP release into the medium was completely blocked by dexamethasone. In conclusion, TSLP is a novel TSH-responsive adipokine. Future studies will be needed to address the potential role of adipocyte-derived TSLP and whether it is linked to TSH-dependent platelet activation.

Randomized Trial of the Effect of Mindfulness-Based Stress Reduction on Pain-Related Disability, Pain Intensity, Health-Related Quality of Life, and A1C in Patients With Painful Diabetic Peripheral Neuropathy.

IN BRIEF Painful diabetic peripheral neuropathy (PDPN) has a large negative impact on patients' physical and mental functioning, and pharmacological therapies rarely provide more than partial relief. Mindfulness-based stress reduction (MBSR) is a group psychosocial intervention that was developed for patients with chronic illness who were not responding to existing medical treatments. This study tested the effects of community-based MBSR courses for patients with PDPN. Among patients whose PDPN pharmacotherapy had been optimized in a chronic pain clinic, those randomly assigned to treatment with MBSR experienced improved function, better health-related quality of life, and reduced pain intensity, pain catastrophizing, and depression compared to those receiving usual care.

Effect of High Glucose Levels on White Adipose Cells and Adipokines-Fuel for the Fire.

White adipocytes release adipokines that influence metabolic and vascular health. Hypertrophic obesity is associated with adipose tissue malfunctioning, leading to inflammation and insulin resistance. When pancreatic islet ß cells can no longer compensate, the blood glucose concentration rises (hyperglycemia), resulting in type 2 diabetes. Hyperglycaemia may further aggravate adipose cell dysfunction in ~90% of patients with type 2 diabetes who are obese or overweight. This review will focus on the effects of high glucose levels on human adipose cells and the regulation of adipokines.

Thyroid-stimulating hormone acutely increases monocyte gene expression in vivo.

OBJECTIVES: Thyroid-stimulating hormone (TSH) acts in an extra-thyroidal fashion and induces a pro-inflammatory, pro-coagulant state. Blood monocytes can be activated by vascular stress, but it is not known if this occurs upon TSH administration. Our aim was to determine if recombinant human (rh) TSH, administered acutely to patients being screened for thyroid cancer recurrence, alters blood monocyte gene expression. DESIGN AND SETTING: Patients (14 women, 1 man) had a mean (±SD) age of 48±10 years, a body mass index of 26±6 kg/m2, a history of total thyroidectomy and radioablation for thyroid cancer, and were on L-thyroxine therapy at a university teaching hospital. They received 2 intramuscular doses of rhTSH (0.9 mg), administered on days 1 and 2. Blood samples were obtained at baseline on day1, and on days 3 and 5. RESULTS: Monocyte MCP-1 mRNA (mean±SE) increased significantly by 1.7±0.3 fold on day 5 following rhTSH stimulation (p=0.03, n=15). IL-1ß and CD36 mRNA expression also increased on day 5 (1.9±0.4 fold, p=0.07, n=14) and 2.5±0.4 fold, p=0.1, n=10), respectively, although did not quite reach statistical significance. Significant correlations were detected between the BMI of patients and their TSH-stimulated monocyte mRNA responses at day 5 for CD11a, (r=0.66, n=14, p=0.01); CD14 (r=0.638, n=13, p=0.019), and CD16, r=0.84, n=13, p=0.0003). CONCLUSION: TSH administration increases pro-atherogenic monocyte gene expression.

Implementation of a consent for chart review and contact and its impact in one clinical centre.

OBJECTIVE: Informed consent and protection of patient confidentiality are central to the conduction of clinical research. Consent for chart review and contact (CCRC) allows a patient chart to be screened for research by persons outside the direct circle-of-care and for the patient to be contacted regarding potential studies. This study describes the process of implementation and benefits of such a consent. DESIGN: We present a descriptive report of a CCRC document that was created and presented to patients over a 3.5-year period at a tertiary care Endocrinology and Metabolism centre. To assess the potential impact of such a document on patient recruitment, the basic demographics of patients who did and did not consent were compared. In addition, we compared the recruitment rate at our centre, using our novel approach, with that at other centres for an ongoing study of patients with type 1 diabetes. RESULTS: A large proportion (6501/8025, or 81%) of patients gave their consent for chart review. Patients who denied consent were more likely to be women and older. Compared with other centres, our centre recruited at the highest rate for a known study of patients with type 1 diabetes. The majority (46/60, or 76.7%) of patients were recruited via the novel approach. CONCLUSIONS: Consent for chart review and contact addresses several important ethical issues regarding the use of patient clinical information for research purposes. Our study demonstrated how such a process can be implemented.

Statin-mediated reduction in mitochondrial cholesterol primes an anti-inflammatory response in macrophages by upregulating JMJD3.

Stains are known to be anti-inflammatory, but the mechanism remains poorly understood. Here we show that macrophages, either treated with statin in vitro or from statin-treated mice, have reduced cholesterol levels and higher expression of Jmjd3, a H3K27me3 demethylase. We provide evidence that lowering cholesterol levels in macrophages suppresses the ATP synthase in the inner mitochondrial membrane (IMM) and changes the proton gradient in the mitochondria. This activates NFkB and Jmjd3 expression to remove the repressive marker H3K27me3. Accordingly, the epigenome is altered by the cholesterol reduction. When subsequently challenged by the inflammatory stimulus LPS (M1), both macrophages treated with statins in vitro or isolated from statin-treated mice in vivo, express lower levels pro-inflammatory cytokines than controls, while augmenting anti-inflammatory Il10 expression. On the other hand, when macrophages are alternatively activated by IL4 (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression is correlated with the statin-induced removal of H3K27me3 from these genes prior to activation. In addition, Jmjd3 and its demethylase activity are necessary for cholesterol to modulate both M1 and M2 activation. We conclude that upregulation of Jmjd3 is a key event for the anti-inflammatory function of statins on macrophages.

Statin-mediated reduction in mitochondrial cholesterol primes an anti-inflammatory response in macrophages by upregulating Jmjd3.

Statins are known to be anti-inflammatory, but the mechanism remains poorly understood. Here, we show that macrophages, either treated with statin in vitro or from statin-treated mice, have reduced cholesterol levels and higher expression of Jmjd3, a H3K27me3 demethylase. We provide evidence that lowering cholesterol levels in macrophages suppresses the adenosine triphosphate (ATP) synthase in the inner mitochondrial membrane and changes the proton gradient in the mitochondria. This activates nuclear factor kappa-B (NF-κB) and Jmjd3 expression, which removes the repressive marker H3K27me3. Accordingly, the epigenome is altered by the cholesterol reduction. When subsequently challenged by the inflammatory stimulus lipopolysaccharide (M1), macrophages, either treated with statins in vitro or isolated from statin-fed mice, express lower levels proinflammatory cytokines than controls, while augmenting anti-inflammatory Il10 expression. On the other hand, when macrophages are alternatively activated by IL-4 (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression is correlated with the statin-induced removal of H3K27me3 from these genes prior to activation. In addition, Jmjd3 and its demethylase activity are necessary for cholesterol to modulate both M1 and M2 activation. We conclude that upregulation of Jmjd3 is a key event for the anti-inflammatory function of statins on macrophages.

Serum fetuin-A levels following recombinant human thyroid-stimulating hormone stimulation.

PURPOSE: Fetuin-A is a hepatokine that is linked to lipid metabolism, obesity, insulin resistance, type 2 diabetes and cardiovascular disease. Elevated thyroid-stimulating hormone (TSH) levels are associated with metabolic and cardiovascular disturbances. Our aim was to determine if TSH can regulate fetuin-A levels. METHODS: Fetuin-A serum levels were examined in 26 subjects (19 women; previous thyroidectomy and radioactive iodine ablation) undergoing recombinant human TSH (rhTSH) stimulation to screen for thyroid cancer recurrence. Their age was 49±10 years, and body mass index (BMI) was 28±6 (both expressed as mean±SD). The patients received two doses of rhTSH (0.9 mg), administered 24 hours apart on days 1 and 2, without discontinuation of ongoing L-thyroxine therapy. Morning blood samples were obtained on days 1 (prior to the first dose of rhTSH), 3 and 5. RESULTS: The baseline value of fetuin-A (mean±SD) for all participants was 527±186 mg/L. Values of fetuin-A did not change in response to rhTSH administration. The lack of response was not dependent on gender, age, baseline free thyroxine level or BMI. CONCLUSION: Fetuin-A has been implicated in metabolic and inflammatory conditions, but there have been no reports on whether fetuin-A is influenced by TSH. Within the context of rhTSH administration for surveillance of thyroid cancer recurrence, there was no effect on serum levels of fetuin-A.

In vitro studies of the renin-angiotensin system in human adipose tissue/adipocytes and possible relationship to SARS-CoV-2: a scoping review.

The renin-angiotensin system (RAS) operates within adipose tissue. Obesity-related changes can affect adipose RAS, predisposing to hypertension, type 2 diabetes, and possibly severe COVID-19. We evaluated the in vitro research on human adipose RAS and identified gaps in the literature. Medline (Ovid), Embase (Ovid), Web of Science, Scopus, and 1findr were searched to identify relevant studies. Fifty primary studies met our inclusion criteria for analysis. Expression of RAS components (n = 14), role in differentiation (n = 14), association with inflammation (n = 15) or blood pressure (n = 7) were investigated. We found (1) obesity-related changes in RAS were frequently studied (30%); (2) an upswing of articles investigating adipose ACE-2 expression since the COVID-19 pandemic; (3) a paucity of papers on AT2R and Ang (1-7)/MasR which counterbalance Ang II/ART1; (4) weight loss lowered adipose ACE-2 mRNA expression; and (5) angiotensin receptor blockers (ARBs) reduced deleterious effects of angiotensin II. Overall, these studies link Ang II/ATR1 signalling to impaired adipogenesis and a pro-inflammatory dysfunctional adipose tissue, with ATR1 blockade limiting these responses. ACE-2 may mitigate Ang II effects by converting it to Ang(1-7) which binds MasR. More work is needed to understand adipose RAS in various pathologic states such as obesity and COVID-19 infection.T.

Macrophage-induced preadipocyte survival depends on signaling through Akt, ERK1/2, and reactive oxygen species.

Obesity is associated with adipose tissue remodeling, characterized by macrophage accumulation, adipocyte hypertrophy, and apoptosis. We previously reported that macrophage-conditioned medium (MacCM) protects preadipocytes from apoptosis, due to serum withdrawal, in a platelet-derived growth factor (PDGF)-dependent manner. We have now investigated the role of intracellular signaling pathways, activated in response to MacCM versus PDGF, in promoting preadipocyte survival. Exposure of 3T3-L1 preadipocytes to J774A.1-MacCM or PDGF strongly stimulated Akt and ERK1/2 phosphorylation from initially undetectable levels. Inhibition of the upstream regulators of Akt or ERK1/2, i.e. phosphoinositide 3-kinase (PI3K; using wortmannin or LY294002) or MEK1/2 (using UO126 or PD98509), abrogated the respective phosphorylation responses, and significantly impaired pro-survival activity. J774A.1-MacCM increased reactive oxygen species (ROS) levels by 3.4-fold, and diphenyleneiodonium (DPI) or N-acetyl cysteine (NAC) significantly inhibited pro-survival signaling and preadipocyte survival in response to J774A.1-MacCM. Serum withdrawal itself also increased ROS levels (2.1-fold), and the associated cell death was attenuated by DPI or NAC. In summary, J774A.1-MacCM-dependent 3T3-L1 preadipocyte survival requires the Akt and ERK1/2 signaling pathways. Furthermore, ROS generation by J774A.1-MacCM is required for Akt and ERK1/2 signaling to promote 3T3-L1 preadipocyte survival. These data suggest potential mechanisms by which macrophages may alter preadipocyte fate.

Macrophage-conditioned medium inhibits the activation of cyclin-dependent kinase 2 by adipogenic inducers in 3T3-L1 preadipocytes.

Macrophage infiltration into adipose tissue, associated with obesity, is thought to contribute to abnormal adipose tissue remodeling, low-grade inflammation, and insulin resistance. Medium conditioned by macrophages (MacCM) inhibits 3T3-L1 and human adipocyte differentiation, as well as early adipogenic cell cycle events including MCE and retinoblastoma protein (Rb) phosphorylation. Our objective was to determine if the inhibition of Rb phosphorylation was linked to changes in cell cycle-related proteins. We treated 3T3-L1 preadipocytes with adipogenic inducers for 24 h in control medium versus J774A.1-MacCM. The differentiation-induced mRNA and protein expression of cyclin A, an activator of cyclin-dependent kinase (cdk) 2 which phosphorylates Rb, was inhibited by 82% and 73%, respectively, by J774A.1-MacCM; adipogenic expression of Myc, a transcriptional regulator of cyclin A, was also suppressed significantly. Consistent with the reduction in cyclin A levels, the activation of cdk2 by adipogenic inducers was inhibited by 75% by J774A.1-MacCM. J774A.1-MacCM also lowered levels of cyclins D1 and D2. Inhibition studies demonstrated that platelet-derived growth factor, an anti-adipogenic factor found in J774A.1-MacCM, was not responsible for the inhibitory effect on differentiation. The anti-adipogenic effect of J774A.1-MacCM was resistant to proteinase K and heat treatment, and was present in a <3 kDa fraction. Our data indicate that J774A.1-MacCM interferes with the upregulation of cyclin A levels and cdk2 activity that are required for Rb phosphorylation and MCE in 3T3-L1 adipogenesis.

Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes.

This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n=3; p<0.001), and BrdU incorporation was impaired by 55% (n=3; p<0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27(kip1), a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n=3; p<0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBPbeta were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.