Shanshan Shao

Thyroid-stimulating hormone regulates hepatic bile acid homeostasis via SREBP-2/HNF-4α/CYP7A1 axis

BACKGROUND & AIMS Bile acids (BAs) play a crucial role in dietary fat digestion and in the regulation of lipid, glucose, and energy metabolism. Thyroid-stimulating hormone (TSH) is a hormone produced by the anterior pituitary gland that directly regulates several metabolic pathways. However, the impact of TSH on BA homeostasis remains largely unknown. METHODS We analyzed serum BA and TSH levels in healthy volunteers under strict control of caloric intake. Thyroidectomized rats were administered thyroxine and injected with different doses of TSH. Tshr(-/-) mice were supplemented with thyroxine, and C57BL/6 mice were injected with Tshr-siRNA via the tail vein. The serum BA levels, BA pool size, and fecal BA excretion rate were measured. The regulation of SREBP-2, HNF-4α, and CYP7A1 by TSH were analyzed using luciferase reporter, RNAi, EMSA, and CHIP assays. RESULTS A negative correlation was observed between the serum levels of TSH and the serum BA levels in healthy volunteers. TSH administration led to a decrease in BA content and CYP7A1 activity in thyroidectomized rats supplemented with thyroxine. When Tshr was silenced in mice, the BA pool size, fecal BA excretion rate, and serum BA levels all increased. Additionally, we found that HNF-4α acts as a critical molecule through which TSH represses CYP7A1 activity. We further confirmed that the accumulation of mature SREBP-2 protein could impair the capacity of nuclear HNF-4α to bind to the CYP7A1 promoter, a mechanism that appears to mediate the effects of TSH. CONCLUSIONS TSH represses hepatic BA synthesis via a SREBP-2/HNF-4α/CYP7A1 signaling pathway. This finding strongly supports the notion that TSH is an important pathophysiological regulator of liver BA homeostasis independently of thyroid hormones.

Expression Profiles of Six Circulating MicroRNAs Critical to Atherosclerosis in Patients With Subclinical Hypothyroidism: A Clinical Study

CONTEXT Increasing evidence shows that subclinical hypothyroidism (SCH) is associated with atherosclerosis (ATH), but the association remains controversial. MicroRNAs (miRNAs) have been proved to be involved in atherosclerosis and dyslipidemia as gene regulators. OBJECTIVE The objective of the study was to determine the expression profiles of six serum miRNAs critical to atherosclerosis in SCH patients and reanalyze the association between atherosclerosis and SCH from a new perspective. OUTCOMES, DESIGN, AND PARTICIPANTS: MicroRNA profiling analysis was performed by real-time PCR in normal control subjects (NC; n = 22); patients with subclinical hypothyroidism alone (SCH; n = 20); SCH patients plus atherosclerosis (SCH+ATH; n = 21); and patients with atherosclerosis but without subclinical hypothyroidism (ATH; n = 22). RESULTS MiR-21-5p was up-regulated in SCH, SCH+ATH, and ATH groups than in the NC group. In addition, expression levels of miR-21-5p in SCH+ATH group were higher than in SCH alone and ATH alone groups, respectively. Both miR-125a-5p and miR-126-3p showed a decreased trend from NC to SCH and then to SCH+ATH or ATH subjects. MiR-221-3p and miR-222-3p were decreased in the SCH+ATH and ATH groups compared with either the NC or SCH groups. No differences were found in the levels of miR125a-5p, miR126-3p, miR221-3p, and miR222-3p between the ATH and SCH+ATH group. CONCLUSIONS MiR-21-5p showed the most specific expression patterns in all patients with subclinical hypothyroidism (SCH and SCH+ATH groups). Down-regulation of miR-125a-5p, miR-126-3p, miR-221-3p, and miR-222-3p may be a manifestation of atherosclerosis either in SCH+ATH or in ATH-alone patients. MiR-126-3p has the most specific expression patterns in all atherosclerosis patients (SCH+ATH and ATH groups).

Dietary high-fat lard intake induces thyroid dysfunction and abnormal morphology in rats

Aim:Excess dietary fat intake can induce lipotoxicity in non-adipose tissues. The aim of this study was to observe the effects of dietary high-fat lard intake on thyroid in rats.Methods:Male Sprague-Dawley rats were fed a high-fat lard diet for 24 weeks, and then the rats were fed a normal control diet (acute dietary modification) or the high-fat lard diet for another 6 weeks. The serum lipid profile, total thyroxine (TT4), free thyroxine (FT4) and thyrotropin (TSH) levels were determined at the 12, 18, 24 and 30 weeks. High-frequency ultrasound scanning of the thyroid glands was performed at the 24 or 30 weeks. After the rats were sacrificed, the thyroid glands were collected for histological and immunohistochemical analyses.Results:The high-fat lard diet significantly increased triglyceride levels in both the serum and thyroid, and decreased serum TT4 and FT4 levels in parallel with elevated serum TSH levels. Ultrasonic imaging revealed enlarged thyroid glands with lowered echotexture and relatively heterogeneous features in the high-fat lard fed rats. The thyroid glands from the high-fat lard fed rats exhibited enlarged follicle cavities and flattened follicular epithelial cells under light microscopy, and dilated endoplasmic reticulum cisternae, twisted nuclei, fewer microvilli and secretory vesicles under transmission electron microscopy. Furthermore, the thyroid glands from the high-fat lard fed rats showed markedly low levels of thyroid hormone synthesis-related proteins TTF-1 and NIS. Acute dietary modification by withdrawal of the high-fat lard diet for 6 weeks failed to ameliorate the high-fat lard diet-induced thyroid changes.Conclusion:Dietary high-fat lard intake induces significant thyroid dysfunction and abnormal morphology in rats, which can not be corrected by short-term dietary modification.

Lipotoxicity, a Potential Risk Factor for the Increasing Prevalence of Subclinical Hypothyroidism?

CONTEXT Subclinical hypothyroidism (SCH) is an important public health problem worldwide for its increasing prevalence and potential deleterious effects, whereas its etiology has not been fully elucidated. Lipotoxicity exerts extensive and serious impact on human health, but so far, the potential effect of lipotoxicity on thyroid is unclear. OBJECTIVE The objective of the study was to assess the association between serum triglyceride levels and the risk for SCH. DESIGN, PARTICIPANTS, AND METHODS We conducted a population-based case-control study. A total of 24 100 subjects with similar and stable iodine nutrition status were recruited from China. Cases of 5033 SCH patients were identified and equal controls were matched by age, gender, and region. Conditional logistic regression was used to analyze the association between serum triglyceride levels and risk for SCH. RESULTS Hypertriglyceridemia was associated with an approximately 35% increased risk for SCH in both men (odds ratio 1.325; 95% confidence interval 1.002-1.753) and women (odds ratio 1.397; 95% confidence interval 1.217-1.604), even after adjustment for potential confounders. Notably, the risk for SCH increased progressively following the elevation of serum triglyceride levels. Compared with individuals with serum triglyceride levels less than 0.97 mmol/L, the risk for SCH increased approximately 1.9-fold in men and 1.4-fold in women, respectively, when triglyceride levels were greater than 1.99 mmol/L. CONCLUSION Our findings suggested that hypertriglyceridemia was positively associated with the risk for SCH.

A novel role for CRTC2 in hepatic cholesterol synthesis through SREBP‐2

Cholesterol synthesis is regulated by the transcription factor sterol regulatory element binding protein 2 (SREBP‐2) and its target gene 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGCR), which is the rate‐limiting enzyme in cholesterol synthesis. Cyclic adenosine monophosphate–responsive element (CRE) binding protein–regulated transcription coactivator (CRTC) 2 is the master regulator of glucose metabolism. However, the effect of CRTC2 on cholesterol and its potential molecular mechanism remain unclear. Here, we demonstrated that CRTC2 expression and liver cholesterol content were increased in patients with high serum cholesterol levels who underwent resection of liver hemangiomas, as well as in mice fed a 4% cholesterol diet. Mice with adenovirus‐mediated CRTC2 overexpression also showed elevated lipid levels in both serum and liver tissues. Intriguingly, hepatic de novo cholesterol synthesis was markedly increased under these conditions. In contrast, CRTC2 ablation in mice fed a 4% cholesterol diet (18 weeks) showed decreased lipid levels in serum and liver tissues compared with those in littermate wild‐type mice. The expression of lipogenic genes (SREBP‐2 and HMGCR) was consistent with hepatic CRTC2 levels. In vivo imaging showed enhanced adenovirus‐mediated HMGCR‐luciferase activity in adenovirus‐mediated CRTC2 mouse livers; however, the activity was attenuated after mutation of CRE or sterol regulatory element sequences in the HMGCR reporter construct. The effect of CRTC2 on HMGCR in mouse livers was alleviated upon SREBP‐2 knockdown. CRTC2 modulated SREBP‐2 transcription by CRE binding protein, which recognizes the half‐site CRE sequence in the SREBP‐2 promoter. CRTC2 reduced the nuclear protein expression of forkhead box O1 and subsequently increased SREBP‐2 transcription by binding insulin response element 1, rather than insulin response element 2, in the SREBP‐2 promoter. Conclusion: CRTC2 regulates the transcription of SREBP‐2 by interfering with the recognition of insulin response element 1 in the SREBP‐2 promoter by forkhead box O1, thus inducing SREBP‐2/HMGCR signaling and subsequently facilitating hepatic cholesterol synthesis. (Hepatology 2017;66:481–497).

Analysis of the correlation between lipotoxicity and pituitary-thyroid axis hormone levels in men and male rats

Lipotoxicity seriously harms human health, but it is unclear whether lipotoxicity is detrimental to the pituitary. We investigated the correlation between serum triglyceride and pituitary axis hormone levels in epidemiological and animal studies. In the epidemiological study, serum thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were greater in male patients with isolated hypertriglyceridemia than in controls, whereas adrenocorticotropin (ACTH) levels were lower in the patients with hypertriglyceridemia. Pituitary hormone levels correlated with triglyceride levels, even after adjustment for potential confounders. In the animal study, male rats were fed a high-fat or control diet for 28 weeks. As the duration of high-fat feeding increased, the serum and pituitary triglyceride concentrations increased. At early times, the high-fat diet elevated serum TSH and triiodothyronine. At later times, much higher serum TSH levels coupled with reduced thyroxine were observed in the high-fat group. Serum levels of pituitary-gonadal and pituitary-adrenal axis hormones were not affected by the diet. The mRNA and protein expression of Tshβ were greater in the high-fat group than in the control group, whereas expression of Fshβ, Lhβ and Acth had no difference between the groups. Overall, serum triglyceride levels were associated with pituitary-thyroid axis hormone levels.

Relative variations of gut microbiota in disordered cholesterol metabolism caused by high-cholesterol diet and host genetics

Recent studies performed provide mechanistic insight into effects of the microbiota on cholesterol metabolism, but less focus was given to how cholesterol impacts the gut microbiota. In this study, ApoE−/− Sprague Dawley (SD) rats and their wild‐type counterparts (n = 12) were, respectively, allocated for two dietary condition groups (normal chow and high‐cholesterol diet). Total 16S rDNA of fecal samples were extracted and sequenced by high‐throughput sequencing to determine differences in microbiome composition. Data were collected and performed diversity analysis and phylogenetic analysis. The influence of cholesterol on gut microbiota was discussed by using cholesterol dietary treatment as exogenous cholesterol disorder factor and genetic modification as endogenous metabolic disorder factor. Relative microbial variations were compared to illustrate the causality and correlation of cholesterol and gut microbiota. It turned out comparing to genetically modified rats, exogenous cholesterol intake may play more effective role in changing gut microbiota profile, although the serum cholesterol level of genetically modified rats was even higher. Relative abundance of some representative species showed that the discrepancies due to dietary variation were more obvious, whereas some low abundance species changed because of genetic disorders. Our results partially demonstrated that gut microbiota are relatively more sensitive to dietary variation. Nevertheless, considering the important effect of bacteria in cholesterol metabolism, the influence to gut flora by “genetically caused cholesterol disorder” cannot be overlooked. Manipulation of gut microbiota might be an effective target for preventing cholesterol‐related metabolic disorders.

High-Cholesterol Diet Disrupts the Levels of Hormones Derived from Anterior Pituitary Basophilic Cells

Emerging evidence shows that elevated cholesterol levels are detrimental to health. However, it is unclear whether there is an association between cholesterol and the pituitary. We investigated the effects of a high‐cholesterol diet on pituitary hormones using in vivo animal studies and an epidemiological study. In the animal experiments, rats were fed a high‐cholesterol or control diet for 28 weeks. In rats fed the high‐cholesterol diet, serum levels of thyroid‐stimulating hormone (TSH; also known as thyrotrophin), luteinising hormone (LH) and follicle‐stimulating hormone (FSH) produced by the basophilic cells of the anterior pituitary were elevated in a time‐dependent manner. Among these hormones, TSH was the first to undergo a significant change, whereas adrenocorticotrophic hormone (ACTH), another hormone produced by basophilic cells, was not changed significantly. As the duration of cholesterol feeding increased, cholesterol deposition increased gradually in the pituitary. Histologically, basophilic cells, and especially thyrotrophs and gonadotrophs, showed an obvious increase in cell area, as well as a potential increase in their proportion of total pituitary cells. Expression of the β‐subunit of TSH, FSH and LH, which controls hormone specificity and activity, exhibited a corresponding increase. In the epidemiological study, we found a similar elevation of serum TSH, LH and FSH and a decrease in ACTH in patients with hypercholesterolaemia. Significant positive correlations existed between serum total cholesterol and TSH, FSH or LH, even after adjusting for confounding factors. Taken together, the results of the present study suggest that the high‐cholesterol diet affected the levels of hormones derived from anterior pituitary basophilic cells. This phenomenon might contribute to the pituitary functional disturbances described in hypercholesterolaemia.

Role of exosome-associated microRNA in diagnostic and therapeutic applications to metabolic disorders

Metabolic disorders are classified clinically as a complex and varied group of diseases including metabolic syndrome, obesity, and diabetes mellitus. Fat toxicity, chronic inflammation, and oxidative stress, which may change cellular functions, are considered to play an essential role in the pathogenetic progress of metabolic disorders. Recent studies have found that cells secrete nanoscale vesicles containing proteins, lipids, nucleic acids, and membrane receptors, which mediate signal transduction and material transport to neighboring and distant cells. Exosomes, one type of such vesicles, are reported to participate in multiple pathological processes including tumor metastasis, atherosclerosis, chronic inflammation, and insulin resistance. Research on exosomes has focused mainly on the proteins they contain, but recently the function of exosome-associated microRNA has drawn a lot of attention. Exosomeassociated microRNAs regulate the physiological function and pathological processes of metabolic disorders. They may also be useful as novel diagnostics and therapeutics given their special features of non-immunogenicity and quick extraction. In this paper, we summarize the structure, content, and functions of exosomes and the potential diagnostic and therapeutic applications of exosome-associated microRNAs in the treatment of metabolic disorders.概要代谢性疾病是指包括代谢综合征、肥胖和糖尿病 在内的一系列复杂疾病。其中脂毒性、慢性炎症 和氧化应激可以通过改变细胞功能,从而在代谢 紊乱的病理进程中发挥重要作用。近期研究发现 细胞可以分泌含有蛋白质、脂质、核酸的纳米级 微小囊泡,介导相邻和远处细胞间的信号传导和 物质转运。外泌体作为这类囊泡的一种,参与包 括肿瘤转移、动脉粥样硬化、慢性炎症和胰岛素 抵抗等多种病理过程。外泌体的研究大多集中于 其所含的蛋白质, 而近期关于外泌体相关 microRNA 的功能研究也日益受到关注。尤其是, 现已证明外泌体相关microRNA 参与了机体代谢 的诸多生理、病理进程,为代谢性疾病的诊断和 治疗提供了新的方向。本文总结了外泌体的结 构、内容物及产生的机制(图1 和图2);体液 和细胞培养液中外泌体所含microRNA 的种类、 靶器官及其功能(表2);外泌体相关microRNA 在代谢性疾病中的作用,以及在诊断和治疗方面 的潜能。

Palmitic Acid Downregulates Thyroglobulin (Tg), Sodium Iodide Symporter (NIS), and Thyroperoxidase (TPO) in Human Primary Thyrocytes: A Potential Mechanism by Which Lipotoxicity Affects Thyroid?

Our previous studies suggested that the thyroid might be a target organ affected by lipotoxicity, which might be partially related to the increasing prevalence of subclinical hypothyroidism. However, the underlying molecular mechanism is not yet clearly established. This study aimed to assess the effect of palmitic acid stimulation on thyrocyte function. Upon palmitic acid stimulation, intracellular contents of lipids, as well as the expression and activity of three key molecules in thyroid hormone synthesis (i.e., thyroglobulin, sodium iodide symporter, and thyroperoxidase), were determined in human primary thyrocytes. The contents of BODIPY® FL C16 (the fluorescently labeled palmitic acid analogue) entering into the thyrocytes were gradually increased with time extending. Accordingly, the intracellular accumulation of both triglyceride and free fatty acids increased in dose- and time-dependent manners. The effect of palmitic acid stimulation on thyroid hormone synthesis was then determined. Both the mRNA and protein levels of thyroglobulin, sodium iodide symporter, and thyroperoxidase were decreased following palmitic acid stimulation. Consistently, upon palmitic acid stimulation, the secreted thyroglobulin levels in supernatants, 131I uptake, and extracellular thyroperoxidase activity were all decreased in a dose-dependent manner. Our results demonstrated that upon palmitic acid stimulation, the expressions of the key molecules (thyroglobulin, sodium iodide symporter, and thyroperoxidase) were reduced and their activities were suppressed, which might lead to impaired thyroid hormone synthesis.