Lipidomics Workflow for Analyzing Lipid Profiles Using Multiple Reaction Monitoring (MRM) in Liver Homogenate of Mice with Non-alcoholic Steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is a condition characterized by inflammation and hepatic injury/fibrosis caused by the accumulation of ectopic fats in the liver. Recent advances in lipidomics have allowed the identification and characterization of lipid species and have revealed signature patterns of various diseases. Here, we describe a lipidomics workflow to assess the lipid profiles of liver homogenates taken from a NASH mouse model. The protocol described below was used to extract and analyze the metabolites from the livers of mice with NASH by liquid chromatography-mass spectrometry (LC-MS); however, it can be applied to other tissue homogenate samples. Using this method, over 1,000 species of lipids from five classes can be analyzed in a single run on the LC-MS. Also, partial elucidation of the identity of neutral lipid (triacylglycerides and diacylglycerides) aliphatic chains can be performed with this simple LC-MS setup. Key features Over 1,000 lipid species (sphingolipids, cholesteryl esters, neutral lipids, phospholipids, fatty acids) are analyzed in one run. Analysis of liver lipids in non-alcoholic steatohepatitis (NASH) mouse model. Normal-phase chromatography coupled to a triple quadrupole mass spectrometer.

Nicotinamide riboside rescues dysregulated glycolysis and fatty acid ß-oxidation in a human hepatic cell model of citrin deficiency.

Citrin deficiency (CD) is an inborn error of metabolism caused by loss-of-function of the mitochondrial aspartate/glutamate transporter, CITRIN, which is involved in both the urea cycle and malate-aspartate shuttle. Patients with CD develop hepatosteatosis and hyperammonemia but there is no effective therapy for CD. Currently, there are no animal models that faithfully recapitulate the human CD phenotype. Accordingly, we generated a CITRIN knockout HepG2 cell line using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 genome editing technology to study metabolic and cell signaling defects in CD. CITRIN KO cells showed increased ammonia accumulation, higher cytosolic ratio of reduced versus oxidized form of nicotinamide adenine dinucleotide (NAD) and reduced glycolysis. Surprisingly, these cells showed impaired fatty acid metabolism and mitochondrial activity. CITRIN KO cells also displayed increased cholesterol and bile acid metabolism resembling those observed in CD patients. Remarkably, normalizing cytosolic NADH:NAD+ ratio by nicotinamide riboside increased glycolysis and fatty acid oxidation but had no effect on the hyperammonemia suggesting the urea cycle defect was independent of the aspartate/malate shuttle defect of CD. The correction of glycolysis and fatty acid metabolism defects in CITRIN KO cells by reducing cytoplasmic NADH:NAD+ levels suggests this may be a novel strategy to treat some of the metabolic defects of CD and other mitochondrial diseases.

Spermidine-mediated hypusination of translation factor EIF5A improves mitochondrial fatty acid oxidation and prevents non-alcoholic steatohepatitis progression.

Spermidine is a natural polyamine that has health benefits and extends life span in several species. Deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) are key enzymes that utilize spermidine to catalyze the post-translational hypusination of the translation factor EIF5A (EIF5AH). Here, we have found that hepatic DOHH mRNA expression is decreased in patients and mice with non-alcoholic steatohepatitis (NASH), and hepatic cells treated with fatty acids. The mouse and cell culture models of NASH have concomitant decreases in Eif5aH and mitochondrial protein synthesis which leads to lower mitochondrial activity and fatty acid ß-oxidation. Spermidine treatment restores EIF5AH, partially restores protein synthesis and mitochondrial function in NASH, and prevents NASH progression in vivo. Thus, the disrupted DHPS-DOHH-EIF5AH pathway during NASH represents a therapeutic target to increase hepatic protein synthesis and mitochondrial fatty acid oxidation (FAO) and prevent NASH progression.

Vitamin B12 and folate decrease inflammation and fibrosis in NASH by preventing syntaxin 17 homocysteinylation.

BACKGROUND & AIMS: Several recent clinical studies have shown that serum homocysteine (Hcy) levels are positively correlated, while vitamin B12 (B12) and folate levels are negative correlated, with non-alcoholic steatohepatitis (NASH) severity. However, it is not known whether hyperhomocysteinemia (HHcy) plays a pathogenic role in NASH. METHODS: We examined the effects of HHcy on NASH progression, metabolism, and autophagy in dietary and genetic mouse models, patients, and primates. We employed vitamin B12 (B12) and folate (Fol) to reverse NASH features in mice and cell culture. RESULTS: Serum Hcy correlated with hepatic inflammation and fibrosis in NASH. Elevated hepatic Hcy induced and exacerbated NASH. Gene expression of hepatic Hcy-metabolizing enzymes was downregulated in NASH. Surprisingly, we found increased homocysteinylation (Hcy-lation) and ubiquitination of multiple hepatic proteins in NASH including the key autophagosome/lysosome fusion protein, Syntaxin 17 (Stx17). This protein was Hcy-lated and ubiquitinated, and its degradation led to a block in autophagy. Genetic manipulation of Stx17 revealed its critical role in regulating autophagy, inflammation and fibrosis during HHcy. Remarkably, dietary B12/Fol, which promotes enzymatic conversion of Hcy to methionine, decreased HHcy and hepatic Hcy-lated protein levels, restored Stx17 expression and autophagy, stimulated ß -oxidation of fatty acids, and improved hepatic histology in mice with pre-established NASH. CONCLUSIONS: HHcy plays a key role in the pathogenesis of NASH via Stx17 homocysteinylation. B12/folate also may represent a novel first-line therapy for NASH. LAY SUMMARY: The incidence of non-alcoholic steatohepatitis, for which there are no approved pharmacological therapies, is increasing, posing a significant healthcare challenge. Herein, based on studies in mice, primates and humans, we found that dietary supplementation with vitamin B12 and folate could have therapeutic potential for the prevention or treatment of non-alcoholic steatohepatitis.

Thyroid Hormone Decreases Hepatic Steatosis, Inflammation, and Fibrosis in a Dietary Mouse Model of Nonalcoholic Steatohepatitis.

Background: Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, lobular inflammation, and fibrosis. Thyroid hormone (TH) reduces steatosis; however, the therapeutic effect of TH on NASH-associated inflammation and fibrosis is not known. This study examined the therapeutic effect of TH on hepatic inflammation and fibrosis during NASH and investigated THs molecular actions on autophagy and mitochondrial biogenesis. Methods: HepG2-TRß cells were treated with bovine serum albumin-conjugated palmitic acid (PA) to mimic lipotoxic conditions in vitro. Mice with NASH were established by feeding C57BL/6J mice Western diet with 15% fructose in drinking water for 16 weeks. These mice were administered triiodothyronine (T3)/thyroxine (T4) supplemented in drinking water for the next eight weeks. Results: In cultured HepG2-TRß cells, TH treatment increased mitochondrial respiration and fatty acid oxidation under basal and PA-treated conditions, as well as decreased lipopolysaccharides and PA-stimulated inflammatory and fibrotic responses. In a dietary mouse model of NASH, TH administration decreased hepatic triglyceride content (3.19 ± 0.68 vs. 8.04 ± 0.42 mM/g liver) and hydroxyproline (1.44 ± 0.07 vs. 2.58 ± 0.30 mg/g liver) when compared with mice with untreated NASH. Metabolomics profiling of lipid metabolites showed that mice with NASH had increased triacylglycerol, diacylglycerol, monoacylglycerol, and hepatic cholesterol esters species, and these lipid species were decreased by TH treatment. Mice with NASH also showed decreased autophagic degradation as evidenced by decreased transcription Factor EB and lysosomal protease expression, and accumulation of LC3B-II and p62. TH treatment restored the level of lysosomal proteins and resolved the accumulation of LC3B-II and p62. Impaired mitochondrial biogenesis was also restored by TH. The simultaneous restoration of autophagy and mitochondrial biogenesis by TH increased ß-oxidation of fatty acids. Additionally, the elevated oxidative stress and inflammasome activation in NASH liver were also decreased by TH. Conclusions: In a mouse model of NASH, TH restored autophagy and mitochondrial biogenesis to increase ß-oxidation of fatty acids and to reduce lipotoxicity, oxidative stress, hepatic inflammation, and fibrosis. Activating thyroid hormone receptor in the liver may represent an effective strategy for NASH treatment.

Caffeine prevents restenosis and inhibits vascular smooth muscle cell proliferation through the induction of autophagy.

Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Although caffeine has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), the mechanism underlying this effect is unknown. Here, we demonstrated that caffeine decreased VSMC proliferation and induced macroautophagy/autophagy in an in vivo vascular injury model of restenosis. Furthermore, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of MTOR signaling in these cells. Genetic deletion of the key autophagy gene Atg5, and the Sqstm1/p62 gene encoding a receptor protein, showed that the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased WNT-signaling and the expression of two WNT target genes, Axin2 and Ccnd1 (cyclin D1). This effect was mediated by autophagic degradation of a key member of the WNT signaling cascade, DVL2, by caffeine to decrease WNT signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and DVL2 were also shown to interact with each other, and the overexpression of DVL2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings demonstrated that caffeine reduced VSMC proliferation by inhibiting WNT signaling via stimulation of autophagy, thus reducing the vascular restenosis. Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.

Thyroid Hormone Receptor α Regulates Autophagy, Mitochondrial Biogenesis, and Fatty Acid Use in Skeletal Muscle.

Skeletal muscle (SM) weakness occurs in hypothyroidism and resistance to thyroid hormone α (RTHα) syndrome. However, the cell signaling and molecular mechanism(s) underlying muscle weakness under these conditions is not well understood. We thus examined the role of thyroid hormone receptor α (TRα), the predominant TR isoform in SM, on autophagy, mitochondrial biogenesis, and metabolism to demonstrate the molecular mechanism(s) underlying muscle weakness in these two conditions. Two genetic mouse models were used in this study: TRα1PV/+ mice, which express the mutant Thra1PV gene ubiquitously, and SM-TRα1L400R/+ mice, which express TRα1L400R in a muscle-specific manner. Gastrocnemius muscle from TRα1PV/+, SM-TRα1L400R/+, and their control mice was harvested for analyses. We demonstrated that loss of TRα1 signaling in gastrocnemius muscle from both the genetic mouse models led to decreased autophagy as evidenced by accumulation of p62 and decreased expression of lysosomal markers (lysosomal-associated membrane protein [LAMP]-1 and LAMP-2) and lysosomal proteases (cathepsin B and cathepsin D). The expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), mitochondrial transcription factor A (TFAM), and estrogen-related receptor α (ERRα), key factors contributing to mitochondrial biogenesis as well as mitochondrial proteins, were decreased, suggesting that there was reduced mitochondrial biogenesis due to the expression of mutant TRα1. Transcriptomic and metabolomic analyses of SM suggested that lipid catabolism was impaired and was associated with decreased acylcarnitines and tricarboxylic acid cycle intermediates in the SM from the mouse line expressing SM-specific mutant TRα1. Our results provide new insight into TRα1-mediated cell signaling, molecular, and metabolic changes that occur in SM when TR action is impaired.

Chronic cold exposure induces autophagy to promote fatty acid oxidation, mitochondrial turnover, and thermogenesis in brown adipose tissue.

Autophagy plays an important role in lipid breakdown, mitochondrial turnover, and mitochondrial function during brown adipose tissue (BAT) activation by thyroid hormone, but its role in BAT during adaptive thermogenesis remains controversial. Here, we examined BAT from mice exposed to 72 h of cold challenge as well as primary brown adipocytes treated with norepinephrine and found increased autophagy as well as increased ß-oxidation, mitophagy, mitochondrial turnover, and mitochondrial activity. To further understand the role of autophagy of BAT in vivo, we generated BAT-specific Atg5 knockout (Atg5cKO) mice and exposed them to cold for 72 h. Interestingly, BAT-specific Atg5cKO mice were unable to maintain body temperature after chronic cold exposure and displayed deranged mitochondrial morphology and reactive oxygen species damage in their BAT. Our findings demonstrate the critical role of autophagy in adaptive thermogenesis, fatty acid metabolism, and mitochondrial function in BAT during chronic cold exposure.

Protocol to Generate Senescent Cells from the Mouse Hepatic Cell Line AML12 to Study Hepatic Aging.

Previously developed senescent primary fibroblast models have limited relevance to study age-related changes in metabolically active tissues such as the liver. Here, we describe a protocol to generate senescent cells from the mouse hepatic cell line, AML12. These senescent cells exhibit molecular and metabolic signatures that are similar to those observed in livers from aged mice. These senescent AML12 cells should be a useful in vitro model to study the metabolic effects of aging in the liver. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020).

Decreased autophagy and fuel switching occur in a senescent hepatic cell model system.

Although aging in the liver contributes to the development of chronic liver diseases such as NAFLD and insulin resistance, little is known about the molecular and metabolic details of aging in hepatic cells. To examine these issues, we used sequential oxidative stress with hydrogen peroxide to induce premature senescence in AML12 hepatic cells. The senescent cells exhibited molecular and metabolic signatures, increased SA-ßGal and γH2A.X staining, and elevated senescence and pro-inflammatory gene expression that resembled livers from aged mice. Metabolic phenotyping showed fuel switching towards glycolysis and mitochondrial glutamine oxidation as well as impaired energy production. The senescent AML12 cells also had increased mTOR signaling and decreased autophagy which likely contributed to the fuel switching from ß-oxidation that occurred in normal AML12 cells. Additionally, senescence-associated secretory phenotype (SASP) proteins from conditioned media of senescent cells sensitized normal AML12 cells to palmitate-induced toxicity, a known pathological effect of hepatic aging. In summary, we have generated senescent AML12 cells which displayed the molecular hallmarks of aging and also exhibited the aberrant metabolic phenotype, mitochondrial function, and cell signaling that occur in the aged liver.

Estrogen-Related Receptor Alpha: An Under-Appreciated Potential Target for the Treatment of Metabolic Diseases.

The estrogen-related receptor alpha (ESRRA) is an orphan nuclear receptor (NR) that significantly influences cellular metabolism. ESRRA is predominantly expressed in metabolically-active tissues and regulates the transcription of metabolic genes, including those involved in mitochondrial turnover and autophagy. Although ESRRA activity is well-characterized in several types of cancer, recent reports suggest that it also has an important role in metabolic diseases. This minireview focuses on the regulation of cellular metabolism and function by ESRRA and its potential as a target for the treatment of metabolic disorders.

Diagnosis and treatment of hypothyroidism in the elderly.

The global population is aging with millions of people today living into their 90 s. Thyroid disease, particularly hypothyroidism, is widespread among all age groups, and it is expected to steadily increase as the population gets older. Clinical diagnosis of hypothyroidism is challenging, as the TSH reference range needs to be evaluated according to age, while evaluation of TSH levels must also take into account body weight and other variants such as polypharmacy, comorbidities, and general health condition. Since thyroid hormone has a potent regulatory effect on cholesterol metabolism, the possibility of thyroid dysfunction should be considered in cases of unexplained dyslipidemia. Once hypothyroidism has been confirmed, treatment requires caution, frequent cardiovascular monitoring, and individualized (precision) medicine. Treatment of subclinical hypothyroidism (SCH) in the elderly should be undertaken with care, guided by age and the degree of SCH: a TSH higher than 10 mU/l seems a reasonable threshold, though it should be regularly re-evaluated, while the LT4 dose needs to be tailored, taking into account the patient's health condition and the potential presence of dyslipidemia as well as other metabolic derangements.

Hyperthyroidism in the personalized medicine era: the rise of mathematical optimization.

Thyroid over-activity or hyperthyroidism constitutes a significant morbidity afflicting the world. The current medical practice of dose titration of anti-thyroid drug (ATD) treatment for hyperthyroidism is relatively archaic, being based on arbitrary and time-consuming trending of thyroid function that requires multiple clinic monitoring visits before an optimal dose is found. This prompts a re-examination into more deterministic and efficient treatment approaches in the present personalized medicine era. Our research project seeks to develop a personalized medicine model that facilitates optimal drug dosing via the titration regimen. We analysed 49 patients' data consisting of drug dosage, time period and serum free thyroxine (FT4). Ordinary differential equation modelling was applied to describe the dynamic behaviour of FT4 concentration. With each patient's data, an optimization model was developed to determine parameters of synthesis rate, decay rate and IC50. We derived the closed-form time- and dose-dependent solution which allowed explicit estimates of personalized predicted FT4. Our equation system involving time, drug dosage and FT4 can be solved for any variable provided the values of the other two are known. Compared against actual FT4 data within a tolerance, we demonstrated the feasibility of predicting the FT4 subsequent to any prescribed dose of ATD with favourable accuracy using the initial three to five patient-visits' data respectively. This proposed mathematical model may assist clinicians in rapid determination of optimal ATD doses within allowable prescription limits to achieve any desired FT4 within a specified treatment period to accelerate the attainment of euthyroid targets.

Novel Transcriptional Mechanisms for Regulating Metabolism by Thyroid Hormone.

The thyroid hormone plays a key role in energy and nutrient metabolisms in many tissues and regulates the transcription of key genes in metabolic pathways. It has long been believed that thyroid hormones (THs) exerted their effects primarily by binding to nuclear TH receptors (THRs) that are associated with conserved thyroid hormone response elements (TREs) located on the promoters of target genes. However, recent transcriptome and ChIP-Seq studies have challenged this conventional view as discordance was observed between TH-responsive genes and THR binding to DNA. While THR association with other transcription factors bound to DNA, TH activation of THRs to mediate effects that do not involve DNA-binding, or TH binding to proteins other than THRs have been invoked as potential mechanisms to explain this discrepancy, it appears that additional novel mechanisms may enable TH to regulate the mRNA expression. These include activation of transcription factors by SIRT1 via metabolic actions by TH, the post-translational modification of THR, the THR co-regulation of transcription with other nuclear receptors and transcription factors, and the microRNA (miR) control of RNA transcript expression to encode proteins involved in the cellular metabolism. Together, these novel mechanisms enlarge and diversify the panoply of metabolic genes that can be regulated by TH.

Changes in Hepatic TRß Protein Expression, Lipogenic Gene Expression, and Long-Chain Acylcarnitine Levels During Chronic Hyperthyroidism and Triiodothyronine Withdrawal in a Mouse Model.

BACKGROUND: Thyroid hormone (TH) has important roles in regulating hepatic metabolism. It was previously reported that most hepatic genes activated by a single triiodothyronine (T3) injection became desensitized after multiple injections, and that approximately 10% of target genes did not return to basal expression levels after T3 withdrawal, despite normalization of serum TH and thyrotropin (TSH) levels. To determine the possible mechanism(s) for desensitization and incomplete recovery of hepatic target gene transcription and their effects on metabolism, mRNA and/or protein expression levels of key regulators of TH action were measured, as well as metabolomic changes after chronic T3 treatment and withdrawal. METHODS: Adult male mice were treated with daily injections of T3 (20 µg/100 g body weight) for 14 days followed by the cessation of T3 for 10 days. Livers were harvested at 6 hours, 24 hours, and 14 days after the first T3 injection, and at 10 days after withdrawal, and then analyzed by quantitative reverse transcription polymerase chain reaction, Western blotting, and metabolomics. RESULTS: Although TH receptor (TRα and TRß) mRNAs decreased slightly after chronic T3 treatment, only TRß protein decreased before returning to basal expression level after withdrawal. The expression of other regulators of TH action was unchanged. TRß protein expression was also decreased in adult male monocarboxylate transporter-8 (Mct8)-knockout mice, an in vivo model of chronic intrahepatic hyperthyroidism. Previously, increased hepatic long-chain acylcarnitine levels were found after acute TH treatment. However, in this study, long-chain acylcarnitine levels were unchanged after chronic T3, and paradoxically increased after T3 withdrawal. Pathway analyses of the previous microarray results showed upregulation of lipogenic genes after acute T3 treatment and withdrawal. Phosphorylation of acetyl-CoA carboxylase also decreased after T3 withdrawal. CONCLUSIONS: Decreased hepatic TRß protein expression occurred after chronic T3 exposure in adult male wild-type and Mct8-knockout mice. Gene array pathway and metabolomics analyses showed abnormalities in hepatic lipogenic gene expression and acylcarnitine levels, respectively, after withdrawal, despite normalization of serum TSH and TH levels. These findings may help explain the variable clinical presentations of some patients during hyperthyroidism and recovery, since TRß protein, target gene expression, and metabolic adaptive changes can occur in individual tissues without necessarily being reflected by circulating TH and TSH concentrations.

Role of thyroid hormone in hepatic gene regulation, chromatin remodeling, and autophagy.

Thyroid hormone (TH) actions on development and metabolism have been studied ever since the discovery of thyroxine almost a century ago. Initial studies focused on the physiological and biochemical actions of TH. Later, the cloning of the thyroid hormone receptor (THR) isoforms and the development of techniques enabled the study of TH regulation of complex cellular processes (such as gene transcription). Recently we found that TH activates secondary transcription factors such as FOXO1, to amplify gene transcription; and also is a potent inducer of autophagy that was critical for fatty acid ß-oxidation in the liver. This review summarizes the recent advancements in our understanding of TH regulation of gene expression of metabolic genes (via co-regulators/transcription factors and epigenetic control) and autophagy in the liver. Our deeper understanding of TH action recently has led to the development of tissue- and THR isoform-specific TH mimetics that may be useful for the treatment of metabolic disorders.

Hyperhomocysteinemia causes ER stress and impaired autophagy that is reversed by Vitamin B supplementation.

Hyperhomocysteinemia (HHcy) is a well-known risk factor for stroke; however, its underlying molecular mechanism remains unclear. Using both mouse and cell culture models, we have provided evidence that impairment of autophagy has a central role in HHcy-induced cellular injury in the mouse brain. We observed accumulation of LC3B-II and p62 that was associated with increased MTOR signaling in human and mouse primary astrocyte cell cultures as well as a diet-induced mouse model of HHcy, HHcy decreased lysosomal membrane protein LAMP2, vacuolar ATPase (ATP6V0A2), and protease cathepsin D, suggesting that lysosomal dysfunction also contributed to the autophagic defect. Moreover, HHcy increased unfolded protein response. Interestingly, Vitamin B supplementation restored autophagic flux, alleviated ER stress, and reversed lysosomal dysfunction due to HHCy. Furthermore, the autophagy inducer, rapamycin was able to relieve ER stress and reverse lysosomal dysfunction caused by HHcy in vitro. Inhibition of autophagy by HHcy exacerbated cellular injury during oxygen and glucose deprivation and reperfusion (OGD/R), and oxidative stress. These effects were prevented by Vitamin B co-treatment, suggesting that it may be helpful in relieving detrimental effects of HHcy in ischemia/reperfusion or oxidative stress. Collectively, these findings show that Vitamin B therapy can reverse defects in cellular autophagy and ER stress due to HHcy; and thus may be a potential treatment to reduce ischemic damage caused by stroke in patients with HHcy.

Physiological and Metabolic Changes During the Transition from Hyperthyroidism to Euthyroidism in Graves' Disease.

BACKGROUND: The serum metabolomic profile and its relationship to physiological changes during hyperthyroidism and restoration to euthyroidism are not known. This study aimed to examine the physiological, adipokine, and metabolomic changes that occur when subjects with Graves' disease transition from hyperthyroidism to euthyroidism with medical treatment. METHODS: Chinese women between 21 and 50 years of age and with newly diagnosed Graves' disease attending the endocrine outpatient clinics in a single institution were recruited between July 2012 and September 2014. All subjects were treated with thioamides to achieve euthyroidism. Clinical parameters (body weight, body composition via bioelectrical impedance analysis, resting energy expenditure and respiratory quotient via indirect calorimetry, and reported total energy intake via 24 h food diary), biochemical parameters (thyroid hormones, lipid profile, fasting insulin and glucose levels), serum leptin, adiponectin, and metabolomics profiles were measured during hyperthyroidism and repeated in early euthyroidism. RESULTS: Twenty four Chinese women with an average age of 36.3 ± 8.6 years were included in the study. The average duration of treatment that was required to reach euthyroidism for these subjects was 38 ± 16.3 weeks. There was a significant increase in body weight (52.6 ± 9.0 kg to 55.3 ± 9.4 kg; p < 0.001) and fat mass (14.3 ± 6.9 kg to 16.8 ± 6.5 kg; p = 0.005). There was a reduction in resting energy expenditure corrected for weight (28.7 ± 4.0 kcal/kg to 21.5 ± 4.1 kcal/kg; p < 0.001) and an increase in respiratory quotient (0.76 to 0.81; p = 0.037). Resting energy expenditure increased significantly with increasing free triiodothyronine levels (p = 0.007). Significant increases in total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were noted. There was no significant change in leptin levels, but adiponectin levels increased significantly (p = 0.018). Significant reductions in fasting C2, medium-chain, long-chain, and total acylcarnitines were observed, but no changes in the fat-free mass, branched chain amino acid levels, or insulin sensitivity during recovery from hyperthyroidism were noted. CONCLUSIONS: Serum metabolomics profile changes complemented the physiological changes observed during the transition from hyperthyroidism to euthyroidism. This study provides a comprehensive and integrated view of the changes in fuel metabolism and energy balance that occur following the treatment of hyperthyroidism.

Desensitization and Incomplete Recovery of Hepatic Target Genes After Chronic Thyroid Hormone Treatment and Withdrawal in Male Adult Mice.

Clinical symptoms may vary and not necessarily reflect serum thyroid hormone (TH) levels during acute and chronic hyperthyroidism as well as recovery from hyperthyroidism. We thus examined changes in hepatic gene expression and serum TH/TSH levels in adult male mice treated either with a single T3 (20 µg per 100 g body weight) injection (acute T3) or daily injections for 14 days (chronic T3) followed by 10 days of withdrawal. Gene expression arrays from livers harvested at these time points showed that among positively-regulated target genes, 320 were stimulated acutely and 429 chronically by T3. Surprisingly, only 69 of 680 genes (10.1%) were induced during both periods, suggesting desensitization of the majority of acutely stimulated target genes. About 90% of positively regulated target genes returned to baseline expression levels after 10 days of withdrawal; however, 67 of 680 (9.9%) did not return to baseline despite normalization of serum TH/TSH levels. Similar findings also were observed for negatively regulated target genes. Chromatin immunoprecipitation analysis of representative positively regulated target genes suggested that acetylation of H3K9/K14 was associated with acute stimulation, whereas trimethylation of H3K4 was associated with chronic stimulation. In an in vivo model of chronic intrahepatic hyperthyroidism since birth, adult male monocarboxylate transporter-8 knockout mice also demonstrated desensitization of most acutely stimulated target genes that were examined. In summary, we have identified transcriptional desensitization and incomplete recovery of gene expression during chronic hyperthyroidism and recovery. Our findings may be a potential reason for discordance between clinical symptoms and serum TH levels observed in these conditions.