Chemical Hybridization of Glucagon and Thyroid Hormone Optimizes Therapeutic Impact for Metabolic Disease.

Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.

Gene therapy targeting the blood-brain barrier improves neurological symptoms in a model of genetic MCT8 deficiency.

A genetic deficiency of the solute carrier monocarboxylate transporter 8 (MCT8), termed Allan-Herndon-Dudley syndrome, is an important cause of X-linked intellectual and motor disability. MCT8 transports thyroid hormones across cell membranes. While thyroid hormone analogues improve peripheral changes of MCT8 deficiency, no treatment of the neurological symptoms is available so far. Therefore, we tested a gene replacement therapy in Mct8- and Oatp1c1-deficient mice as a well-established model of the disease. Here, we report that targeting brain endothelial cells for Mct8 expression by intravenously injecting the vector AAV-BR1-Mct8 increased tri-iodothyronine (T3) levels in the brain and ameliorated morphological and functional parameters associated with the disease. Importantly, the therapy resulted in a long-lasting improvement in motor coordination. Thus, the data support the concept that MCT8 mediates the transport of thyroid hormones into the brain and indicate that a readily accessible vascular target can help overcome the consequences of the severe disability associated with MCT8 deficiency.

Pharmacological targeting of α3ß4 nicotinic receptors improves peripheral insulin sensitivity in mice with diet-induced obesity.

AIMS/HYPOTHESIS: Treatment with the α3ß4 nicotinic acetylcholine receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), improves glucose tolerance in diet-induced obese (DIO) mice, but the physiological and molecular mechanisms are unknown. METHODS: DMPP (10 mg/kg body weight, s.c.) was administered either in a single injection (acute) or daily for up to 14 days (chronic) in DIO wild-type (WT) and Chrnb4 knockout (KO) mice and glucose tolerance, tissue-specific tracer-based glucose metabolism, and insulin signalling were assessed. RESULTS: In WT mice, but not in Chrnb4 KO mice, single acute treatment with DMPP induced transient hyperglycaemia, which was accompanied by high plasma adrenaline (epinephrine) levels, upregulated hepatic gluconeogenic genes, and decreased hepatic glycogen content. In contrast to these acute effects, chronic DMPP treatment in WT mice elicited improvements in glucose tolerance already evident after three consecutive days of DMPP treatment. After seven days of DMPP treatment, glucose tolerance was markedly improved, also in comparison with mice that were pair-fed to DMPP-treated mice. The glycaemic benefit of chronic DMPP was absent in Chrnb4 KO mice. Chronic DMPP increased insulin-stimulated glucose clearance into brown adipose tissue (+69%), heart (+93%), gastrocnemius muscle (+74%) and quadriceps muscle (+59%), with no effect in white adipose tissues. After chronic DMPP treatment, plasma adrenaline levels did not increase following an injection with DMPP. In glucose-stimulated skeletal muscle, we detected a decreased phosphorylation of the inhibitory Ser640 phosphorylation site on glycogen synthase and a congruent increase in glycogen accumulation following chronic DMPP treatment. CONCLUSIONS/INTERPRETATION: Our data suggest that DMPP acutely induces adrenaline release and hepatic glycogenolysis, while chronic DMPP-mediated activation of ß4-containing nAChRs improves peripheral insulin sensitivity independently of changes in body weight via mechanisms that could involve increased non-oxidative glucose disposal into skeletal muscle.

Persistent Organic Pollutants in Human Breast Milk and Associations with Maternal Thyroid Hormone Homeostasis.

Epidemiological studies have indicated the thyroid-disrupting effects of persistent organic pollutants (POPs). However, the association of low-exposure POPs with thyroid hormones (THs) remains unclear. Here, we aim to assess the association of low exposure of POPs, including polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), and polybrominated dibenzo-p-dioxins and furans, with THs [total L-thyroxine (TT4), total 3,3',5-triiodo-L-thyronine (TT3), and total 3,3',5'-triiodo-L-thyronine (TrT3)] measured in human breast milk. Ninety-nine breast milk samples were collected from the LUPE cohort (2015-2016, Bavaria, Germany). Fourteen PBDEs, 17 PCBs, and 5 PCDD/Fs had quantification rates of >80%. Nonmonotonic associations were observed. In adjusted single-pollutant models, (1) TT4 was inversely associated with BDE-99, -154, and -196; (2) TT3 was inversely associated with BDE-47, -99, -100, -197, -203, -207, and OCDD; and (3) TrT3 was inversely associated with BDE-47, -99, -183, and -203. Multipollutant analysis using principal component analysis and hierarchical clustering revealed inverse associations of PBDEs (BDE-28, -47, -99, -100, -154, -183, and -197) with TT4 and TrT3. These results indicate that POPs at low levels might be related to reduced THs. This study shows that human breast milk might be an appropriate specimen to evaluate the thyroid disruption of POPs.

Comprehensive analysis of nine monoamines and metabolites in small amounts of peripheral murine (C57Bl/6 J) tissues.

Monoamines, acting as hormones and neurotransmitters, play a critical role in multiple physiological processes ranging from cognitive function and mood to sympathetic nervous system activity, fight-or-flight response and glucose homeostasis. In addition to brain and blood, monoamines are abundant in several tissues, and dysfunction in their synthesis or signaling is associated with various pathological conditions. It was our goal to develop a method to detect these compounds in peripheral murine tissues. In this study, we employed a high-performance liquid chromatography method using electrochemical detection that allows not only detection of catecholamines but also a detailed analysis of nine monoamines and metabolites in murine tissues. Simple tissue extraction procedures were optimized for muscle (gastrocnemius, extensor digitorum longus and soleus), liver, pancreas and white adipose tissue in the range of weight 10-200 mg. The system allowed a limit of detection between 0.625 and 2.5 pg µL-1 for monoamine analytes and their metabolites, including dopamine, 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine, homovanillic acid, norepinephrine, epinephrine, 3-methoxy-4-hydroxyphenylglycol, serotonin and 5-hydroxyindoleacetic acid. Typical concentrations for different monoamines and their metabolization products in these tissues are presented for C57Bl/6 J mice fed a high-fat diet.

Novel methyl substituted 1-(5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanones are P2X7 antagonists.

The optimization efforts that led to a novel series of methyl substituted 1-(5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanones that are potent rat and human P2X7 antagonists are described. These efforts resulted in the discovery of compounds with good drug-like properties that are capable of high P2X7 receptor occupancy in rat following oral administration, including compounds 7n (P2X7 IC50 = 7.7 nM) and 7u (P2X7 IC50 =7 .7 nM). These compounds are expected to be useful tools for characterizing the effects of P2X7 antagonism in models of depression and epilepsy, and several of the compounds prepared are candidates for effective P2X7 PET tracers.

HDAC5 controls a hypothalamic STAT5b-TH axis, the sympathetic activation of ATP-consuming futile cycles and adult-onset obesity in male mice.

With age, metabolic perturbations accumulate to elevate our obesity burden. While age-onset obesity is mostly driven by a sedentary lifestyle and high calorie intake, genetic and epigenetic factors also play a role. Among these, members of the large histone deacetylase (HDAC) family are of particular importance as key metabolic determinants for healthy ageing, or metabolic dysfunction. Here, we aimed to interrogate the role of class 2 family member HDAC5 in controlling systemic metabolism and age-related obesity under non-obesogenic conditions. Starting at 6 months of age, we observed adult-onset obesity in chow-fed male global HDAC5-KO mice, that was accompanied by marked reductions in adrenergic-stimulated ATP-consuming futile cycles, including BAT activity and UCP1 levels, WAT-lipolysis, skeletal muscle, WAT and liver futile creatine and calcium cycles, and ultimately energy expenditure. Female mice did not differ between genotypes. The lower peripheral sympathetic nervous system (SNS) activity in mature male KO mice was linked to higher dopaminergic neuronal activity within the dorsomedial arcuate nucleus (dmARC) and elevated hypothalamic dopamine levels. Mechanistically, we reveal that hypothalamic HDAC5 acts as co-repressor of STAT5b over the control of Tyrosine hydroxylase (TH) gene transactivation, which ultimately orchestrates the activity of dmARH dopaminergic neurons and energy metabolism in male mice under non-obesogenic conditions.

Discovery of a new series of [1,2,4]triazolo[4,3-a]quinoxalines as dual phosphodiesterase 2/phosphodiesterase 10 (PDE2/PDE10) inhibitors.

The synthesis, preliminary evaluation and structure-activity relationship (SAR) of a series of 1-aryl-4-methyl[1,2,4]triazolo[4,3-a]quinoxalines as dual phosphodiesterase 2/phosphodiesterase 10 (PDE2/PDE10) inhibitors are described. From this investigation compound 31 was identified, showing good combined potency, acceptable brain uptake and high selectivity for both PDE2 and PDE10 enzymes. Compound 31 was subjected to a microdosing experiment in rats, showing preferential distribution in brain areas where both PDE2 and PDE10 are highly expressed. These promising results may drive the further development of highly potent combined PDE2/PDE10 inhibitors, or even of selective inhibitors of PDE2 and/or PDE10.

Perinatal effects of persistent organic pollutants on thyroid hormone concentration in placenta and breastmilk.

Thyroid hormones (TH) are known to play a critical role in regulating many biological processes including growth and development, energy homeostasis, thermogenesis, lipolysis and metabolism of cholesterol. Severe TH deficiency especially during fetal development results in cretinism, but can also lead to an imbalance in metabolism with, among others, an alteration in body weight composition. Over the past two decades, increasing evidence has shown that certain persistent organic pollutants (POP) can interfere with the endocrine system. These POP referred to as "endocrine disrupting chemicals" are widely present in the environment and populations are exposed globally. Moreover, epidemiological studies have shown that a particularly sensitive period is the pre- and postnatal time. Indeed, perinatal exposure to such chemicals could lead to the onset diseases in later life. It is known, that, maternal thyroid hormones are transported by the placenta to the fetus from 6 weeks of gestation and it seems that during the first trimester, and part of the second, the fetus is entirely dependent on maternal TH supply for its development. Interferences in the TH-network as a consequence of the exposure to such pollutants could cause variations in TH concentration. Only small changes in maternal thyroid hormone levels in early stages of pregnancy can influence fetal neurological and cardiovascular development, as well as according to recent studies, have effect on childhood body composition. With this review, we will report the most recent and important studies concerning the association between thyroid hormone concentration and POP levels measured during the perinatal period. We will mostly focus on the data recently reported on placenta and breastmilk as main sources for understanding the potential consequences of exposure. The possible link between exposure to pollutants, TH dysregulation and possible adverse outcome will also be briefly discussed. From our literature search, several studies support the hypothesis that pre- and postnatal exposure to different pollutants might play a role in causing variation in thyroid hormone concentration. However, few research papers have so far studied the relationship linking exposure to pollutants, TH concentration and possible health consequences. Therefore, this review highlights the need for further research in this direction.

Mediobasal hypothalamic FKBP51 acts as a molecular switch linking autophagy to whole-body metabolism.

The mediobasal hypothalamus (MBH) is the central region in the physiological response to metabolic stress. The FK506-binding protein 51 (FKBP51) is a major modulator of the stress response and has recently emerged as a scaffolder regulating metabolic and autophagy pathways. However, the detailed protein-protein interactions linking FKBP51 to autophagy upon metabolic challenges remain elusive. We performed mass spectrometry-based metabolomics of FKBP51 knockout (KO) cells revealing an increased amino acid and polyamine metabolism. We identified FKBP51 as a central nexus for the recruitment of the LKB1/AMPK complex to WIPI4 and TSC2 to WIPI3, thereby regulating the balance between autophagy and mTOR signaling in response to metabolic challenges. Furthermore, we demonstrated that MBH FKBP51 deletion strongly induces obesity, while its overexpression protects against high-fat diet (HFD)-induced obesity. Our study provides an important novel regulatory function of MBH FKBP51 within the stress-adapted autophagy response to metabolic challenges.

Development and validation of an LC-MS/MS methodology for the quantification of thyroid hormones in dko MCT8/OATP1C1 mouse brain.

The Allan-Herndon Dudley Syndrome (AHDS) is a rare disease caused by the progressive loss of monocarboxylate transporter 8 (MCT8). In patients with AHDS, the absence of MCT8 impairs transport of thyroid hormones (TH) through the blood brain barrier, leading to a central state of TH deficiency. In mice, the AHDS is mimicked by simultaneous deletion of the TH transporters MCT8 and the solute carrier organic anion transporter family member 1c1 (OATP1C1). To support preclinical mouse studies, an analytical methodology was developed and successfully applied for quantifying selected thyroid hormones in mouse whole brain and in specific regions using liquid chromatography tandem mass-spectrometry (LC-MS/MS). An important requirement for the methodology was its high sensitivity since a very low concentration of THs was expected in MCT8/OATP1C1 double-knockout (dko) mouse brain. Seven THs were targeted: L-thyroxine (T4), 3,3´,5-triiodo-L-thyronine-thyronine (T3), 3,3´,5´-triiodo-L-thyronine-thyronine (rT3), 3,3´-diiodo-L-thyronine (3,3´-T2, T2), 3,5-diiodo-L-thyronine (rT2, 3,5-T2), 3-iodo-L-thyronine (T1), 3-iodothyronamine (T1AM). Isotope dilution liquid chromatography triple-quadrupole mass spectrometry methodology was applied for detection and quantification. The method was validated in wild-type animals for mouse whole brain and for five different brain regions (hypothalamus, hippocampus, prefrontal cortex, brainstem and cortex). Instrumental calibration curves ranged from 0.35 to 150 pg/µL with good linearity (r2 >0.996). The limit of quantification was from 0.08 to 0.6 pg/mg, with an intra- and inter-day precision of 4.2-14.02% and 0.4-17.9% respectively, and accuracies between 84.9% and 114.8% when the methodology was validated for the whole brain. In smaller, distinct brain regions, intra- and inter-day precision were 0.6-20.7% and 2.5-15.6% respectively, and accuracies were 80.2-128.6%. The new methodology was highly sensitive and allowed for the following quantification in wild-type mice: (i) for the first time, four distinct thyroid hormones (T4, T3, rT3 and 3,3´-T2) in only approximately 100 mg of mouse brain were detected; (ii) the quantification of T4 and T3 for the first time in distinct mouse brain regions were reported. Further, application of our method to MCT8/OATP1C1 dko mice revealed the expected, relative lack of T3 and T4 uptake into the brain, and confirmed the utility of our analytical method to study TH transport across the blood brain barrier in a preclinical model of central TH deficiency.

Determination of 3-iodothyronamine (3-T1AM) in mouse liver using liquid chromatography-tandem mass spectrometry.

3-iodothyronamine (3-T1AM) has been suggested as a novel chemical messenger and potent trace amine-associated receptor 1 ligand in the CNS that occurs naturally as endogenous metabolite of the thyroid hormones. Discrepancies and variations in 3-T1AM plasma and tissue concentrations have nonetheless caused controversy regarding the existence and biological role of 3-T1AM. These discussions are at least partially based on potential analytical artefacts caused by differential decay kinetics of 3-T1AM and the widely used deuterated quantification standard D4-T1AM. Here, we report a novel LC-MS/MS method for the quantification of 3-T1AM in biological specimens using stable isotope dilution with 13C6-T1AM, a new internal standard that showed pharmacodynamic properties comparable to endogenous 3-T1AM. The method detection limit (MDL) and method quantification limit (MQL) of 3-T1AM were 0.04 and 0.09 ng/g, respectively. The spike-recoveries of 3-T1AM were between 85.4% and 94.3%, with a coefficient of variation of 3.7-5.8%. The intra-day and inter-day variations of 3-T1AM were 8.45-11.2% and 3.58-5.73%, respectively. Endogenous 3-T1AM liver values in C57BL/6J mice were 2.20 ± 0.49 pmol/g with a detection frequency of 50%. Higher liver 3-T1AM values were found when C57BL/6J mice were treated with N-acetyl-3-iodothyronamine or O-acetyl-3-iodothyronamine. Overall, our new stable isotope dilution LC-MS/MS method improves both the sensitivity and selectivity compared with existing methods. The concomitant possibility to quantify additional thyroid hormones such as thyroxine, 3,5,3'-triiodo-L-thyronine, 3,3',5'-triiodo-L-thyronine, 3,3'-diiodo-L-thyronine, and 3,5-diiodo-L-thyronine further adds to the value of our novel method in exploring the natural occurrence and fate of 3-T1AM in biological tissues and fluids.

Multiple persistent organic pollutants in mothers' breastmilk: Implications for infant dietary exposure and maternal thyroid hormone homeostasis in Uganda, East Africa.

Persistent organic pollutants (POPs) are ubiquitous contaminants with adverse health effects in the ecosystem. One of such effects is endocrine disruption in humans and wildlife even at background exposure concentrations. This study assessed maternal breastmilk concentrations of POPs; brominated flame retardants (BFRs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), and the potential health risks posed to the nursing infants. We also evaluated the association of these POPs with total 3,3',5-triiodo-L-thyronine (T3), L-thyroxine (T4), and 3,3',5'-triiodo-L-thyronine (rT3) levels measured in human breast milk. Thirty breastmilk samples were collected from Kampala, Uganda between August and December 2018. Hexabromobenzene was not detected while the maximum level of 2,2',4,4',5,5'-hexabrombiphenyl was 64.7 pg/g lw. The median levels of total indicator PCBs, PBDEs, dioxin-like PCBs, and PCDD/Fs in the samples were 159 pg/g lw, 511 pg/g lw, 1.16 pg TEQ/g lw, and 0.4 pg TEQ/g lw, respectively. These levels were lower than those reported in other countries. Owing to their bio accumulative nature, PCBs -81, -169, and ∑PCDD/Fs increased with increase in maternal age. Estimated dietary intakes for dioxin-like PCBs and PCDD/Fs were lower than those reported elsewhere but were higher than the WHO tolerable daily intakes suggesting potential health risks to nursing infants. In adjusted single pollutant models, PCB-126, PCB-169, and ∑PCBTEQ were negatively associated with T3, while 1,2,3,4,5,7,8-HpCDF was positively associated with rT3. Although these associations did not persist in multipollutant models, our findings suggest potential thyroid hormone disruption by POPs in mothers. This may reduce the levels of thyroid hormones transferred from the mother to the neonates and, hence, adversely influence infant growth. A temporal study with a bigger sample size is required to corroborate these findings.

Detection and quantification of the anti-obesity drug celastrol in murine liver and brain.

Celastrol is a natural pentacyclic triterpene extracted from the roots of Tripterygium wilfordi (thunder god vine). Celastrol was reported as a powerful anti-obesity drug with leptin sensitizing properties that decreases food consumption and mediates body weight loss when administered to diet-induced obese mice at 100 µg/kg body weight. The weight lowering properties of celastrol are likely mediated by the CNS, in particular, by the hypothalamus, but the final proof for the accumulation of celastrol in the brain and hypothalamus remains to be established. Here, we aimed to demonstrate that intraperitoneal celastrol administration at 100 µg/kg can rapidly reach the brain and, in particular, the hypothalamus of mice. We developed and validated a sensitive liquid chromatography mass spectrometry method for the quantitative determination of celastrol in murine tissues, namely liver, brain and hypothalamus. Chow-fed lean mice were randomly assigned to the vehicle vs. celastrol groups, injected with saline or 100 µg/kg body weight of celastrol, and sacrificed 30 min or 120 min post injection. Celastrol was extracted from homogenized tissue using ethyl acetate as organic solvent, and quantified using a matrix-matched calibration curve with glycyrrhetinic acid as internal standard. Liver celastrol concentrations were 32.60 ± 8.21 pg/mg and 40.52 ± 15.6 pg/mg, 30 and 120 min after injection, respectively. We found 4.70 ± 0.31 pg/mg celastrol after 30 min, and 16.22 ± 3.33 pg/mg after 120 min in whole brain lysates, and detectable amounts in the hypothalamus. These results corroborate the validity of our methodology, demonstrate the accumulation of celastrol in the brain of mice injected intraperitoneally with a dose of 100 µg/kg, and confirm the CNS as possible site of action for the weight lowering properties of celastrol.

Placental distribution of endogenous and exogenous substances: A pilot study utilizing cryo-sampled specimen off delivery room.

INTRODUCTION: Reliability in the use of placentome (including placenta, umbilical cord, and cord blood) biomarkers requires an understanding of their distributions. Here we aim to develop a simple and proper placenta sampling scheme, and to evaluate the placental distributions of biomarkers. METHODS: We developed a continuous cooling chain protocol off delivery room and cryo-subsampling method for placenta sampling. The levels of thyroid hormones (THs), elements, persistent organic pollutants (POPs), monoamines, and vitamin E were measured using UPLC-Q-TOF-MS, HPLC-ICP-MS, HPLC-EcD, and HRGC-HRMS, respectively. The distributions of biomarkers were assessed. RESULTS: In human placentome, l-thyroxine (T4), Cd, Se, Zn, Cu, Fe, Ca, K, Mg, α-tocopherol, ß-tocopherol, and ß-tocotrienol levels were higher in placenta than in umbilical cord, while Pb and Mn were concentrated in human cord. In porcine placentome, T4, 3,3',5'-triiodo-l-thyronine (rT3), 3,3'-diiodo-l-thyronine, Cd, Pb, Zn, K, and Al levels were higher in the cord. The intraclass correlation coefficient (ICC) was <0.4 for 3,3',5-triiodo-l-thyronine, rT3, α-tocopherol, and 7 elements in human basal plate, indicating low reliability. rT3, Cd, Zn, Mn, and Cu were significantly concentrated in the central region in human placenta, while higher levels of As, Cd, Cr, and Al were found in the periphery region in porcine placenta. Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) showed moderate reliability (ICC: 0.40-0.98) except PCB-81, -126, and BDE-208, while polychlorinated dibenzo-p-doixins/furans (PCDD/Fs) showed poor reliability (ICC: 0.07-0.31). DISCUSSION: These results highlight the complexity of placenta sampling. This study provides a novel and simple sampling approach in investigating placental exposomics.

Type 2 diabetes risk gene Dusp8 regulates hypothalamic Jnk signaling and insulin sensitivity.

Recent genome-wide association studies (GWAS) identified DUSP8, encoding a dual-specificity phosphatase targeting mitogen-activated protein kinases, as a type 2 diabetes (T2D) risk gene. Here, we reveal that Dusp8 is a gatekeeper in the hypothalamic control of glucose homeostasis in mice and humans. Male, but not female, Dusp8 loss-of-function mice, either with global or corticotropin-releasing hormone neuron-specific deletion, had impaired systemic glucose tolerance and insulin sensitivity when exposed to high-fat diet (HFD). Mechanistically, we found impaired hypothalamic-pituitary-adrenal axis feedback, blunted sympathetic responsiveness, and chronically elevated corticosterone levels driven by hypothalamic hyperactivation of Jnk signaling. Accordingly, global Jnk1 ablation, AAV-mediated Dusp8 overexpression in the mediobasal hypothalamus, or metyrapone-induced chemical adrenalectomy rescued the impaired glucose homeostasis of obese male Dusp8-KO mice, respectively. The sex-specific role of murine Dusp8 in governing hypothalamic Jnk signaling, insulin sensitivity, and systemic glucose tolerance was consistent with functional MRI data in human volunteers that revealed an association of the DUSP8 rs2334499 risk variant with hypothalamic insulin resistance in men. Further, expression of DUSP8 was increased in the infundibular nucleus of T2D humans. In summary, our findings suggest the GWAS-identified gene Dusp8 as a novel hypothalamic factor that plays a functional role in the etiology of T2D.

Aging Is Associated with Low Thyroid State and Organ-Specific Sensitivity to Thyroxine.

Background: Serum thyroid state in older adults correlates with extended longevity. We hypothesized that age impacts not only systemic but also organ-specific thyroid state and response to thyroxine (T4). Methods: Young (3 months) and old (23 months) male mice were analyzed at baseline and after acute T4 challenge. Age effects on circulating thyrotropin (TSH) and thyroid hormone (TH) concentrations, transcript expression in the pituitary and thyroid were compared with organ-specific responses characterized by hepatic and cardiac content of TH and TH metabolites and expression of TH-target genes, as well as hepatic deiodinase 1 activity. Results: Circulating TH concentrations and hepatic and cardiac TH content were lower in old versus young mice. After injection with T4, conversion of T4 to triiodothyronine was decreased in old mice while TH transport in liver and heart was not affected. Organ-specific TH response was augmented in old mice in liver but not heart, indicating age- and tissue-specific sensitivity to TH. A compensatory increase of thyroid stimulating hormone subunit beta expression in the pituitary and increased serum TSH concentrations, but reduced expression of thyroid differentiation markers were found in old mice. Conclusions: We suggest that a reduced activity of the aged thyroid is responsible for the systemic low TH state in old mice. Further, divergent TH metabolism and tissue response in liver and heart occur after T4 treatment in an aged organism. These rodent data are in agreement with a much narrower window for T4 substitution in the older adults to avoid overtreatment.

Association of In Utero Persistent Organic Pollutant Exposure With Placental Thyroid Hormones.

In utero exposure to persistent organic pollutants (POPs) can result in thyroid function disorder, leading to concerns about their impact on fetal and neonatal development. The associations between placental levels of various POPs and thyroid hormones (THs) were investigated. In a prospective Danish study initially established for assessing congenital cryptorchidism, 58 placenta samples were collected from mothers of boys born with (n = 28) and without (n = 30) cryptorchidism. The concentrations of polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), organotin chemicals (OTCs), organochlorine pesticides (OCPs), T4, T3, and rT3 were measured. The associations between placental THs and various POPs were analyzed using multiple linear regression. Five PBDEs, 35 PCBs, 14 PCDD/Fs, 3 OTCs, 25 OCPs, T4, T3, and rT3 were measured. No correlation between THs and the odds of cryptorchidism was found. Several POPs were significantly associated with THs: (1) T4 was inversely associated with BDEs 99, 100, ΣPBDE, and 2378-TeCDD, and positively associated with 1234678-HpCDF; (2) T3 was positively associated with 2378-TeCDF and 12378-PeCDF; and (3) rT3 was positively associated with PCB 81, 12378-PeCDF, and 234678-HxCDF, and inversely associated with tributyltin, ΣOTC, and methoxychlor. These results revealed that POP exposures were associated with TH levels in placenta, which may be a possible mechanism for the impacts of POP exposures on children's growth and development. This study provides new insight into the complexity of thyroid-disrupting properties of POPs. More research is needed to elucidate the biological consequences of POP exposures.

Determination of thyroid hormones in placenta using isotope-dilution liquid chromatography quadrupole time-of-flight mass spectrometry.

The transplacental passage of thyroid hormones (THs) is of great significance since the maternal THs are vitally important in ensuring the normal fetal development. In this paper, we determined the concentrations of seven THs, viz. L-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3), 3,3',5'-triiodo-l-thyronine (rT3), 3,3'-diiodo-l-thyronine (T2), 3,5-diiodo-l-thyronine (rT2), 3-iodo-l-thyronine (T1) and 3-iodothyronamine (T1AM), in placenta using isotope dilution liquid chromatography quadrupole time-of-flight mass spectrometry. We optimized the method using isotopically labeled quantification standards (13C6-T4, 13C6-T3, 13C6-rT3 and 13C6-T2) and recovery standard (13C12-T4) in combination with solid-liquid extraction, liquid-liquid extraction and solid phase extraction. The linearity was obtained in the range of 0.5-150 pg uL-1 with R2 values >0.99. The method detection limits (MDLs) ranged from 0.01 ng g-1 to 0.2 ng g-1, while the method quantification limits (MQLs) were between 0.04 ng g-1 and 0.7 ng g-1. The spike-recoveries for THs (except for T1 and T1AM) were in the range of 81.0%-112%, with a coefficient of variation (CV) of 0.5-6.2%. The intra-day CVs and inter-day CVs were 0.5%-10.3% and 1.19%-8.88%, respectively. Concentrations of the THs were 22.9-35.0 ng g-1 T4, 0.32-0.46 ng g-1 T3, 2.86-3.69 ng g-1 rT3, 0.16-0.26 ng g-1 T2, and < MDL for other THs in five human placentas, and 2.05-3.51 ng g-1 T4, 0.37-0.62 ng g-1 T3, 0.96-1.3 ng g-1 rT3, 0.07-0.13 ng g-1 T2 and < MDL for other THs in five mouse placentas. The presence of T2 was tracked in placenta for the first time. This method with improved selectivity and sensitivity allows comprehensive evaluation of TH homeostasis in research of metabolism and effects of environmental contaminant exposures.

Celastrol Promotes Weight Loss in Diet-Induced Obesity by Inhibiting the Protein Tyrosine Phosphatases PTP1B and TCPTP in the Hypothalamus.

Celastrol is a natural pentacyclic triterpene used in traditional Chinese medicine with significant weight-lowering effects. Celastrol-administered mice at 100 µg/kg decrease food consumption and body weight via a leptin-dependent mechanism, yet its molecular targets in this pathway remain elusive. Here, we demonstrate in vivo that celastrol-induced weight loss is largely mediated by the inhibition of leptin negative regulators protein tyrosine phosphatase (PTP) 1B (PTP1B) and T-cell PTP (TCPTP) in the arcuate nucleus (ARC) of the hypothalamus. We show in vitro that celastrol binds reversibly and inhibits noncompetitively PTP1B and TCPTP. NMR data map the binding site to an allosteric site in the catalytic domain that is in proximity of the active site. By using a panel of PTPs implicated in hypothalamic leptin signaling, we show that celastrol additionally inhibited PTEN and SHP2 but had no activity toward other phosphatases of the PTP family. These results suggest that PTP1B and TCPTP in the ARC are essential for celastrol's weight lowering effects in adult obese mice.