GLIS3 expression in the thyroid gland in relation to TSH signaling and regulation of gene expression.

Loss of GLI-Similar 3 (GLIS3) function in mice and humans causes congenital hypothyroidism (CH). In this study, we demonstrate that GLIS3 protein is first detectable at E15.5 of murine thyroid development, a time at which GLIS3 target genes, such as Slc5a5 (Nis), become expressed. This, together with observations showing that ubiquitous Glis3KO mice do not display major changes in prenatal thyroid gland morphology, indicated that CH in Glis3KO mice is due to dyshormonogenesis rather than thyroid dysgenesis. Analysis of GLIS3 in postnatal thyroid suggested a link between GLIS3 protein expression and blood TSH levels. This was supported by data showing that treatment with TSH, cAMP, or adenylyl cyclase activators or expression of constitutively active PKA enhanced GLIS3 protein stability and transcriptional activity, indicating that GLIS3 activity is regulated at least in part by TSH/TSHR-mediated activation of PKA. The TSH-dependent increase in GLIS3 transcriptional activity would be critical for the induction of GLIS3 target gene expression, including several thyroid hormone (TH) biosynthetic genes, in thyroid follicular cells of mice fed a low iodine diet (LID) when blood TSH levels are highly elevated. Like TH biosynthetic genes, the expression of cell cycle genes is suppressed in ubiquitous Glis3KO mice fed a LID; however, in thyroid-specific Glis3 knockout mice, the expression of cell cycle genes was not repressed, in contrast to TH biosynthetic genes. This indicated that the inhibition of cell cycle genes in ubiquitous Glis3KO mice is dependent on changes in gene expression in GLIS3 target tissues other than the thyroid.

Maintaining the thyroid gland in mutant thyroglobulin-induced hypothyroidism requires thyroid cell proliferation that must continue in adulthood.

Congenital hypothyroidism with biallelic thyroglobulin (Tg protein, encoded by the TG gene) mutation is an endoplasmic reticulum (ER) storage disease. Many patients (and animal models) grow an enlarged thyroid (goiter), yet some do not. In adulthood, hypothyroid TGcog/cog mice (bearing a Tg-L2263P mutation) exhibit a large goiter, whereas adult WIC rats bearing the TGrdw/rdw mutation (Tg-G2298R) exhibit a hypoplastic thyroid. Homozygous TG mutation has been linked to thyroid cell death, and cytotoxicity of the Tg-G2298R protein was previously thought to explain the lack of goiter in WIC-TGrdw/rdw rats. However, recent studies revealed that TGcog/cog mice also exhibit widespread ER stress-mediated thyrocyte death, yet under continuous feedback stimulation, thyroid cells proliferate in excess of their demise. Here, to examine the relative proteotoxicity of the Tg-G2298R protein, we have used CRISPR-CRISPR-associated protein 9 technology to generate homozygous TGrdw/rdw knock-in mice in a strain background identical to that of TGcog/cog mice. TGrdw/rdw mice exhibit similar phenotypes of defective Tg protein folding, thyroid histological abnormalities, hypothyroidism, and growth retardation. TGrdw/rdw mice do not show evidence of greater ER stress response or stress-mediated cell death than TGcog/cog mice, and both mouse models exhibit sustained thyrocyte proliferation, with comparable goiter growth. In contrast, in WIC-TGrdw/rdw rats, as a function of aging, the thyrocyte proliferation rate declines precipitously. We conclude that the mutant Tg-G2298R protein is not intrinsically more proteotoxic than Tg-L2263P; rather, aging-dependent difference in maintenance of cell proliferation is the limiting factor, which accounts for the absence of goiter in adult WIC-TGrdw/rdw rats.

Motor-sparing peripatellar plexus block provides noninferior block duration and complete block area of the peripatellar region compared with femoral nerve block: a randomized, controlled, noninferiority study.

BACKGROUND: Developing adequate regional anaesthesia for knee surgeries without affecting lower limb mobilization is crucial to perioperative analgesia. However, reports in this regard are limited. We proposed a technique for ultrasound-guided peripatellar plexus (PP) block. Compared with the femoral nerve (FN) block, we hypothesized that this technique would provide a noninferior block duration and a complete cutaneous sensory block in the peripatellar region without affecting lower limb mobilization. An investigation was conducted to verify our hypothesis in cadavers and volunteers. METHODS: The study was designed in two parts. First, eight cadaveric lower limbs were dissected to verify the feasibility of PP block after methylene blue injection under ultrasound. Second, using a noninferiority study design, 50 healthy volunteers were randomized to receive either a PP block (PP group) or an FN block (FN group). The primary outcome was the duration of peripatellar cutaneous sensory block, with the prespecified noninferiority margin of -3.08 h; the secondary outcome was the area of peripatellar cutaneous sensory block; in addition, the number of complete anaesthesias of the incision line for total knee arthroplasty and the Bromage score 30 min after block were recorded. RESULTS: The PP was successfully dyed, whereas the FN and saphenous nerve were unstained in all cadaveric limbs. The mean difference of the block duration between the two groups was - 1.24 (95% CI, -2.81 - 0.33) h, and the lower boundary of the two-sided 95% CI was higher than the prespecified noninferiority margin (Pnoninferiority = 0.023), confirming the noninferiority of our technique over FN block. The cutaneous sensory loss covered the entire peripatellar region in the PP group. PP block achieved complete anaesthesia of the incision line used for total knee arthroplasty and a Bromage score of 0 in 25 volunteers, which differed significantly from that of volunteers who underwent FN block. CONCLUSION: Ultrasound-guided PP block is a feasible technique. Compared with FN block, PP block provides noninferior block duration and complete blocking of the peripatellar region without affecting lower limb mobilization. TRIAL REGISTRATION: This study was registered in the Chinese Clinical Trial Register (registration no. ChiCTR2000041547, registration date 28/12/2020).

Noncanonical thyroid hormone signaling mediates cardiometabolic effects in vivo.

Thyroid hormone (TH) and TH receptors (TRs) α and ß act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger signaling pathways independently of gene expression (noncanonical or type 3 TR signaling). To test the physiological relevance of noncanonical TR signaling, we generated knockin mice with a mutation in the TR DNA-binding domain that abrogates binding to DNA and leads to complete loss of canonical TH action. We show that several important physiological TH effects are preserved despite the disruption of DNA binding of TRα and TRß, most notably heart rate, body temperature, blood glucose, and triglyceride concentration, all of which were regulated by noncanonical TR signaling. Additionally, we confirm that TRE-binding-defective TRß leads to disruption of the hypothalamic-pituitary-thyroid axis with resistance to TH, while mutation of TRα causes a severe delay in skeletal development, thus demonstrating tissue- and TR isoform-specific canonical signaling. These findings provide in vivo evidence that noncanonical TR signaling exerts physiologically important cardiometabolic effects that are distinct from canonical actions. These data challenge the current paradigm that in vivo physiological TH action is mediated exclusively via regulation of gene transcription at the nuclear level.

Augmenter of liver regeneration protects the kidney from ischaemia-reperfusion injury in ferroptosis.

Acute kidney injury (AKI) is a common and severe clinical condition with high morbidity and mortality. Ischaemia-reperfusion (I/R) injury remains the major cause of AKI in the clinic. Ferroptosis is a recently discovered form of programmed cell death (PCD) that is characterized by iron-dependent accumulation of reactive oxygen species (ROS). Compelling evidence has shown that renal tubular cell death involves ferroptosis, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR) is a widely distributed multifunctional protein that is expressed in many tissues. Our previous study demonstrated that ALR possesses an anti-oxidant function. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. Here, to elucidate the role of ALR in ferroptosis, ALR expression was inhibited using short hairpin RNA lentivirals (shRNA) in vitro model of I/R-induced AKI. The results suggest that the level of ferroptosis is increased, particularly in the shRNA/ALR group, accompanied by increased ROS and mitochondrial damage. Furthermore, inhibition of system xc- with erastin aggravates ferroptosis, particularly silencing of the expression of ALR. Unexpectedly, we demonstrate a novel signalling pathway of ferroptosis. In summary, we show for the first time that silencing ALR aggravates ferroptosis in an in vitro model of I/R. Notably, we show that I/R induced kidney ferroptosis is mediated by ALR, which is linked to the glutathione-glutathione peroxidase (GSH-GPx) system.

Overexpression of augmenter of liver regeneration (ALR) mitigates the effect of H2O2-induced endoplasmic reticulum stress in renal tubule epithelial cells.

Ischemia/reperfusion is a major cause of acute kidney injury and can induce apoptosis in renal epithelial tubule (HK-2) cells. Accumulating evidence indicates that endoplasmic reticulum (ER) stress is a major contributor to apoptosis in acute kidney injury. We previously reported that augmenter of liver regeneration (ALR) functions as an anti-apoptotic factor in H2O2-treated HK-2 cells although the precise mechanism underlying this action remains unclear. In the present study, we investigate the role of ALR in H2O2-induced ER stress-mediated apoptosis. We overexpressed ALR and established a H2O2-induced ER stress model in HK-2 cells. Overexpression of ALR reduced the level of reactive oxygen species and the rate of apoptosis in H2O2-treated HK-2 cells. Using confocal microscopy and western blot, we observed that ALR colocalized with the ER and mitochondria compartment. Moreover, ALR suppressed ER stress by maintaining the morphology of the ER and reducing the levels of the ER-related proteins, glucose-regulated protein 78 (GRP78), phospho-protein kinase-like ER kinase (p-PERK), phospho-eukaryotic initiation factor 2α (p-eIF2α) and C/EBP-homologous protein (CHOP) significantly (p < 0.05). Mechanistically, ALR promoted Bcl-2 expression and suppressed Bax and cleaved-caspase-3 expression significantly during ER-stress induced apoptosis (p < 0.05). Furthermore, ALR attenuated calcium release from the ER, and transfer to mitochondria, under ER stress. To conclude, ALR alleviates H2O2-induced ER stress-mediated apoptosis in HK-2 cells by suppressing ER stress response and by maintaining calcium homeostasis. Consequently, ALR may protect renal tubule epithelial cells from ischemia/reperfusion induced acute kidney injury.

Augmenter of liver regeneration promotes mitochondrial biogenesis in renal ischemia-reperfusion injury.

Mitochondria are the center of energy metabolism in the cell and the preferential target of various toxicants and ischemic injury. Renal ischemia-reperfusion (I/R) injury triggers proximal tubule injury and the mitochondria are believed to be the primary subcellular target of I/R injury. The promotion of mitochondrial biogenesis (MB) is critical for the prevention I/R injury. The results of our previous study showed that augmenter of liver regeneration (ALR) has anti-apoptotic and anti-oxidant functions. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. To gain further insight into the role of ALR in MB, human kidney (HK)-2 cells were treated with lentiviruses carrying ALR short interfering RNA (siRNA) and a model of hypoxia reoxygenation (H/R) injury in vitro was created. We observed that knockdown of ALR promoted apoptosis of renal tubular cells and aggravated mitochondrial injury, as evidenced by the decrease in the mitochondrial respiratory proteins adenosine triphosphate (ATP) synthase subunit ß, cytochrome c oxidase subunit 1, and nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) beta subcomplex 8. Meanwhile, the production of reactive oxygen species was increased and ATP levels were decreased significantly in HK-2 cells, as compared with the siRNA/control group (p < 0.05). In addition, the mitochondrial DNA copy number and membrane potential were markedly decreased. Furthermore, critical transcriptional regulators of MB (i.e., peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, mitochondrial transcription factor A, sirtuin-1, and nuclear respiratory factor-1) were depleted in the siRNA/ALR group. Taken together, these findings unveil essential roles of ALR in the inhibition of renal tubular cell apoptosis and attenuation of mitochondrial dysfunction by promoting MB in AKI.

Generation of functional thyroid from embryonic stem cells.

The primary function of the thyroid gland is to metabolize iodide by synthesizing thyroid hormones, which are critical regulators of growth, development and metabolism in almost all tissues. So far, research on thyroid morphogenesis has been missing an efficient stem-cell model system that allows for the in vitro recapitulation of the molecular and morphogenic events regulating thyroid follicular-cell differentiation and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription factors NKX2-1 and PAX8 is sufficient to direct mouse embryonic stem-cell differentiation into thyroid follicular cells that organize into three-dimensional follicular structures when treated with thyrotropin. These in vitro-derived follicles showed appreciable iodide organification activity. Importantly, when grafted in vivo into athyroid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mouse embryonic stem cells can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue.

Augmenter of liver regeneration regulates autophagy in renal ischemia-reperfusion injury via the AMPK/mTOR pathway.

Autophagy may have protective effects in renal ischemia-reperfusion (I/R) injury, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR), a widely distributed multifunctional protein that is originally identified as a hepatic growth factor, may participate in the process of autophagy. To investigate the role of ALR in autophagy, ALR expression is knocked-down in human kidney 2 (HK-2) cells with short hairpin RNA lentivirals. Then, the level of autophagy is measured in the shRNA/ALR group and the shRNA/control group in an in vitro model of ischemia-reperfusion (I/R). The results indicate that the level of autophagy in two groups increase, accompanied by increased reactive oxygen species production, especially in the shRNA/ALR group. The AMPK/mTOR signaling pathway is hyperactive in the shRNA/ALR group. Inhibition of autophagy with the AMPK inhibitor compound C induce apoptosis, especially in the shRNA/ALR group. These findings collectively indicate that ALR negatively regulates the autophagy process through an association with the AMPK/mTOR signaling pathway. Autophagy inhibit apoptosis and play a protective role under conditions of oxidative stress.

Screen detection of ductal carcinoma in situ and subsequent incidence of invasive interval breast cancers: a retrospective population-based study.

BACKGROUND: The value of screen detection and treatment of ductal carcinoma in situ (DCIS) is a matter of controversy. At present, the extent to which the diagnosis and treatment of DCIS could prevent the occurrence of invasive breast cancer in the future is not clear. We sought to estimate the association between detection of DCIS at screening and invasive interval cancers subsequent to the relevant screen. METHODS: We obtained aggregate data for screen-detected cancers from 84 local screening units within 11 regional Quality Assurance Reference Centres in England, Wales, and Northern Ireland from the National Health Service Breast Screening Programme. Data for DCIS diagnoses were obtained for women aged 50-64 years who were invited to and attended mammographic breast screening from April 1, 2003, to March 31, 2007 (4 screening years). Patient-level data for interval cancer arising in the 36 months after each of these were analysed by Poisson regression with invasive interval cancer screen detection rate as the outcome variable; DCIS detection frequencies were fitted first as a continuous and then as a categorical variable. We repeated this analysis after adjustment with both small size and high-grade invasive screen-detected cancers. FINDINGS: We analysed data for 5,243,658 women and on interval cancers occurring in the 36 months after the relevant screen. The average frequency of DCIS detected at screening was 1·60 per 1000 women screened (median 1·50 [unit range 0·54-3·56] [corrected to] per 1000 women). There was a significant negative association of screen-detected DCIS cases with the rate of invasive interval cancers (Poisson regression coefficient -0·084 [95% CI -0·13 to -0·03]; p=0·002). 90% of units had a DCIS detection frequency within the range of 1·00 to 2·22 per 1000 women; in these units, for every three screen-detected cases of DCIS, there was one fewer invasive interval cancer in the next 3 years. This association remained after adjustment for numbers of small screen-detected invasive cancers and for numbers of grade 3 invasive screen-detected cancers. INTERPRETATION: The association between screen-detected DCIS and subsequent invasive interval cancers suggests that detection and treatment of DCIS is worthwhile in prevention of future invasive disease. FUNDING: UK Department of Health Policy Research Programme and NHS Cancer Screening Programmes.

Augmenter of liver regeneration plays a protective role against hydrogen peroxide-induced oxidative stress in renal proximal tubule cells.

Oxidative stress plays an important role in cellular destruction. Augmenter of liver regeneration (ALR) is an anti-apoptotic factor that is expressed in all mammalian cells and functions as an anti-oxidant by stimulating the expression of a secretory isoform of clusterin and inhibiting reactive oxygen species (ROS) generation. Previous work from our group showed that ALR expression is upregulated in acute kidney injury (AKI) rats, and recombinant human ALR reduces tubular injury. In the present study, we used small interfering RNA (siRNA) silencing of ALR to examine its role in H2O2 induced mitochondrial injury and apoptosis. Knockdown of ALR increased ROS levels, reduced mitochondrial membrane potential, and increased the release of mitochondrial proteins and the rate of apoptosis in response to H2O2. In addition, the ratio of Bax/Bcl-2 was increased in siRNA/ALR groups treated with H2O2. These data confirm the protective role of ALR against oxidative stress-induced mitochondrial injury and suggest a potential mechanism underlying the protective role of ALR in AKI.

Augmenter of liver regeneration inhibits TGF-ß1-induced renal tubular epithelial-to-mesenchymal transition via suppressing TßR II expression in vitro.

Tubular epithelial-to-mesenchymal transition (EMT) plays a crucial role in the progression of renal tubular interstitial fibrosis (TIF), which subsequently leads to chronic kidney disease (CKD) and eventually, end-stage renal disease (ESRD). We propose that augmenter of liver regeneration (ALR), a member of the newly discovered ALR/Erv1 protein family shown to ameliorate hepatic fibrosis, plays a similar protective role in renal tubular cells and has potential as a new treatment option for CKD. Here, we showed that recombinant human ALR (rhALR) inhibits EMT in renal tubular cells by antagonizing activation of the transforming growth factor-ß1 (TGF-ß1) signaling pathway. Further investigation revealed that rhALR suppresses the expression of TGF-ß receptor type II (TßR II) and significantly alleviates TGF-ß1-induced phosphorylation of Smad2 and nuclear factor-κB (NF-κB). No apparent adverse effects were observed upon the addition of rhALR alone to cells. These findings collectively suggest that ALR plays a role in inhibiting progression of renal tubular EMT, supporting its potential utility as an effective antifibrotic strategy to reverse TIF in CKD.

Thyrotrophin receptor signaling dependence of Braf-induced thyroid tumor initiation in mice.

Mutations of BRAF are found in ∼45% of papillary thyroid cancers and are enriched in tumors with more aggressive properties. We developed mice with a thyroid-specific knock-in of oncogenic Braf (LSL-Braf(V600E)/TPO-Cre) to explore the role of endogenous expression of this oncoprotein on tumor initiation and progression. In contrast to other Braf-induced mouse models of tumorigenesis (i.e., melanomas and lung), in which knock-in of Braf(V600E) induces mostly benign lesions, Braf-expressing thyrocytes become transformed and progress to invasive carcinomas with a very short latency, a process that is dampened by treatment with an allosteric MEK inhibitor. These mice also become profoundly hypothyroid due to deregulation of genes involved in thyroid hormone biosynthesis and consequently have high TSH levels. To determine whether TSH signaling cooperates with oncogenic Braf in this process, we first crossed LSL-Braf(V600E)/TPO-Cre with TshR knockout mice. Although oncogenic Braf was appropriately activated in thyroid follicular cells of these mice, they had a lower mitotic index and were not transformed. Thyroid-specific deletion of the Gsα gene in LSL-Braf(V600E)/TPO-Cre/Gnas-E1(fl/fl) mice also resulted in an attenuated cancer phenotype, indicating that the cooperation of TshR with oncogenic Braf is mediated in part by cAMP signaling. Once tumors were established in mice with wild-type TshR, suppression of TSH did not revert the phenotype. These data demonstrate the key role of TSH signaling in Braf-induced papillary thyroid cancer initiation and provide experimental support for recent observations in humans pointing to a strong association between TSH levels and thyroid cancer incidence.

Role of type 2 deiodinase in response to acute lung injury (ALI) in mice.

Thyroid hormone (TH) metabolism, mediated by deiodinase types 1, 2, and 3 (D1, D2, and D3) is profoundly affected by acute illness. We examined the role of TH metabolism during ventilator-induced lung injury (VILI) in mice. Mice exposed to VILI recapitulated the serum TH findings of acute illness, namely a decrease in 3,5,3'-triiodothyronine (T(3)) and thyroid-stimulating hormone and an increase in reverse T(3). Both D2 immunoreactivity and D2 enzymatic activity were increased significantly. D1 and D3 activity did not change. Using D2 knockout (D2KO) mice, we determined whether the increase in D2 was an adaptive response. Although similar changes in serum TH levels were observed in D2KO and WT mice, D2KO mice exhibited greater susceptibility to VILI than WT mice, as evidenced by poorer alveoli integrity and quantified by lung chemokine and cytokine mRNA induction. These data suggest that an increase in lung D2 is protective against VILI. Similar findings of increased inflammatory markers were found in hypothyroid WT mice exposed to VILI compared with euthyroid mice, indicating that the lungs were functionally hypothyroid. Treatment of D2KO mice with T(3) reversed many of the lung chemokine and cytokine profiles seen in response to VILI, demonstrating a role for T(3) in the treatment of lung injury. We conclude that TH metabolism in the lung is linked to the response to inflammatory injury and speculate that D2 exerts its protective effect by making more TH available to the injured lung tissue.

Mutations in SECISBP2 result in abnormal thyroid hormone metabolism.

Incorporation of selenocysteine (Sec), through recoding of the UGA stop codon, creates a unique class of proteins. Mice lacking tRNA(Sec) die in utero, but the in vivo role of other components involved in selenoprotein synthesis is unknown, and Sec incorporation defects have not been described in humans. Deiodinases (DIOs) are selenoproteins involved in thyroid hormone metabolism. We identified three of seven siblings with clinical evidence of abnormal thyroid hormone metabolism. Their fibroblasts showed decreased DIO2 enzymatic activity not linked to the DIO2 locus. Systematic linkage analysis of genes involved in DIO2 synthesis and degradation led to the identification of an inherited Sec incorporation defect, caused by a homozygous missense mutation in SECISBP2 (also called SBP2). An unrelated child with a similar phenotype was compound heterozygous with respect to mutations in SECISBP2. Because SBP2 is epistatic to selenoprotein synthesis, these defects had a generalized effect on selenoproteins. Incomplete loss of SBP2 function probably causes the mild phenotype.

STR mutations on chromosome 15q cause thyrotropin resistance by activating a primate-specific enhancer of MIR7-2/MIR1179.

Thyrotropin (TSH) is the master regulator of thyroid gland growth and function. Resistance to TSH (RTSH) describes conditions with reduced sensitivity to TSH. Dominantly inherited RTSH has been linked to a locus on chromosome 15q, but its genetic basis has remained elusive. Here we show that non-coding mutations in a (TTTG)4 short tandem repeat (STR) underlie dominantly inherited RTSH in all 82 affected participants from 12 unrelated families. The STR is contained in a primate-specific Alu retrotransposon with thyroid-specific cis-regulatory chromatin features. Fiber-seq and RNA-seq studies revealed that the mutant STR activates a thyroid-specific enhancer cluster, leading to haplotype-specific upregulation of the bicistronic MIR7-2/MIR1179 locus 35 kb downstream and overexpression of its microRNA products in the participants' thyrocytes. An imbalance in signaling pathways targeted by these micro-RNAs provides a working model for this cause of RTSH. This finding broadens our current knowledge of genetic defects altering pituitary-thyroid feedback regulation.

Thyroid hormone receptor-ß agonists prevent hepatic steatosis in fat-fed rats but impair insulin sensitivity via discrete pathways.

Liver-specific thyroid hormone receptor-ß (TRß)-specific agonists are potent lipid-lowering drugs that also hold promise for treating nonalcoholic fatty liver disease and hepatic insulin resistance. We investigated the effect of two TRß agonists (GC-1 and KB-2115) in high-fat-fed male Sprague-Dawley rats treated for 10 days. GC-1 treatment reduced hepatic triglyceride content by 75%, but the rats developed fasting hyperglycemia and hyperinsulinemia, attributable to increased endogenous glucose production (EGP) and diminished hepatic insulin sensitivity. GC-1 also increased white adipose tissue lipolysis; the resulting increase in glycerol flux may have contributed to the increase in EGP. KB-2115, a more TRß- and liver-specific thyromimetic, also prevented hepatic steatosis but did not induce fasting hyperglycemia, increase basal EGP rate, or diminish hepatic insulin sensitivity. Surprisingly, insulin-stimulated peripheral glucose disposal was diminished because of a decrease in insulin-stimulated skeletal muscle glucose uptake. Skeletal muscle insulin signaling was unaffected. Instead, KB-2115 treatment was associated with a decrease in GLUT4 protein content. Thus, although both GC-1 and KB-2115 potently treat hepatic steatosis in fat-fed rats, they each worsen insulin action via specific and discrete mechanisms. The development of future TRß agonists must consider the potential adverse effects on insulin sensitivity.

Role of GLIS3 in thyroid development and in the regulation of gene expression in thyroid specific Glis3KO mice.

Loss of GLI-Similar 3 (GLIS3) function in mice and humans causes congenital hypothyroidism (CH). In this study, we demonstrate that GLIS3 protein is first detectable at E15.5 of murine thyroid development, a time when GLIS3 target genes, such as Slc5a5 (Nis), become also expressed. We further show that Glis3KO mice do not display any major changes in prenatal thyroid gland morphology indicating that CH in Glis3KO mice is due to dyshormonogenesis rather than thyroid dysgenesis. Analysis of thyroid-specific Glis3 knockout (Glis3-Pax8Cre) mice fed either a normal or low-iodine diet (ND or LID) revealed that, in contrast to ubiquitous Glis3KO mice, thyroid follicular cell proliferation and the expression of cell cycle genes were not repressed suggesting that the inhibition of thyroid follicular cell proliferation in ubiquitous Glis3KO mice is related to loss of GLIS3 function in other cell types. However, the expression of several thyroid hormone biosynthesis-, extracellular matrix (ECM)-, and inflammation-related genes was still suppressed in Glis3-Pax8Cre mice particularly under conditions of high blood levels of thyroid stimulating hormone (TSH). We further demonstrate that treatment with TSH, protein kinase A (PKA) or adenylyl cyclase activators or expression of constitutively active PKA enhances GLIS3 protein and activity, suggesting that GLIS3 transcriptional activity is regulated in part by TSH/TSHR-mediated activation of the PKA pathway. This mechanism of regulation provides an explanation for the dramatic increase in GLIS3 protein expression and the subsequent induction of GLIS3 target genes, including several thyroid hormone biosynthetic genes, in thyroid follicular cells of mice fed a LID.

ASIC1a affects hypothalamic signaling and regulates the daily rhythm of body temperature in mice.

The body temperature of mice is higher at night than during the day. We show here that global deletion of acid-sensing ion channel 1a (ASIC1a) results in lower body temperature during a part of the night. ASICs are pH sensors that modulate neuronal activity. The deletion of ASIC1a decreased the voluntary activity at night of mice that had access to a running wheel but did not affect their spontaneous activity. Daily rhythms of thyrotropin-releasing hormone mRNA in the hypothalamus and of thyroid-stimulating hormone ß mRNA in the pituitary, and of prolactin mRNA in the hypothalamus and pituitary were suppressed in ASIC1a-/- mice. The serum thyroid hormone levels were however not significantly changed by ASIC1a deletion. Our findings indicate that ASIC1a regulates activity and signaling in the hypothalamus and pituitary. This likely leads to the observed changes in body temperature by affecting the metabolism or energy expenditure.