Relacorilant or surgery improved hemostatic markers in Cushing syndrome.

PURPOSE: Glucocorticoid-mediated hypercoagulability can persist in patients with endogenous Cushing syndrome (CS) after curative surgery and may transiently worsen early postoperatively. These studies aimed to characterize coagulation markers at baseline in patients with CS and the impact of relacorilant or remission post-surgery in an open-label, phase 2 study (NCT02804750) and a retrospective, longitudinal, surgical cohort study. METHODS: In the relacorilant study, 34 patients received relacorilant (100-200 mg/day for up to 12 weeks or 250-400 mg/day for up to 16 weeks) and had postbaseline data. Coagulation markers were assessed before and during treatment. In the surgical study, conducted at "Federico II" University of Naples, Italy, coagulation markers were assessed in 30 patients before surgery and after biochemical remission. RESULTS: In the relacorilant study, significant mean changes from baseline to last observed visit were reported in factor VIII (- 18.9%, P = 0.022), activated partial thromboplastin time (aPTT) (+ 1.5 s, P = 0.046), and platelet count (- 68.8*109/L, P < 0.0001), whereas von Willebrand factor was unchanged. In the surgical study, the mean time to hemostasis assessment was 6.2 months. Significant mean changes from baseline to hemostasis assessment were reported in factor VIII (- 24.2%, P = 0.044), von Willebrand factor (- 20.6%, P = 0.018), and aPTT (+ 2.0 s, P = 0.031), whereas platelet count was unchanged. CONCLUSIONS: Several coagulation markers improved in patients with CS after 3-4 months of relacorilant treatment and within an average of 6 months after surgery. Relacorilant's positive effects on coagulation markers support further investigation of its use preoperatively in patients with CS or in patients who are not eligible for surgery. CLINICAL TRIAL REGISTRATION NUMBER: NCT0280475 (registration date: 15 June 2016).

Spot 14 gene deletion increases hepatic de novo lipogenesis.

Previous studies have investigated the relationship between the Spot 14 gene and hepatic lipogenesis. Those studies found that the Spot 14 protein was induced when lipogenesis was induced and suggested that induction of the Spot 14 protein was required for induction of hepatic lipogenesis by thyroid hormone and dietary carbohydrate. Analysis of those findings led us to hypothesize that the Spot 14 gene is required for induced hepatic de novo lipogenesis in vivo. To test this hypothesis, we created an in vivo deletion of the Spot 14 gene in mice using gene-targeting technology. Southern blot analysis showed that the Spot 14 gene was disrupted. Northern blot analysis showed that this disruption ablated expression of intact hepatic Spot 14 mRNA. In contrast to our hypothesis, acute thyroid hormone administration led to comparable induction of hepatic lipogenic enzyme mRNAs between the wild-type and knockout mice. Furthermore, long-term treatment with both thyroid hormone and a diet promoting lipogenesis led to enhanced lipogenic enzyme activity and a greater rate of hepatic de novo lipogenesis in the knockout, compared with the wild-type, mice. Although these data indicate that the Spot 14 protein is not required for induced hepatic de novo lipogenesis, they also suggest that Spot 14 plays some role in this process. It is possible that alternative pathways that complement the loss of the Spot 14 protein are present, and in the absence of Spot 14, these alternative pathways overcompensate to produce an enhanced rate of induced lipogenesis.

Quantitative assessment of pituitary resistance to thyroid hormone from plots of the logarithm of thyrotropin versus serum free thyroxine index.

Previous studies have shown that, in patients with primary alterations in thyroid hormone secretion, the level of the natural logarithm of serum TSH (lnTSH) is negatively related to the level of free T4. Because such patients can generally be assumed to exhibit normal tissue responsivity to thyroid hormone, we were interested in determining whether the lnTSH/free T4 index (FTI) relationship in patients with established thyroid hormone resistance (THR) exhibit a lower slope than patients with normal tissue sensitivity to thyroid hormone. We have therefore analyzed the relationship between the lnTSH and the FTI in members of three families with documented THR. In these patients, a given dose of T4 was maintained for a 1- to 2-month period, to achieve hormonal equilibration. Two of the families, though not related, exhibited the same mutation, E460K. The third was identified as A317T. As anticipated, the slope of the lnTSH/FTI ratio was significantly lower in the patients with THR than in T4-treated patients who were presumed to have normal sensitivity to thyroid hormone. The slope of the lnTSH/FTI relationship seemed to be characteristic of the specific mutation involved in the three genotypes (wild-type and two mutations) examined. Further, the in vivo slope of the lnTSH/FTI relationship seemed to be linearly related to the T3 association constant of the in vitro translated receptor. These findings support the potential usefulness of measuring the slope of lnTSH, as a function of the FTI, in quantitating pituitary THR.

The orphan nuclear receptor Ear-2 is a negative coregulator for thyroid hormone nuclear receptor function.

Thyroid hormone (T3) nuclear receptors (TR) are ligand-dependent transcription factors which regulate growth, differentiation, and development. One emerging hypothesis suggests that TR mediate these diverse effects via a large network of coregulators. Recently, we found that TR-mediated transcriptional responses varied in six cell lines derived from different tissues. We therefore used human TR subtype beta1 (TRbeta1) as bait to search for coregulators in human colon carcinoma RKO cells with a yeast two-hybrid system. RKO cells exhibited T3-dependent and -independent transcriptional activation. One of the three positive clones was identified as Ear-2, which is a distant member of the chick ovalbumin upstream promoter-transcription factors of the orphan nuclear receptor family. The physical interaction between Ear-2 and TRbeta1 was further confirmed by specific binding of Ear-2 to glutathione S-transferase-TRbeta1. In addition, Ear-2 was found to associate with TRbeta1 in cells. As a result of this physical interaction, binding of TRbeta1 to the T3 response elements was inhibited. Using reporter systems, we found that both the basal activation and the T3-dependent activation mediated by TRbeta1 were repressed by Ear-2 in CV1 cells. In RKO cells, however, the T3-independent transcriptional activity was more sensitive to the repression effect of Ear-2 than the T3-dependent transcriptional activity. The repression effect of Ear-2 was reversed by steroid hormone receptor coactivator 1. These results suggest that TR-mediated responses reflect a balance of corepressors and coactivators in cells. These findings further strengthen the hypothesis that the diverse activities of TR are achieved via a large network of coregulators that includes Ear-2.

Two different gene elements are required for glucose regulation of S14 transcription.

Carbohydrate feeding increases the transcriptional activity of the hepatic S14 gene. The region of the S14 promoter between -1384/-1275 contributes to the transcriptional regulation by carbohydrate. A previously identified element (-1303/-1289) within this region is required but is not sufficient for the carbohydrate effect. Therefore, we ligated -1384/-1275 to a heterologous promoter and created mutants in this region to identify other potential responsive sequences. We found that mutation within -1365/-1350 eliminated the response to high glucose (27.5 mM). However, three copies of this element ligated to a mouse mammary tumor virus-luciferase vector did not respond to glucose indicating the -1365/-1350 element is insufficient to confer a glucose response in isolation. Nevertheless. mutating the -1365/-1350 element in the native promoter led to a loss of response to glucose, proving this element is necessary. Electrophoretic mobility shift assays (EMSA) using three copies of the element showed significant binding to rat hepatic nuclear extracts, but no difference between the dietary states. Competition EMSA studies showed that the previously identified element at -1303/-1289 was unable to compete for proteins that bind to the -1365/-1350 element. Therefore, we have demonstrated two separate elements within the -1384/-1275 region of the S14 gene that bind different proteins and interact to elicit the carbohydrate effect.

Adipose S14 mRNA is abnormally regulated in obese subjects.

In rat hepatocyte culture, the S14 gene is necessary for induction of lipogenesis by carbohydrate metabolism and thyroid hormone. To determine if this gene plays a role in regulation of lipid storage in humans we compared the response to fasting of the human S14 gene between obese and nonobese subjects. We measured the relative content of human S14 mRNA in abdominal subcutaneous fat before and after a 48-hour fast. We found that mRNA-S14 is strongly downregulated in nonobese subjects in response to the fast, but only minimally down-regulated in obese subjects. There is an excellent correlation between the body mass index, and the fasting induced fall in S14 mRNA. There was no difference in the postfasting glucose, insulin, and ketone levels between the 2 groups of subjects. Therefore, the S14 gene is abnormally downregulated during fasting in adipose tissue of obese individuals. Further study of this gene could provide important information on the mechanism of the acquisition or maintenance of obesity in humans.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) modulates expression of the Purkinje cell protein-2 gene. A potential role for COUP-TF in repressing premature thyroid hormone action in the developing brain.

The cerebellar Purkinje cell-specific PCP-2 gene is transcriptionally activated by thyroid hormone during the 2nd and 3rd weeks of postnatal life in the rat. In contrast, thyroid hormone has no detectable effects on PCP-2 expression in the fetal rat. We now present data that suggest that the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor (COUP-TF) represses triiodothyronine (T3)-dependent transcriptional activation of PCP-2 in the immature Purkinje cell. Gel shift assays show that the PCP-2 A1TRE and adjoining sequences (-295/-199 region) bind to rat and mouse brain nucleoproteins in a developmentally regulated fashion and that one of these nucleoproteins could be the orphan nucleoprotein COUP-TF. In support of this hypothesis, in vitro translated COUP-TF binds to the -295/-199 region and COUP-TF represses T3-dependent activation of the PCP-2 promoter in transient transfection analyses. Finally, immunohistochemical studies reveal that COUP-TF is specifically expressed in the immature fetal and early neonatal Purkinje cell and that this expression diminishes coincident with thyroid hormone induction of PCP-2 expression. Our findings are consistent with the hypothesis that the presence or absence of inhibitory proteins bound to the thyroid hormone response element of T3-responsive genes governs the responsivity of these genes to thyroid hormone during brain development.

Beta receptor isoforms are not essential for thyroid hormone-dependent acceleration of PCP-2 and myelin basic protein gene expression in the developing brains of neonatal mice.

In rat pups, thyroid hormone dependent brain development coincides with the appearance of the thyroid hormone receptor (TR)beta1 isoform. This finding led to the suggestion that TRbeta1 plays an essential role in brain development. The recent availability of a mouse TRbeta knockout strain allowed us to test this possibility by determining whether TRbeta is essential for the normal developmental pattern of expression of two thyroid hormone regulated brain genes, myelin basic protein (MBP), and Purkinje cell protein 2 (Pcp-2). Northern analysis of total mRNA from the brains of wild-type mice established that, as in the rat pup, the initial rate of rise of the MBP and Pcp-2 mRNA is slowed in the hypothyroid state. Supporting the effectiveness of TRbeta gene deletion was the finding that the thiiodothyronine (T3) nuclear binding capacity in the livers and brains of knockout animals was consistent with the fractional contribution of TRbeta1 to total binding capacity in the wild-type tissues. Further, no TRbeta1 could be detected by isoform-specific immunoprecipitation of nuclear receptor extracts. However, deletion of the functional TRbeta in the TRbeta knockout mice did not affect the normal ontogeny of expression of the Pcp-2 and MBP genes in the postnatal pup. We conclude that TRbeta is not essential for the normal developmental expression of these T3 dependent brain genes.

Purkinje cell protein-2 cis-elements mediate repression of T3-dependent transcriptional activation.

Previous studies in our laboratory show that triiodothyronine upregulates expression of the cerebellar Purkinje cell-specific gene Pcp-2 during the first 2 weeks of rat neonatal life. A specific thyroid hormone response element, the A1 TRE, mediates this regulation. The finding that the contiguous 68 bases (-267/ -199) of the Pcp-2 promoter 3' to the A1 TRE repressed T3 response in transactivation studies suggested that this sequence could play a role in preventing premature T3-dependent activation of Pcp-2 in the fetus. We now show that deletion of this region resulted in enhanced T3-dependent activation of the native Pcp-2 promoter. The sequence is not a generalized silencer since it does not alter basal activity of mouse mammary tumor virus (MMTV) or thymidine kinase (TK) promoters. Deletion and linker scanning studies indicate that the 5' 30 bases of the -267/ -199 region mediate most of the response silencing activity. The -267/ -199 region also attenuates T3-induced transactivation mediated by other TREs. Gel shift analysis reveals that nuclear proteins from fetal but not adult brains complex with the -267/ -199 region, supporting the hypothesis that this region binds proteins that suppress Pcp-2 expression early in brain development.

Cloning, expression and regulation of the human S14 gene.

The rat S14 gene has been a useful model to study carbohydrate and triiodothyronine (T3) regulation of hepatic gene expression. To gain insight into the regulation and function of the S14 gene, we isolated the human S14 gene and studied its sequence, tissue specific expression, and transcriptional regulation by glucose and T3. The deduced amino acid sequence of the human S14 protein is 78% identical to that of the rat. Northern blot analysis showed that the S14-mRNA is a single species in human liver and is not present in human brain or HepG2 cells. Transfection studies in primary hepatocytes revealed that transcription of the human S14 gene is regulated by glucose and T3 in a similar manner to that of the rat gene. However, in HepG2 cells, T3 and glucose did not affect the transcription of the human S14 gene. These observations suggest that the S14 gene is highly conserved in mammals and is similarly regulated by carbohydrate and T3 in vivo. More importantly, the function of the human S14 gene may be critical in lipid metabolism in human liver as the rat S14 gene is in rodents.

Lowering glucose depletes a thapsigargin-sensitive calcium pool and inhibits transcription of the S14 gene.

S14 is a nuclear protein that is rapidly and synergistically induced by glucose and thyroid hormone, and the level of it's messenger RNA correlates with hepatocyte and adipocyte lipogenesis. We previously reported that the calcium ionophore A23187 markedly inhibits the carbohydrate response of the S14 gene without inhibiting glucose metabolism. Because the calcium ionophore not only increased intracellular cytosolic free calcium but also depletes intracellular calcium stores, we examined which of these two possibilities accounts for the regulation of S14 gene transcription. We found that increasing cytosolic calcium with arginine vasopressin is insufficient to inhibit S14 gene transcription. Furthermore, reduction of intracellular calcium by addition of EGTA to medium containing A23187 leads to further inhibition of S14 transcription. Measurement of intracellular free calcium in indo-1-loaded hepatocytes showed no significant changes induced by high glucose. These results suggested that depletion of an intracellular pool of calcium by A23187 causes the inhibition of S14 transcription. Addition of thapsigargin, which depletes intracellular calcium pools by inhibition of endoplasmic reticulum Ca2+-ATPase, led to significant inhibition of glucose-regulated S14 transcription. Lastly, continuous incubation in 5.5 mM glucose depletes the thapsigargin-sensitive calcium pool. These studies imply that the ability of glucose to induce S14 transcription is related to a thapsigargin-sensitive calcium pool, and depletion of this pool by lowering glucose inhibits S14 transcription.

Identification of a carbohydrate response element in rat S14 gene.

To characterize the mechanism by which carbohydrate feeding regulates S14 gene transcription, we created mutations within footprinted sequences of the carbohydrate response region of the S14 gene and either transiently transfected these mutations into rat primary hepatocytes or end-labeled them for gel mobility shift assays. The wild type DNA gave a 2.9 +/- 1.0-fold response to raising the media glucose concentration. Mutations within the 3' footprint eliminated the glucose response and also eliminated hepatic nuclear factor binding. However, mutations within the 5' footprint did not inhibit the glucose responsiveness and had no effect on hepatic nuclear binding. The gel mobility shift assays were confirmed by appropriate competition gel assays. The 3' footprint contains a sequence similar to the USF (upstream stimulating factor) binding site (CACGTG) that has been suggested to play a role in carbohydrate regulation. However, the gel shift pattern with purified USF1 is distinctly different from that of rat hepatic nuclear extract. These studies demonstrate that the sequence GGCACCCGTGT is required for the carbohydrate response in the S14 gene.

Insulin increases the processing efficiency of messenger ribonucleic acid-S14 nuclear precursor.

Feeding a lipogenic diet increases transcription and enhances processing of the rat hepatic messenger RNA (mRNA)-S14 gene. To determine the separate roles of insulin and increased glucose in these processes, we used the streptozotocin-induced diabetic rat model. Diabetes caused a reduction in mature mRNA-S14 in chow- and lipogenic diet-fed animals (P < 0.006 and P < 0.001, respectively). Insulin restored these levels to normal. Despite the known effects of insulin and carbohydrate on the transcription of this gene, we were unable to demonstrate significant changes in the nuclear proteins that bind to carbohydrate response regions. Yet, insulin restored the content of the mRNA by increasing the ratio of mature to precursor mRNA-S14. Insulin significantly increased this ratio (P < 0.0001) independent of diet and diabetes, further supporting the action of insulin on increasing processing from precursor to mature mRNA. The mechanism of the enhanced processing was studied by ribonuclease mapping and primer extension analysis. Ribonuclease mapping showed that lipogenic diet feeding increases the efficiency of processing at a step before formation of the branched form of the precursor mRNA. Taken together, our data demonstrate for the first time that insulin significantly enhances the efficiency of processing of a pre-mRNA.

Detecting graves' disease: presentations in young athletes.

Graves' disease has a multitude of presentations, and certain symptoms can mimic sports-related concerns such as overtraining. A review of three cases of Graves' disease in young athletes illustrates the spectrum of symptoms and the pathophysiology. Diagnosis involves a detailed patient history, physical exam, and appropriate lab studies, including a thyroid radioactive iodine uptake scan. Treatment consists of symptom management and antithyroid medication, radioactive iodine thyroid ablation, or, rarely, thyroidectomy.

Direct evidence for a role of the "spot 14" protein in the regulation of lipid synthesis.

"Spot 14" is a nuclear protein that is rapidly induced by thyroid hormone (T3) and dietary carbohydrate in liver. We used an antisense oligonucleotide to inhibit induction of spot 14 protein by T3 and glucose in primary cultures of rat hepatocytes to test the hypothesis that the protein could function in the regulation of lipid synthesis. Spot 14 protein was undetectable in hepatocytes maintained in 5.5 mM glucose without T3, and was induced within 4 h after addition of 27.5 mM glucose and 50 nM T3 to the culture medium, reaching a maximal level within 24 h. Accumulation of spot 14 protein was markedly inhibited in hepatocytes transfected with a spot 14 antisense oligonucleotide, but not in those treated with a control oligonucleotide. Transfection of the antisense, but not control, oligonucleotide also abrogated the increase in lipogenesis induced by T3 and glucose. Reduced triglyceride formation accounted for the diminished net lipid synthesis. In contrast to lipogenesis, glucose uptake was not significantly affected by the transfections. Antisense transfection inhibited the induction of both ATP-citrate lyase and fatty acid synthase immunoreactivities, as well as malic enzyme activity, indicating that the observed reduction in lipogenesis could be explained by diminished cellular content of lipogenic enzymes. Reduced malic enzyme activity in antisense-transfected hepatocytes was accompanied by lowered relative abundance of malic enzyme mRNA, suggesting that the antisense effects on lipogenic enzymes were mediated at the pretranslational level. The oligonucleotides did not significantly affect lipogenesis in a rat hepatoma cell line that does not express detectable spot 14 mRNA or protein. These data directly implicate the spot 14 protein in the transduction of hormonal and dietary signals for increased lipid metabolism in hepatocytes.

Two glucose-signaling pathways in S14 gene transcription in primary hepatocytes: a common role of protein phosphorylation.

Transcription of the rat S14 gene is induced in response to increased carbohydrate metabolism in the liver. Because carbohydrate-induced changes in lipogenesis are mediated in part by changes in phosphorylation of multiple proteins, we investigated the role of protein phosphorylation on transcriptional regulation of the two carbohydrate response elements, a thyroid hormone receptor-independent carbohydrate response element and a thyroid receptor-dependent glucose response element located up-stream of the S14 gene. S14 reporter constructs were transiently transfected into rat primary hepatocytes and incubated with the protein or phosphatase inhibitor okadaic acid calyculin-A, or one of several protein kinase activators. Low dose okadaic acid blocked glucose induction from both elements without inhibiting glucose metabolism. Calyculin-A, a preferential phosphatase-1 inhibitor, only blocked the glucose response when glucose metabolism was inhibited. The protein kinase-C activator, 12-myristate 13-acetate, did not change the glucose responses, whereas the protein kinase-A activator, 8-(4-chlorophenylthio)cAMP, inhibited S14 transcription by inhibiting glucose metabolism. In contrast, the calcium ionophore A23187, a calmodulin kinase activator, mimicked the effect of low dose okadaic acid, but had no effect on glucose metabolism. We conclude that protein phosphatase-2A and calmodulin kinases may be involved in the glucose signaling pathway of the S14 gene. A similar phosphorylation step may be involved in the two distinct glucose response pathways.

Kinetic model of the response of precursor and mature rat hepatic mRNA-S14 to thyroid hormone.

We found in preliminary experiments that multiple daily injections of triiodothyronine (T3) resulted in an apparent prolongation in the half time (t1/2) of mRNA-S14 decay. To appropriately interpret these observations, we developed a mathematical model of the fluctuations of mRNA-S14 and its nuclear precursor after a single injection or multiple daily injections of T3. The model parameters include 1) the effect of plasma protein binding and metabolic clearance rates on receptor-bound nuclear T3, 2) the threefold circadian variation in mRNA-S14, 3) a 12-min t1/2 for the nuclear precursor and a 1.5-h t1/2 for the mature mRNA-S14, 4) previously derived relationships between the level of plasma T3 and nuclear occupancy, and 5) direct proportionality between nuclear transcription of the S14 gene and T3 nuclear occupancy. The model faithfully predicted the excursions of the mature mRNA-S14 and its nuclear precursor. Nuclear retention of T3 and the effects of circadian variation on S14 gene transcription explain the apparent prolongation in the t1/2 of decay of mature mRNA. Our findings illustrate the feasibility of incorporating parameters at the molecular level into a comprehensive kinetic analysis of hormone action.

Location of a glucose-dependent response region in the rat S14 promoter.

The rat S14 gene provides an excellent model to examine the DNA sequences associated with carbohydrate regulation of hepatic gene transcription. We constructed internal deletions within 5 kilobases of the 5'-up-stream region and ligated these to a luciferase reporter gene. The constructs were transfected into primary hepatocytes and pancreatic HIT cells. In hepatocytes, an increase in the medium glucose concentration led to a parallel increase in endogenous mRNA S14 content and transfected luciferase reporter activity driven by 5 kilobases of the S14 promoter. Internal deletions of several sequences from -2706 to -285 each led to a decrease in glucose-stimulated activity, suggesting that multiple elements are necessary for the transcriptional response to glucose. Deletion from -1583 to -1069 nearly abolished the glucose effect in both cell types and delineated the carbohydrate response element (CHORE). The CHORE deletion was specific for glucose, because it did not alter the response to thyroid hormone, another known regulator of this gene. Although the CHORE sequence did not confer glucose activation to either a heterologous promoter or the basal S14 promoter (bases -285 to +19), a 5-fold enhanced response was observed when two copies of the CHORE were ligated to the first 2110 basepairs of the S14 promoter. The results suggest that the CHORE contains a carbohydrate regulatory element and operates as an enhancer in concert with other sequences within the S14 gene.