Severe Hypothyroidism and Large Goiter due to Iodine Deficiency in an Adolescent Male in the United States: A Case Report and Review of the Literature.

Acquired hypothyroidism due to iodine deficiency is extremely rare in the United States due to the introduction of table salt iodization in the 1920s (Leung et al., 2012). We present the case of an adolescent male with a history of mild autism spectrum disorder and an extremely restrictive diet who was found to have iodine deficiency as the etiology for his rapidly enlarging goiter and antibody-negative hypothyroidism. Thyroid-stimulating hormone (TSH) was 416 µIU/mL (0.350-5.500 µIU/mL), free thyroxine (T4) was <0.1 ng/dL (0.80-1.80 ng/dL), and triiodothyronine (T3) was 41 ng/dL (82-213 mg/dL) at diagnosis. The patient's 24-hour urinary iodine was undetectable. He was started on iodine supplementation with rapid visible improvement of goiter within two weeks and normalization of thyroid function tests within four weeks. Thorough dietary history and nutritional screening are important in cases of acquired hypothyroidism and/or goiter. Alternatively, diets that are low in iodized salt, dairy, bread, and seafood should raise concern for iodine deficiency, and patients with suspected or proven iodine deficiency should be screened for hypothyroidism.

Clinical Thyrotoxicosis Resulting from Liothyronine Augmentation of Antidepressant Therapy in an Adolescent.

Background/Objective. Thyrotoxicosis, a condition resulting from excessive peripheral thyroid hormone, is typically accompanied by thyroid function tests demonstrating a high free thyroxine (free T4) with appropriate suppression of thyroid-stimulating hormone (TSH). Case report. We describe a 17-year-old female presenting with symptoms of thyrotoxicosis along with suppressed TSH and low free T4, a laboratory pattern concerning for central hypothyroidism. Further history revealed that she was prescribed liothyronine as an adjunct therapy for depression. Discussion. Due to the short half-life of liothyronine, clinical signs and symptoms of thyrotoxicosis may develop before detection by interval lab monitoring. Conclusion. This case highlights the need for close monitoring and caution when treating adolescents with liothyronine and the importance of interpreting atypical laboratory findings within clinical context.

Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy.

Eukaryotic elongation factor 2 kinase (eEF2K) inhibits the elongation stage of protein synthesis by phosphorylating its only known substrate, eEF2. eEF2K is tightly regulated by nutrient-sensitive signalling pathways. For example, it is inhibited by signalling through mammalian target of rapamycin complex 1 (mTORC1). It is therefore activated under conditions of nutrient deficiency. Here we show that inhibiting eEF2K or knocking down its expression renders cancer cells sensitive to death under nutrient-starved conditions, and that this is rescued by compounds that block protein synthesis. This implies that eEF2K protects nutrient-deprived cells by inhibiting protein synthesis. Cells in which signalling through mTORC1 is highly active are very sensitive to nutrient withdrawal. Inhibiting mTORC1 protects them. Our data reveal that eEF2K makes a substantial contribution to the cytoprotective effect of mTORC1 inhibition. eEF2K is also reported to promote another potentially cytoprotective process, autophagy. We have used several approaches to test whether inhibition or loss of eEF2K affects autophagy under a variety of conditions. We find no evidence that eEF2K is involved in the activation of autophagy in the cell types we have studied. We conclude that eEF2K protects cancer cells against nutrient starvation by inhibiting protein synthesis rather than by activating autophagy.

Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival.

Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca(2+)/calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis.

Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia.

Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.

Regulated stability of eukaryotic elongation factor 2 kinase requires intrinsic but not ongoing activity.

Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical protein kinase which negatively regulates protein synthesis, is activated under stress conditions and plays a role in cytoprotection, e.g. in cancer cells. It is regarded as a possible target for therapeutic intervention in solid tumours. Earlier studies showed that eEF2K is degraded by a proteasome-dependent pathway in response to genotoxic stress and that this requires a phosphodegron that includes an autophosphorylation site. Thus, application of eEF2K inhibitors would stabilize eEF2K, partially negating the effects of inhibiting its activity. In the present study, we show that under a range of other stress conditions, including acidosis or treatment of cells with 2-deoxyglucose, eEF2K is also degraded. However, in these settings, the previously identified phosphodegron is not required for its degradation. Nevertheless, kinase-dead and other activity-deficient mutants of eEF2K are stabilized, as is a mutant lacking a critical autophosphorylation site (Thr348 in eEF2K), which is thought to be required for eEF2K and other α-kinases to achieve their active conformations. In contrast, application of small-molecule eEF2K inhibitors does not stabilize the protein. Our data suggest that achieving an active conformation, rather than eEF2K activity per se, is required for its susceptibility to degradation. Additional degrons and E3 ligases beyond those already identified are probably involved in regulating eEF2K levels. Our findings have significant implications for therapeutic targeting of eEF2K, e.g. in oncology.

Eukaryotic elongation factor 2 kinase activity is controlled by multiple inputs from oncogenic signaling.

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.

Exendin-4 stimulates islet cell replication via the IGF1 receptor activation of mTORC1/S6K1.

Glucagon-like peptide 1 receptor (GLP1R) agonists, such as exendin-4, potentiate glucose-stimulated insulin secretion and are currently used in the management of type 2 diabetes. Interestingly, GLP1R agonists also have the ability to augment ß-cell mass. In this report, we provide evidence that in the presence of glucose, exendin-4 stimulates rodent islet cell DNA replication via the activation of ribosomal protein S6 kinase 1 (S6K1) and that this is mediated by the protein kinase B (PKB)-dependent activation of mTOR complex 1 (mTORC1). We show that activation of this pathway is caused by the autocrine or paracrine activation of the IGF1 receptor (IGF1R), as siRNA-mediated knockdown of the IGF1R effectively blocked exendin-4-stimulated PKB and mTORC1 activation. In contrast, pharmacological inactivation of the epidermal growth factor receptor has no discernible effect on exendin-4-stimulated PKB or mTORC1 activation. Therefore, we conclude that GLP1R agonists stimulate ß-cell proliferation via the PKB-dependent stimulation of mTORC1/S6K1 whose activation is mediated through the autocrine/paracrine activation of the IGF1R. This work provides a better understanding of the molecular basis of GLP1 agonist-induced ß-cell proliferation which could potentially be exploited in the identification of novel drug targets that increase ß-cell mass.

Eukaryotic elongation factor 2 kinase, an unusual enzyme with multiple roles.

Eukaryotic elongation factor 2 kinase (eEF2K) is a member of the small group of atypical 'α-kinases'. It phosphorylates and inhibits eukaryotic elongation factor 2, to slow down the elongation stage of protein synthesis, which normally consumes a great deal of energy and amino acids. The activity of eEF2K is normally dependent on calcium ions and calmodulin. eEF2K is also regulated by a plethora of other inputs, including inhibition by signalling downstream of anabolic signalling pathways such as the mammalian target of rapamycin complex 1. Recent data show that eEF2K helps to protect cancer cells against nutrient starvation and is also cytoprotective in other settings, including hypoxia. Growing evidence points to roles for eEF2K in neurological processes such as learning and memory and perhaps in depression.

A conserved loop in the catalytic domain of eukaryotic elongation factor 2 kinase plays a key role in its substrate specificity.

Eukaryotic elongation factor 2 kinase (eEF2K) is the best-characterized member of the α-kinase family. Within this group, only eEF2K and myosin heavy chain kinases (MHCKs) have known substrates. Here we have studied the roles of specific residues, selected on the basis of structural data for MHCK A and TRPM7, in the function of eEF2K. Our data provide the first information regarding the basis of the substrate specificity of α-kinases, in particular the roles of residues in the so-called N/D loop, which appears to occupy a position in the structure of α-kinases similar to that of the activation loop in other kinases. Several mutations in the EEF2K gene occur in tumors, one of which (Arg303Cys) is at a highly conserved residue in the N/D loop. This mutation greatly enhances eEF2K activity and may be cytoprotective. Our data support the concept that the major autophosphorylation site (Thr348 in eEF2K) docks into a binding pocket to help create the kinase-competent conformation. This is similar to the situation for MHCK A and is consistent with this being a common feature of α-kinases.

Molecular mechanisms of muscarinic acetylcholine receptor-stimulated increase in cytosolic free Ca(2+) concentration and ERK1/2 activation in the MIN6 pancreatic ß-cell line.

Muscarinic acetylcholine receptor (mAChR) activation of pancreatic ß-cells elevates intracellular Ca(2+) and potentiates glucose-stimulated insulin secretion. In addition, it activates a number of signaling molecules, including ERK1/2, whose activation has been shown to play an important role in regulating pancreatic ß-cell function and mass. The aim of this work was to determine how mAChR activation elevates intracellular Ca(2+) concentration ([Ca(2+)]( i )) and activates ERK1/2 in the pancreatic ß-cell line MIN6. We demonstrate that agonist-stimulated ERK1/2 activation is dependent on the activation of phospholipase C and an elevation in [Ca(2+)]( i ), but is independent of the activation of diacylglycerol-dependent protein kinase C isoenzymes. Using a pharmacological approach, we provide evidence that agonist-induced increases in [Ca(2+)]( i ) and ERK activity require (1) IP(3) receptor-mediated mobilization of Ca(2+) from the endoplasmic reticulum, (2) influx of extracellular Ca(2+) through store-operated channels, (3) closure of K(ATP) channels, and (4) Ca(2+) entry via L-type voltage-operated Ca(2+) channels. Moreover, this Ca(2+)-dependent activation of ERK is mediated via both Ras-dependent and Ras-independent mechanisms. In summary, this study provides important insights into the multifactorial signaling mechanisms linking mAChR activation to increases in [Ca(2+)]( i ) and ERK activity.

Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains.

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called 'α-kinases' which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured 'linker' region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

cAMP inhibits mammalian target of rapamycin complex-1 and -2 (mTORC1 and 2) by promoting complex dissociation and inhibiting mTOR kinase activity.

cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP](i) in mouse embryonic fibroblasts (MEFs) and human embryonic kidney 293 (HEK293) cells inhibits mTORC1 activation via a PKA-dependent mechanism. Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades (i.e. PKB, MAPK and AMPK) that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2(-/-)) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP](i) can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.

PERK activation at low glucose concentration is mediated by SERCA pump inhibition and confers preemptive cytoprotection to pancreatic ß-cells.

Protein kinase R-like ER kinase (PERK) is activated at physiologically low glucose concentrations in pancreatic ß-cells. However, the molecular mechanisms by which PERK is activated under these conditions and its role in ß-cell function are poorly understood. In this report, we investigated, in dispersed rat islets of Langerhans and mouse insulinoma-6 (MIN6) cells, the relationship between extracellular glucose concentration, the free endoplasmic reticulum (ER) calcium concentration ([Ca(2+)](ER)) measured directly using an ER targeted fluorescence resonance energy transfer-based calcium sensor, and the activation of PERK. We found that a decrease in glucose concentration leads to a concentration-dependent reduction in [Ca(2+)](ER) that parallels the activation of PERK and the phosphorylation of its substrate eukaryotic initiation factor-2α. We provide evidence that this decrease in [Ca(2+)](ER) is caused by a decrease in sarcoplasmic/ER Ca(2+)-ATPase pump activity mediated by a reduction in the energy status of the cell. Importantly, we also report that PERK-dependent eukaryotic initiation factor-2α phosphorylation at low glucose concentration plays a significant role in 1) the regulation of both proinsulin and global protein synthesis, 2) cell viability, and 3) conferring preemptive cytoprotection against ER stress. Taken together, these results provide evidence that a decrease in the ATP/energy status of the cell in response to a decrease in glucose concentration results in sarcoplasmic/ER Ca(2+)-ATPase pump inhibition, the efflux of Ca(2+) from the ER, and the activation of PERK, which plays an important role in both pancreatic ß-cell function and survival.

Identification of cAMP-dependent kinase as a third in vivo ribosomal protein S6 kinase in pancreatic beta-cells.

Ribosomal protein S6 (rpS6) is phosphorylated in vivo by isoforms of p70 S6 protein kinase and p90 ribosomal S6 kinase, and there is good evidence that it plays a positive role in controlling pancreatic beta-cell size and function. In this report, we demonstrate in the pancreatic beta-cell line MIN6 (mouse insulinoma cell line 6) and islets of Langerhans that agents which stimulate increases in cAMP, such as glucagon-like peptide-1 and forskolin, lead to the phosphorylation of rpS6 at Ser235/Ser236 independently of the activation of the currently known in vivo rpS6 kinases via a pathway that is sensitive to inhibitors of cAMP-dependent protein kinase [protein kinase A (PKA)]. This cAMP-dependent rpS6 kinase activity is also sensitive to PKI in vitro, and PKA exclusively phosphorylates recombinant rpS6 on Ser235/Ser236 in vitro. With these data taken together, we conclude that PKA can phosphorylate rpS6 exclusively at Ser235/Ser236 in vivo in pancreatic beta-cells, thus providing a potentially important link between cAMP signalling and the regulation of protein synthesis. Lastly, we provide evidence that PKA is also likely to phosphorylate rpS6 on Ser235/Ser236 in vivo in a number of other mammalian cell types.

Evaluation of the Litron In Vitro MicroFlow Kit for the flow cytometric enumeration of micronuclei (MN) in mammalian cells.

We have evaluated the performance of the prototype In Vitro MicroFlow Kit (Litron Laboratories), which offers a flow cytometric method for scoring micronuclei (MN). This method uses sequential staining to differentiate MN from chromatin fragments derived from apoptotic or necrotic cells. Data were generated using the genotoxins methylmethane sulphonate (MMS), dimethylbenzanthracene (DMBA) and vinblastine, and the non-genotoxins dexamethasone and staurosporine, which are known to induce apoptosis in vitro. The results obtained with these agents were compared with conventional microscopy. For short-duration exposures (3-4h) both manual and flow methodologies demonstrated good concordance, with concentration-related increases in the percentage of MN for MMS, DMBA and vinblastine. Statistically significant increases were observed at > or = 20 and 40 microg/mL, for manual and flow analysis, respectively, for MMS; at 0.5 and 0.75 microg/mL for DMBA; and at 0.035 and 0.04 microg/mL, respectively, for vinblastine. Dexamethasone showed clear negative responses by manual and flow cytometric analysis, with comparable results for both methodologies (all <1.7-fold compared with concurrent vehicle controls). Data for staurosporine, however, were less consistent showing significantly higher flow cytometric MN frequencies compared with those seen after manual analysis. Continuous (24 h) treatments were also conducted with MMS, vinblastine, dexamethasone and staurosporine. There was good concordance between the methodologies for MMS, staurosporine and vinblastine. However, dexamethasone generated discordant results, i.e. microscopic analysis was clearly negative at all doses tested, whereas flow cytometry produced significant increases in MN frequency (up to 8.1-fold at 100 microg/mL compared with the concurrent vehicle control). The inconsistencies observed between flow cytometry and standard microscopy, and the differences in assay sensitivity, particularly for apoptosis-inducing compounds, suggest that the prototype In Vitro MicroFlow Kit requires further refinement. Studies to investigate new parameters to address these issues are now under way and will be reported separately.

Distinct glucose-dependent stress responses revealed by translational profiling in pancreatic beta-cells.

In pancreatic beta-cells, following an acute (within 1 h) increase in glucose concentration, there are rapid changes in the expression of a large subset of proteins. The change in the expression of many of these proteins is mediated by a post-transcriptional mechanism through either increases or decreases in the rate of translation from pre-existing transcripts. These proteins, whose synthesis is rapidly up- or down-regulated in response to glucose, are likely important in mounting the correct response to changes in plasma glucose concentrations. However, the vast majority of these proteins remain unidentified. Therefore, in order to identify these proteins, we analysed changes in the levels of mRNAs associated with polysomes (i.e. actively translating mRNAs) isolated from mouse insulinoma 6 cells incubated at either 0.5 or 20 mM glucose for 1 h. Changes in the levels of polysomal mRNAs in response to glucose were analysed using affymetrix oligonucleotide microarrays (translational profiling). This work revealed that, in response to a change in glucose concentration, the abundance of 313 transcripts associated with polysomes changed by more than 1.5-fold, of which the abundance of 37 changed by more than twofold. The majority of these transcripts encoded proteins associated with metabolism or gene expression. More detailed analysis showed that a number of mRNAs encoding proteins associated with the induction of oxidative stress, including thioredoxin-2 and thioredoxin-interacting protein were rapidly redistributed onto heavier polysomes at high glucose concentration, indicating an increase in their expression. At low glucose concentration, when the general rate of protein synthesis is low, a number of mRNAs encoding integrated stress response proteins, including ATF4 and CHOP10, associate with heavier polysomes, indicating that their expression is up-regulated. In conclusion, translational profiling has revealed that, at either low or at high glucose concentration, beta-cells rapidly increase the synthesis of a specific subset of proteins that are likely important in maintaining beta-cell integrity and survival during conditions of nutritional stress.

The selective recruitment of mRNA to the ER and an increase in initiation are important for glucose-stimulated proinsulin synthesis in pancreatic beta-cells.

Glucose acutely stimulates proinsulin synthesis in pancreatic beta-cells through a poorly understood post-transcriptional mechanism. In the present study, we demonstrate in pancreatic beta-cells that glucose stimulates the recruitment of ribosome-associated proinsulin mRNA, located in the cytoplasm, to the ER (endoplasmic reticulum), the site of proinsulin synthesis, and that this plays an important role in glucose-stimulated proinsulin synthesis. Interestingly, glucose has greater stimulatory effect on the recruitment of proinsulin mRNA to the ER compared with other mRNAs encoding secretory proteins. This, as far as we are aware, is the first example whereby mRNAs encoding secretory proteins are selectively recruited to the ER and provides a novel regulatory mechanism for secretory protein synthesis. Contrary to previous reports, and importantly in understanding the mechanism by which glucose stimulates proinsulin synthesis, we demonstrate that there is no large pool of 'free' proinsulin mRNA in the cytoplasm and that glucose does not increase the rate of de novo initiation on the proinsulin mRNA. However, we show that glucose does stimulate the rate of ribosome recruitment on to ribosome-associated proinsulin mRNA. In conclusion, our results provide evidence that the selective recruitment of proinsulin mRNA to the ER, together with increases in the rate of initiation are important mediators of glucose-stimulated proinsulin synthesis in pancreatic beta-cells.