DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency.

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.

The G protein-coupled taste receptor T1R1/T1R3 regulates mTORC1 and autophagy.

Cells continually assess their energy and nutrient state to maintain growth and survival and engage necessary homeostatic mechanisms. Cell-autonomous responses to the fed state require the surveillance of the availability of amino acids and other nutrients. The mammalian target of rapamycin complex 1 (mTORC1) integrates information on nutrient and amino acid availability to support protein synthesis and cell growth. We identify the G protein-coupled receptor (GPCR) T1R1/T1R3 as a direct sensor of the fed state and amino acid availability. Knocking down this receptor, which is found in most tissues, reduces the ability of amino acids to signal to mTORC1. Interfering with this receptor alters localization of mTORC1, downregulates expression of pathway inhibitors, upregulates key amino acid transporters, blocks translation initiation, and induces autophagy. These findings reveal a mechanism for communicating amino acid availability through a GPCR to mTORC1 in mammals.

Time to failure of oral therapy in children with type 2 diabetes: a single center retrospective chart review.

There are no data in children with type 2 diabetes (T2D) regarding the durability of glycemic control with oral medication. Therefore, we assessed the likelihood of and time to failure of oral therapy in children and adolescents diagnosed with T2D. Charts of patients presenting to our large tertiary-care children's hospital between January 2000 and June 2007 with a new diagnosis of diabetes (n = 1625) were reviewed to identify those with T2D (n = 184). Subjects' initial therapy, hemoglobin A1c (HbA1c), body mass index, age, gender, and antibody status were documented, as well as subsequent therapies and HbA1c values, to determine whether baseline characteristics predicted future insulin dependence. Kaplan-Meier survival curves and Cox proportional hazards analysis demonstrated time to failure of oral therapy. Eighty-nine patients remained on insulin throughout the study. Baseline characteristics that determined future insulin dependence included being placed on insulin initially, initial HbA1c and race (whites less likely to be insulin dependent at study conclusion). Patients who failed oral therapy were more often reported to be non-compliant or unable to tolerate metformin than those who continued on oral therapy. The median time to failure of oral therapy (metformin monotherapy in 84/95) was not significantly different for patients initially treated with oral therapy (42 months) and insulin (35 months). Thus, children with T2D appear to fail oral therapy more quickly than what is reported in adults. It is not yet known if improving compliance with treatment might allow more children to remain on oral medications.

Insulin-like growth factor-I and fibroblast growth factor, but not growth hormone, affect growth plate chondrocyte proliferation.

Of the many factors that regulate linear growth, IGF-I has a central role in epiphyseal chondrocyte development. Whether IGF-I is solely of systemic or also of local origin is uncertain, as is how other growth factors interact with IGF-I at the growth plate. We studied the proliferative effects of IGF-I on juvenile bovine epiphyseal chondrocytes fractionated by density gradient centrifugation. Cell density correlated with size, glycogen content, and gene expression patterns. There was a gradient of response to IGF-I, with the greatest proliferative response in high-density cells corresponding to the reserve zone, as measured by [3H]thymidine uptake. Low-density (hypertrophic zone) cells proliferated only when exposed to IGF-I and basic fibroblast growth factor (FGF). The gradient of IGF-I response correlated with [125I]IGF-I binding as determined by Scatchard analysis: IGF-I receptor number was 10-fold greater in reserve zone cells than in hypertrophic cells. When exposed to basic FGF for 24 hours, IGF-I binding in hypertrophic cells increased 3-fold. In contrast, no specific binding of GH was demonstrated in juvenile bovine chondrocytes. GH produced neither signal transducer and activator of transcription phosphorylation, increased proliferation, nor increased IGF-I mRNA levels in any chondrocyte fraction. IGF-I mRNA levels were extremely low at 800-1100 copies/microg 18S RNA in bovine chondrocytes. These results suggest that the major regulator of chondrocyte proliferation is systemic IGF-I; FGFs may influence the actions of IGF-I at the growth plate by altering its receptor number in chondrocytes.

Isoxazole Alters Metabolites and Gene Expression, Decreasing Proliferation and Promoting a Neuroendocrine Phenotype in ß-Cells.

Novel strategies are needed to modulate ß-cell differentiation and function as potential ß-cell replacement or restorative therapies for diabetes. We previously demonstrated that small molecules based on the isoxazole scaffold drive neuroendocrine phenotypes. The nature of the effects of isoxazole compounds on ß-cells was incompletely defined. We find that isoxazole induces genes that support neuroendocrine and ß-cell phenotypes and suppresses genes important for proliferation. Isoxazole alters ß-cell metabolites and protects glucose-responsive signaling pathways under lipotoxic conditions. Finally, we show that isoxazole improves glycemia in a mouse model of ß-cell regeneration. Isoxazole is a prime candidate to alter cell fate in different contexts.

Prostacyclin regulates bone growth via the Epac/Rap1 pathway.

Prostaglandins, particularly PGE2, are important to adult bone and joint health, but how prostaglandins act on growth plate cartilage to affect bone growth is unclear. We show that growth plate cartilage is distinct from articular cartilage with respect to cyclooxygenase (COX)-2 mRNA expression; although articular chondrocytes express very little COX-2, COX-2 expression is high in growth plate chondrocytes and is increased by IGF-I. In bovine primary growth plate chondrocytes, ATDC5 cells, and human metatarsal explants, inhibition of COX activity with nonsteroidal antiinflammatory drugs (NSAIDs) inhibits chondrocyte proliferation and ERK activation by IGF-I. This inhibition is reversed by prostaglandin E2 and prostacyclin (PGI2) but not by prostaglandin D2 or thromboxane B2. Inhibition of COX activity in young mice by ip injections of NSAIDs causes dwarfism. In growth plate chondrocytes, inhibition of proliferation and ERK activation by NSAIDs is reversed by forskolin, 8-bromoadenosine, 3',5'-cAMP and a prostacyclin analog, iloprost. The inhibition of proliferation and ERK activation by celecoxib is also reversed by 8CPT-2Me-cAMP, an activator of Epac, implicating the small G protein Rap1 in the pathway activated by iloprost. These results imply that prostacyclin is required for proper growth plate development and bone growth.

Mice with a conditional deletion of the neurotrophin receptor TrkB are dwarfed, and are similar to mice with a MAPK14 deletion.

Long bone growth results from ordered chondrocyte development within the cartilagenous growth plate. Chondrocytes are recruited from a resting pool to proliferate along the long axis of the bone, until various signals trigger differentiation and hypertrophy. We have shown previously that the neurotrophin receptor TrkB is expressed in growth plate chondrocytes, where the tyrosine kinase receptor regulates the pace of hypertrophic differentiation by modulating the activities of ERK and p38 MAP kinases. To investigate the physiological relevance of TrkB to bone growth in vivo, we generated mice with a targeted disruption of the receptor, and compared them to mice targeted for MAPK14, the gene for p38α. The TrkB mutant and p38α mutant mice showed a similar degree of dwarfism and delayed hypertrophic differentiation. To extend these findings, we showed that both the TrkB and p38α mutant mice have altered expression of Runx2 and Sox9, two key transcription factors required for skeletogenesis. The data provides in vivo evidence for the role of TrkB in bone growth, supports the role of p38 downstream of TrkB, and suggests that Runx2 and Sox9 expression is regulated by this pathway at the growth plate.

BDNF alters ERK/p38 MAPK activity ratios to promote differentiation in growth plate chondrocytes.

The ERK and p38 MAPK pathways are well-known transducers of signals that regulate proliferation and differentiation, but precisely how these pathways control growth plate chondrocyte development is unclear. For example, the ERK pathway has been reported to be required by some investigators but inhibitory to chondrocyte development by others. Moreover, how these two pathways interact to regulate chondrocyte development is even less clear. Using primary bovine growth plate chondrocytes and murine ATDC5 cells, we demonstrate that the ERK and p38 pathways have opposing effects on proliferation but are both absolutely required for differentiation. Two factors that promote chondrocyte differentiation, brain-derived neurotrophic factor (BDNF) and C-type natriuretic peptide, increase p38 activity while decreasing, but not completely inhibiting, ERK activity. The attenuation of ERK activity by BDNF occurs via p38-dependent raf-1 inhibition. The inhibition of raf-1 by p38 is direct, because purified p38 protein inhibits the kinase activity of purified active raf-1 as well as raf-1 immunoprecipitated from chondrocyte lysates. Moreover, IGF-I, which stimulates proliferation, suppresses p38 activation. This work describes a model wherein unopposed IGF-I promotes high ERK/p38 activity ratios favoring proliferation, whereas BDNF signals a transition to differentiation by decreasing the ERK/p38 activity ratio without completely inhibiting ERK, which involves the direct inhibition of raf-1 by p38.

Vitamin D deficiency in obese children and its relationship to glucose homeostasis.

OBJECTIVES: The aim of the study was to compare the prevalence of vitamin D deficiency in obese and non-overweight children in North Texas, to examine relationships between dietary habits and 25-hydroxyvitamin D [25(OH)D] level in obese children, and to examine the relationship between 25(OH)D level and markers of abnormal glucose metabolism and blood pressure. PATIENTS AND METHODS: Using a cross-sectional design, systolic and diastolic blood pressure, dietary information, serum 25(OH)D, fasting glucose and insulin, 2-h glucose from oral glucose tolerance test, hemoglobin A1c, and homeostasis model assessment of insulin resistance were recorded for 411 obese subjects (6-16 yr old) at an obesity referral clinic. 25(OH)D was also obtained from 87 control non-overweight subjects (6-16 yr old). RESULTS: Ninety-two percent of obese subjects had a 25(OH)D level below 75 nmol/liter, and 50% were below 50 nmol/liter. Among non-overweight subjects, these frequencies were 68 and 22%, respectively (both P < 0.01 compared with obese subjects). 25(OH)D was negatively associated with soda intake (P < 0.001), juice intake (P = 0.009), and skipping breakfast (P < 0.001). 25(OH)D was negatively correlated with homeostasis model assessment of insulin resistance (r = -0.19; P = 0.001) and 2-h glucose (r = -0.12; P = 0.04) after adjustment for body mass index and age but was not correlated with hemoglobin A1c, systolic blood pressure Z score, or diastolic blood pressure Z score. CONCLUSIONS: Vitamin D deficiency is common in children in this southern United States location and is significantly more prevalent in obese children. Lower 25(OH)D level is associated with risk factors for type 2 diabetes in obese children.

SCF, BDNF, and Gas6 are regulators of growth plate chondrocyte proliferation and differentiation.

We previously demonstrated that bovine epiphyseal chondrocytes separated by density gradient centrifugation differ in proliferative response to IGF-I and IGF-I receptor number. To identify novel modifiers of IGF-I action at the growth plate, we used microarray analyses to compare bovine hypertrophic and reserve zones and identified several receptors differentially expressed across the growth plate: NTRK2 [receptor for brain-derived neurotrophic factor (BDNF)], KIT [receptor for stem cell factor (SCF)], and MER and AXL [two receptors for growth arrest-specific 6 (Gas6)]. The corresponding ligands were tested for their ability to stimulate either proliferation of isolated chondrocytes or differentiation in ATDC5 cells. Each factor inhibited IGF-I-mediated proliferation in isolated chondrocytes by attenuating ERK1/2 activation. SCF, BDNF, Gas6, and C-type natriuretic peptide promoted differentiation in ATDC5 cells, each factor producing different expression patterns for collagen X, collagen 2, aggrecan, and lysyl oxidase. Whereas multiple factors stimulated ATDC5 differentiation, only IGF-I and high-dose insulin, out of several factors implicated in chondrocyte maturation, stimulated proliferation of isolated chondrocytes. IGF-I appears to be the primary proliferative signal in growth plate chondrocytes, whereas multiple factors including SCF, BDNF, and Gas6 regulate the pace of differentiation at the growth plate.