Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins.

Selective macroautophagy (autophagy) of ubiquitinated protein is implicated as a compensatory mechanism of the ubiquitin-proteasome system. p62/SQSTM1 is a key molecule managing autophagic clearance of polyubiquitinated proteins. However, little is known about mechanisms controlling autophagic degradation of polyubiquitinated proteins. Here, we show that the specific phosphorylation of p62 at serine 403 (S403) in its ubiquitin-associated (UBA) domain increases the affinity between UBA and polyubiquitin chain, resulting in efficiently targeting polyubiquitinated proteins in "sequestosomes" and stabilizing sequestosome structure as a cargo of ubiquitinated proteins for autophagosome entry. Casein kinase 2 (CK2) phosphorylates S403 of p62 directly. Furthermore, CK2 overexpression or phosphatase inhibition reduces the formation of inclusion bodies of the polyglutamine-expanded huntingtin exon1 fragment in a p62-dependent manner. We propose that phosphorylation of p62 at S403 regulates autophagic clearance of ubiquitinated proteins and protein aggregates that are poorly degraded by proteasomes.

Comprehensive screening for monogenic diabetes in 89 Japanese children with insulin-requiring antibody-negative type 1 diabetes.

BACKGROUND: Mutations in causative genes for neonatal diabetes or maturity-onset diabetes of the young have been identified in multiple patients with autoantibody-negative type 1 diabetes (T1D). OBJECTIVES: We aimed to clarify the prevalence and phenotypic characteristics of monogenic abnormalities among 89 children with autoantibody-negative insulin-requiring T1D. METHODS: Mutations in 30 genes were screened using next-generation sequencing, and copy-number alterations of 4 major causative genes were examined using multiplex-ligation-dependent probe amplification. We compared the clinical characteristics between mutation carriers and non-carriers. RESULTS: We identified 11 probable pathogenic substitutions (6 in INS , 2 in HNF1A , 2 in HNF4A , and 1 in HNF1B ) in 11 cases, but no copy-number abnormalities. Only 2 mutation carriers had affected parents. De novo occurrence was confirmed for 3 mutations. The non-carrier group, but not the carrier group, was enriched with susceptible HLA alleles. Mutation carriers exhibited comparable phenotypes to those of non-carriers, except for a relatively normal body mass index (BMI) at diagnosis. CONCLUSIONS: This study demonstrated significant genetic overlap between autoantibody-negative T1D and monogenic diabetes. Mutations in INS and HNF genes, but not those in GCK and other monogenic diabetes genes, likely play critical roles in children with insulin-requiring T1D. This study also suggests the relatively high de novo rates of INS and HNF mutations, and the etiological link between autoimmune abnormalities and T1D in the non-carrier group. Carriers of monogenic mutations show non-specific phenotypes among all T1D cases, although they are more likely to have a normal BMI at diagnosis than non-carriers.

Depletion of p62 reduces nuclear inclusions and paradoxically ameliorates disease phenotypes in Huntington's model mice.

Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine (polyQ) tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here, we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer life spans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyQ length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions.

A randomized crossover study of the efficacy and safety of switching from insulin glargine to insulin degludec in children with type 1 diabetes.

This study implemented a randomized crossover design to evaluate the efficacy and safety of switching from insulin glargine (IGlar) to insulin degludec (IDeg) in 18 children (11 males, 7 females; age 11.0 ± 0.5 years) with type 1 diabetes. All subjects had previously used IGlar once daily at bedtime. We compared fasting plasma glucose (FPG) and HbA1c levels, frequencies of overall and nocturnal (2200 h - 0659 h) hypoglycemia, and basal insulin dose at the baseline with those measured during a 24-week period during which IGlar or IDeg was administered in combination with pre-meal rapid acting insulin analogues. IDeg was initially given at the same dose as IGlar but was subsequently titrated to achieve FPG levels of 90-140 mg/dL. There were no significant changes in FPG and HbA1c levels from the baseline during the 24-week study period with IGlar or IDeg. The daily basal insulin dose did not significantly differ with IGlar or IDeg. Although the frequencies of overall hypoglycemia were similar, nocturnal hypoglycemia significantly decreased at 12 and 24 weeks from the baseline with IDeg use (2 ± 0.4 vs. 0 ± 0.3, 0 ± 0.5 episodes/month, both P <0.05), whereas no significant change in the frequency of nocturnal hypoglycemia was observed with IGlar. No severe hypoglycemia occurred during the study period with either basal insulin analogues. These results suggest that IDeg, injected once at bedtime, may provide similar glycemic control as IGlar while better reducing the risk of nocturnal hypoglycemia in children with type 1 diabetes.

Insulin glulisine for continuous subcutaneous insulin infusion in pediatric type 1 diabetes.

We evaluated the efficacy and safety of insulin glulisine (GLU) used for continuous s.c. insulin infusion (CSII) in 20 children with type 1 diabetes after 1 year of GLU treatment. There were no significant differences in mean plasma glucose before breakfast and before dinner between before and after using GLU, but the levels after breakfast and after dinner significantly improved, from 192.5 ± 31.7 to 162.0 ± 27.3 mg/dL for breakfast, and from 191.1 ± 33.3 to 161.1 ± 24.5 mg/dL for dinner (P < 0.01). Mean hemoglobin A1c significantly decreased (from 8.0 ± 0.8 to 7.7 ± 0.8%, P < 0.05), and the mean frequency of hypoglycemia significantly reduced after using GLU (from 8.3 ± 4.9 to 6.0 ± 3.4/month, P < 0.05). In conclusion, the use of GLU rather than other rapid-acting analogues for CSII might be an effective treatment option in children with type 1 diabetes.

Efficacy and safety of switching from insulin glargine to insulin degludec in young people with type 1 diabetes.

We evaluated the efficacy and safety of switching to insulin degludec (IDeg) from insulin glargine (IGlar) as basal-bolus therapy in young people with type 1 diabetes. The subjects were 36 patients, 21.3±1.0 years of age, with type 1 diabetes. IGlar had previously been injected once daily in 25 patients and twice daily in 11. They were then switched from IGlar to once-daily injection of IDeg. Both fasting plasma glucose (FPG) and HbA1c levels decreased significantly from 134±3.9 mg/dL and 7.9±0.2% at baseline to 116±2.2 mg/dL and 7.4±0.2% at 12 months after starting IDeg (P<0.0001 and P≤0.001, respectively). Overall and nocturnal hypoglycemia (PG<70 mg/dL) frequencies also decreased significantly from 4.9±0.7 and 2.0±0.3 times/month to 2.4±0.3 and 0.4±0.1 times/month at 12 months after starting IDeg (P≤0.005 and P<0.0005, respectively). The daily basal insulin dose was significantly reduced from 0.48±0.04 units/kg/day at baseline to 0.38±0.03 units/kg/day at the end of the study period (P<0.0001), which corresponded to 79.2% of the baseline value. Trends were similar in patients receiving the once-daily injection and those given twice-daily injections, but basal-insulin value reductions from baseline were more marked in patients receiving twice-daily injections of basal insulin (76.0% vs. 82.6% of the baseline value). These results suggest that switching from IGlar to an appropriate dose of IDeg may effectively control hyperglycemia while reducing the frequency of hypoglycemia episodes in young Japanese people with type 1 diabetes.

Intracellular localization and splicing regulation of FUS/TLS are variably affected by amyotrophic lateral sclerosis-linked mutations.

TLS (translocated in liposarcoma), also known as FUS (fused in sarcoma), is an RNA/DNA-binding protein that plays regulatory roles in transcription, pre-mRNA splicing and mRNA transport. Mutations in TLS are responsible for familial amyotrophic lateral sclerosis (ALS) type 6. Furthermore, TLS-containing intracellular inclusions are found in polyglutamine diseases, sporadic ALS, non-SOD1 familial ALS and a subset of frontotemporal lobar degeneration, indicating a pathological significance of TLS in a wide variety of neurodegenerative diseases. Here, we identified TLS domains that determine intracellular localization of the murine TLS. Among them, PY-NLS located in the C-terminus is a strong determinant of intracellular localization as well as splicing regulation of an E1A-derived minigene. Disruption of PY-NLS promoted the formation of cytoplasmic granules that were partially overlapped with stress granules and P-bodies. Some of the ALS-linked mutations altered both intracellular localization and splicing regulation of TLS, while most mutations alone did not affect splicing regulation. However, phospho-mimetic substitution of Ser505 (or Ser513 in human) could enhance the effects of ALS mutations, highlighting interplay between post-translational modification and ALS-linked mutations. These results demonstrate that ALS-linked mutations can variably cause loss of nuclear functions of TLS depending on the degree of impairment in nuclear localization.

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb.

The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb.

EphrinB-EphB signalling regulates clathrin-mediated endocytosis through tyrosine phosphorylation of synaptojanin 1.

Recent studies show that Eph receptors act mainly through the regulation of actin reorganization. Here, we show a novel mode of action for EphB receptors. We identify synaptojanin 1 - a phosphatidylinositol 5'-phosphatase that is involved in clathrin-mediated endocytosis - as a physiological substrate for EphB2. EphB2 causes tyrosine phosphorylation in the proline-rich domain of synaptojanin 1, and inhibits both the interaction with endophilin and the 5'-phosphatase activity of synaptojanin 1. Treatment with the EphB ligand, ephrinB2, elevates the cellular level of phosphatidylinositol 4,5-bisphosphate and promotes transferrin uptake. A kinase inactive mutant of EphB2 and a phosphorylation site mutant of synaptojanin 1 both neutralize the increase of transferrin uptake after ephrinB2 treatment. These mutants also inhibit AMPA glutamate receptor endocytosis in hippocampal neurons. Interestingly, incorporated transferrin does not reach endosomes, suggesting dual effects of EphB signalling on the early and late phases of clathrin-mediated endocytosis. Our results indicate that ephrinB-EphB signalling regulates clathrin-mediated endocytosis in various cellular contexts by influencing protein interactions and phosphoinositide turnover through tyrosine phosphorylation of synaptojanin 1.

Genetic ablation and chemical inhibition of IP3R1 reduce mutant huntingtin aggregation.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expansion of the polyglutamine (polyQ) stretch in huntingtin (htt). Previously, it has been shown that inhibition of the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) activity reduced aggregation of pathogenic polyQ proteins. Experimentally, this effect was achieved by modification of the intracellular IP3 levels or by application of IP3R1 inhibitors, such as 2-aminoethyl diphenylborinate (2-APB). Unfortunately, there are certain concerns about the 2-APB specificity and cytotoxicity. Moreover, a direct link between IP3R1 and polyQ aggregation has not been shown yet. In this study we show, that down-regulation of the IP3R1 levels by shRNA reduced the aggregation of mutant htt. We tested 2-APB analogs in an attempt to identify less toxic and more IP3R1-specific compounds and found that the effect of these analogs on the reduction of the mutant htt aggregation did weakly correlate with their inhibitory action toward the IP3-induced Ca(2+) release (IICR). Their effect on aggregation was not correlated with the store-operated Ca(2+) entry (SOCE), which is another target of the 2-APB related compounds. Our findings suggest that besides functional contribution of the IP3R inhibition on the mutant htt aggregation there are additional mechanisms for the anti-aggregation effect of the 2-APB related compounds.

Heparan sulfate regulates ephrin-A3/EphA receptor signaling.

Increasing evidence indicates that many signaling pathways involve not only ligands and receptors but also various types of coreceptors and matrix components as additional layers of regulation. Signaling by Eph receptors and their ephrin ligands plays a key role in a variety of biological processes, such as axon guidance and topographic map formation, synaptic plasticity, angiogenesis, and cancer. Little is known about whether the ephrin-Eph receptor signaling system is subject to such additional layers of regulation. Here, we show that ephrin-A3 binds to heparan sulfate, and that the presence of cell surface heparan sulfate is required for the full biological activity of ephrin-A3. Among the ephrins tested, including ephrin-A1, -A2, -A5, -B1, and -B2, only ephrin-A3 binds heparin or heparan sulfate. Ephrin-A3-dependent EphA receptor activation is reduced in mutant cells that are defective in heparan sulfate synthesis, in wild-type cells from which cell surface heparan sulfate has been removed, and in the hippocampus of conditional knockout mice defective in heparan sulfate synthesis. Ephrin-A3-dependent cell rounding is impaired in CHO cells lacking heparan sulfate, and cortical neurons lacking heparan sulfate exhibit impaired growth cone collapse. In contrast, cell rounding and growth cone collapse in response to ephrin-A5, which does not bind heparan sulfate, are not affected by the absence of heparan sulfate. These results show that heparan sulfate modulates ephrin/Eph signaling and suggest a physiological role for heparan sulfate proteoglycans in the regulation of ephrin-A3-dependent biological processes.

The polymicrogyria-associated GPR56 promoter preferentially drives gene expression in developing GABAergic neurons in common marmosets.

GPR56, a member of the adhesion G protein-coupled receptor family, is abundantly expressed in cells of the developing cerebral cortex, including neural progenitor cells and developing neurons. The human GPR56 gene has multiple presumptive promoters that drive the expression of the GPR56 protein in distinct patterns. Similar to coding mutations of the human GPR56 gene that may cause GPR56 dysfunction, a 15-bp homozygous deletion in the cis-regulatory element upstream of the noncoding exon 1 of GPR56 (e1m) leads to the cerebral cortex malformation and epilepsy. To clarify the expression profile of the e1m promoter-driven GPR56 in primate brain, we generated a transgenic marmoset line in which EGFP is expressed under the control of the human minimal e1m promoter. In contrast to the endogenous GPR56 protein, which is highly enriched in the ventricular zone of the cerebral cortex, EGFP is mostly expressed in developing neurons in the transgenic fetal brain. Furthermore, EGFP is predominantly expressed in GABAergic neurons, whereas the total GPR56 protein is evenly expressed in both GABAergic and glutamatergic neurons, suggesting the GABAergic neuron-preferential activity of the minimal e1m promoter. These results indicate a possible pathogenic role for GABAergic neuron in the cerebral cortex of patients with GPR56 mutations.

Digital gene atlas of neonate common marmoset brain.

Interest in the common marmoset (Callithrix jacchus) as a primate model animal has grown recently, in part due to the successful demonstration of transgenic marmosets. However, there is some debate as to the suitability of marmosets, compared to more widely used animal models, such as the macaque monkey and mouse. Especially, the usage of marmoset for animal models of human cognition and mental disorders, is still yet to be fully explored. To examine the prospects of the marmoset model for neuroscience research, the Marmoset Gene Atlas (https://gene-atlas.bminds.brain.riken.jp/) provides a whole brain gene expression atlas in the common marmoset. We employ in situ hybridization (ISH) to systematically analyze gene expression in neonate marmoset brains, which allows us to compare expression with other model animals such as mouse. We anticipate that these data will provide sufficient information to develop tools that enable us to reveal marmoset brain structure, function, cellular and molecular organization for primate brain research.

Efficacy and safety of alogliptin in a pediatric patient with maturity-onset diabetes of the young type 1.

The first-line pharmacological treatment for patients with maturity-onset diabetes of the young type 1 (MODY1) and maturity-onset diabetes of the young type 3 (MODY3) are sulfonylureas (SUs) or insulin. However, several reports have suggested the possibility of using incretin-associated drugs, including dipeptidyl-peptidase-4 (DPP-4) inhibitors, for the treatment of patients with these types of MODY. Here we report a case of a pediatric patient with MODY1 who was successfully treated with a DPP-4 inhibitor, alogliptin. A 13-yr-old Japanese girl with diabetes was initially treated with insulin for 5 mo. After diagnosis of MODY1, confirmed via a genetic analysis, treatment was changed from insulin to alogliptin. SUs were prescribed temporarily, but monotherapy with alogliptin finally resulted in good glycemic control. After changing to alogliptin, the patient maintained optimal glycemic control with glycated hemoglobin levels of 6.3-7.0% while maintaining substantial ß-cell function. No adverse events associated with alogliptin were observed. These results suggest that DPP-4 inhibitors may be a potential treatment for patients with MODY1 at the early stage of the disease when residual insulin secretion is still being sustained.

Nucleotide substitutions in CD101, the human homolog of a diabetes susceptibility gene in non-obese diabetic mouse, in patients with type 1 diabetes.

AIMS/INTRODUCTION: Although genome-wide association studies have identified more than 50 susceptibility genes for type 1 diabetes, low-frequency risk variants could remain unrecognized. The present study aimed to identify novel type 1 diabetes susceptibility genes by newly established methods. MATERIALS AND METHODS: We carried out whole-exome sequencing and genome-wide copy-number analysis for a Japanese family consisting of two patients with type 1 diabetes and three unaffected relatives. Further mutation screening was carried out for 127 sporadic cases. The functional consequences of identified substitutions were evaluated by in silico analyses and fluorescence-activated cell sorting of blood samples. RESULTS: Whole-exome sequencing and genome-wide copy-number analysis of familial cases showed co-segregation of the p.V863L substitution in CD101, the human homolog of an autoimmune diabetes gene in the non-obese diabetic mouse, with type 1 diabetes. Mutation screening of CD101 in 127 sporadic cases detected the p.V678L and p.T944R substitutions in two patients. The p.V863L, p.V678L and p.T944R substitutions were absent or extremely rare in the general population, and were assessed as 'probably/possibly damaging' by in silico analyses. CD101 expression on monocytes, granulocytes and myeloid dendritic cells of mutation-positive patients was weaker than that of control individuals. CONCLUSIONS: These results raise the possibility that CD101 is a susceptibility gene for type 1 diabetes.

Endocrinopathies in a boy with cryptic copy-number variations on 4q, 7q and Xp.

We report a male patient with three copy-number variations (CNVs) and unique phenotype. He carried ~11.2 Mb terminal duplication on 4q, ~13.4 Mb terminal deletion on 7q and ~1.7 Mb interstitial duplication on Xp22.31, which were identified by array-based comparative genomic hybridization. He manifested mental retardation, mild brain anomalies and skeletal deformities ascribable to these CNVs, together with central precocious puberty and mild adrenocorticotropic hormone overproduction of unknown etiologies.

Three years of liraglutide treatment offers continuously optimal glycemic control in a pediatric patient with maturity-onset diabetes of the young type 3.

Sulfonylureas (SUs) are recommended as the first-line pharmacological treatment in patients with uncontrolled maturity-onset diabetes of the young type 3 (MODY3). In contrast, glucagon-like peptide-1 (GLP-1) receptor agonists have the advantages of a low risk of hypoglycemia and maintained ß-cell function. We report a pediatric patient with MODY3 treated with a GLP-1 receptor agonist, liraglutide. A 12-year-old Japanese girl with MODY3 had been treated with insulin for 6 months since the time of diagnosis. After genetic analysis, we switched her treatment from insulin to liraglutide. After switching to liraglutide, the patient maintained optimal glycemic control with hemoglobin A1c levels of 6.8%-7.5% and had postprandial C-peptide levels >3.0 ng/mL during a 3-year treatment period. No adverse events associated with liraglutide were observed. GLP-1 receptor agonists are the potential medications for patients with MODY3 who maintain residual insulin secretion.

Efficacy and safety of switching to insulin glulisine from other rapid-acting insulin analogs in children with type 1 diabetes.

We investigated the efficacy and safety of switching to insulin glulisine (GLU) from other rapid-acting insulin analogs (Ra) in children with type 1 diabetes treated with multiple daily injections of insulin or continuous subcutaneous insulin infusion. A total of 26 children with type 1 diabetes were included. Ra in all of these patients was changed to GLU, and they were observed for a 6-month period after having previously finished treatment with other Ra. The mean glycated hemoglobin value decreased from 7.6 ± 1.0 to 7.4 ± 0.9% (P = 0.0034), and mean plasma glucose values after breakfast and supper also improved from 183 ± 50 to 153 ± 32 mg/dL (P = 0.0035), and from 203 ± 29 to 164 ± 23 mg/dL (P < 0.0001), respectively. Furthermore, the mean frequency of hypoglycemia was reduced from 7 ± 6 to 4 ± 4/month (P = 0.0004), while insulin doses and obesity degree were stable with statistically non-significant differences. In conclusion, switching to GLU might be a good treatment option for improving glycemic control in children with type 1 diabetes.

Characterization of a multipotent neural progenitor cell line cloned from an adult p53-/- mouse cerebellum.

Here we report developmental characteristics of clonal cell line 2Y6f1, which was established from an adult p53(-/-) mouse cerebellum. 2Y6f1 began as a homogeneous population of small polygonal epithelial cells, but during passages it gradually became heterogeneous, containing cells of varying size and shape that expressed either neuron- or astrocyte-specific proteins. Supplements to the culture medium altered the levels of some of the cell type markers. For example, addition of insulin increased expression of neurofilaments, while cholera toxin increased that of glial fibrillary acidic protein. In a colony assay, 2Y6f1 cells gave rise to both homogeneous and heterogeneous colonies, consistent with the idea that they contained multipotent neural progenitor cells. Establishment of subclones that were exclusively neuronal or astroglial in differentiation further supported the conclusion that 2Y6f1 cells have many features that may qualify them as bona fide stem cells and make them a useful new model in neural stem cell biology.

Basal insulin requirement of youth with type 1 diabetes differs according to age.

We investigated the percentage of total basal insulin dose to total daily insulin dose (%TBD) among Japanese youth of different ages with type 1 diabetes. The study enrolled 69 patients with type 1 diabetes who were treated with multiple daily injections of insulin. The participants were divided into the following age groups: group A, 0 to <10 years (n = 18); group B, 10 to <20 years (n = 31) and group C, 20 to <25 years (n = 20). We found no difference in the sex ratio, body mass index, and glycated hemoglobin and 2-h postprandial C-peptide levels among the three groups. Participants assigned to group B had a significantly higher percentage of total daily insulin dose than those in group A and group C (49.7 ± 10.4% vs 38.5 ± 13.7% and 38.3 ± 8.2%, P = 0.0005). In conclusion, the basal insulin requirements of Japanese youth with type 1 diabetes might have an age effect that is associated with puberty.