Short-term Insulin Research Articles

FoxO Transcription Factors Are Critical Regulators of Diabetes-Related Muscle Atrophy

Insulin deficiency and uncontrolled diabetes lead to a catabolic state with decreased muscle strength, contributing to disease-related morbidity. FoxO transcription factors are key mediators of insulin action. To study their role in diabetic muscle wasting, we created mice with musclespecific triple knockout of FoxO1/3/4 and induced diabetes in these M-FoxO-TKO mice with streptozotocin (STZ). Muscle mass and myofiber area were decreased 20-30% in STZ-Diabetes mice due to increased ubiquitinproteasome degradation and autophagy, characterized by increased pULK1 and LC3-II. Both the muscle loss and markers of increased degradation/autophagy were completely prevented in STZ-FoxO-TKO mice. Transcriptomic analyses revealed FoxO-dependent increases in ubiquitin-mediated proteolysis pathways in STZ-Diabetes, including regulation of Fbxo32 (Atrogin1), Trim63 (MuRF1), Bnip3L, and Gabarapl. These same genes were increased 1.4- to 3.3-fold in muscle from type 1 diabetics after shortterm insulin deprivation. Thus, FoxO-regulated genes play a rate-limiting role in increased protein degradation and muscle atrophy in insulin-deficient diabetes.

Inzulinová pumpa u diabetu 2. typu: léčba zaměřená na B-buňku

Type 2 diabetes is a disorder characterized by insulin resistance and progressive deterioration of B-cell insulin secretion. B-cell protective strategies for lowering glucolipotoxicity by rapid achievement of normoglycemia using exogenous insulin improve their function and prolong diabetes remission. Insulin pump is an effective treatment method in newly diagnosed diabetes, where even short-term pump therapy is B-cell protective. Combination therapy with insulin pump and antidiabetics targeting the incretin system acts in synergy to protect the B-cell. While the positive effect of insulin pump is apparent even a year after stopping the therapy, the effect of incretins lasts only while on the medication. Short-term insulin treatment, especially delivered by insulin pump, is an effective method of B-cell protection in recent type 2 diabetes.Key words: B-cell function - diabetes mellitus - insulin pump - insulin resistance - type 2 diabetes.

Regulation of Tight-Junction Integrity by Insulin in an In Vitro Model of Human Blood–Brain Barrier

Although insulin receptor is expressed at the human blood–brain barrier (BBB), the physiological and pathologic roles of insulin signaling in biologic responses at the BBB remain unclear. Here, we investigate insulin signaling at the human BBB using human cerebral microvascular endothelial cell line (hCMEC/D3) as a well-established in vitro model. Western blot analysis showed that insulin induced phosphorylation of extracellular signal-regulated kinase and insulin receptor substrate-1 in hCMEC/D3 cells. Short-term insulin stimulation increased cell proliferation via the canonical phosphoinositide-3 kinase/protein kinase B and mitogen-activated protein kinase signaling pathways, suggesting that insulin signaling is involved in the regulation of biologic responses in the human BBB. We also found that insulin rapidly increased tight-junction integrity of hCMEC/D3 cells via the phosphoinositide-3 kinase/protein kinase B/glycogen synthase kinase-3 β signaling pathway. Inhibition of insulin/insulin-like growth factor-1 receptor kinase by AG1024 blocked the increase of tight-junction integrity. In addition, high-insulin/high-glucose treatment (as a model of hyperglycemia and hyperinsulinemia) synergistically reduced the tight-junction integrity in hCMEC/D3 cells, although either condition alone had little or no effect. Our findings suggest that, in addition to the established role of interactions of astrocytes and pericytes with brain capillary endothelial cells, insulin signaling from the blood side of the BBB contributes to maintenance of homeostasis by regulating cell proliferation and tight-junction integrity.

Reactive oxygen species are involved in insulin-dependent regulation of autophagy in primary rat podocytes

Autophagy is an intracellular defense mechanism responsible for the turnover of damaged or non-functional cellular constituents. This process provides cells with energy and essential compounds under unfavorable environmental conditions—such as oxidative stress and hyperglycemia, which are both observed in diabetes. The most common diabetes complication is diabetic nephropathy (DN), which can lead to renal failure. This condition often includes impaired podocyte function. Here we investigated autophagic activity in rat podocytes cultured with a high insulin concentration (300nM). Autophagy was activated after 60min of insulin stimulation. Moreover, this effect was abolished following pharmacological (apocynin) or genetic (siRNA) inhibition of NAD(P)H oxidase activity, indicating that insulin-dependent autophagy stimulation involved reactive oxygen species (ROS). We also observed a continuous and time-dependent increase of podocyte albumin permeability in response to insulin, and this process was slightly improved by autophagy inhibition following short-term insulin exposure. Our results suggest that insulin may be a factor affecting the development of diabetic nephropathy.

Effect of short-term insulin pump therapy on left ventricular systolic function in type 2 diabetes mellitus patients evaluated by three-dimensional speckle tracking imaging

Objective To assess the improvements of left ventricular systolic function by three-dimensional speckle tracking imaging (3D-STI) in type 2 diabetes mellitus (T2DM) patients after short-term insulin pump intensive therapy. Methods Thirty-five T2DM patients complicated with microangiopathy and thirty-two healthy volunteers were studied, underwent the dynamic image of the four-chamber view, three-dimensional images of left ventricle were obtained for all the individuals. The left ventricular global longitudinal strain (LVGLS), left ventricular global circumferential strain (LVGCS), left ventricular ejection fraction (LVEF), peak basal and apical rotation (LV-ProtB, LV-ProtA), peak LV twist (LV-tw ) were calculated using TomTec software.After insulin pump intensive therapy for two weeks, all the indexes were reexamined in T2DM patients. Results Compared with control group, the LVGLS, LVGCS, LV-tw and LV-ProtA were significantly decreased in diabetes mellitus group before and after treatment (P<0.01 or P<0.05). Compared with diabetes mellitus patients before treatment, the LVGLS, LVGCS had significant higher level after treatment(P<0.05). The LVGLS, LVGCS, LV-tw and LV-ProtA were significantly correlated with LVEF in type 2 diabetes mellitus patients and normal controls. Conclusions Insulin pump intensive treatment could improve left ventricular systolic function in type 2 diabetes patients complicated with microangiopathy. 3D-STI can be sensitive to accurately assess the therapeutic effect and has the important clinical value. Key words: Echocardiography; Type 2 diabetes mellitus; Insulin pump intensive therepy; Three dimensional speckle tracking imaging

Long-term insulin treatment restores cardioprotection induced by sufentanil postconditioning in diabetic rat heart

Sufentanil, a commonly used opioid analgesic, could mimic ischemia postconditioning to attenuate ischemia reperfusion injury, but this effect might be hindered in diabetic animals by inhibition of glycogen synthase kinase-3β phosphorylation. Also, diabetes can abrogate the cardioprotection of sevoflurane (an inhaled anesthetic) against ischemia reperfusion injury, and short-term insulin treatment does not restore protection by sevoflurane postconditioning. We hypothesized that long-term insulin treatment might restore the cardioprotective effect of sufentanil postconditioning in diabetic rats via phosphorylation of glycogen synthase kinase-3β. Streptozotocin (55 mg/kg)-induced diabetic rats received insulin (Novolin N, 6-8 u/d) for two days or two weeks, then were exposed to 30-min ischemia and 120-min reperfusion. Sufentanil postconditioning was performed 5 min before the onset of reperfusion. Controls included non-diabetic rats, sham surgery for ischemia/reperfusion, and sufentanil vehicle. Infarct size, cardiac troponin I, and phosphorylated glycogen synthase kinase-3β were examined. Sufentanil postconditioning reduced infarct size by 46% in non-diabetic rats (P < 0.001), but diabetes prevented this protective effect. Two-day insulin treatment was not effective, but two-week treatment reduced infarct size by 45% (P < 0.001), reduced cardiac troponin I by 33% (P < 0.001), and increased phosphorylated glycogen synthase kinase-3β levels (P < 0.001) in the diabetic sufentanil postconditioning group. In conclusion, sufentanil-induced cardioprotection was restored by long-term insulin treatment. The underlying mechanism may be increased phosphorylation of glycogen synthase kinase-3β.

Novel Strategies for Inducing Glycemic Remission during the Honeymoon Phase of Type 2 Diabetes.

A lofty goal in the management of type 2 diabetes is the achievement of glycemic remission. Glycemic remission can be defined as the sustained maintenance of normoglycemia without antidiabetic therapy for variable periods of time after stopping an initial disease-modifying intervention. Although this goal remains largely elusive at this time, growing recognition of the potential reversibility of pancreatic beta-cell dysfunction early in the course of type 2 diabetes has yielded a target for such disease modification. Furthermore, short-term intensive insulin therapy for 2 to 5 weeks has emerged as an intervention that could be applied as a biologic agent for this purpose during a window of opportunity that we have called the honeymoon phase of type 2 diabetes. This recognition has led to a novel therapeutic paradigm consisting of initial induction therapy to improve reversible beta-cell dysfunction during the honeymoon phase, followed by maintenance therapy aimed at preserving this beneficial beta-cell effect. This concept of induction and maintenance therapy is being applied in a series of recent and ongoing clinical trials, toward the goal of ultimately preserving beta-cell function and thereby modifying the natural history of type 2 diabetes.

Activated pancreatic stellate cells can impair pancreatic islet function in mice

Background. Pancreatic or islet fibrosis is often associated with activated pancreatic stellate cells (PSCs). PSCs are considered not only to promote fibrosis, but also to be associated with glucose intolerance in some diseases. We therefore evaluated morphological and functional relationships between islets and PSCs in the normal mouse pancreas and transplanted islets.Methods. Immunohistochemistry was used to map the presence of PSCs in the normal mouse pancreas and islets implanted under the renal capsule. We isolated and cultured mouse PSCs and characterized them morphologically by immunofluorescence staining. Furthermore, we measured their cytokine production and determined their effects on insulin release from simultaneously cultured islets.Results. PSCs were scattered throughout the pancreas, with occasional cells within the islets, particularly in the islet capsule. In islet transplants they were found mainly in the graft periphery. Cultured PSCs became functionally activated and produced several cytokines. Throughout the culture period they linearly increased their production of interleukin-6 and mammalian keratinocyte-derived chemokine. PSC cytokine production was not affected by acute hyperglycemia. Syngeneic islets co-cultured with PSCs for 24–48 h increased their insulin release and lowered their insulin content. However, short-term insulin release in batch-type incubations was unaffected after 48 h of co-culture. Increased islet cell caspase-3 activation and a decreased islet cell replication were consistently observed after co-culture for 2 or 7 days.Conclusion. Activated PSCs may contribute to impaired islet endocrine function seen in exocrine pancreatitis and in islet fibrosis associated with some cases of type 2 diabetes.

Abstract No. 501 - Islet cell transplantation - an update for interventional radiologists

To provide an update on the current progress of islet cell transplantation and its future relevance for interventional radiologists. Interest in islet cell transplantation as a cure for T1DM surged after the landmark “Edmonton Protocol” trial in 2000. Since then, clinical trials have shown consistent achievement of insulin independence. However, clinical implementation has faced many obstacles, forcing the IR and transplant fields to continually reevaluate its clinical utility. A total of 571 patients have received 1,072 infusions at a total of 33 centers from 1999-2009. While the insulin independence rate at 1 year has more than quadrupled since before the Edmonton protocol, the long-term value remains unclear. Graft function dramatically decreases over time (Table 1), and up to 70% of patients require multiple infusions. Currently, 16 centers are listed as active with open protocols, and 11 centers performed at least 1 islet cell transplant in 2009. Interestingly, from 2009 to 2010 there was a 40% increase in recipients (159) and a 30% increase in infusions (244). It is expected that a few leading centers will soon apply for a Biologic License Application (BLA), which would allow insurance coverage and widespread implementation of this procedure. To date, islet transplantation has consistently demonstrated short-term insulin independence. Long-term outcomes are less clear, but are currently being investigated. FDA approval of BLA at experienced centers will undoubtedly advance the field. Interventional radiologists are positioned to play a significant role in the future of islet transplantation, as well as future innovative cellular therapies.

Diet is critical for prolonged glycemic control after short-term insulin treatment in high-fat diet-induced type 2 diabetic male mice.

BackgroundClinical studies suggest that short-term insulin treatment in new-onset type 2 diabetes (T2DM) can promote prolonged glycemic control. The purpose of this study was to establish an animal model to examine such a “legacy” effect of early insulin therapy (EIT) in long-term glycemic control in new-onset T2DM. The objective of the study was to investigate the role of diet following onset of diabetes in the favorable outcomes of EIT.MethodologyAs such, C57BL6/J male mice were fed a high-fat diet (HFD) for 21 weeks to induce diabetes and then received 4 weeks of daily insulin glargine or sham subcutaneous injections. Subsequently, mice were either kept on the HFD or switched to a low-fat diet (LFD) for 4 additional weeks.Principal FindingsMice fed a HFD gained significant fat mass and displayed increased leptin levels, increasing insulin resistance (poor HOMA-IR) and worse glucose tolerance test (GTT) performance in comparison to mice fed a LFD, as expected. Insulin-treated diabetic mice but maintained on the HFD demonstrated even greater weight gain and insulin resistance compared to sham-treated mice. However, insulin-treated mice switched to the LFD exhibited a better HOMA-IR compared to those mice left on a HFD. Further, between the insulin-treated and sham control mice, in spite of similar HOMA-IR values, the insulin-treated mice switched to a LFD following insulin therapy did demonstrate significantly better HOMA-B% values than sham control and insulin-treated HFD mice.Conclusion/InterpretationEarly insulin treatment in HFD-induced T2DM in C57BL6/J mice was only beneficial in animals that were switched to a LFD after insulin treatment which may explain why a similar legacy effect in humans is achieved clinically in only a portion of cases studied, emphasizing a vital role for diet adherence in diabetes control.

Research progress on atypical diabetes mellitus

Atypical diabetes mellitus(ADM)is characterized by severe hyperglycemia and unprovoked ketosis or ketoacidosis,with insulin resistance but with negative insulin-related antibody.The treatment of ADM firstly needs a shortterm insulin therapy,which can be replaced by oral glycemic agent therapy or lifestyle intervention to control the blood glucose.ADM is regarded as a new subtype of diabetes different from typical type 1diabetes mellitus and type 2diabetes mellitus.In this paper,we reviewed the latest research progress on ADM,including its prevalence,clinical presentation,pathogenesis,diagnosis and management,hoping to help clinicians to better understand ADM and avoid the missed diagnosis and misdiagnosis.

Short-term intensive insulin therapy at diagnosis in type 2 diabetes: plan for filling the gaps.

Short-term intensive insulin therapy is unique amongst therapies for type 2 diabetes because it offers the potential to preserve and improve beta-cell function without additional pharmacological treatment. On the basis of clinical experience and the promising results of a series of studies in newly diagnosed patients, mostly in Asian populations, an expert workshop was convened to assess the available evidence and the potential application of short-term intensive insulin therapy should it be advocated for inclusion in clinical practice. Participants included primary care physicians and endocrinologists. We endorse the concept of short-term intensive insulin therapy as an option for some patients with type 2 diabetes at the time of diagnosis and have identified the following six areas where additional knowledge could help clarify optimal use in clinical practice: (1) generalizability to primary care, (2) target population and biomarkers, (3) follow-up treatment, (4) education of patients and providers, (5) relevance of ethnicity, and (6) health economics.

Insulin resistance induced by hyperinsulinemia coincides with a persistent alteration at the insulin receptor tyrosine kinase domain.

Insulin resistance, the diminished response of target tissues to insulin, is associated with the metabolic syndrome and a predisposition towards diabetes in a growing proportion of the worldwide population. Under insulin resistant states, the cellular response of the insulin signaling pathway is diminished and the body typically responds by increasing serum insulin concentrations to maintain insulin signaling. Some evidence indicates that the increased insulin concentration may itself further dampen insulin response. If so, insulin resistance would worsen as the level of circulating insulin increases during compensation, which could contribute to the transition of insulin resistance to more severe disease. Here, we investigated the consequences of excess insulin exposure to insulin receptor (IR) activity. Cells chronically exposed to insulin show a diminished the level of IR tyrosine and serine autophosphorylation below that observed after short-term insulin exposure. The diminished IR response did not originate with IR internalization since IR amounts at the cell membrane were similar after short- and long-term insulin incubation. Förster resonance energy transfer between fluorophores attached to the IR tyrosine kinase (TK) domain showed that a change in the TK domain occurred upon prolonged, but not short-term, insulin exposure. Even though the altered ‘insulin refractory’ IR TK FRET and IR autophosphorylation levels returned to baseline (non-stimulated) levels after wash-out of the original insulin stimulus, subsequent short-term exposure to insulin caused immediate re-establishment of the insulin-refractory levels. This suggests that some cell-based ‘memory’ of chronic hyperinsulinemic exposure acts directly at the IR. An improved understanding of that memory may help define interventions to reset the IR to full insulin responsiveness and impede the progression of insulin resistance to more severe disease states.

Liraglutide reverses pronounced insulin-associated weight gain, improves glycaemic control and decreases insulin dose in patients with type 2 diabetes: a 26 week, randomised clinical trial (ELEGANT).

The best treatment strategy for a patient with type 2 diabetes who shows pronounced weight gain after the introduction of insulin treatment is unclear. We determined whether addition of a glucagon-like peptide-1 (GLP-1) analogue could reverse pronounced insulin-associated weight gain while maintaining glycaemic control, and compared this with the most practised strategy, continuation and intensification of standard insulin therapy. In a 26-week, randomised controlled trial (ELEGANT), conducted in the outpatient departments of one academic and one large non-academic teaching hospital in the Netherlands, adult patients with type 2 diabetes with ≥ 4% weight gain during short-term (≤ 16 months) insulin therapy received either open-label addition of liraglutide 1.8 mg/day (n = 26) or continued standard therapy (n = 24). A computer-generated random number list was used to allocate treatments. Participants were evaluated every 4-6 weeks for weight, glycaemic control and adverse events. The primary endpoint was between-group weight difference after 26 weeks of treatment (intention to treat). Of 50 randomised patients (mean age 58 years, BMI 33 kg/m(2), HbA1c 7.4% [57 mmol/mol]), 47 (94%) completed the study; all patients were analysed. Body weight decreased by 4.5 kg with liraglutide and increased by 0.9 kg with standard therapy (mean difference -5.2 kg [95% CI -6.7, -3.6 kg]; p < 0.001). The respective changes in HbA1c were -0.77% (-8.4 mmol/mol) and +0.01% (+0.1 mmol/mol) (difference -0.74% [-8.1 mmol/mol]) ([95% CI -1.08%, -0.41%] [-11.8, -4.5 mmol/mol]; p < 0.001); respective changes in insulin dose were -29 U/day and +5 U/day (difference -33 U/day [95% CI -41, -25 U/day]; p < 0.001). In five patients (19%), insulin could be completely discontinued. Liraglutide was well tolerated; no severe adverse events or severe hypoglycaemia occurred. In patients with pronounced insulin-associated weight gain, addition of liraglutide to their treatment regimen reverses weight, decreases insulin dose and improves glycaemic control, and hence seems a valuable therapeutic option compared with continuation of standard insulin treatment. Trial registration ClinicalTrials.gov NCT01392898. Funding The study was funded by Novo Nordisk.

Intensive Therapy in Newly Diagnosed Type 2 Diabetes

BackgroundThis study aimed to assess the efficacy of early intensive diabetes therapy with either insulin plus metformin (INS) or triple oral therapy (TOT) with metformin, glyburide, and pioglitazone on glycemic...

Short-Term Insulin Requirements Following Gastric Bypass Surgery in Severely Obese Women with Type 1 Diabetes

BackgroundIn severely obese type 2 diabetes patients, gastric bypass surgery (GB) reduces body mass index (BMI) and hemoglobin A1c (HbA1c) and allows reduced doses of insulin and other medications. Data regarding the effects of GB on severely obese patients with type 1 diabetes are limited.MethodsSeverely obese women with type 1 diabetes (n = 9) were studied immediately before and after GB (7.7 ± 5.8 weeks, mean ± SD).ResultsOn average, GB reduced mean BMI by 11 % and mean HbA1c by 0.9 % (from 8.0 to 7.1 %), with a parallel 38 % decrease in basal insulin requirements (expressed per kilogram of body weight).ConclusionGB rapidly decreased BMI, HbA1c, and insulin requirements in severely obese women with type 1 diabetes. However, physiologic insulin replacement remains necessary in patients with type 1 diabetes.

Cytokines and Insulin Resistance after Zoledronic Acid-Induced Acute Phase Response

Zoledronic acid is known to induce a transient acute phase response (APR). The aim of the study was to investigate whether an APR caused by zoledronic acid administration can induce insulin resistance in post-menopausal osteoporotic women and the potential involvement of different inflammatory markers, cytokines and adipokines to this response. Insulin resistance was calculated by the homeostasis model assessment (HOMA-IR). APR symptoms appeared in 30 post-menopausal osteoporotic women within 24 h and attenuated on day 3 after zoledronic acid infusion. Twenty-eight age- and body mass index-matched, patients without an APR following zoledronic acid administration, served as a control group. In patients with APR, concurrently with a significant increase in serum high sensitive C-reactive protein (hsCRP), interleukin-6 (hsIL-6), tumour necrosis factor-alpha (hsTNF-α) and cortisol levels on days one and two, serum insulin was also significantly elevated, resulting in an increased HOMA-IR. Leptin and resistin significantly increased on day two in contrast to adiponectin which declined, though not statistically significant. The alterations in HOMA-IR were mainly associated to the increase of hsCRP and leptin. In conclusion, zoledronic acid induces an acute, short term insulin resistance, due to an APR, by altering the levels of various adipokines and cytokines.

Effect of short-term intensive insulin pump therapy on the secretion of islet β cells in patients with type 2 diabetes mellitus

Objective To investigate the effect of short-term intensive insulin pump therapy on the secretion of islet β cells in patients with type 2 diabetes mellitus.Methods In our hospital in 2009 January-2012 year in December 360 cases of patients with newly diagnosed type 2 diabetes,according to the principle of voluntary divided into study group and control group,180 cases in each group,the group received continuous subcutaneous insulin infusion of insulin aspart,is before meals and at bedtime insulin aspart Novolin N subcutaneous injection control group,appeared after 2 weeks of treatment two groups of patients,by which oral hypoglycemic drugs for treatment,compared two groups of fasting blood glucose,postprandial blood glucose,fasting serum C-peptide (C-P),2h postprandial serum C-peptide (C-P2h),the incidence of hypoglycemia,and observe the changes of HbAlC,HOMA-IR,and TC,TG,LDL-C,HDL-C before and after the groups of patients.Results The two groups after treatment for 2 weeks,6 months treatment,FPG,PG2h,C-P,C-P2h significant difference,the study group patients two monitoring point in time the incidence of hypoglycemia were lower than the control group,P＜ 0.05,there were no serious hypoglycemia.Study groups before and after the treatment of HbA1C,HOMA-IR,TC,LDL-C,TG significant difference,HDL-C no difference.Conclusion The short-term intensive insulin pump treatment of newly diagnosed T2DM,can be more rapid,more smoothly to control blood sugar,improved the function of islet beta cells,and has good clinical application value. Key words: Type 2 diabetes mellitus; Insulin pump; Intensive therapy; insulin aspartate

Effects of short-term intensive glycemic control on insulin, glucagon, and glucagon-like peptide-1 secretion in patients with Type 2 diabetes.

Short-term intensive insulin therapy (IIT) in patients with Type 2 diabetes mellitus (T2DM) has beneficial effects on insulin secretion. However, IIT effect on glucagon and glucagon-like peptide-1 (GLP-1) secretion is unknown. We evaluated short-term intensive glycemic control effects on insulin, glucagon, and GLP-1 secretory dynamics in T2DM. Twenty-six patients with T2DM were hospitalized and treated with IIT for 10-14 days. A meal tolerance test was performed before and after IIT and the differences in serum immunoreactive insulin (IRI) and C-peptide immunoreactivity (CPR) as well as plasma glucagon and active GLP-1 levels were evaluated. Glycoalbumin levels decreased significantly from 23.0% before to 19.6% after IIT (p<0.001). However, pre- and post-IIT, IRI and CPR levels were not significantly different; post-IIT glucose levels were significantly decreased. The post-IIT glucagon levels at 0 and 60 min were lower than pre-IIT levels. Moreover, post- IIT area under the curve (AUC) of glucagon significantly reduced from 6755 ± 996 pg/dl · 60 min to 5796 ± 1074 pg/dl · 60 min (p<0.001). Furthermore, post-IIT GLP-1 levels and AUC were significantly higher than pre-IIT values. Our results suggest that patients with T2DM who received shortterm IIT demonstrated decreased postprandial glucagon levels and increased GLP-1 levels following a meal tolerance test.

Type 2 Diabetes in Children and Adolescents

Type 2 Diabetes in Children and Adolescents