RESUMO
Beta-cell dedifferentiation as shown by cellular colocalization of insulin with glucagon and/or vimentin, and decreased expression of MAFA and/or urocortin3 has been suggested to contribute to metabolic decompensation in type 2 diabetes, and was recently described postimplantation in islet allotransplant patients. Dysglycaemia and diabetes mellitus are often encountered preoperatively in patients undergoing pancreatectomy and islet autotransplantation (PIAT). In this series of case reports, we document variation in islet phenotypic identity in three patients with chronic pancreatitis (CP) without diabetes or significant insulin resistance who subsequently underwent PIAT. Pancreas histology was examined using colocalization of endocrine hormones, mesenchymal and pan-endocrine markers in islets, and the relative expression of MAFA and urocortin3 in insulin-expressing cells as compared to that of nondiabetic and type 2 diabetic donors. We present results of pre- and posttransplant clinical metabolic testing. Varying degrees of islet-cell dedifferentiation are identified in nondiabetic patients with CP at the time of PIAT, and may need further investigation.
Assuntos
Células Endócrinas/patologia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Pancreatite Crônica/terapia , Adulto , Biomarcadores/metabolismo , Células Endócrinas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Transplante Autólogo , Adulto JovemRESUMO
Pancreatic ß-cell expansion is a highly regulated metabolic adaptation to increased somatic demands, including obesity and pregnancy; adult ß cells otherwise rarely proliferate. We previously showed that high-fat diet (HFD) feeding induces mouse ß-cell proliferation in less than 1 wk in the absence of insulin resistance. Here we metabolically profiled tissues from a short-term HFD ß-cell expansion mouse model to identify pathways and metabolite changes associated with ß-cell proliferation. Mice fed HFD vs. chow diet (CD) showed a 14.3% increase in body weight after 7 days; ß-cell proliferation increased 1.75-fold without insulin resistance. Plasma from 1-wk HFD-fed mice induced ß-cell proliferation ex vivo. The plasma, as well as liver, skeletal muscle, and bone, were assessed by LC and GC mass-spectrometry for global metabolite changes. Of the 1,283 metabolites detected, 159 showed significant changes [false discovery rate (FDR) < 0.1]. The majority of changes were in liver and muscle. Pathway enrichment analysis revealed key metabolic changes in steroid synthesis and lipid metabolism, including free fatty acids and other bioactive lipids. Other important enrichments included changes in the citric acid cycle and 1-carbon metabolism pathways implicated in DNA methylation. Although the minority of changes were observed in bone and plasma (<20), increased p-cresol sulfate was increased >4 fold in plasma (the largest increase in all tissues), and pantothenate (vitamin B5) decreased >2-fold. The results suggest that HFD-mediated ß-cell expansion is associated with complex, global metabolite changes. The finding could be a significant insight into Type 2 diabetes pathogenesis and potential novel drug targets.
Assuntos
Proliferação de Células/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica , Células Secretoras de Insulina/citologia , Lipídeos/sangue , Animais , Glicemia , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Obesidade/metabolismoRESUMO
Metabolic syndrome (MetS) is an increasing global health threat and strong risk factor for type 2 diabetes (T2D). MetS causes both hyperinsulinemia and islet size overexpansion, and pancreatic ß-cell failure impacts insulin and proinsulin secretion, mitochondrial density, and cellular identity loss. The low-density lipoprotein receptor knockout (LDLr-/-) model combined with high-fat diet (HFD) has been used to study alterations in multiple organs, but little is known about the changes to ß-cell identity resulting from MetS. Osteocalcin (OC), an insulin-sensitizing protein secreted by bone, shows promising impact on ß-cell identity and function. LDLr-/- mice at 12 months were fed chow or HFD for 3 months ± 4.5 ng/h OC. Islets were examined by immunofluorescence for alterations in nuclear Nkx6.1 and PDX1 presence, insulin-glucagon colocalization, islet size and %ß-cell and islet area by insulin and synaptophysin, and mitochondria fluorescence intensity by Tomm20. Bone mineral density (BMD) and %fat changes were examined by Piximus Dexa scanning. HFD-fed mice showed fasting hyperglycemia by 15 months, increased weight gain, %fat, and fasting serum insulin and proinsulin; concurrent OC treatment mitigated weight increase and showed lower proinsulin-to-insulin ratio, and higher BMD. HFD increased %ß and %islet area, while simultaneous OC-treatment with HFD was comparable to chow-fed mice. Significant reductions in nuclear PDX1 and Nkx6.1 expression, increased insulin-glucagon colocalization, and reduction in ß-cell mitochondria fluorescence intensity were noted with HFD, but largely prevented with OC administration. OC supplementation here suggests a benefit to ß-cell identity in LDLr-/- mice and offers intriguing clinical implications for countering metabolic syndrome.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperinsulinismo , Células Secretoras de Insulina , Ilhotas Pancreáticas , Síndrome Metabólica , Animais , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glucagon/metabolismo , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Lipoproteínas LDL , Síndrome Metabólica/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteocalcina/metabolismo , Proinsulina/metabolismo , Aumento de PesoRESUMO
Islet and ß-cell function is intrinsic to glucose homeostasis. Pancreatectomy and islet autotransplantation (PIAT) for chronic pancreatitis (CP) treatment is a useful model for assessing islet function in the absence of immune-suppression and to perform extensive presurgical metabolic evaluations not possible from deceased donors. We recently showed that in CP-PIAT patients, preoperative islet identity loss presented with postoperative glycemic loss. Here, we examine presurgical islet function using Homeostatic Model Assessment-Beta Cell Function (%) (HOMA-ß) and glycemic variables and compared them with postsurgical insulin independence and their predicted alignment with Secretory Unit of Islet Transplant Objects (SUITO) and beta cell score after transplantation (BETA-2) scores. Methods: Seven CP-PIAT patients were assessed for ß-cell function metrics, including pretransplant and 6-mo posttransplant HOMA-ß using insulin and C-peptide and evaluations of proposed insulin independence by SUITO and BETA-2 graft function equations. These were compared with oral glucose tolerance tests and pancreas histological samples taken at the time of transplant, examined for ß-cell maturity markers. Results: Pre-PIAT, HOMA-ß (60%-100%) associated with post-PIAT insulin independence. This association was only moderately supported by post-PIAT SUITO threshold scores (≥26) but robustly by BETA-2 scores (≥16.2). Appropriate posttransplant oral glucose tolerance test curves were found in those patients with normal pretransplant HOMA-ß values. Preoperative low serological ß-cell function was displayed by concurrent evidence of ß-cell identity alterations including colocalization of insulin and glucagon, loss of urocortin-3, and increased intra-islet vimentin in patients who were insulin-dependent post-PIAT. Conclusions: These data encourage HOMA-ß assessment before PIAT for estimating posttransplant insulin independence.
RESUMO
PURPOSE: Compared with photon cranial radiation therapy (X-CRT), proton cranial radiation therapy (P-CRT) offers potential advantages in limiting radiation-induced sequalae in the treatment of pediatric brain tumors. This study aims to identify cognitive, functional magnetic resonance and positron emission tomography imaging markers and molecular differences between the radiation modalities. METHODS AND MATERIALS: Juvenile rats received a single faction of 10 Gy (relative biological effectiveness-weighted dose) delivered with 6 MV X-CRT or at the midspread out Bragg peak of a 100 MeV P-CRT beam. At 3, 6, and 12 months post-CRT, executive function was measured using 5-choice serial reaction time task. At â¼12 months post-CRT, animals were imaged with 18F-Flurodeoxy-glucose positron emission tomography imaging followed by functional ex vivo magnetic resonance imaging and stained for markers of neuroinflammation. RESULTS: Irradiated animals had cognitive impairment with a higher number of omissions and lower incorrect and premature responses compared with sham (P ≤ .05). The accuracy of the animals' X-CRT was less than that of sham (P ≤ .001). No significant difference in rates of cognitive change were found between the radiation modalities. At 12 months post-CRT, glucose metabolism was significantly higher than sham in X-CRT (P = .04) but not P-CRT. Using diffusion tensor imaging, P-CRT brains were found to have higher white matter volume and fiber lengths compared with sham (P < .03). Only X-CRT animals had higher apparent diffusion coefficient values compared with sham (P = .04). P-CRT animals had more connectomic changes compared with X-CRT. Correlative analysis identified several imaging features with cognitive performance. Furthermore, microgliosis (P < .05), astrogliosis (P < .01), and myelin thinning (P <.05) were observed in both radiation modalities, with X-CRT showing slightly more inflammation. CONCLUSIONS: Both P-CRT and X-CRT lead to neurocognitive changes compared with sham. Although no significant difference was observed in neuroinflammation between the irradiated groups, differences were found in late-term glucose metabolism and brain connectome. Our results indicate that despite relative biological effectiveness weighting of the proton dose there are still differential effects which warrants further investigation.
Assuntos
Imagem de Tensor de Difusão , Prótons , Animais , Encéfalo/patologia , Cognição/efeitos da radiação , Irradiação Craniana/efeitos adversos , Imagem de Tensor de Difusão/métodos , RatosRESUMO
PURPOSE: Cranial radiation therapy (CRT) is a common treatment for pediatric brain tumor patients. However, side effects include significant neurobehavioral dysfunction in survivors. This dysfunction may in part be caused by inflammation, including increased production of tumor necrosis factor alpha (TNFα) and its receptor TNFR1, which can activate the nuclear factor kappa light-chain enhancer of activated B cells (NF-κB). The TNFα blockade abrogates this inflammatory response, although it presents immunologic risks. Thus, modulation of pathway subsets may be preferable. Here, we test whether inhibition of NF-κB activation using an NF-κB essential modulator binding domain (NBD) peptide mitigates CRT-induced neuroinflammation and improves behavioral outcomes. METHODS AND MATERIALS: Male C57BL/6J 28-day old mice were randomized to saline (sham), 5 Gy whole-brain CRT, or CRT + NBD-peptide. Brain tissue was collected after 4 hours or 3 months for Western blot or immunohistochemistry. The cortex, corpus callosum (CC), and dentate gyrus were variably imaged for NF-κB-p65, IκBα, proliferation, apoptosis, necroptosis, TNFα, TNFR1, IBA-1, doublecortin, CD11c, and GFAP. Neurobehavioral changes were assessed by open field and elevated plus maze tests 3 months post-CRT. RESULTS: NF-κB expression increased in whole and nuclear fractions 4 hours after CRT and was abrogated by NBD treatment. Cell death increased and proliferation decreased after CRT, including within neuronal progenitors, with some loss mitigated by NBD. Increased levels of TNFα, IBA-1, and GFAP were found in the CC and cortex months after CRT and were limited by NBD. The anti-NF-κB peptide also improved neurobehavioral assessments, yielding improvements in anxiety and exploration. CONCLUSIONS: Results suggest a role for NF-κB modulation by NBD peptide in the reduction of neuroinflammation and mitigation of behavioral complications after pediatric radiation therapy.
Assuntos
Comportamento Animal/efeitos da radiação , Irradiação Craniana/efeitos adversos , Encefalite/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Fatores Etários , Animais , Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Canais de Cloreto/metabolismo , Irradiação Craniana/métodos , Encefalite/etiologia , Encefalite/metabolismo , Encefalite/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/prevenção & controle , Proteína HMGB1/metabolismo , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Inibidor de NF-kappaB alfa , Doses de Radiação , Distribuição Aleatória , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: We explored the association of C-peptide (marker of secreted insulin), proinsulin and proinsulin /C-peptide ratio (PI/C) (markers of beta-cell endoplasmic reticulum [ER] stress) with undercarboxylated (uOC) and carboxylated osteocalcin (cOC) and their ratio (uOC/cOC) in children with recently diagnosed type 1 (T1D) or type 2 diabetes (T2D), and the correlation of these variables with partial remission (PR) in children with T1D. METHODS: Demographic and clinical data of children with new-onset diabetes (n = 68; median age = 12.2 years; 33.8% non-Hispanic White, 45.6% Hispanic/Latino, 16.2% African American and 4.4% other) were collected at diagnosis and during the first (V1), second (V2) and third clinical visits at 9.0, 32.0 and 175.7 weeks, respectively. Serum proinsulin, C-peptide, uOC and cOC values were measured 7.0 weeks after diagnosis. PR was defined as insulin dose-adjusted HbA1c (IDAA1c) ≤9. RESULTS: In children with new-onset T1D with DKA (33.3%) or T2D (29.4%), Spearman's correlation coefficient revealed a positive association between the C-peptide levels and both uOC and uOC/cOC ratio. In T1D (n = 48), both higher serum C-peptide levels and low PI:C ratio were associated with higher BMI percentile (ß = 0.02, P = .001; ß = -0.01, P = .02, respectively) and older age at diagnosis (ß = 0.13, P = .001; ß = -0.12, P = .001, respectively). Furthermore, in children with T1D, C-peptide levels at V1 correlated with IDAA1c ≤ 9 at V1 (P = .04). CONCLUSION: C-peptide levels are associated with a higher uOC and uOC/cOC ratio in paediatric diabetes. In new-onset T1D children, older age and higher BMI were associated with lower beta-cell stress and higher preserved function, which was predictive of PR on follow-up.
RESUMO
BACKGROUND: Cranial radiotherapy (CRT) is an important part of brain tumor treatment, and although highly effective, survivors suffer from long-term cognitive side effects. In this study we aim to establish late-term imaging markers of CRT-induced brain injury and identify functional markers indicative of cognitive performance. Specifically, we aim to identify changes in executive function, brain metabolism, and neuronal organization. METHODS: Male Sprague Dawley rats were fractionally irradiated at 28 days of age to a total dose of 30 Gy to establish a radiation-induced brain injury model. Animals were trained at 3 months after CRT using the 5-choice serial reaction time task. At 12 months after CRT, animals were evaluated for cognitive and imaging changes, which included positron emission tomography (PET) and magnetic resonance imaging (MRI). RESULTS: Cognitive deficit with signs of neuroinflammation were found at 12 months after CRT in irradiated animals. CRT resulted in significant volumetric changes in 38% of brain regions as well as overall decrease in brain volume and reduced gray matter volume. PET imaging showed higher brain glucose uptake in CRT animals. Using MRI, irradiated brains had an overall decrease in fractional anisotropy, lower global efficiency, increased transitivity, and altered regional connectivity. Cognitive measurements were found to be significantly correlated with six image features that included myelin integrity and local organization of the neural network. CONCLUSIONS: These results demonstrate that CRT leads to late-term morphological changes, reorganization of neural connections, and metabolic dysfunction. The correlation between imaging markers and cognitive deficits can be used to assess late-term side effects of brain tumor treatment and evaluate efficacy of new interventions.
RESUMO
A compensatory increase in ß-cell mass occurs during pregnancy to counter the associated insulin resistance, and a failure in adaptation is thought to contribute to gestational diabetes. Insulin-expressing but glucose-transporter-2-low (Ins+Glut2LO) progenitor cells are present in mouse and human pancreas, being predominantly located in extra-islet ß-cell clusters, and contribute to the regeneration of the endocrine pancreas following induced ablation. We therefore sought to investigate the contribution of Ins+Glut2LO cells to ß-cell mass expansion during pregnancy. Female C57Bl/6 mice were time mated and pancreata were collected at gestational days (GD) 6, 9, 12, 15, and 18, and postpartum D7 (n = 4/time-point) and compared to control (non-pregnant) animals. Beta cell mass, location, proliferation (Ki67+), and proportion of Ins+Glut2LO cells were measured using immunohistochemistry and bright field or confocal microscopy. Beta cell mass tripled by GD18 and ß-cell proliferation peaked at GD12 in islets (≥6 ß-cells) and small ß-cell clusters (1-5 ß-cells). The proportion and fraction of Ins+Glut2LO cells undergoing proliferation increased significantly at GD9 in both islets and clusters, preceding the increase in ß-cell mass and proliferation, and their proliferation within clusters persisted until GD15. The overall number of clusters increased significantly at GD9. Quantitative PCR showed a significant increase in Pdx1 presence at GD9 vs. GD18 or control pancreas, and Pdx1 was visualized by immunohistochemistry within both Ins+Glut2LO and Ins+Glut2HI cells within clusters. These results indicate that Ins+Glut2LO cells are likely to contribute to ß-cell mass expansion during pregnancy.
Assuntos
Diabetes Gestacional/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Feminino , Idade Gestacional , Transportador de Glucose Tipo 2/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , GravidezRESUMO
The presence and location of resident pancreatic ß-cell progenitors is controversial. A subpopulation of insulin-expressing but glucose transporter-2-low (Ins+Glut2LO) cells may represent multipotent pancreatic progenitors in adult mouse and in human islets, and they are enriched in small, extra-islet ß-cell clusters (<5 ß cells) in mice. Here, we sought to identify and compare the ontogeny of these cells in mouse and human pancreata throughout life. Mouse pancreata were collected at postnatal days 7, 14, 21, 28, and at 3, 6, 12, and 18 months of age, and in the first 28 days after ß-cell mass depletion following streptozotocin (STZ) administration. Samples of human pancreas were examined during fetal life (22-30 weeks gestation), infancy (0-1 year), childhood (2-9), adolescence (10-17), and adulthood (18-80). Tissues were analyzed by immunohistochemistry for the expression and location of insulin, GLUT2 and Ki67. The proportion of ß cells within clusters relative to that in islets was higher in pancreas of human than of mouse at all ages examined, and decreased significantly at adolescence. In mice, the total number of Ins+Glut2LO cells decreased after 7 days concurrent with the proportion of clusters. These cells were more abundant in clusters than in islets in both species. A positive association existed between the appearance of new ß cells after the STZ treatment of young mice, particularly in clusters and smaller islets, and an increased proportional presence of Ins+Glut2LO cells during early ß-cell regeneration. These data suggest that Ins+Glut2LO cells are preferentially located within ß-cell clusters throughout life in pancreas of mouse and human, and may represent a source of ß-cell plasticity.
Assuntos
Envelhecimento/fisiologia , Transportador de Glucose Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica , Transportador de Glucose Tipo 2/genética , Humanos , Lactente , Recém-Nascido , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Pessoa de Meia-Idade , Maturidade Sexual , Adulto JovemRESUMO
Regeneration of insulin-producing ß-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare ß-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that ß-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of ß-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing ß-cells in culture, but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of ß-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive, glucose-transporter-2-low (Ins(+)Glut2(LO)) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 ß-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature ß-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new, functional ß-cells, and which may be potentially exploited for regenerative therapies in the future.
Assuntos
Plasticidade Celular , Proteínas Facilitadoras de Transporte de Glucose/análise , Células Secretoras de Insulina/fisiologia , Insulina/análise , Animais , Diferenciação Celular , Linhagem da Célula/fisiologia , Citometria de Fluxo , Queratina-19/análise , Camundongos , Microscopia de FluorescênciaRESUMO
Low birth weight and poor foetal growth following low protein (LP) exposure are associated with altered islet development and glucose intolerance in adulthood. Additionally, LP-fed offspring fail to regenerate their ß-cells following depletion with streptozotocin (STZ) in contrast to control-fed offspring that restore ß-cell mass. Our objective was to identify signalling pathways and cellular functions that may be critically altered in LP offspring rendering them susceptible to developing long-term glucose intolerance and decreased ß-cell plasticity. Pregnant Balb/c mice were fed a control (C; 20% protein) or an isocaloric LP (8% protein) diet throughout gestation and C diet thereafter. Female offspring were injected intraperitoneally with 35 mg/kg STZ or vehicle on days 1 to 5 for each dietary treatment. At 30 days of age, total RNA was extracted from pancreatic tissue for microarray analysis using the Affymetrix GeneChip Mouse Genome 430 2.0. Gene and protein expression were quantified from isolated islets. Finally, ß-cell proliferation was determined in vitro following REG1α treatment. The microarray data and GO enrichment analysis indicated that foetal protein restriction alters the early expression of genes necessary for many cell functions, such as oxidative phosphorylation and free radical scavenging. Expression of Reg1 was upregulated following STZ, whereas protein content was decreased in LP + STZ islets. Furthermore, REG1α failed to stimulate ß-cell proliferation in vitro in LP + STZ islets. Therefore, early nutritional insults may programme the Reg1 pathway resulting in a limited ability to increase ß-cell mass during metabolic stress. In conclusion, this study implicates the Reg1 pathway in ß-cell regeneration and describes altered programming of gene expression in LP offspring, which underlies later development of cell dysfunction and glucose intolerance in adulthood.