Design, synthesis, and biological evaluation of novel sesquiterpene lactone derivatives as PKM2 activators with potent anti-ulcerative colitis activities.

Pyruvate kinase isoform 2 (PKM2) is closely related to the regulation of Th17/Treg balance, which is considered to be an effective strategy for UC therapy. Parthenolide (PTL), a natural product, only possesses moderate PKM2-activating activity. Thus, five series of PTL derivatives are designed and synthesized to improve PKM2-activated activities and anti-UC abilities. Through detailed structure optimization, B4 demonstrates potent T-cell anti-proliferation activity (IC50 = 0.43 µM) and excellent PKM2-activated ability (AC50 = 0.144 µM). Subsequently, through mass spectrometry analysis, B4 is identified to interact with Cys423 of PKM2 via covalent-bond. Molecular docking and molecular dynamic simulation results reveal that the trifluoromethoxy of B4 forms a stronger hydrophobic interaction with Ala401, Pro402, and Ile403. In addition, B4 has a significant effect only on Th17 cell differentiation, thereby regulating the Th17/Treg balance. The effect of B4 on Th17/Treg imbalance can be attributed to inhibition of PKM2 dimer translocation and suppression of glucose metabolism. Finally, B4 can notably ameliorate the symptoms of dextran sulfate sodium (DSS)-induced colitis in mouse model in vivo. Thus, B4 is confirmed as a potent PKM2 activator, and has the potential to develop as a novel anti-UC agent.

Effect of Iodine Nutrition Status on Thyroid Function and Pregnancy Outcomes.

Iodine is essential for normal thyroid function, supporting healthy fetal and child development. The relevance between maternal iodine nutrition status and pregnancy outcomes remains controversial. The aim was to explore whether urinary iodine concentrations (UIC)/urinary creatinine (UCr) was associated with thyroid function and adverse pregnancy outcomes. This study was performed in the Department of Endocrinology and Metabolism of the First Affiliated Hospital of Nanjing Medical University. A total of 212 pregnant women were enrolled from May 2018 to November 2021, from the first visit until postpartum. Maternal serum samples were obtained in the second half of pregnancy, and then thyroid-stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), UIC, and UCr were tested. The correlation of UIC/UCr, which represented maternal iodine nutrition status, with TSH, FT4, and FT3 was studied using linear regression. And we assessed associations between UIC/UCr and pregnancy outcomes. Notably, we explored consistency between UIC/UCr and the incidence of low birth weight (LBW) by application of logistic regression analysis. A total of 212 women were divided into 3 groups according to the upper and lower quartiles of UIC/UCr. There were 53 women in group 1 (UIC/UCr < 106.96 ug/g), 106 women in group 2 (UIC/UCr 106.96-259.08 ug/g), and 53 women in group 3 (UIC/UCr > 259.08 ug/g). The level of UIC/UCr had a negative correlation with FT4 (r = - 0.139, p = 0.043) but a positive correlation with TSH (r = 0.096, p = 0.162 > 0.05). There was a significant difference in the incidence of LBW among the 3 groups (p = 0.007). Logistic regression analysis found that the level of UIC/UCr was an independent factor for LBW (p = 0.048, OR = 0.991, 95%CI (0.982, 0.999)). The receiver operating characteristic (ROC) curve showed that the area under the curve (AUC) for UIC/UCr predicting the incidence of low birth weight was 0.687 (p = 0.013, 95%CI 0.575, 0.799). Lower UIC/UCr during pregnancy was associated with higher FT4 and lower TSH. And iodine deficiency during pregnancy is a risk factor for low birth weight. Our findings indicated that more attention should be paid to the appropriate iodine nutrition status in pregnant women, which can help prevent suffering from adverse pregnancy outcomes.

Effects of Thyroperoxidase Antibody and Thyroglobulin Antibody on Maternal and Neonatal Outcomes in Pregnant Women.

The aim of the study was to evaluate the effects of thyroperoxidase antibody (TPOAb) and thyroglobulin antibody (TgAb) on maternal and neonatal adverse outcomes in pregnant women. A total of 296 singleton pregnant women were classified into four groups according to the thyroid auto-antibody in the first trimester. Finally, there were 97 women in TPOAb positive group (TPOAb+/TgAb-), 35 in TgAb positive group (TPOAb-/TgAb+), 85 in TPOAb and TgAb positive group (TPOAb+/TgAb+), and 79 in TPOAb and TgAb negative group (TPOAb-/TgAb-). Thyroid function, TPOAb, and TgAb were checked during pregnancy and followed up at 6 weeks, 3 months, 6 months, 9 months, and 12 months postpartum. Levothyroxine sodium tablets could be taken to maintain euthyroid antepartum. Thyroid function of women with postpartum thyroiditis (PPT) were followed up at 2 and 3 years postpartum. We observed the incidence of PPT, premature rupture of membranes (PROM), placental abruption, placenta previa, polyhydramnios, oligohydramnios, postpartum hemorrhage, preterm birth, and low birth Weight in the four groups. 19.93% of the women had PPT. The incidence of PPT in TPOAb+/TgAb-, TPOAb-/TgAb+, TPOAb+/TgAb+groups was significantly higher than that in TPOAb-/TgAb- group, respectively (16.49 vs. 6.33%, 22.86 vs. 6.33%, 35.29 vs. 6.33%, p <0.05). The incidence of PPT in TPOAb+/TgAb+group was significantly higher than that in TPOAb+/TgAb- group (35.29 vs. 16.49%, p <0.01). PPT occurred as early as 6 weeks postpartum, but mainly at 3 and 6 months postpartum in the four groups (62.50%, 75.00%, 70.00%, 80.00%). All PPT in TPOAb-/TgAb- group occurred within 6 months postpartum, while it was found at 9 months or 12 months postpartum in other three groups. There was no classical form of PPT in TPOAb-/TgAb- group, while in the other three groups, all three types (classical form, isolated thyrotoxicosis, isolated hypothyroidism) existed. At 2 years postpartum of the women with PPT, the rate of euthyroidism in TPOAb+/TgAb+group was significantly lower than that in TPOAb-/TgAb- group (p <0.05). At 3 years postpartum of the women with PPT, the rate of euthyroidism in TPOAb+/TgAb-, TPOAb-/TgAb+, and TPOAb+/TgAb+groups were significantly lower than that in TPOAb-/TgAb- group (p <0.05). The values of TPOAb and TgAb postpartum were significantly higher than those during pregnancy (p <0.05). The incidence of PROM in TPOAb+/TgAb- group was significantly higher than that in TPOAb-/TgAb- group (32.99 vs. 17.72%, p <0.05). The binary logistic regression for PPT showed that the OR value of TPOAb was 2.263 (95% CI 1.142-4.483, p=0.019). The OR value of TgAb was 3.112 (95% CI 1.700-5.697, p=0.000). In conclusion, pregnant women with positive thyroid auto-antibodies had an increased risk of PPT and a reduced rate of euthyroidism at 2 and 3 years postpartum. TPOAb is associated with the incidence of PROM. Both of TPOAb and TgAb were independent risk factors for PPT. TgAb deserves more attention when studying autoimmune thyroid disease (AITD) combined with pregnancy.

The mechanisms of lncRNA Tug1 in islet dysfunction in a mouse model of intrauterine growth retardation.

Taurine upregulated gene 1 (Tug1) is a novel lncRNA that participates in growth, and the abnormal expression of Tug1 related to mouse islet cell dysfunction. A recent study revealed that intrauterine growth retardation (IUGR) related to the pathogenesis of diabetes. Here, we aimed to explore the role and mechanism of Tug1 in IUGR-mediated islet dysfunction. We observed that newborn IUGR mice had lower body and pancreas weight and smaller islets than newborn control mice. After IUGR mice were given a normal diet, they showed catch-up growth and abnormal glucose tolerance; however, the pancreas/body weight ratio remained low. Blood glucose, serum insulin and related gene expression showed mild recovery after overexpression of Tug1 in IUGR mice. Furthermore, Tug1 was enriched in the nuclei of MIN6 cells. Using RIP and CHIP analyses we found that Tug1 could regulate Hes1 expression by binding to EZH2 to affect insulin synthesis in MIN6 cells. These findings indicate that lncRNA Tug1 could regulate the expression of Hes1 via EZH2-driven H3K27 methylation and affect insulin production. SIGNIFICANCE OF THE STUDY: This study suggests Tug1 as a novel biomarker, as it was shown to regulate ß cell function and is worthy of further investigation due to its potential for diabetes treatment.

A theoretical study of several fully hydrogenated borophenes.

Several recently synthesized two dimensional borophene monolayers are almost all metallic with a strong anisotropic character, but their structural instability and the need to explore their novel physical properties are still ongoing issues. We present a detailed study of four fully hydrogenated borophenes (ß12, δ3, δ5 and α borophanes) by first-principles calculations. According to phonon dispersion relations and ab initio molecular dynamics simulations, δ3 and δ5 borophanes are dynamically and thermally stable. The structural, mechanical, and electronic properties of δ3 and δ5 borophanes are analyzed. The results indicate that charge transfer from B to H atoms is crucial for the stability of two borophane phases. The HSE06 calculations predict that both δ3 and δ5 borophanes are semiconductors with indirect band gaps of 1.51 and 1.99 eV, respectively. These findings indicate that δ3 and δ5 borophanes are ideal for applications in nanoelectronics.

Inhibition of Lincpint expression affects insulin secretion and apoptosis in mouse pancreatic ß cells.

LncRNAs have been reported to maintain islet function and are associated with the development of diabetes. Here, we investigated Lincpint biological functions in mouse pancreatic ß cells both in vivo and in vitro. We observed that Lincpint was highly expressed in BALB/c mouse islets and downregulated in db/db mouse islets by using qRT-PCR. Lincpint could be regulated by different concentrations of glucose in MIN6 cells. MTT and flow cytometry showed that silencing Lincpint expression in vitro increased cell apoptosis. In a GSIS assay, we detected that inhibition of the expression of Lincpint suppressed insulin synthesis and secretion. In male BALB/c mouse, blood glucose and serum insulin were decreased after knockdown of Lincpint expression as detected by IPGTT and ELISA. Immunohistochemistry indicated that positive islet area and expression of Pdx1 and Glut2 was reduced after Lincpint silencing in male BALB/c mouse. These findings suggest that Lincpint may play an important role in maintaining islet ß cell function and is worthy of further investigation due to its potential in diabetes treatment.

Dynamic transcriptome profile in db/db skeletal muscle reveal critical roles for long noncoding RNA regulator.

T2DM is a global health problem that seriously lowers the quality of life and insulin resistance makes a considerable contribution to the pathophysiology of T2DM. Long noncoding RNAs (lncRNAs) have emerged as important regulators in glucose and lipid metabolism. However, comprehensive analysis of lncRNAs in db/db mice skeletal muscle and their potential roles involved in skeletal muscle insulin resistance (IR) remains poorly characterized. Here, we identified 331 lncRNAs, 172 upregulated and 159 downregulated (|fold change|>2, q<0.05), differentially expressed in db/db mice skeletal muscle. Gene Ontology analysis, Pathway analysis and Gene Set Enrichment Analysis of network gene expression revealed the potential functions of dysregulated lncRNAs may involve skeletal muscle function, fatty acid metabolism and the PPAR signaling pathway. In addition, differentially expressed lncRNAs were verified in skeletal muscle from the widely known IR mouse models (db/db and ob/ob mice). Further validation of lncRNAs in C2C12 myotubes exposed with various concentrations of palmitate uncovered that lncRNAs were responsive to palmitate exposure at the high concentrations (0.5mM and 0.75mM). Coexpression analysis revealed the key lncRNA-mRNA interactions and indicated a potential regulatory role of lncRNAs. Moreover, we characterized two candidate lncRNAs Gm15441 and 3110045C21Rik by a comprehensive examination of their genomic context and validated their expression with neighboring genes (Txnip and Ddr2) by the Spearman correlation analysis. Collectively, these findings improve our understanding of lncRNAs that mediate skeletal muscle insulin resistance in diabetes and represent potential molecular therapeutic targets to improve insulin sensitivity and associated metabolic diseases.

Long Noncoding RNA Meg3 Regulates Mafa Expression in Mouse Beta Cells by Inactivating Rad21, Smc3 or Sin3α.

BACKGROUND/AIMS: The main pathogenic mechanism of diabetes is a decrease in the number of islet beta cells or a decline in their function. Recent studies have shown that pancreatic long noncoding RNAs (lncRNAs) have a high degree of tissue specificity and may be involved in the maintenance of islet cells function and the development of diabetes. The aim of this study was to investigate the molecular regulatory mechanism of mouse maternal expressed gene 3 (Meg3) in insulin biosynthesis in pancreatic islets. METHODS: Chromatin immunoprecipitation-quantitative polymerase chain reaction (qPCR) and RNA immunoprecipitation-qPCR were used to investigate the molecular mechanism of lncRNA Meg3 in insulin biosynthesis by regulating v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), a mature beta cell marker in the MIN6 beta cell line. Further, the expression levels of Meg3, Ezh2, MafA, Rad21, Smc3, and Sin3α were analyzed in vivo and in vitro by RT-PCR and western blotting. RESULTS: Intranuclear lncRNA Meg3 can bind EZH2, a methyltransferase belonging to the Polycomb repressive complex-2, in pancreatic islet cells. In addition, knockdown of Ezh2 can also inhibit the expression of MafA and Ins2, while expression levels of Rad21, Smc3, and Sin3α are upregulated, by interfering with Ezh2 or Meg3 in pancreatic beta cells. Knockdown of Meg3 resulted in the loss of EZH2 binding and H3K27 trimethylation occupancy of Rad21, Smc3, and Sin3α promoter regions. The inhibition of Rad21, Smc3, or Sin3α, which directly act on the MafA promoter, leads to upregulated expression of MafA in both MIN6 cells and mouse islets. Moreover, the synthesis and secretion of insulin were increased by inhibition of these transcription factors. CONCLUSIONS: Pancreatic lncRNA Meg3 can epigenetically regulate the expression of Rad21, Smc3, and Sin3α via EZH2-driven H3K27 methylation. By inhibiting the expression of Rad21, Smc3, or Sin3α, Meg3 promotes the expression of MafA and affects the production of insulin.

Exenatide substantially improves proinsulin conversion and cell survival that augment Ins2+/Akita beta cell function.

Proinsulin folding imperfections cause extensive beta-cell defects known in diabetes. Here, we investigated whether exenatide can alleviate such defects in proinsulin conversion, beta-cell survival, and insulin secretion, in the Ins2+/Akita beta-cells that have a spontaneous mutation (Cys 96 Tyr) in the insulin 2 gene caused dominant negative misfolding problem. 15 or 120 min exenatide administration substantially improves glucose-stimulated insulin secretion, marked in the secreted insulin levels and proinsulin/insulin ratio. This improvement is mainly due to enhanced conversion of proinsulin to insulin, having nothing to do with the prohormone convertase PC1/3 and PC2 levels. The 15 min improvement is calcium-independent. The 120 min improvement is linked to calcium and/or cAMP dependent mechanisms. This efficacy is validated during longer treatment and in Akita islets. Exenatide improves Ins2+/Akita beta-cell survival and Akita mouse's glucose tolerance. The results suggest a potential of incretin mimetics in alleviating defective proinsulin conversion and other proinsulin misfolding consequences.

Effects of Thyroid Peroxidase Antibody on Maternal and Neonatal Outcomes in Pregnant Women in an Iodine-Sufficient Area in China.

Purposes. To evaluate the effects of thyroid peroxidase antibodies (TPOAb) on maternal and neonatal adverse outcomes in pregnant women. Methods. 208 pregnant women at 24-28 weeks were divided into two groups, TPOAb-positive and TPOAb-negative groups. Thyroid function and TPOAb were determined in all subjects until 12 months postpartum. Levothyroxine was supplemented to maintain euthyroid with periodical checking of thyroid functions. The prevalence of postpartum thyroiditis (PPT), placenta previa, placental abruption, premature rupture of membrane, postpartum haemorrhage, polyhydramnios, oligohydramnios, preterm birth, low birth weight, congenital hypothyroidism, and neonatal diseases were observed in two groups. Results. Of all women, 11.54% had a PPT. The prevalence of PPT was significantly higher in TPOAb-positive than TPOAb-negative group (42.31% versus 7.14%, P < 0.001), with 45.46% and 53.85% of PPT happening at 6 weeks postpartum in TPOAb-positive and TPOAb-negative groups. The incidence of polyhydramnios was significantly higher in TPOAb-positive than TPOAb-negative group (15.38% versus 2.74%, P = 0.02). Conclusion. Pregnant women with TPOAb-positive had increased risk of PPT, predominantly happening at 6 weeks postpartum. TPOAb was associated with increased incidence of polyhydramnios and the underlying mechanisms required further investigation. Earlier screening of thyroid function during pregnancy and postpartum was warranted in our region.

Downregulation of Long Noncoding RNA Meg3 Affects Insulin Synthesis and Secretion in Mouse Pancreatic Beta Cells.

Increasing evidence indicates that long noncoding RNAs (lncRNAs) are involved in diverse biological process. Mouse maternal expressed gene 3 (Meg3) is an imprinted gene and essential for development. Here, we explored the relationship between Meg3 and the function of mouse beta cells in vitro and in vivo. Real-time PCR analyses revealed that Meg3 was more abundantly expressed in Balb/c mouse islets than exocrine glands. Moreover, the expression of Meg3 in islets was decreased in T1DM (NOD female mice) and T2DM (db/db mice) models. Meg3 expression was modulated dynamically by glucose in Min6 cells and isolated mouse islets. The function role of Meg3 was investigated in Min6 cells and normal mouse by knockdown of Meg3 using small interfering RNA. After suppression of Meg3 expression in vitro, insulin synthesis and secretion were impaired and the rate of beta cells apoptosis was increased. Moreover, knockdown of Meg3 in vivo led to the impaired glucose tolerance and decreased insulin secretion, consisted with the reduction of insulin positive cells areas by immunochemistry assays. Notably, islets from Meg3 interference groups showed significant decrease of Pdx-1 and MafA expression in mRNA and protein levels. These results indicate that Meg3 may function as a new regulator of maintaining beta cells identity via affecting insulin production and cell apoptosis. J. Cell. Physiol. 231: 852-862, 2016. © 2015 Wiley Periodicals, Inc.

Downregulation of lncRNA TUG1 affects apoptosis and insulin secretion in mouse pancreatic ß cells.

BACKGROUND: Increasing evidence indicates that long noncoding RNAs (IncRNAs) perform specific biological functions in diverse processes. Recent studies have reported that IncRNAs may be involved in ß cell function. The aim of this study was to characterize the role of IncRNA TUG1 in mouse pancreatic ß cell functioning both in vitro and in vivo. METHODS: qRT-PCR analyses were performed to detect the expression of lncRNA TUG1 in different tissues. RNAi, MTT, TUNEL and Annexin V-FITC assays and western blot, GSIS, ELISA and immunochemistry analyses were performed to detect the effect of lncRNA TUG1 on cell apoptosis and insulin secretion in vitro and in vivo. RESULTS: lncRNA TUG1 was highly expressed in pancreatic tissue compared with other organ tissues, and expression was dynamically regulated by glucose in Nit-1 cells. Knockdown of lncRNA TUG1 expression resulted in an increased apoptosis ratio and decreased insulin secretion in ß cells both in vitro and in vivo . Immunochemistry analyses suggested decreased relative islet area after treatment with lncRNA TUG1 siRNA. CONCLUSION: Downregulation of lncRNA TUG1 expression affected apoptosis and insulin secretion in pancreatic ß cells in vitro and in vivo. lncRNA TUG1 may represent a factor that regulates the function of pancreatic ß cells.

Mesothelin promotes cell proliferation in the remodeling of neonatal rat pancreas.

AIM: To investigate the effect of mesothelin in the remodeling of the endocrine pancreas in neonatal rats. METHODS: Overexpression or downregulation of mesothelin expression in INS-1 cells was carried out to investigate the effect of mesothelin during cell proliferation and cell apoptosis in vitro. Adenovirus-mediated RNA interference was performed to block mesothelin in vivo to directly assess the role of mesothelin in the remodeling of the endocrine pancreas in neonatal rats. RESULTS: Exogenous overexpression of mesothelin promoted cell proliferation, cell colony formation and enhanced cell resistance to apoptosis of INS-1 cells. Down-regulation of mesothelin made no difference in cell proliferation and apoptosis compared with that in the control group. After an injection of adenovirus-mesothelin, a significantly increased number of small islets appeared, and the expression of PCNA was decreased on day 7 and day 14 compared with the Ad-EGFP group. CONCLUSION: Mesothelin was able to promote ß cell proliferation in the remodeling stage of neonatal rats. Mesothelin may have an important role in the remodeling of the endocrine pancreas in neonatal rats.

Proinsulin atypical maturation and disposal induces extensive defects in mouse Ins2+/Akita ß-cells.

Because of its low relative folding rate and plentiful manufacture in ß-cells, proinsulin maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) through the integration of maturation and disposal processes. PIHO is susceptible to genetic and environmental influences, and its disorder has been critically linked to defects in ß-cells in diabetes. To explore this hypothesis, we performed polymerase chain reaction (PCR), metabolic-labeling, immunoblotting, and histological studies to clarify what defects result from primary disorder of PIHO in model Ins2(+/Akita) ß-cells. We used T antigen-transformed Ins2(+/Akita) and control Ins2(+/+) ß-cells established from Akita and wild-type littermate mice. In Ins2(+/Akita) ß-cells, we found no apparent defect at the transcriptional and translational levels to contribute to reduced cellular content of insulin and its precursor and secreted insulin. Glucose response remained normal in proinsulin biosynthesis but was impaired for insulin secretion. The size and number of mature insulin granules were reduced, but the size/number of endoplasmic reticulum, Golgi, mitochondrion, and lysosome organelles and vacuoles were expanded/increased. Moreover, cell death increased, and severe oxidative stress, which manifested as increased reactive oxygen species, thioredoxin-interacting protein, and protein tyrosine nitration, occurred in Ins2(+/Akita) ß-cells and/or islets. These data show the first clear evidence that primary PIHO imbalance induces severe oxidative stress and impairs glucose-stimulated insulin release and ß-cell survival as well as producing other toxic consequences. The defects disclosed/clarified in model Ins2(+/Akita) ß-cells further support a role of the genetic and stress-susceptible PIHO disorder in ß-cell failure and diabetes.

Substrate-favored lysosomal and proteasomal pathways participate in the normal balance control of insulin precursor maturation and disposal in ß-cells.

Our recent studies have uncovered that aggregation-prone proinsulin preserves a low relative folding rate and maintains a homeostatic balance of natively and non-natively folded states (i.e., proinsulin homeostasis, PIHO) in ß-cells as a result of the integration of maturation and disposal processes. Control of precursor maturation and disposal is thus an early regulative mechanism in the insulin production of ß-cells. Herein, we show pathways involved in the disposal of endogenous proinsulin at the early secretory pathway. We conducted metabolic-labeling, immunoblotting, and immunohistochemistry studies to examine the effects of selective proteasome and lysosome or autophagy inhibitors on the kinetics of proinsulin and control proteins in various post-translational courses. Our metabolic-labeling studies found that the main lysosomal and ancillary proteasomal pathways participate in the heavy clearance of insulin precursor in mouse islets/ß-cells cultured at the mimic physiological glucose concentrations. Further immunoblotting and immunohistochemistry studies in cloned ß-cells validated that among secretory proteins, insulin precursor is heavily and preferentially removed. The rapid disposal of a large amount of insulin precursor after translation is achieved mainly through lysosomal autophagy and the subsequent basal disposals are carried out by both lysosomal and proteasomal pathways within a 30 to 60-minute post-translational process. The findings provide the first clear demonstration that lysosomal and proteasomal pathways both play roles in the normal maintenance of PIHO for insulin production, and defined the physiological participation of lysosomal autophagy in the protein quality control at the early secretory pathway of pancreatic ß-cells.

Postnatal pancreatic islet ß cell function and insulin sensitivity at different stages of lifetime in rats born with intrauterine growth retardation.

Epidemiological studies have linked intrauterine growth retardation (IUGR) to the metabolic diseases, consisting of insulin resistance, type 2 diabetes, obesity and coronary artery disease, during adult life. To determine the internal relationship between IUGR and islet ß cell function and insulin sensitivity, we established the IUGR model by maternal nutrition restriction during mid- to late-gestation. Glucose tolerance test and insulin tolerance test (ITT) in vivo and glucose stimulated insulin secretion (GSIS) test in vitro were performed at different stages in IUGR and normal groups. Body weight, pancreas weight and pancreas/body weight of IUGR rats were much lower than those in normal group before 3 weeks of age. While the growth of IUGR rats accelerated after 3 weeks, pancreas weight and pancreas/body weight remained lower till 15 weeks of age. In the newborns, the fasting glucose and insulin levels of IUGR rats were both lower than those of controls, whereas glucose levels at 120 and 180 min after glucose load were significantly higher in IUGR group. Between 3 and 15 weeks of age, both the fasting glucose and insulin levels were elevated and the glucose tolerance was impaired with time in IUGR rats. At age 15 weeks, the area under curve of insulin (AUCi) after glucose load in IUGR rats elevated markedly. Meanwhile, the stimulating index of islets in IUGR group during GSIS test at age 15 weeks was significantly lower than that of controls. ITT showed no significant difference in two groups before 7 weeks of age. However, in 15-week-old IUGR rats, there was a markedly blunted glycemic response to insulin load compared with normal group. These findings demonstrate that IUGR rats had both impaired pancreatic development and deteriorated glucose tolerance and insulin sensitivity, which would be the internal causes why they were prone to develop type 2 diabetes.

Control of precursor maturation and disposal is an early regulative mechanism in the normal insulin production of pancreatic ß-cells.

The essential folding and maturation process of proinsulin in ß-cells is largely uncharacterized. To analyze this process, we improved approaches to immunoblotting, metabolic labeling, and data analysis used to determine the proportion of monomers and non-monomers and changes in composition of proinsulin in cells. We found the natural occurrence of a large proportion of proinsulin in various non-monomer states, i.e., aggregates, in normal mouse and human ß-cells and a striking increase in the proportion of proinsulin non-monomers in Ins2(+/Akita) mice in response to a mutation (C96Y) in the insulin 2 (Ins2) gene. Proinsulin emerges in monomer and abundant dual-fate non-monomer states during nascent protein synthesis and shows heavy and preferential ATP/redox-sensitive disposal among secretory proteins during early post-translational processes. These findings support the preservation of proinsulin's aggregation-prone nature and low relative folding rate that permits the plentiful production of non-monomer forms with incomplete folding. Thus, in normal mouse/human ß-cells, proinsulin's integrated maturation and degradation processes maintain a balance of natively and non-natively folded states, i.e., proinsulin homeostasis (PIHO). Further analysis discovered the high susceptibility of PIHO to cellular energy and calcium changes, endoplasmic reticulum (ER) and reductive/oxidative stress, and insults by thiol reagent and cytokine. These results expose a direct correlation between various extra-/intracellular influences and (a)typical integrations of proinsulin maturation and disposal processes. Overall, our findings demonstrated that the control of precursor maturation and disposal acts as an early regulative mechanism in normal insulin production, and its disorder is crucially linked to ß-cell failure and diabetes pathogenesis.