Rat Insulin Research Articles

Repression of insulin gene transcription by indirect genomic signaling via the estrogen receptor in pancreatic beta cells

The mechanism whereby 17β-estradiol (E2) mediates insulin gene transcription has not been fully elucidated. In this study, exposure of hamster insulinoma (HIT-T15) cells to 5 × 10−9 to 1 × 10−7 M E2 led to a concentration-dependent decrease of insulin mRNA levels. Transient expression of the estrogen receptor (ER) in HIT-T15 cells revealed that estrogen receptor α (ERα) repressed transcription of the rat insulin II promoter in both ligand-dependent and ligand-independent manners. The N-terminal A/B domain of ERα was not required for either activity. However, the repression was absent with mutated ER lacking the DNA-binding domain. Moreover, introducing mutations in the D-box and P-box of the zinc finger of ER (C227S, C202L) also abolished the repression. Deletion of the insulin promoter region revealed that nucleotide positions − 238 to − 144 (relative to the transcriptional start site) were needed for ER repression of the rat insulin II gene. PDX1- and BETA2-binding sites were required for the repression, but an estrogen response element-like sequence or an AP1 site in the promoter was not involved. In conclusion, we found that estrogen repressed insulin mRNA expression in a beta cell line. In addition, the ER suppressed insulin gene transcription in a ligand-independent matter. These observations suggest ER may regulate insulin transcription by indirect genomic signaling.

AAV8-mediated gene transfer of microRNA-132 improves beta cell function in mice fed a high-fat diet.

MicroRNAs have emerged as essential regulators of beta cell function and beta cell proliferation. One of these microRNAs, miR-132, is highly induced in several obesity models and increased expression of miR-132 in vitro modulates glucose-stimulated insulin secretion. The aim of this study was to investigate the therapeutic benefits of miR-132 overexpression on beta cell function in vivo. To overexpress miR-132 specifically in beta cells, we employed adeno-associated virus (AAV8)-mediated gene transfer using the rat insulin promoter in a double-stranded, self-complementary AAV vector to overexpress miR-132. Treatment of mice with dsAAV8-RIP-mir132 increased miR-132 expression in beta cells without impacting expression of miR-212 or miR-375. Surprisingly, overexpression of miR-132 did not impact glucose homeostasis in chow-fed animals. Overexpression of miR-132 did improve insulin secretion and hence glucose homeostasis in high-fat diet-fed mice. Furthermore, miR-132 overexpression increased beta cell proliferation in mice fed a high-fat diet. In conclusion, our data show that AAV8-mediated gene transfer of miR-132 to beta cells improves beta cell function in mice in response to a high-fat diet. This suggests that increased miR-132 expression is beneficial for beta cell function during hyperglycemia and obesity.

Maternal High-Protein Diet during Pregnancy Modifies Rat Offspring Body Weight and Insulin Signalling but Not Macronutrient Preference in Adulthood.

Diet of mothers during gestation may impact offspring phenotype. This study evaluated the consequences of a maternal High-Protein (HP) diet during gestation on food preferences and phenotypic characteristics in adult rat offspring. Dams were fed a HP or a Normal-Protein (NP) isocaloric diet during gestation only. Weaned female pups were divided into 3 diet groups: NP control or one of two dietary self-selection (DSS) conditions. In DSS1, offspring had a free choice between proteins (100%) or a mix of carbohydrates (88%) and lipids (12%). In DSS2, the choice was between proteins (100%), carbohydrate (100%) or lipids (100%). DSS2 groups consumed more of their energy from protein and lipids, with a decreased carbohydrate intake (p < 0.0001) compared to NP groups, regardless of the maternal diet. Offspring from HP gestation dams fed the DSS2 diet (HPDSS2) had a 41.2% increase of total adiposity compared to NPDSS2 (p < 0.03). Liver Insulin receptor and Insulin substrate receptor 1 expression was decreased in offspring from HP compared to NP gestation dams. These results showed the specific effects of DSS and maternal diet and data suggested that adult, female offspring exposed to a maternal HP diet during foetal life were more prone to adiposity development, in response to postweaning food conditions.

Cellular stress is a prerequisite for glucose-induced mitochondrial matrix alkalinization in pancreatic β-cells

Changes in mitochondrial and cytosolic pH alter the chemical gradient across the inner mitochondrial membrane. The proton chemical gradient contributes to mitochondrial ATP synthesis as well as the uptake and release of metabolites and ions from the organelle. Here mitochondrial pH and ΔpH were studied for the first time in human pancreatic β-cells. Adenoviruses were used for rat insulin promoter dependent expression of the pH sensor SypHer targeted to either the mitochondrial matrix or the cytosol. The matrix pH in resting human β-cells is low (pH = 7.50 ± SD 0.17) compared to published values in other cell types. Consequently, the ΔpH of β-cells mitochondria is small. Glucose stimulation consistently resulted in acidification of the matrix pH in INS-1E insulinoma cells and β-cells in intact human islets or islet monolayer cultures. We registered acidification with similar kinetics but of slightly smaller amplitude in the cytosol of β-cells, thus glucose stimulation further reduced the ΔpH. Infection of human islets with high levels of adenoviruses caused the mitochondrial pH to increase. The apoptosis inducer and broad-spectrum kinase inhibitor staurosporine had similar effects on pH homeostasis. Although staurosporine alone does not affect the mitochondrial pH, glucose slightly increases the matrix pH of staurosporine treated cells. These two cellular stressors alter the normal mitochondrial pH response to glucose in pancreatic β-cells.

Four weeks of exercise early in life reprograms adult skeletal muscle insulin resistance caused by a paternal high-fat diet.

A paternal high-fat diet/obesity before mating can negatively influence the metabolism of offspring. Exercise only early in life has a remarkable effect with respect to reprogramming adult rat offspring exposed to detrimental insults before conception. Exercise only early in life normalized adult whole body and muscle insulin resistance as a result of having a high-fat fed/obese father. Unlike the effects on the muscle, early exercise did not normalize the reduced adult pancreatic beta cell mass as a result of having a high-fat fed/obese father. Early-life exercise training may be able to reprogram an individual whose father was obese, inducing long-lasting beneficial effects on health. A paternal high-fat diet (HFD) impairs female rat offspring glucose tolerance, pancreatic morphology and insulin secretion. We examined whether only 4weeks of exercise early in life could reprogram these negative effects. Male Sprague-Dawley rats consumed a HFD for 10weeks before mating with chow-fed dams. Female offspring remained sedentary or performed moderate intensity treadmill exercise (5daysweek-1 , 60minday-1 , 20mmin-1 ) from 5 to 9weeks of age. Paternal HFD impaired (P< 0.05) adult offspring whole body insulin sensitivity (i.p. insulin sensitivity test), as well as skeletal muscle ex vivo insulin sensitivity and TBC1D4 phosphorylation. It also lowered β-cell mass and reduced in vivo insulin secretion in response to an i.p. glucose tolerance test. Early-life exercise in offspring reprogrammed the negative effects of a paternal HFD on whole body insulin sensitivity, skeletal muscle ex vivo insulin-stimulated glucose uptake and TBC1D4 phosphorylation and also increased glucose transporter 4 protein. However, early exercise did not normalize the reduced pancreatic β-cell mass or insulin secretion. In conclusion, only 4weeks of exercise early in life in female rat offspring reprograms reductions in insulin sensitivity in adulthood caused by a paternal HFD without affecting pancreatic β-cell mass or insulin secretion.

Vanadate inhibits transcription of the rat insulin receptor gene via a proximal sequence of the 5′flanking region

Vanadate, a protein tyrosine phosphatase inhibitor which elicits insulin-like effects, has previously been shown to inhibit expression of the insulin receptor gene at the transcriptional level in rat hepatoma cells. In an attempt to identify the DNA sequence and transcription factors potentially involved in this effect, a fragment of the proximal 5′flanking region of the IR gene (−1143/−252 upstream the ATG codon) has been cloned and functionally characterized. RNase protection allowed the identification of several transcription start sites in the conserved region of the gene, among which two major sites at −455 and −396. Upon fusion to the luciferase gene and transient transfection into hepatoma cells, the −1143/−252 fragment showed promoter activity. This was unaffected by deletion of the −1143/−761 sequence, but markedly decreased (90%) by additional deletion of the −760/−465 sequence. Treatment of hepatoma cells with vanadate led to a dose-dependent decrease in promoter activity of the 1143/−252, −760/−252 and −464/−252 constructs (change relative to untreated cells, 40, 55 and 23% at 125 μM, and 70, 85 and 62% at 250 μM, respectively). These data suggest that although the entire DNA sequence upstream the transcription start sites is probably involved in vanadate-induced inhibition, the short sequence downstream of position −464 and is sufficient for inhibition. Potential targets of vanadate are the transcription factors FoxO1 and HMGA1, two downstream targets of the insulin signaling pathway which have been shown to mediate the inhibitory effect of insulin on IR gene expression.

Proinsulin-mediated induction of type 1 diabetes in HLA-DR4-transgenic mice

Antigen-specific immunotherapy of autoimmune disease currently remains the only potentially curative approach. However, translation of promising pre-clinical results into successful clinical application has proven challenging. In part, this is because pre-clinical findings in mouse models have to be redesigned for human application due to differences in MHC II. To reduce the gap between pre-clinical and clinical studies, we have created a novel mouse model that expresses human HLA-DR4, but no endogenous MHC on antigen-presenting cells. Moreover, human B7.1 (CD80) is expressed in the pancreatic islets under the control of the rat insulin promoter. Although this model does not develop diabetes spontaneously, it is susceptible to the induction of type 1 diabetes by challenging mice with overlapping peptides derived from murine proinsulin-2 in adjuvant. Unlike the NOD model of spontaneous type 1 diabetes, but akin to the human condition, this model does not have a gender bias. Furthermore, similar to the human condition, the disease is characterised by a diverse leucocyte infiltration of the pancreatic islets and the formation of anti-proinsulin auto-antibodies. The model that we report here offers detailed insights into type-1 diabetes and is expected to prove instrumental when studying the mechanism of action in translational, antigen-specific immunotherapy.

The role of calcium sensing receptors in GLP-1 and PYY secretion after acute intraduodenal administration of L-Tryptophan in rats

Objectives: The calcium-sensing receptor (CaSR), the major sensor of extracellular Ca2+, is expressed in various tissues, including the gastrointestinal tract. Although the essential ligand of CaSR is calcium, its activity can be regulated by aromatic L-amino acids. The expression of CaSR on enteroendocrine cells suggests that CaSR functions as a physiological amino acid sensor for gut hormone release. Here, we investigated the effects of L-tryptophan (L-Trp) on rat glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and insulin secretion, and the role of CaSR in this mechanism in vivo.Methods: The effects of intraduodenal L-Trp on GLP-1, PYY, and insulin secretion were investigated. A CaSR antagonist, NPS 2143, was administered to determine whether CaSR plays a role in L-Trp-mediated gut hormone release. Male Wistar rats were divided into L-Trp, L-Trp+NPS 2143, and L-Trp+vehicle groups. Blood samples were collected, before and after the intraduodenal infusions, for determining plasma glucose, L-Trp, insulin, GLP-1, and PYY levels.Results: Our study showed a significant increase in plasma GLP-1 and insulin levels, but not plasma PYY and glucose levels, following the acute intraduodenal administration of L-Trp. We demonstrated that CaSR plays a role in L-Trp-mediated GLP-1 secretion due to attenuation of GLP-1 release with the CaSR antagonist NPS 2143.Discussion: We demonstrated that GLP-1, but not PYY, secretion following intraduodenal L-Trp administration was mediated through calcium-sensing receptors. This mechanism underlying protein sensing in the gastrointestinal system may be important for the development of new therapeutic strategies without side effects for obesity and diabetes.

Glucocorticoid Signaling Enhances Expression of Glucose-Sensing Molecules in Immature Pancreatic Beta-Like Cells Derived from Murine Embryonic Stem Cells In Vitro

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription-polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GRflox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.

Prevention of autoimmune diabetes and islet allograft rejection by beta cell expression of XIAP: Insight into possible mechanisms of local immunomodulation.

Overexpression of the X-linked inhibitor of apoptosis (XIAP) prevents islet allograft rejection. We constructed an adeno-associated virus expressing XIAP driven by the rat insulin promoter (dsAAV8-RIP-XIAP) for long-term beta-cell gene expression in vivo. Pancreatic delivery of dsAAV8-RIP-XIAP prevented autoimmune diabetes in 70% of non-obese diabetic (NOD) mice, associated with decreased insulitis. Islets from Balb/c mice transduced with dsAAV8-RIP-XIAP were protected following transplantation into streptozotocin (STZ)-diabetic Bl/6 recipients, associated with decreased graft infiltration. Interestingly, dsAAV8-RIP-XIAP transduction induced expression of lactate dehydrogenase (LDHA) and monocarboxylate transporter 1 (MCT1), two genes normally suppressed in beta cells and involved in production and release of lactate, a metabolite known to suppress local immune responses. Transduction of Balb/c islets with AAV8-RIP-LDHA-MCT1 tended to prolong allograft survival following transplant into STZ-diabetic Bl/6 recipients. These findings suggest that XIAP has therapeutic potential in autoimmune diabetes and raise the possibility that local lactate production may play a role in XIAP-mediated immunomodulation.

The protective effect of Azadirachta excelsa leaves extract and quercetin treatment on the learning and memory impairments in relation with insulin and amylin levels in the brain of streptozotocin-induced diabetic rats

Azadirachta excelsa increases the secretion of plasma insulin with subsequent reduction of fasting blood glucose. However, its protective effect towards brain amylin and insulin levels as well as the memory and learning functions remain elusive. Thus, this study aimed to ascertain the effect of A. excelsa and quercetin consumptions on the brain amylin and insulin concentrations as well as memory and learning abilities of the streptozotocin (STZ)-induced diabetic rats. Diabetic condition was induced in the male Sprague Dawley rats by the intraperitoneal injection of 60 mg/kg body weight (bwt) STZ. The experimental animals were divided into normal control (saline), diabetic control (saline), Metformin-treated diabetic (1000 mg/kg bwt) (positive control), A. excelsa-treated diabetic (250 mg/kg bwt) and Quercetin-treated diabetic (40 mg/kg bwt) rats. All treatments were administered orally by oral gavage once daily throughout the 8 weeks of the treatment period. Amylin and insulin concentrations were determined by using the commercial rat amylin and insulin immunoassay kits, while learning and memory consolidation parameters were conducted using Morris Water Maze (MWM) paradigm. Treatment with A. excelsa had significantly (p < 0.05) improved the concentration of both brain amylin and insulin. In MWM test, the administration of A. excelsa and quercetin significantly (p < 0.05) attenuated the learning and memory impairments in STZ-induced diabetic rats. These analytical results provide evidence that A. excelsa can improve learning and memory impairments due to diabetes by increasing the amylin and insulin levels. These also suggest that, quercetin might only improve the learning and memory impairment, but not be the one that contributed to the improvement in the brain amylin and insulin concentrations.

The liver surface as a favorable site for islet cell sheet transplantation in type 1 diabetes model mice.

IntroductionIslet transplantation is one of the most promising therapeutic approaches for patients with severe type 1 diabetes mellitus (T1DM). Transplantation of engineered islet cell sheets holds great potential for treating T1DM as it enables the creation of stable neo-islet tissues. However, a large mass of islet cell sheets is required for the subcutaneous transplantation to reverse hyperglycemia in diabetic mice. Here, we investigated whether the liver surface could serve as an alternative site for islet cell sheet transplantation.MethodsDispersed rat islet cells (0.8 × 106 cells) were cultured on laminin-332-coated thermoresponsive culture dishes. After 2 days of cultivation, we harvested the islet cell sheets by lowering the culture temperature using a support membrane with a gelatin gel. We transplanted two recovered islet cell sheets into the subcutaneous space or onto the liver surface of severe combined immunodeficiency (SCID) mice with streptozocin-induced diabetes.ResultsIn the liver surface group, the non-fasting blood glucose level decreased rapidly within several days after transplantation. In marked contrast, the hyperglycemia state was maintained in the subcutaneous space transplantation group. The levels of rat C-peptide and insulin in the liver surface group were significantly higher than those in the subcutaneous space group. An immunohistological analysis confirmed that most of the islet cells engrafted on the liver surface were insulin-positive. The CD31-positive endothelial cells formed vascular networks within the neo-islets and in the surrounding tissues. In contrast, viable islet cells were not found in the subcutaneous space group.ConclusionsCompared with the subcutaneous space, a relatively small mass of islet cell sheets was enough to achieve normoglycemia in diabetic mice when the liver surface was selected as the transplantation site. Our results demonstrate that the optimization of the transplantation site for islet cell sheets leads to significant improvements in the therapeutic efficiency for T1DM.

Atrophied thymus produces altered repertoire of tTregs with potential to break the balance of peripheral tolerance

Abstract We have previously shown that in the age-related atrophied thymus, there is an increased ratio of thymic T regulatory (tTreg) cells to thymic T conventional (tTcon) cells, suggesting that the aged thymus has an enhanced tTreg generation. It is also known that in the periphery of aged mice and humans, there is an accumulation of peripheral Tregs. We raise the following question: why is the increased Treg population unable to suppress self-reactivity in the elderly? We utilized a sub-lethally irradiated chimeric mouse model in which OT-II TCR transgenic bone-marrow cells were transplanted into RIP-mOVA host mice. Therefore, OVA serves as a mock self-antigen, and because this transgene is driven by the rat insulin promotor, the pancreas of these mice expresses OVA. Further, the mOVA mice also carry a FoxN1-floxed gene, which can be conditionally knocked-out via CreERT transgene activation by injection(s) with tamoxifen to induce thymic atrophy. We observed that chimera mice with induced thymic atrophy show total Tregs (termed pan-Tregs) were increased but OT-II specific Tregs were slightly decreased in the spleen. Additionally, there is a dramatic decrease in the OTII-specific Tregs found in the pancreas, and pancreatic atrophy was observed in these mice compared to those with normal thymus. Our initial results suggest that thymic atrophy leads to a biased tTreg generation that manifests in decreased selftissue specific-Tregs. Therefore, although pan-Tregs may accumulate and be enhanced with age, tissue-specific peripheral tolerance may be poised to fail.

Beta‐cell‐specific loss of the inhibitory G protein, Gαz, prevents development of Type 1 Diabetes in NOD mice.

The mechanisms that underlie the beta‐cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our lab has discovered a role for the unique inhibitory G‐protein, Gαz, in beta‐cell function, survival, and replication in both normal conditions and those of pathological stress. Gαz, when acting as a canonical inhibitory G‐protein, reduces production of cyclic adenosine monophosphate (cAMP) through inhibition of adenylate cyclase. cAMP acts as essential player in the amplification of glucose‐stimulated insulin secretion, while also mediating pathways that regulate beta‐cell death and replication. We've shown previously that removal of this inhibitory protein is sufficient to promote insulin secretion, beta‐cell growth and survival using chemically‐induced beta‐cell death and diabetes in C57BL/6J mice. Previous work in our lab has also shown that non‐obese diabetic (NOD) mice with global loss of Gαz are protected from developing T1D‐like hyperglycemia through a multi‐faceted maintenance of beta‐cell health. Whole body Gαznull animals exhibited a significant increase in beta‐cell function and replication and decreased beta‐cell death. Most interestingly, islets from Gαz‐null NOD animals exhibited a marked decrease in immune infiltration. In order to determine if Gαz in the beta‐cell was responsible for the protected phenotype, we generated a beta‐cell‐specific Gαz knockout by breeding Gz Flox/Flox mice with Rat Insulin Promoter (RIP) driven CreHerr mice, on the NOD background. After 10 generations of backcrossing, we began by determining if loss of beta cell Gαz was sufficient to protect NOD animals from developing T1D‐like hyperglycemia. Weekly random‐fed blood glucose measurements were taken beginning at weaning. In the initial cohort, we saw a 12% penetrance of hyperglycemia in male Cre‐positive NOD mice and a 23% penetrance of hyperglycemia in female Cre‐positive NOD mice by 30 weeks of age. In contrast, Gαzβ‐cell‐KO males and females were entirely protected from developing hyperglycemia during this period. Despite this stark contrast, fasting blood glucose was no different between genotypes, grouped by sex, at either 12 or 28 weeks of age. Glucose tolerance was also not different between Cre‐positive NOD controls and Gαzβ‐cell‐KO animals. Experiments are ongoing to further investigate other facets of beta‐cell health, including function, replication and survival. Additionally, it will be crucial to evaluate the presence and extent of insulitis in our Gαzβ‐cell‐KO animals. Thus far, our results show a critical role for beta‐cell Gαz in promoting T1D pathophysiology, although the precise mechanism remains to be determined.Support or Funding InformationNIH F31 DK109698 to RFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel synthetic human insulin super promoter for targeting PDX-1-expressing pancreatic cancer

Our previous studies have shown that a rat insulin promoter II fragment (RIP) was used to effectively target pancreatic adenocarcinoma (PDAC) and insulinoma that over-express pancreatic and duodenal homeobox-1 (PDX-1). To enhance the activity and specificity of the human insulin promoter, we engineered a synthetic human insulin super-promoter (SHIP). Reporter assay demonstrated that SHIP1 was the most powerful promoter among all of the SHIPs and had far greater activity than the endogenous human insulin promoters and RIP in PDAC expressing PDX-1. Over-expression, knockdown and competitive inhibition of PDX-1 expression assay proved that PDX-1 is a critical transcript factor to regulate the activity of SHIP1. SHIP1-driven viral thymidine kinase followed by ganciclovir (SHIP1-TK/GCV) resulted in cytotoxicity to PDAC cells in vitro. Systemic delivery of SHIP1-TK/GCV in PDAC xenograft mice significantly suppressed PANC-1 tumor growth in vivo greater than RIP-TK/GCV and CMV-TK/GCV controls (p < .05). These preclinical data suggest that SHIP1 is a powerful novel promoter that can be used to target human PDAC expressing PDX-1 in clinical trials. Furthermore, this novel strategy of engineering synthetic super-promoters could be used for other cancer targets.

β-cell-specific overexpression of adiponectin receptor 1 does not improve diabetes mellitus in Akita mice.

Adiponectin, a metabolically-active cytokine secreted from adipose tissue, is reported to have anti-apoptotic effects on β-cells as well as anti-hyperglycemic effects through adiponectin receptor signaling. However, the anti-apoptotic effects of adiponectin on β-cells have not been confirmed in established diabetic models, and the anti-hyperglycemic effects and their associated signal cascades remain controversial. To investigate the effects of adiponectin on β-cell protection and its down-stream signaling events, we have generated β-cell-specific rat insulin promoter (RIP)-AdipoR1 transgenic mice (AdipoR1 mice), in which the adiponectin receptor, AdipoR1, is overexpressed in β-cells in a manner synchronous with insulin demand. AdipoR1 mice were then mated with Akita mice, a diabetes model in which β-cell apoptosis results from endoplasmic reticulum (ER) stress. AdipoR1 protein expression and localization in islets from AdipoR1 mice as well as in an AdipoR1-transfected mouse insulinoma cell line were confirmed, as was the activation of both AMPK and Akt in AdipoR1 mice by adiponectin. Nevertheless, there were no significant differences in Ad lib feed and fasting blood glucose levels, or in glucose tolerance tests, between Akita mice [Ins2Akita (C96Y) +/- mouse model] and AdipoR1/Akita and from 4 weeks to 10 weeks of age. Similarly, pancreatic insulin contents of AdipoR1/Akita mice were not significantly different from those in Akita mice from 15 to 20 weeks of age, but they were significantly lower than in wild-type mice. Immunostaining for insulin and subsequent electron microscopy showed that β-cell destruction in AdipoR1/Akita mice was not markedly improved in comparison with that in Akita mice. Serum adiponectin concentrations were confirmed to be extremely high (> 30 μg / ml) compared with the Kd value (0.06 μg / ml) in all mouse groups at 15 to 20 weeks of age. Therefore, although the physiological levels of adiponectin are sufficient to activate AMPK and Akt when AdipoR1 is overexpressed in β-cells, yet adiponectin cannot protect β-cells in Akita mice from ER stress-induced destruction.

Nuclear factor E2-related factor expression and its relationship with oxidative stress in the placenta of pregnant diabetic rats

Objective: The aim of this study is to investigate Nrf2 expression in placenta of gestational diabetes mellitus rat (GDM) and the relationship between oxidative stress. Methods: Thirty pregnancy SD rats were chosen in our study, and randomly divided into 3 groups: normal control group, GDM group and GDM+Oltipraz (5 mg/kg, via intragastric administration 1 hour before STZ treatment) group. GDM and GDM group were treatment with Streptozotocin (STZ) via intraperitoneal injection once a week, the control group was treatment with the same amount of citric acid buffer. On day 20 of pregnant period, anesthesia and dissect rats, placenta tissue and blood were prepared for experiment. The expression of Nrf2 in placenta was determined by western blotting. Insulin expression and Total antioxidative capability and MDA were detection by ELISA Kit. Results: Blood glucose has been increased significantly when the GDM group and control group were compared with the difference was statistically significant. With the GDM group, the intervention group was compared so that the placental insulin levels were significantly increased and the difference was statistically significant. GDM group Nrf2 protein expression levels were significantly increased and the difference was statistically significant (P<0.05) when it is compared with normal group. Conclusion: GDM is a problem of great clinical importance core for the development of numerous adverse maternal and perinatal outcomes. Through the placenta tissue of rat insulin levels, total antioxidant capacity (total antioxidant capacity, TAC), lipid peroxidation levels and protein expression of Nrf2, this study recognized a gestational diabetic rat model to explore the possible pathogenesis of gestational diabetes, and provided a theoretical basis for clinical treatment.

Astragaloside IV prevents kidney injury caused by iatrogenic hyperinsulinemia in a streptozotocin‑induced diabetic rat model.

Diabetic patients are able to manage their blood glucose with exogenous insulin but, ultimately, remain at risk of diabetic nephropathy (DN). Long‑term use of insulin may lead to iatrogenic hyperinsulinemia, which has been suggested to cause kidney injury. However, there are no effective interventions for iatrogenic hyperinsulinemia leading to kidney damage. In the present paper, the hypothesis that astragalosideIV (AS‑IV), a novel saponin purified from Astragalus membranaceus (Fisch) Bunge, may prevent DN in iatrogenic hyperinsulinemic diabetic rats through antioxidative and anti‑inflammatory mechanisms was investigated. Diabetes was induced with streptozotocin (STZ) (55mg/kg) by intraperitoneal injection in rats. At 1week following STZ injection, the diabetic rats were treated with Levemir subcutaneously for4weeks. Diabetic rat insulin levels >30µU/ml were considered as iatrogenic hyperinsulinemia. Rats were divided into six groups (n=8 per group): Iatrogenic hyperinsulinemic rats, and iatrogenic hyperinsulinemic rats treated with Tempol and AS‑IV at 2.5, 5 and 10mg/kg/day, intragastric infusion, for 12weeks. The normal rats were used as a non‑diabetic control group. AS‑IV ameliorated albuminuria, mesangial cell proliferation, basement membrane thickening and podocyte foot process effacement in iatrogenic hyperinsulinemic rats. In iatrogenic hyperinsulinemic rat renal tissues, malondialdehyde, interleukin‑1β(IL‑1β), tumor necrosis factor‑α (TNF‑α), typeIV collagenand laminin levels were increased, whereas glutathione peroxidaseand superoxide dismutase activity levels were decreased. Nicotinamide adenine dinucleotide phosphate oxidase4 expression and extracellular signal‑regulated kinase1/2(ERK1/2) activation were upregulated, and canonical transient receptor potential cation channel6 (TRPC6) protein expression was downregulated. However, all these abnormalities were attenuated by AS‑IV. These findings suggested that AS‑IV prevented rat kidney injury caused by iatrogenic hyperinsulinemia by inhibiting oxidative stress, IL‑1β and TNF‑α overproduction, downregulating ERK1/2 activation, and upregulating TRPC6 expression.

Glycyrrhizin ameliorates high fat diet-induced obesity in rats by activating NrF2 pathway

AimObesity based on insulin resistance is a state of chronic oxidative stress and inflammation that are highly regulated through nuclear factor Erythroid 2-related factor 2 (NrF2) pathway. Materials and methods70 male Wistar rats were randomized into two models. The prophylactic model was 10weeks and rats were grouped into: normal group, GL group (received glycyrrhizin 50mg/kg/day orally along with normal pellet diet), HFD group and HFD+ GL group (received glycyrrhizin along with HFD). The treatment model was 14weeks and rats were grouped into: normal group, HFD group and HFD+GL group (received glycyrrhizin from the week 10). Key findingsGlycyrrhizin decreased significantly rat weights and insulin resistance, normalized lipid profile and reduced significantly the adipocytes size in adipose tissue and lipid deposition in the liver tissue through histopathologic examination. Furthermore, glycyrrhizin ameliorated obesity-induced oxidative stress which indicated by significant decrease in liver malondialdehyde level (P<0.001) and increase in the total antioxidant capacity (P<0.001). Interestingly, molecular mechanism of glycyrrhizin was explored, that included significant reduction of liver gluconeogenic enzymes mRNA expression (P<0.001), a significant increase of liver insulin receptor, NrF2 and homooxygenase-1 mRNA expressions (P<0.001) and significant increase and nuclear translocation of NrF2 in liver tissue. SignificanceGlycyrrhizin ameliorates HFD-induced obesity in rats that may be attributed to its ability to increase insulin receptor expression and to activate NrF2 and subsequent homooxygenase-1 pathway. Thus, this work represents a safe natural compound (glycyrrhizin) that has a great role either as prophylaxis or treatment for insulin resistance related to obesity.

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model.

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis and to identify novel therapeutic targets. To investigate molecular events that drive the progression from primary cancer to metastasis, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva. In this mouse model, the rat insulin promoter (RIP) drives the expression of the SV40 T antigen (Tag) and the receptor for subgroup A avian leukosis virus (tva) in pancreatic β cells. The mice develop pancreatic neuroendocrine tumors with 100% penetrance through well-defined stages that are similar to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. Because RIP-Tag; RIP-tva mice do not develop metastatic disease, genetic alterations that promote metastasis can be identified easily. Somatic gene transfer into tva-expressing, proliferating pancreatic β premalignant lesions is achieved through intracardiac injection of avian retroviruses harboring the desired genetic alteration. A titer of >1 x 108 infectious units per ml is considered appropriate for in vivo infection. In addition, avian retroviruses can infect cell lines derived from tumors in RIP-Tag; RIP-tva mice with high efficiency. The cell lines can also be used to characterize the metastatic factors. Here we demonstrate how to utilize this mouse model and cell lines to assess the functions of candidate genes in tumor metastasis.