Characterization of NLRP3 Inflammasome Activation in the Onset of Diabetic Retinopathy.

The aim of this study was to characterize the role of nucleotide-binding oligomerization domain- (NOD-) like receptor (NLR) protein 3 (NLRP3) inflammasome activation in the onset of diabetic retinopathy (DR) using retina and vitreous from donors without diabetes mellitus (CTL), with diabetes mellitus alone (DM), and with DR. Retinal expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1), the key markers of retinal inflammation, connexin43 (Cx43) which is involved in upstream inflammasome regulation, as well as NLRP3 and cleaved caspase-1, the main markers of inflammasome activation, were evaluated using immunohistochemistry and Western blotting. Vitreous interleukin (IL)-1ß and IL-18, biomarkers of the activated inflammasome, were measured using a Luminex multiplex assay. Results showed a significant increase in the number and size of Iba-1+ cells and NLRP3 expression in DM, while a significant increase in GFAP, Cx43, cleaved caspase-1 and vitreous IL-18, as well as a further increase in Iba-1 and NLRP3 was found in DR. This suggests that the inflammasome is already primed in DM before its activation in DR. Furthermore, IL-18 may act as the major effector of inflammasome activation in DR while nuclear translocation of cleaved caspase-1 may play a role in gene transcription contributing to DR onset.

Differentiation of Islet Progenitors Regulated by Nicotinamide into Transcriptome-Verified ß Cells That Ameliorate Diabetes.

Developmental stage-specific differentiation of stem or progenitor cells into safe and functional cells is of fundamental importance in regenerative medicine, including ß-cell replacement. However, the differentiation of islet progenitor cells (IPCs) into insulin-secreting ß cells remains elusive. Here, we report that the multifunctional molecule nicotinamide (NIC) is a specific differentiation regulator of mouse IPCs. The differentiated cells regulated by NIC exhibited many characteristics of adult ß cells, including ameliorating preclinical diabetes and a highly comparable transcriptome profile. Gene set enrichment analysis showed that during differentiation, numerous IPC transcription factor genes, including Ngn3, Pax4, Fev, and Mycl1, were all down regulated. Pharmacological, biochemical, and gene knockdown analyses collectively demonstrated that NIC regulated the differentiation via inhibiting Sirt1 (silent information regulator transcript 1). Finally, NIC also regulates human IPC differentiation. Thus, our study advances islet developmental biology and impacts on translational research and regenerative therapies to diabetes and other diseases. Stem Cells 2017;35:1341-1354.

The effect of molecular weights of microencapsulating polymers on viability of mouse-cloned pancreatic ß-cells: biomaterials, osmotic forces and potential applications in diabetes treatment.

INTRODUCTION: Ideal cell-containing microcapsules should be capable of maintaining cell viability and exhibit significant structural stability to support cellular functionality. To date, such microcapsules remain unavailable; thus, this study used our well-established microencapsulating methods to examine a total of 32 different microencapsulating formulations and correlate polymers' molecular weights (Mwt) and UDCA addition, with cell viability and microcapsules' stability, postmicroencapsulation. METHODS: MIN6 mouse-cloned pancreatic ß-cells were microencapsulated using control (n = 16; without UDCA) and test (n = 16; with UDCA) different polymers. Confocal microscopic imaging, cell viability, and microcapsules' stability were assessed. RESULTS: Best cell viability (>50%) was obtained at average Mwt of 50,000 g/mol (poly-l-ornithine), followed by 110,000 g/mol (poly-l-lysine). There was no linear correlation between Mwt and viability. Confocal imagining showed similar microcapsules' shape and cell distribution among all different polymers' molecular weights, which suggests that the microencapsulating method was efficient and maintained microcapsules' uniformity. UDCA addition resulted in enhanced osmotic stability of the microcapsules and improved cell viability, when the formulation contained 1% polylornithine, 1% polyethylene glycol, 20% Eudragit® NM30D, 1% polytetrafluoroethylene, or 5% pentamethylcyclopentasiloxane. CONCLUSIONS: UDCA addition improved microenvironmental conditions within the microcapsules but this effect was largely dependent on the polymer systems used.

New Biotechnological Microencapsulating Methodology Utilizing Individualized Gradient-Screened Jet Laminar Flow Techniques for Pancreatic ß-Cell Delivery: Bile Acids Support Cell Energy-Generating Mechanisms.

In previous studies, we developed a new technique (ionic gelation vibrational jet flow; IGVJF) in order to encapsulate pancreatic ß-cells, for insulin in vivo delivery, and diabetes treatment. The fabricated microcapsules showed good morphology but limited cell functions. Thus, this study aimed to optimize the IGVJF technique, by utilizing integrated electrode tension, coupled with high internal vibration, jet-flow polymer stream rate, ionic bath-gelation concentrations, and gelation time stay. The study also utilized double inner/outer nozzle segmented-ingredient flow of microencapsulating dispersion, in order to form ß-cell microcapsules. Furthermore, a microcapsule-stabilizing bile acid was added, and microcapsule's stability and cell functions measured. Buchi-based built-in system utilizing IGVJF technology was screened to produce best microcapsule-containing ß-cells with or without a stabilizing-enhancing bile acid. Formed microcapsules were examined, for physical characteristics, and encapsulated cells were examined for survival, insulin release, and inflammatory profiles. Optimized microencapsulating parameters, using IGJVF, were: 1000 V voltage, 2500 Hz frequency, 1 mL/min flow rate, 3% w/v ionic-bath gelation concentration, and 20 min gelation time. Microcapsules showed good morphology and stability, and the encapsulated cells showed good survival, and insulin secretion, which was optimized by the bile acid. Deployed IGVJF-based microencapsulating parameters utilizing stability-enhancing bile acid produced best microcapsules with best pancreatic ß-cells functions and survival rate, which, suggests potential application in cell transplantation.

Innovative Microcapsules for Pancreatic ß-Cells Harvested from Mature Double-Transgenic Mice: Cell Imaging, Viability, Induced Glucose-Stimulated Insulin Measurements and Proinflammatory Cytokines Analysis.

PURPOSE: Recently we demonstrated that microencapsulation of a murine pancreatic ß-cell line using an alginate-ursodeoxycholic acid (UDCA) matrix produced microcapsules with good stability and cell viability. In this study, we investigated if translation of this formulation to microencapsulation of primary ß-cells harvested from mature double-transgenic healthy mice would also generate stable microcapsules with good cell viability. METHODS: Islets of Langerhans were isolated from Ngn3-GFP/RIP-DsRED mice by intraductal collagenase P digestion and density gradient centrifugation, dissociated into single cells and the ß-cell population purified by Fluorescence Activated Cell Sorting. ß-cells were microencapsulated using either alginate-poly-l-ornithine (F1; control) or alginate-poly-l-ornithine-UDCA (F2; test) formulations. Microcapsules were microscopically examined and microencapsulated cells were analyzed for viability, insulin and cytokine release, 2 days post-microencapsulation. RESULTS: Microcapsules showed good uniformity and morphological characteristics and even cell distribution within microcapsules with or without UDCA. Two days post microencapsulation cell viability, mitochondrial ATP and insulin production were shown to be optimized in the presence of UDCA whilst production of the proinflammatory cytokine IL-1ß was reduced. Contradictory to our previous studies, UDCA did not reduce production of any other pro-inflammatory biomarkers. CONCLUSIONS: These results suggest that UDCA incorporation improves microcapsules' physical and morphological characteristics and improves the viability and function of encapsulated mature primary pancreatic ß-cells.

Oral glucose tolerance test to diagnose gestational diabetes mellitus: Impact of variations in specimen handling.

To improve birth outcomes, all pregnant women without known diabetes are recommended for an oral glucose tolerance test (OGTT) to screen for hyperglycaemia in pregnancy (diabetes in pregnancy or gestational diabetes mellitus (GDM)). This narrative review presents contemporary approaches to minimise preanalytical glycolysis in OGTT samples with a focus on GDM diagnosis using criteria derived from the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study. The challenges of implementing each approach across a diverse Australian healthcare setting were explored. Many Australian sites currently collect and transport OGTT samples at ambient temperature in sodium fluoride (NaF) tubes which is likely to lead to missed diagnosis of GDM in a significant proportion of cases. Alternative preanalytical solutions should be pragmatic and tailored to individual settings and as close as possible to the preanalytical conditions of the HAPO study for correct interpretation of OGTT results. Rapid centrifugation of barrier tubes to separate plasma could be suitable in urban settings provided time to centrifugation is strictly controlled. Tubes containing NaF and citrate could be useful for remote or resource poor settings with long delays to analysis but the impact on the interpretation of OGTT results should be carefully considered. Testing venous blood glucose at the point-of-care bypasses the need for glycolytic inhibition but requires careful selection of devices with robust analytical performance. Studies to evaluate the potential error of each solution compared to the HAPO protocol are required to assess the magnitude of misdiagnosis and inform clinicians regarding the potential impact on patient safety and healthcare costs.

Underestimation of risk for large babies in rural and remote Australia: Time to change plasma glucose collection protocols.

AIMS: Preanalytical glycolysis in oral glucose tolerance tests (OGTT) leads to substantial underestimation of gestational diabetes mellitus (GDM) and hence risk for large-for-gestational-age (LGA) babies. This paper quantified the impact of glycolysis on identification of LGA risk in a prospective rural and remote Australian cohort. METHODS: For 495 women, OGTT results from room temperature fluoride-oxalate (FLOX) tubes were algorithmically corrected for estimated glycolysis compared to 1) the Hyperglycaemia and Adverse Pregnancy Outcomes (HAPO) study protocol (FLOX tubes in ice-slurry); and 2) room temperature fluoride-citrate (FC) tubes. GDM was defined by International Association of the Diabetes and Pregnancy Study Groups (IADPSG) criteria. Unadjusted and corrected OGTT were related to LGA outcome. RESULTS: Correction for FC tubes increased GDM incidence from 9.7% to 44.6%. After correction for HAPO protocol, GDM incidence was 27.7% and prediction of LGA risk (RR 1.82, [1.11-2.99]) improved compared to unadjusted rates (RR 1.12, [0.51-2.47]). To provide similar results for FC tube correction (29.3% GDM; RR 1.81, [1.11-2.96]) required + 0.2 mmol/L adjustment of IADPSG criteria. CONCLUSIONS: FC tubes present a practical alternative to the HAPO protocol in remote settings but give + 0.2 mmol/L higher glucose readings. Modification of IADPSG criteria would reduce perceived 'overdiagnosis' and improve LGA risk-assessment.

Real-world screening for diabetes in early pregnancy: Improved screening uptake using universal glycated haemoglobin.

AIMS: To improve perinatal outcomes, screening for hyperglycaemia using 75 g oral glucose tolerance test (OGTT) is recommended for all pregnant women at 24-28 weeks gestation (routine), and earlier if high-risk. Screening coverage for remote and Aboriginal Australian women is less than ideal. This study examined OGTT completion (early and routine) by women from rural and remote Western Australia compared with early glycated haemoglobin (HbA1c). METHODS: In 2015-2018, 27 primary health care sites recruited 600 (233 Aboriginal) women aged ≥16-years, without pre-existing diabetes, who delivered >30-weeks gestation. All women presenting <20-weeks gestation (541) were offered an early study HbA1c. Early OGTTs were requested at the discretion of the local clinician, with routine OGTT offered at 24-28 weeks. RESULTS: HbA1c uptake was high (85.7% Aboriginal, 86.4% non-Aboriginal); OGTT completion in Aboriginal women was low (early OGTT: 38.6% v 69.6% non-Aboriginal, P < 0.001; routine OGTT: 44.5% v 84.7% non-Aboriginal, P < 0.001). Aboriginal women with both early tests had HbA1c completed 3-weeks prior to OGTT (9.6 ± 3.5 v 12.5 ± 3.5 weeks gestation, P < 0.001). CONCLUSIONS: Universal early pregnancy HbA1c appears feasible as an early screening test for women at risk of hyperglycaemia in pregnancy and would expedite and increase screening in Aboriginal women compared to an early OGTT.

Prediabetes and pregnancy: Early pregnancy HbA1c identifies Australian Aboriginal women with high-risk of gestational diabetes mellitus and adverse perinatal outcomes.

AIMS: To assess whether early pregnancy HbA1c can predict gestational diabetes mellitus (GDM) and adverse birth outcomes in Australian women. METHODS: Prospective study of 466 women without diabetes, aged ≥16-years at first antenatal presentation. Recruitment was from 27 primary healthcare sites in rural and remote Australia from 9-January 2015 to 31-May 2018. HbA1c was measured with first antenatal investigations (<20-weeks gestation). Primary outcome measure was predictive value of HbA1c for GDM, by routine 75 g oral glucose tolerance test (OGTT; ≥24-weeks gestation), and for large-for-gestational-age (LGA) newborn. RESULTS: Of 396 (129 Aboriginal) women with routine OGTT, 28.8% had GDM (24.0% Aboriginal). HbA1c ≥5.6% (≥38 mmol/mol) was highly predictive (71.4%, 95% CI; 47.8-88.7%) for GDM in Aboriginal women, and in the total cohort increased risk for LGA newborn (RR 2.04, 95% CI; 1.03-4.01, P = 0.040). There were clear differences between Aboriginal and non-Aboriginal women: 16.3% v 5.2% (P < 0.001) had elevated HbA1c whereas 12.4% v 29.6% (P < 0.001) developed hyperglycemia during pregnancy. CONCLUSIONS: Early pregnancy HbA1c ≥5.6% (≥38 mmol/mol) identifies Aboriginal women with apparent prediabetes and elevated risk of having an LGA newborn. Universal HbA1c at first antenatal presentation could facilitate earlier management of hyperglycemia and improved perinatal outcome in this high-risk population.

A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance.

Mitochondrial energy metabolism plays an important role in the pathophysiology of insulin resistance. Recently, a missense N437S variant was identified in the MRPP3 gene, which encodes a mitochondrial RNA processing enzyme within the RNase P complex, with predicted impact on metabolism. We used CRISPR-Cas9 genome editing to introduce this variant into the mouse Mrpp3 gene and show that the variant causes insulin resistance on a high-fat diet. The variant did not influence mitochondrial gene expression markedly, but instead, it reduced mitochondrial calcium that lowered insulin release from the pancreatic islet ß cells of the Mrpp3 variant mice. Reduced insulin secretion resulted in lower insulin levels that contributed to imbalanced metabolism and liver steatosis in the Mrpp3 variant mice on a high-fat diet. Our findings reveal that the MRPP3 variant may be a predisposing factor to insulin resistance and metabolic disease in the human population.

Real-World Gestational Diabetes Screening: Problems with the Oral Glucose Tolerance Test in Rural and Remote Australia.

Gestational diabetes mellitus (GDM) is the most common antenatal complication in Australia. All pregnant women are recommended for screening by 75 g oral glucose tolerance test (OGTT). As part of a study to improve screening, 694 women from 27 regional, rural and remote clinics were recruited from 2015-2018 into the Optimisation of Rural Clinical and Haematological Indicators for Diabetes in pregnancy (ORCHID) study. Most routine OGTT samples were analysed more than four hours post fasting collection (median 5.0 h, range 2.3 to 124 h), potentially reducing glucose levels due to glycolysis. In 2019, to assess pre-analytical plasma glucose (PG) instability over time, we evaluated alternative sample handling protocols in a sample of participants. Four extra samples were collected alongside routine room temperature (RT) fluoride-oxalate samples (FLOXRT): study FLOXRT; ice slurry (FLOXICE); RT fluoride-citrate-EDTA (FC Mix), and RT lithium-heparin plasma separation tubes (PST). Time course glucose measurements were then used to estimate glycolysis from ORCHID participants who completed routine OGTT after 24 weeks gestation (n = 501). Adjusting for glycolysis using FLOXICE measurements estimated 62% under-diagnosis of GDM (FLOXRT 10.8% v FLOXICE 28.5% (95% CI, 20.8-29.5%), p < 0.001). FC Mix tubes provided excellent glucose stability but gave slightly higher results (Fasting PG: +0.20 ± 0.05 mmol/L). While providing a realistic alternative to the impractical FLOXICE protocol, direct substitution of FC Mix tubes in clinical practice may require revision of GDM diagnostic thresholds.

Electrokinetic potential-stabilization by bile acid-microencapsulating formulation of pancreatic ß-cells cultured in high ratio poly-L-ornithine-gel hydrogel colloidal dispersion: applications in cell-biomaterials, tissue engineering and biotechnological applications.

INTRODUCTION: Current trials for ß-cell transplantation have been hindered by poor cell viability and function post-transplantation. Recently, electric charges of the microencapsulating formulation carrying ß-cells have shown significant effects on cell survival and function. Thus, this study aimed at investigating the effects of electric charge, of novel colloidal formulation containing ß-cells, on cell viability, biological activity and insulin release. METHODS: A new formulation, containing high ratios of poly-L-ornithine, suspending electrical-stimulation hydrogel and polystyrene sulphone (1:1:0.1 ratio), was used to form microcapsules utilizing 800 V and 2000 Hz encapsulating conditions. The bile acid, ursodeoxycholic acid, was added into the microcapsules to measure its effects on electric charges. RESULTS: The electric charge of the microencapsulating formulation was enhanced by bile acid addition, and resulted in better cell viability and function. CONCLUSION: Ursodeoxycholic acid microencapsulated with poly-L-ornithine, suspending electrical-stimulation hydrogel and polystyrene sulphone at 1:1:0.1 ratio, using 800 V and 2000 Hz microencapsulating conditions, produced enhanced electrokinetic parameters of microcapsules with optimized cell functions. This suggests that electric charge of formulations containing pancreatic ß-cell may have significant effects on cell mass and functions, post-transplantation.

Influence of Biotechnological Processes, Speed of Formulation Flow and Cellular Concurrent Stream-Integration on Insulin Production from ß-cells as a Result of Co-Encapsulation with a Highly Lipophilic Bile Acid.

INTRODUCTION: We have shown that incorporation of the hydrophilic bile acid, ursodeoxycholic acid, into ß-cell microcapsules exerted positive effects on microcapsules' morphology and size, but these effects were excipient and method dependent. Cell viability remained low which suggests low octane-water solubility and formation of highly hydrophilic dispersion, which resulted in low lipophilicity dispersion and compromised cellular permeation of the incorporated bile acid. Thus, this study aimed at investigating various microencapsulating methodologies using highly lipophilic bile acid (LPBA), in order to optimise viability and functions of microencapsulated ß-cells. METHODS: Four different types of microcapsules were produced with (test) and without (control) LPBA, totalling eight different microcapsules. Microencapsulating methodologies were screened for best microcapsule-cell functions and microencapsulating processes were examined in terms of frequency, formulation flow, total bath-gelation time and cellular concurrent stream-integration rate, cell-viability, insulin production and inflammatory profile. RESULTS: Optimum biotechnological processes include formation frequency (Hz) of 2350, formulation flow (ml/min) of 1.2, total gelation time (min) of 18 and cellular concurrent stream-integration rate (ml/min) of 0.7. In all formulations, LPBA consistently improved cell viability, insulin production, mitochondrial activities and ameliorated inflammation. CONCLUSION: The deployed biotechnological processes and LPBA optimised formation and functions of ß-cell microcapsules, which suggests potential applications in diabetes mellitus via the creation of more stable ß-cell microcapsules capable of delivering adequate levels of insulin to control glycaemia and potentially curing diabetes.

Alginate-combined cholic acid increased insulin secretion of microencapsulated mouse cloned pancreatic ß cells.

AIM: A semisynthetic primary bile acid (PBA) has exerted hypoglycemic effects in Type 1 diabetic animals, which were hypothesized to be due to its anti-inflammatory and cellular glucose-regulatory effects. Thus, the research purpose aimed to examine antidiabetic effects of a PBA, in terms of cellular inflammation and survival and insulin release, in the context of supporting ß-cell delivery and Type 1 diabetic treatment. MATERIALS & METHODS: 10 formulations were prepared, five without PBA (control) and five with PBA (test). Formulations were used to microencapsulate pancreatic ß cells and the microcapsules were examined for morphology, cell viability, insulin release and inflammation. RESULTS & CONCLUSION: PBA improved cell viability, insulin release and reduced inflammation in a formulation-dependent manner, which suggests potential use in cell delivery and diabetes treatment. [Formula: see text].

Designing anti-diabetic ß-cells microcapsules using polystyrenic sulfonate, polyallylamine, and a tertiary bile acid: Morphology, bioenergetics, and cytokine analysis.

PURPOSE: Recently sodium alginate (SA)-poly-l-ornithine (PLO) microcapsules containing pancreatic ß-cells that showed good morphology but low cell viability (<27%) was designed. In this study, two new polyelectrolytes, polystyrenic sulfonate (PSS; at 1%) and polyallylamine (PAA; at 2%) were incorporated into a microencapsulated-formulation, with the aim of enhancing the physical properties of the microcapsules. Following incorporation, the structural characteristics and cell viability were investigated. The effects of the anti-inflammatory bile acid, ursodeoxycholic acid (UDCA), on microcapsule morphology, size, and stability as well as ß-cell biological functionality was also examined. METHODS: Microcapsules were prepared using PLO-PSS-PAA-SA mixture and two types of microcapsules were produced: without UDCA (control) and with UDCA (test). Microcapsule morphology, stability, and size were examined. Cell count, microencapsulation efficiency, cell bioenergetics, and activity were also examined. RESULTS: The new microcapsules showed good morphology but cell viability remained low (29% ± 3%). UDCA addition improved cell viability post-microencapsulation (42 ± 5, P < 0.01), reduced swelling (P < 0.01), improved mechanical strength (P < 0.01), increased Zeta-potential (P < 0.01), and improved stability. UDCA addition also increased insulin production (P < 0.01), bioenergetics (P < 0.01), and decreased ß-cell TNF-α (P < 0.01), IFN-gamma (P < 0.01), and IL-6 (P < 0.01) secretions. CONCLUSIONS: Addition of 4% UDCA to a formulation system consisting of 1.8% SA, 1% PLO, 1% PSS, and 2% PAA enhanced cell viability post-microencapsulation and resulted in a more stable formulation with enhanced encapsulated ß-cell metabolism, bioenergetics, and biological activity with reduced inflammation. This suggests potential application of UDCA, when combined with SA, PLO, PSS, and PAA, in ß-cell microencapsulation and diabetes treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:501-509, 2016.

Glucose induces pancreatic islet cell apoptosis that requires the BH3-only proteins Bim and Puma and multi-BH domain protein Bax.

OBJECTIVE: High concentrations of circulating glucose are believed to contribute to defective insulin secretion and beta-cell function in diabetes and at least some of this effect appears to be caused by glucose-induced beta-cell apoptosis. In mammalian cells, apoptotic cell death is controlled by the interplay of proapoptotic and antiapoptotic members of the Bcl-2 family. We investigated the apoptotic pathway induced in mouse pancreatic islet cells after exposure to high concentrations of the reducing sugars ribose and glucose as a model of beta-cell death due to long-term metabolic stress. RESEARCH DESIGN AND METHODS: Islets isolated from mice lacking molecules implicated in cell death pathways were exposed to high concentrations of glucose or ribose. Apoptosis was measured by analysis of DNA fragmentation and release of mitochondrial cytochrome c. RESULTS: Deficiency of interleukin-1 receptors or Fas did not diminish apoptosis, making involvement of inflammatory cytokine receptor or death receptor signaling in glucose-induced apoptosis unlikely. In contrast, overexpression of the prosurvival protein Bcl-2 or deficiency of the apoptosis initiating BH3-only proteins Bim or Puma, or the downstream apoptosis effector Bax, markedly reduced glucose- or ribose-induced killing of islets. Loss of other BH3-only proteins Bid or Noxa, or the Bax-related effector Bak, had no impact on glucose-induced apoptosis. CONCLUSIONS: These results implicate the Bcl-2 regulated apoptotic pathway in glucose-induced islet cell killing and indicate points in the pathway at which interventional strategies can be designed.

Perturbations in nuclear factor-kappaB or c-Jun N-terminal kinase pathways in pancreatic beta cells confer susceptibility to cytokine-induced cell death.

Pro-inflammatory cytokines have been implicated in the death of pancreatic beta cells leading to type 1 diabetes. NIT-1 cells are an insulinoma cell line derived from mice expressing the SV40 large T antigen. These cells are a useful tool in analysis of beta cell death. NIT-1 cells are highly susceptible to caspase-dependent apoptosis induced by TNF-alpha alone. Primary islets are not susceptible to cell death induced by TNF-alpha alone; however, they are killed by TNF-alpha and IFN-gamma in a nitric oxide-dependent manner. We examined signal transduction in NIT-1 cells in response to cytokines to determine the mechanism for TNF-alpha-induced apoptosis. We found that NIT-1 cells are defective in the activation of nuclear factor-kappaB (NFkappaB) as a result of functionally deficient RelA activity, because overexpression of RelA protected NIT-1 cells from apoptosis. TNF-alpha also did not induce phosphorylation of c-Jun N-terminal kinase in NIT-1 cells. Together, these defects prevent expression of anti-apoptotic genes in NIT-1 cells and make them susceptible to TNF-alpha. To determine whether similar defects in primary beta cells would induce the same effect, we examined TNF-alpha-induced apoptosis in islets isolated from mice deficient in NFkappaB p50. These islets were as susceptible as wild-type islets to TNF-alpha and IFN-gamma-induced cell death. In contrast to wild-type islets, cell death was not prevented by inhibition of nitric oxide in p50-deficient islets. Blocking NFkappaB has been proposed as a mechanism for protection of beta cells from cytokine-induced cell death in vivo. Our results suggest that this would make beta cells equally or more sensitive to cytokines.

Suppressor of cytokine signaling-1 in T cells and macrophages is critical for preventing lethal inflammation.

The balance between pro- and anti-inflammatory cytokines modulates inflammation. Intracellular inhibitors of signaling, in turn, contribute to the negative regulation of cytokines. One of these inhibitors is suppressor of cytokine signaling-1 (SOCS-1). Socs1(-/-) mice die by 3 weeks of age with inflammation and fatty necrosis of the liver. Here, cre/loxP deletion of Socs1 was used to investigate the contribution of specific cells/tissues to inflammatory disease. Mice with SOCS-1 deficiency in myeloid and lymphoid cells, but not lymphoid alone, became ill at 50 to 250 days of age. These mice developed splenomegaly and T-cell/macrophage infiltration of many organs, including liver, lung, pancreas, and muscle. There were also abnormally high levels of the proinflammatory cytokines interferon gamma (IFN-gamma), tumor necrosis factor (TNF), and interleukin-12 (IL-12), and activated T cells circulating in these mice. Socs1(null) T cells were found to be hypersensitive to multiple cytokines, including IL-1, IL-2, and IL-12, resulting in IFN-gamma production without requiring T-cell receptor (TCR) ligation. Additionally, Socs1(null) macrophages produced excessive amounts of IL-12 and TNF in response to other cytokines, including IFN-gamma. A dysregulated cytokine network between T cells and macrophages is thus associated with this inflammatory disease. These findings indicate that SOCS-1 is critical in both T cells and macrophages for preventing uncontrolled inflammation.

Socs1 deficiency enhances hepatic insulin signaling.

Suppressor of cytokine signaling 1 (SOCS1) is an intracellular inhibitor of cytokine, growth factor, and hormone signaling. Socs1-/- mice die before weaning from a multiorgan inflammatory disease. Neonatal Socs1-/- mice display severe hypoglycemia and hypoinsulinemia. Concurrent interferon gamma gene deletion (Ifng-/-) prevented inflammation and corrected the hypoglycemia. In hyperinsulinemic clamp studies, however, Socs1-/- Ifng-/- mice had enhanced hepatic insulin sensitivity demonstrated by greater suppression of endogenous glucose production compared with controls with no difference in glucose disposal. Socs1-/- Ifng-/- mice had elevated liver insulin receptor substrate 2 expression (IRS-2) and IRS-2 tyrosine phosphorylation. This was associated with lower phosphoenolpyruvate carboxykinase mRNA expression. These effects were not associated with elevated hepatic AMP-activated protein kinase activity. Hepatic insulin sensitivity and IRS-2 levels play central roles in the pathogenesis of type 2 diabetes. Socs1 deficiency increases IRS-2 expression and enhances hepatic insulin sensitivity in vivo indicating that inhibition of SOCS1 may be a logical strategy in type 2 diabetes.