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1.
Diabetes Metab J ; 45(5): 629-640, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34610718

RESUMO

The year 2021 marks the 100th anniversary of the discovery of insulin, which has greatly changed the lives of people with diabetes and become a cornerstone of advances in medical science. A rapid bench-to-bedside application of the lifesaving pancreatic extract and its immediate commercialization was the result of a promising idea, positive drive, perseverance, and collaboration of Banting and colleagues. As one of the very few proteins isolated in a pure form at that time, insulin also played a key role in the development of important methodologies and in the beginning of various fields of modern science. Since its discovery, insulin has evolved continuously to optimize the care of people with diabetes. Since the 1980s, recombinant DNA technology has been employed to engineer insulin analogs by modifying their amino acid sequence, which has resulted in the production of insulins with various profiles that are currently used. However, unmet needs in insulin treatment still exist, and several forms of future insulins are under development. In this review, we discuss the past, present, and future of insulin, including a history of ceaseless innovations and collective intelligence. We believe that this story will be a solid foundation and an unerring guide for the future.


Assuntos
Diabetes Mellitus , Insulinas , Sequência de Aminoácidos , Diabetes Mellitus/tratamento farmacológico , Humanos , Insulina/uso terapêutico , Insulina Regular Humana
2.
Nat Commun ; 12(1): 6185, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34702819

RESUMO

The circadian system cyclically regulates many physiological and behavioral processes within the day. Desynchronization between physiological and behavioral rhythms increases the risk of developing some, including metabolic, disorders. Here we investigate how the oscillatory nature of metabolic signals, resembling feeding-fasting cycles, sustains the cell-autonomous clock in peripheral tissues. By controlling the timing, period and frequency of glucose and insulin signals via microfluidics, we find a strong effect on Per2::Luc fibroblasts entrainment. We show that the circadian Per2 expression is better sustained via a 24 h period and 12 h:12 h frequency-encoded metabolic stimulation applied for 3 daily cycles, aligned to the cell-autonomous clock, entraining the expression of hundreds of genes mostly belonging to circadian rhythms and cell cycle pathways. On the contrary misaligned feeding-fasting cycles synchronize and amplify the expression of extracellular matrix-associated genes, aligned during the light phase. This study underlines the role of the synchronicity between life-style-associated metabolic signals and peripheral clocks on the circadian entrainment.


Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/genética , Comportamento Alimentar/fisiologia , Animais , Ciclo Celular/genética , Linhagem Celular , Relógios Circadianos/genética , Meios de Cultura/metabolismo , Matriz Extracelular/genética , Jejum/fisiologia , Glucose/metabolismo , Insulinas/metabolismo , Dispositivos Lab-On-A-Chip , Camundongos , Proteínas Circadianas Period/genética , Transcriptoma
5.
Theriogenology ; 175: 100-110, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34534687

RESUMO

The role of anti-Müllerian hormone (AMH) and insulin-like peptide 3 (INSL3) in male infertility is not fully understood. We used the downregulated testis as a model of gonadotropin-dependent infertility. Serum testosterone and AMH concentrations were studied in five adult male Beagles implanted (day 0) with 4.7 mg deslorelin (Suprelorin®, Virbac) (DES group). Testicular expression of LH receptor (LHR) and androgen receptor (AR), AMH, type 2 AMH receptor (AMHR2), INSL3 and its receptor (RXFP2) was evaluated 112 days (16 weeks) after deslorelin treatment by qPCR and immunohistochemistry, and compared to untreated adult (CON, n = 6) and prepubertal (PRE, n = 8) dogs. Serum testosterone concentration decreased significantly by the onset of aspermia on study day 14 (four dogs) or day 21 (one dog), and was baseline on day 105 (week 15). In contrast, serum AMH started to increase only after the onset of aspermia and reached the maximum detectable concentration of the assay by day 49-105 in individual dogs. Testicular LHR gene expression in DES was lower than in CON and PRE (P < 0.0001), while AR gene expression in DES was similar to CON and significantly higher than PRE (P < 0.0001). Testicular AMH expression in DES was intermediate compared to the lowest mRNA levels found in CON and the highest in PRE (P ≤ 0.006). AMHR2 gene expression was similar between groups. AMH protein was detected in Sertoli cells only, while AMHR2 immunoreactivity was principally detected in Leydig cells which appeared to be increased in DES. INSL3 and RXFP2 gene expression was significantly downregulated in the DES testis along with noticeably weak Leydig cell immunosignals compared to CON. In conclusion, deslorelin treatment caused testicular LH insensitivity without affecting androgen sensitivity, and de-differentiation of Sertoli and Leydig cells. In DES, upregulation of the AMH-AMHR2 feed-back loop and downregulation of the INSL3-RXFP2 feed-forward loop are paracrine-autocrine mechanisms that may additionally regulate testosterone production independent of gonadotropins. Our results support AMH and INSL3 as unique biomarkers and paracrine-autocrine regulators of testis function involved in the intimate interplay between Sertoli and Leydig cells.


Assuntos
Hormônio Antimülleriano , Insulina , Insulinas , Células Intersticiais do Testículo , Proteínas , Testículo/efeitos dos fármacos , Testosterona , Animais , Hormônio Antimülleriano/genética , Hormônio Antimülleriano/metabolismo , Biomarcadores , Cães , Regulação para Baixo , Insulina/genética , Insulina/metabolismo , Células Intersticiais do Testículo/metabolismo , Masculino , Peptídeos , Proteínas/genética , Proteínas/metabolismo , Testículo/metabolismo , Pamoato de Triptorrelina/análogos & derivados
6.
J Dairy Sci ; 104(12): 12616-12627, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34538499

RESUMO

Our objective was to determine whether abomasal infusions of increasing doses of oleic acid (cis-9 C18:1; OA) improved fatty acid (FA) digestibility and milk production of lactating dairy cows. Eight rumen-cannulated multiparous Holstein cows (138 d in milk ± 52) were randomly assigned to treatment sequence in a replicated 4 × 4 Latin square design with 18-d periods consisting of 7 d of washout and 11 d of infusion. Production and digestibility data were collected during the last 4 d of each infusion period. Treatments were 0, 20, 40, or 60 g/d of OA. We dissolved OA in ethanol before infusions. The infusate solution was divided into 4 equal infusions per day, occurring every 6 h, delivering the daily cis-9 C18:1 for each treatment. Animals received the same diet throughout the study, which contained (percent diet dry matter) 28% neutral detergent fiber, 17% crude protein, 27% starch, and 3.3% FA (including 1.8% FA from a saturated FA supplement containing 32% C16:0 and 52% C18:0). Infusion of OA did not affect intake or digestibility of dry matter and neutral detergent fiber. Increasing OA from 0 to 60 g/d linearly increased the digestibility of total FA (8.40 percentage units), 16-carbon FA (8.30 percentage units), and 18-carbon FA (8.60 percentage units). Therefore, increasing OA linearly increased absorbed total FA (162 g/d), 16-carbon FA (26.0 g/d), and 18-carbon FA (127 g/d). Increasing OA linearly increased milk yield (4.30 kg/d), milk fat yield (0.10 kg/d), milk lactose yield (0.22 kg/d), 3.5% fat-corrected milk (3.90 kg/d), and energy-corrected milk (3.70 kg/d) and tended to increase milk protein yield. Increasing OA did not affect the yield of mixed milk FA but increased yield of preformed milk FA (65.0 g/d) and tended to increase the yield of de novo milk FA. Increasing OA quadratically increased plasma insulin concentration with an increase of 0.18 µg/L at 40 g/d OA, and linearly increased the content of cis-9 C18:1 in plasma triglycerides by 2.82 g/100 g. In conclusion, OA infusion increased FA digestibility and absorption, milk fat yield, and circulating insulin without negatively affecting dry matter intake. In our short-term infusion study, most of the digestion and production measurements responded linearly, indicating that 60 g/d OA was the best dose. Because a quadratic response was not observed, improvements in FA digestibility and production might continue with higher doses of OA, which deserves further attention.


Assuntos
Ácidos Graxos , Insulinas , Ração Animal/análise , Animais , Bovinos , Dieta/veterinária , Suplementos Nutricionais , Digestão , Feminino , Lactação , Ácido Oleico , Ácido Palmítico
7.
Elife ; 102021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34403334

RESUMO

To investigate the role of the vasculature in pancreatic ß-cell regeneration, we crossed a zebrafish ß-cell ablation model into the avascular npas4l mutant (i.e. cloche). Surprisingly, ß-cell regeneration increased markedly in npas4l mutants owing to the ectopic differentiation of ß-cells in the mesenchyme, a phenotype not previously reported in any models. The ectopic ß-cells expressed endocrine markers of pancreatic ß-cells, and also responded to glucose with increased calcium influx. Through lineage tracing, we determined that the vast majority of these ectopic ß-cells has a mesodermal origin. Notably, ectopic ß-cells were found in npas4l mutants as well as following knockdown of the endothelial/myeloid determinant Etsrp. Together, these data indicate that under the perturbation of endothelial/myeloid specification, mesodermal cells possess a remarkable plasticity enabling them to form ß-cells, which are normally endodermal in origin. Understanding the restriction of this differentiation plasticity will help exploit an alternative source for ß-cell regeneration.


Assuntos
Diferenciação Celular , Células Secretoras de Insulina/fisiologia , Mesoderma/embriologia , Regeneração , Peixe-Zebra/embriologia , Animais , Endotélio/fisiologia , Insulinas/metabolismo , Peixe-Zebra/fisiologia
8.
Sci Rep ; 11(1): 17395, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34462500

RESUMO

Prolonged periods of forced social isolation is detrimental to well-being, yet we know little about which genes regulate susceptibility to its effects. In the fruit fly, Drosophila melanogaster, social isolation induces stark changes in behavior including increased aggression, locomotor activity, and resistance to ethanol sedation. To identify genes regulating sensitivity to isolation, I screened a collection of sixteen hundred P-element insertion lines for mutants with abnormal levels of all three isolation-induced behaviors. The screen identified three mutants whose affected genes are likely central to regulating the effects of isolation in flies. One mutant, sex pistol (sxp), became extremely aggressive and resistant to ethanol sedation when socially isolated. sxp also had a high level of male-male courtship. The mutation in sxp reduced the expression of two minor isoforms of the actin regulator hts (adducin), as well as mildly reducing expression of CalpA, a calcium-dependent protease. As a consequence, sxp also had increased expression of the insulin-like peptide, dILP5. Analysis of the social behavior of sxp suggests that these minor hts isoforms function to limit isolation-induced aggression, while chronically high levels of dILP5 increase male-male courtship.


Assuntos
Proteínas de Drosophila/genética , Drosophila/fisiologia , Isolamento Social , Agressão , Animais , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Calpaína/genética , Calpaína/metabolismo , Corte , Regulação para Baixo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Insulinas/genética , Insulinas/metabolismo , Locomoção , Masculino , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Regulação para Cima
9.
Science ; 373(6554): 522-527, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326234

RESUMO

Despite innovations in insulin therapy since its discovery, most patients living with type 1 diabetes do not achieve sufficient glycemic control to prevent complications, and they experience hypoglycemia, weight gain, and major self-care burden. Promising pharmacological advances in insulin therapy include the refinement of extremely rapid insulin analogs, alternate insulin-delivery routes, liver-selective insulins, add-on drugs that enhance insulin effect, and glucose-responsive insulin molecules. The greatest future impact will come from combining these pharmacological solutions with existing automated insulin delivery methods that integrate insulin pumps and glucose sensors. These systems will use algorithms enhanced by machine learning, supplemented by technologies that include activity monitors and sensors for other key metabolites such as ketones. The future challenges facing clinicians and researchers will be those of access and broad clinical implementation.


Assuntos
Automonitorização da Glicemia , Glicemia/análise , Diabetes Mellitus Tipo 1/tratamento farmacológico , Controle Glicêmico , Hipoglicemiantes/uso terapêutico , Insulinas/uso terapêutico , Absorção Fisiológica , Algoritmos , Automação , Diabetes Mellitus Tipo 1/sangue , Humanos , Sistemas de Infusão de Insulina , Insulinas/administração & dosagem , Insulinas/sangue , Insulinas/farmacocinética , Refeições
10.
J Dairy Sci ; 104(10): 11306-11316, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34275626

RESUMO

Trans-10,cis-12 conjugated linoleic acid (t10,c12 CLA) is well recognized as a key CLA isomer responsible for the reduction in milk fat synthesis that leads to milk fat depression in dairy cows. Sterol regulatory element binding protein-1 (SREBP1) is a key transcription factor in bovine mammary gland coordinating transcription of the genes for fatty acid synthesis. SREBP1 activation requires the removal of insulin-induced gene-1 (Insig1) that serves as a repressor of SREBP1 in the endoplasmic reticulum (ER). We hypothesized that t10,c12 CLA reduced SREBP1 activation by delaying Insig1 degradation. In the present study, we used undifferentiated bovine mammary epithelial cells (MAC-T cells) and treated them with t10,c12 CLA for 6 h. We found that SREBP1 protein expression declined over 56% when cells were treated with 60 µM or greater concentration of t10,c12 CLA. Such inhibitory effects were also observed in the mRNA expression of SREBP1-regulated genes including SREBP1, fatty acid synthetase, stearoyl-CoA desaturase, and Insig1. Compared with no CLA group, 60 µM or higher concentration of t10,c12 CLA increased Insig1 protein expression over 2-fold in cells transfected with FLAG-tagged Insig1. This stimulatory effect was not specific to t10,c12 CLA but also other polyunsaturated fatty acids including cis-9,trans-11 CLA and linoleic acid. Oleic acid had no effect on Insig1 protein expression, whereas palmitic acid decreased Insig1 protein expression. Further investigation revealed that increased abundance of FLAG-Insig1 with t10,c12 CLA was due to the inhibition of the proteasomal degradation of Insig1. The t10,c12 CLA delayed the Insig1 decay when protein synthesis was blocked. Immunoprecipitation also confirmed that the interaction between ubiquitin-like domain-containing protein 8 and Insig1, the key step of removing Insig1 from ER and freeing SREBP1 for proteolytic processing, was inhibited by t10,c12 CLA, but not palmitic acid. These findings suggested that t10,c12 CLA played a role in regulating SREBP1 activation by reducing proteasomal degradation of Insig1. We concluded that stabilized Insig1 retained SREBP1 in the ER from activation, thus reducing lipogenic gene transcription.


Assuntos
Insulinas , Ácidos Linoleicos Conjugados , Animais , Bovinos , Células Epiteliais , Ácidos Graxos , Feminino , Ácidos Linoleicos Conjugados/farmacologia , Glândulas Mamárias Animais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética
13.
Obes Res Clin Pract ; 15(4): 362-367, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34148808

RESUMO

BACKGROUND: The present study aimed to evaluate whether mothers with obesity/central obesity and metabolic syndrome before gestation are at higher risk of insulin administration in gestational diabetes mellitus (GDM) to diminish the burden of insulin use during pregnancy. METHODS: This was a population-based retrospective cohort study conducted using data from the National Health Information Database of Korea. We identified all deliveries from January 1, 2011 to December 31, 2015 (N = 1,214,655). Among the deliveries, we identified mothers with pre-pregnancy health checkup records and without previous diabetes history (N = 325,208). Hazards of insulin use in GDM were calculated based on pre-pregnancy obesity/central obesity and metabolic syndrome. RESULTS: Hazards of insulin use in GDM increased proportionately with an increase in the pre-pregnancy body mass index (BMI) and waist circumference (WC). After the adjustment for clinical factors, high BMI group (≥30 kg/m2) and high WC group (≥100 cm) were significantly associated with higher hazard ratios (HRs) (HR 4.161, 95% Confidence interval [CI] 3.381-5.121, P < 0.001 and HR 2.563, 95% CI 1.769-3.712, P < 0.001, respectively). The presence of pre-pregnancy metabolic syndrome significantly increased the hazard of insulin use in GDM (0.54% vs. 5.04%). In the presence of obesity (BMI ≥ 25 kg/m2) or central obesity (WC ≥ 85 cm), HRs of insulin use in GDM were 2.637 (95% CI 2.275-3.056) and 1.603 (95% CI 1.023-2.511), respectively, after adjustment for clinical factors. CONCLUSIONS: The presence of pre-pregnancy obesity/central obesity and metabolic syndrome in Korean mothers is associated with increased risk of insulin use in GDM.


Assuntos
Diabetes Gestacional , Insulinas , Síndrome Metabólica , Índice de Massa Corporal , Estudos de Coortes , Diabetes Gestacional/epidemiologia , Feminino , Humanos , Síndrome Metabólica/induzido quimicamente , Síndrome Metabólica/epidemiologia , Gravidez , Estudos Retrospectivos , Fatores de Risco
14.
Circ Heart Fail ; 14(6): e008075, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34129365

RESUMO

BACKGROUND: Patients with heart failure with reduced ejection fraction (HFrEF) and insulin-treated diabetes have a high risk of cardiovascular complications. Mineralocorticoid receptor antagonists may mitigate this risk. We aim to explore the effect of eplerenone on cardiovascular outcomes and all-cause mortality in HFrEF patients with diabetes, including those treated with insulin in the EMPHASIS-HF trial (Eplerenone in Patients with Systolic Heart Failure and Mild Symptoms). METHODS: The primary outcome was the composite of heart failure hospitalization or cardiovascular death. Cox models with treatment-by-diabetes subgroup interaction terms were used. RESULTS: The median follow-up was 21 (10-33) months. Of the 2737 patients included, 623 (23%) had non-insulin-treated diabetes, 236 (9%) had insulin-treated diabetes and 1878 did not have diabetes. Patients with insulin-treated diabetes were younger, more often women, with higher body mass index, waist circumference, more frequent ischemic heart failure cause, impaired kidney function, and longer diabetes duration. Compared with patients without diabetes, those with insulin-treated diabetes had a 2-fold higher risk of having a primary outcome event. The hazard ratio (95% CI) for the effect of eplerenone, compared with placebo, on the primary outcome was 0.31 (0.19-0.50) in insulin-treated diabetes, 0.69 (0.50-0.93) in non-insulin-treated diabetes, and 0.72 (0.58-0.88) in patients without diabetes; interaction P=0.007. The annualized number needed-to-treat-to-benefit with regards to the primary outcome was 3 (95% CI, 3-4) in patients with insulin-treated diabetes, 16 (13-19) in patients with diabetes not receiving insulin, and 26 (24-28) in patients without diabetes. CONCLUSIONS: Patients with insulin-treated diabetes experienced a greater benefit from eplerenone than those with diabetes not treated with insulin and people without diabetes. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00232180.


Assuntos
Eplerenona/farmacologia , Insuficiência Cardíaca Sistólica/tratamento farmacológico , Insuficiência Cardíaca/tratamento farmacológico , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Idoso , Diabetes Mellitus/tratamento farmacológico , Feminino , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca Sistólica/fisiopatologia , Humanos , Insulinas/uso terapêutico , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Espironolactona/uso terapêutico , Resultado do Tratamento
15.
Sci Transl Med ; 13(596)2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078744

RESUMO

Transplantation of stem cell-derived ß (SC-ß) cells represents a promising therapy for type 1 diabetes (T1D). However, the delivery, maintenance, and retrieval of these cells remain a challenge. Here, we report the design of a safe and functional device composed of a highly porous, durable nanofibrous skin and an immunoprotective hydrogel core. The device consists of electrospun medical-grade thermoplastic silicone-polycarbonate-urethane and is soft but tough (~15 megapascal at a rupture strain of >2). Tuning the nanofiber size to less than ~500 nanometers prevented cell penetration while maintaining maximum mass transfer and decreased cellular overgrowth on blank (cell-free) devices to as low as a single-cell layer (~3 micrometers thick) when implanted in the peritoneal cavity of mice. We confirmed device safety, indicated as continuous containment of proliferative cells within the device for 5 months. Encapsulating syngeneic, allogeneic, or xenogeneic rodent islets within the device corrected chemically induced diabetes in mice and cells remained functional for up to 200 days. The function of human SC-ß cells was supported by the device, and it reversed diabetes within 1 week of implantation in immunodeficient and immunocompetent mice, for up to 120 and 60 days, respectively. We demonstrated the scalability and retrievability of the device in dogs and observed viable human SC-ß cells despite xenogeneic immune responses. The nanofibrous device design may therefore provide a translatable solution to the balance between safety and functionality in developing stem cell-based therapies for T1D.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Insulinas , Transplante das Ilhotas Pancreáticas , Nanofibras , Animais , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Cães , Insulina , Camundongos
17.
J Trace Elem Med Biol ; 67: 126785, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34015661

RESUMO

BACKGROUND & AIMS: Pervious epidemiological evidence on the associations of selenium, zinc with lipid profile and glycemic indices was contradictory. The aim of this study was to investigate whether selenium and zinc were casually associated with lipid profile and glycemic indices using mendelian randomization (MR) analysis. METHOD: A two-sample MR was used to evaluate the causal-effect estimations. Summary statistics for selenium, zinc, lipids and glycemic indices were retrieved from previous large-scale genome-wide association study (GWAS). Single nucleotide polymorphisms (SNPs) that independently and strongly associated with the selenium and zinc were selected as the instrumental variables. The casual estimates were calculated using inverse variance weighted method (IVW), with weighted median, MR-Egger, and MR-PRESSO test as sensitivity analysis, respectively. RESULTS: In the standard IVW analysis, per SD increment in selenium was associated with an 0.077 mmol/L decrease of TC (95 %CI: -0.102,-0.052) and 0.074 mmol/L of LDL-C (95 %CI: -0.1,-0.048). Suggestive casual associations were found between selenium and insulin or HbA1c. With IVW method, per SD increase in selenium was associated with an 0.023 mmol/L increase of insulin (95 %CI: 0.001,0.045), and an 0.013 mmol/L increase of HbA1c (95 %CI: 0.003,0.023). The results were robust in the sensitivity analysis. Zinc was not casually associated with any of lipid and glycemic markers. CONCLUSION: Our MR analysis provides evidence of the potential causal effect of Se on beneficial lipid profile, including decreased TC and LDL-C. Furthermore, suggestive casual evidence was suggested between Se and increased serum HbA1c levels. Careful consideration is required for the protective effects of Se supplementation. No casual-effect association was found between Zn and any indices of the lipid and glucose parameters.


Assuntos
Selênio/sangue , Colesterol , LDL-Colesterol , Estudo de Associação Genômica Ampla , Hemoglobina A Glicada , Insulinas , Lipídeos , Zinco
18.
Mater Sci Eng C Mater Biol Appl ; 123: 112009, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33812628

RESUMO

Different bioinks have been used to produce cell-laden alginate-based hydrogel constructs for cell replacement therapy but some of these approaches suffer from issues with print quality, long-term mechanical instability, and bioincompatibility. In this study, new alginate-based bioinks were developed to produce cell-laden grid-shaped hydrogel constructs with stable integrity and immunomodulating capacity. Integrity and printability were improved by including the co-block-polymer Pluronic F127 in alginate solutions. To reduce inflammatory responses, pectin with a low degree of methylation was included and tested for inhibition of Toll-Like Receptor 2/1 (TLR2/1) dimerization and activation and tissue responses under the skin of mice. The viscoelastic properties of alginate-Pluronic constructs were unaffected by pectin incorporation. The tested pectin protected printed insulin-producing MIN6 cells from inflammatory stress as evidenced by higher numbers of surviving cells within the pectin-containing construct following exposure to a cocktail of the pro-inflammatory cytokines namely, IL-1ß, IFN-γ, and TNF-α. The results suggested that the cell-laden construct bioprinted with pectin-alginate-Pluronic bioink reduced tissue responses via inhibiting TLR2/1 and support insulin-producing ß-cell survival under inflammatory stress. Our study provides a potential novel strategy to improve long-term survival of pancreatic islet grafts for Type 1 Diabetes (T1D) treatment.


Assuntos
Bioimpressão , Insulinas , Alginatos , Animais , Camundongos , Pectinas , Impressão Tridimensional , Engenharia Tecidual , Tecidos Suporte
19.
Biosensors (Basel) ; 11(5)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924867

RESUMO

Organ-on-a-chip (OOC) devices offer new approaches for metabolic disease modeling and drug discovery by providing biologically relevant models of tissues and organs in vitro with a high degree of control over experimental variables for high-content screening applications. Yet, to fully exploit the potential of these platforms, there is a need to interface them with integrated non-labeled sensing modules, capable of monitoring, in situ, their biochemical response to external stimuli, such as stress or drugs. In order to meet this need, we aim here to develop an integrated technology based on coupling a localized surface plasmon resonance (LSPR) sensing module to an OOC device to monitor the insulin in situ secretion in pancreatic islets, a key physiological event that is usually perturbed in metabolic diseases such as type 2 diabetes (T2D). As a proof of concept, we developed a biomimetic islet-on-a-chip (IOC) device composed of mouse pancreatic islets hosted in a cellulose-based scaffold as a novel approach. The IOC was interfaced with a state-of-the-art on-chip LSPR sensing platform to monitor the in situ insulin secretion. The developed platform offers a powerful tool to enable the in situ response study of microtissues to external stimuli for applications such as a drug-screening platform for human models, bypassing animal testing.


Assuntos
Técnicas Biossensoriais , Secreção de Insulina , Animais , Diabetes Mellitus Tipo 2 , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Humanos , Insulinas , Dispositivos Lab-On-A-Chip , Análise de Sequência com Séries de Oligonucleotídeos , Ressonância de Plasmônio de Superfície
20.
Redox Biol ; 43: 101962, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33892285

RESUMO

Peroxiporins are distinct aquaporins (AQP) which, beside water, also facilitate the bidirectional transport of hydrogen peroxide (H2O2) across cellular membranes. H2O2 serves as the major reactive oxygen species that mediates essential cell signaling events. In pancreatic ß-cells, H2O2 has been associated with the regulation of cell growth but in excess it leads to failure of insulin secretion, making it important for diabetes mellitus (DM) pathogenesis. In the present study, the role of aquaporin-8 (AQP8) as a peroxiporin was investigated in RINm5F cells. The role of AQP8 was studied in an insulin-producing cell model, on the basis of stable AQP8 overexpression (AQP8↑) and CRISPR/Cas9-mediated AQP8 knockdown (KD). A complete AQP8 knock-out was found to result in cell death, however we demonstrate that mild lentiviral re-expression through a Tet-On-regulated genetically modified AQP8 leads to cell survival, enabling functional characterization. Proliferation and insulin content were found to be increased in AQP8↑ cells underlining the importance of AQP8 in the regulation of H2O2 homeostasis in pancreatic ß-cells. Colocalization analyses of V5-tagged AQP8 proteins based on confocal microscopic imaging revealed its membrane targeting to both the mitochondria and the plasma membrane, but not to the ER, the Golgi apparatus, insulin vesicles, or peroxisomes. By using the fluorescence H2O2 specific biosensor HyPer together with endogenous generation of H2O2 using d-amino acid oxidase, live cell imaging revealed enhanced H2O2 flux to the same subcellular regions in AQP8 overexpressing cells pointing to its importance in the development of type-1 DM. Moreover, the novel ultrasensitive H2O2 sensor HyPer7.2 clearly unveiled AQP8 as a H2O2 transporter in RINm5F cells. In summary, these studies establish that AQP8 is an important H2O2 pore in insulin-producing RINm5F cells involved in the transport of H2O2 through the mitochondria and cell membrane and may help to explain the H2O2 transport and toxicity in pancreatic ß-cells.


Assuntos
Aquaporinas , Insulinas , Animais , Membrana Celular/metabolismo , Peróxido de Hidrogênio/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo
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