RESUMO
Parathyroid hormone-related protein (PTHrP) is a pleiotropic hormone essential for morphogenesis, tissue differentiation, as well as cell regulation and function. PTHrP is expressed by pancreatic beta cells which are responsible for insulin secretion. Previous studies have reported that N-terminal PTHrP stimulated proliferation in beta cells in rodents. We have developed a knockin mouse model (PTHrP Δ/Δ) lacking the C-terminal and nuclear localization sequence (NLS) of PTHrP. These mice die at â¼day 5, are severely stunted in growth, weigh 54% less than control mice at day 1-2 and eventually fail to grow. PTHrP Δ/Δ mice are also hypoinsulinemic and hypoglycemic yet have nutrient intake proportional to size. To characterize the pancreatic islets in these mice, islets (â¼10-20) were isolated from 2 to 5 day-old-mice using collagenase digestion. Islets from PTHrP Δ/Δ mice were smaller in size but secreted more insulin than littermate controls. PTHrP Δ/Δ and control mice islets were exposed to various glucose concentrations and intracellular calcium, the trigger for insulin release, was elevated for glucose concentrations of 8-20 mM. Immunofluorescence staining showed less glucagon-stained area in islets from PTHrP Δ/Δ mice (â¼250 µm2) compared to islets from control mice (â¼900 µm2), and ELISA confirmed there was reduced glucagon content. These data collectively demonstrate increased insulin secretion and reduced glucagon at the islet level, which may contribute to the observed hypoglycemia and early death in PTHrP Δ/Δ mice. Thus, the C-terminus and NLS of PTHrP are crucial to life, including regulation of glucose homeostasis and islet function.
Assuntos
Ilhotas Pancreáticas , Proteína Relacionada ao Hormônio Paratireóideo , Animais , Camundongos , Glucagon , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismoRESUMO
Obesity-related insulin resistance is a highly prevalent and growing health concern, which places stress on the pancreatic islets of Langerhans by increasing insulin secretion to lower blood glucose levels. The glucose transporters GLUT1 and GLUT3 play a key role in glucose-stimulated insulin secretion in human islets, while GLUT2 is the key isoform in rodent islets. However, it is unclear whether other glucose transporters also contribute to insulin secretion by pancreatic islets. Herein, we show that SLC2A6 (GLUT6) is markedly upregulated in pancreatic islets from genetically obese leptin-mutant (ob/ob) and leptin receptor-mutant (db/db) mice, compared to lean controls. Furthermore, we observe that islet SLC2A6 expression positively correlates with body mass index in human patients with type 2 diabetes. To investigate whether GLUT6 plays a functional role in islets, we crossed GLUT6 knockout mice with C57BL/6 ob/ob mice. Pancreatic islets isolated from ob/ob mice lacking GLUT6 secreted more insulin in response to high-dose glucose, compared to ob/ob mice that were wild type for GLUT6. The loss of GLUT6 in ob/ob mice had no adverse impact on body mass, body composition, or glucose tolerance at a whole-body level. This study demonstrates that GLUT6 plays a role in pancreatic islet insulin secretion in vitro but is not a dominant glucose transporter that alters whole-body metabolic physiology in ob/ob mice.
Assuntos
Diabetes Mellitus Tipo 2 , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Obesidade/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos ObesosRESUMO
In the endocrine pancreas, growth hormone (GH) is known to promote pancreatic islet growth and insulin secretion. In this study, we show that GH receptor (GHR) loss in the germline and in adulthood impacts islet mass in general but more profoundly in male mice. GHR knockout (GHRKO) mice have enhanced insulin sensitivity and low circulating insulin. We show that the total cross-sectional area of isolated islets (estimated islet mass) was reduced by 72% in male but by only 29% in female GHRKO mice compared with wild-type controls. Also, islets from GHRKO mice secreted â¼50% less glucose-stimulated insulin compared with size-matched islets from wild-type mice. We next used mice with a floxed Ghr gene to knock down the GHR in adult mice at 6 mo of age (6mGHRKO) and examined the impact on glucose and islet metabolism. By 12 mo of age, female 6mGHRKO mice had increased body fat and reduced islet mass but had no change in glucose tolerance or insulin sensitivity. However, male 6mGHRKO mice had nearly twice as much body fat, substantially reduced islet mass, and enhanced insulin sensitivity, but no change in glucose tolerance. Despite large losses in islet mass, glucose-stimulated insulin secretion from isolated islets was not significantly different between male 6mGHRKO and controls, whereas isolated islets from female 6mGHRKO mice showed increased glucose-stimulated insulin release. Our findings demonstrate the importance of GH to islet mass throughout life and that unique sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose metabolism.NEW & NOTEWORTHY Growth hormone (GH) is important for more than just growth. GH helps to maintain pancreatic islet mass and insulin secretion throughout life. Sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose regulation despite losing islet mass.
Assuntos
Células Germinativas/metabolismo , Hormônio do Crescimento/deficiência , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/fisiologia , Receptores da Somatotropina/genética , Fatores Etários , Animais , Proliferação de Células/genética , Feminino , Células Germinativas/fisiologia , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão/genética , Receptores da Somatotropina/deficiência , Receptores da Somatotropina/metabolismo , Caracteres Sexuais , Transdução de Sinais/genéticaRESUMO
Insufficient insulin secretion is a key component of both type 1 and type 2 diabetes. Since insulin is released by the islets of Langerhans, obtaining viable and functional islets is critical for research and transplantation. The effective and efficient isolation of these small islands of endocrine cells from the sea of exocrine tissue that is the rest of the pancreas is not necessarily simple or quick. Choosing and administering the digestive enzyme, separation of the islets from acinar tissue, and culture of islets are all things that must be considered. The purpose of this review is to provide a history of the development of islet isolation procedures and to serve as a practical guide to rodent islet research for newcomers to islet biology. We discuss key elements of mouse islet isolation including choosing collagenase, the digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews techniques for assessing islet viability and function such as visual assessment, glucose-stimulated insulin secretion and intracellular calcium measurements. A detailed protocol is provided that describes a common method our laboratory uses to obtain viable and functional mouse islets for in vitro study. This review thus provides a strong foundation for successful procurement and purification of high-quality mouse islets for research purposes.
RESUMO
Insulin pulsatility is important to hepatic response in regulating blood glucose. Growing evidence suggests that insulin-secreting pancreatic ß-cells can adapt to chronic disruptions of pulsatility to rescue this physiologically important behavior. We determined the time scale for adaptation and examined potential ion channels underlying it. We induced the adaptation both by chronic application of the ATP-sensitive K+ [K(ATP)] channel blocker tolbutamide and by application of the depolarizing agent potassium chloride (KCl). Acute application of tolbutamide without pretreatment results in elevated Ca2+ as measured by fura-2AM and the loss of endogenous pulsatility. We show that after chronic exposure to tolbutamide (12-24 h), Ca2+ oscillations occur with subsequent acute tolbutamide application. The same experiment was conducted with potassium chloride (KCl) to directly depolarize the ß-cells. Once again, following chronic exposure to the cell stimulator, the islets produced Ca2+ oscillations when subsequently exposed to tolbutamide. These experiments suggest that it is the chronic stimulation, and not tolbutamide desensitization, that is responsible for the adaptation that rescues oscillatory ß-cell activity. This compensatory response also causes islet glucose sensitivity to shift rightward following chronic tolbutamide treatment. Mathematical modeling shows that a small increase in the number of K(ATP) channels in the membrane is one adaptation mechanism that is compatible with the data. To examine other compensatory mechanisms, pharmacological studies provide support that Kir2.1 and TEA-sensitive channels play some role. Overall, this investigation demonstrates ß-cell adaptability to overstimulation, which is likely an important mechanism for maintaining glucose homeostasis in the face of chronic stimulation.
Assuntos
Adaptação Fisiológica , Sinalização do Cálcio , Ilhotas Pancreáticas/metabolismo , Canais de Potássio/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Hiperinsulinismo Congênito/metabolismo , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Canais KATP/metabolismo , Masculino , Camundongos , Modelos Teóricos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Cloreto de Potássio , Estimulação Química , Tolbutamida/farmacologiaRESUMO
BACKGROUND: Cells are continuously exposed to changes in their environment. Endocrine systems, in particular, communicate by rhythms and feedback loops. In this study, we developed an automated system to produce such conditions for cultured cells in a precisely timed manner. We utilized a programmable pair of syringe pumps for inflow and a peristaltic pump for outflow to create rhythmic pulses at 5-min intervals in solutions that mimic the endogenous patterns of insulin produced by pancreatic islets as a test case. RESULTS: This perifusion system was first tested by measuring trypan blue absorbance, which was intermittently added and washed out at 3:3 and 2:3 min (in:out). Absorbance corresponded with patterns of trypan blue delivery. We then created patterns of forced oscillations in islets by intermittently switching between solutions containing 28 millimolar (mM) glucose (producing high levels of intracellular calcium ([Ca2+]i) and insulin secretion) and 28 mM glucose + calcium-channel blocker nifedipine (producing low levels of [Ca2+]i and insulin secretion). Forced perifusion effects were monitored by fura-2 AM fluorescence measurements of [Ca2+]i. Islets showed uniform oscillations in [Ca2+]i at time intervals consistent with the perifusion pattern, mimicking endogenous pulsatility. CONCLUSIONS: This study highlights a valuable method to modify the environment of the cell culture over a period of hours to days.
RESUMO
Apolipoprotein A-IV (apoA-IV) is secreted by the small intestine in response to fat absorption. Here we demonstrate a potential role for apoA-IV in regulating glucose homeostasis. ApoA-IV-treated isolated pancreatic islets had enhanced insulin secretion under conditions of high glucose but not of low glucose, suggesting a direct effect of apoA-IV to enhance glucose-stimulated insulin release. This enhancement involves cAMP at a level distal to Ca(2+) influx into the ß cells. Knockout of apoA-IV results in compromised insulin secretion and impaired glucose tolerance compared with WT mice. Challenging apoA-IV(-/-) mice with a high-fat diet led to fasting hyperglycemia and more severe glucose intolerance associated with defective insulin secretion than occurred in WT mice. Administration of exogenous apoA-IV to apoA-IV(-/-) mice improved glucose tolerance by enhancing insulin secretion in mice fed either chow or a high-fat diet. Finally, we demonstrate that exogenous apoA-IV injection decreases blood glucose levels and stimulates a transient increase in insulin secretion in KKAy diabetic mice. These results suggest that apoA-IV may provide a therapeutic target for the regulation of glucose-stimulated insulin secretion and treatment of diabetes.
Assuntos
Apolipoproteínas A/fisiologia , Glucose/metabolismo , Homeostase , Insulina/metabolismo , Animais , Apolipoproteínas A/genética , Teste de Tolerância a Glucose , Secreção de Insulina , Camundongos , Camundongos KnockoutRESUMO
We used the patch clamp technique in situ to test the hypothesis that slow oscillations in metabolism mediate slow electrical oscillations in mouse pancreatic islets by causing oscillations in KATP channel activity. Total conductance was measured over the course of slow bursting oscillations in surface ß-cells of islets exposed to 11.1 mM glucose by either switching from current clamp to voltage clamp at different phases of the bursting cycle or by clamping the cells to -60 mV and running two-second voltage ramps from -120 to -50 mV every 20 s. The membrane conductance, calculated from the slopes of the ramp current-voltage curves, oscillated and was larger during the silent phase than during the active phase of the burst. The ramp conductance was sensitive to diazoxide, and the oscillatory component was reduced by sulfonylureas or by lowering extracellular glucose to 2.8 mM, suggesting that the oscillatory total conductance is due to oscillatory KATP channel conductance. We demonstrate that these results are consistent with the Dual Oscillator model, in which glycolytic oscillations drive slow electrical bursting, but not with other models in which metabolic oscillations are secondary to calcium oscillations. The simulations also confirm that oscillations in membrane conductance can be well estimated from measurements of slope conductance and distinguished from gap junction conductance. Furthermore, the oscillatory conductance was blocked by tolbutamide in isolated ß-cells. The data, combined with insights from mathematical models, support a mechanism of slow (â¼5 min) bursting driven by oscillations in metabolism, rather than by oscillations in the intracellular free calcium concentration.
Assuntos
Relógios Biológicos/fisiologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/fisiologia , Canais KATP/metabolismo , Animais , Relógios Biológicos/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Técnicas de Patch-ClampRESUMO
BACKGROUND: Activation of innate immunity through polyinosinic:polycytidylic acid [poly(I:C)] causes acute salivary gland hypofunction. As a major consequence of poly(I:C) treatment is type I interferon (IFN) production, this study was undertaken to investigate their role in salivary gland dysfunction. METHODS: Different strains of mice deficient in either interferon alpha receptor (IFNAR1(-/-)) or IL-6(-/-), or IL-10(-/-), or EBI3(-/-) were treated with poly(I:C). Salivary gland function was determined by measuring pilocarpine-induced saliva volume. Gene expression levels were measured by real-time PCR. Ca(2+) mobilization studies were performed using ex-vivo acinar cells. RESULTS: A single injection of poly(I:C) rapidly induced salivary gland hypofunction in wild-type B6 mice (41% drop in saliva volumes compared to PBS-treated mice). In contrast, the loss of function in poly(I:C)-treated IFNAR(-/-) mice was only 9.6%. Gene expression analysis showed reduced levels of Il-6, Il-10, and Il-27 in submandibular glands of poly(I:C)-treated IFNAR(-/-) mice. While salivary gland dysfunction in poly(I:C)-treated IL-10(-/-) and EBI3(-/-) mice was comparable to wild-type mice, the IL-6(-/-) mice were more resistant, with only a 21% drop in function. Pilocarpine-induced Ca(2+) flux was significantly suppressed in acinar cells obtained from poly(I:C)-treated wild-type mice. CONCLUSIONS: Our data demonstrate that a combined action of type I IFNs and IL-6 contributes toward salivary gland hypofunction. This happens through interference with Ca(2+) mobilization within acinar cells. Thus, in acute viral infections and diseases like Sjögren's syndrome, elevated levels of type I IFNs and IL-6 can directly affect glandular function.
Assuntos
Sinalização do Cálcio/fisiologia , Imunidade Inata , Interferon Tipo I/fisiologia , Interleucina-6/fisiologia , Glândula Submandibular/efeitos dos fármacos , Xerostomia/imunologia , Animais , Feminino , Injeções Intraperitoneais , Interferon Tipo I/biossíntese , Interleucina-10/biossíntese , Interleucina-10/fisiologia , Interleucina-17/biossíntese , Interleucina-17/fisiologia , Interleucina-6/biossíntese , Camundongos , Camundongos Mutantes , Poli I-C/farmacologia , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/imunologia , Saliva/metabolismo , Glândula Submandibular/metabolismo , Xerostomia/induzido quimicamenteRESUMO
Synchrotron X-ray fluorescence microscopy (SXRF) presents a valuable opportunity to study the metallome of single cells because it simultaneously provides high-resolution subcellular distribution and quantitative cellular content of multiple elements. Different sample preparation techniques have been used to preserve cells for observations with SXRF, with a goal to maintain fidelity of the cellular metallome. In this case study, mouse pancreatic beta-cells have been preserved with optimized chemical fixation. We show that cell-to-cell variability is normal in the metallome of beta-cells due to heterogeneity and should be considered when interpreting SXRF data. In addition, we determined the impact of several immunofluorescence (IF) protocols on metal distribution and quantification in chemically fixed beta-cells and found that the metallome of beta-cells was not well preserved for quantitative analysis. However, zinc and iron qualitative analysis could be performed after IF with certain limitations. To help minimize metal loss using samples that require IF, we describe a novel IF protocol that can be used with chemically fixed cells after the completion of SXRF.
Assuntos
Metais , Síncrotrons , Animais , Camundongos , Raios X , Espectrometria por Raios X/métodos , Metais/análise , Ferro/análiseRESUMO
Type 1 diabetes (T1D) is a chronic autoimmune disorder that damages beta cells in the pancreatic islets of Langerhans and results in hyperglycemia due to the loss of insulin. Exogenous insulin therapy can save lives but does not halt disease progression. Thus, an effective therapy may require beta-cell restoration and suppression of the autoimmune response. However, currently, there are no treatment options available that can halt T1D. Within the National Clinical Trial (NCT) database, a vast majority of over 3000 trials to treat T1D are devoted to insulin therapy. This review focuses on non-insulin pharmacological therapies. Many investigational new drugs fall under the category of immunomodulators, such as the recently FDA-approved CD-3 monoclonal antibody teplizumab. Four intriguing candidate drugs fall outside the category of immunomodulators, which are the focus of this review. Specifically, we discuss several non-immunomodulators that may have more direct action on beta cells, such as verapamil (a voltage-dependent calcium channel blocker), gamma aminobutyric acid (GABA, a major neurotransmitter with effects on beta cells), tauroursodeoxycholic acid (TUDCA, an endoplasmic reticulum chaperone), and volagidemab (a glucagon receptor antagonist). These emerging anti-diabetic drugs are expected to provide promising results in both beta-cell restoration and in suppressing cytokine-derived inflammation.
Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Humanos , Diabetes Mellitus Tipo 1/terapia , Citoproteção , Insulina/farmacologia , Fatores Imunológicos/farmacologia , Insulina Regular HumanaRESUMO
Although type 1 diabetes cannot be prevented or reversed, replacement of insulin production by transplantation of the pancreas or pancreatic islets represents a definitive solution. At present, transplantation can restore euglycemia, but this restoration is short-lived, requires islets from multiple donors, and necessitates lifelong immunosuppression. An emerging paradigm in transplantation and autoimmunity indicates that systemic inflammation contributes to tissue injury while disrupting immune tolerance. We identify multiple barriers to successful islet transplantation, each of which either contributes to the inflammatory state or is augmented by it. To optimize islet transplantation for diabetes reversal, we suggest that targeting these interacting barriers and the accompanying inflammation may represent an improved approach to achieve successful clinical islet transplantation by enhancing islet survival, regeneration or neogenesis potential, and tolerance induction. Overall, we consider the proinflammatory effects of important technical, immunological, and metabolic barriers including: 1) islet isolation and transplantation, including selection of implantation site; 2) recurrent autoimmunity, alloimmune rejection, and unique features of the autoimmune-prone immune system; and 3) the deranged metabolism of the islet transplant recipient. Consideration of these themes reveals that each is interrelated to and exacerbated by the other and that this connection is mediated by a systemic inflammatory state. This inflammatory state may form the central barrier to successful islet transplantation. Overall, there remains substantial promise in islet transplantation with several avenues of ongoing promising research. This review focuses on interactions between the technical, immunological, and metabolic barriers that must be overcome to optimize the success of this important therapeutic approach.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Inflamação/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/cirurgia , Sobrevivência de Enxerto/imunologia , Humanos , Tolerância Imunológica/imunologia , Inflamação/metabolismoRESUMO
Pulsatility is important to islet function. As islets mature into fully developed insulin-secreting micro-organs, their ability to produce oscillatory intracellular calcium ([Ca2+]i) patterns in response to glucose also matures. In this study, we measured [Ca2+]i using fluorescence imaging to characterize oscillations from neonatal mice on postnatal (PN) days 0, 4, and 12 in comparison to adult islets. Under substimulatory (3-mM) glucose levels, [Ca2+]i was low and quiescent for adult islets as expected, as well as for PN day 12 islets. In contrast, one-third of islets on PN day 0 and 4 displayed robust [Ca2+]i oscillations in low glucose. In stimulatory glucose (11 mM) conditions, oscillations were present on all neonatal days but differed from patterns in adults. By PN day 12, [Ca2+]i oscillations were approaching characteristics of fully developed islets. The immature response pattern of neonatal islets was due, at least in part, to differences in adenosine 5'-triphosphate (ATP)-sensitive K+-channel activity estimated by [Ca2+]i responses to KATP channel agents diazoxide and tolbutamide. Neonatal [Ca2+]i patterns were also strikingly similar to patterns observed in mature islets exposed to hyperglycemic conditions (20 mM glucose for 48 hours): elevated [Ca2+]i and oscillations in low glucose along with reduced pulse mass in high glucose. Since a hallmark of diabetic islets is dedifferentiation, we propose that diabetic islets display features of "reverse maturation," demonstrating similar [Ca2+]i dynamics as neonatal islets. Pulsatility is thus an important emergent feature of neonatal islets. Our findings may provide insight into reversing ß-cell dedifferentiation and to producing better functioning ß cells from pluripotent stem cells.
Assuntos
Hiperglicemia , Ilhotas Pancreáticas , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Sinalização do Cálcio , Glucose/metabolismo , Glucose/farmacologia , Hiperglicemia/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , CamundongosRESUMO
Islets of Langerhans, found in the pancreas, are microorgans essential for glucose homeostasis within the body. Many cells are found with an islet, such as beta cells (~70%), alpha cells (~20%), delta cells (~5%), F cells (~4%), and epsilon cells (1%), each with its own unique function. To better understand the roles of these cells and how cell communication alters their function, several techniques have been established such as islet isolation and beta cell dispersion. Here we describe how to isolate primary rodent islets, disperse pancreatic islets, measure intracellular calcium, and use immunofluorescent staining to distinguish beta cells and alpha cells.
Assuntos
Comunicação Celular , Separação Celular , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Animais , Cálcio/análise , Imunofluorescência , Ratos , Coloração e RotulagemRESUMO
Insulin secretion is widely thought to be maximally stimulated in glucose concentrations of 16.7-to-30 mM (300-to-540 mg/dL). However, insulin secretion is seldom tested in hyperglycemia exceeding these levels despite the Guinness World Record being 147.6 mM (2656 mg/dL). We investigated how islets respond to 1-h exposure to glucose approaching this record. Insulin secretion from human islets at 12 mM glucose intervals dose-dependently increased until at least 72 mM glucose. Murine islets in 84 mM glucose secreted nearly double the insulin as in 24 mM (p < 0.001). Intracellular calcium was maximally stimulated in 24 mM glucose despite a further doubling of insulin secretion in higher glucose, implying that insulin secretion above 24 mM occurs through amplifying pathway(s). Increased osmolarity of 425-mOsm had no effect on insulin secretion (1-h exposure) or viability (48-h exposure) in murine islets. Murine islets in 24 mM glucose treated with a glucokinase activator secreted as much insulin as islets in 84 mM glucose, indicating that glycolytic capacity exists above 24 mM. Using an incretin mimetic and an adenylyl cyclase activator in 24 mM glucose enhanced insulin secretion above that observed in 84 mM glucose while adenylyl cyclase inhibitor reduced stimulatory effects. These results highlight the underestimated ability of islets to secrete insulin proportionally to extreme hyperglycemia through adenylyl cyclase activity.
RESUMO
Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158 ± 57.7 femtograms (fg) to 65.7 ± 29.7 fg; calcium decreased from 216 ± 67.4 to 154.3 ± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4 ± 12.2 to 47.2 ± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.
Assuntos
Cálcio/metabolismo , Inflamação/metabolismo , Células Secretoras de Insulina/metabolismo , Ferro/metabolismo , Imagem Óptica/métodos , Síncrotrons , Zinco/metabolismo , Animais , Mediadores da Inflamação/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Masculino , Camundongos , Frações Subcelulares/metabolismoRESUMO
Pancreatic islet cells develop mature physiological responses to glucose and other fuels postnatally. In this study, we used fluorescence imaging techniques to measure changes in intracellular calcium ([Ca2+]i) to compare islets isolated from mice on postnatal days 0, 4, and 12 with islets from adult CD-1 mice. In addition, we used publicly available RNA-sequencing data to compare expression levels of key genes in ß-cell physiology with [Ca2+]i data across these ages. We show that islets isolated from mice on postnatal day 0 displayed elevated [Ca2+]i in basal glucose (≤4â¯mM) but lower [Ca2+]i responses to stimulation by 12-20â¯mM glucose compared to adult. Neonatal islets displayed more adult-like [Ca2+]i in basal glucose by day 4 but continued to show lower [Ca2+]i responses to 16 and 20â¯mM glucose stimulation up to at least day 12. A right shift in glucose sensing (EC50) correlated with lower fragment-per-kilobase-of-transcript-per-million-reads-mapped (FPKM) of Slc2a2 (glut2) and Actn3 and increased FPKM for Galk1 and Nupr1. Differences in [Ca2+]i responses to additional stimuli were also observed. Calcium levels in the endoplasmic reticulum were elevated on day 0 but became adult-like by day 4, which corresponded with reduced expression in Atp2a2 (SERCA2) and novel K+-channel Ktd17, increased expression of Pml, Wfs1, Thada, and Herpud1, and basal [Ca2+]i maturing to adult levels. Ion-channel activity also matured rapidly, but RNA sequencing data mining did not yield strong leads. In conclusion, the maturation of islet [Ca2+]i signaling is complex and multifaceted; several possible gene targets were identified that may participate in this process.
Assuntos
Sinalização do Cálcio , Ilhotas Pancreáticas/metabolismo , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diabetes Mellitus/tratamento farmacológico , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Glicólise/efeitos dos fármacos , Glicólise/genética , Homeostase/efeitos dos fármacos , Homeostase/genética , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Nifedipino/farmacologia , Cloreto de Potássio/farmacologiaRESUMO
PURPOSE: Previously we showed that natural compound α-penta-galloyl-glucose (α-PGG) and its synthetic derivative 6-chloro-6-deoxy-1,2,3,4-tetra-O-galloyl-α-D-glucopyranose (6Cl-TGQ) act to improve insulin signaling in adipocytes by increasing glucose transport. In this study, we investigated the mechanism of actions of α-PGG and 6Cl-TGQ on insulin secretion. METHODS: Mouse islets and/or INS-1832/13 beta-cells were used to test the effects of our compounds on glucose-stimulated insulin secretion (GSIS), intracellular calcium [Ca2+]i using fura-2AM, glucose transport activity via a radioactive glucose uptake assay, intracellular ATP/ADP, and extracellular acidification (ECAR) and mitochondrial oxygen consumption rates (OCAR) using Seahorse metabolic analysis. RESULTS: Both compounds reduced GSIS in beta-cells without negatively affecting cell viability. The compounds primarily diminished glucose uptake into islets and beta-cells. Despite insulin-like effects in the peripheral tissues, these compounds do not act through the insulin receptor in islets. Further interrogation of the stimulus-secretion pathway showed that all the key metabolic factors involved in GSIS including ECAR, OCAR, ATP/ADP ratios, and [Ca2+]i of INS-1832/13 cells were diminished after the compound treatment. CONCLUSION: The compounds suppress glucose uptake of the beta-cells, which consequently slows down the rates of glycolysis and ATP synthesis, leading to decrease in [Ca2+]i and GSIS. The difference between adipocytes and beta-cells in effects on glucose uptake is of great interest. Further structural and functional modifications could produce new compounds with optimized therapeutic potentials for different target cells. The higher potency of synthetic 6Cl-TGQ in enhancing insulin signaling in adipocytes but lower potency in reducing glucose uptake in beta-cells compared to α-PGG suggests the feasibility of such an approach.
RESUMO
The role of mitochondria in apoptosis is well known; however, the mechanisms linking mitochondria to the proapoptotic effects of proinflammatory cytokines, hyperglycemia, and glucolipotoxicity are not completely understood. Complex Ca2+ signaling has emerged as a critical contributor to these proapoptotic effects and has gained significant attention in regulating the signaling processes of mitochondria. In pancreatic ß-cells, Ca2+ plays an active role in ß-cell function and survival. Prohibitin (PHB), a mitochondrial chaperone, is actively involved in maintaining the architecture of mitochondria. However, its possible interaction with Ca2+-activated signaling pathways has not been explored. The present review aims to examine potential crosstalk between Ca2+ signaling and PHB function in pancreatic ß-cells. Moreover, this review will focus on the effects of cytokines and glucolipotoxicity on Ca2+ signaling and its possible interaction with PHB. Improved understanding of this important mitochondrial protein may aid in the design of more targeted drugs to identify specific pathways involved with stress-induced dysfunction in the ß-cell.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose , Glicemia/metabolismo , Citocinas/metabolismo , Diabetes Mellitus/patologia , Humanos , Células Secretoras de Insulina/patologia , Metabolismo dos Lipídeos , Mitocôndrias/patologia , ProibitinasRESUMO
Recruiting talented high school and college students to consider a career in the biomedical or biophysical sciences is important, yet often difficult. Encouraging students in regions like Appalachia adds additional challenges due to socioeconomic hurdles and misperceptions. This brief report contains the reflections of a research mentor engaging with students as a high school physics teacher, a principal investigator at research-intensive university, and as a principal investigator at a predominantly undergraduate-focused research university, as well as the viewpoint of a former undergraduate student in the mentor's lab. Different hurdles stand in the way of success at each level. A key issue at the high school level is engaging students in 'real science', the discovery of new knowledge and ideas. With undergraduate students at a larger research institution, a key issue is for the student to have opportunities to engage in meaningful scientific research. At a smaller and more rural research institution, especially in Appalachia, many students have socioeconomic concerns and misconceptions of what scientific careers entail. Regardless of background and environment, there are certain students who thrive on the scientific curiosity to discover new things. All they need is that opportunity to engage in meaningful scientific discovery to become interested in a scientific career.