ABSTRACT
Over the past few years, the implementation of mass spectrometry (MS) in QC laboratories has become a more common occurrence. The multi-attribute method (MAM), and emerging intact multi-attribute method (iMAM), are powerful analytical tools utilising liquid chromatography-mass spectrometry (LC-MS) methods that enable the monitoring of critical quality attributes (CQAs) in biotherapeutic proteins in compliant settings. Both MAM and iMAM are intended to replace or supplement several conventional assays with a single LC-MS method utilising MS data in combination with robust, semi-automated data processing workflows. MAM and iMAM workflows can also be implemented into current Good Manufacturing Practices environments due to the availability of CFR 11 compliant chromatography data system software. In this study, MAM and iMAM are employed for the analysis of 4 batches of a glucagon-like peptide-Fc fusion protein. MAM approach involved a first the discovery phase for the identification of CQAs and second, the target monitoring phase of the selected CQAs in other samples. New peak detection was performed on the data set to determine the appearance, absence or change of any peak. For native iMAM workflow both size exclusion and strong cation exchange chromatography were optimized for the identification and monitoring of CQAs at the intact level.
Subject(s)
Recombinant Fusion Proteins , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Chromatography, Liquid/methods , Mass Spectrometry/methods , Workflow , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/analysis , Glucagon/analysis , Glucagon/chemistry , Humans , Tandem Mass Spectrometry/methods , Liquid Chromatography-Mass SpectrometryABSTRACT
Little is known about the adaptor protein FAM159B. To determine whether FAM159B expression findings in rats or mice can be extrapolated to humans, we compared FAM159B expression in healthy tissue samples from all three species using immunohistochemistry. Despite variations in expression intensity, similar FAM159B expression patterns were observed in most organs across species. The most prominent species difference was noted in pancreatic islets; while FAM159B expression was limited to single cells on the outer edges in mice and rats, it was detectable across entire islets in humans. Double-labeling immunohistochemistry revealed partial overlap of FAM159B expression with that of insulin, glucagon, and somatostatin in human islets. By contrast, FAM159B showed complete colocalization with only somatostatin in rats and mice. An additional analysis of FAM159B expression in lean and obese Zucker rats revealed larger islet areas due to increased ß-cell mass in obese rats, which was accompanied by a smaller percentage of FAM159B-positive δ-cells per islet area. Beyond the known differences in islet architecture across species, our results point to larger dissimilarities in blood glucose regulation between rodents and humans than generally assumed. Moreover, findings regarding FAM159B expression (and function) cannot be directly transferred between rodents and humans.
Subject(s)
Islets of Langerhans , Rats, Zucker , Species Specificity , Animals , Humans , Rats , Male , Islets of Langerhans/metabolism , Mice , Female , Mice, Inbred C57BL , Immunohistochemistry , Glucagon/metabolism , Glucagon/analysis , Adult , Insulin/metabolism , Insulin/analysis , Somatostatin/metabolism , Somatostatin/analysis , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Middle AgedABSTRACT
Recently, the first generic glucagon for injection was approved for the treatment of severe hypoglycemia. Unlike its brand name recombinant glucagon, the generic glucagon is synthetic. Since glucagon has a high propensity to form aggregates in solution, it is essential to assess the aggregation profile of the synthetic glucagon compared to the recombinant glucagon. In this study, two robust separation methods, size-exclusion chromatography (SEC-HPLC) and field-flow fractionation coupled with a multi-angle light scattering detector (FFF-MALS), were employed to characterize generic and brand glucagon aggregation in six lots (three newly released, three expired). The presence of aggregation in samples was determined from the generated chromatograms and analyzed. The study showed that both products have comparable aggregation profiles. The SEC-HPLC demonstrated that in both glucagon versions, the expired lots had a higher percentage of dimers than the newly released lots, but even at expiration, the amount was negligible (â¼0.1%). The FFF-MALS method did not detect any dimers or higher molecular weight aggregates. Further evaluation of the detection limit found that FFF-MALS was unable to detect aggregates at amounts lower than 0.5% of total glucagon. The negligible amounts of dimer detected in the generic and brand glucagon indicate that both versions are physically stable and are not prone to aggregation under clinically relevant conditions.
Subject(s)
Chromatography, Gel , Glucagon , Protein Aggregates , Glucagon/chemistry , Glucagon/analysis , Chromatography, High Pressure Liquid/methods , Chromatography, Gel/methods , Scattering, Radiation , Humans , LightABSTRACT
Peripheral serotonin regulates energy metabolism in several mammalian species, however, the potential contribution of serotonergic mechanisms as metabolic and endocrine regulators in growing dairy calves remain unexplored. Objectives were to characterize the role of serotonin in glucose and insulin metabolism in dairy calves with increased serotonin bioavailability. Milk replacer was supplemented with saline, 5-hydroxytryptophan (90 mg/d), or fluoxetine (40 mg/d) for 10-d (n = 8/treatment). Blood was collected daily during supplementation and on days 2, 7, and 14 during withdrawal. Calves were euthanized after 10-d supplementation or 14-d withdrawal periods to harvest liver and pancreas tissue. 5-hydroxytryptophan increased circulating insulin concentrations during the supplementation period, whereas both treatments increased circulating glucose concentration during the withdrawal period. The liver and pancreas of preweaned calves express serotonin factors (ie, TPH1, SERT, and cell surface receptors), indicating their ability to synthesize, uptake, and respond to serotonin. Supplementation of 5-hydroxytryptophan increased hepatic and pancreatic serotonin concentrations. After the withdrawal period, fluoxetine cleared from the pancreas but not liver tissue. Supplementation of 5-hydroxytryptophan upregulated hepatic mRNA expression of serotonin receptors (ie, 5-HTR1B, -1D, -2A, and -2B), and downregulated pancreatic 5-HTR1F mRNA and insulin-related proteins (ie, Akt and pAkt). Fluoxetine-supplemented calves had fewer pancreatic islets per microscopic field with reduced insulin intensity, whereas 5-hydroxytryptophan supplemented calves had increased islet number and area with greater insulin and serotonin and less glucagon intensities. After the 14-d withdrawal of 5-hydroxytryptophan, hepatic mRNA expression of glycolytic and gluconeogenic enzymes were simultaneously downregulated. Improving serotonin bioavailability could serve as a potent regulator of endocrine and metabolic processes in dairy calves.
Subject(s)
Cattle/metabolism , Serotonin/physiology , 5-Hydroxytryptophan/administration & dosage , Animals , Blood Glucose/analysis , Fluoxetine/administration & dosage , Fluoxetine/blood , Gene Expression Regulation/drug effects , Glucagon/analysis , Insulin/analysis , Insulin/blood , Liver/chemistry , Liver/drug effects , Liver/metabolism , Male , Pancreas/chemistry , Pancreas/drug effects , Pancreas/metabolism , Serotonin/analysis , Serotonin/bloodSubject(s)
Insulin-Secreting Cells/metabolism , Interleukin-4 Receptor alpha Subunit/biosynthesis , Interleukin-4/physiology , Adolescent , Glucagon/analysis , Glucagon-Secreting Cells/metabolism , Glucagon-Secreting Cells/ultrastructure , Humans , Insulin/analysis , Insulin-Secreting Cells/ultrastructure , Interleukin-4/pharmacology , Microscopy, Fluorescence , Protein SubunitsABSTRACT
In recent years, the prevalence of obesity, metabolic syndrome and type 2 diabetes is increasing dramatically. They share pathophysiological mechanisms and often lead to cardiovascular diseases. The ZDSD rat was suggested as a new animal model to study diabetes and the metabolic syndrome. In the current study, we have further characterized metabolic and hepatic gene expression changes in ZDSD rats. Immuno-histochemical staining of insulin and glucagon on pancreas sections of ZDSD and control SD rats revealed that ZDSD rats have severe damage to their islet structures as early as 15 weeks of age. Animals were followed till they were 26 weeks old, where they exhibited obesity, hypertension, hyperglycemia, dyslipidemia, insulin resistance and diabetes. We found that gene expressions involved in glucose metabolism, lipid metabolism and amino acid metabolism were changed significantly in ZDSD rats. Elevated levels of ER stress markers correlated with the dysregulation of hepatic lipid metabolism in ZDSD rats. Key proteins participating in unfolded protein response pathways were also upregulated and likely contribute to the pathogenesis of dyslipidemia and insulin resistance. Based on its intact leptin system, its insulin deficiency, as well as its timeline of disease development without diet manipulation, this insulin resistant, dyslipidemic, hypertensive, and diabetic rat represents an additional, unique polygenic animal model that could be very useful to study human diabetes.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Disease Models, Animal , Hypertension/metabolism , Metabolic Syndrome/metabolism , Obesity/metabolism , Amino Acids/metabolism , Animals , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/pathology , Glucagon/analysis , Glucagon/metabolism , Humans , Hypertension/genetics , Hypertension/pathology , Insulin/analysis , Insulin/metabolism , Lipid Metabolism/genetics , Liver/pathology , Male , Metabolic Syndrome/genetics , Metabolic Syndrome/pathology , Multifactorial Inheritance , Obesity/genetics , Obesity/pathology , Pancreas/pathology , Rats , Rats, Inbred Strains , Rats, Sprague-Dawley , Rats, ZuckerABSTRACT
BACKGROUND: An increased access of research laboratories to isolated human islets has improved our understanding of the biology of the endocrine pancreas and hence the mechanisms causing diabetes. However, in vitro studies of human islets remain technically challenging, and optimal use of such precious material requires a minimum of rigor and coordination to optimize the reliability and share of the information. A detailed report of the demographics of pancreas donors and of the procedures of islet handling after isolation is important but insufficient. Correct characterization of islet basic functions (a token of quality) at the time of experimentation is also crucial. SCOPE OF REVIEW: I have analyzed the literature reporting measurements of insulin and glucagon in the human pancreas or isolated human islets. The published information is often fragmentary. Elementary features such as islet size, insulin content, or rate of hormone secretion are either unreported or incorrectly reported in many papers. Although internal comparisons between control and test groups may remain valid, comparisons with data from other laboratories are problematic. The drawbacks, pitfalls and errors of common ways of expressing hormone content or secretion rates are discussed and alternatives to harmonize data presentation are proposed. MAJOR CONCLUSIONS: Greater coherence and rigor in the report of in vitro studies using human islets are necessary to ensure optimal progress in our understanding of the pathogenesis of diabetes.
Subject(s)
Islets of Langerhans/metabolism , Islets of Langerhans/physiology , Specimen Handling/methods , Diabetes Mellitus/metabolism , Glucagon/analysis , Glucagon/metabolism , Glucose/metabolism , Humans , Insulin/analysis , Insulin/metabolism , Insulin Secretion , Pancreas/metabolism , Reproducibility of ResultsABSTRACT
Glucagon circulates in concentrations in the low picomolar range, which is demanding regarding the sensitivity of the methods for quantification applied. In addition, the differential and tissue specific proteolytic processing of the glucagon precursor and the presence in of several glucagon-like sequences, not only in the precursor of glucagon, but also in a number of other peptides of the glucagon-secretin family of peptides, put special demands on the specificity of the assays. Finally, experience has shown that unspecific interference of plasma components has presented additional problems. All of these problems have resulted in a lot of diverging results concerning measured and reported glucagon responses in both humans and experimental animals that have and still are causing considerable debate and controversy. There is very solid evidence that glucagon is an important hormone in human and mammalian metabolism, but its precise physiological role in glucose and lipid metabolism and in metabolic disease has been difficult to establish, not least because of these difficulties. It was our purpose with this review to discuss the methods of glucagon quantification and discuss pitfalls and sources of error. We also reviewed some of the dogmas regarding glucagon secretion in the light of the methodological difficulties.
Subject(s)
Glucagon/analysis , Plasma/chemistry , Animals , Glucagon/blood , Guidelines as Topic , Humans , Lipid Metabolism , Sensitivity and SpecificityABSTRACT
Most studies in type 1 diabetes (T1D) have focused on the loss of the pancreatic beta-cell population. However, despite the involvement of the alpha-cell in the aetiology and complications of T1D, little is known about the regulation of the pancreatic alpha-cell mass in this disease. The need for a better understanding of this process is further emphasized by recent findings suggesting that alpha-cells may constitute a potential reservoir for beta-cell regeneration. In this study, we characterized the pancreatic alpha-cell mass and its regulatory processes in the transgenic RIP-B7.1 mice model of experimental autoimmune diabetes (EAD). Diabetic mice presented insulitis, hyperglycaemia, hypoinsulinemia and hyperglucagonemia along with lower pancreatic insulin content. While alpha-cell mass and pancreatic glucagon content were preserved at the early-onset of EAD, both parameters were reduced in the advanced phase. At both stages, alpha-cell size, proliferation and ductal neogenesis were up-regulated, whereas apoptosis was almost negligible. Interestingly, we found an increase in the proportion of glucagon-containing cells positive for insulin or the beta-cell transcription factor PDX1. Our findings suggest that pancreatic alpha-cell renewal mechanisms are boosted during the natural course of EAD, possibly as an attempt to maintain the alpha-cell population and/or to increase beta-cell regeneration via alpha-cell transdifferentiation.
Subject(s)
Diabetes Mellitus, Experimental/pathology , Animals , B7-1 Antigen/deficiency , B7-1 Antigen/genetics , Cell Proliferation , Cell Transdifferentiation , Diabetes Mellitus, Experimental/complications , Disease Models, Animal , Glucagon/analysis , Glucagon/metabolism , Glucagon-Secreting Cells/cytology , Glucagon-Secreting Cells/metabolism , Homeodomain Proteins/metabolism , Hyperglycemia/complications , Hyperglycemia/pathology , Insulin/analysis , Insulin/blood , Mice , Mice, Inbred C57BL , Mice, Transgenic , Trans-Activators/metabolismABSTRACT
Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay-which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates-can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5-1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.
Subject(s)
Amyloid/analysis , Antibodies/chemistry , Glucagon/analysis , Amyloid/ultrastructure , Enzyme-Linked Immunosorbent Assay/methods , HEK293 Cells , Humans , Protein Aggregates , SolubilityABSTRACT
In this work, we demonstrate the potential use of SPRi for secretion-monitoring of pancreatic islets, small micro-organs that regulate glucose homeostasis in the body. In the islets, somatostatin works as a paracrine inhibitor of insulin and glucagon secretion. However, this inhibitory effect is lost in diabetic individuals and little is known about its contribution to the pathology of diabetes. Thus, the simultaneous detection of insulin, glucagon and somatostatin could help understand such communications. Previously, the authors introduced an SPRi biosensor to simultaneously monitor insulin, glucagon and somatostatin using an indirect competitive immunoassay. However, our sensor achieved a relatively high LOD for somatostatin detection (246 nM), the smallest of the three hormones. For this reason, the present work aimed to improve the performance of our SPRi biosensor using gold nanoparticles (GNPs) as a means of ensuring somatostatin detection from a small group of islets. Although GNP amplification is frequently reported in the literature for individual detection of analytes using SPR, studies regarding the optimal strategy in a multiplexed SPR setup are missing. Therefore, with the aim of finding the optimal GNP amplification strategies for multiplex sensing we compared three architectures: (1) GNPs immobilized on the sensor surface, (2) GNPs conjugated with primary antibodies (GNP-Ab1) and (3) GNPs conjugated with a secondary antibody (GNP-Ab2). Among these strategies an immunoassay using GNP-Ab2 conjugates was able to achieve multiplex detection of the three hormones without cross-reactivity and with 9-fold LOD improvement for insulin, 10-fold for glucagon and 200-fold for somatostatin when compared to the SPRi biosensor without GNPs. The present work denotes the first report of the simultaneous detection of such hormones with a sensitivity level comparable to ELISA assays, particularly for somatostatin.
Subject(s)
Glucagon/analysis , Gold/chemistry , Insulin/analysis , Metal Nanoparticles/chemistry , Somatostatin/analysis , Antibodies, Monoclonal/immunology , Biosensing Techniques/methods , Calibration , Glucagon/immunology , Humans , Immunoassay/methods , Insulin/immunology , Limit of Detection , Somatostatin/immunologyABSTRACT
Currently, the clinical dynamics of glucagon need to be revised based on previous data obtained from conventional glucagon radioimmunoassays. In the present study, we evaluated plasma glucagon levels in type 1 diabetes patients using a newly-developed sandwich enzyme-linked immunosorbent assay, and its association with clinical parameters and markers of diabetes complications were statistically assessed. The plasma glucagon level in 77 Japanese type 1 diabetes patients was 28.1 ± 17.7 pg/mL, and comparable with that reported previously for type 2 diabetes patients. However, the values were widely spread and did not correlate with plasma glucose values. Additionally, the average glucagon levels in patients in a hypoglycemic state (glucose level <80 mg/dL) did not increase (21.7 ± 12.2 pg/mL). The average glucagon level of patients experiencing hypoglycemia unawareness was significantly lower. Plasma glucagon levels evaluated using the new enzyme-linked immunosorbent assay were dysregulated in type 1 diabetes patients in respect to plasma glucose levels, suggesting dysregulation of secretion.
Subject(s)
Diabetes Mellitus, Type 1/blood , Glucagon/blood , Adult , Asian People , Enzyme-Linked Immunosorbent Assay/methods , Female , Glucagon/analysis , Humans , Hypoglycemia/blood , Japan , MaleABSTRACT
Context: A better understanding of nocturnal regulation of glucose homeostasis will provide the framework for designing rational therapeutic strategies to improve the management of overnight glucose in patients with type 2 diabetes (T2D). Objective: To establish the nocturnal pattern and regulation of glucose production (EGP) in humans and to determine whether the pattern is dysregulated in people with T2D. Design: Subjects were infused with [3-3H] glucose overnight. Arterial blood samples were drawn for hormones and analytes to estimate EGP throughout the night. Deuterium-labeled water was provided to measure gluconeogenesis (GNG) using the hexamethylenetetramine method of Landau. Setting: Mayo Clinic Clinical Research Trials Unit, Rochester, MN, USA. Participants and Interventions: A total of 43 subjects [23 subjects with T2D and 20 nondiabetic (ND) subjects comparable for age and body mass index] were included in this study. Main Outcome(s) Measure(s): Glucose and EGP. Results: Plasma glucose, C-peptide, and glucagon concentrations were higher throughout the night, whereas insulin concentrations were higher in subjects with T2D vs ND subjects at 1:00 and 4:00 am but similar at 7:00 am. EGP was higher in the subjects with T2D than in the ND subjects throughout the night (P < 0.001). Glycogenolysis (GGL) fell and GNG rose, resulting in significantly higher (P < 0.001) rates of GNG at 4:00 and 7:00 am and significantly (P < 0.001) higher rates of GGL at 1:00, 4:00, and 7:00 am in T2D as compared with ND. Conclusions: These data imply that optimal therapies for T2D for nocturnal/fasting glucose control should target not only the absolute rates of EGP but also the contributing pathways of GGL and GNG sequentially.
Subject(s)
Biomarkers/blood , Blood Glucose/analysis , C-Peptide/analysis , Circadian Rhythm , Diabetes Mellitus, Type 2/prevention & control , Glucagon/analysis , Insulin/analysis , Blood Glucose Self-Monitoring , Body Mass Index , Case-Control Studies , Diabetes Mellitus, Type 2/physiopathology , Female , Follow-Up Studies , Homeostasis , Humans , Male , Middle Aged , Prognosis , Sleep/physiologyABSTRACT
BACKGROUND Emerging evidence suggests the potential of Dendrobium officinale Kimura et Migo (DO) in treating the complications of diabetes mellitus (DM). We evaluated the therapeutic potential of DO in treating diabetic nephropathy (DN) by preventing insulin resistance. MATERIAL AND METHODS A DN model was established. Mean glomerular volume of rats was estimated by the method of Weibel-Gomez. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression of mRNAs and we used Western blot assay to determine the expression of proteins. The levels of fasting insulin (FINS) and glucagon (GLU) were measured and we assessed the levels of high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) using the enzyme-linked immunosorbent assay (ELISA). RESULTS Compared with the Normal rats, the levels of urinary glucose, albuminuria, Scr, albuminuria/Scr and BUN, and the expression levels of CaN, TLR-2, TLR-4, MyD88, hs-CRP, TNF-a, and IL-6, the level of FINS, GLU, and HOMAIR were increased in DN, DO 1.0, DO 2.0, and DMBG groups. Compared with the DN rats, in DO 1.0, DO 2.0, and DMBG groups the glomerular volume was smaller, the levels of urinary glucose, albuminuria, Scr, albuminuria/Scr, and BUN, the expression levels of CaN, TLR-2, TLR-4, MyD88, hs-CRP, TNF-a, and IL-6, the level of FINS, GLU, and HOMA-IR were decreased. CONCLUSIONS We found that DO prevents insulin resistance in rats with DN. This may be associated with reduction of TLRs and inflammatory response, which should be further verified by loss of DO effects on DN after treatment of inhibitors of TLRs.
Subject(s)
Dendrobium/metabolism , Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/drug therapy , Animals , C-Reactive Protein/analysis , Cytokines/drug effects , Female , Glucagon/analysis , Glucagon/blood , Insulin/metabolism , Insulin Resistance/physiology , Interleukin-6/analysis , Interleukin-6/blood , Rats , Rats, Sprague-Dawley , Toll-Like Receptors/drug effects , Tumor Necrosis Factor-alpha/analysis , Tumor Necrosis Factor-alpha/bloodABSTRACT
Neprilysin (NEP) is an endopeptidase known to modulate nervous, cardiovascular, and immune systems via inactivation of regulatory peptides. In addition, it may also contribute to impaired glucose homeostasis as observed in type 2 diabetes (T2D). Specifically, we and others have shown that NEP is upregulated under conditions associated with T2D, whereas NEP deficiency and/or inhibition improves glucose homeostasis via enhanced glucose tolerance, insulin sensitivity, and pancreatic ß-cell function. Whether increased ß-cell mass also occurs with lack of NEP activity is unknown. We sought to determine whether NEP deficiency confers beneficial effects on ß- and α-cell mass in a mouse model of impaired glucose homeostasis. Wild-type and NEP-/- mice were fed low- or high-fat diet for 16 weeks, after which pancreatic ß- and α-cell mass were assessed by immunostaining for insulin and glucagon, respectively. Following low-fat feeding, NEP-/- mice exhibited lower ß- and α-cell mass compared with wild-type controls. A high-fat diet had no effect on these parameters in wild-type mice, but in NEP-/- mice, it resulted in the expansion of ß-cell mass. Our findings support a role for NEP in modulating ß-cell mass, making it an attractive T2D drug target that acts via multiple mechanisms to affect glucose homeostasis.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Diet, High-Fat , Gene Deletion , Insulin-Secreting Cells/pathology , Neprilysin/genetics , Animals , Blood Glucose/analysis , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/pathology , Diet, High-Fat/adverse effects , Glucagon/analysis , Glucagon-Secreting Cells/metabolism , Glucagon-Secreting Cells/pathology , Insulin/analysis , Insulin/blood , Insulin-Secreting Cells/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Size , Pancreas/metabolism , Pancreas/pathologyABSTRACT
Pancreatic islets are endocrine micro-organs scattered throughout the exocrine pancreas. Islets are surrounded by a network of vasculature, ducts, neurons, and extracellular matrix. Three-dimensional imaging is critical for such structural analyses. We have adapted transparent tissue tomography to develop a method to image thick pancreatic tissue slices (1 mm) with multifluorescent channels. This method takes only 2 to 3 days from specimen preparation and immunohistochemical staining to clearing tissues and imaging. Reconstruction of the intact pancreas visualizes islets with ß, α, and δ cells together with their surrounding networks. Capturing several hundred islets at once ensures sufficient power for statistical analyses. Further surface rendering provides clear views of the anatomical relationship between islets and their microenvironment as well as the basis for volumetric quantification. As a proof-of-principle demonstration, we show an islet size-dependent increase of intraislet capillary density and an inverse decrease in sphericity.
Subject(s)
Imaging, Three-Dimensional/methods , Islets of Langerhans/diagnostic imaging , Pancreas/diagnostic imaging , Animals , Antibodies, Monoclonal , Arterioles/diagnostic imaging , Capillaries/diagnostic imaging , Frozen Sections , Glucagon/analysis , Humans , Immunohistochemistry , Insulin/analysis , Islets of Langerhans/blood supply , Islets of Langerhans/chemistry , Mice , Pancreas/chemistry , Platelet Endothelial Cell Adhesion Molecule-1/analysis , Somatostatin/analysisABSTRACT
Diabetes arises from secretory defects in vascularized micro-organs known as the islets of Langerhans. Recent studies indicated that furthering our understanding of the paracrine effect of somatostatin on glucose-induced insulin secretion could represent a novel therapeutic avenue for diabetes. While many research groups are interested in insulin and glucagon secretion, few are particularly focused on studying the paracrine interaction in islets' cells, and none on monitoring a secretory fingerprint that contemplates more than two hormones. Surface plasmon resonance imaging can achieve high-throughput and multiplexed biomolecule quantification, making it an ideal candidate for detection of multiple islet's secretion products if arrays of hormones can be properly implemented on the sensing surface. In this study, we introduced a multiplex surface plasmon resonance imaging-based biosensor for simultaneous quantification of insulin, glucagon, and somatostatin. Performing this multiplex biosensing of hormones was mainly the result of the design of an antifouling sensing surface comprised by a mixed self-assembly monolayer of CH3O-PEG-SH and 16-mercaptohexadecanoic acid, which allowed it to operate in a complex matrix such as an islet secretome. The limit of detection in multiplex mode was 1 nM for insulin, 4 nM for glucagon, and 246 nM for somatostatin with a total analysis time of 21 min per point, making our approach the first reporting a label-free and multiplex measurement of such a combination of human hormones. This biosensor holds the promise of providing us with a mean for the further understanding of the paracrine effect of somatostatin on glucose-induced insulin secretion and consequently help develop novel therapeutic agents for diabetes.
Subject(s)
Biosensing Techniques/methods , Glucagon/analysis , Insulin/analysis , Somatostatin/analysis , Surface Plasmon Resonance/methods , Animals , Antibodies/immunology , Biofouling/prevention & control , Cattle , Glucagon/immunology , Humans , Immunoassay/methods , Insulin/immunology , Limit of Detection , Muramidase/chemistry , Palmitic Acids/chemistry , Polyethylene Glycols/chemistry , Serum Albumin, Bovine/chemistry , Somatostatin/immunologyABSTRACT
Crossbreeding is an effective method of improving the efficiency and profit of production in commercial pig operations. To understand the effect of crossbreeding on meat and nutrient quality, a combination including three purebred (Duroc, D; Landrace, L; Yorkshire, Y) and two crossbred pig lines (Landrace × Yorkshire, LY; Duroc × (Landrace × Yorkshire), DLY) frequently used internationally were studied. The results showed that meat from the LY and DLY crosses had lower values for lightness L24h∗, shear force and epinephrine and higher values for drip loss, C18:1, insulin, glucagon and monounsaturated fatty acids than D, L and Y pigs. Moreover, LY had higher values for post mortem pH and lower values for a* and b* than the purebreds. In contrast, DLY had lower values for pH and higher values for a* and b* than the purebreds. Meat quality-related gene analysis showed that the CAST, IGF2 and MC4R gene expression levels in the LY and DLY pigs were significantly higher than those in the D, L and Y pigs. These results indicate that crossbreeding can alter the meat quality, nutritive value, energy metabolism and gene expression of pigs. Future research should focus on microRNA expression and DNA methylation that regulate gene expression and thus affect the meat quality.
Subject(s)
Gene Expression , Hybridization, Genetic , Meat/analysis , Nutritive Value , Swine/genetics , Swine/metabolism , Animals , Cytoskeletal Proteins/genetics , Energy Metabolism , Epinephrine/analysis , Fatty Acids, Monounsaturated/analysis , Glucagon/analysis , Hybridization, Genetic/genetics , Hydrogen-Ion Concentration , Insulin/analysis , Insulin-Like Growth Factor II/genetics , Receptor, Melanocortin, Type 4/genetics , Shear StrengthABSTRACT
Mammalian cells contain various macromolecules that can be investigated non-invasively with Raman spectroscopy. The particular mixture of major macromolecules present in a cell being probed are reflected in the measured Raman spectra. Determining macromolecular identities and estimating their concentrations from these mixture Raman spectra can distinguish cell types and otherwise enable biological research. However, the application of canonical multivariate methods, such as principal component analysis (PCA), to perform spectral unmixing yields mathematical solutions that can be difficult to interpret. Non-negative matrix factorization (NNMF) improves the interpretability of unmixed macromolecular components, but can be difficult to apply because ambiguities produced by overlapping Raman bands permit multiple solutions. Furthermore, theoretically sound methods can be difficult to implement in practice. Here we examined the effects of a number of empirical approaches on the quality of NNMF results. These approaches were evaluated on simulated mammalian cell Raman hyperspectra and the results were used to develop an enhanced procedure for implementing NNMF. We demonstrated the utility of this procedure using a Raman hyperspectral data set measured from human islet cells to recover the spectra of insulin and glucagon. This was compared to the relatively inferior PCA of these data.
Subject(s)
Cytological Techniques/methods , Spectrum Analysis, Raman/methods , Algorithms , Animals , Cells, Cultured , Glucagon/analysis , Glucagon/chemistry , Humans , Insulin/analysis , Insulin/chemistry , Islets of Langerhans/chemistry , Macromolecular Substances/analysis , Macromolecular Substances/chemistry , Multivariate AnalysisABSTRACT
In type-2 diabetes, both insufficient insulin and excessive glucagon secretion contribute to hyperglycemia. We compared insulin, glucagon and somatostatin stores in pancreas obtained at autopsy of 20 lean and 19 obese non-diabetic (ND), and 18 type-2 diabetic (T2D) subjects. From concentrations and pancreas weight, total content of hormones was calculated. Insulin content was 35% lower in T2D than ND subjects (7.4 versus 11.3 mg), whereas glucagon content was similar (0.76 versus 0.81 mg). The higher ratio of glucagon/insulin contents in T2D was thus explained by the decrease in insulin. With increasing BMI of ND subjects, insulin and glucagon contents respectively tended to increase and decrease, resulting in a lower glucagon/insulin ratio in obesity. With aging, insulin and glucagon contents did not significantly change in ND subjects but declined in T2D subjects, without association with the duration of diabetes or type of treatment. The somatostatin content was lower in T2D than ND subjects (0.027 versus 0.038 mg), but ratios somatostatin/insulin and somatostatin/glucagon were not different. In conclusion, insulin stores are about 1/3 lower in T2D than ND subjects, whereas glucagon stores are unchanged. Abnormal secretion of each hormone in type-2 diabetes cannot be attributed to major alterations in their pancreatic reserves.