Human proinsulin production in the milk of transgenic cattle.

BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

Insulin and aging - a disappointing relationship.

Experimental studies in animal models of aging such as nematodes, fruit flies or mice have observed that decreased levels of insulin or insulin signaling promotes longevity. In humans, hyperinsulinemia and concomitant insulin resistance are associated with an elevated risk of age-related diseases suggestive of a shortened healthspan. Age-related disorders include neurodegenerative diseases, hypertension, cardiovascular disease, and type 2 diabetes. High ambient insulin concentrations promote increased lipogenesis and fat storage, heightened protein synthesis and accumulation of non-functional polypeptides due to limited turnover capacity. Moreover, there is impaired autophagy activity, and less endothelial NO synthase activity. These changes are associated with mitochondrial dysfunction and oxidative stress. The cellular stress induced by anabolic activity of insulin initiates an adaptive response aiming at maintaining homeostasis, characterized by activation of the transcription factor Nrf2, of AMP activated kinase, and an unfolded protein response. This protective response is more potent in the long-lived human species than in short-lived models of aging research resulting in a stronger pro-aging impact of insulin in nematodes and fruit flies. In humans, resistance to insulin-induced cell stress decreases with age, because of an increase of insulin and insulin resistance levels but less Nrf2 activation. These detrimental changes might be contained by adopting a lifestyle that promotes low insulin/insulin resistance levels and enhances an adaptive response to cellular stress, as observed with dietary restriction or exercise.

Simultaneous monitoring of multiple hormones from human islets of Langerhans using solid-phase extraction-mass spectrometry.

Islets of Langerhans release peptide hormones in controlled amounts and patterns to ensure proper maintenance of blood glucose levels. The overall release of the hormones is shaped by external factors and by autocrine and paracrine interactions occurring within the islets. To better understand what controls the secretion of islet-secreted peptides, and how these processes go awry in diabetes, methods to monitor the release of multiple hormones simultaneously are needed. While antibody-based assays are typically used, they are most often applied to quantification of a single hormone. Mass spectrometry (MS), on the other hand, is well suited for quantifying multiple hormones simultaneously but typically requires time-consuming separation steps with biological samples. In this report, response surface methodology was used to identify a set of optimal solid-phase extraction (SPE) conditions for the islet-secreted peptides, insulin, C-peptide, glucagon, and somatostatin. The optimized SPE method was used with multiple reaction monitoring and isotopically labeled standards to quantify secretion levels. Calibrations were linear from 0.5 to 50 nM with < 15% RSD peak area ratios. A microfluidic system was used to perfuse 30 human islets with different glucose conditions, and fractions were collected every 2 min for SPE-MS analysis. Results showed the release dynamics of the individual peptides, as well as patterns, such as positively and negatively correlated release and oscillations. This rapid SPE-MS method is expected to be useful for examining other peptide and small-molecule secretions from islets and could be applied to a number of other biological systems for investigating cellular communication.

Comparative analysis of biochemical, hormonal, and mineral compositions of preovulatory and cystic ovarian follicles in buffalo during the non-breeding season.

This study is a comparative analysis of the biochemical, hormonal, and mineral compositions of follicular fluid in preovulatory and cystic follicles of water buffalo (Bubalus bubalis). In total, reproductive tracts from 215 buffalo along with intact ovaries were collected randomly from an abattoir. The incidence of cystic conditions found in this study was 3.72% (8/215), involving the right ovary in 62.5% of instances and the left ovary in 37.5% of instances during the non-breeding season. Follicular fluid was aspirated from preovulatory follicles (12-15 mm diameter, oestrogen-active, follicular phase or stage IV corpus luteum on one of the two ovaries, n = 10) and cystic follicles (at least 20 mm diameter, no corpus luteum on any one of the two ovaries, n = 8). The follicular fluid samples were assayed for biochemical components (uric acid, creatinine, blood urea nitrogen, cholesterol, total protein, glucose, ascorbic acid, and alkaline phosphatase), hormones (progesterone, estradiol, and insulin), and minerals (calcium, magnesium, phosphorus, copper, zinc, and cobalt). Cystic follicles had greater (P < 0.05) concentrations of creatinine, blood urea nitrogen, cholesterol, progesterone, copper, zinc, and cobalt, and lesser (P < 0.05) concentrations of uric acid, glucose, ascorbic acid, estradiol, insulin, calcium, magnesium, and phosphorus compared with preovulatory follicles. These results indicated the marked differences in follicular fluid composition between preovulatory and cystic follicles in buffalo. Some of the changes were indicative of oxidative stress and disturbed steroidogenesis, two important mechanisms shown to be associated with cystic ovarian disease in various species. Further studies are warranted to investigate whether these differences are directly or indirectly involved in the formation of cystic follicles or are mere manifestations of the condition.

Maternal hair cortisol concentrations and its association with increased insulin resistance in midpregnancy.

PURPOSE: Stress and elevated maternal glycemia have negative effects on pregnancy. We evaluated the association of hair cortisol concentrations (HCC), a marker of chronic stress, with insulin resistance and gestational diabetes (GDM). METHODS: In total, 527 women from Lima, Peru, provided a hair sample in the second trimester of their pregnancy to measure HCC using liquid chromatography-tandem mass spectrometry. Each 6 cm of hair captured HCC in early (T1=1-12 weeks) and midpregnancy (T2 = 13-24 weeks). GDM diagnosis was conducted in midpregnancy. Multivariable regression models adjusted for putative risk factorsincluding maternal sociodemographic factors, diabetes history, and hair characteristics, were used to estimate the association of HCC with GDM and various glycemic traits. RESULTS: GDM was diagnosed in 122 (23%) women. Mean HCC across pregnancy was T1 = 3.7 (±3.4) pg/mg and T2 = 4.8 (±3.4) pg/mg. HCC was associated with increased log-transformed units of fasting insulin (T1 = 0.15 [0.03, 0.27], T2 = 0.17 [0.04, 0.30]), homeostasis model assessment for insulin resistance (T1 = 0.14 [0.01, 0.26], T2 = 0.17 [0.03, 0.30]), and homeostasis model assessment for ß-cell function (T1 = 0.20 [0.05, 0.34], T2 = 0.20 [0.04, 0.36]), but not with GDM (T1 = 0.95 [0.63, 1.40], T2 = 1.11 [0.74, 1.67]). CONCLUSIONS: Elevated maternal HCC was associated with abnormal insulin homeostasis in pregnancy. Dysregulation of the hypothalamic-pituitary-adrenal axis, as reflected by high HCC, may also contribute to insulin resistance syndrome in pregnancy.

Effects of Exercise Training on Inflammatory and Cardiometabolic Risk Biomarkers in Patients With Type 2 Diabetes Mellitus: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: The interaction between type 2 diabetes mellitus (T2DM) and cardiometabolic morbidity and mortality stems from the progressive nature of inflammation underpinning both diseases. Exercise training is considered an effective treatment strategy for T2DM and cardiometabolic diseases. OBJECTIVE: The current systematic review and meta-analysis investigated the effects of exercise training on inflammatory and cardiometabolic risk biomarkers in patients with T2DM. DATA SOURCES: Electronic databases (PubMed/Medline, Embase, Cochrane Library, CINAHL, Google Scholar, Scopus, and Web of Science) were searched for randomized controlled trials (RCTs) from inception to January 2022. We used random effects models to estimate weighted mean differences with 95% confidence intervals. STUDY SELECTION: Twenty-five RCTs were included (N = 1257 participants; mean age = 52 years). Included studies had moderate to good overall methodological quality (TESTEX = 9 (range 7-13). RESULTS: Meta-analysis indicated that exercise training significantly increased adiponectin and decreased fasting insulin, homeostatic model assessment for insulin resistance, tumor necrosis factor-α, interleukin-6, and C-reactive protein (ps ≤ 0.05). Subgroup analysis by type of training indicated that aerobic exercise had the most consistent beneficial effects as compared to other types of exercise training; however, there was high heterogeneity among studies. CONCLUSION: Different types of exercise training increase adiponectin levels and decrease pro-inflammatory cytokines such as TNF-α, IL-6, and CRP, as well as fasting insulin and insulin resistance markers in patients with T2DM. However, these effects were not beneficial for more commonly measured cardiometabolic risk factors (i.e., lipid profiles). Additional relevant clinical trials are required to confirm these results. TRIAL REGISTRATION: This systematic review and meta-analysis was prospectively registered in the PROSPERO database (CRD42022307396).

Monitoring hormone and small molecule secretion dynamics from islets-on-chip.

Tissue functions such as hormone secretion involve the interplay of multiple chemical signals and metabolic processes over time. Measuring the different components involved is useful in unraveling the interactions, but often requires use of multiple analytical techniques. The challenge of measuring the necessary components with temporal resolution is greater when tissue samples are limited. Here, an accessible microfluidic platform compatible with multiple measurement techniques to monitor cell secretions has been developed. The platform is applied to islets of Langerhans, micro-organs involved in glucose homeostasis and diabetes. The device houses 1 to 8 islets and the perfusion fluid can be controlled to change conditions, e.g., glucose concentration, in seconds. Samples are collected in fractions and split for offline analysis. The device is paired with a scaled-down immunoassay, AlphaLISA, for hormone quantification and liquid chromatography-mass spectrometry for small molecule quantification to study secretion dynamics. The combined system allows the first simultaneous measurement of insulin, glucagon, biogenic amines, and amino acids from islet secretions. The combined measurements revealed correlation in secretion events and differences in timing of release between hormones and biogenic amines and amino acids. These efforts decreased the number of islets required compared to standard approaches, thus decreasing necessary animal use, reagent use, and cost, while increasing information content achievable from one sample. The microfluidic device is a suitable platform for in-depth characterization of secretion from small tissue samples.

Pitfalls in Diagnosing Hypoglycemia Due to Exogenous Insulin: Validation and Utility of an Insulin Analog Assay.

OBJECTIVE: To overcome the limitations of commercially available insulin immunoassays which have variable detection of analog insulin and can lead to clinically discordant results and misdiagnosis in the workup of factitious hypoglycemia. PATIENTS AND METHODS: We performed analytical validation of a liquid chromatography high resolution accurate mass (LC-HRAM) immunoassay to detect insulin analogs. We completed clinical assessment using a large cohort of human serum samples from 78 unique individuals, and subsequently used the assay in the evaluation of eight individuals with high diagnostic suspicion for factitious hypoglycemia. RESULTS: The performance characteristics show that the LC-HRAM immunoassay can be applied to detect five commonly used synthetic insulin analogs (lispro, glulisine, aspart, glargine metabolite, and detemir) in human serum. Our clinical cases show that this assay could be used in the diagnosis of factitious hypoglycemia by identifying the analog insulin(s) in question. CONCLUSION: The LC-HRAM immunoassay reported here overcomes a gap in our diagnostic pathway for hypoglycemia. The results obtained from our studies suggest that this method is appropriate for use in clinical laboratories when factitious hypoglycemia is considered as a differential diagnosis.

Diabetes Mellitus gestacional: perfil epidemiológico de maternidade de alto risco / Gestational Diabetes Mellitus: epidemiological profile of high- risk maternity / Diabetes Mellitus gestacional: perfil epidemiológico de una maternidad de alto riesgo

O Diabetes Mellitus Gestacional é definido como doença que se caracteriza pelos altos níveis de glicemia sanguínea, diagnosticada durante a gestação. Este adoecimento pode acarretar várias complicações maternas e fetais, muitas vezes, necessitando de internamento precoce e cuidados avançados. Objetivou-se caracterizar o perfil epidemiológico de gestantes com diabetes mellitus gestacionais atendidas em serviço de referência. Trata-se de estudo descritivo, documental, retrospectivo, de caráter quantitativo, realizado com gestantes atendidas na maternidade do Hospital Regional do Sudoeste ­ PR, Francisco Beltrão. A amostra foi constituída por 216 gestantes, cujos dados foram coletados dos prontuários das pacientes. Incluíram-se as gestantes atendidas e diagnosticadas com diabetes mellitus gestacional no período de 2020 e com pelo menos um exame de glicose em jejum ou um teste de tolerância oral à glicose para comprovação diagnóstica. Foram exclusas as gestantes dos anos de 2019 e 2021 e oito transferências. A amostra teve maior porcentual do Diabetes mellitus gestacional (90,7%), com prevalência na raça branca (69,9%), faixa etária de 15- 35 anos (68,5%). Ademais,65,7% realizaram controle com dieta e 32,4 % necessitaram realizar o uso de insulina e 51,9%delas eram obesas. A presente pesquisateve considerável relevância, pois permitiu obter perfil epidemiológico de gestantes diagnosticadas com diabetes mellitus, trazendo benefícios, como identificação precocemente da doença, de modo a evitar complicações para gestantes e bebês. PALAVRAS-CHAVE: Gestacional; Diabetes; Prevalência; Maternidade.

Gestational Diabetes Mellitus is defined as a disease characterized by high levels of blood glucose, which is diagnosed for the first time during pregnancy. It can cause several maternal and fetal complications, often requiring early hospitalization and advanced care. The aim of thestudy was to characterize the epidemiological profile of pregnant women with gestational diabetes mellitus seen at a reference service. This is a descriptive, documentary, retrospective, quantitative study, carried out with pregnant women attended at the maternity hospital of the Hospital Regional do Sudoeste - PR in the city of Francisco Beltrão. The sample consisted of216 pregnant women, and data were collected from the patients' medical records. The study included all pregnant women who were attended and diagnosed with GDM in the period described, and who had at least one fasting glucose test or an oral glucose tolerance test for diagnostic confirmation. All pregnant women from the year 2019 and 2021 were excluded fromthe study. The sample had a higher percentage of GDM 90.7% according to race 69.9% werewhite, aged 15-35 years 68 , 5%, while 65.7% performed control with diet and 32.4% neededto use insulin and 51.9% of them were obese. This research had great results because it had an epidemiological profile of pregnant women diagnosed with Diabetes Mellitus, bringing benefitsand thus being able to identify gestational Diabetes mellitus early, aiming to avoid complications for the pregnant woman and the baby.

La diabetes mellitus gestacional se define como una enfermedad caracterizada por niveles elevados de glucosa en sangre, diagnosticada durante el embarazo. Esta enfermedad puede dar lugar a varias complicaciones maternas y fetales, que a menudo requieren una hospitalización temprana y cuidados avanzados. El objetivo es caracterizar el perfil epidemiológico de las gestantes con diabetes mellitus atendidas en el servicio de referencia. Se trata de un estudio descriptivo, documental, retrospectivo, de carácter cuantitativo, realizado con gestantes atendidas en la maternidad del Hospital Regional del Sudoeste - PR, Francisco Beltrão. La muestra estaba formada por 216 mujeres embarazadas, cuyos datos se recogieron de las historias clínicas de las pacientes. Se incluyeron las mujeres embarazadas atendidas y diagnosticadas de diabetes mellitus gestacional en el periodo 2020 y con al menos una prueba de glucosa en ayunas o una prueba de tolerancia oral a la glucosa para su diagnóstico. Se excluyeron las embarazadas de los años 2019 y 2021 y ocho traslados. La muestra tuvo un mayor porcentaje de Diabetes mellitus gestacional (90,7%), con prevalencia en la raza blanca (69,9%), grupo de edad 15-35 años (68,5%). Además, el 65,7% se controlaba con la dieta y el 32,4% necesitaba utilizar insulina y el 51,9% era obeso. La presente investigación tiene una relevancia considerable, ya que permite obtener el perfil epidemiológico de las gestantes diagnosticadas con diabetes mellitus, lo que conlleva beneficios, como la identificación precoz de la enfermedad, a fin de evitar complicaciones para las gestantes y los bebés.

Quantitative 3D OPT and LSFM datasets of pancreata from mice with streptozotocin-induced diabetes.

Mouse models for streptozotocin (STZ) induced diabetes probably represent the most widely used systems for preclinical diabetes research, owing to the compound's toxic effect on pancreatic ß-cells. However, a comprehensive view of pancreatic ß-cell mass distribution subject to STZ administration is lacking. Previous assessments have largely relied on the extrapolation of stereological sections, which provide limited 3D-spatial and quantitative information. This data descriptor presents multiple ex vivo tomographic optical image datasets of the full ß-cell mass distribution in mice subject to single high and multiple low doses of STZ administration, and in glycaemia recovered mice. The data further include information about structural features, such as individual islet ß-cell volumes, spatial coordinates, and shape as well as signal intensities for both insulin and GLUT2. Together, they provide the most comprehensive anatomical record of the effects of STZ administration on the islet of Langerhans in mice. As such, this data descriptor may serve as reference material to facilitate the planning, use and (re)interpretation of this widely used disease model.

Quantitative, in situ visualization of intracellular insulin vesicles in pancreatic beta cells.

Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.

The latest trends in the design of electrochemical biosensors for the diagnosis and monitoring of diabetes mellitus.

RESULTS: The overworld health problem, the incurable disease, the global burden on health insurers and society, and above all one of the leading causes of death - all characterize diabetes mellitus, a lifelong chronic disease that affects hundreds of millions of people around the world. The new types of biosensors bring new opportunities in the care of diabetic patients and improve current methods. The practical relevance of the recent findings is expected in medicine in next years. CONCLUSIONS: The authors summarized the modern possibilities of biosensing, their pros and cons, and their perspectives for the future. The discussion outcome from the current literature (Tab. 4, Fig. 1, Ref. 63).

Serum Orosomucoid Is Associated with Serum Adiponectin, Adipose Tissue Insulin Resistance Index, and a Family History of Type 2 Diabetes in Young Normal Weight Japanese Women.

INTRODUCTION: Adipose tissue (AT) expandability may be facilitated by adiponectin and suppressed by orosomucoid, and reduced AT expandability may be associated with first-degree relatives of type 2 diabetes. We tested the hypothesis that orosomucoid may be associated not only with adiponectin and adipose tissue insulin resistance but also with a family history of type 2 diabetes (FHD). Research Design and Methods. Anthropometric and metabolic variables, adipokines, and measures of inflammatory and insulin resistance were cross-sectionally investigated in 153 young normal weight Japanese women. Stepwise multivariate linear regression analyses were used to identify the most important determinants of orosomucoid. RESULTS: Orosomucoid was higher in women with positive (n = 57) compared to women with negative FHD and was associated positively with FHD (both p = 0.01). Orosomucoid also showed positive associations with fasting glucose (p < 0.001), free fatty acids (p = 0.001), and HbA1c (p = 0.007), whereas there was no association with fasting insulin and serum lipids. In addition, orosomucoid was associated inversely with adiponectin (p = 0.02) and positively with adipose tissue-insulin resistance index (AT-IR, the product of fasting insulin and free fatty acids; p = 0.001) but not with homeostasis model assessment-insulin resistance, leptin, and high-sensitivity C-reactive protein. In multivariate analyses, AT-IR (standardized ß, 0.22; p = 0.003), serum adiponectin (standardized ß, -0.163; p = 0.032), FHD+ (standardized ß, 0.178; p = 0.029), and HbA1c (standardized ß, 0.213; p = 0.005) emerged as independent determinants of orosomucoid and explained 15.2% of its variability. CONCLUSIONS: These results are the first to demonstrate that orosomucoid is associated not only with adipose tissue-insulin resistance and adiponectin but also with FHD.

Gain of Function of Malate Dehydrogenase 2 and Familial Hyperglycemia.

CONTEXT: Genes causing familial forms of diabetes mellitus are only partially known. OBJECTIVE: We set out to identify the genetic cause of hyperglycemia in multigenerational families with an apparent autosomal dominant form of adult-onset diabetes not due to mutations in known monogenic diabetes genes. METHODS: Existing whole-exome sequencing (WES) data were used to identify exonic variants segregating with diabetes in 60 families from the United States and Italy. Functional studies were carried out in vitro (transduced MIN6-K8 cells) and in vivo (Caenorhabditis elegans) to assess the diabetogenic potential of 2 variants in the malate dehydrogenase 2 (MDH2) gene linked with hyperglycemia in 2 of the families. RESULTS: A very rare mutation (p.Arg52Cys) in MDH2 strongly segregated with hyperglycemia in 1 family from the United States. An infrequent MDH2 missense variant (p.Val160Met) also showed disease cosegregation in a family from Italy, although with reduced penetrance. In silico, both Arg52Cys and Val160Met were shown to affect MDH2 protein structure and function. In transfected HepG2 cells, both variants significantly increased MDH2 enzymatic activity, thereby decreasing the NAD+/NADH ratio-a change known to affect insulin signaling and secretion. Stable expression of human wild-type MDH2 in MIN6-K8 cell lines enhanced glucose- and GLP-1-stimulated insulin secretion. This effect was blunted by the Cys52 or Met160 substitutions. Nematodes carrying equivalent changes at the orthologous positions of the mdh-2 gene showed impaired glucose-stimulated insulin secretion. CONCLUSION: Our findings suggest a central role of MDH2 in human glucose homeostasis and indicate that gain of function variants in this gene may be involved in the etiology of familial forms of diabetes.

Human Milk Lactose, Insulin, and Glucose Relative to Infant Body Composition during Exclusive Breastfeeding.

Human milk (HM) components may influence infant growth and development. This study aimed to investigate relationships between infant body composition (BC) and HM lactose, insulin, and glucose (concentrations and calculated daily intakes (CDI)) as well as 24-h milk intake and maternal BC at 3 months postpartum. HM samples were collected at 2 months postpartum. Infant and maternal BC was assessed with bioimpedance spectroscopy. Statistical analysis used linear regression accounting for infant birth weight. 24-h milk intake and CDI of lactose were positively associated with infant anthropometry, lean body mass and adiposity. Higher maternal BC measures were associated with lower infant anthropometry, z-scores, lean body mass, and adiposity. Maternal characteristics including BC and age were associated with concentrations and CDI of HM components, and 24-h milk intake. In conclusion, 24-h intake of HM and lactose as well as maternal adiposity are related to development of infant BC.

Insulin stimulates ß-alanine uptake in skeletal muscle cells in vitro.

We evaluated whether insulin could stimulate ß-alanine uptake by skeletal muscle cells in vitro. Mouse myoblasts (C2C12) (n = 3 wells per condition) were cultured with ß-alanine (350 or 700 µmol·L-1), with insulin (100 µU·mL-1) either added to the media or not. Insulin stimulated the ß-alanine uptake at the lower (350 µmol·L-1) but not higher (700 µmol·L-1) ß-alanine concentration in culture medium, indicating that transporter saturation might blunt the stimulatory effects of insulin.

Thermoultrasonication, ultraviolet-C irradiation, and high-pressure processing: Novel techniques to preserve insulin in donor human milk.

BACKGROUND & AIMS: Donor human milk (DHM) is recommended as the first alternative for preterm infants if their mother's own milk is not available or if the quantity is not sufficient. The most commonly used technique to eliminate microbial contaminants in DHM is holder pasteurization (HoP). However, the heating process during HoP partially destroys milk bioactive factors such as insulin. Therefore, innovative techniques have been developed as alternatives to HoP. The objective of this study was to determine the effect of HoP, high-temperature-short-time (HTST), thermoultrasonication (TUS), ultraviolet-C irradiation (UV-C), and high-pressure processing (HPP) on the insulin concentration in DHM. METHODS: Milk samples from 28 non-diabetic mothers were collected. The milk samples were aliquoted and either left untreated or treated with HoP (62.5 °C; 30 min), HTST (72 °C; 15 s), TUS (60 W; 6 min), UV-C (4863 J/L), or HPP (500 MPa; 5 min). RESULTS: The mean insulin concentration in untreated milk was 79 ± 41 pmol/L. The mean insulin retention rate was 67% for HoP, 78% for HTST, 97% for TUS, 94% for UV-C, and 106% for HPP. The mean insulin concentration in milk treated with HoP was significantly lower compared to untreated milk (p = 0.01). CONCLUSION: TUS, UV-C, and HPP preserve insulin in DHM. The insulin concentration in DHM is affected to a larger extent by HoP than by HTST. These results indicate that TUS, UV-C, and HPP may serve as alternatives to HoP.

Insulin-Binding Peptide Probes Provide a Novel Strategy for Pancreatic ß-Cell Imaging.

Type 1 diabetes develops in childhood and adolescence, with peak incidence in the early teenage years. There is an urgent need for an accurate method to detect insulin-producing ß-cells in patients that is not affected by alterations in ß-cell function. As part of our research program to design specific probes to measure ß-cell mass, we recently developed a novel insulin-binding peptide probe (IBPP) for the detection of ß-cells in vivo. Here, we applied our innovative method to show specific labeling of this IBPP to human and mouse fixed ß-cells in pancreatic islets. Importantly, we showed staining of human and mouse islets in culture without any negative functional or cell viability impact. Moreover, the IBPP-stained mouse islets after tail vein injection in vivo, albeit with batch differences in staining efficiency. In conclusion, we provide evidence showing that the IBPP can be used for future accurate detection of ß-cell mass in a variety of preclinical models of diabetes.

Novel Human Insulin Isoforms and Cα-Peptide Product in Islets of Langerhans and Choroid Plexus.

Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells, where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains ß-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP, and its amyloid formation was inhibited in vitro more efficiently by Cα-peptide than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase-processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus autopsy donors. Intriguingly, 100 years after the discovery of insulin, we found that INS isoforms are present in ChP from insulin-deficient autopsy donors.

Glucose Tolerance after Pancreatectomy: A Prospective Observational Follow-Up Study of Pancreaticoduodenectomy and Distal Pancreatectomy.

BACKGROUND: Effects of pancreatectomy on glucose tolerance have not been clarified, and evidence regarding the difference in postoperative glucose tolerance between pancreaticoduodenectomy (PD) and distal pancreatectomy (DP) is lacking. STUDY DESIGN: This prospective, single-center observational study analyzed 40 patients undergoing PD and 29 patients undergoing DP (Clinical trial registry number UMIN000008122). Glucose tolerance, including insulin secretion (Δ C-peptide immunoreactivity, ΔCPR) and insulin resistance (homeostasis model assessment of insulin resistance, HOMA-IR) were assessed before and 1 month after pancreatectomy using the oral glucose tolerance test (OGTT) and glucagon stimulation test. We assessed long-term hemoglobin A1c (HbA1c) levels in patients, with a follow-up time of 3 years. RESULTS: Percentages of patients diagnosed with abnormal OGTT decreased after PD (from 12 [30%] to 7 [17.5%] of 40 patients, p = 0.096); however, they increased after DP (from 4 [13.8%] to 8 [27.6%] of 29 patients, p = 0.103), although the changes were not statistically significant. ΔCPR decreased after both PD (from 3.2 to 1.0 ng/mL, p < 0.001) and DP (from 3.3 to 1.8 ng/mL, p < 0.001). HOMA-IR decreased after PD (from 1.10 to 0.68, p < 0.001), but did not change after DP (1.10 and 1.07, p = 0.42). Median HbA1c level was higher after DP than after PD for up to 3 years, but the differences were not statistically significant. CONCLUSIONS: In comparisons of pre- and 1 month post-pancreatectomy data, glucose tolerance showed improvement after PD, whereas it worsened after DP. Insulin secretion decreased after both PD and DP. Insulin resistance improved after PD, but did not change after DP. Further studies are warranted to clarify mechanisms of improved insulin resistance after PD.