Diabetic Human Subjects Research Articles

Enhancing the accuracy of blood-glucose tests by upgrading FTIR with multiple-reflections, quantum cascade laser, two-dimensional correlation spectroscopy and machine learning

The accuracy of screening diabetes from non-diabetes is drastically enhanced by strategically upgrading the bench-marking infrared spectroscopy technique for non-invasive tests of blood-glucose, both with state-of-the-art instrumentation-retrofits and with intelligent spectral-datamining tools. First, the signal-to-noise performance of FTIR in measuring the spectral features of a glucose solution containing bovine serum albumin is improved by 2–3 times with the common single-pass attenuated total-reflection setup replaced by a multi-passes-reflections setup. Second, replacing the ordinary infrared lamp with a quantum cascade laser further improves the signal-to-noise by 3 times. The performance of the upgraded spectrometer in screening hyperglycemia is gauged by the accuracy of such screens derived from 100 repetitive spectral-measurements per glucose concentration, for 24 glucose concentrations spanning the range of 70–300 mg/dL, a range which covers the blood-glucose contents of all non-diabetic and diabetic human-subjects. Third, intelligent datamining methods are exploited to implement decision trees for screening hyperglycemia. Their decisions are mapped into a confusion matrix and the matrix-elements are used to calculate the accuracy merits of each method. Evidently, the accuracy of the multi-passes-FTIR with the standard principal-components datamining method is 80 %. The adoptions of the quantum cascade laser and two-dimensional correlation spectroscopy datamining technique raises this to 96.3 %. Finally, a novel machine learning method, which comprises three different decision-tree tools to generate trial screening decisions and a “majority-voting” datamining tool to reach a final screening decision, yields the best accuracy of 98.8 % ever reported in the literature.

The influence of insulin and incretin-based therapies on renal tubular transport.

The tubular function of the kidney is very complex and is finely regulated by many factors. These include a variety of hormonal signaling pathways which are involved in the expression, activation and regulation of renal transporters responsible for the handling of electrolytes. Glucose-lowering drugs such as insulin and incretin-based therapies, exert a well-known renal protective role in diabetic kidney disease, mainly acting at the glomerular level. In the literature, several studies have described the effect of insulin and the incretin hormones on tubular transport. Most of these studies focused on the variations in excretion and clearance of sodium but did not extensively and systematically investigate the possible variations that these hormones may induce in the tubular regulation of all the other electrolytes, urea metabolism, acid-base balance and urinary pH. While insulin action on the kidney is very well-described, the renal tubular impact of incretin-based therapies is less consistent and the results available are scarce. To our knowledge, this is the first review summarizing the effects induced on renal tubules by insulin, glucagon-like peptide-1 (GLP-1) receptor agonists and serine protease dipeptidyl peptidase-4 (DPP4) inhibitors in both healthy and diabetic human subjects. This is significant because it highlights the existence of a renal-gut and pancreas axis which also has a direct tubular effect and enables a deeper understanding of renal physiology.

CREB activates the MafA promoter through proximal E-boxes and a CCAAT motif in pancreatic β-cells.

MafA is a key transcriptional regulator of pancreatic islet β-cell function. Its target genes include those encoding preproinsulin and the glucose transporter Glut2 (Slc2a2); thus, MafA function is essential for glucose-stimulated insulin secretion. Expression levels of MafA are reduced in β-cells of diabetic mouse models and human subjects, suggesting that β-cell dysfunction associated with type 2 diabetes is attributable to the loss of MafA. On the other hand, MafA is transcriptionally upregulated by incretin hormones through activation of CREB and its co-activator CRTC2. β-cell-specific expression of MafA relies on a distal enhancer element. However, the precise mechanism by which CREB-CRTC2 regulates the enhancer and proximal promoter regions of MafA remains unclear. In this report, we analyzed previously published ChIP-seq data and found that CREB and NeuroD1, a β-cell-enriched transactivator, bound to both the promoter and enhancer regions of human MAFA. A series of reporter assays revealed that CREB activated the enhancer through a conserved cAMP-responsive element (CRE) but stimulated MAFA promoter activity even when the putative CRE was deleted. Two E-box elements and a CCAAT motif, which bind NeuroD1 and ubiquitous NF-Y transcription factors, respectively, were necessary for transcriptional activation of the MAFA promoter by CREB. Genome-wide analysis of CREB-bound loci in β-cells revealed that they were enriched with CCAAT motifs. Furthermore, promoter analysis of the Isl1 gene encoding a β-cell-enriched transcription factor revealed that a CRE-like element and two CCAAT motifs, but not the E-box, were necessary for activation by CREB. These results provide clues to elucidate the detailed mechanism by which CREB regulates MafA as well as β-cell-specific genes.

Adropin Treatment Restores Cardiac Function and Metabolic Flexibility in Diabetic Cardiomyopathy

Background: Diabetic cardiomyopathy (DCM) is a major complication of diabetes, and has been recognized as a cause of heart failure independent of other common risk factors. Metabolic inflexibility is a hallmark feature, where increased free fatty acid availability and decreased myocardial glucose uptake lead to an over-reliance on fatty acid oxidation, reducing cardiac work effciency. Energetic ineffciency in diabetic hearts may have profound implications for cardiac function under conditions of increased workload, and therefore therapeutic approaches are warranted. Adropin is a liver- and brain-secreted peptide hormone shown to regulate fuel metabolism in the heart. Adropin levels are significantly reduced in obese and diabetic human subjects, and this decrease is linked to increased adiposity, insulin resistance, and impaired glucose tolerance. Our lab recently showed that acute adropin treatment restores glucose oxidation activity in the hearts of prediabetic obese mice. However, the effects of chronic adropin exposure in the heart remains unknown. Hypothesis: We hypothesize that adropin treatment will exhibit beneficial effects on cardiac fuel metabolism that may lead to long-term improvements in functional output. Methods: We used a preclinical model of DCM to investigate the effects of long-term adropin treatment on cardiac structural and metabolic remodeling. Mice were treated with adropin daily for four weeks, and subjected to echocardiography, glucose tolerance tests, histopathology, RNA-sequencing, and metabolomics. Results: We report that long-term adropin treatment restores normal cardiac structure and metabolic function in the diabetic heart, by inhibiting flux of non-oxidative glycolytic intermediates into the hexosamine biosynthetic pathway. Conclusions: Our findings highlight the therapeutic potential of adropin signaling to attenuate cardiac remodeling in DCM. The project described was supported by NIDDK 1F31DK134089 and NHLBI R01HL147861. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High-protein, low glycemic index snack from optimized blend of three wholegrains exhibits nutraceutical quality and elicits low glycemic response in diabetic human subjects

Snack products are evolving as new carriers of functional ingredients with nutritional and health-promoting benefits. A blend of whole grains is increasingly being utilized to harness the functional potential of the grain mix. Amaranth, acha, and pearl millet grains flours were optimized using response surface methodology (RSM), to obtain optimum blends (90:5:5 and 47.98:26.68:25.34) with high protein content and low glycemic index. Snack bar products from the blends were labelled MBY and MBZ. A total of 40 diabetic and 10 non-diabetic subjects were recruited. Of the diabetic, about 42% were overweight while 40% were obese, the non-diabetic had normal weights. Each was allowed to consume snacks containing the equivalent of 50 g of carbohydrates. Finger prick was employed to evaluate the postprandial glucose response of snack products while venous blood was evaluated for antioxidant enzymes, carbohydrate-hydrolyzing activities, and insulin using standard methods. Consumption of the multigrain snacks elicited a stable postprandial response (133–141 mg/dL) with 16 and 24% postprandial decline. In addition, snacks had low to intermediate glycemic index (52 and 56) in diabetic and low glycemic index (43 and 45) in non-diabetics; likewise reduced α-amylase/α-glucosidase activities compared to control snacks. Similarly, glutathione level, glutathione peroxidase, superoxide dismutase, and catalase activities in serum from subjects that consumed multigrain snacks were upregulated compared to control and market sample groups. Moreso, snack products promoted a reduction in serum insulin levels in diabetic subjects (45 and 17% for MBY and MBZ respectively). Following the nutraceutical properties displayed by the formulated snack especially MBY, it can be promoted as a functional snack for the management of diabetes while solving the limited snack product choice of diabetes sufferers.Graphical

Hypoglycemic Potential of Wild Plants in Diabetic and Non-Diabetic Human Subjects: Double-blind Randomized-controlled Trials

Hypoglycemic Potential of Wild Plants in Diabetic and Non-Diabetic Human Subjects: Double-blind Randomized-controlled Trials

322-OR: Proinflammatory Macrophage Infiltration in Sural Nerve Is Associated with Reduction of Myelinated Nerve Fiber Density but Not IENFD in Human Diabetic Subjects

Background: The etiology of diabetic polyneuropathy (DPN) is multifactorial, and other than hyperglycemia, circulatory disturbance such as hypertension can be involved. Pro-inflammatory macrophages (M1) infiltration in the peripheral nerve is known to be the pathogenesis of DPN. However, it is still unclear how the inflammation and circulatory disturbance are involved in the pathological changes of human DPN. Methods: Autopsied cases at Hirosaki University Hospital were evaluated: 7 nondiabetic cases (nDM), 11 nondiabetic cases with hypertension (nDM+HT), 6 diabetes cases (DM), 9 cases with hypertension and diabetes (DM+HT). Intraepidermal nerve fiber density (IENFD) was examined by immunofluorescent staining with the skin tissue 10 cm above external condyle. Dissected sural nerves were pathologically examined. Macrophage infiltration into the endoneurium of sural nerves was evaluated by double immunostaining using anti-CD68 and anti-CD163 antibodies. Results: HbA1c level was significantly higher in DM and DM+HT than in nDM (p<0.01, respectively). IENFD was significantly decreased in DM compared to nDM (p<0.05) and further decreased in DM+HT (p<0.05 vs DM). The infiltration of M1 was significantly increased in DM (p<0.05). M1 infiltration was comparable between DM and DM+HT. Regression analysis showed that the M1/anti-inflammatory macrophages (M2) ratio was not significantly correlated with IENFD (r=0.3, p=0.09). Morphometric measurements of the sural nerve using semithin sections revealed that myelinated nerve fiber density (MNFD) was significantly decreased in DM compared with nDM (p<0.05). MNFD was comparable between DM and DM+HT. MNFD was inversely correlated with M1/M2 ratio (r=0.37, p<0.05). Conclusions: Our results suggest that the factors other than sural nerve inflammation, such as hypertension, are possibly more involved in the loss of IENFD in DPN. Disclosure H. Mizukami: None. S. Osonoi: None. Y. Takeuchi: None. T. Sasaki: None.

Hypoglycemic Potential of African Medicinal Plants in Diabetic and Non-Diabetic Human Subjects: A Review

Africa is blessed with several plants and marine organisms used to treat various diseases including diabetes. The folkloric use of some of these plants in treating diabetes has been documented in various databases and publications, and validated using various diabetes models. However, most of the investigations on African medicinal plants as antidiabetic agents are not taken beyond proof-of-concept stage. Besides, limited number of clinical trials were conducted to validate the efficacy in humans and the few available clinical studies have not been critically reviewed. Therefore, this review is intended to document the antidiabetic potential of African medicinal plants investigated in human subjects as well their safety implications. Relevant articles were harvested from the major scientific databases (Scopus, PubMed, and Google Scholar). According to the present search, eighteen (18) African medicinal plants and one herbal formulation were the only plants investigated for antidiabetic activity in humans. The plants showed no noticeable toxicity throughout the study period. Interestingly, all the 18 plant species were traditionally used to treat diabetes in various parts of Africa. Despite variations in the study period and dosages used, Laportea ovalifolia (Schumach. & Thonn.) Chew. (Urticaceae) and Momordica charantia L. (Cucurbitaceae) were the most effective as they reduced fasting blood glucose by >50% after 10 days and 4 weeks treatment periods, respectively. The available information revealed that African medicinal plants especially L. ovalifolia and M. charantia should be subjected to phase III clinical trial, aimed at getting the natural product-based antidiabetic drugs from Africa.

Abstract 546: MicroRNA-1282 As A Cis-Antisense Regulator Of Angiogenesis In Diabetic Critical Limb Ischemia

Introduction: Peripheral artery disease (PAD) is an atherosclerotic occlusive disease that leads to reduced blood flow to the lower extremities and endothelial dysfunction. Some patients develop critical limb ischemia (CLI), a severe manifestation of PAD defined as chronic ischemic rest pain with higher risk of amputation. Major risk factors for both PAD and CLI is type 2 diabetes. However, the pathophysiological contribution of microRNAs (miRNAs), in diabetic CLI progression remains poorly elucidated. Methods: By overlapping plasma miRNA sequencing in PAD patients that develop CLI with a mouse model, miRNAs were identified in the progression of diabetic limb ischemia. In this study, we describe miR-1282, a previously uninvestigated miRNA that was the highest upregulated miRNA in high-risk diabetic CLI human subjects. Results: Preliminary studies uncover a mouse ortholog of human miR-1282, a cis-antisense miRNA, that strongly inhibits the expression of SERF2 (small EDRK-rich factor 2), a protein-coding gene located on the opposite DNA strand in both human and mouse endothelial cells. SERF2 is predicted to affect the formation of aggresomes, the inclusion bodies of misfolded proteins; however, its molecular function is also poorly understood. MiR-1282 expression was decreased by diabetic stimuli such as hypoxia and glucose; and its expression inversely correlated with SERF2 expression in endothelial cells. Our in vitro studies indicate that miR-1282 mimic overexpression or SERF2 siRNA-mediated inhibition reduced a range of endothelial angiogenic assays and inhibits apoptosis. Proteomics by tandem mass-tag mass-spectrometry and transcriptomics by RNA-Seq suggest that miR-1282 likely regulates protein quality control system (SERF2, BAG1, BAG3, and HSPB8) and regulated cell death (CASP3, SLC7A11, and CTH), among others. Intriguingly, intramuscular administration of miR-1282 significantly improved blood flow recovery by laser doppler imaging in the hindlimbs of diabetic db/db mice compared to control injected mice. Conclusion: Taken together, the cis-antisense regulatory network of miR-1282-SERF2 axis reveals novel insights in the progression of diabetic CLI.

Profiling of G-Protein Coupled Receptors in Adipose Tissue and Differentiating Adipocytes Offers a Translational Resource for Obesity/Metabolic Research.

G protein-coupled receptors (GPCRs) are expressed essentially on all cells, facilitating cellular responses to external stimuli, and are involved in nearly every biological process. Several members of this family play significant roles in the regulation of adipogenesis and adipose metabolism. However, the expression and functional significance of a vast number of GPCRs in adipose tissue are unknown. We used a high-throughput RT-PCR panel to determine the expression of the entire repertoire of non-sensory GPCRs in mouse white, and brown adipose tissue and assess changes in their expression during adipogenic differentiation of murine adipocyte cell line, 3T3-L1. In addition, the expression of GPCRs in subcutaneous adipose tissues from lean, obese, and diabetic human subjects and in adipocytes isolated from regular chow and high-fat fed mice were evaluated by re-analyzing RNA-sequencing data. We detected a total of 292 and 271 GPCRs in mouse white and brown adipose tissue, respectively. There is a significant overlap in the expression of GPCRs between the two adipose tissue depots, but several GPCRs are specifically expressed in one of the two tissue types. Adipogenic differentiation of 3T3-L1 cells had a profound impact on the expression of several GPCRs. RNA sequencing of subcutaneous adipose from healthy human subjects detected 255 GPCRs and obesity significantly changed the expression of several GPCRs in adipose tissue. High-fat diet had a significant impact on adipocyte GPCR expression that was similar to human obesity. Finally, we report several highly expressed GPCRs with no known role in adipose biology whose expression was significantly altered during adipogenic differentiation, and/or in the diseased human subjects. These GPCRs could play an important role in adipose metabolism and serve as a valuable translational resource for obesity and metabolic research.

Epigenetic Modifications in Obesity and Type 2 Diabetes

Obesity is a chronic condition that is also a risk factor of several other chronic conditions including type 2 diabetes. The effects of maternal obesity and type 2 diabetes on fetal development and offspring health are mediated through the transmission of epigenetic modifications in addition to the possible permanent changes of the organs caused by the intrauterine environment hypothesized by the Developmental Origins of Health and Disease (DOHaD) theory. Epigenetic modifications can be altered by environmental factors including dietary and lifestyle factors. The current priorities include identification and confirmation of the specific epigenetic biomarkers associated with obesity and type 2 diabetes in human subjects and identification of the dietary and lifestyle factors that contribute to each of the identified specific epigenetic biomarkers.

Abstract P3050: The Adropin-GPR19 Signaling Axis In Diabetic Cardiomyopathy

Background: Diabetic cardiomyopathy (DCM) is a major complication of diabetes and has been recognized as a cause of heart failure independent of other common risk factors for cardiac disease. In DCM, increased free fatty acid availability and decreased myocardial glucose uptake lead to a reliance on fatty acid oxidation for ATP generation. This loss of metabolic flexibility can reduce cardiac plasticity in response to stress. Energetic inefficiency in diabetic hearts may have profound implications for cardiac function under conditions of increased workload, and therefore therapeutic approaches are warranted. Alleviating metabolic inflexibility, by promoting cardiac glucose utilization, has been proposed as a potential strategy to reverse cardiomyopathy in diabetic and obese patients. Adropin is a liver- and brain-secreted peptide hormone shown to regulate fuel metabolism in a number of tissues, including the heart. Adropin levels are significantly reduced in obese and diabetic human subjects, and this decrease is linked to increased adiposity, insulin resistance, and impaired glucose tolerance. Our lab recently showed that adropin treatment restores glucose oxidation activity in vivo by reducing the expression of the mitochondrial acetyltransferase enzyme GCN5L1. The reduction in GCN5L1 abundance altered the acetylation and activity of fuel metabolism enzymes to favor glucose utilization; however, the mechanism that underlies this effect remains unknown. Our data suggests that the class A orphan G-coupled protein receptor, GPR19, acts as a putative cellular receptor for adropin in cardiac cells to regulate mitochondrial bioenergetic output. Hypothesis: We hypothesize that adropin-GPR19 signaling regulates GCN5L1 expression, which in turn promotes the deacetylation and activity of pyruvate dehydrogenase to restore glucose utilization in the diabetic heart. Methods: We used a novel whole-body GPR19 knockout mouse to perform a preliminary characterization of the effects of GPR19 depletion on cardiac and whole-body physiology. Using in vivo metabolic approaches, we report that GPR19 KO mice exhibit sex-dependent glucose intolerance, and display markers of altered cardiac energy metabolism. Conclusions: These findings further define the fundamental role of the adropin-GPR19 signaling pathway in the regulation of metabolic homeostasis, and thus highlight a potential target for therapeutic interventions in DCM.

Assessment of the risk of developing cardiovascular disease in prediabetic and diabetic subjects with Insulinaemia

Introduction: This examination investigated the risk of developing cardiovascular disease in pre-diabetic and diabetic subjects with insulinaemia. The assessment solidified the human diabetic subjects in the evaluation of the model and took the blood analytes to screen for diabetes. Methodology: 120 male and female human subjects containing forty subjects each for control, pre-diabetics and diabetics facilitated for age, sex, height, weight, and BMI were enrolled into the assessment reliant upon decided measures. All of the three sets of human subjects were males and females independently. Every illustration of blood serum and plasma was explored using Randox and Accubind packs and an autoanalyser to test for various biochemical and hematological limits. Results: The overall results revealed a colossal differentiation (p≤0.05) in the limits except for that of Na+. The 500mg/kg body weight part of the concentrate was ideal while there was a much basic development in the HOMA-IR with potential gains of 0.94±0.04, 2.28±0.17, and 3.25±0.44 for the three sets. The HOMA-IR is an alluring model, as compared to various models previously investigated. This assessment revealed that HOMA-IR works best in the management of diabetes in connection with various models.

Hormonal and Hematologic Profile of Pre-Diabetic and Diabetic Patients in the University of Port Harcourt Teaching Hospital

This examination investigated haematologic and hormonal profile in human diabetic subjects. The assessment solidified the human diabetic subjects and took the blood analytes to screen for diabetes. 120 male and female human subjects containing forty subjects each for control, pre-diabetics, and diabetics (three sets) facilitated for age, sex, height, weight and BMI were enrolled into the assessment reliant upon decided measures. Twenty each of the three sets of human subjects were males and females independently. Every illustration of blood serum and plasma was explored using Randox and Accubind packs and an autoanalyser to test for various biochemical and hematological limits. The overall results revealed a colossal differentiation (p≤0.05) in the limits of the analytes, except for that of Na+. Contributions to Knowledge: Based on the findings, analytes are good for predicting and managing DM, by comparing the blood analytes of non-diabetics, pre-diabetics, and diabetics in order to monitor the onset of diabetes and proffer possible solutions to enhance early detection and manage diabetes.

DNA Methylation Patterning and the Regulation of Beta Cell Homeostasis.

Pancreatic beta cells play a central role in regulating glucose homeostasis by secreting the hormone insulin. Failure of beta cells due to reduced function and mass and the resulting insulin insufficiency can drive the dysregulation of glycemic control, causing diabetes. Epigenetic regulation by DNA methylation is central to shaping the gene expression patterns that define the fully functional beta cell phenotype and regulate beta cell growth. Establishment of stage-specific DNA methylation guides beta cell differentiation during fetal development, while faithful restoration of these signatures during DNA replication ensures the maintenance of beta cell identity and function in postnatal life. Lineage-specific transcription factor networks interact with methylated DNA at specific genomic regions to enhance the regulatory specificity and ensure the stability of gene expression patterns. Recent genome-wide DNA methylation profiling studies comparing islets from diabetic and non-diabetic human subjects demonstrate the perturbation of beta cell DNA methylation patterns, corresponding to the dysregulation of gene expression associated with mature beta cell state in diabetes. This article will discuss the molecular underpinnings of shaping the islet DNA methylation landscape, its mechanistic role in the specification and maintenance of the functional beta cell phenotype, and its dysregulation in diabetes. We will also review recent advances in utilizing beta cell specific DNA methylation patterns for the development of biomarkers for diabetes, and targeting DNA methylation to develop translational approaches for supplementing the functional beta cell mass deficit in diabetes.

Ocular surface complications result from dysregulation of the OGF-OGFr signaling pathway in female diabetic rats.

Approximately 4.5 million women in the United States exhibit diabetes-associated ocular complications. The time course and magnitude of these complications, and their association with the dysregulation of the opioid growth factor (OGF)-OGF receptor (OGFr) signaling pathway are unknown. The present study investigated the onset and magnitude of ocular surface complications and the association with a dysregulated OGF-OGFr signaling pathway in diabetic female rats. Adult female Sprague-Dawley rats were injected with streptozotocin in order to establish a model of type 1 diabetes (T1D), and a subset received insulin (T1D-INS). Blood glucose, body weight, tear production and corneal sensitivity, as well as serum and tissue expression levels of OGF and OGFr, were assessed. Corneal epithelial wound healing was also evaluated. In a second study, female T1D rats were treated with topical naltrexone (NTX) to determine whether blockade of the OGF-OGFr signaling pathway by NTX altered development of corneal surface complications. Female T1D rats had elevated glucose levels and reduced body weight compared with control and T1D-INS rats. In both diabetic groups, tear production was decreased within 2 weeks and corneal sensitivity was decreased 2.5-fold within 5 weeks, while corneal epithelial wound healing was delayed only in T1D rats. Serum and tissue levels of OGF and OGFr were elevated in diabetes. Twice daily NTX treatment reversed most ocular surface complications in the diabetic female rats. The present data demonstrated a seminal discovery in female T1D rats, in which the onset and magnitude of diabetes-associated ocular surface complications were associated with dysregulation of the OGF-OGFr regulatory pathway. Blockade of the OGF-OGFr pathway with the opioid receptor antagonist NTX prevented the onset and/or decreased the magnitude of these deficits. The current data support the need for translational research on this therapeutic approach for diabetic human subjects.

Effects of Chuju Polysaccharides on Glucose Metabolism in Diabetic Mice

Chuju is a special Chinese flower tea with extensive health benefits. In this study, we have examined the effects of crude Chuju polysaccharides on alloxan-induced diabetic mice by measuring fasting blood glucose, insulin-related index, activities of superoxide dismutase, glutathione peroxidase, and content of malondialdehyde in the serum and liver. The results show that the treatment led to increased insulin sensitivity and decreased fasting glucose. The blood and liver levels of malondialdehyde decreased, whereas superoxide dismutase and glutathione peroxidase levels were elevated by crude Chuju polysaccharides in alloxan-induced diabetic mice. These data support a basis for clinical investigations on crude Chuju polysaccharides in diabetic human subjects.

Bone Morphogenetic Protein-2 Induces Non-Canonical Inflammatory and Oxidative Pathways in Human Retinal Endothelial Cells.

The mechanisms of diabetic retinopathy (DR), are not yet fully understood. We previously demonstrated an upregulation of retinal bone morphogenetic protein-2 (BMP2) in experimental diabetes and in retinas of diabetic human subjects. The purpose of current study was to investigate the role of non-canonical inflammatory pathway in BMP2-induced retinal endothelial cell (REC) barrier dysfunction. For this purpose, we used RT-PCR and western blotting to evaluate the levels of BMP2 signaling components (BMP2, BMP4, BMP receptors), VEGF, phosphorylated p38 MAPK and NFκB, and oxidative stress markers in cultured human retinal endothelial cells (HRECs) subjected to BMP2 (50ng/ml) for up to 24 h. Also, effect of high glucose (HG, 30mM D-glucose) on the expression of BMP2 and its downstream genes was examined in HRECs. H2-DCF is a fluorogenic dye that measures the levels of cellular reactive oxygen species (ROS) was used to measure the pro-oxidative effect of BMP2. Moreover, we evaluated the effect of inhibiting p38 and VEGF signaling on BMP2-induced HRECs barrier dysfunction by measuring the trans-endothelial cell electrical resistance (TER) using electric cell-substrate impedance sensing (ECIS). We also tested the effect of HG on the integrity of HRECs barrier in the presence or absence of inhibitors of BMP2 signaling. Our data reveals that BMP2 and high glucose upregulates BMP components of the BMP signaling pathway (SMAD effectors, BMP receptors, and TGFβ ligand itself) and induces phosphorylation of p38 MAPK and NFκB with nuclear translocation of NFκB. Inhibition of p38 or NFκB attenuated BMP2-induced VEGF expression and barrier dysfunction in HRECs. Also, inhibition of VEGFR2 attenuated BMP2-induced barrier dysfunction. Moreover, BMP2 induces generation of ROS and endothelial nitric oxide synthase (eNOS) expression and activity in HRECs. Finally, HG upregulated BMP2 and its downstream genes (SMAD, BMP4, ALKs, and TGF-β) in HRECs and BMP2 inhibitors attenuated HG-induced HRECs barrier dysfunction. Our results suggest that in addition to the regular canonical SMAD signaling BMP2 induces non-canonical inflammatory pathway in HRECs via activation of p38/NFκB pathway that causes the upregulation of VEGF and the disruption of HRECs. Inhibition of BMP2 signaling is a potential therapeutic intervention to preserve endothelial cell barrier function in DR.

Diabetic condition induces hypertrophy and vacuolization in glomerular parietal epithelial cells

Diabetic nephropathy (DN) is accompanied by characteristic changes in the glomerulus, but little is known about the effect of diabetes on parietal epithelial cells (PECs). In this study, a descriptive analysis of PECs was undertaken in diabetic db/db mice and in diabetic patients. PEC hypertrophy was significantly more prominent in diabetic mice than in nondiabetic mice, and this was evident even at the early stage. Additionally, the number of vacuoles in PECs was markedly increased in diabetic mice, suggesting the presence of cellular injury in PECs in DN. Although rare, binuclear cells were observed in mice with early diabetes. In cultured PECs, a high glucose condition, compared with normal glucose condition, induced cellular hypertrophy and apoptosis. Flow cytometry showed that some PECs in the G0 phase reentered the cell cycle but got arrested in the S phase. Finally, in human diabetic subjects, hypertrophy and vacuolization were observed in the PECs. Our data showed that PECs undergo substantial changes in DN and may participate in rearrangement for differentiation into podocytes.

β-carotene oxygenase 2 deficiency-triggered mitochondrial oxidative stress promotes low-grade inflammation and metabolic dysfunction

Low-grade inflammation is a critical pathological factor contributing to the development of metabolic disorders. β-carotene oxygenase 2 (BCO2) was initially identified as an enzyme catalyzing carotenoids in the inner mitochondrial membrane. Mutations in BCO2 are associated with inflammation and metabolic disorders in humans, yet the underlying mechanisms remain unknown. Here, we used loss-of-function approaches in mice and cell culture models to investigate the role of BCO2 in inflammation and metabolic dysfunction. We demonstrated decreases in BCO2 mRNA and protein levels and suppression of mitochondrial respiratory complex I proteins and mitochondrial superoxide dismutase levels in the liver of type 2 diabetic human subjects. Deficiency of BCO2 caused disruption of assembly of the mitochondrial respiratory supercomplexes, such as supercomplex III2+IV in mice, and overproduction of superoxide radicals in primary mouse embryonic fibroblasts. Further, deficiency of BCO2 increased protein carbonylation and populations of natural killer cells and M1 macrophages, and decreased populations of T cells, including CD4+ and/or CD8+ in the bone marrow and white adipose tissues. Elevation of plasma inflammatory cytokines and adipose tissue hypertrophy and inflammation were also characterized in BCO2 deficient mice. Moreover, BCO2 deficient mice were more susceptible to high-fat diet-induced obesity and hyperglycemia. Double knockout of BCO2 and leptin receptor genes caused a significantly greater elevation of the fasting blood glucose level in mice at 4 weeks of age, compared to the age- and sex-matched leptin receptor knockout. Finally, administration of Mito-TEMPO, a mitochondrial specific antioxidant attenuated systemic low-grade inflammation induced by BCO2 deficiency. Collectively, these findings suggest that BCO2 is essential for mitochondrial respiration and metabolic homeostasis in mammals. Loss or decreased expression of BCO2 leads to mitochondrial oxidative stress, low-grade inflammation, and the subsequent development of metabolic disorders.