Diabetic Human Subjects Research Articles

120-LB: Small Molecule Activator of Pyruvate Kinase Regulates In Vivo Glucose Homeostasis

Background: Nonalcoholic fatty liver disease (NAFLD) is a major factor in the pathogenesis of type-2 diabetes (T2D). Liver pyruvate kinase (PK) inactivating mutations are associated with diabetes in human subjects and PK deficiency associated with severe liver dysfunction and fat accumulation. Aim: To determine whether small molecule activators of PK (PKa) could improve liver metabolism in rats fed a high fat diet. Methods: Sprague Dawley regular chow (RC) or high fat diet (HFD) rats were either treated acutely (IV bolus 11mg/kg/min then primed continuous 0.06 mg/kg/min) or chronically (50 mg/kg/day x 3 weeks). Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp studies. Results: PKa dose dependently suppressed glucose production from primary rat hepatocytes. Acute PKa infusion suppressed endogenous glucose production (EGP) by 20% in RC rats. Chronic PKa exposure did not affect body weight in HFD-fed rats. Three-week PKa treatment did not change activity, food intake, whole-body energy expenditure, or body weight in HFD-fed mice. Nevertheless, PKa improved whole-body insulin sensitivity compared with control HFD-fed rats (glucose infusion rate: control 11.8±1.4 vs. PKa 15.3±0.7 mg/(kg-min), P<0.05). Differences in whole-body insulin sensitivity were accounted for by a 15% reduction in EGP and increased insulin-stimulated peripheral glucose uptake (21.2±0.6 vs. 26.4±1.7 mg/(kg-min), P<0.05). Insulin-stimulated hepatic AKT phosphorylation was also significantly improved. Consistent with improving of peripheral insulin sensitivity, liver and muscle triglyceride content (liver: 27.2±2.6 vs. 16.9±1.5 mg/g, P<0.05. muscle: 9.8 ±1.4 vs. 6.7±1.0 mg/g, P<0.05) were improved in PKa-treated rats. In conclusion, activation of pyruvate kinase in rodents protects against HFD-induced insulin resistance. By reducing EGP and insulin resistance, activation of PK may be an exciting potential therapeutic target for T2D. Disclosure A. Abulizi: None. R.L. Cardone: None. S. Siebel: None. C. Kung: Employee; Self; Agios Pharmaceuticals. Stock/Shareholder; Self; Agios Pharmaceuticals. R. Kibbey: None. Funding American Diabetes Association (1-16-IBS-158 to R.K.); National Institutes of Health (R01DK108283-01A1)

Hepatic F-box Only Protein 2 (FBXO2) Might Not Mediate Glucose Homeostasis in Obese Diabetic Mice

Abstract Objectives F-box only protein 2 (FBXO2) is an E3 ubiquitin protein ligase highly expressed in the brain and cochlea, hepatic FBXO2 protein levels are very low in healthy mice. It was reported that hepatic FBXO2 elevation causes hyperglycemia in obese mice. Here, we investigated whether hepatic FBXO2 expression was enhanced in diabetic human samples; and if so, whether deficiency of FBXO2 could improve blood glucose control in diet-induced obese diabetic mice. Methods Human liver specimen samples from healthy and type 2 diabetic human subjects were subject to transcriptomics and proteomics. The results were confirmed by real-time QPCR and immunoblotting, respectively. Male FBXO2 whole-body knockout (KO) and C57BL/6 J mice (WT, genetic background) at 6 weeks of age were fed a high fat (26% kCal)/high sucrose (15% kCal)/high fructose (15% kCal) diet for 8 weeks. Food intake and body weight gain were monitored weekly. Glucose tolerance and insulin tolerance tests were performed at the 7th week of dietary intervention. At the termination of intervention, mice were euthanized, and blood and liver tissues were collected for further assessment. Primary mouse embryonic fibroblasts (MEF) cells were isolated from WT and KO mice. Mitochondrial superoxide was detected by MitoSOX live cell staining. Results Transcriptomics and proteomics results showed vast shifts in pathways involved in energy metabolism and mitochondrial function, and significant elevation of FBXO2 expression at mRNA and protein levels in diabetic human livers. However, deficiency of FBXO2 did not alter mitochondrial superoxide production in MEF cells and did not cause an elevation of the fasting blood glucose level before a diet feeding. Moreover, both strains had significantly elevated fasting blood glucose levels after the high-fat diet intervention but did not differ by strains. The insulin sensitivity had no significant difference between strains after diet intervention. Conclusions The results suggested that FBXO2 depletion did not alter glucose homeostasis in diet-induced obese mouse models, though diabetic humans had a highly elevated hepatic FBXO2 level. Funding Sources N/A.

MD2 activation by direct AGE interaction drives inflammatory diabetic cardiomyopathy

Hyperglycemia activates toll-like receptor 4 (TLR4) to induce inflammation in diabetic cardiomyopathy (DCM). However, the mechanisms of TLR4 activation remain unclear. Here we examine the role of myeloid differentiation 2 (MD2), a co-receptor of TLR4, in high glucose (HG)- and diabetes-induced inflammatory cardiomyopathy. We show increased MD2 in heart tissues of diabetic mice and serum of human diabetic subjects. MD2 deficiency in mice inhibits TLR4 pathway activation, which correlates with reduced myocardial remodeling and improved cardiac function. Mechanistically, we show that HG induces extracellular advanced glycation end products (AGEs), which bind directly to MD2, leading to formation of AGEs-MD2-TLR4 complex and initiation of pro-inflammatory pathways. We further detect elevated AGE-MD2 complexes in heart tissues and serum of diabetic mice and human subjects with DCM. In summary, we uncover a new mechanism of HG-induced inflammatory responses and myocardial injury, in which AGE products directly bind MD2 to drive inflammatory DCM.

Dopamine metabolite levels in the vitreous of diabetic and non-diabetic humans

Animal studies suggest that the retinal dysfunction in diabetic subjects that precedes overt clinical vasculopathy may be due to a retinal dopamine deficit. We analyzed levels of dopamine (DA) and its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in the vitreous of diabetic and non-diabetic human subjects. Adult patients undergoing pars plana vitrectomy for non-hemorrhagic indications were prospectively recruited from the Emory Eye Center in Atlanta, GA. Vitreous samples were analyzed using high performance liquid chromatography (HPLC) to measure levels of DOPAC and DA in the vitreous specimens. Vitreous samples from 9 diabetic patients and 20 from non-diabetic patients were analyzed. No eyes had apparent diabetic retinopathy. Mean normalized DA concentration in vitreous of diabetic subjects was 0.76 ± 0.12 pg/μL vs. 0.73 ± 0.08 pg/μL in non-diabetic vitreous (p = 0.849). DOPAC concentration was 8.84 ± 0.74 pg/μL in vitreous of diabetic subjects vs. 9.22 ± 0.56 pg/μL in vitreous of non-diabetic subjects (p = 0.691). No difference was observed in the concentrations of DA and DOPAC in the vitreous of people without diabetes compared to those with diabetes without retinopathy.

Enhanced skeletal muscle growth in myostatin-deficient transgenic pigs had improved glucose uptake in stretozotocin-induced diabetes.

The size of skeletal muscle mass plays a significant role in glucose uptake in healthy and diabetic human subjects. Previously, we have generated myostatin-deficient (MSTN-/-) transgenic pigs via animal cloning technology. MSTN-/- pigs had dramatic phenotype with individual muscle mass increase by 100% over their wild-type controls, which provides a unique large animal model to investigate how enhanced skeletal muscles are beneficial to glucose update in diabetes. We employed intravenous administration of stretozotocin (STZ) to male MSTN-/- and wild-type pigs (100mg/kg body weight). One month later, blood glucose and insulin concentrations and pancreas histology were examined, STZ-induced diabetes occurred in both MSTN transgenic and wild-type pigs. Histology of pancreas, analysis of pAKT and Glut4 transporter proteins by Western blotting, and real-time qPCR for MSTN gene expression were used in the study. The STZ-treated pigs had increased levels of fasting plasma glucose and insulin levels in comparison with animals receiving sodium citrate buffer, their pancreas also had reduced beta cells and slight increases in lymphocyte. There are significant lower concentrations of fasting plasma glucose and insulin in MSTN-/- pigs than that of wild-type pigs after STZ administration. Detections of pAKT and Glut4 transporter proteins by Western blotting in muscle tissue indicates significant elevations of both proteins in MSTN-/- pigs compared with the wild-type pigs. The results from this pig model suggest that enhanced skeletal muscle by manipulation of myostatin function can improve glucose uptake even in the status of diabetes.

Circulatory miR-98-5p levels are deregulated during diabetes and it inhibits proliferation and promotes apoptosis by targeting PPP1R15B in keratinocytes

ABSTRACTAlthough deregulated circulatory miRNA signatures during diabetes have been identified for some years now, the effects of such miRNAs on several target tissues are not yet thoroughly investigated. The skin that is nourished by components present in the circulation exhibits several notable abnormal features during diabetes. We, therefore, hypothesized that such altered circulatory miRNA levels might be critical in the onset and progression of impaired skin health during diabetes. RNA sequencing from blood samples of normal and type 2 diabetic human subjects identified 9 upregulated and 19 downregulated miRNAs. miR-98-5p was significantly downregulated and its overexpression down-regulated PPP1R15B levels in HaCaT cells and this was prevented by the miR-98-5p inhibitor. This was validated in human primary epidermal keratinocytes and further supported by a dual reporter luciferase assay of the PPP1R15B 3ʹUTR where miR-98-5p significantly decreased the luciferase activity which was prevented in the presence of the miRNA inhibitor and by mutation in the miRNA binding site. By targeting PPP1R15B, miR-98-5p increases levels of p-eIF2α, BiP and CHOP. Consequently, there was induction of apoptosis accompanied with decreased proliferation in the presence of miR-98-5p. Conversely, miR-98-5p inhibition alone inhibited apoptosis and promoted proliferation. Taken together, our data suggest that by targeting PPP1R15B, miR-98-5p induces apoptosis and decreases proliferation. As opposed to this since circulatory miR-98-5p levels are decreased in diabetes, we believe that this decrease in the circulation that feeds the skin layers might be a major contributor of hyperproliferation as seen in the skin during diabetes.Abbreviations: miRNAs: MicroRNAs; PPP1R15B: PPP1R15B: Protein Phosphatase 1 Regulatory Subunit 15B; TGFβR1: Transforming Growth Factor Beta Receptor 1; ER: Endoplasmic Reticulum; Bip: Binding Immunoglobulin Protein; Chop: CCAAT-enhancer-binding protein homologous protein; p-eIF2α: Eukaryotic Translation Initiation Factor 2a; Bax: Bcl2-associated X protein; Bcl-2: B-cell CLL/lymphoma 2; PCNA: Proliferating Cell Nuclear Antigen; K5: Cytokeratin 5; qRT-PCR: Quantitative Real-Time PCR; ESCC: Oesophageal squamous cell carcinoma; HCC: Hepatocellular carcinoma; CTHRC1: Collagen triple helix repeat containing 1; SALL4: Sal-like protein 4; TNFα: Tumour Necrosis Factor alpha; PGC-1β: Peroxisome Profilerator-activated receptor-γ coactivator-1β; IGF2BP1: Insulin-like growth factor 2 mRNA binding protein 1.

Evaluating the Effects of Kombucha as a Hyperglycemic Therapeutic Agent Within Diabetic Human Subjects

Evaluating the Effects of Kombucha as a Hyperglycemic Therapeutic Agent Within Diabetic Human Subjects

Pu-erh Tea Regulates Fatty Acid Metabolism in Mice Under High-Fat Diet.

Pu-erh tea has been extensively reported to possess lipid lowering effects but the underlying mechanisms remained unclear. Free fatty acids (FFAs) are generally correlated with the development of obesity, leading to increased risk for type 2 diabetes mellitus and cardiovascular diseases. To investigate whether Pu-erh tea treatment alters FA metabolism, we treated HFD induced obese mice with Pu-erh tea for 22 weeks and analyzed FFA profiles of experimental mice using a UPLC-QTOF-MS platform. Results showed remarkable changes in metabolic phenotypes and FFA compositions in mice treated with or without Pu-erh tea. HFD induced a marked obese phenotype in mice as revealed by significantly increased body weight, liver and adipose tissue weight, lipid levels in serum and liver, and these parameters were markedly reduced by Pu-erh tea treatment. Several FFA or FFA ratios, such as DGLA, palmitoleic acid, and OA/SA ratio, were significantly increased while the levels of SA/PA and AA/DGLA were significantly reduced in HFD-induced obese mice. Interestingly, these differential FFAs or FFA ratios were previous identified as key markers in human obese subjects, and their changes observed in the HFD group were reversed by Pu-erh tea treatment. Moreover, a panel of FFA markers including C20:3 n6/C18:3 n6 and C20:3 n6/C20:2 n6, C18:3 n6/C18:2 n6, C18:3 n3/C18:2 n6 and C24:1 n9/C22:1 n9, which were previously identified as biomarkers in predicting the remission of obesity and diabetes in human subjects who underwent metabolic surgery procedures, were reversed by Pu-erh tea intervention. Pu-erh tea significantly improved glucose homeostasis and insulin tolerance compared to the HFD group. Additionally, Pu-erh tea treatment significantly decreased FFA synthesis genes and increased the expression of genes involved in FFA uptake and β-oxidation including FATP2, FATP5, PPARα, CPT1α, and ACOX-1. These finding confirmed the beneficial effects of Pu-erh tea on regulating lipid and glucose metabolism, and further validated a panel of FFA markers with diagnostic and prognostic value for obesity and diabetes.

Loss of Angiotensin-Converting Enzyme 2 Exacerbates Diabetic Retinopathy by Promoting Bone Marrow Dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2-/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2-/y -Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis toward myelopoiesis, and an impairment of lineage- c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared with Akita mice, ACE2-/y -Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represents a therapeutic strategy for prevention of diabetic retinopathy. Stem Cells 2018;36:1430-1440.

Focusing on Sodium Glucose Cotransporter-2 and the Sympathetic Nervous System: Potential Impact in Diabetic Retinopathy.

The prevalence of diabetes is at pandemic levels in today's society. Microvascular complications in organs including the eye are commonly observed in human diabetic subjects. Diabetic retinopathy (DR) is a prominent microvascular complication observed in many diabetics and is particularly debilitating as it may result in impaired or complete vision loss. In addition, DR is extremely costly for the patient and financially impacts the economy as a range of drug-related therapies and laser treatment may be essential. Prevention of microvascular complications is the major treatment goal of current therapeutic approaches; however, these therapies appear insufficient. Presently, sodium glucose cotransporter-2 (SGLT2) inhibitors may offer a novel therapy beyond simple glucose lowering. Excitingly, the EMPA-REG clinical trial, which focuses on the clinically used SGLT2 inhibitor empagliflozin, has been extremely fruitful and has highlighted beneficial cardiovascular and renal outcomes. The effects of SGLT2 inhibitors on DR are currently a topic of much research as outlined in the current review, but future studies are urgently needed to fully gain mechanistic insights. Here, we summarize current evidence and identify gaps that need to be addressed.

Unaltered Fatty Acid Uptake, Utilization, and Mitochondrial Respiration in Right Atrial Appendage of Type 2 Diabetic Human Heart

Previous studies on diabetic human heart have shown decreased utilization of fatty acid (FA)/carbohydrate substrates. We hypothesized that increased mitochondrial dysfunction with concomitant decrease in fatty acid utilization would be found in diabetic hearts of patients from the Indian subcontinent that would explain their increased propensity of cardiovascular complications. We have compared mitochondrial respiration in atrial appendage tissue from nondiabetic and diabetic human subjects undergoing elective coronary artery bypass graft (CABG) surgery using high-resolution respirometry with FA and carbohydrate substrate combinations. In the presence of FA, diabetic mitochondria showed unimpaired complex I and electron transferring flavoprotein (ETF) mediated respiration while significantly lower oxygen consumption was noted when succinate was added, indicating the possible derangement of complex II respiration. Unlike other human studies, we report no significant reduction in FA- mediated mitochondrial respiration. However, the carbohydrate protocol indicated impairment of complex I mediated respiration. There was no significant change in the expression of OXPHOS proteins. Unchanged levels of FA uptake proteins like CD36 and FABP, FA oxidation enzyme, long chain acyl CoA deydrogenase (LCAD), regulators of FA metabolism like sirtuins and nuclear receptors, PPARα and transcription coactivators, PGC1α/β were observed in diabetic heart. The acetylation status of mitochondrial specific proteins involved in fatty acid oxidation (acetylated LCAD and PGC1α/β) also showed no significant changes. Diabetic patients showed equal expression of the antioxidant enzymes and other markers of oxidative stress. The unimpaired FA uptake, oxidation and mitochondrial respiration indicates absence of overt mitochondrial dysfunction in type 2 diabetic human heart. Disclosure N. Rj: None. R. Sr: None. V.V. Pillai: None. J. K.: None. S. Gopala: None.

Novel oxolane derivative DMTD mitigates high glucose-induced erythrocyte apoptosis by regulating oxidative stress

Chronic hyperglycemia is one of the characteristic conditions associated with Diabetes Mellitus (DM), which often exerts deleterious effects on erythrocyte morphology and hemodynamic properties leading to anemia and diabetes-associated vascular complications. High glucose-induced over production of reactive oxygen species (ROS) can alter the blood cell metabolism and biochemical functions subsequently causing eryptosis (red blood cell death), yet another complication of concern in DM. Therefore, blocking high glucose-induced oxidative damage and subsequent eryptosis is of high importance in the better management of DM and associated vascular complications. In this study, we synthesized an oxolane derivative 1-(2,2-dimethyltetrahydrofuro[2,3][1,3]dioxol-5-yl)ethane-1,2-diol (DMTD), and demonstrated its efficacy to mitigate hyperglycemia-induced ROS generation and subsequent eryptosis. We showed that DMTD effectively inhibits high glucose-induced ROS generation, intracellular calcium levels, phosphaditylserine (PS) scrambling, calpain and band 3 activation, LDH leakage, protein glycation and lipid peroxidation, meanwhile enhances the antioxidant indices, osmotic fragility and Na+/K+-ATPase activity in erythrocytes. DMTD dose dependently decreased the glycated hemoglobin level and enhances the glucose utilization by erythrocytes in vitro. Further, DMTD alleviated the increase in ROS production, intracellular Ca2+ level and PS externalization in the erythrocytes of human diabetic subjects and enhanced the Na+/K+-ATPase activity. Taken together, the synthesized oxolane derivative DMTD could be a novel synthetic inhibitor of high glucose-induced oxidative stress and eryptosis. Considering the present results DMTD could be a potential therapeutic to treat DM and associated complications and open new avenues in developing synthetic therapeutic targeting of DM-associated complications.

Impaired Cerebral Autoregulation-A Common Neurovascular Pathway in Diabetes may Play a Critical Role in Diabetes-Related Alzheimer’s Disease

Alzheimer's disease (AD) is the leading cause of progressive degenerative dementia. The hallmark pathological features include beta amyloid deposition and neurofibrillary tangles. There has been a strong association of AD with Diabetes (DM) based on human studies and animal experiments. The hallmark features of AD seem to have an exaggerated presence in AD with DM, especially type 2 diabetes (T2D). In addition, insulin resistance is a common feature in both diseases and as such AD has been called type 3 diabetes. Furthermore, impairment of cerebral autoregulation has been reported in both animal and human diabetic subjects. Cerebral vascular impairment has also been implicated in the pathophysiology of AD. There is an urgent need to develop animal models of AD and DM to explore the neuropathological mechanisms of these disease and utilize such models to develop treatment strategies.

Abstract 372: PPARα/γ Agonist Tesaglitazar Reduces Inflammatory Macrophage Numbers And Induces Adipose Ucp-1 Expression In Diabetic Ob/ob Mice

Rationale: PPARα/γ agonist tesaglitazar effectively improves insulin sensitivity and dyslipidemia in diabetic mice and human subjects. The cellular and molecular mechanisms whereby this occurs remain unclear. Given the established expression of PPARs in macrophages and in vitro findings demonstrating attenuated inflammation with PPAR agonism, we hypothesized that tesaglitazar attenuates macrophage-mediated inflammation within the adipose to improve metabolic outcomes in diabetic mice. Methods & Results: To validate that tesaglitazar reduces diabetic symptoms, we treated Ob/ob mice with tesaglitazar or vehicle control for one week and observed significant reductions in fasting blood glucose, insulin, and triglyceride levels with drug treatment when compared to vehicle treatment. Using flow cytometry, we determined that treatment with tesaglitazar results in reduced numbers of total macrophages (CD45+CD11b+F4/80+) in the epididymal and subcutaneous adipose depots. Furthermore, this reduction in macrophages numbers is primarily due to reduced numbers within the M1, or pro-inflammatory, compartment (CD11b+F4/80+CD11c+CD206-) of the macrophage population. Given the established role of inflammation in inhibiting adipocyte Ucp-1 expression, we measured Ucp-1 expression levels in epididymal adipocytes and observed a significant induction of Ucp-1 mRNA expression with tesaglitazar treatment. Conclusion: As anticipated, treatment with tesaglitazar improves insulin sensitivity and dyslipidemia in Ob/ob mice. Interestingly, tesaglitazar treatment also reduced the number of inflammatory macrophages in adipose and induced adipocyte Ucp-1 mRNA expression. Taken together, our findings suggest that tesaglitazar has anti-inflammatory effects indicated by reduced numbers of pro-inflammatory macrophages and increased thermogenic capacity as seen by increased Ucp-1 expression.

The Antidiabetic Potential of Quercetin: Underlying Mechanisms.

The dramatic increase in modern lifestyle diseases such as cancer, cardiovascular diseases and diabetes has renewed researchers' interest to explore nature as a source of novel therapeutic agents. Flavonoids are a large group of polyphenols that are widely present in the human diet. They have shown promising therapeutic activities against a wide variety of ailments. One of the most widely distributed and most extensively studied flavonoid is the flavonol quercetin. Its powerful antioxidant and anti-inflammatory activities are well documented and are thought to play a role in treating and protecting against diseases including diabetes, cancer, neurodegenerative and cardiovascular diseases. The purpose of this review is to shed light on quercetin therapeutic potential as an antidiabetic agent. Quercetin was reported to interact with many molecular targets in small intestine, pancreas, skeletal muscle, adipose tissue and liver to control whole-body glucose homeostasis. Mechanisms of action of quercetin are pleiotropic and involve the inhibition of intestinal glucose absorption, insulin secretory and insulin-sensitizing activities as well as improved glucose utilization in peripheral tissues. Initial studies suggested poor bioavailability of quercetin. However, recent reports have shown that quercetin was detected in the plasma after food or supplements consumption and has a long half-life in human body. Despite the wealth of in vitro and in vivo results supporting the antidiabetic potential of quercetin, its efficacy in diabetic human subjects is yet to be explored.

Combining laser-Doppler flowmetry measurements with spectral analysis to study different microcirculatory effects in human prediabetic and diabetic subjects.

We aimed to identify the microcirculatory regulatory mechanisms in diabetic and prediabetic humans using a noninvasive method combining spectral analysis with laser-Doppler flowmetry (LDF) measurements on the skin surface. LDF signals were measured by a moorVMS-LDF device to measure the microcirculatory blood flow flux with a time constant of 0.001s, a cutoff frequency of 14.9kHz, and a sampling frequency of 40Hz. The laser operating wavelength and output power were 400-700nm and 6mW, respectively. LDF signals were obtained noninvasively in 115 subjects, who were assigned to three groups (diabetic, prediabetic, and normal) according to the results of the oral glucose tolerance tests. A Morlet mother wavelet transform was applied to the measured 20-min LDF signals, and periodic oscillations with five characteristic frequency peaks were obtained within the following frequency bands: 0.0095-0.02, 0.02-0.06, 0.06-0.15, 0.15-0.4, and 0.4-1.6Hz (defined as FR1-FR5), respectively. The relative energy contribution (REC) of FR1 was significantly smaller (by using the Kruskal-Wallis test followed by Dunn's multiple-comparison tests) in diabetic subjects than in normal subjects. The REC of FR2-FR3 was significantly smaller in diabetic and prediabetic subjects than in normal subjects. The REC of FR1-FR3 from normal to prediabetic and diabetic subjects showed a progressive decrease. The present findings may aid in the development of a noninvasive method for the early detection of prediabetes and the monitoring of disease progression. This may be useful in preventing disease progression and reducing the risk of concomitant end-organ damage.

Role of Pro-Inflammatory Cytokines and Biochemical Markers in the Pathogenesis of Type 1 Diabetes: Correlation with Age and Glycemic Condition in Diabetic Human Subjects.

BackgroundType 1 diabetes mellitus is a chronic inflammatory disease involving insulin producing β-cells destroyed by the conjoined action of auto reactive T-cells, inflammatory cytokines and monocytic cells. The aim of this study was to elucidate the status of pro-inflammatory cytokines and biochemical markers and possible correlation of these factors towards outcome of the disease.MethodsThe study was carried out on 29 T1D subjects and 20 healthy subjects. Plasma levels of oxidative stress markers, enzymatic and non-enzymatic antioxidants were estimated employing biochemical assays. The levels of pro-inflammatory cytokines such as by IL-1β & IL-17 in the serum were determined by ELISA, while the expression of TNF-α, IL-23 & IFN-γ was ascertained by qRT-PCR.ResultsThe onset of T1D disease was accompanied with elevation in levels of Plasma malondialdehyde, protein carbonyl content and nitric oxide while plasma vitamin C, reduced glutathione and erythrocyte sulfhydryl groups were found to be significantly decreased in T1D patients as compared to healthy control subjects. Activity of antioxidant enzymes, superoxide dismutase, catalase, glutathione reductase and glutathione-s-transferase showed a significant suppression in the erythrocytes of T1D patients as compared to healthy subjects. Nevertheless, the levels of pro-inflammatory cytokines IL-1β and IL-17A were significantly augmented (***p≤.001) on one hand, while expression of T cell based cytokines IFN-γ, TNF-α and IL-23 was also up-regulated (*p≤.05) as compared to healthy human subjects.ConclusionThe level of pro-inflammatory cytokines and specific biochemical markers in the serum of the patient can be exploited as potential markers for type 1 diabetes pathogenesis. The study suggests that level of inflammatory markers is up-regulated in T1D patients in an age dependent manner.

An accurate and efficient experimental approach for characterization of the complex oral microbiota.

BackgroundCurrently, taxonomic interrogation of microbiota is based on amplification of 16S rRNA gene sequences in clinical and scientific settings. Accurate evaluation of the microbiota depends heavily on the primers used, and genus/species resolution bias can arise with amplification of non-representative genomic regions. The latest Illumina MiSeq sequencing chemistry has extended the read length to 300 bp, enabling deep profiling of large number of samples in a single paired-end reaction at a fraction of the cost. An increasingly large number of researchers have adopted this technology for various microbiome studies targeting the 16S rRNA V3–V4 hypervariable region.ResultsTo expand the applicability of this powerful platform for further descriptive and functional microbiome studies, we standardized and tested an efficient, reliable, and straightforward workflow for the amplification, library construction, and sequencing of the 16S V1–V3 hypervariable region using the new 2 × 300 MiSeq platform. Our analysis involved 11 subgingival plaque samples from diabetic and non-diabetic human subjects suffering from periodontitis. The efficiency and reliability of our experimental protocol was compared to 16S V3–V4 sequencing data from the same samples. Comparisons were based on measures of observed taxonomic richness and species evenness, along with Procrustes analyses using beta(β)-diversity distance metrics. As an experimental control, we also analyzed a total of eight technical replicates for the V1–V3 and V3–V4 regions from a synthetic community with known bacterial species operon counts. We show that our experimental protocol accurately measures true bacterial community composition. Procrustes analyses based on unweighted UniFrac β-diversity metrics depicted significant correlation between oral bacterial composition for the V1–V3 and V3–V4 regions. However, measures of phylotype richness were higher for the V1–V3 region, suggesting that V1–V3 offers a deeper assessment of population diversity and community ecology for the complex oral microbiota.ConclusionThis study provides researchers with valuable experimental evidence for the selection of appropriate 16S amplicons for future human oral microbiome studies. We expect that the tested 16S V1–V3 framework will be widely applicable to other types of microbiota, allowing robust, time-efficient, and inexpensive examination of thousands of samples for population, phylogenetic, and functional crossectional and longitutidal studies.

Optical Coherence Tomography Angiography of Diabetic Retinopathy in Human Subjects.

Optical coherence tomography angiography (OCTA) is a novel, non-invasive OCT technique capable of imaging the retinal vasculature. This study aims to evaluate the retinal microvasculature in diabetic human subjects with OCTA and assess potential clinical applications. Cross-sectional study of 33 subjects with diabetic retinopathy. OCTA was performed on 3 mm × 3 mm sections using a swept-source OCTA prototype and a phase- and intensity-based contrasting algorithm. OCT angiograms were studied with corresponding clinical examination and fluorescein angiograms, when available, to assess accuracy and clinical utility. OCTA was able to demonstrate most clinically relevant vascular changes in subjects with diabetic retinopathy, including microaneurysms, impaired vascular perfusion, some forms of intraretinal fluid, vascular loops, intraretinal microvascular abnormalities, neovascularization, and cotton-wool spots that were largely consistent with fluorescein angiography. OCTA generates high-resolution angiograms that illustrate many of the clinically relevant findings in diabetic retinopathy and offers a novel complement or alternative to fluorescein angiography. Although currently an investigational technique, OCTA in combination with standard OCT imaging is at least as good as fluorescein angiography in the evaluation of the macular complications of diabetic retinopathy.

Advances in pathology of diabetes from pancreatic islets to neuropathy--a tribute to Paul Langerhans.

There emerges a world epidemic of diabetes, afflicting over 3.8 billion people globally. The socio-economic burden of this disorder is tremendous and there is an urgent need to solve the problems incurred from this disorder and to establish an efficient way of prevention and treatment. Fundamental pathology of diabetes has been too diverse to reach a simple etiology and the mechanisms of how the lesions specific to diabetes develop are yet to be clear. Nevertheless, there has been slow but significant advancement in the understanding of the disease based on characterization of the salient features of pathological lesions in human diabetic subjects. Progressive decline of islet β cells associated with increased α cell volume density was found to account for clinical manifestation of hypoinsulinemia and hyperglucagonemia in type 2 diabetes. Concurrently, signs of complications represented by distal nerve fiber loss in the skin commences from the beginning of this disease. Thus the pathological studies disclosed the major attributes in this disorder targeting the islet of pancreas and epidermal nerve, both of which were discovered by Paul Langerhans more than 140 years ago. In this review, I attempt to summarize the progress in pathology of diabetes which Langerhans opened this field.