Association of POC-based Measurement of Skin AGEs with Serum AGEs as well as AGE Biomarkers in Individuals with and without Glucose Intolerance

Aim: Accumulation of advanced glycation end products (AGEs) occurs with aging and in various disease states. There are no reliable screening techniques to measure AGEs in clinical settings. In this study, a point-of-care (POC) device was used to validate skin AGE measurements with serum AGE levels and to assess its usefulness to identify individuals with abnormal glucose tolerance (AGT). Materials and methods: The study group comprised individuals with normal glucose tolerance (NGT: n = 47) and with AGT, that is, either diabetes or prediabetes (n = 68). Intrinsic AGE fluorescence was measured spectrofluorimetrically using multimode plate reader in the serum by exciting the samples at 370 nm and emission readouts at 440 nm. Skin AGEs were acquired using a CE-marked Scout DS commercial device. Serum levels of biomarkers carboxymethyl lysine (CML), carboxyethyl lysine (CEL), and pentosidine were analyzed by enzyme-linked immunosorbent assay (ELISA). Results: In subjects with AGT, the skin AGEs [61.3 vs 53.7 arbitrary units (AU), p<0.0001] and serum AGEs (3.5 vs 2.8 AU, p<0.0001) were significantly higher than in individuals with NGT. The levels of CML, CEL, and pentosidine were also significantly higher in the subjects with AGT when compared with NGT (138 vs 89 pg/mL; 2.4 vs 1.4 nmol/mL, and 64 vs 48 pmol/mL, p<0.0001), respectively. Pearson correlation analysis showed a significant positive association of skin AGEs with serum AGEs (r = 0.344) (p<0.001), CML (r = 0.323) (p<0.001), CEL (r = 0.308) (p<0.001), and pentosidine (r = 0.251) (p<0.001). In addition, it also showed a positive correlation with fasting plasma glucose (FPG) (p<0.001), 2-hour post-glucose (p<0.001), glycated hemoglobin (HbA1c) (p<0.001), and body mass index (BMI) (p<0.05). Multiple logistic regression analysis using AGT as a dependent variable showed that skin AGE scores were significantly (p<0.001) associated with AGT (odds ratio: 1.133, confidence intervals: 1.067–1.203). Conclusion: This study shows that the measurement of skin AGEs using a POC device may be suitable for mass screening of AGT even in low-resource settings.

Metformin Reduces the Progression of Atherogenesis by Regulating the Sestrin2-mTOR Pathway in Obese and Diabetic Rats

Objective@#In previous research, we found that Sestrin2 has a strong association with plasma atherogenicity and combats the progression of atherogenesis by regulating the AMPK-mTOR pathway. Metformin, an activator of AMPK, is widely used as a first-line therapy for diabetes, but its role in preventing atherosclerosis and cardiac outcomes is unclear. Hence, we aimed to assess the effect of metformin on preventing atherosclerosis and its regulatory role in the Sestrin2-AMPK -mTOR pathway in obese/diabetic rats. @*Methods@#Animals were fed a high-fat diet to induce obesity, administered streptozotocin to induce diabetes, and then treated with metformin (150 mg/kg body weight) for 14 weeks. Aorta and heart tissues were analyzed for Sestrin2 status by western blotting and immunohistochemistry, AMPK and mTOR activities were investigated using western blotting, and atherogenicity-related events were evaluated using reverse transcription quantitative polymerase chain reaction and histology. @*Results@#Obese and diabetic rats showed significant decrease in Sestrin2 levels and AMPK activity, accompanied by increased mTOR activity in the heart and aorta tissues. Metformin treatment significantly restored Sestrin2 and AMPK levels, reduced mTOR activity, and restored the altered expression of inflammatory markers and adhesion molecules in obese and diabetic rats to normal levels. A histological analysis of samples from obese and diabetic rats showed atherosclerotic lesions both in aorta and heart tissues. The metformin-treated rats showed a decrease in atherosclerotic lesions, cardiac hypertrophy, and cardiomyocyte degeneration. @*Conclusion@#This study presents further insights into the beneficial effects of metformin and its protective role against atherosclerosis through regulation of the Sestrin2-AMPK-mTOR pathway.

Picroside II attenuates fatty acid accumulation in HepG2 cells via modulation of fatty acid uptake and synthesis

BACKGROUND/AIMS: Hepatic steatosis is caused by an imbalance between free fatty acids (FFAs) uptake, utilization, storage, and disposal. Understanding the molecular mechanisms involved in FFAs accumulation and its modulation could drive the development of potential therapies for Nonalcoholic fatty liver disease. The aim of the current study was to explore the effects of picroside II, a phytoactive found in Picrorhiza kurroa, on fatty acid accumulation vis-à-vis silibinin, a known hepatoprotective phytoactive from Silybum marianum. METHODS: HepG2 cells were loaded with FFAs (oleic acid:palmitic acid/2:1) for 20 hours to mimic hepatic steatosis. The FFAs concentration achieving maximum fat accumulation and minimal cytotoxicity (500 μM) was standardized. HepG2 cells were exposed to the standardized FFAs concentration with and without picroside II pretreatment. RESULTS: Picroside II pretreatment inhibited FFAs-induced lipid accumulation by attenuating the expression of fatty acid transport protein 5, sterol regulatory element binding protein 1 and stearoyl CoA desaturase. Preatreatment with picroside II was also found to decrease the expression of forkhead box protein O1 and phosphoenolpyruvate carboxykinase. CONCLUSIONS: These findings suggest that picroside II effectively attenuated fatty acid accumulation by decreasing FFAs uptake and lipogenesis. Picroside II also decreased the expression of gluconeogenic genes.

Spheniodal mucocele causing bilateral optic neuropathy and ophthalmoplegia.

Sphenoid sinus mucocele comprises only 2% of all paranasal sinus mucoceles. In literature, there is a case report on sphenoidal mucocele causing bilateral optic neuropathy, with unilateral partial recovery and cranial nerve palsy, but we did not come across any literature with bilateral optic neuropathy and ophthalmoplegia together caused by spheno-ethmoidal mucocele. We present such a rare case of spheno-ethmoidal mucocele causing bilateral optic neuropathy and unilateral sixth nerve palsy who had postsurgery, unilateral good vision recovery, and complete resolution of sixth nerve palsy.

Accelerated fat cell aging links oxidative stress and insulin resistance in adipocytes.

Telomere shortening is emerging as a biological indicator of accelerated aging and aging-related diseases including type 2 diabetes. While telomere length measurements were largely done in white blood cells, there is lack of studies on telomere length in relation to oxidative stress in target tissues affected in diabetes. Therefore, the aim of this study is to induct oxidative stress in adipocytes and to test whether these adipocytes exhibit shortened telomeres, senescence and functional impairment. 3T3-L1 adipocytes were subjected to oxidative stress and senescence induction by a variety of means for 2 weeks (exogenous application of H2O2, glucose oxidase, asymmetric dimethylarginine (ADMA) and glucose oscillations). Cells were probed for reactive oxygen species generation (ROS), DNA damage, mRNA and protein expression of senescent and pro-inflammatory markers, telomere length and glucose uptake. Compared to untreated cells, both ROS generation and DNA damage were significantly higher in cells subjected to oxidative stress and senescence. Adipocytes subjected to oxidative stress also showed shortened telomeres and increased mRNA and protein expression of p53, p21, TNFα and IL-6. Senescent cells were also characterized by decreased levels of adiponectin and impaired glucose uptake. Briefly, adipocytes under oxidative stress exhibited increased ROS generation, DNA damage, shortened telomeres and switched to senescent/pro-inflammatory phenotype with impaired glucose uptake.

Benefits of early glycemic control by insulin on sensory neuropathy and cataract in diabetic rats.

While there is an emphasis on the early glycemic control for its long-term benefits in preventing microvascular complications of diabetes, the biochemical mechanisms responsible for the long-lasting effects are not clearly understood. Therefore the impact of early insulin (EI) versus late insulin (LI) treatment on diabetic sensory neuropathy and cataract in streptozotocin-induced diabetic Wistar male rats were evaluated. EI group received insulin (2.5 IU/animal, once daily) treatment from day 1 to 90 while LI group received insulin from day 60 to 90. Early insulin treatment significantly reduced the biochemical markers like glucose, triglyceride, glycated hemoglobin, thiobarbituric acid reactive substances, advanced glycation end products and ratio of reduced glutathione and oxidized glutathione in diabetic rats. The late insulin treatment failed to resist the biochemical changes in diabetic rats. Diabetic rats developed sensory neuropathy as evidenced by mechanical and thermal hyperalgesia and showed a higher incidence and severity of cataract as revealed by slit lamp examination. Early insulin treatment protected the rats from the development of neuropathy and cataract, but late insulin administration failed to do so. The results demonstrate the benefits of early glycemic control in preventing neuropathy and cataract development in diabetic rats.

Quantitative physical variations in ripening of honey of indigenous hive bee apis cerana indica.

Physical factors of refractive index, optical density, viscosity and surface tension demonstrated quantitative change throughout five stages of honey ripening process in indigenous hive honeybee Apis cerana indica. The five stages of nectar to honey transformations include floral nectar (fn), honey crop of foragers (hf), honey crop of house bees (hh), unsealed honey cells (uh) and sealed honey cells (sh). All the four physical factors gradually augmented through successive stages of honey ripening phenomena. The refractive index of floral nectaries and sealed honey cells was 1.23 and 1.49 respectively. Similarly, the optical density of house bees and unsealed honey cells was 0.39 and 0.54 respectively. Viscosity and surface tension of honey crop of foragers and sealed honey cells were 0.15 poise, 0.76 dynes/cms. and 76.65 poise, 102.01 dynes/cms. respectively. The analysis of variance (ANOVA) of viscosity and surface tension in honey formation was significant at P<0.01% levels and refractive index and optical density of honey in ripening process was not significant at P<0.01% levels.

Quantitative chemical variations in ripening of honey of indigenous hive bee apis cerana indica.

Chemical characteristics of TRS, NRS, ash, acidity and pH were detected in five stages of honey ripening process of indigenous hive honeybee Apis cerana indica. Five stages of nectar to honey transformation comprise floral nectar (fn), honey crop of foragers (hf), honey crop of house bees (hh), unsealed honey cells (uh) and sealed honey cells (sh). The TRS of fn and sh cells was 2.12% and 73.01% respectively. Correspondingly NRS of hh and uh cells was 14.90% and 7.50%. The ash content of hf and sh cells was 0.20% and 1.49% respectively, whereas the acidity of sh cells and hf was 0.503 and 0.06 respectively. Similarly pH of hh and uh cells had a value of 1.03 and 2.61. All the five parameters viz., TRS, NRS, ash, acidity and pH tested apart from NRS were less in floral nectaries and maximum in sealed honey cells. The analysis of variance (ANOVA) of TRS of ripening of honey was significant at P<0.01% levels, while NRS, ash, acidity and pH in honey formation was not significant at P<0.01% levels.

Association of leukocyte count and hsCRP with metabolic abnormalities in subjects with normal glucose tolerance (CURES – 64).

Objective : The aim of the present study was to assess the association of leukocyte count and high sensitivity C-Reactive protein (hsCRP) with metabolic abnormalities in subjects with normal glucose tolerance. Methods : Subjects with Normal Glucose Tolerance (NGT) (n = 865) were recruited from the Chennai Urban Rural Epidemiology Study [CURES]. Standard methods were used for assessing hsCRP [Nephelometry, in a subset] and leukocytes [Flowcytometry, Sysmex SF-3000]. Insulin resistance was calculated using the Homeostasis Assessment model (HOMA-IR). Results : Body mass index, waist circumference, systolic and diastolic blood pressure, fasting plasma glucose, HbA1c, serum cholesterol, LDL cholesterol, HOMA IR and hsCRP increased significantly with increasing tertiles of leukocyte count [p for trend < 0.001]. Both leukocyte count and hsCRP showed a positive correlation with cardiovascular risk factors. Leukocyte count showed a positive correlation with hsCRP [p=0.008]. Both mean leukocyte count [p<0.001] and hsCRP [p=0.04] were higher in subjects with Metabolic Syndrome (MS), which increased with increase in number of metabolic abnormalities [p for trend <0.001]. Regression models showed leukocyte count [p<0.001] and hsCRP [p=0.03] to be associated with MS, even after adjusting for age and gender. Conclusion : A significant association exists between systemic inflammation [leukocyte count and hsCRP] and MS/ cardiovascular risk factors in Asian Indians even among non-diabetic subjects. ©

Telomere shortening & metabolic/vascular diseases.

Telomeres are specialized DNA-protein structures located at the ends of eukaryotic chromosomes whose length is progressively reduced in most somatic cells during ageing. Over the past decade, emerging evidence has shown that the telomeres are essential regulators of cellular life span and chromosome integrity in a dynamic fashion. By inducing genomic instability, replicative senescence and apoptosis, shortening of telomeres is thought to contribute to organismal ageing. While the aetiology of cardiovascular diseases and diabetes represent a complex interaction between various risk factors overlaid on different genetic backgrounds, the conventional risk factors often did not explain the inter-individual variability related to predisposition of disease states. This underscores the need for biological indicators of ageing in evaluating the aetiology of several age-related disorders, and recent studies indicate that telomere length could qualify as an ideal marker of biological ageing. Short telomeres have been detected in senescent endothelial cells and vascular smooth muscle cells from human atherosclerotic plaque as well as in myocardial tissue from patients with end-stage heart failure and cardiac hypertrophy. In addition, telomere shortening has been demonstrated in WBCs from patients with coronary heart disease, premature myocardial infarction, hypertension and diabetes mellitus. In this review, we discuss the telomere hypothesis of ageing as well as human studies that address the role of telomeres in cardiovascular, diabetes and other cardio-metabolic pathologies.

Endoplasmic reticulum (ER) stress & diabetes.

The endoplasmic reticulum (ER) is a central organelle entrusted with lipid synthesis, protein folding and protein maturation. It is endowed with a quality control system that facilitates the recognition and targeting of aberrant proteins for degradation. When the capacity of this quality control system is exceeded, a stress response (ER stress) is switched on. Prolonged stress leads to apoptosis and may thus be an important factor in the pathogenesis of many diseases. A complex homeostatic signaling pathway, known as the unfolded protein response (UPR), has evolved to maintain a balance between the load of newly synthesized proteins and the capacity of the ER to aid in their maturation. Dysfunction of the UPR plays an important role in certain diseases, especially those involving tissues dedicated to extracellular protein synthesis. Diabetes is an example of such a disease, since pancreatic beta-cells depend on efficient UPR signaling to meet the demands for constantly varying levels of insulin synthesis. Recent studies have indicated that the importance of the UPR in diabetes is not restricted to the beta-cell but also to tissues of peripheral insulin resistance such as liver and adipose tissue. Better understanding of the basic mechanisms of ER stress and development of insulin resistance/type 2 diabetes is pivotal for the identification of newer molecular targets for therapeutic interventions.

Fluctuating asymmetry in dermatoglyphics of non-insulin-dependent diabetes mellitus in Bangalore-based population.

Background: The aetiology of NIDDM is believed to be as a consequence of genetic and environmental factors that impair metabolism. While little can be done on the genetic component, much can be done as a preventive measure in NIDDM. Because nothing much can be done prenatally, researchers have resorted to studying physical variables like dermatoglyphics (DGs). Dermatoglyphic patterns form on the finger pad and the palm prenatally and remain unchanged throughout life, thus these features may serve as markers for fetal origin of adult disease like NIDDM. Thus the concept of fluctuating asymmetry (FA) which has been defined as random differences between the right (R) and left (L) sides of a morphological trait has gained prominence in diseases like schizophrenia. When the distribution of R-L differences in a population sample approximates a normal curve with a mean approximately equal to zero, the variance of distributions of R-L difference is a measure of FA. Studies have shown that genetic factors may also have a link to FA in finger and a-b ridge counts. No studies have been reported on FA in NIDDM. FA derived from quantitative parameters in DGs of NIDDM may throw light on fetal origins of an adult disease. Hence this study has been undertaken. Aim: The present study aims at deriving FA from quantitative parameters in DGs of NIDDM compared to controls in the Bangalore based population. Materials and Methods: Bilateral rolled finger and palm prints of 150 NIDDM patients (Males - 75, Females - 75) were compared to 120 controls (Males - 60, females -60) from Bangalore based population. FA measures derived from quantitative parameters (finger ridge counts, a-b ridge counts, main line index and palmar angles) were analysed. Results: Comparisons were made in all parameters between homologous fingers of both hands using Pearson's product moment correlation coefficients (r). The difference in correlation coefficients between cases and controls was calculated using Fisher's Z transformation. 1-r2 an estimate of error variance thus measures FA. FA measures were significantly higher in NIDDM males for the 5th finger (FA=2.04) and for the palmar angle 'dat' (FA=2.24); for the NIDDM female a high FA was found in the 2nd finger (FA=2.17) compared to controls. CONCLUSION: Overall measures of the above ridge counts and angles and their derived measures of FA were prominent features of NIDDM in this sample.

Variations of the location of the greater palatine foramina in dry human skulls.

Seventy-one adult skulls (58 dentulous skulls: 56 bilateral and 2 unilateral, 13 edentulous: 11 bilateral and 2 unilateral) of unknown sex were examined for the variations of the location of the greater palatine foramen. The location of greater palatine foramen (GPF) was noted with relation to mid sagittal suture (MSS), posterior palatal border (PPB), and maxillary molars by measuring the distances using a sliding caliper to the nearest millimeter. Difference in the distance of the foramen from MSS and PPG in both the skull types was found to be statistically insignificant. Commonest location of the foramen was found to be opposite to third molar tooth (85.95%), followed by the interval between second and third molar teeth (13.15%), and opposite to the second molar tooth (only one case - 0.88%). Well-formed bony canals were found along the groove for the palatine nerves and vessels unilaterally in the right side in two skulls. These observation may be of help to the dental surgeon during the peripheral block of maxillary nerve for the maxillo-facial surgery.

Retromolar foramen.

Of the many variants in the mandible, the retromolar foramen is the most important. The incidence of retromolar foramen was studied in a collection of 157 south Indian mandibles available at the department of Anatomy, St. John's Medical College, Bangalore. On each side of the mandible, the area behind the last molar tooth was inspected for the presence of retro molar foramen. Wherever present, their distance from the last molar tooth and their size if they were sufficiently large were measured with a sliding caliper to the nearest millimeter. Retromolar foramen were found to be bilateral in 8 mandibles (5.1%) and unilateral 20 [right: 9 (5.7%); left: 11 (7%)] mandibles. The diameter of the foramina was 2 mm on both sides.

The varying shape of the coronoid process of the mandible.

The coronoid process (meaning crow) is a flattened triangular bony process of the ramus of the mandible. It is usually directed upwards and slightly forwards in the living subject. The coronoid process is of clinical significance to the maxillofacial surgeon for reconstructive purposes. Several authors have described the varying shapes of the process. In the present study, the variable shape and surface area of the coronoid process were observed in a collection of 154 south Indian mandibles. Using the criterion proposed by Isaac and Holla (2001), the coronoid process of each side was inspected for the shape and direction, and surface area of the ramus and the coronoid process was also calculated. The shape of the coronoid process was bilaterally triangular in 16.2%, hook-shaped in 45.5%, rounded in 18.2% rounded and hook-shaped in 4.5%, and unilaterally triangular in 12.3%, hook-shaped in 11.7%, and rounded in 7.1% of the mandibles. The coronoid processes faced directly upward in 55.4%, forward in 40.7% and backward in 3.8% of the mandible.

Morphological variations of the lung fissures and lobes.

BACKGROUND: Knowledge of the position of fissures of the lungs is necessary for the appreciation of lobar anatomy and thus locating the bronchopulmonary segments. METHODS: Morphological variations of fissures and lobes of the lungs were studied in 30 pairs of lungs from cadavers. RESULTS: Five right-sided lungs showed absence of horizontal fissure, 19 showed incomplete horizontal fissure. Eleven right-sided and 14 left-sided lungs showed incomplete oblique fissure and two right-sided lungs showed both absence of horizontal fissure and an incomplete oblique fissure. Accessory fissure was seen in three left-sided and one right-sided lungs. CONCLUSIONS: Awareness regarding anatomical variations is essential for performing lobectomies and segmental resection and interpreting radiological images.

Curcumin-induced inhibition of cellular reactive oxygen species generation: novel therapeutic implications.

There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric, Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca2+ influx suggest that a rise in cytosolic Ca2+ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca2+ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.