Fenfuro®-mediated arrest in the formation of protein-methyl glyoxal adducts: a new dimension in the anti-hyperglycemic potential of a novel fenugreek seed extract.

The fenugreek plant (Trigonella foenum-graecum) is traditionally known for its anti-diabetic properties owing to its high content of furostanolic saponins, which can synergistically treat many human ailments. Non-enzymatic protein glycation leading to the formation of Advanced Glycation End products (AGE) is a common pathophysiology observed in diabetic or prediabetic individuals, which can initiate the development of neurodegenerative disorders. A potent cellular source of glycation is Methyl Glyoxal, a highly reactive dicarbonyl formed as a glycolytic byproduct. We demonstrate the in vitro glycation arresting potential of Fenfuro®, a novel patented formulation of Fenugreek seed extract with clinically proven anti-diabetic properties, in Methyl-Glyoxal (MGO) adducts of three abundant amyloidogenic cellular proteins, alpha-synuclein, Serum albumin, and Lysozyme. A 0.25% w/v Fenfuro® was able to effectively arrest glycation by more than 50% in all three proteins, as evidenced by AGE fluorescence. Glycation-induced amyloid formation was also arrested by more than 36%, 14% and 15% for BSA, Alpha-synuclein and Lysozyme respectively. An increase in MW by attachment of MGO was also partially prevented by Fenfuro® as confirmed by SDS-PAGE analysis. Glycation resulted in enhanced aggregation of the three proteins as revealed by Native PAGE and Dynamic Light Scattering. However, in the presence of Fenfuro®, aggregation was arrested substantially, and the normal size distribution was restored. The results cumulatively indicated the lesser explored potential of direct inhibition of glycation by fenugreek seed in addition to its proven role in alleviating insulin resistance. Fenfuro® boosts its therapeutic potential as an effective phytotherapeutic to arrest Type 2 diabetes.

Fenfuro^® is a novel patented formulation of Fenugreek seed extract with more than 45% furostanolic saponins and anti-diabetic property free from any side effect as established through clinical study.In the present study, the role of Fenfuro® in arresting in vitro AGE formation and glycation-induced amyloid formation has been demonstrated with the help of three amyloidogenic proteins, namely Human Lysozyme, Human alpha-synuclein and Bovine Serum Albumin using Methyl Glyoxal as the glycating agent.A 0.25% (w/v) ethanolic solution of Fenfuro® resulted in more than 50% arrest in glycation with simultaneous prevention of aggregation as demonstrated by native PAGE, DLS and inhibition of development of Thio-T positive amyloid like entities.The studies collectively aim toward the development of a safe therapeutic method for arresting protein glycation through direct physical intervention.

A randomized double blind placebo controlled trial to assess the safety and efficacy of a patented fenugreek (Trigonella foenum-graecum) seed extract in Type 2 diabetics.

Background: Fenugreek plant (Trigonella foenum-graecum) constitutes a traditionally acclaimed herbal remedy for many human ailments including diabetes, obesity, neurodegenerative diseases, and reproductive disorders. It is also used as an effective anti-oxidative, anti-inflammatory, antibacterial, and anti-fungal agent. The seed of the plant is especially enriched in several bioactive molecules including polyphenols, saponins, alkaloids, and flavonoids and has demonstrated potential to act as an antidiabetic phytotherapeutic. A novel patented formulation (Fenfuro®) was developed in our laboratory from the fenugreek seeds which contained >45% furostanolic saponins (HPLC). Objective: A placebo-controlled clinical compliance study was designed to assess the effects of complementing Fenfuro® on a randomized group of human volunteers on antidiabetic therapy (Metformin and sulphonylurea) in controlling the glycemic index along with simultaneous safety assessment. Study methodology and trial design: In a randomized double-blind, placebo-controlled trial, 42 individuals (21 male and 21 female volunteers) in the treatment group (out of 57 enrolled) and 39 individuals (17 male and 22 female volunteers) in the placebo group (out of 47 enrolled), all on antidiabetic therapy with Metformin/Metformin with sulphonyl urea within the age group of 18-65 years were administered either 1,000 mg (500 mg × 2) (Fenfuro®) capsules or placebo over a period of 12 consecutive weeks. Fasting and postprandial glucose along with glycated hemoglobin were determined as primary outcomes to assess the antidiabetic potential of the formulation. Moreover, in order to evaluate the safety of the formulation, C-peptide and Thyroid Stimulating Hormone (TSH) levels as well as immunohematological parameters were assessed between the treatment and placebo groups at the completion of the study. Results: After 12 weeks of administration, both fasting as well as postprandial serum glucose levels decreased by 38 and 44% respectively in the treatment group. Simultaneously, a significant reduction in glycated hemoglobin by about 34.7% was also noted. The formulation did not have any adverse effect on the study subjects as there was no significant change in C- peptide level and TSH level; liver, kidney, and cardiovascular function was also found to be normal as assessed by serum levels of key immunohematological parameters. No adverse events were reported. Conclusion: This clinical compliance study re-instated and established the safety and efficacy of Fenfuro® as an effective phytotherapeutic to treat hyperglycemia.

Current Insights on the Photoprotective Mechanism of the Macular Carotenoids, Lutein and Zeaxanthin: Safety, Efficacy and Bio-Delivery.

Ocular health has emerged as one of the major issues of global health concern with a decline in quality of life in an aging population, in particular and rise in the number of associated morbidities and mortalities. One of the chief reasons for vision impairment is oxidative damage inflicted to photoreceptors in rods and cone cells by blue light as well as UV radiation. The scenario has been aggravated by unprecedented rise in screen-time during the COVID and post-COVID era. Lutein and Zeaxanthin are oxygenated carotenoids with proven roles in augmentation of ocular health largely by virtue of their antioxidant properties and protective effects against photobleaching of retinal pigments, age-linked macular degeneration, cataract, and retinitis pigmentosa. These molecules are characterized by their characteristic yellow-orange colored pigmentation and are found in significant amounts in vegetables such as corn, spinach, broccoli, carrots as well as fish and eggs. Unique structural signatures including tetraterpenoid skeleton with extensive conjugation and the presence of hydroxyl groups at the end rings have made these molecules evolutionarily adapted to localize in the membrane of the photoreceptor cells and prevent their free radical induced peroxidation. Apart from the benefits imparted to ocular health, lutein and zeaxanthin are also known to improve cognitive function, cardiovascular physiology, and arrest the development of malignancy. Although abundant in many natural sources, bioavailability of these compounds is low owing to their long aliphatic backbones. Under the circumstances, there has been a concerted effort to develop vegetable oil-based carriers such as lipid nano-emulsions for therapeutic administration of carotenoids. This review presents a comprehensive update of the therapeutic potential of the carotenoids along with the challenges in achieving an optimized delivery tool for maximizing their effectiveness inside the body.

Lutein and zeaxanthin are the two most abundant natural xanthophylls (oxygenated carotenoids) with a linear C40 tetraterpene/isoprenoid lycopene-based backbone.Presence of extensive conjugation (more than 10 double bonds) enable these molecules to act as accessory light harvesting pigments apart from chlorophyll.More importantly, the xanthophylls prevent photobleaching of the pigments and proteins in the Light Harvesting Complex (LHC) by sequestering the excess unutilized blue light and preventing triplet chlorophyll associated formation of Reactive Oxygen Species.In human eye, lutein, zeaxanthin along with mesozeaxanthin constitute the three macular pigments forming the so called "yellow spot" of the macula and are implicated in maintaining the redox balance, homeostasis and normal physiology of the eyes.However, unlike plants, xanthophylls must be acquired from dietary sources such as colored leafy vegetables and egg yolk.Increase in the number of eye diseases in the aging population coupled with insufficient bioavailability of xanthophylls has mandated the industrial production of supplements enriched in xanthophylls.The bioavailability and delivery of xanthophylls can be significantly enhanced by suspension in a blend of extra-virgin olive oil and other vegetable oils.

Clinical Evaluation of a Novel, Patented Green Coffee Bean Extract (GCB70®), Enriched in 70% Chlorogenic Acid, in Overweight Individuals.

OBJECTIVE: Obesity and overweight are challenging health problems of the millennium that lead to diabetes, hypertension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), and atherosclerosis. Green coffee bean exhibited significant promise in healthy weight management, potentiating glucose-insulin sensitization and supporting liver health. The safety and efficacy of a novel, patented water-soluble green coffee bean extract (GCB70® enriched in 70% total chlorogenic acid and <1% caffeine) was investigated in 105 participants for 12 consecutive weeks. An institutional review board and Drugs Controller General (India) (DCGI) approvals were obtained, and the study was registered at ClinicalTrials.gov. METHOD: Body weight, body mass index (BMI), waist circumference, lipid profile, plasma leptin, glycosylated hemoglobin (HbA1c), and total blood chemistry were assessed over a period of 12 weeks of treatment. Safety was affirmed. RESULTS: GCB70 (500 mg BID) supplementation significantly reduced body weight (approximately 6%; p = 0.000**) in approximately 97% of the study population. About a 5.65% statistically significant reduction (p = 0.000**) in BMI was observed in 96% of the study volunteers. Waist circumference was significantly reduced by 6.77% and 6.62% in 98% of the male and female participants, respectively. Plasma leptin levels decreased by 13.6% in 99% of the study population as compared to the baseline value. Upon completion of 12 weeks' treatment, fasting glucose levels decreased by 13.05% (p = 0.000**) in 79% of the study population. There was a statistically significant decrease in HbA1c levels in both male and female participants (p = 0.000**), while 86.7% of the study participants showed a statistically significant decrease in thyroid-stimulating hormone (TSH) levels (p = 0.000**). The mean decrease in TSH levels on completion of the treatment was 14.07% in the study population as compared to baseline levels. Total blood chemistry analysis exhibited broad-spectrum safety. CONCLUSIONS: This investigation demonstrated that GCB70 is safe and efficacious in healthy weight management.

A Placebo-Controlled, Double-Blind Clinical Investigation to Evaluate the Efficacy of a Patented Trigonella foenum-graecum Seed Extract "Fenfuro®" in Type 2 Diabetics.

BACKGROUND: Trigonella foenum-graecum (Fenugreek) is an extensively researched phytotherapeutic for the management of Type 2 diabetes without any associated side effects. The major anti-diabetic bioactive constituents present in the plant are furostanolic saponins, which are more abundantly available in the seed of the plant. However, the bioavailability of these components depends on the method of extraction and hence formulation of the phytotherapeutic constitutes a critical step for its success. OBJECTIVE: The present study reports the efficacy of a novel, patented fenugreek seed extract, Fenfuro®, containing significant amount of furostanolic saponins, in an open-labelled, two-armed, single centric study on a group of 204 patients with Type 2 diabetes mellitus over a period of twelve consecutive weeks. RESULTS: Administration of Fenfuro® in the dosage of 500 mg twice daily along with metformin and/or sulfonylurea-based prescribed antidiabetic drug resulted in a reduction of post-prandial glucose by more than 33% along with significant reduction in fasting glucose, both of which were greater than what resulted for the patient group receiving only Metformin and/or Sulfonylurea therapy. Fenfuro® also resulted in reduction in mean baseline HOMA index from 4.27 to 3.765, indicating restoration of insulin sensitivity which was also supported by a significant decrease in serum insulin levels by >10% as well as slight reduction in the levels of C-peptide. However, in the case of the Metformin and/or Sulfonylurea group, insulin levels were found to increase by more than 14%, which clearly indicated that drug-induced suppression of glucose levels instead of restoration of glucose homeostasis. Administration of the formulation was also found to be free from any adverse side effects as there were no changes in hematological profile, liver function and renal function. CONCLUSION: The study demonstrated the promising potential of this novel phytotherapeutic, Fenfuro®, in long-term holistic management of type-2 diabetes.

Revisiting the Multifaceted Therapeutic Potential of Withaferin A (WA), a Novel Steroidal Lactone, W-ferinAmax Ashwagandha, from Withania Somnifera (L) Dunal.

Withania somnifera (L.) Dunal, abundant in the Indian subcontinent as Ashwagandha or winter cherry, is a herb of unprecedented therapeutic value. The number of ailments for which crude Ashwagandha extract can be used as a preventive or curative is practically limitless; and this explains why its use has been in vogue in ancient Ayurveda since at-least about four thousand years. The therapeutic potential of Ashwagandha mainly owes from its reservoir of alkaloids (isopelletierine, anaferine), steroidal lactones (withanolides) and saponins with an extra acyl group (sitoindoside VII and VIII). Withaferin A is an exceptionally potent withanolide which is found in high concentrations in W. somnifera plant extracts. The high reactivity of Withaferin A owes to the presence of a C-28 ergostane network with multiple sites of unsaturation and differential oxygenation. It interacts with the effectors of multiple signaling pathways involved in inflammatory response, oxidative stress response, cell cycle regulation and synaptic transmission and has been found to be significantly effective in inducing programmed cell death in cancer cells, restoring cognitive health, managing diabetes, alleviating metabolic disorders, and rejuvenating the overall body homeostasis. Additionally, recent studies suggest that Withaferin A (WA) has the potential to prevent viral endocytosis by sequestering TMPRSS2, the host transmembrane protease, without altering ACE-2 expression. The scope of performing subtle structural modifications in this multi-ring compound is believed to further expand its pharmacotherapeutic horizon. Very recently, a novel, heavy metal and pesticide free formulation of Ashwagandha whole herb extract, with a significant amount of WA, termed W-ferinAmax Ashwagandha, has been developed. The present review attempts to fathom the present and future of this wonder molecule with comprehensive discussion on its therapeutic potential, safety and toxicity.Key teaching pointsWithania somnifera (L.) Dunal is a medicinal plant with versatile therapeutic values.The therapeutic potential of the plant owes to the presence of withanolides such as Withaferin A.Withaferin A is a C-28 ergostane based triterpenoid with multiple reactive sites of therapeutic potential.It is effective against a broad spectrum of ailments including neurodegenerative disorders, cancer, inflammatory and oxidative stress disorders and it also promotes cardiovascular and sexual health.W-ferinAmax Ashwagandha, is a heavy metal and pesticide free Ashwagandha whole herb extract based formulation with significant amount of Withaferin A.

Pathogenesis of Neurodegenerative Diseases and the Protective Role of Natural Bioactive Components.

Neurodegenerative diseases are a serious problem throughout the world. There are several causes of neurodegenerative diseases; these include genetic predisposition, accumulation of misfolded proteins, oxidative stress, neuroinflammation, and excitotoxicity. Oxidative stress increases the production of reactive oxygen species (ROS) that advance lipid peroxidation, DNA damage, and neuroinflammation. The cellular antioxidant system (superoxide dismutase, catalase, peroxidase, and reduced glutathione) plays a crucial role in scavenging free radicals. An imbalance in the defensive actions of antioxidants and overproduction of ROS intensify neurodegeneration. The formation of misfolded proteins, glutamate toxicity, oxidative stress, and cytokine imbalance promote the pathogenesis of Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Antioxidants are now attractive molecules to fight against neurodegeneration. Certain vitamins (A, E, C) and polyphenolic compounds (flavonoids) show excellent antioxidant properties. Diet is the major source of antioxidants. However, diet medicinal herbs are also rich sources of numerous flavonoids. Antioxidants prevent ROS-mediated neuronal degeneration in post-oxidative stress conditions. The present review is focused on the pathogenesis of neurodegenerative diseases and the protective role of antioxidants. KEY TEACHING POINTSThis review shows that multiple factors are directly or indirectly associated with the pathogenesis of neurodegenerative diseases.Failure to cellular antioxidant capacity increases oxidative stress that intensifies neuroinflammation and disease progression.Different vitamins, carotenoids, and flavonoids, having antioxidant capacity, can be considered protective agents.

A critical assessment of the whole plant-based phytotherapeutics from Withania somnifera (L.) Dunal with respect to safety and efficacy vis-a-vis leaf or root extract-based formulation.

Withania somnifera (L.) Dunal, popularly known as Ashwagandha or Indian ginseng, is well acclaimed for its health-enhancing effects, including its potent immunomodulatory, anti-inflammatory, neuroprotective, and anti-tumorigenic properties. The prime biological effectors of these attributes are a diverse group of ergostane-based steroidal lactones termed withanolides. Withanones and withanosides are distributed differentially across the plant body, whereas withanolides and withanones are known to be more abundant in leaves, while withanosides are found exclusively in the roots of the plants. Standardized W. somnifera extract is Generally Recognized as Safe (GRAS)-affirmed, however, moderate to severe toxic manifestations may occur at high dosages. Withaferin A, which also happens to be the primary bioactive ingredient for the effectiveness of this plant. There have been contrasting reports regarding the distribution of withaferin A in W. somnifera. While most reports state that the roots of the plant have the highest concentrations of this phytochemical, several others have indicated that leaves can accumulate withaferin A in proportionately higher amounts. A comprehensive survey of the available reports suggests that the biological effects of Ashwagandha are grossly synergistic in nature, with many withanolides together mediating the desired physiological effect. In addition, an assorted formulation of withanolides can also neutralize the toxic effects (if any) associated with withaferin A. This mini-review presents a fresh take on the recent developments regarding the safety and toxicity of the plant, along with a critical assessment of the use of roots against leaves as well as whole plants to develop therapeutic formulations. Going by the currently available scientific evidence, it is safe to infer that the use of whole plant formulations instead of exclusively root or leaf recipes may present the best possible option for further exploration of therapeutic benefits from this novel medicinal plant.HighlightsTherapeutic potential of withanolides owes to the presence of α,ß unsaturated ketone which binds to amines, alcohols, and esters and 5ß, 6ß epoxy group which react with side chain thiols of proteins.At concentrations above NOAEL (no observed adverse effect level), the same mechanisms contribute towards toxicity of the molecule.Although withanosides are found exclusively in roots, whole plants have higher contents of withanones and withanolides.Whole plant-based formulations have other metabolites which can nullify the toxicity associated with roots.Extracts made from whole plants, therefore can holistically impart all therapeutic benefits as well as mitigate toxicity.

Differential Evolution and Its Applications in Image Processing Problems: A Comprehensive Review.

Differential evolution (DE) is one of the highly acknowledged population-based optimization algorithms due to its simplicity, user-friendliness, resilience, and capacity to solve problems. DE has grown steadily since its beginnings due to its ability to solve various issues in academics and industry. Different mutation techniques and parameter choices influence DE's exploration and exploitation capabilities, motivating academics to continue working on DE. This survey aims to depict DE's recent developments concerning parameter adaptations, parameter settings and mutation strategies, hybridizations, and multi-objective variants in the last twelve years. It also summarizes the problems solved in image processing by DE and its variants.

Comparative Performance Analysis of Differential Evolution Variants on Engineering Design Problems.

Because of their superior problem-solving ability, nature-inspired optimization algorithms are being regularly used in solving complex real-world optimization problems. Engineering academics have recently focused on meta-heuristic algorithms to solve various optimization challenges. Among the state-of-the-art algorithms, Differential Evolution (DE) is one of the most successful algorithms and is frequently used to solve various industrial problems. Over the previous 2 decades, DE has been heavily modified to improve its capabilities. Several DE variations secured positions in IEEE CEC competitions, establishing their efficacy. However, to our knowledge, there has never been a comparison of performance across various CEC-winning DE versions, which could aid in determining which is the most successful. In this study, the performance of DE and its eight other IEEE CEC competition-winning variants are compared. First, the algorithms have evaluated IEEE CEC 2019 and 2020 bound-constrained functions, and the performances have been compared. One unconstrained problem from IEEE CEC 2011 problem suite and five other constrained mechanical engineering design problems, out of which four issues have been taken from IEEE CEC 2020 non-convex constrained optimization suite, have been solved to compare the performances. Statistical analyses like Friedman's test and Wilcoxon's test are executed to verify the algorithm's ability statistically. Performance analysis exposes that none of the DE variants can solve all the problems efficiently. Performance of SHADE and ELSHADE-SPACMA are considerable among the methods used for comparison to solve such mechanical design problems.

Assessing the Capability of Chemical Ameliorants to Reduce the Bioavailability of Heavy Metals in Bulk Fly Ash Contaminated Soil.

In-situ rehabilitation of fly ash at dumping sites has rarely been addressed for crop production due to growth-related constraints, largely of heavy metal (HM) contamination in soils and crops. Current communication deals with a novel approach to identify a suitable management option for rejuvenating the contaminated soils. In this background, a 60-days incubation experiment was conducted with different fly ash-soil mixtures (50 + 50%, A1; 75 + 25%, A2; 100 + 0%, A3) along with four ameliorants, namely, lime (T1), sodium sulphide (T2), di-ammonium phosphate (T3), and humic acid (T4) at 30 ± 2 °C to assess the ability of different fly ash-soil-ameliorant mixtures in reducing bio-availability of HMs. Diethylenetriaminepentaacetic acid (DTPA)-extractable bio-available HM contents for lead (Pb), cadmium (Cd), nickel (Ni), and chromium (Cr) and their respective ratios to total HM contents under the influence of different treatments were estimated at 0, 15, 30, 45, and 60 days of incubation. Further, the eco-toxicological impact of different treatments on soil microbial properties was studied after 60 days of experimentation. A1T1 significantly recorded the lowest bio-availability of HMs (~49-233% lower) followed by A2T1 (~35-133%) among the treatments. The principal component analysis also confirmed the superiority of A1T1 and A2T1 in this regard. Further, A1T1 achieved low contamination factor and ecological risk with substantial microbial biomass carbon load and dehydrogenase activity. Thus, liming to fly ash-soil mixture at 50:50 may be considered as the best management option for ameliorating metal toxicity. This technology may guide thermal power plants to provide the necessary package of practices for the stakeholders to revive their contaminated lands for better environmental sustainability.

COVID-19 X-ray image segmentation by modified whale optimization algorithm with population reduction.

Coronavirus disease 2019 (COVID-19) has caused a massive disaster in every human life field, including health, education, economics, and tourism, over the last year and a half. Rapid interpretation of COVID-19 patients' X-ray images is critical for diagnosis and, consequently, treatment of the disease. The major goal of this research is to develop a computational tool that can quickly and accurately determine the severity of an illness using COVID-19 chest X-ray pictures and improve the degree of diagnosis using a modified whale optimization method (WOA). To improve the WOA, a random initialization of the population is integrated during the global search phase. The parameters, coefficient vector (A) and constant value (b), are changed so that the algorithm can explore in the early stages while also exploiting the search space extensively in the latter stages. The efficiency of the proposed modified whale optimization algorithm with population reduction (mWOAPR) method is assessed by using it to segment six benchmark images using multilevel thresholding approach and Kapur's entropy-based fitness function calculated from the 2D histogram of greyscale images. By gathering three distinct COVID-19 chest X-ray images, the projected algorithm (mWOAPR) is utilized to segment the COVID-19 chest X-ray images. In both benchmark pictures and COVID-19 chest X-ray images, comparisons of the evaluated findings with basic and modified forms of metaheuristic algorithms supported the suggested mWOAPR's improved performance.

The Synergism in Hormonal and Cellular Changes in Male Mice on Long Term High Fat Exposure.

OBJECTIVE: To determine the hormonal changes that occur as a result of the long-term intake of a very-high-fat diet (VHFD) that leads to simultaneous changes in the islets of Langerhans and adipocyte cell size. METHODS: Male mice were fed with a normal chow diet (ND, n = 15) and a VHFD (n = 30) for 2, 12, and 24 weeks. Body weight, food intake, caloric intake (fat [saturated and unsaturated], protein, and carbohydrate), hormone levels (leptin and insulin), and islet of Langerhans/adipocyte size were quantitatively recorded. RESULTS: In VHFD-fed animals, body weight showed a significant percent increase within the first 12 weeks and then plateaued with time. VHFD-fed animals consumed significantly less food than ND at all time periods, indicating that it was the quality of food and not the quantity that caused this increase in body weight. Male mice on VHFD showed a significant increase in leptin and insulin levels, along with accompanying growth in islet and adipocyte size within the first 12 weeks, which plateaued as the mice aged. The increases in the islet and adipocyte size in VHFD-fed animals were similar to the analogous increases in hormonal levels (2 vs. 12 vs. 24 weeks). These results, therefore, suggest that in diet-induced obesity changes, shifts in hormonal levels works hand-in-hand with metabolic adjustments at the cellular level to combat the effect of fat. CONCLUSION: Thus, mechanisms like hormonal resistance, changes in adiposity, islet size, and caloric intake with prolonged exposure to high fat are probably defensive mechanisms employed to protect against diabetes. In order to understand these complicated and nuanced effects of high fat and to comprehend the underlying mechanism associated with it, it is important to focus on long-term studies that emphasize the synergy between cellular and hormonal changes, in addition to an analysis of individual components.

Estrogen is neuroprotective against hypoglycemic injury in murine N38 hypothalamic cells.

Estrogen (E2) has been demonstrated to possess protective effects from hypoglycemic toxicity, particularly in the pancreas. In the central nervous system, several brain regions, such as the hypothalamus, are highly vulnerable to hypoglycemic injuries that may lead to seizures, coma, and mortality. The present study performed a novel in vitro assay of hypoglycemic injury to hypothalamic cells, and is the first study, to the best of our knowledge, to demonstrate that E2 protects hypothalamic cells from hypoglycemic toxicity. The toxic effects of hypoglycemia on hypothalamic cells in vitro was determined by performing cell counts, together with MTT and lactate dehydrogenase assays, using the N38 murine hypothalamic cell line. Following 24 and 48 h in hypoglycemic conditions, a 60 and 75% reduction in cell number and mitochondrial function was observed, which reached 80 and ~100% by 72 and 96 h, respectively. E2 treatment prevented the hypoglycemia­induced loss in cell number and mitochondrial toxicity at 24 and 48 h. However at 72 and 96 h of hypoglycemic conditions, the neuroprotective effects of E2 on cell number or mitochondrial function was not significant or not present at all. In order to determine whether E2 exerted its effects through the AKT signaling pathway, the expression of proline­rich AKT substrate of 40 kDa (PRAS40) was analyzed. No alterations in PRAS40 expression were observed when N38 cells were exposed to hypoglycemic shock. From the biochemical and molecular data obtained, the authors speculated that E2 exhibits neuroprotective effects against hypoglycemic shock in hypothalamic cells, which dissipates with time. Despite demonstrating no significant effect on total AKT/PRS40 activity, it is possible that E2 may mediate these neuroprotective effects by upregulating the phosphorylated­AKT/pPRAS40 signaling pathway. The present study presented, to the best of our knowledge, the first in vitro model for hypoglycemic toxicity to hypothalamic cells, and provided evidence to suggest that E2 may protect hypothalamic cells from the damaging effects of hypoglycemia.

Long-Term High Fat Diet Has a Profound Effect on Body Weight, Hormone Levels, and Estrous Cycle in Mice.

BACKGROUND Obesity causes several health complications along with disruption of the reproductive system. The aim of the current study was to determine how long-term intake of very high fat diet (VHFD) changes the hormonal milieu, affecting the cellular morphology and reproductive cycle in female mice. MATERIAL AND METHODS Mice were fed on normal diet (ND) and VHFD for 2 weeks, 12 weeks, and 25-27 weeks. We assessed changes in body weight, food consumption, energy intake, cellular and tissue morphology, hormonal levels (leptin, insulin, and estradiol), and vaginal smears were performed at various time points to determine the length and cellularity at each stage of the estrous cycle. RESULTS Mice fed on VHFD showed a significant increase in weight gain, reduction in food intake, and increase in energy intake compared to animals fed on ND, indicating that the caloric density of the diet is responsible for the differences in weight gain. Hormonal analysis showed hyperleptinemia, hyperinsulinemia, and increases in estrogen levels, along with increases in size of the islet of Langerhans and adipocytes. After 25-27 weeks, all animals fed on VHFD showed complete acyclicity; elongation of phases (e.g., diestrous), skipping of phases (e.g., metestrous), or a combination of both, indicating disruption in the reproductive cycle. Quantitative analysis showed that in the diestrous phase there was a 70% increase in cell count in VHFD compared to animals fed on ND. CONCLUSIONS The above results show that morphological and hormonal changes caused by VHFD probably act via negative feedback to the hypothalamic-pituitary axis to shut down reproduction, which has a direct effect on the estrous cycle, causing acyclicity in mice.

Relationships between urinary biomarkers of phytoestrogens, phthalates, phenols, and pubertal stages in girls.

Phytoestrogens, phthalates, and phenols are estrogen-disrupting chemicals that have a pronounced effect at puberty. They are exogenous chemicals that are either plant-derived or man-made, and can alter the functions of the endocrine system and cause various health defects by interfering with the synthesis, metabolism, binding, or cellular responses of natural estrogens. Phytoestrogens, phthalates, and phenols are some of the potent estrogens detectable in urine. Phytoestrogens are plant-derived xenestrogens found in a wide variety of food products, like soy-based food, beverages, several fruits, and vegetables. Exposure to phytoestrogens can delay breast development and further lead to precocious puberty. The effect of phytoestrogens is mediated through estrogen receptors α and ß or by binding with early immediate genes, such as jun and fos. Phthalates are multifunctional synthetic chemicals used in plastics, polyvinyl chloride products, cosmetics, hair spray, and children's toys. Phthalates have been shown to cause defeminization, thelarche, precocious puberty, and an increase in breast and pubic hair in pubertal girls. However, reports are also available that show no association of phthalates with precocious puberty in girls. Phthalates can act through a receptor-mediated signaling pathway or affect the production of luteinizing hormone and follicle-stimulating hormone that has a direct effect on estrogen formation. Phenols like bisphenol A are industrial chemicals used mainly in the manufacture of polycarbonates and plastic materials. Bisphenol A has been shown to cause precocious puberty and earlier menarche in pubertal girls. Reports suggest that the neurotoxic effect of bisphenol A can be mediated either by competing with estradiol for binding with estrogen receptors or via the ERK/NK-kappa or ERRγ pathway. This review demonstrates the effects of phytoestrogens, phthalates, and phenols on the development of girls during puberty.

Estrogen-like endocrine disrupting chemicals affecting puberty in humans--a review.

Estrogen-like endocrine disrupting chemicals (EEDC) are exogenous, man-made chemicals that alter the functions of the endocrine system and cause various health defects by interfering with the synthesis, metabolism, binding or cellular responses of natural estrogens. EEDCs have been found in various plastic products, flame retardants, pesticides and many other products that are needed for daily use. Some of the greatest effects of EEDCs are on puberty, a period of rapid physiological changes like growth spurt, maturation of the gonads and the brain. Estrogen, one of the key hormones required in puberty is crucial for the sexual differentiation. The structural similarity of estrogen disruptors with estrogen allow them to bind and activate estrogen receptors and show a similar response even in the absence of estrogen that can lead to precocious puberty (PP). Major EEDCs found abundantly in our environment include; dichlorodiphenyltrichloroethane (DDT), dioxin, polychlorinated biphenyls (PCBs), bisphenol A (BPA), polybrominated biphenyls (PBB), phthalate esters, endosulfan, atrazine and zeranol. In girls, DDT has been linked to earlier menarche. Dioxin causes abnormal breast development in pre-pubertal girls. BPA has shown to cause PP in pubertal girls. PBB causes earlier menarche, thelarche and earlier pubic hair stage in pubertal girls. PCB's showed a significant delay in puberty in pubertal boys. De-feminization, thelarche, or early secondary breast development are shown in pubertal girls when exposed to phthalate esters. Endosulfan affects pubertal boys by slowing down the timing of reproductive maturation. This article provides a possible structure-function relation of the above mentioned EEDCs which interfere with sexual development during puberty.

Stereological analysis of estrogen receptor expression in the hypothalamic arcuate nucleus of ob/ob and agouti mice.

Circulating gonadal steroid levels affect metabolic homeostasis by regulating appetite and food intake. The actions of estrogen are mediated through its two receptors ERalpha and ERbeta. ERalpha expression is necessary to maintain normal food intake, body weight and adiposity. Leptin plays a central role in regulating feeding behavior, homeostasis and reproduction. It is known that there is an effect of estrogen and leptin on feeding behavior. The present study was undertaken 1) to assess the changes in the reproductive cycle in obese, infertile ob/ob mice with no circulating leptin and infertile, obese, agouti (Ay/a) mice with high circulating leptin levels, 2) to evaluate the hypothalamic distribution of ERalpha and ERbeta, and 3) to analyze the differences in expression of ERs related to leptin and beta-estradiol levels in these mouse lines. The results show that the ob/ob and Ay/a mice were acyclic and were at a persistent estrous phase. The beta-estradiol levels were similar between WT, ob/ob and Ay/a mice. Stereologic analysis showed that there were significantly higher numbers of ERalpha-immunoreactive cells in ob/ob mice irrespective of sex when compared to wild-type (WT) in arcuate nucleus (ARH) and no significant change in ERbeta immunoreactive cell numbers in ARH or paraventricular nucleus (PVN). Ovariectomy in female wild-type mice caused a 50% increase of ERalpha-immunoreactive cells. Results suggest that leptin and estrogen act via the same neuronal circuits to affect reproduction, neuroendocrine and behavioral processes. However, estrogen levels and acyclicity have more profound effect on the regulation of ERalpha cell numbers in the ARH than circulating leptin levels.

The presence of distal and proximal promoters for rat mitochondrial glycerol-3-phosphate acyltransferase.

Sequence analysis using the Promoser program predicted two promoter-like regions for rat mtGPAT: a distal promoter approximately 30kb upstream and a proximal promoter near the first translational codon. Rat liver cells transfected with pGL3-basic vector containing the distal and proximal promoter resulted in 10.8- and 4.8-fold increase in the luciferase activity, respectively. Results of electromobility shift assay and chromatin immunoprecipitation suggested binding of transcription factors to the distal and proximal promoter regions. 5' RACE PCR showed two transcripts with different transcriptional start sites. When transfected rat liver cells were starved and refed, there was about 2.7-fold increase in the luciferase activity with cells transfected with the distal promoter while the proximal promoter showed no change. Thus, the two promoters could be functionally distinguished. Taken together, the results suggest that there are two promoters for rat mtGPAT gene and that the transcriptional regulation is mediated through the distal promoter.

Phosphorylation of rat liver mitochondrial glycerol-3-phosphate acyltransferase by casein kinase 2.

We have previously shown rat liver mitochondrial glycerol-3-phosphate acyltransferase (mtGAT), which catalyzes the first step in de novo glycerolipid biosynthesis, is stimulated by casein kinase 2 (CK2) and that a phosphorylated protein of approximately 85 kDa is present in CK2-treated mitochondria. In this paper, we have identified the (32)P-labeled 85-kDa protein as mtGAT. We have also investigated whether the phosphorylation of mtGAT is because of CK2. Mitochondria were treated with CK2 and [gamma-(32)P]GTP as the phosphate donor. Autoradiography, Western blot, and immunoprecipitation results showed mtGAT was phosphorylated by CK2. Next, we incubated mitochondria with CK2 and either ATP or GTP, in the presence of heparin, a known inhibitor of CK2. Heparin inhibited CK2-induced stimulation of mtGAT activity; this inhibition resulted in decreased (32)P-labeling of mtGAT. Additionally, mitochondria were treated with CK2 and [gamma-(32)P]ATP in the presence of staurosporine (a serine/threonine protein kinase inhibitor), genistein (a tyrosine kinase inhibitor), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB, a CK2 inhibitor). Only DRB, the CK2 inhibitor, greatly reduced the amount of (32)P-incorporation into mtGAT by CK2. Finally, isolated mitochondrial outer membrane was incubated with cytosol in the presence of [gamma-(32)P]GTP; (32)P-labeled mtGAT was detected. Collectively, these data suggest that CK2 phosphorylates mtGAT. The impact of our results in the regulation of mtGAT and other anabolic processes is discussed.