#water under the bridge atp | Explore Tumblr posts and blogs

7nsomnia · 3 months ago

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can i ask, what’s wrong with dcc? i always hear that they kinda suck as a company, but from the vlogs i’ve seen, they’re one of the better companies. i’m not really as into dreamcatcher as some of the blogs on here even though i consider myself a stan, so i might not have the right information

okay. I feel like this is like opening my personal pandora box so this might be long. I'm pretty tired today so apologies in advance if this isn't very coherent asdkjh

dcc are a pretty decent company on a surface level, they treat the members well (which should be like the bare minimum for any company but I know that in this industry that's something to genuinely praise) and they actually change according/respond to negative feedback from the fandom etc when they or the members mess up (or they used to anyway).

for me it started in 2020 and how they handled handong's return. like the way they handled her absence was fine (good even, I would say), but the lack of hype for her actual return made things feel so underwhelming even though it was supposed to feel like a relief that she was finally back. I can't remember all the details anymore, but I do remember that the first time I felt like things were actually alright with dc was when they did the online concert crossroads in march of 2021. on that note I think most ppl were expecting ttol and dlm to be repackaged with ot7 versions and yet it's 2024 and they still haven't released them.

the handong stuff atp is water under the bridge tho, the group is fine, the members are fine, etc, I'm only mentioning it because that's when things started to feel really off for me.

so now we get into the actual things that happened that have left the fandom feeling burned out/frustrated/disconnected etc etc, whereas this happened to me at the end of 2022, I'm seeing more people now going through what I did back then:

I think the most pressing thing was that dcc didn't capitalize at all on dc's first win. they got their first win in april 2022 and didn't even do anything special in korea to commemorate it. it was a HUGE moment and they did nothing with it. usually after a group gets a first win you'll see them getting more promotions in korea, magazine photoshoots, mc deals, etc but dc just went on ahead to do festivals in europe and have a usa tour, these things are not bad but it was the lack of promotion in korea that in turn just made it all feel useless. that year dc also weren't invited to any end of year awards if I'm not mistaken so it all felt really disappointing and like all of the work we had as a fandom had been for nothing. I have to reiterate, dc/insomnias had been getting screwed over on music shows since 2019 with deja vu to get that first win, like I don't want to talk about the injustices the group and this fandom suffered through the years but it was a true story of resilience, so getting that first win in 2022 was a huge relief. to see it all going to waste was just... heartbreaking honestly.

when it comes to tours...... god I don't wanna get too much into it, but 4 tours in the usa in the span of 2 years is not normal. specially when they're prioritizing that over having a proper asia tour and the likes (AND promoting in korea??). latam tour is practically sold out rn and they're getting no merch or m&g benefits like the usa tour. I don't think doing exclusive things for a specific tour is bad per say, but you have to treat all your fans semi equally at least, specially for a group whose fanbase is majorly international (this will be important later), or it will happen what is happening rn which is ppl will leave the fandom. This is the first latam tour since 2019 (2017 for brazil!)... they've waited a really long time so personally (even tho this doesn't affect me bc I'm european) I feel like it's really disrespectful but wtv, onto other things.

now, speaking of the fanbase being majorly international, if this is the case, you'd think the company would make an effort to stream important events to their fans, like hmm the 7th anniversary concert perhaps? but nop, that didn't get streamed. a repetition of the dumbassery they did in 2022 where they split the concert and the members' solos in 2 days and only streamed one and so intl fans couldn't watch half the solo stages? and don't get me wrong, I think it's important that they have events that are korea only like they have the fansigns etc, but something as major as their 7th anniversary? when they've gotten here thanks to their international fans? that stings a little.

and lastly (maybe), we have dcc's usual lack of promotion during comebacks. fans always paying for ads, intl fans always doing the most for digitals even when it's Not their place (because this is smth that the korean fandom and dcc should be responsible for), fans having to reach out for vendors etc... Justice cb truly has been the culmination of the very worst promotions dcc has done tho and there have been some really bad promotions before... no radio shows, minimum interviews, barely any variety... were there even any ads? usually it's always fans paying out of pocket for ads. it just feels like throwing the members' and the company's work out the window for no good reason? Virtuous is one of their best albums and yet it feels like they just dumped it to go on tour again. I don't think that's necessarily a bad thing btw, having short promotions in korea is fine but like... promote for real? give your fandom content that they can watch and rewatch for however long it takes your group to have another cb? specially now that it seems that they're shifting to one album per year (not sure this is their wisest decision tho all things considered), you have to make sure that you promote that album properly? which kinda also goes with like, giving your fandom enough time to save for what you release and put out, specially if you're not trying to grow the fandom anymore. if they're dropping an album then don't announce a tour on top of that, and if they're announcing a tour then don't announce a photobook on top of that, and if they've just released an album then wait longer than a month to announce a photobook, and if they've just dropped a photobook then wait a bit longer until announcing the re print of albums the fans have been begging you for 6 years to re print LOL bc all this does is frustrate fans who can't make that much money in such a short time and it's stupid. like. in 2018 I dropped like 200 euros for like their very first photobook BECAUSE I had time to save that amount from their you and I cb (may) to whenever it was announced (I think it was august), and that was the highest tier (so you could get it for much cheaper) and bc back then it was like. well they barely release anything other than albums, so it's fine (also shipping was sooooooo much cheaper I miss it everyday, ofc this is not their fault tho but anyways).

lastly actually, oh my god. that stupid ass app where fans pay a subscription to message the members privately? has been the fucking worst thing to happen to this fandom and the members imo. if fans weren't respecting their boundaries before, it's even worse now. but it's also like. yeah the members should be reinforcing those boundaries, and I get wanting to at least make a buck of those problematic type of fans but I just don't think it has been good for the members at all. I won't elaborate too much on this because it will genuinely piss me the hell off but bottom line: that app has been hell for everyone genuinely there is no bright side to it other than dcc makes money out of it. and there's better ways to make money :))))))))

anyway this is over 1k words atp and somehow I feel like this all just the tip of the iceberg and I probably have forgotten many things bc tbh in the past year I've just. been trying to make peace with it all and just accept things for what they are because dc have been really special to me for such a long time and I just don't want dcc's decisions to make me throw all of that away (like I almost did). I love their music, I love the members, and so I will continue to celebrate wtv right decisions dcc makes but I'm not going to pretend that they're a good company when it comes to business decisions bc they're really not

#not gonna proof read all of this but yeah #hope this all makes some sense at least #ask

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kikker-oma · 4 months ago

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It’s 2:10 am as I’m typing this but here is the result of a late night writing session

He could only cover his ears, fingers digging into the cartilage as his mind screamed in protest. The eyes in front of him were deep and lively, a trait he lacked. His chest boiled, his throat tightening yet letting go at the same time. Kill me, he prayed, which he vowed to never do again. He would never pray again. Her disgrace broke him, shattered his soul, burned his mind and held his heart captive. The heart that rotted away in her cell, kill me now.

But you’re alive she whispered back, her voice the thorns of roses, a biting cold, yet forged from the fire at the same time, you’re alive, and will continue to live until your purpose has been filled. Their was no bargaining. No way to be free. He was stuck.

“You’re okay,” The teen reached out as he extended a hand towards him, “You’re okay.” His other hand’s grip slowly tightened around the younger boy’s smooth palm as he looked into his eyes.

Hatred and anger only swelled further beneath the surface, chunks of skin and earwax collecting under his fingers. He hated those eyes. “No!” His hands trembled, tears splashing and soaking in the dirt below the three, “It’s never okay-they all say it is,” the salty water made its way to his lips, “but it never is? We’re all lied to? Can’t you see? We’re pawns in this world, sacrificed for nothing!” His face flushed with intensity, “I am not a pawn!” His eyes flickered back to his palm, its wound scabbed over. A little bridge to connect the broken sides of the skin, “We were never meant to fight. I was never meant to fight.”

The boy slowly squirmed his grip out of the other’s hands, “But it’s destiny!” He called out, his voice rising to an insistent pitch.

He couldn’t tell if he was laughing or crying anymore, the loud heartbeat of his blurring the lines of emotion, “There’s no such thing.” He smiled, gazing up at the clouded sky, “You hear that? I can’t be manipulated by you anymore! But I’m still a slave, a plaything to your world!” The knees buckled as he crumpled to the ground, caving in on himself, “I can never be free.”

and I still haven’t found names for the three. they were originally based off of LU/LOZ characters but atp in writing I’ve merged and broke and created a whole new personalities for them and I need to stop calling them 1 2 and 3 in my storyboarding (writing storyboard is a thing right? Or is it called something else) they’re also the same dude just one from like far past , past ,and present so it can’t be that hard to pick-it’s just that none of the ones I’ve seen suit the personality

-blareandhee2:00amshenanagins

👀👀👀

BLARRREEE oh gosh this was so VISCERAL

Dang girl

Ooohh your poor blorbos

Thing 1,2, and 3 are having a BAD TIME

#sweet blarefordaglare #we love early am shenanigans hehe

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indigosprite · 3 years ago

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So Tsuna just beat the dog shit out of Mukuros body and NOBODY but chrome goes over to check on him. Which makes complete sense bc they don’t even know him or that he like daemon can body hop SKSKKSKS. But it makes me kinda warm that Tsuna also is like hey I know what this means and goes over there too and even apologizes for hurting him that bad despite Mukuro yelling at him to literally kill his body if he had to. Also this is chromes first time meeting him in person and...I need a moment.

#khr #manga cap #Rokudo Mukuro #chrome dokuro #tsuna sawada #I love this page #Mukuro about to feel the outside world again in his own body right after getting memories of being in that prison for a decade #chrome meeting the mythical voice in her head #Tsuna having any remorse like Mukuro didn’t try to kill him #water under the bridge atp #REUNION #Mukuro is so fuckin sick for getting up a week later and declaring war on him jsdkjdkdjdnfn #sawada tsunayoshi

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lupinepublishers-ado · 4 years ago

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Potentiation of Activity of Benfotiamine Co Administered with Thyroxine in Diabetes Induced Peripheral Neuropathy in Rats

Lupine Publishers| Archives of Diabetes & Obesity

Abstract

Diabetic peripheral neuropathy (DPN) is a multi-etiological microvascular complication. Prolong hyperglycemia leads to formation of advance glycation end product (AGE) and oxidative stress which are contributors of nerve dysfunction. DPN manifests as pain, slowing of nerve conduction velocity (NCV), sensory loss etc. The aim of the present study is to evaluate the individual and combined protective effect of benfotiamine (BT) and thyroxine (T4) against Streptozotocin (STZ) induced DPN in rats. After 48 hours of a single injection of STZ (60 mg/kg bw i.p) diabetic rats were administered BT (100 mg/kg p.o.), T4 (1mg/kg.s.c,) and their combination. Diabetic rats at 5th week, exhibited significant decrease in body weight, hyperalgesia, decreased muscle coordination, grip strength and NCV. Antioxidant activity of reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) was also found to be significantly decreased. Significant higher levels of glycosylated hemoglobin (GHb) and Malondialdehyde (MDA) were also observed in diabetic rats. Treatment with BT, T4 and their combination attenuated the decrease in level of nociceptive threshold, muscle coordination, grip strength.

NCV and antioxidant activity. Significant decrease in the elevated levels of GHb and MDA was also observed. A histopathological study of sciatic nerve also confirmed the improvement in cell architecture as compared to diabetic rats and has strengthened the neuroprotective effect of BT and T4 combination group. An improved In Vitro AGE inhibitory activity of BT, T4 and their combination was observed. These finding suggested that BT, T4 and their combination exerts a protective effect in progression of diabetic neuropathy by decreasing GHb, AGE formation and oxidative stress.

Keywords:Micro vascular; NCV; Antioxidant; AGE; Thyroxine; Benfotiamine

Abbreviations:DM: Diabetes mellitus; DPN: Diabetic Peripheral Neuropathy; MAPK: Mitogen Activated Protein Kinase; NCV: Nerve Conduction Velocity; TH: Thyroid Hormones; BT: Benfotiamine; AGE: Advance Glycation End Product; LPO: Lipid Peroxidation; SOD: Superoxide Dismutase; CAT: Catalase; GSH: Glutathione; TBARS: Thiobarbituric Acid Reactive Substances; BSA: Bovine Serum Albumin; PKC: Protein Kinase C; ROS: Reactive Oxygen Species

Introduction

Diabetes mellitus (DM) is a worldwide major health problem and it is a chronic metabolic disorder characterized by hyperglycemia resulting from inadequate secretion or impaired action of endogenous insulin. The prevalence of diabetes is increasing worldwide and is believed to increase to 300 million by the year 2030 [1]. Uncontrolled or persistent hyperglycemia in DM leading to several microvascular and microvascular complications. Diabetic peripheral neuropathy (DPN) is a common microvascular complication of DM affecting more than 50% of the diabetic patients [2]. The pathogenesis of DPN is considered to be complex and multifactorial resulting from contributions of various pathways including metabolic and vascular factors, which consists of activation of polyol pathway, advanced glycation end products pathway, hexosamine pathway, increased activity of mitogen-activated protein kinase (MAPK), protein kinase C, poly (ADP-ribose) polymerase, oxidative stress, apoptosis, impaired neurotrophic support, autoimmunity, inflammation, up regulation of endothelin [3]. The impairment of nerve function is a well-established early manifestation of diabetes both clinically and in experimental animal models. DPN causes dysfunction of small and large nerve fibers and negatively impacts quality of life in diabetic patients. Small-fiber peripheral neuropathy is characterized by behavioral abnormalities (cold, thermal hyperalgesia, loss of grip strength and motor incoordination and burning or lancinating pain, and predisposition to foot ulceration). Large-fiber dysfunctions include loss of position and vibration sensation, nerve-conduction abnormalities, and distal muscle weakness. Early disorders of nerve function include slowing in nerve conduction velocity (NCV), followed by axonal degeneration, axoglial disjunction, paranodal and loss of fibre density. Microangiopathy [4]. A number of different agents from diverse chemical classes have entered clinical trials for the treatment of metabolic abnormalities in DPN, but only few approved for clinical use while other drugs either ineffective or withdrawn [2]. Current treatment options for symptomatic treatment of DPN include antidepressants, anticonvulsants. These agents are modestly effective for symptomatic relief, but they neither affect the underlying pathology nor do they slow progression of the disease [5]. Hence a novel approach to bridge the gap in selecting the compound in treatment of DPN was used .The discovery of use of a drug for a new indication is an arbitrary process, as shown by many past examples like the use of zinc acetate for the treatment of Wilson’s disease [6], arsenic for acute promyelocytic leukemia [7], amphotericin B for leishmaniasis [8], and thalidomide for multiple myeloma [9]. The discovery of these “alternative” uses for drugs different from originally intended drug development process is referred to as drug repurposing or repositioning. Repositioning of drug efforts has many advantages, because the pharmacokinetics and pharmacodynamics of the drug are known, repositioning discoveries are less costly and quicker than traditional discovery efforts [10], which usually take 10-15 years [11], and cost upward of $1 billion [12], Although physicians and pharmaceutical/ biotechnology companies have manual methods and prior knowledge that enable them to carry out drug repositioning clinical trials, such successful repositioning of drugs is often serendipitous and rare. In this study we have selected thyroxine to explore for its activity in DPN. Thyroid hormones (TH) [T4 (tetraiodothyronine) and T3 (triiodothyronine)], the only iodine-containing compounds with biological activity [13]. TH stimulate synthesis of Na+/K+ ATPase and also regulates metabolism by stimulating protein synthesis and increase the use of glucose and fatty acids for ATP production. They also increase lipolysis and enhance cholesterol excretion [14]. The cardiac side effect of D isomer of thyroxine resulted discontinuation of the clinical uses of this hormone. Under normal conditions, about 41%of thyroxine is converted to T3, and about 21% is converted to metabolically inactive 3,3,5-triiodothyronine (reverseT3, rT3). T3 is a powerful inducer of pancreatic acinar cell proliferation in rodents [15]. In Vitro studies of human and rodent insulinoma cell lines showed that T3 protected from apoptosis and induces β-cell growth and proliferation [16]. A serendipitous, positive association between serum-FT3 (free tri iodothyronine) and an estimate of insulin production was found in euthyroid, lean, healthy individuals [17].Treatment of Human pancreatic duct cells (hPANC-1)with T3 induced changes in cell morphology, promotes cell differentiation into insulin-producing β-cells, unregulated insulin and glucose transporter protein-2 transcripts, and increases insulin release into the medium. TH receptor has been identified in pancreatic β -cell lines [18,19], T3-enhanced insulin release in isolated rat pancreatic islets exposed to glucose concentrations of 2-8 mmol/l, had no effect at concentrations of 12 mmol/l, and inhibited insulin release at concentrations of 16.6 mmol/l [20]. T3 promoted expression of important proteins involved in both glucose and lipid metabolism that may influence insulin secretion [21]. Benfotiamine (BT), a lipid soluble vitaminB1 with much higher bioavailability than thiamine [5] Benfotiamine was shown to prevent experimental diabetic retinopathy and In Vitro hyperglycemia-induced endothelial dysfunction. The effects of benfotiamine on in vivo endothelial function remained unknown [22]. Therefore, the present study was designed to evaluate whether diabetes induced DPN can be reversed by treatment with thyroxine and BT.

Materials and Methods

Experimental Animals

In-house laboratory bred healthy Wistar rats of weighing 200-250g were included for the study. Animals were housed in polypropylene cages on clean paddy husk bedding. Animals were maintained under controlled temperature at 25°C± 2°C with 12hr light/dark cycle with food and water provided ad labium. Animals which do not comply with above criteria, and which are found to be diseased will be excluded from the study. Before conducting the experiment, ethical clearance was obtained from “Institutional animal ethics committee” Al-Ameen College of Pharmacy”, Bangalore. Approval No: AACP/P-48.

Drugs and Reagents

Thyroxine (gift sample from Apotex Pharmachem India Pvt. Ltd.), benfotiamine (gift sample from Strides Arco Lab Pvt.Ltd.) were used in the present investigation. Streptozotocin (STZ) was purchased from Sigma Aldrich. Commercial diagnostic kit for the estimation of serum glucose was obtained from Span Diagnostics Ltd. Glycosylated hemoglobin kit was obtained from Crest Biosystems Pvt. Ltd. Other chemicals and reagents were of analytical grade and purchased from local suppliers.

Design of the Experiment

Induction of Diabetic Peripheral Neuropathy: After an overnight fast, Wistar rats were administered a single injection of streptozotocin [60 mg/kg of body weight i.p.in 100 mM sodium citrate buffers, pH 4.5] [23]. After 48 hours, animals with fasting blood glucose levels higher than 250mg/dl were selected for the study.

Experimental Procedure

Rats were randomly assigned in six groups (n=6)

Group1: Normal Control

Group2: Diabetic Control

Group3: Diabetic Control+ T4 (1mg/kg.s.c) [24] thrice a week for 5 weeks

Group4: Diabetic Control+ BT (100 mg/kg p.o.) [25] daily for 5 weeks

Group5: Diabetic Control +BT (100 mg/kg p.o.)+T4 (1mg/ kg.s.c,) for 5 weeks

For preventive studies treatment was started from day 2 of STZ administration along with insulin (3IU/kg, s.c) [26]. 5 weeks post treatment various behavioral, biochemical, electrophysiological and histopathological parameters were studied.

Behavioral Studies

Thermal and Cold Hyperalgesia [27,28]: Thermal and cold hyperalgesia were measured using the tail-immersion test in water maintained at high (46ºC) or low (4ºC) temperature. The duration of tail immersion was recorded, and a cut-off time of 15 s was used.

Measurement of Muscle Incoordination Using Rota Rod [28,29]: Rota rod has been used to evaluate motor coordination by testing the ability of rats to remain on a revolving rod. The apparatus has a horizontal rough metal rod of 3 cm diameter attached to a motor with variable speed. This 70 cm long rod was divided into four sections by wooden partitions. The rod was placed at a height of 50 cm to discourage the animals to jump from the rotating rod. The rate of rotation was adjusted in such a manner that it allowed the normal rats to stay on it for 5 min. Each rat was given five trials before the actual reading was taken. The readings were taken at15, 25, rpm after treatment in all groups of rats.

Measurement of grip strength [29,30]: Grip strength meter was used for evaluating grip strength of animals. Before commencement of experiment, the animals were acclimatized by placing on the instrument for some time to train, and then rats were held by the tail above the grid of grip strength meter. The animal was moved until its front legs were grasped the grid and it was brought to an almost horizontal position. The base of the tail was then pulled following the axle of the sensor until it released the grid. The force achieved by the animal was then displayed on the screen and was recorded as new tons or kg units.

Biochemical Studies

Estimation of GHb [30,31]: At the end of study (5 weeks) blood was withdrawn through retroorbital plexus of overnight fasted rat under light ether anesthesia using a glass capillary and collected in EDTA tubes. The glycosylated hemoglobin was determined by using kit. A hemolysed preparation of whole blood is mixed continuously for 5 minutes with a weakly binding cation-exchange resin. The labile fraction is eliminated during the hemolysate preparation and during the binding. During this mixing, HbAo binds to the ion exchange resin leaving GHb free in the supernatant. After the mixing period, a filter separator is used to remove the resin from the supernatant. The percent GHb is determined by measuring absorbance of the GHb fraction & the total hemoglobin (THb) fraction. The ratio of the absorbance of the GHb & the THb fraction of the Control and the Test is used to calculate the percent GHb of the sample using below formula.

Preparation of Nerve Homogenate: A segment of sciatic nerve, approximately 1.5 cm in length, 5mm proximal and 5 mm distal was used for preparing the 10% w/v homogenates for biochemical estimation. Tissue homogenates were prepared in 0.1M phosphate buffer (pH 7.4). The homogenate was centrifuged at 1000 rpm 4ºC for 3 min and the supernatant divided into two portions, one of which was used for measurement of lipid peroxidation (LPO) and the remaining supernatant was again centrifuged at 12,000 rpm at 4ºC for 15 min and used for the measurement of lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH).

Measurement of Lipid Peroxidation: The extent of lipid peroxidation in terms of thiobarbituric acid reactive substances (TBARS) formation was measured according to the method of Esterbauer and Cheeseman. Tissue extracts were mixed separately with 1ml TCA (20%), 2ml TBA (0.67%) and heated for 1h at 100°C, after cooling, the precipitate was removed by centrifugation. The absorbance of each sample was measured at 535 nm using a blank containing all the reagents except the sample. As 99% TBARS are malondialdehyde (MDA), so TBARS concentrations of the samples were calculated using the extinction coefficient of MDA, which is1.56 x 105 M-1 cm-1 and were expressed as μM of malondialdehyde per mg protein [31-34].

Estimation of Superoxide Dismutase (SOD) and Catalase Activity: Sciatic nerve homogenate was centrifuged at 4°C, 17,500×g for 10min. Supernatant was used for the measurement of SOD activity by pyrogallol autooxidation method [35,32] and catalase activity by H2O2 degradation method, which is a quantitative spectroscopic method developed for following the breakdown of H2O2 at 240nm in unit time. The sample readings were taken by placing 1ml of phosphate buffer and 100 μl of tissue homogenate in the reference cuvette and 1 ml of H2O2 and 100 μl of homogenate in the test cuvette in the spectrophotometer. For each measurement, the reading was taken at 240nm 1min after placing the cuvettes in Shimadzu spectrophotometer [36,33].

Measurement of Reduced Glutathione Activity: Reduced glutathione was assayed by the method of Van Dooran [37,34]. Briefly1.0 ml of sciatic nerve homogenate (10%w/v) was precipitated with 1.0 ml of sulphosalicylic acid (4%). The samples were kept at 4°C for at least 1h and then subjected to centrifugation at 1200g for 15min at 4°C.The assay mixture contained 0.1 ml supernatant,2.7 ml phosphate buffer (0.1M, pH 7.4) and 0.2 ml 5,5, dithiopyrs (2-nitro benzoic acid) (Ellman’s reagent, 0.1 mM, pH 8.0) in a total volume of 3.0 ml. The yellow color developed was read immediately at 412nm and the reduced GSH levels were expressed as μg/mg protein.

Electrophysiological Studies

Isolation of Sciatic Nerve: The rats were sacrificed by administration of overdose of ketamine/xylazine i.p. After anesthesia, rat backs were shaved and NCV was recorded. Briefly incision was made at L4-L6 spinal segments. The sciatic nerves were surgically exposed from sciatic notch to the gastrocnemius tendon and the left and right sciatic nerves were rapidly removed, carefully impregnated on fine filter paper to remove any accompanying blood soaked for 10 minutes in Ringer-Locke buffer to prevent spontaneous firing of the nerve.

Measurement of Nerve Conduction Velocity (NCV): The rats were anesthetized by administration of thiopentone sodium, 30mg/kg, and i.p [1]. After anesthesia, rat backs were shaved and motor NCV was recorded. Briefly incision was made at L4-L6 spinal segments. The sciatic nerves were surgically exposed from sciatic notch to the gastrocnemius tendon and the left and right sciatic nerves were rapidly removed, carefully impregnated on fine filter paper to remove any accompanying blood soaked for 10 minutes in Ringer-Locke buffer to prevent spontaneous firing of the nerve [32].The left sciatic nerves were then placed in a moist nerve chamber (MLT016/B AD Instruments, Australia) to measure NCV. NCV was measured by stimulating proximally at the sciatic notch by stimulating electrode (MLA270 AD Instruments, Australia) with 10 mV at 1Hz to 5 Hz and the action potential was measured using recording electrodes (MLA 285) by placing distally to the sciatic knottins was calculated by distance between stimulating and recording electrodes divided by the latency. Right sciatic nerves were transferred into Glutaraldehyde solution for histopathological studies and rinsed with ice cold saline homogenized in chilled phosphate buffer (pH 7.4) and used to assay lipid peroxidation, reduced glutathione and catalase [33,35].

Histopathological Studies

The right sciatic nerves were isolated and transferred in to 0.05mol/L phosphate buffered (30g/L) glutaraldehyde solution for histopathological studies (H&E staining, Kulchitsky Pal staining and Massion’s trichome) [36,37].

To determine In Vitro glycation of protein bovine serum albumin (BSA)-glucose assay was performed based on the procedure of Brownlee et al. BSA (l0mg/mL) was incubated with glucose (500mM) in phosphate buffered-saline (pH 7.4) and extract containing sodium azide (0.02%) at 37ºC with a final concentrations of BSA (2mg/mL), glucose (40mM), sample (0.1 to 0.5mg/mL). Sterilization of reagents and samples were done by filtration through 0.2μm membrane filters. Aminoguanidine was used as an inhibitor positive control and a reactions without any inhibitor were also setup. All the samples and positive control were kept for incubation at 37ºC for 15 days. After 15 days of incubation, fluorescence intensity (excitation wavelength of 370nm and emission wave-length of 440nm) was measured for the test solutions. Percent inhibition was calculated as follows: Inhibition %= Inhibition % = (1− ( As − Ab) ( Ac − Ab) ×100

Where As = fluorescence of the incubated mixture with sample, Ac, Ab = are the fluorescence of the incubated mixture without sample as a positive control and the fluorescence of incubated mixture without sample as a blank control.

Statistical Analysis

Statistical evaluations were done by ANOVA, expressed as mean± S.E.M. followed by Bonferroni comparison test using Graph Pad In Stat (Ver.3.10) and Graph Pad Prism 5 computer programme.

Results

Assessment of General Toxicity

The percentage body weight of normal and diabetic rats treated with T4, BT and combination at 5th week was found to be 26.87±1.74g, -15.90 ± 0.769g,-6.433±0.493g.-6.635 ± 0.661,-5.150± 0.4366.The percentage of change of body weight of diabetic rats significantly less (<P0.001) as compared to normal control, similarly the percentage of change of body weight of diabetic treated rats was significantly less (<P0.001) as compared to diabetic control rats control (Figure 1).

Behavioral Studies

Thermal and Cold Hyperalgesia

The tail flick latencies in both hot and cold immersion test of diabetic rats significantly changed at 5th week in diabetic rats as compared to normal rats (P<0.001) . 5 weeks treatment with T4, BT and combination significantly improved P<0.001 cold and hot immersion performance. (Figures 2&3).

Measurement of Muscle Incoordination using Rota Rod

Time latencies at both 15, 25 rpm of normal rats was found to be 103.8±1.74,67.67±1.687 respectively, time latencies of diabetic rats at both 15,25 rpm was found to 75.5±1.176 38.83±0.307 and same were significantly reduced (P<0.001) as compared to normal control. Time latencies of diabetic rats treated with T4, BT and Combination at both 15, 25rpm was found to be 86.67±0.7149, 44.83±0.477, 86.83±0.792, 52.17±0.872, 91.5±0.846, 57.17±0.792 respectively and same were significantly P<0.001 improved (Figures 4&5).

Measurement of Grip Strength

The grip strength of normal rats was found to be 9.822±0.1332,27.97±0.1171 respectively, The grip strength of diabetic rats was found to be 75.5±1.176, 38.83±0.307 and same were significantly reduced (P<0.001) as compared to normal control. Time latencies of diabetic rats treated with T4, BT and combination at both 15,25 rpm was found to be 86.67±0.7149, 44.83±0.477,86.83±0.792,52.17±0.872,91.5±0.846,57.17±0.792 respectively and same were significantly P<0.001 improved (Figure 6).

Biochemical Studies

Estimation of GHb: The percentage of GHb of normal rats was found to be 4.577±0.0249, The percentage of GHb of diabetic rats was found to be 9.537±0.066, and same was significantly reduced ( P<0.001) as compared to normal control. The percentage of GHb of diabetic rats treated with T4, BT and combination was found to be 9.357±0.01838, 5.698±0.02561, 5.277±0.0261, respectively and same were significantly improved. P<0.05 when compared T4 treated diabetic rats with diabetic control. P<0.001 when compared BT, combination treated diabetic rats with diabetic control (Figure 7).

Measurement of Lipid Peroxidation: The sciatic nerve MDA levels of normal rats was found to 1.627±0.008433, The sciatic nerve of MDA levels diabetic rats was found to be significantly high (P<0.001) i.e 3.553±0.02860, The sciatic nerve MDA levels of diabetic rats treated with T4, BT and combination was found to be 3.235±0.008466, 3.368±0.009098, 4.080±0.01807, respectively and same were significantly (P<0.001) reduced (Figure 8).

Estimation of Superoxide Dismutase (SOD): Sciatic nerve SOD activity in normal rats was found to be 205.7±0.1078, Sciatic nerve SOD activity was significantly (P.001) low 106.8±0.2798 in 5 weeks diabetic rats. SOD activity of diabetic rats treated with T4, BT and combination were found to be 130.0±0.2540, 135.6±0.1474 and 128.5±3.212 treatment significantly (P<0.001) restored SOD activity when compared to diabetic control (Figure 9).

Estimation of Catalase: Sciatic nerve catalase activity in normal rats was found to be 0.1058± 0.0004, Sciatic nerve catalase activity was significantly (P.001) low 0.0545±0.0013 in 5 weeks diabetic rats. Catalase activity of diabetic rats treated with T4, BT and combination were found to be 0.08833±0.00230, 0.0950±0.00096and 128.5±0.00047 treatment significantly (P<0.001) restored catalase activity when compared to diabetic control (Figure 10).

Measurement of Reduced Glutathione Activity: Sciatic nerve glutathione content in normal rats was found to be 0.1058± 0.0004, Sciatic nerve catalase activity was significantly (P.001) low 0.0545±0.0013 in 5 weeks diabetic rats. Catalase activity of diabetic rats treated with T4, BT and combination were found to be 0.08833±0.00230, 0.0950±0.00096and 128.5±0.00047 treatment significantly (P<0.001) restored catalase activity when compared to diabetic control. (Figure 11).

Measurement of Nerve Conduction Velocity (NCV)

Sciatic nerve conduction velocity was significantly (<P0.001) was significantly reduced in 5 weeks diabetic rats 44.11± 0.2907 when compared to normal 53.13±0.4599, T4 treated diabetic rats significantly (P<0.01) exhibited improved NCV 45.72±0.1954. BT and combination both have also significantly improved (P<0.001) NCV 48.01±0.1954, 48.64±0.1876 (Figure 12).

In Vitro

Glycation of Proteins

AGEs formation after incubation at 37ºC for 15 days, with an IC50 value of BT, T4, combination and standard aminoguanidine was found out to be 166.6±0.45μg/ml,410.25±0.32μg/ml, 162.7±0.37 μg/ml, 322.4± 2.23 μg/ml respectively. The combination exhibited higher inhibitory activity i.e.162.7±0.37μg/ml against AGEs formation after incubation compare to aminoguanidine (Figure 13).

Discussion

The major findings of the present study were that STZ induced diabetic rats showed significant weight loss, muscle incoordination, thermal and cold hyperalgesia, decreased grip strength, increased % GHb, electrophysiological abnormalities like decreased NCV and histological abnormalities when compared to normal rats. Treatment of diabetic rats with T4, BT and combination significantly improved the diabetes induced above deficits. Our results indicated that rats with diabetes induced by STZ showed body weight reduction during the experimental period. T4, BT and combination improved body weight from the initial value. Rats treated with combination exhibited less percentage of loss of body weight compared to T4 and BT alone thus the combination improved general health of rats by improving the body weight. DPN is associated with neuropathic pain which is most common in DPN, thus we evaluated the nociception in diabetic rats. Nociception was evaluated by thermal, cold hyperalgesia and was well evident in diabetic rats, which is in accordance with several other reports [39,37]. In the present study a significant reduction in nociception with T4, BT and combination treatment for five weeks was observed. The combination of T4, and BT exhibited synergistic effect on reducing nociception. The effectiveness of T4, BT and combination in neuropathic pain is further assessed by measurement of grip strength and muscle incoordination by grip strength meter and rotarod apparatus. Rotarod test was performed to examine the motor incoordination [40,38]. The Rotarod experiment demonstrated the impairment of the motor function and coordination in the diabetic rats. Diabetic rats showed shorter fall off time from the rotating rod when compared to control, suggesting impairment in their ability to integrate sensory inputs with appropriate motor commands to balance their posture. The T4, BT and combination treated diabetic rats increased the fall off time from the rotating rod compared to STZ-induced diabetic rats. Our results showed that T4, BT and combination normalize the motor function and coordination thus helps to maintain their posture during movement on the rod. The severity of diabetic neuropathy has been associated with decreased muscle strength in both type 1 and type 2 diabetes [41,39]. In our study we observed significant improvement in motor behavior particularly grip strength in addition to motor incordination after treatment with T4, BT and combination. The combination of T4, and BT thus exhibited synergistic effect on muscle incordination and gripstrength Marked increase inpercentage of Glycosylated haemoglobin (GHb) has been reported in previous studies in diabetic rats [42,40].

The levels of GHb is the marker of state of diabetes over a period of 90 days. Similar observations were found in diabetic control rats. In our study T4, BT and combination treated diabetic rats showed decreased HbA1levles. The decrease levels of HbA1 in T4 treated ratscould be due to decreasing elevated glucose by promoting cell differentiation into insulin-producing β-cells, upregulation of insulin, glucose transporter protein-2 transcripts, and insulin release [19], thus less glucose available for glycation with hemoglobin. The inhibition activity of T4 on glycation of proteins In Vitro was also measured in our study. T4 inhibited AGE formation In Vitro could be the contributing factor in inhibition of GHb in diabetic rats. Similar trend was observed in BT and combination treated rats. BT is a transketolase (TK) activator [43,41]. BT was shown to prevent experimental diabetic retinopathy and In Vitro hyperglycemia-induced endothelial dysfunction. The effects of benfotiamine on in vivo endothelial function remained unknown. BT has shown to inhibit hexosamine pathway, advanced glycation end product (AGE) formation pathway and the diacylglycerol (DAG) protein kinase C (PKC) pathways. BT was significantly decreased levels of HbA1 as discussed earlier could be due to inhibition of AGE formation as it was evidence by inhibition of In Vitro AGE formation in our study. The protective role of BT also probably due to its activity as co enzyme in various biological pathways [44,42]. The combination of BT and T4 has also shown synergistic effect in decreasing GHb.

A number of reports indicate that DPN is a hypoxic neuropathy. Decreased nerve blood flow may lead to decreased nerve conduction velocity (NCV) due to lower Na+-K+-exchanging ATPase activity [4]. Reactive oxygen species (ROS) such as superoxides and hydroxyl radicals cause vascular endothelial damage and reduced nitric oxide mediated vasodilatation. Studies have also showed evidence that superoxides and proximitized impairs endothelium dependent vascular relaxation of epineural arterioles of the sciatic nerve in diabetic rats [45,43]. In addition to vascular mechanisms nonvascular mechanisms like impairment of neurotrophic support have also been reported to cause nerve conduction deficits in DPN [46,44]. Enhancement of neurotrophic factors by prosaposinderived peptide was reported to preserve nerve conduction [47,45].The deficit in nerve conduction velocity was completely prevented by T4 treatment could be due to increased Na+-K+- exchanging ATPase activity, providing neurotrophic support and improving micro vascular circulation [48,46] further, T4 improved the endogenous antioxidants and decreased LPO in sciatic nerve homogenate. BT, combination treated rats were also exhibited augmented NCV could be due to the ability of BT to inhibit AGE formation, improved endogenous sciatic nerve antioxidant thus inhibiting AGE, free radicals induced nerve damage in our study. Thus these two vascular and nonvascular effects of BT and T4 would be the contributing factors for the synergistic activity in combination treated rats Morphological study of siatic nerve of normal rats showed closely packed nerve fibers normal endoneuria matrix separating the nerve fibers (Figure 14A), admixture of large and small diameter myelinated fibers and the thickness of the myelin sheath is proportionate the width of the axonal diameter (Figure 14 B). Conversly, diabetic rats displayed the histological damages like reduced nerve fiber density in the endoneurium, (loss of more of small myelinated fibers while large diameter fibers are better preserved), (endoneurial (Figure 15A), vascular thickening (diabetic microangiopathy) (Figure 15 B), However, myelin sheath was unaffected (Figure 14 B) by streptozotocin in DPN [49,47]. The altered sodium cell gradient due to impairment of Na+/K+ ATPase activity leading to altered membrane environment which in turn causes histological damages, and decrease myelin protein expression.

T4, BT and combination treated rats sections showed absence of diabetic vascular changes, no vascular thickening to mild vascular thickening (Figure 15B), normal density of fibers with preservation of small and large diameter fibers, presence of regenerating nerve clusters (Figures 16-18 ).Treatment with T4, BT and combination for five weeks almost completely prevented the histological damages induced by diabetes. T4, BT could probably prevent the histological damages induced by diabetes due to prevention of hyperglycemia induced vascular and non-vascular mechanisms.

Conclusion

Treatment with T4, BT, and combination effectively prevented many of the behavioural, electrophysiological and histological manifestations of diabetes induced peripheral neuropathy by decreasing thermal and cold hyperalgesia, improving motor incoordination, grip strength, NCV, fiber density.

Highlights of the Study

a) Diabetic control rats displayed behavioral, biochemical electrophysiological and histological deficits; however, myelin sheath was unaffected.

b) T4, BT and combination of both exhibited beneficial effects in diabetes induced peripheral neuropathy in rats.

c) T4, BT and combination also inhibited In Vitro AGE formation, however IC 50 values of thyroxine was found to be high compared to BT, standard.

d) T4 was found less effective compare to BT in reducing GHb in diabetic rats.

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xpaylife · 5 years ago

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The USP of XPay Life that is changing Technological pastures

The XPay Life company is started by the co-founders Rohit Kumar(CEO) and Bohitesh Misra(COO and CTO) of the startup. They were bonded over the passion of solving the nation's concerns related to village e-commerce and empowering people digitally. HEnce under their stewardship, XPay Life, the brainchild has seen a great purpose and providing the best digital solutions for Easy water bill payment in the country. For this venture, the company was recognized from the technical committees and is covered by popular online magazines like economic times, yourstory, Times Of India and many more.

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cash payment machine also known as the bill payment ATP kiosk is a state-of-art machine that provides bill payment facility through the cash acceptors that accepts cash and makes bill payment digital. Hence aiding in the digital India mission, by tapping rural India, with the roust infrastructures and the important factor of bridging the socio-economic digital gap between the rural and urban India. Hence pay all your utility bills through XPay Life kiosk and make life simpler.

Electricity bill online payment app is very user-friendly and provides easy navigation through the application. Hence the online bill payment is easy and adds another advantage by providing bill payment lucrative discounts upto Rs 200 on any recurring bill payment through XPay Life. Stay updated on all the discount updates through the social media of the XPay Life. big tv recharge online and offline can be done through the XPay Life services that include the bill payment through Mobile App, online website, ATP kiosk, PoS machine, and the mobile Van. It is catering to all sectors devoid of digital awareness or the geographies and thus digitizing the whole of India which was a colossal challenge till now.

Source: http://www.apsense.com/article/the-usp-of-xpay-life-that-is-changing-technological-pastures.html

#bill payment #bill #Online Bill Payment #water bill #jio postpaid bill payment #bsnl postpaid bill payment #BSNL broadband postpaid bill payment

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juniperpublishers-endocrinology · 5 years ago

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Potential Role of Mitochondrial Dysfunction in Diabetic Hypertriglyceridemia- Juniper Publishers

Abstract

Type 2 diabetes patients have increased oxidative stress and hypertriglyceridemia. We tested the hypothesis that these two are related and the latter could be the result of peroxide-mediated mitochondrial dysfunction and increased acetate production. Treatment of isolated liver mitochondria or primary hepatocytes with oxidized linoleic acid (LOOH) or hydrogen peroxide (H2O2) resulted in a drastic decrease in activities of pyruvate dehydrogenase (PDHC), aconitase and α-ketoglutarate dehydrogenase (KDHC). In contrast, the incorporation of 14C-acetate into lipids was not affected by peroxides suggesting that fatty acid synthesis was not affected. The livers of Db/db diabetic mice showed reduced enzyme activities as compared to control non-diabetic mice. In vitro reaction of pyruvate in the presence of LOOH or H2O2showed that pyruvate was non-enzymatically converted to acetate together with the release of carbon dioxide (CO2).

These results show that diabetic mice may convert more pyruvate non-enzymatically into acetate in the cytoplasm in the presence of peroxides. In addition, mitochondria in diabetic state may have poor capacity to utilize acetate by TCA cycle to generate energy. Combined with the findings that peroxides did not affect acetate incorporation of acetate into fatty acids, one could expect a net increase in fatty acid triacylglycerol (TG) production.

Abbreviations: EDTA: Ethylene Diamine Tetraacetic Acid; EGTA: Ethylene Glycol-Bis (β-aminoethyl Ether)-N,N,N’,N’-Tetraacetic Acid; FCCP: Carbonyl Cyanide-P-Trifluoromethoxy Phenylhydrazone; HBSS: Hanks Balnced Salt Solution; HNE: Hydroxy Non Enal; KDHC: Α-Ketoglutarate Dehydrogenase; LMB: Leukomethylene Blue Reaction; LOH: Lipid Hydroxide; LOOH: Lipid Peroxide; MSH: Mannitol Sucrose Hepes; NADH: Nicotinamide Adenine Dinucleotide Hydrogenase; NMR: Nuclear Magnetic Resonance; PBS: Phosphate Buffer Saline; PDHC: Pyruvate Dehydrogenase; RNS: Reactive Nitrogen Species; ROS: Reactive Oxygen Species; TBARS: Thiobarbituric Acid Reactive Substances; TCA: Tricarboxylic Acid Cycle

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Introduction

Increased oxidative stress observed in both clinical and experimental diabetes mellitus has been implicated in the etiology of chronic diabetic complications [1-4]. Hyperglycemia leads to an increase in lipid peroxidation in diabetic patients and animals reflecting a rise in reactive oxygen species production [5-8]. Association of diabetic pathology to mitochondrial dysfunction and oxidative stress have been well documented [9-12]. Various studies point to generalized mitochondrial dysfunction in type 2 diabetes patients [13]. For example, mitochondria of type 2 diabetes patients have been shown to possess reduced electron transport chain capacities and reduced citrate synthase activity [14]. Type 2 diabetes patient’s show reduced fatty acid oxidative capacities [15]. Free fatty acid levels are increased together with decreases in fat oxidative capacity in obese, and diabetic patients, and in time this can result in accumulation of fatty acids and acyl glycerols in tissues [13,16,17]. Under both normal and pathological conditions, mitochondria are considered as the major endogenous source of ROS [18-20]. During normal metabolism 1-2% of the electrons that flow into the respiratory chain catalyze the incomplete reduction of O2 generating superoxide anion and hydrogen peroxide. However, under certain pathophysiological conditions the generation of these oxidants dramatically increases, leading to an imbalance between the pro-oxidant and the antioxidant systems. In addition, the high content of polyunsaturated fatty acids in mitochondrial membranes enhances mitochondrial susceptibility to lipid peroxidation, leading to alterations in major enzymes involved in energy production. Liver is heavily dependent on mitochondrial oxidative catabolism for the majority of their ATP requirements.Elevated levels of ROS in liver cells are particularly dangerous because they mediate mitochondrial damage, which in turn can generate further oxidative stress in the cells [21]. Fatty acids are particularly sensitive to ROS/RNS oxidation, resulting in the formation of lipid peroxides, which are cytotoxic and lead to free-radical damage to other lipids, proteins and DNA [13].

Lipid abnormalities are commonly associated with diabetes, especially high risk for hyperlipidemia, most commonly in the form of elevated triacylglycerol levels and decreased highdensity lipoprotein (HDL) levels. The most important pathogenic mechanisms, such as increased oxidative stress and increase free fatty acids and triacylglycerols followed by inactivation of mitochondrial enzymes have been identified in experimental studies [4,22-27]. Although multiple oxidant moieties may participate, there is in vitro and ex vivo evidence to support a role for superoxide anion and H2O2 in the pathogenesis of vascular dysfunction, lipid peroxidation, and formation of glycooxidation products in diabetes [23,26-30]. Various markers of oxidative stress such as increases in oxidized lipoproteins, red cell membrane lipid peroxidation, advanced glycation end products have been documented in blood and tissues of human and experimental diabetic subjects [31-34].

Recent studies shown defects in TCA cycle enzymes are associated with accumulation of very long chain fatty acids and may be accompanied by alterations in the intracellular pool of fatty acid and fatty acyl CoAs, which are known to alter mitochondrial function [35]. Most importantly, mitochondrial alterations in TCA cycle enzymes directly cause defects in electron transport chain (ETC). The pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme located exclusively in the mitochondrial matrix that catalyzes the oxidative decarboxylation of pyruvate and represents the sole bridge between anaerobic and aerobic cerebral energy metabolism. α-Ketoglutarate dehydrogenase complex (KGDHC) located in the inner mitochondrial matrices is crucial in the cellular production of reducing equivalents (NADH) and in the maintenance of the mitochondrial redox state [36]. Both pyruvate dehydrogenase [37] and α-Ketoglutarate dehydrogenase [38] are highly susceptible to oxidants inactivation in vitro. Aconitase contains an (4Fe-4S) cluster and is present in two isoforms. The mitochondrial isoform catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle, and the cytosolic isoform is involved in iron metabolism [39]. The (4Fe-4S) cluster confers a marked sensitivity to oxidative stress, and the enzyme is inactivated by reactive oxygen species (ROS) [40-42].

Because mitochondria are a site of free radical production and oxidative damage in diabetes [43,44], it is likely that free radical events contribute to declines in mitochondrial function in those subjects. Since mitochondria are the major cellular site involved in fatty acid metabolism, and the main source of reactive oxygen species (ROS), they could play a key role in fat storage and related complications. However, only limited dataexist on the involvement of the mitochondrial compartment in this process.

The present study show increased oxidative stress in diabetic compared to age matched normal mice. Diabetic mice show an inactivation of mitochondrial energy producing enzymes. In addition, these mitochondrial enzymes are very susceptible to inactivation by LOOH (HPODE) or H2O2. There is little doubt that one of the most reactive products of fat oxidation is lipid hydroperoxide. Our in vitro studies show that under oxidative stress pyruvate is converted non-enzymatically into acetate that further converted into free fatty acids and triacylglycerols. The goals of this study were to

Determine the mechanism of increased production of free fatty acids and triglycerides in diabetic subjects and

To identify mitochondrial enzymes inactivated in diabetes under oxidative influence of LOOH or H2O2.

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Materials And Methods

Animals

Male BKS.Cg-m +/+ Leprdb/J (Db/db) mice (Jackson Laboratories) of 7-9 months of age were housed in a temperature controlled room with 12:12hr light cycle and maintained with access to food and water ad libitum. Db/db mice have a spontaneous Leprdb mutation and develop hyperinsulinemia, hyperglycemia and obesity by 1 to 2 months of age. Aged matched, C57BKS/J mice were used as controls (BKS). All procedures were conducted with the approval of the Institutional Animal Care and Use Committee of the Ohio State University, Columbus, OH, USA and in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Blood glucose was checked via tail stick with a freestyle glucose monitor in order to verify the presence of hyperglycemia in the Db/db mice.

Isolation of mitochondria

Liver mitochondria were isolated from male BKS.Cg-m +/+ Leprdb/J (Db/db) or C57BKS/J mice and Sprague-Dawley rats by differential centrifugation and purified by Percoll purification as described [45]. In brief, liver was removed, washed, and homogenized in MSH EDTA (mannitol, 220mm; sucrose, 70mm; HEPES, 5mm; EDTA, 1mm; pH 7.40). The homogenate was centrifuged at 1,000 x g for 10min, and the supernatant was re-centrifuged at 10,000 x g for 10min to obtain crude mitochondrial fraction. The enriched mitochondrial pellet was layered on a Percoll solution (25%) and centrifuged for 30min at 100,000×g. The middle layer was extracted and washed twice in MSH (mannitol, 220mm; sucrose, 70mm; HEPES, 5mm; pH 7.40) and centrifuged for 10 min at 10,000×g. The purified mitochondria pellet was resuspended in 0.5ml MSH and the purity of the mitochondrial preparation was determined. All steps were carried out at 4 °C. Purity of the isolated mitochondria was assessed by measuring cytochrome a using ε605-630nm 12mm-1 cm-1 and by measuring transmembrane potential that is rapidly reversed by the uncoupler FCCP. Only mitochondria with less than 5% impurity were used.

Human HepG2 culture and treatments

Human HepG2 cells (ATCC) were cultured in DMEM containing 10% fetal bovine serum, 2mm glutamine, 100 units/ml penicillin and 100μg/ml streptomycin and 4.5mg/L D-glucose. The cells were cultured in 75cm2 flask s for 5 days at 37 °C under a humidified atmosphere of 95% air and 5% CO2 to about 80% confluence. Cells were treated with 50μM LOOH and 1mm H2O2 for a period of 4 hours. After treatment, cells were washed and supplemented with fresh DMEM and incubated with (13C) Na-Acetate (1mm) for 2 hours. At the end of incubation, the medium was aspirated, cells were washed twice with PBS. For the determination of de novo fatty acid synthesis, cells were scrapped from flasks, homogenized with glass homogenizer and protein content was assayed by Bradford method (1976) [46]. The cells were saponified with aqueous KOH at 37°C for 120min. The total fatty acids were extracted with chloroform. Fatty acid synthesis was determined by LC/MS.

Liver perfusion and culture of primary hepatocytes

Sprague-Dawley rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50mg/kg b.w.). Initially inferior venacava was cannulated and the liver was perfused in situ with an oxygenated Hank’s buffer salt solution (HBSS; pH 7.4) containing penicillin/streptomycin (100U/ml) at the rate of 98ml/min for 10min 37 °C. Liver was further perfused with oxygenated HBSS containing penicillin/streptomycin (100U/ ml), and insulin (1 x 10-7 M) followed by another perfusion with 0.04 % collagenase type IV (pH 7.4) for 10min. After perfusion the liver was gently removed and minced in HBSS containing CaCl2 (1mm), MgCl2 (1mm), penicillin/streptomycin (100U/ ml), and insulin (1 x 10-7 M) (pH 7.4). The liver cell suspension was filtered with Falcon cell strainers and centrifuged at 50 x g for 4min.

Rat hepatocytes were cultured in Williams medium supplemented with penicillin/streptomycin (100U/ml) and insulin (1 x 10-7M) for 1-2 days and treated with LOOH or H2O2 for 4 hours. After treatments hepatocytes were scrapped from wells. Mitochondria were isolated from hepatocytes as described under, broken down by freeze and thaw followed by sonication and centrifugation at 10,000 x g to get clear mitoplasts.

Preparation of mitochondria from cultured hepatocytes

Mitochondria from hepatocytes were isolated as described [47]. Briefly, cells were homogenized in lysis buffer (250mm sucrose, 10mm Tris/HCl (pH 7.4), 1mm EDTA, 10μg/ml leupeptin, 40 komberg international units/ml aprotinin, 10μg/ ml pepstatin A, 0.2mm phenylmethylsulfonyl fluoride) and the homogenate was centrifuged at 1500 × g for 10min at 4 °C, and the supernatant was kept on ice. The pellet was re-homogenized with a further 3ml of isolation buffer (320mm sucrose, 1mm K+-EDTA,10mm Tris-HCl, pH 7.4), and the homogenate was centrifuged at 1500 × g for 5min at 4 °C. The two supernatants were pooled and centrifuged at 1500 × g for 10min at 4 °C. Supernatant was further centrifuged at 17,000 × g for 10min at 4 °C. The pellet, i.e. the mitochondrial fraction, was then resuspended in 200μl of isolation buffer to obtain about 4mg of mitochondrial protein/ ml. These mitochondria, incubated in PBS, were checked for their intactness and coupling by measuring the cytochrome a using ε605-630nm 12mm-1 cm-1 and by measuring transmembrane potential that is rapidly reversed by the uncoupler FCCP.

Broken mitochondria and mitoplasts were prepared by 5 times freezing the intact mitochondria in liquid nitrogen followed by thawing and sonication (3min with 30s interval). Sonicated mitochondria were centrifuged at 25,000 x g for 10min and the supernatant was used for assay of PDHC, aconitase and KGDHC activities.

Preparation of LOOH (HPODE)

Linoleic acid hydroperoxide (HPODE) was prepared by oxidation of linoleic acid by soybean lipoxidase [48]. The isolated hydroperoxide was estimated by U.V. spectrophotometry [49] using the Leukomethylene Blue assay.

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Assays Of Mitochondrial Enzymes

Pyruvate dehydrogenase complex (PGDHC)

PGDHC activity was measured spectrophotometrically using a modified method of Hinman and Blass [50] by following the formation of NADH at 340nm at 37 °C. Isolated mitochondria were resuspended in the assay buffer (50mm phosphate buffer, pH 7.4). The reaction mixture contained the assay buffer, 0.2mm thiamine pyrophosphate, 1mm MgCl2, 2mm NAD+, 0.2mm EGTA, 2.6mm L-cysteine, 0.5mm CaCl2, 0.3mm dithiotreitol, 2mm pyruvate, and mitochondria (60μg/ml). The reaction was initiated by the addition of 0.2mm coenzyme A. Blank samples containing no pyruvate were included in all assays. The activity of the pyruvate dehydrogenase complex was expressed as nanomoles of NADH produced per minute per milligram of mitochondrial protein.

Aconitase

Aconitase activity was assessed by the method of Drapier JC and Hibbs (1976) [51]. The mitochondria (60μg/ml) was resuspended in 100mm Tris•HCl buffer, pH 7.4, containing 1mm MgCl2, 1mm NADP, and 1mm potassium citrate. The reaction was started by adding isocitrate dehydrogenase (2U/ml), carried out at 37°C. Blank samples containing no isocitrate dehydrogenase were included in all assays. The activity of aconitase was expressed as nanomoles of NADPH formed per minute per milligram of protein.

α-Ketoglutarate dehydrogenase complex (KGDHC)

KGDHC activity was measured according to the method of Tretter and Adam-Vizi, (2000) [52] by following the formation of NADH at 340nm at 37 °C. Mitochondrial aliquots (60μg/ml)were added to a 50mm phosphate buffer (pH 7.4) containing 0.2mm thiamine pyrophosphate, 1mm MgCl2, 2mm NAD+, 0.2mm EGTA, 2.6mm L-cysteine, 0.5mm CaCl2, 0.3mm dithiotreitol, and 2mm α-ketoglutarate. The reaction was initiated by the addition of 0.2mm coenzyme A. Blank samples containing no α-ketoglutarate were included in all assays. The activity of α-ketoglutarate dehydrogenase complex was expressed as nanomoles of NADH produced per minute per milligram of mitochondrial protein.

Statistical analysis

Unless otherwise indicated, data are shown as mean±SD. Data were compared by Student’s t-test. For all experiments, p<0.05 denoted statistical significance.

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Results

Body and weight in diabetic mice.

Db/db mice displayed significantly increased body weight as compared to BKS mice (in g: 56.2±1.2 vs. 27.5±0.4, respectively, p<0.05).

Lipid peroxidation

To assess oxidative damage in liver mitochondria, TBARS was tested. Our results showed an increase in TBARS levels in liver mitochondria of diabetic mice compared to normal mice (data not shown).

Decreased activities of PDHC, aconitase and KGDHC in diabetic mice

Next we tested the possibility that increased lipid peroxides and consequently increased oxidative stress may cause alterations in mitochondrial energy producing enzymes, making it potentially difficult to maintain energy production in mitochondria. We measured activities of liver PDHC, aconitase and KGDHC in diabetic and normal mice. Our study show decrease in activities of PDHC, aconitase and KGDHC in liver of diabetic mice compared to normal (Figure 1).

Decreased activities of PDHC, aconitase and KGDHC in rat liver isolated mitochondria and hepatocytes by LOOH and H2O2

In order to verify potential effects of lipid peroxides on mitochondrial energy producing enzymes, we treated isolated rat liver mitochondria with LOOH or H2O2 for 1hr at room temperature followed by 1hr in ice. Treated mitochondria were disrupted by freeze and thaw followed by sonication and clear homogenous liquid of mitoplasts was obtained for assay. As shown in Figure 2 treatment of mitochondria with both LOOH and H2O2 caused drastic inactivation of PDHC, aconitase and KGDHC. We further verified inactivation of these mitochondrial enzymes using cultured primary hepatocytes treated with LOOH or H2O2. After treatment mitochondria were isolated from cells, disrupted by freeze and thaw followed by sonication for determination of enzymes activities. As shown in Figure 2, our results show a similar decrease in PDHC, aconitase and KGDHC activities in mitochondria obtained from hepatocytes.

Increased fatty acid after treatment of LOOH

We anticipate that peroxides, in contrast to mitochondrial enzymes, would not adversely affect fatty acid synthesis in the liver cells. Cells were incubated with 13C -acetate in the presence of hydrogen peroxide for two hours, lipids were extracted and saponified. Tandem MS conditions were optimized with pure palmitic acid (PA). The results presented in Figure 3, shows that 13C-acetate was incorporated very efficiently. The number of 13C-atoms increased up to 4 acetate units in the 2hr incubation and then slowly decreased, indicating that the acetate was efficiently utilized for FA synthesis even in the presence of peroxides.

Loss of pyruvate and LOOH

First, we tested loss of pyruvate in the presence of H2O2. Incubation of 50mm pyruvate with 50mm H2O2 causes disappearance of pyruvate with the lapse of time. We noted a decrease of 37% to 22% with increase in incubation time from 30min to 90min (Figure 4A). The reaction was stoichiometric with 1:1 mole equivalents of reactants. We employed mill molar concentrations of reactants in order to follow spectrophotometric detection.

LOOH contains both peroxide component as well as conjugated diene structure (Figure 4B). When LOOH is reduced to LOH, the peroxide component (as measured by LMB reaction) will be lost while the conjugated diene structure will be unaffected. Incubation of LOOH alone did not result in the loss of either peroxide or conjugated diene content As shown in Figure 4, both the open and closed bars remained at 100 % of the initial levels at the end of incubation. Similar incubations of LOOH with pyridine for 60min at 37 °C resulted in a complete loss of LMB assay activity (P<0.05). The levels of conjugated diene however, remained at original levels. We used acetate instead of pyruvate as control in these studies and there was no reduction in either LMB reactivity or conjugated diene in these incubations (data not shown). We used several other α-keto acids (phenyl pyruvic acid, α-ketoglutarate and dehydroascorbate) with result similar to that obtained using pyridine. Thus α-ketoacids are readily decarboxylated by LOOH or H2O2 to yield acetate. As increased cytoplasmic glucose has been reported to generate increased levels of pyruvate, the results can be interpreted to suggest that increased levels of acetate might be generated when there is a oxidative stress.

Non-enzymatic conversion of pyruvate into acetate

Next we tested non-enzymatic conversion of pyruvate into acetate. In order to demonstrate that acetate was indeed formed in the reaction, we used proton NMR as separation of acetate and pyruvate cannot be achieved by simple means. We incubated 1μmole of LOOH with 5μmoles of pyruvate and the products were analyzed by a Varian 400MHz NMR spectrometer. The 3H-NMR spectrum of sodium pyruvate in deuterated water exhibited a sharp singlet signal at δ 2.309 (Figure 5) showing the presence of methyl group whereas methyl group in sodiumacetate resonated at δ 1.861 (Figure 5). Due to presence of electron withdrawing keto group adjacent to methyl group in sodium pyruvate deshielded the methyl protons and shifted the signal toward downfield. The 3H-NMR spectrum of pyruvate and LOOH reaction mixture in deuterated water (D2O) gave two signals indicating the presence of both the reactant (pyruvate) and the product acetate (the reactant pyruvate was added at 5 times excess). The proton NMR spectrum of LOOH did not yield any of those signals indicating the change in chemical shift value and it must have occurred due to loss of keto group in sodium pyruvate catalyzed by LOOH or pyruvate yielding sodium acetate. Controls that lacked either LOOH or PYR did not show the generation of acetate.

Release of CO2 by decarboxylation of α-keto acids

Carbon dioxide evolved in H2O2 and pyruvate reaction was measured using radioactive calcium carbonate trapping method. Pyruvate was incubated alone, with H2O2 or H2O2 alone in a vial with a cap lined with 45CaCO3 (Figure 6A). Carbon dioxide released in experiment where pyruvate was treated with hydrogen peroxide was overwhelming (100%) compared to pyruvate (8%; p<0.001) or H2O2 alone (16%; p<0.002). Similarly formation of carbon dioxide evolution was higher when pyruvate was treated with LOOH (Figure 6B) (100%) when compared to pyruvate (10%; p<0.005) or LOOH alone (52%). The increased production of CO2 from LOOH alone was perplexing but interesting and reproducible. This suggest a direct peroxide mediated decarboxylation reaction. Such reactions are not unprecedented as it might suggest the formation of a per-acid step.

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Discussion

In this study, we establish for the first time that a mouse model of type II diabetes, Db/db mice exhibits not only oxidative stress in liver but also inactivation of three major mitochondrial enzymes, PDHC, aconitase and KGDHC activities.

ROS production is indicated as one of the potential causes leading to insulin resistance and hepatic disease in diabetic animals [53]. For these reasons, we also assessed the oxidative status of liver mitochondria by measuring TBARS reactive substance. Our results show higher TBARS in diabetic mice that indicate an increased oxidative damage compared to normalmice. Increased ROS production in fatty liver has already been documented [54,55], and has been indicated as one of the causes rendering the liver cell more vulnerable to further injury. The main cellular site of fatty acid oxidation is the mitochondrion. It is possible that excess fat deposition in the liver is partly due to alteration in mitochondrial function. Using the diabetic mice, in the present paper, we determined whether mitochondrial function is altered in the liver compared to age-matched normal mice. Increase in oxidative stress can inactivate mitochondrial enzymes. In an attempt to determine whether diabetic mice show inactivation of mitochondrial enzymes we measured PDH, aconitase and KGDHC. These dehydrogenases were chosen because the activities of these enzymes are required for the synthesis of NADH and because these enzymes have been shown to be highly susceptible to free radical-mediated inactivation. We found that all three mitochondrial enzymes are inactivated in mitochondria isolated from diabetic mice (Figure 2). A direct consequence of inactivation of key mitochondrial enzymes and increased oxidative stress as has been obtained in the present study is that all NADH producing steps in the TCA cycle are inhibited that may be reflected by dramatically reduced TCA cycle flux. These observations show that diabetic mice have lower capacity of mitochondrial NADH production. A low level of mitochondrial NADH may stimulate β-oxidation and TCA cycle flux and decrease triacylglyceride formation [56]. However, a high level of pyruvate that accumulate because of inactivation of PDHC, aconitase and KGDHC could form acetate non-enzymatically that may convert into fatty acids and triacylglycerol. Unutilized pyruvate and inactivation of mitochondrial enzymes may be related to an increase in triacylglycerols in two possible ways. First, as shown in Figure 5 pyruvate non-enzymatically converted to acetate and triacylglycerol and secondly, enzymes that lead to formation of free fatty acids and triacylglycerol are stable in oxidative environments in contrast to PDHC, aconitase and KGDHC that are highly susceptible to oxidative environments. Thus inactivation of mitochondrial enzymes and consequently over accumulation of pyruvate may be an alternative mechanism of triacylglycerol deposition in diabetic mice.

These diabetic mice have been reported to have higher triacylglycerols level. Under the conditions that found in those diabetic mice liver, we anticipate that treating isolated mitochondria or primary hepatocytes with LOOH or H2O2 reflect a combination of oxidative stress and increased lipid peroxides. Our study clearly show that PDH, aconitase and KGDHC are exquisitely sensitive to LOOH or H2O2.

Oxidative stress has been implicated in liver mitochondrial dysfunction in diabetic subjects [57-60]. Oxidative stress is associated with increases in lipid hydroperoxides, lipid peroxidation, and production of lipid peroxidation products such as and 4-hydroxy-2-nonenal (HNE) [61-63], and modification to mitochondrial protein by oxidized lipids [64,65]. HNE is a major product of lipid peroxidation that readily reacts withand inactivates protein [66-69]. Lipid peroxides and H2O2 have been viewed primarily from the perspective of the damage they may impart. It is becoming increasingly apparent, however, that by virtue of the ability to alter protein function [70] they can modify mitochondrial functions. To determine whether observed declines in mitochondrial enzymes could, at least in part, due to increased reactive oxygen species or oxidized lipids, mitochondria were isolated from perfused rat liver and treated with LOOH or H2O2. The inactivation of enzyme was then determined after two hours of incubation. As shown in Figure 2, PDH, aconitase and KGDHC activities are drastically inhibited with LOOH and H2O2. We further verified these results in primary hepatocytes treated with LOOH or H2O2. Our results show similar results in mitochondria isolated with hepatocytes treated with LOOH or H2O2. Based on results presented in Figure 2, we conclude that inactivation of PDH, aconitase and KGDHC could affect supply of NADH to the electron transport chain that controls the rate of NADH-linked mitochondrial respiration. The results of this study indicate that, LOOH and H2O2 interact with PDH, aconitase and KGDHC resulting in enzyme inactivation.

Previous studies showed the susceptibility of aconitase to free radical inactivation [71-75]. Aconitase contains an active site iron-sulfur (4Fe-4S)2+ complex. Using electronic spin resonance, it was determined that when purified mitochondrial aconitase is treated with superoxide the (4Fe-4S)2+ cluster is oxidized to (3Fe-4S)1+, resulting in the release of Fe(II) and H2O2 [76]. Similarly KGDHC is also very susceptible to oxidative species. In vitro studies show that lipid peroxidation and lipid peroxidation products induced inactivation of KGDHC involves modification of essential lipoic acid residues covalently linked to E2 subunits of the enzyme [77]. One consequence of inactivation of KGDHC may be oxidation of glutamate by glutamate dehydrogenase that result in the accumulation of α-ketoglutarate. The conversion of glutamate to α-ketoglutarate by glutamate dehydrogenase is thermodynamically unfavorable (Keq = 1.8 × 10-13), and a buildup of α-ketoglutarate would be expected to reduce the rate of glutamate utilization. In addition, pyruvate dehydrogenase, an enzyme that shares structural and functional similarities with KGDH and contains covalently bound lipoic acid residues [78], may exhibit a similar response to LOOH or H2O2 inactivation. Together inactivation of these mitochondrial enzymes in diabetic mice as well as in vitro experiments in isolated mitochondria and hepatocytes treated with LOOH or H2O2 suggesting impairment of NADH production.

During the course of present study, we noted an increase of 13C acetate as detected by 13C LC/MS of the hepatocytes treated with LOOH or H2O2. The impact of LOOH or H2O2 on increase in the fatty acid concentration is a direct reflection of its importance as a catastrophic pathway that lead to accumulation of triacylglycerols in the tissue. Under normal conditions, fatty acid synthesis and fatty acid oxidation must always balanced. However, with the inactivation of PDH, aconitase and KGDHC,pyruvate and TCA cycle intermediates may accumulate. Increase in these intermediates may cause allosteric inhibition of TCA cycle activity and fatty acid oxidation [79].

Thus, inactivation of PDHC, aconitase and KGDHC appears to be the primary mechanism by which LOOH or H2O2 may cause an increase in free fatty acids and triacylglycerols in tissues. Because the diabetic mice has more oxidative stress as shown by increased TBARS substances, therefore these mice would be expected to be more sensitive to mitochondrial enzyme inactivation.

It is noteworthy that, under the conditions of our experiments, the magnitude to which PDH, aconitase and KGDH were inactivated was similar, regardless of the concentration of LOOH or H2O2 utilized (data not shown).

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Conclusion

Maintenance of mitochondrial function depends on the ability of mitochondria to sense changes in redox status and respond in a manner commensurate with metabolic requirements. In this study, we have provided clear evidence that overall mitochondrial enzymes are inactivated by the addition of LOOH and H2O2 that promotes formation of fatty acids and triacylglycerols in a switch over mechanism from catabolic pathway to anabolic pathway. Whereas these observations suggest a role of oxidative stress in the liver mitochondrial impairments, this study points out a mechanisms whereby mitochondrial enzyme inactivation leads to formation of free fatty acids and triacylglycerols in diabetic subjects. In addition, experiments designed to test the effects of exogenously added LOOH and H2O2 will further enhance our understanding of the process and its physiological significance. In conclusion, our results indicate that alterations in the mitochondrial enzymes induced by LOOH or H2O2 are associated with the ectopic fat storage in the liver. Although this association cannot distinguish between causes and effects, it is interesting that our results fit with the emerging idea that mitochondrial dysfunction can lead to the development of metabolic diseases, such as obesity, type 2 diabetes mellitus. These results suggest that LOOH or H2O2 production may serve to regulate mitochondrial function.

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meredithjpierre · 6 years ago

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Best Alternative Treatments for Cervical Cancer

Cervical cancer is one of the most treatable forms of cancer, especially in its early stages. These days, the strong push for women to being to do regular pap-smear tests means that early detection is a real possibility and the rate of cervical cancer worldwide has seen a sharp decline. For any woman who finds themselves diagnosed with this condition, the mistake that is most often made, is following the conventional course of surgery, radiation and chemotherapy – which varies in degree depending on how far advanced the cancer tissues are determined to be.

Even if the patient chose to undergo conventional treatments to “remove” the cancer from their bodies, the stem cells would remain, and so too would the underlying causes that lead to the formation of cancerous cells and tissues in the first place. If we were able to adapt our mindsets, realising that the condition is metabolic in nature – we would quickly realise that these forms of treatment are not the answer and start looking for alternative treatments.

Cancer is systemic by nature, even if it only shows up in the “weakest link” of our body, it will quickly spread if left untreated. In order to stop this from occurring, we must first cleanse and detoxify our systems, removing the scourge that lead to inflammation and a state of dis-ease. We do this via cleansing with green juices and water – switching off digestion and allowing the body a chance to heal. Combined with powerful physical therapies – such as colon hydrotherapy, lymphatic drainage, EWOT, hyperthermia and meditation and yoga, our bodies are suddenly allowed the opportunity to reset through each of our systems of elimination – the colon, lungs, liver, kidneys, lymph, skin and importantly, the mind.

Simultaneously, we learn how to starve the cancer cells – taking away their most efficient fuel source – sugar. By switching to a plant-based diet that is nutrient dense, with a focus on good fats maintaining a ratio of double omega 3’s to 6’s and 9’s, moderate protein and minimal carbohydrates. We flood the body with adequate alkalising minerals and fluids, and allow it the chance to begin the process of healing on a profound level.

The use of metabolic treatments is one of the least invasive of ways of killing cancer cells, whilst maintaining the integrity of our healthy cells and tissues. Treatments like high dose vitamin c, B17, venofer, artusenate and curcumin are nature’s artillery against cancer cells – potently delivered in the form of IV therapy, we are able to build up the levels required in the tissues in order to markedly target cancer cells, enzymatically challenging them in a fatal manner.

We activate and enhance the immune system with cutting-edge immune therapies, sourced from the most highly regarded labs in the world. These therapies activate enhanced functioning of the patients own lymphocytes, macrophages and NK cells, in order to encourage the body to heal itself, rather than administering a medicine to “kill” rogue cells – which usually causes them to come back, often in trickier locations than they originated.

Sometimes, medical intervention is required in the short-term to quickly exert control over the growth of cancer. In these cases we use IPT, a low dose, targeted form of chemotherapy which averts the usual side effects of the high dose, systemic variety. By administering a small amount of insulin when the patient is in a fasted state, we are able to biologically take advantage of cancer cells and create a “therapeutic moment” – when the insulin has bound to the cancer cells much more quickly than healthy cells, they become permeable whilst the healthy cells remain impermeable. Stimulating delta-9 desaturase, the cancer cells effectively open up so that when we administer very small doses of chemotherapy, it becomes directly absorbed into those cells – leaving the healthy cells relatively untouched. Unlike high-dose chemotherapy, there are few side effects – our patients don’t lose their hair, have endless bouts of vomiting and diarrhea nor do they get long term side effects. We only use this therapy when absolutely necessary, and usually only for a short period until the cancer cells are deemed under control.

The bridge between all of these treatments is knowledge and education – because these are the only things that will allow you to live a life which can prevent cancer from returning. It is often said that getting rid of cancer is the easy part – keeping it at bay is where it becomes difficult. Our program is an active experiential learning process, where our patients become experts in metabolic disease prevention. They learn about the wonder of the human body, and its fervent quest for homeostasis. Once we realise that the body is always seeking to protect us, striving for healing – we are able to see how we can assist it to be able to efficiently defend us, rather than being permanently stuck on maintenance mode. Learning the fundamentals of healthy nutrition, and the vital ingredients for promoting cellular integrity and producing ATP – our life force. The importance of quietening our inner voices so that we are able to listen to our innate wisdom – through meditation and yoga, and other workshops encouraging creativity and exploring long neglected parts of our psyche all lead to healing on a spiritual level.

All in all, the alternate healing journey for a cervical cancer patient need not be an invasive, toxic and painful experience. It could be a life affirming voyage into healing – not just the physical manifestation of the disease, but a deeply holistic and spiritual awakening. Rather than becoming sick with treatments that not only kill cancer cells, but destroy the immune system – thereby creating the perfect environment for cancer to emerge again, why not embark on a journey that allows you to start feeling well as your body heals.

The post Best Alternative Treatments for Cervical Cancer appeared first on Akesis Life - Integrative Oncology.

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kellyjrpeterson · 6 years ago

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Best Alternative Treatments for Cervical Cancer

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shirleyjgolding · 6 years ago

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Best Alternative Treatments for Cervical Cancer

The post Best Alternative Treatments for Cervical Cancer appeared first on Akesis Life – Integrative Oncology.

Best Alternative Treatments for Cervical Cancer published first on https://akesislife.wordpress.com

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akesislife · 6 years ago

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Best Alternative Treatments for Cervical Cancer

The post Best Alternative Treatments for Cervical Cancer appeared first on Akesis Life - Integrative Oncology.

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robertawilliams · 6 years ago

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Report warns of Britain's ability to impose sanctions on countries post-Brexit

The UK must urgently address how it plans to maintain sanctions on , North Korea, and other countries post-Brexit or risk losing influence in the world, a report has warned.

The EU Select Committee found the Government’s planned approach to sanctions after leaving the European Union was “untested” and needed to be reconsidered.

It found sanctions were most effective when implemented on a multilateral basis and urged the Government to consider staying aligned with the EU27’s sanction regime.

South Korea imposes strong new sanctions on North Korea

Official data shows the UK has sanctions of sorts against more than two dozen countries around the world, sometimes under the auspices of the UN, EU or other international partners.

But the committee’s report said: “The influence of the UK on the sanctions policy of its international partners will depend on the extent to which it is able to retain its authority and leadership on key foreign policy dossiers after Brexit.

“Further consideration of the impact of leaving the EU on the UK’s ability to pursue and achieve its foreign policy objectives will be urgently required.”

Setting out how the UK could choose to impose sanctions after Brexit, the committee noted that Norway and Switzerland align themselves with the EU regime.

UK news in pictures

15 December 2017

Jonny Bairstow of England headbutts his helmet to celebrate his century during day two of the Third Test match in the 2017/18 Ashes Series between Australia and England at the WACA in Perth, Australia. Bairstow was embroiled in controversy at the beginning of the tour after lightly headbutting Australian opening batsman Cameron Bancroft in an exchange in a bar

14 December 2017

People at the Grenfell Tower National Memorial Service

13 December 2017

Wax figures of Prime Minister Theresa May and Foreign Secretary Boris Johnson wearing a Christmas Jumper at Madame Tussauds

EPA

12 December 2017

Victims and family of victims of the Grenfell Tower fire, Nicholas Burton (left), Sandra Ruiz (second right), Karim Mussilhy (right) and a girl who asked not be named (second left), hand in a petition to Downing Street, asking for an overhaul of the public inquiry.

11 December 2017

A homeless man on the streets of Manchester. Many people are spending the night on the streets in freezing temperatures as the Met Office continues to issue weather warnings across the country. The Shelter charity has said that more than 300,000 are now homeless across Britain, equating to the population of a city the size of Newcastle

Getty

10 December 2017

Pedestrians walk over the Millennium Bridge with St Paul's Cathedral pictured in the background as snow falls

AFP/Getty Images

9 December 2017

British Foreign Secretary Boris Johnson, left, and Secretary of Iran's Supreme National Security Council Ali Shamkhani, right, with interpreter at centre, during their meeting in Tehran, Iran. Johnson is expected to discuss the fate of detained British-Iranian woman Nazanin Zaghari-Ratcliffe, who is serving a five-year prison sentence for allegedly plotting to overthrow Iran's government.

8 December 2017

British Prime Minister Theresa May (L) and European Commission President Jean-Claude Juncker address a press conference at the European Commission in Brussels

AFP/Getty Images

7 December 2017

Nick Dunn, one of the so-called Chennai Six is greeted by his sister Lisa as he arrives at Newcastle Airport after being released from India after serving four years in jail on weapons charges.

6 December 2017

Britain's Queen Elizabeth II (L) greets Nigeria's ambassador to the United Kingdom, George Adesola Oguntade (C), and his wife, Modupe Oguntade, during a private audience at Buckingham Palace in central London

AFP/Getty

5 December 2017

800 abandoned buckets appear at Potters Field Park, London, in a moving tribute to the 800 children who die every day, on average, due to a lack of clean water and sanitation. Just one bucket in the installation, part of WaterAid’s #Untapped appeal, could hold almost enough safe drinking water for one child for a week. Every £1 donated to the #Untapped appeal until 31st January 2018 will be matched by the UK Government.

WaterAid / Ollie Dixon

4 December 2017

British Prime Minister Theresa May smiles to European Union President Donald Tusk as she attends Brexit negotiations' meetings

AFP/Getty

3 December 2017

The last Supermoon of 2017 sets over Whitley Bay, Northumberland

2 December 2017

The crowd reacts as England's Dawid Malan fails to stop a boundary during the first day of the second Ashes test match

REUTERS

1 December 2017

England manager, Gareth Southgate, jokes with Belgium manager, Roberto Martinez, after their sides were drawn in the same group during the Final Draw for the 2018 FIFA World Cup in Russia

Getty Images

30 November 2017

A supporter of Lauri Love, who is accused of hacking into U.S. government computers, wears a Donald Trump mask as he protests in front of the Royal Courts of Justice in London

29 November 2017

A sign reading 'We want our future back' is displayed in front of Westminster during an Anti-Brexit Demonstration

Rex Features

28 November 2017

This year's innovative V&A Christmas Tree, The Singing Tree, is shaped from a cloud of floating words, contributed by visitors, and is created by leading stage and performance designer Es Devlin.

Rex Features

27 November 2017

David Jones and Margaret Tyler wait outside Kensington Palace after hearing about the engagement of Prince Harry and Meghan Markle

Rex

26 November 2017

Sailors from the Royal Navy perform the Changing of the Guard ceremony at Buckingham Palace for the first time in its 357-year history

25 November 2017

Arlene Foster gives her leader's speech during the annual DUP party conference at La Mon House

Getty Images

24 November 2017

Ex-England footballer Michael Owen prior to riding in a charity race at Ascot racecourse

Rex

23 November 2017

Shoppers pass a promotional sign for 'Black Friday' sales discounts on Oxford Street

AFP/Getty

22 November 2017

Britain's Chancellor of the Exchequer Phillip Hammond poses with the budget box at 11 Downing Street

EPA

21 November 2017

Protestors hold up a banner during a protest held in solidarity with the University of London cleaners' strike

Petros Elia

20 November 2017

British Prime Minister Theresa May greets Ghana's President Nana Akufo-Addo outside number 10 Downing Street

Getty

19 November 2017

Grigor Dimitrov reacts to winning the Men's Singles Final with the trophy, during day eight of the NITTO ATP World Tour Finals at the O2 Arena in London

18 November 2017

Central Scotland MSP Richard Leonard is congratulated by Glasgow MSP Anas Sarwar at the Glasgow Science Centre after he was announced as the new leader of Scottish Labour

Jane Barlow/PA

17 November 2017

British Military Working Dog Mali poses for a photograph with his handler, Cpl. Daniel Hatley, after receiving the PDSA Dickin Medal, the animal equivalent of the Victoria Cross, for his heroic action in Afghanistan

Reuters

16 November 2017

Theresa May chats with resident Val Lay during a visit to a housing estate in London

AFP/Getty

15 November 2017

Richard Radcliffe leaves the Foreign Office with his local MP Tulip Siddiq, following a meeting with Foreign Secretary Boris Johnson

Marc Ward/REX

32/46 14 November 2017

Four-time Olympic champion Sir Mo Farah after being awarded a Knighthood by Queen Elizabeth II

33/46 13 November 2017

Restoration work continues on the Palace of Westminster

Photographs by Reuters/Getty/iStock

34/46 12 November 2017

A veteran takes his hat off during the Remembrance Sunday Cenotaph wreathe laying ceremony

REUTERS

35/46 11 November 2017

Members of the Western Front Association during a service at the Cenotaph to mark the Armistice Day

EPA

36/46 10 November 2017

David Davis and Michel Barnier

REUTERS

37/46 9 November 2017

Britain's newly appointed Secretary of State for International Development, Penny Mordaunt, leaves Downing Street

38/46 8 November 2017

Priti Patel leaves number 10 Downing street through the back entrance

EPA

39/46 7 November 2017

School children and their teacher from Thomas Tallis School look at pictures on display at the Red Star Over Russia exhibition at the Tate Modern in London

Philip Toscano/PA

40/46 6 November 2017

A cast of The Wrestlers, two men taking part in the Greek sport pankration, is lowered into place at Natural Trust's Stowe Landscape Garden near Buckingham

41/46 5 November 2017

Protesters in Trafalgar Square, London, during the Million Mask March bonfire night protest

42/46 4 November 2017

Protestors take part in the 'Justice Now: Make it Right for Palestine' march, organised by the Palestine Solidarity Campaign, in central London

43/46 3 November 2017

People queue outside an Apple store in London to purchase the new iPhone X upon its release in the U.K. The iPhone X is positioned as a high-end, model intended to showcase advanced technologies such as wireless charging, OLED display, dual cameras and a face recognition unlock system

Getty

44/46 2 November 2017

British Prime Minister Theresa May greets Israeli Prime Minister Benjamin Netanyahu outside 10 Downing Street in London. The pair are today celebrating the centenary of a British declaration that ultimately led to the foundation of the state of Israel

Getty

45/46 1 November 2017

Mammatus clouds over St Mary's Lighthouse in Whitley Bay, Northumberland

Owen Humphreys/PA

46/46 31 October 2017

Women protest outside Downing Street as they join a demonstration demanding rights for working mothers

Getty Images

But while this would preserve the current unity, it would require the UK to implement decisions taken by the remaining 27 EU members without any say over the design of the sanctions.

Following the US approach of “informal engagement” with the EU on sanctions could be valuable but “it is no substitute for the influence that can be exercised through formal inclusion in EU meetings”.

The Government’s preferred approach is an “unprecedented” UK-EU partnership on sanctions, it said.

Their report went on: “The UK has some leverage in that it currently plays a leading role in developing EU sanctions policy, is most active in proposing individuals and entities to be listed, and is home to the largest international financial centre in the bloc.

“But we note that the Government’s approach is untested and it is not yet clear what its proposed arrangements would involve.

“Future co-operation could also be limited by the UK’s new legal framework for sanctions and its post-Brexit position outside the EU’s single market and customs union.

“More broadly, the extent to which the UK and the EU co-operate on sanctions will depend on their future relationship in the wider foreign policy arena. This needs urgent consideration.”

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