Researchers show how insulin helps fat cells take in glucose

Using high-resolution microscopy, researchers have shown how insulin prompts fat cells to take in glucose in a rat model. The findings were reported in the 8 September 2010 in Cell Metabolism.

By studying the surface of healthy, live fat cells in rats, researchers were able to understand the process by which cells take in glucose. Next, they plan to observe the fat cells of people with varying degrees of insulin sensitivity, including insulin resistance- considered a precursor to type 2 diabetes. These observations may help identify the interval when someone becomes at risk for developing diabetes.

“What we’re doing here is actually trying to understand how glucose transporter proteins called GLUT4 work in normal, insulin-sensitive cells,” said Karin G. Stenkula, PhD, a researcher at the US National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and a lead author of the paper. “With an understanding of how these transporters in fat cells respond to insulin, we could detect the differences between an insulin-sensitive cell and an insulin-resistant cell, to learn how the response becomes impaired. We hope to identify when a person becomes pre-diabetic, before they go on to develop diabetes.”

Like a key fits into a lock, insulin binds to receptors on the cell’s surface, causing GLUT4 molecules to come to the cell’s surface. As their name implies, glucose transporter proteins act as vehicles to ferry glucose inside the cell.

To get detailed images of how GLUT4 is transported and moves through the cell membrane, the researchers used high-resolution imaging to observe GLUT4 that had been tagged with a fluorescent dye.

The researchers then observed fat cells suspended in a neutral liquid and later soaked the cells in an insulin solution, to determine the activity of GLUT4 in the absence of insulin and in its presence. In the neutral liquid, the researchers found that individual molecules of GLUT4 as well as GLUT4 clusters were distributed across the cell membrane in equal numbers. Inside the cell, GLUT4 was contained in balloon-like vesicles, which transported GLUT4 to the cell membrane and merged with the membrane, a process known as fusion.

After fusion, the individual molecules of GLUT4 are the first to enter the cell membrane, moving at a continuous but relatively infrequent rate. The researchers termed this process fusion with release.

When exposed to insulin, however, the rate of total GLUT4 entry into the cell membrane peaked, quadrupling within three minutes. The researchers saw a dramatic rise in fusion with release – 60 times more often on cells exposed to insulin than on cells not exposed to insulin.

After exposure to insulin, a complex sequence occurred, with GLUT4 shifting from clusters to individual GLUT4 molecules. Based on the total amount of glucose the cells took in, the researchers deduced that glucose was taken into the cell by individual GLUT4 molecules as well as by clustered GLUT4. The researchers also noted that after four minutes, entry of GLUT4 into the cell membrane started to decrease, dropping to levels observed in the neutral liquid in 10 to 15 minutes.

“The magnitude of this change shows just how important the regulation of this process is for the survival of the cell and for the normal function of the whole body,” said Joshua Zimmerberg, PhD, MD, the paper’s senior author and director of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program in Physical Biology.

The research team next plans to examine the activity of glucose transporters in human fat cells, Zimmerberg said. “Understanding how insulin prepares the cell for glucose uptake may lead to ideas for stimulating this activity when the cells become resistant to insulin.”

New drug may make some eye surgeries unnecessary

In the course of participating in a Phase 2 trial for the new drug microplasmin, opthamologist Dr Matthew Benz and his colleagues made an exciting discovery: a pharmaceutical that had been developed to make surgery easier actually had the ability to make it unnecessary in some patients.

Patients selected for the trial had been diagnosed with one of two conditions. The first was vitreomacular traction, in which the vitreous (the central fluid part of the eye) develops an abnormally strong adhesion to the macula (a light-sensitive layer of tissue at the back of the eye). The second was the appearance of small holes in the macula, which is usually age-related. Both conditions can cause blurred and distorted vision, and both are traditionally treated with a vitrectomy, surgical removal of some or all of the vitreous.

“As one of the top five retinal research sites in the United States, Methodist Hospital was chosen as one of the sites for the Phase 2 trial of microplasmin,” explains Dr Benz. “It was going to be used as a pre-surgery treatment with the idea of making the vitrectomy easier.”

Administering the drug was a fairly simple procedure that could be carried out in a doctor’s office. First the white of the eye was numbed with a local anesthesia, then a tiny amount of microplasmin (between 1/10 and 1/20 cc) was injected directly into the vitreous with a small needle. “Intravitreal injections are actually quite common,” says Dr Benz. “We do them fairly often in treating patients with macular degeneration or eye infections.”

Much to the surprise of Dr Benz and his colleagues, about 44% of the trial patients ended up not needing surgery at all. “We found out that microplasmin actually has the potential to be a nonsurgical intervention for what have up to now been surgical problems,” he says. “In some of the patients, we saw even better outcomes than we would have expected from the surgery.” Microplasmin is not yet approved by the US FDA or in Europe. Trials are on-going.

Malaria mosquito becoming new species, say scientists

Two strains of the type of mosquito responsible for most of malaria transmission in Africa have evolved such substantial genetic differences that they are becoming different species, according to researchers behind two new studies published recently in the journal Science.

Over 200 million people globally are infected with malaria, according to the World Health Organisation, and the majority of these people are in Africa. Malaria kills one child every 30 seconds.

The international research effort, co-led by scientists from Imperial College London, carried out a detailed look at the genomes of two strains of the Anopheles gambiae mosquito, the type of mosquito primarily responsible for transmitting malaria in sub- Saharan Africa. These strains, known as M and S, are physically identical. However, the new research shows that their genetic differences are such that they appear to be becoming different species, so efforts to control mosquito populations may be effective against one strain of mosquito but not the other.

The scientists argue that when researchers are developing new ways of controlling malarial mosquitoes, for example by creating new insecticides or trying to interfere with their ability to reproduce, they need to make sure that they are effective in both strains. The authors also suggest that mosquitoes are evolving more quickly than previously thought, meaning that researchers need to continue to monitor the genetic makeup of different strains of mosquitoes very closely, in order to watch for changes that might enable the mosquitoes to evade control measures in the future.

Nanoscale transducers could revolutionise ultrasound

Scientists and engineers at the University of Nottingham have built the world’s smallest ultrasonic transducers capable of generating and detecting ultrasound. These revolutionary transducers which are orders of magnitude smaller than current systems — are so tiny that up to 500 of the smallest ones could be placed across the width of one human hair.

While at an early stage these devices offer a myriad of possibilities for imaging and measuring at scales a thousand times smaller than conventional ultrasonics. They can be made so small they could be placed inside cells to perform intracellular ultrasonics. They can produce ultrasound of such a high frequency that its wavelength is smaller than that of visible light. Theoretically they make it possible for ultrasonic images to take finer pictures than the most powerful optical microscopes.

The work, by the Applied Optics Group in the Division of Electrical Systems and Optics has been deemed so potentially innovative that last year it was awarded a £850,000 (about $1.36 million) five year Platform Grant by the Engineering and Physical Sciences Research Council (EPSRC) to develop advanced ultrasonic techniques.

The team has also been supported by additional funding of £350,000 from an EPSRC grant to underpin aerospace research. Matt Clark, of the Applied Optics Group, said: “With the rise of nanotechnology you need more powerful diagnostic tools, especially ones that can operate non-destructively and ones which can be used to access the mechanical and chemical properties of the samples at this scale.

These new transducers are hugely exciting and bring the power of ultrasonics to the nanoscale.” The ultrasonic transducers consist of sandwich or shell like structures carefully engineered to possess both optical and ultrasonic resonances. When they are hit by a pulse of laser light they are set ringing at high frequency which launches ultrasonic waves into the sample.

When they are excited by ultrasound the transducers are very slightly deformed and this changes their optical resonances which are detected by a laser. The devices can be constructed either by micro/nano lithography techniques similar to those used for microchips or by molecular self assembly where the transducers are constructed chemically. Perhaps the most familiar application of ultrasonics is medical imaging but it is also widely used in engineering applications and for chemical sensing.

These tiny transducers open up the possibility of using these techniques on the smallest scales, for instance inside cells and on nano-engineered components. Dr Clark said: “Imagine imaging inside cells in the same way that ultrasonic imaging is performed inside bodies.

Theoretically we could get higher resolution with the nano-ultrasonics than you can with optical microscopes and the contrast would be very interesting.”

New malaria drug shows promise

A chemical that rid mice of malariacausing parasites after a single oral dose may eventually become a new malaria drug if further tests in animals and people uphold the promise of early findings. The compound, NITD609, was developed by an international team of researchers including Elizabeth A. Winzeler, PhD, a grantee of the US National Institute of Allergy and Infectious Diseases (NIAID), part of the US National Institutes of Health.

“Although significant progress has been made in controlling malaria, the disease still kills nearly 1 million people every year, mostly infants and young children,” says NIAID Director Anthony S. Fauci, MD. “It has been more than a decade since the last new class of antimalarials – artemisinins – began to be widely used throughout the world. The rise of drug-resistant malaria parasites further underscores the need for novel malaria therapies.”

Dr Fauci adds: “The compound developed and tested by Dr Winzeler and her colleagues appears to target a parasite protein not attacked by any existing malaria drug, and has several other desirable features. This research is also a notable example of successful collaboration between government-supported scientists and private sector researchers."

The study, in the 3 September issue of Science, was led by Thierry T. Diagana, PhD, of the Novartis Institute for Tropical Diseases (NITD), and Dr Winzeler. Dr Winzeler is affiliated with The Scripps Research Institute and the Genomic Institute of the Novartis Research Foundation, La Jolla, California.

Work began o in Dr Winzeler’s lab in 2007 where scientists screened 12,000 compounds active against Plasmodium falciparum, the most deadly malaria parasite. “From the beginning, NITD609 stood out because it looked different, in terms of its structure and chemistry, from all other currently used antimalarials,” says Dr Winzeler. “The ideal new malaria drug would not just be a modification of existing drugs, but would have entirely novel features and mechanism of action. NITD609 does.”

NITD609, which could be formulated as a tablet and manufactured in large quantities, is one of a new class of chemicals, the spiroindolones, which have been described in recently published research by Dr Winzeler and colleagues as having potent effects against two kinds of malaria parasites.

In the current study, the scientists detail attributes of NITD609 that suggest it could be a good malaria drug. For example – In test-tube experiments, NITD609 killed two species of parasites in their blood-stage form and also was effective against drug-resistant strains. In humans, malaria parasites spend part of their life cycle in the blood and part in the liver. – The compound worked faster than some older malaria drugs, although not as quickly as the best current malaria drug, artemisinin. – Other laboratory tests showed that NITD609 is not toxic to a variety of human cells.

When given orally to rodents, the compound stayed in circulation long enough to reach levels predicted to be effective against malaria parasites. According to Dr Winzeler, if NITD609 behaves similarly in people, it might be possible to develop the compound into a drug that could be taken just once. Such a dosage regimen, she says, would be substantially better than the current standard treatment in much of the world in which uncomplicated malaria infections are treated for three to seven days with drugs that are taken between one and four times daily.

Typically, she says, rodents infected with the mouse malaria parasite, Plasmodium berghei, die within a week. But a single large dose of NITD609 cured all five infected mice that received it, while half of six mice receiving a single smaller dose were cured of infection. Three doses of the smaller amount of NITD609 upped the cure rate to 90%. Additional tests in animals are under way and NITD609 could enter early-stage safety testing in humans later this year, says Dr Winzeler.

But, she adds, many drug candidates fail in clinical trials and thus it will be important for the community to continue to work on developing other potential antimalarial compounds.

Study unravels clues to infertility among obese women

Obese women have a well-known risk for infertility, but a new Johns Hopkins Children’s Center study has unraveled what investigators there believe is the mechanism that accounts for the risk.

The research conducted in mice and published online 8 September 2010 in Cell Metabolism, shows that the pituitary gland actively responds to chronically high insulin levels, triggering a cascade of hormonal changes that disrupt ovarian function and impair fertility. The findings challenge the widely held belief that infertility is a result of insulin resistance and suggest a new culprit: heightened sensitivity to insulin’s effects on the pituitary gland.

“What we propose is a fundamentally new model showing that different tissues respond to obesity differently, and that while cells in the liver and muscle do become insulin resistant, cells in the pituitary remain sensitive to insulin,” says principal investigator Andrew Wolfe, PhD, of Hopkins Children’s.

Scientists traditionally have focused on treating infertility by lowering insulin levels as a way to treat insulin resistance. However, the new model suggests that decreasing the pituitary’s sensitivity to insulin could be an important new target for treatment instead. Insulin resistance, marked by persistently elevated insulin, abnormal regulation of blood sugar and insensitivity to insulin in the liver and muscle cells, occurs in type 2 diabetes, metabolic syndrome and polycystic ovary syndrome (PCOS). PCOS is the most common cause of infertility, affecting up to one in 10 women.

Because ovarian function and fertility are mostly regulated by the pituitary, the body's master gland, the Hopkins team set out to find out exactly how elevated insulin levels affect the pituitaries of obese women to render them infertile.

The investigators focused on a class of pituitary cells called gonadotrophs, which secrete luteinizing hormone (LH), critical for ovulation and fertility. The researchers suspected that when awash with too much circulating insulin, the gonadotrophs of obese mice start pumping out large amounts of LH, thus disrupting ovulation.

To test their hypothesis, the scientists engineered mice with missing insulin receptors in their pituitary glands and compared them to mice with intact insulin receptors. After three months on a high-fat diet, the obese mice with intact insulin receptors developed all the classic symptoms of PCOS: elevated LH levels, high testosterone, irregular reproductive cycles and fewer ovulations. The mice with missing insulin receptors, however, maintained nearnormal LH levels, regular cycles and normal ovulation despite their obesity.

To further clarify the effect of insulin on the pituitary, the researchers compared the gonadotrophs of obese mice to those of lean mice by injecting the animals with gonadotropin-releasing hormone (GnRH), which stimulates LH and is critical for ovulation and fertility. Lean mice, with and without pituitary insulin receptors, had normal elevations of LH. Obese mice with intact insulin receptors experienced increases of LH twice as high. Yet, the obese mice with missing insulin receptors in their pituitaries had nearnormal LH elevations.

These results, the researchers say, show that the high levels of insulin seen in obesity make the pituitary more sensitive to GnRH and help initiate a hormonal chainreaction that disrupts fertility. To demonstrate insulin’s direct effects on the pituitary, the scientists injected mice with insulin. Mice with intact insulin receptors, lean or obese, had mild LH elevations, while mice with deleted insulin receptors, lean or obese, experienced none.

To determine whether these hormonal differences would carry over into actual differences in fertility, the researchers allowed the mice to mate. The pregnancy outcomes mirrored the hormonal findings. Lean mice, with or without pituitary insulin receptors, had six times the number of successful pregnancies as obese mice. However, obese mice with missing pituitary insulin receptors had near-normal pregnancy outcomes, with five times more successful pregnancies than obese mice whose pituitary insulin receptors were intact.

By deleting the insulin receptors in the pituitary cells of mice, the researchers managed to restore normal LH levels, maintain ovulation and near-intact fertility even in obese mice with elevated insulin levels. Despite normal hormonal levels and ovulation, the obese mice with missing insulin receptors were not as fertile as lean mice with normal insulin levels. The finding suggests that since the ovaries share partial control of ovulation and fertility with the pituitary, they too may be affected by high insulin levels.

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