Study shows estrogen works in the brain to keep weight in check

Study shows estrogen works in the brain to keep weight in check
DALLAS – Oct. 20, 2011 – A recent UT Southwestern Medical Center
study found that estrogen regulates energy expenditure, appetite and
body weight, while insufficient estrogen receptors in specific parts of
the brain may lead to obesity. “Estrogen has a profound effect on metabolism,” said Dr. Deborah Clegg, associate professor of internal medicine and senior author of the study published Oct. 5 in Cell Metabolism. “We hadn’t previously thought of sex hormones as being critical regulators of food intake and body weight.”
The mouse study is the first to show that estrogen, acting through two
hypothalamic neural centers in the brain, keeps female body weight in
check by regulating hunger and energy expenditure. Female mice lacking
estrogen receptor alpha – a molecule that sends estrogen signals to
neurons – in those parts of the brain became obese and developed related
diseases, such as diabetes and heart disease. Similar results
were not seen in male mice, although researchers suspect other unknown
estrogen receptor sites in the brain play a similar role in regulating
metabolism for males as well. Estrogen receptors are located
throughout the body, but researchers found two specific populations of
estrogen receptors that appear to regulate energy balance for female
mice.
Dr. Deborah Clegg
The findings are potentially important for millions of postmenopausal
women, many of whom have decided against hormonal replacement therapy.
The study could lead to new hormonal replacement therapies in which
estrogen is delivered to specific parts of the brain that regulate body
weight, thereby avoiding the risks associated with full-body estrogen
delivery, such as breast cancer and stroke. Doctors stopped
routinely recommending long-term estrogen therapy for menopausal women
in 2002 when a Women’s Health Initiative study showed the hormone also
led to increased risk of cardiovascular disease. “The role of
estrogen in postmenopausal women continues to remain uncertain,” Dr.
Clegg said. “Current research is focused on the timing and the type of
estrogen supplementation that would be most beneficial to women. Our
findings further support a role for estrogens in regulating body weight
and energy expenditure, suggesting a benefit of estrogen supplementation
in postmenopausal women.” Other UT Southwestern researchers
involved in the study included lead author Dr. Yong Xu, a former
postdoctoral researcher in Dr. Clegg’s lab; Dr. Carol Elias, assistant professor of internal medicine; and Dr. Joel Elmquist, professor of internal medicine.
The research was supported by grants from the National Institutes of
Health, the American Heart Association and the American Diabetes
Association. Visit http://www.utsouthwestern.org/nutrition
to learn more about clinical services in nutrition at UT Southwestern,
including treatments for diabetes, kidney disease and obesity.
###
Media Contact: Debbie Bolles 214-648-3404 debbie.bolles@utsouthwestern.eduTo automatically receive news releases from UT Southwestern via email,subscribe at www.utsouthwestern.edu/receivenews

Proposal for Reducing the Fat Content of Fried Snacks

Proposal for Reducing the Fat Content
of Fried Snacks
Executive
Summary:
The Seeker stated that: any proposed
solution should address the following Technical Requirements:

Any additive or treatment should enable reduction of
fat content by 20% or more
Any additive or treatment should be stable at 360 - 370
° F
Any additive or treatment should not affect the
organoleptic (taste, aroma, texture, or appearance) properties of the
finished product
Any additive should meet Generally Regarded As Safe
(GRAS) Standards
Any proposed additive or process should not negatively
influence consumer acceptance of the product (e.g. label perception).
Any proposed additive or treatment should be
cost-effective for use in the large scale production of snack foods.
The proposed method should offer the Seeker client
"freedom to practice" or be available for potential licensing.
There should be no third party patent art preventing the use of specific
equipment and materials for their commercial application.
.
Legumes and their blends are widely used
for the production of papads. Papads with low fat content would be a boon to
populations looking for low-calorie foods with retention of organoleptic
profile. Judicious blending of legumes such as black gram, green gram, bengal
gram, red gram and cowpea revealed that low-fat fried papads could be prepared
from a blend of 40 : 36 : 24 blend of bengal gram: black gram : green gram
flours. The blend had 15.6% lower fat content as compared to the control
prepared from black gram flour alone. Other quality parameters such as expansion
ratio, texture in terms of crispness, colour and overall organoleptic quality
were also evaluated. Cereals Legumes, legumes and their blends are widely used
for the production of a range of fried products with low fat content would be a
boon to populations looking for low-calorie foods with retention of organoleptic
profile. Judicious blending of cereals and legumes such as chickpea, cowpea,
green gram, black gram, rice, wheat and amaranth revealed that low-fat fried
snacks could be prepared from a blend of 64: 16: 20 of cowpea : chickpea :
green gram flour. The blend had 27.8%
lower fat content as compared to the control prepared from checkpea flour alone.(Ref.1,
and 3). Barley has emerged as a new
source of dietary fiber with promising evidence of health benefits, viz. both
hypoglycemic effects in vivo. In the present study, incorporation of both
pearled and whole barley at varying levels (5
to 25%) on the quality characteristics of bread – including physical,
rheological and sensory attributes, (viz. loaf volume, texture, taste and
flavour), breads having 10% whole barley (WB) flour, or 15% pearled barley (PB)
flour were found to be acceptable. Both WB and PB bread resulted in
significantly lower post-prandial blood glucose than standard white bread. Two
hours after the test meal, both the experimental breads showed a higher satiety
score than did the white bread. The results reveal that by incorporation of
barley at suitable levels, it is possible to formulate breads that would cater
to the therapeutic needs of various targeted population such as diabetics and persons
suffering from coronary heart diseases or other disorders with impaired
carbohydrate /lipid metabolism.(Ref.2).

Akara, a fried
finger food made from cowpeas (Vigna unguiculata),
is popular in West
Africa and has been
shown to be acceptable to American consumers. Akara is, however, a high-fat
food (about 31%, dry wt basis). We determined the effects of incorporating two modifiers,
high amylose cornstarch or extruded cowpea flour, on akara fat content and consumer
acceptability. The modifiers were used at the 10% level. Akara fat content was reduced
by 26.1% with cornstarch and by 36.8% with extruded cowpea flour. There were no
significant differences in sensory ratings among samples, and all samples
received acceptable ratings (6 = like
slightly) for overall liking.(Ref.4). .
Pre-drying and subsequent dipping in a sugar solution
(‘sugar dipping’) is a potentially effective process for pre-treatment of
potato crisps. In this study, potato crisps were blanched, pre-dried, and
dipped in the solution of sugar (23.07 wt%) 2 s before frying at
180 °C. There was a significant reduction in the oil content of crisps
observed. Crisps that had been treated had about 30% less oil than the samples
that were not treated. The treatment did not affect the final moisture content
of crisps. A linear oil–moisture relationship of samples during frying was
obtained. The effect of pre-drying followed by sugar dipping on color scores of
potato crisps, and the kinetics of the color changes were also evaluated. The
potato crisps with dipping had more shrinkage compared with the non-dipping
ones. What has emerged is that the pre-drying and subsequent sugar dipping is
an effective technique for reducing the oil contents of potato crisps. Soaking
in NaCl can also decrease fat content and improve the quality of French-fries
though rather salty taste is expected. Effect of osmotic dehydration pretreatment on
quality of French fries, used osmotic dehydration as a pre-treatment to
produce low-fat French fries where potato strips were immersed into different
solutions, which are sugar (40% w/w), NaCl, maltodextrine 12 and maltodextrine
21, for 3 h before frying(Ref.5).

Introduction:
This Challenge is looking for an additive, or other
method to reduce the fat content of a vegetable substrate fried in sunflower
oil. The process or technology should not influence the taste, odor, or texture
of the seasoning or the finished food product.
Potato tubers were stored in
darkness at about 10 °C with a relative humidity of 60%. Potatoes were
taken out of storage 12 h before frying to allow to let them reach room
temperature and for the reducing sugar contents to decrease . The potatoes were
washed, peeled and sliced with the flat ridge cutter. A 40 cm cylinder was cut from the centre
region of the slices with 2 mm thickness. To minimize enzymatic browning,
slices were blanched 5 min in an 80 °C thermostatically-controlled
stirred water bath and then cooled down to 25 °C. A single layer of
blanched potato slices was placed on stainless steel trays, which were covered
with aluminum foil, and then dried in a convection oven at a temperature of
60 °C to approximately 60% of their initial weight (a flow diagram of the
procedures is shown in Fig. 1)(Ref.5)


















Fig. 1. Potato
pre-treatment and frying processes(Ref.5).


Also with preference to Fig. 1, after air drying, the potato slices
were left to cool down to ambient conditions for 1 h. Slices were
individually dipped in 23.1 wt% sugar solution for 2 s. The sugar
solution was prepared with boiling water that had been cooled to ambient
temperature 1 h before being made into sugar solution (to prevent
microorganism contamination). After dipping, about 100 g of slices were
submerged in 3 l of canola oil in an immersion fryer (Kambrook 6 l
Deep Fryer, Kmart, Auckland, New Zealand) at 180 °C. To optimize the heat
diffusion, a Citence Type KQPS/29 Mixer (Griffen & George Limited, Great
Britain) was placed in the fryer and set at 250 rpm. During frying, 10
slices were removed every minute for 6 min. After frying, samples were
wrapped on absorbing tissue paper for 5 min prior to testing. This was done to minimize the absorption of oil due to the
cooling down period, when most of the oil content of potatoes is affected by
this process. Southern,
2000 Southern, C. R. (2000). Heat and mass transfer in the potato crisp
frying process. PhD thesis, Department of Chemical and Materials
Engineering, The University of Auckland, New Zealand. In relation to this issue, also declared that
oil uptake was a post-frying phenomenon, where surface oil was absorbed during
the cooling of the crisp; with little or no oil absorbed during frying (Ref.5)
Results
and Discussion:
There was a significant difference (P < 0.05)
between the final oil contents of pre-dried and sugar dipped and the control
samples (Fig. 2).
The oil content was, dependent on the frying time. The pre-dried and dipped
samples had lower oil contents compared with the untreated samples (the
control) and pre-dried samples. On average, 0.05 g of sugar was added to
each potato slice after dipping. To calculate the amount of sugar attached into
each potato slice, the weights of potato slice before and after dipping were
determined. The difference in weight of the potato slice before and after
dipping was the weight of sugar solution added into each slice. With the
concentration of sugar solution being 23.07 wt%, the amount of sugar added
in each slice of potato was calculated. On average, after dipping, the
percentage of sugar increased to about 3% on wet basis with pre-dried potatoes;
compared with fresh potatoes without pre-drying, the increase is only about 2%
on wet basis. The sugar addition may induce a higher specific gravity or high
solids thus leaving less space for oil uptake. In order to establish whether
the sugar-dipping affects much of the oil content or not, separate experiments
which are pre-dried non-dipping and pre-dried with sugar dipping were carried
out on the crisps. The results show that there was a significant reduction in
oil content of pre-dried and dipping compared with pre-dried and non-dipping
(as well as with the control samples). Oil reduction of 30% was obtained in this
research study. This is a considerable reduction compared with previous
techniques. For example, the NaCl soaking method only had 22.2% reduction in
oil content. This indicates that effect of sugar solution on the pre-treated
samples is not a simple additive effect on the solids density which effects the
oil uptake (Ref.5).




Fig. 2. Effect of pre-drying followed
by sugar dipping on the oil content of potato crisps during frying process
(Ref.5).
The moisture content of raw potato slices varies between 75% and 85%, depending on the environmental growing conditions and varieties.
When potato slices are fried in oil at a high temperature, the moisture would
boil explosively. This may result in cell wall bursting and damages, and
consequently, the formation of capillary holes and voids. Oil adheres to the
surfaces of the chips and is also absorbed into the pores or the voids in the
porous slices. This is particularly pronounced if the chips which just leave
the frying oil are exposed to the atmosphere, and cooled creating a vacuum
within. For these reasons, regular potato chips can have high oil contents,
ranging from 35% to 39%, and even as high as 42%. This mass transfer process has been characterized
by the movement of oil into the product and water, in the form of vapor, from
the product. The relationship of oil uptake and moisture loss of the
thin crisps during immersion frying has been studied previously by. A linear
moisture–oil relationship reported for crisps) was also obtained in this study
(see Fig. 3). The data have also demonstrated that the
initial moisture content (85.5 ± 1 wt%) and the final oil
content of control samples (37 ± 4 wt%) are in the range shown
by. Lee
et al., 1988 Lee, Y., Bretch, E. E., Bath, C. K., & Merritt, C. G.
(1988). Process for preparing low oil potato chips. United States Patent,
Patent number: 4,721,625. The pre-dried and then sugar-dipped crisps had
much lower oil contents than the control samples. It was found that the
pre-drying and sugar-dipping were the crucial steps in reducing the oil
content. Each of the two processes alone can not yield such a good overall
result. This means that the oil reduction did not happen when dipping without
pre-drying (data not shown) or pre-drying without dipping (Ref.5).

Fig. 3. Effect of the pre-treatment
on the relationships of oil uptake and moisture loss(Ref.5)
Potato
strips were soaked in 3, 5 or 7% NaCl solutions (25 _C)
previous to frying, to study the effect on oil uptake. Sensory responses
indicated the best texture and lowest oil uptake at 3% NaCl solution for 50
min. Soaking had no effect (P<0.05)
on color (L*, a*
and b*) or moisture loss during
deep fat frying at 180 _C. Soaking
significantly reduced oil uptake from 0.13 to 0.10 g oil/g dry matter and
increased the measured texture parameters (hardness to penetrate both crusts,
and both work and initial rigidity). Sensory acceptability was not different (P<0.05)
between the soaked product and a commercial sample. #
2002 Elsevier Science Ltd. All rights reserved(Ref.6).
In vacuum frying operations, food is heated under reduced
pressure(<6.67 kPa (50 Torr)) causing a reduction in the boiling point of
the oil and the moisture in the foods. It is an efficient method to produce
fruit and vegetable snacks with the necessary degree of dehydration without
excessive darkening or scorching Vacuum frying is excellent
to maintain product’s nutritional quality (phytochemicals), the color is
enhanced (less oxidation) , and it reduces oil degradation. However, a
de-oiling mechanism is necessary to remove the excessive oil absorption at the
surface of the product. Perez-Tinoco et al. (2008) produced
high quality pineapple chips by vacuum frying the fresh product at 24 kPa for
120 _C for 7 min. The chips had a golden yellow color with low oil
content
(18% w.b.), high residual content of vitamin C, presence of
phenolic compounds, and antioxidant capacity. Ref.7 observed that mango, blue potato chips,
green beans, and sweet potato chips fried under atmospheric conditions were of
lower quality than the products fried under vacuum (1.33 kPa and 120 _C), though texture characteristics
of the fried products were not affected by the frying method. Anthocyanin (mg/100g
d.b.) of vacuum fried blue potato chips was 60%
higher. Final total carotenoids (mg/g d.b.) was higher by 18% for green
beans, 19% for mango chips, and by 51% for sweet potato chips. Sensory
panelists overwhelmingly preferred (P < 0.05) the vacuum fried products for color, texture, taste,
and overall quality. Most of the products retained or accentuated their
original colors
when fried under vacuum.
The traditional fried products showed
excessive darkening and scorching. Ref.7 used paper towels to remove the
excess of oil at the surface of potato chips after the product was
removed
from the fryer. They concluded that vacuum frying (_3.12 kPa) could produce potato
chips with lower oil content (30% less) and the same texture and color
characteristics of those fried in conventional (atmospheric) fryers. They
observed that one major difference between potato slices fried under vacuum and
atmospheric conditions was the surface structure of the potato chips formed
during the process. A vacuum fried potato chips had less expansion and the
surface had numerous small bubbles, as opposed to a potato chips fried under
atmospheric pressure(Ref.7)
Fig. 4. The lab-scale
vacuum frying equipment with the de-oiling mechanism.(Ref.7


Vacuum
frying was tested as an alternative technique to develop low oil content potato
chips. The effect of oil temperature (118, 132, 144 _C)
and vacuum pressure (16.661, 9.888, and 3.115 kPa) on the drying rate and oil
absorption of potato chips and on the product quality attributes such as
shrinkage, color, and texture was investigated. Furthermore, the
characteristics of the vacuumfried
potato
chips (3.115 kPa and 144 _C)
were compared to potato chips fried under atmospheric conditions (165 _C).
During vacuum frying, oil temperature and vacuum pressure had a significant
effect on the drying rate and oil absorption rate of potato chips. Potato chips
fried at lower vacuum pressure and higher temperature had less volume
shrinkage. Color was not significantly affected by the oil temperature and
vacuum pressure. Hardness values increased with increasing oil temperature and decreasing
vacuum levels. Potato chips fried under vacuum (3.115 kPa and 144 _C)
had more volume shrinkage, were slightly softer, and lighter in color than the
potato chips fried under atmospheric conditions (165 _C).
It was concluded that vacuum frying is a process that could be a feasible
alternative to produce potato chips with lower oil content and desirable color
and texture(Ref.8).

Fig. 5 Schematic of the vacuum frying
system(Ref.8).



Fig.
6. Flow diagram of the vacuum frying process(Ref.8)..




Recommendation:

I recommend vacuum frying. The main purpose of using vacuum
frying in this study was to evaluate its feasibility for production of low
oil content potato chips.Oil absorption rate during vacuum frying of
potato chips was related to the moisture loss rate. The highest drying
loss rate (thus the highest the oil absorption rate) was obtained with the
highest oil temperature (Toil
= 144_C)
and lowest vacuum pressure (Pvac
= 3:115
kPa). The fryer operating conditions did not affect the final oil content
of the vacuum-fried chips. However, results showed that the faster the
water loss rate, the higher the oil adhesion at the chips surface and then
the higher oil absorption. In addition, as the percentage of free water is
depleted in the product, less oil is absorbed. The pressurization step
plays an important role in reducing the oil absorption during vacuum frying
(Ref.7, and 8). Vacuum frying
is a dehydration process that produces healthy fruit snacks which
partially preserve the fruit's original colour and nutritional compounds
and have a high hydrophilic antioxidant capacity(Ref.9,and 10).
I recommend
judicious blending for cereals and legumes such as chickpea, cowpea, green
gram, black gram, rice, wheat and amaranth revealed that Low-Fat fried
snacks could be prepared from a blend of 64 : 16 : 20 of cowpea : chickpea
: green gram flours. The blend had 27.8% lower fat content as compared to
the control prepared from chickpea flour alone(Ref.1, 3, and 4).
I recommend A
single layer of blanched potato slices is placed on stainless steel trays,
which are covered with aluminum foil, and then dried in a convection oven
at a temperature of 60 °C to approximately 60% of their initial
weight(Fig.1), this is for drying, the dipping before drying in sugar
solution or3,or 5, or7%NaCl solution for one hour approximately, produces about 35% reduction of oil as
described in references 5 and 6(Ref.5, and 6). Go to figures,1,2,3.4.5.
and 6. And references,1,2,3,4,5,6,7,8,9, and 10.

References:
1. Low Fat Snacks From Judicious blending of Cereal
and Legumes, by Uday S. Annapure, et al., International Journal of Food
Sciences and Nutrition, Vol.49, pp 309-314 (1998).
2. Effect of barley incorporation in bread on its
quality and glycemic responses in diabetics, by Asna Urooj, et al.,
International Journal of Food Science and Nutrition, Vol. 49 pp 265-270 (1998).
3. Influence of Legume blends on fried papad
quality by, Sunita J. Patil, et al., International Journal of Food Science and
Nutrition, Vol.51, pp 381-388 (2000).
4. Fat reduction affects quality ofakara (fried
cowpea paste),by Sara P. Patterson, R. Dixon Phillips, et al., International
Journal of Food Science and Technology, Vol. 39 pp 681-689 (2004).
5. Reducing Oil Content of Fried Potato Crisps
Considerably Using a Sweet pre-treatment Technique, by T.T. Mai Tran, Journal
of Food Engineering, Vol.80, Issue2 pp719-726 (2007).
6. NaCl Soaking Treatment for Imroving The Quality
of Fresh-Fried Potatoes, by Andrea Bunger, et al., Food Research Vol.36 pp
161-166 (2003).
7. The effect of a de-oiling mechanism on
production of high quality vacuum fried potato chips by, Rosana G. Moreira, et
al. Journal of Food Engineering, Vol.92 pp 297-304 (2009).
8. Vacuum Frying of Potato Chips, by Jagoba Gouryo,
et al., Journal of Food Engineering Vol. 55, pp 181-191 (2002).
9. Effect of Vacuum Frying on main physicochemical
and nutritional quality parameters of pineapple chips, by Maria Rosalba
Perez-Tinoco, et al. Journal of the Science of Food and Agriculture, Vol., 88
Issue6 pp945-953 (2008).
Sorption Isotherms of Vacuum-Fried Carrot Chips by, Fan Liu-Ping, et
al., Drying Technolo

H1N1 Patient Education

H1N1 Patient Education
The novel influenza A (H1N1) is spread from person to person through droplets from coughing and sneezing. You also may get the H1N1 virus if you touch something with the virus on it and then touch your nose or mouth. Close contact (within 6 feet) of an infected person increases your risk of getting the virus. Symptoms of the H1N1 virus include fever, cough, sore throat, and a runny nose. You may feel very tired and have body aches and a headache. You also may have a decreased appetite, nausea, vomiting, and diarrhea.
Ways to help prevent getting or spreading the H1N1 virus and other infections include:
Cover your mouth and nose when coughing or sneezing.
Wash your hands with soap and water or use an alcohol‐based hand cleaner often. Always wash your hands after coughing and sneezing, or after touching sick people or their personal items.
Use paper towels to dry your hands after washing when you are sick, and throw the paper towels away. If cloth towels are used, each person in a household should have his own towel.
Get plenty of rest.
Get an H1N1 vaccine.
If you have the H1N1 virus, do the following:
Stay home for at least 24 hours after your fever is gone or after you have no fever without using fever medications. Wear a surgical mask if you must leave home before your symptoms go away.
If you live with others, stay in one room away from common areas in your home. Keep the door closed to your room.
If possible, use a different bathroom than others in your home. The bathroom you use should be cleaned daily with household products that kill germs.
Avoid close contact with others. If you need to leave your home or be near others, wear a mask that covers your nose and mouth.
If you have a fever, diarrheas, or vomiting, drink plenty of liquids to help prevent dehydration.
Wash all dishes with hot soapy water.
Continue to wash your hands with soap often or use an alcohol‐based cleaner.
If you are caring for someone with the H1N1 virus, do the following:
Avoid close contact with the infected person. Always wear a mask that covers your nose and mouth when entering the room. Wearing gloves and eye protection also may help decrease your risk for getting the virus.
Do not allow visitors into the sick person’s room. Only those people who are caring for the sick person should enter the room. If possible, only one adult in the home should be the caregiver.
If possible, keep the windows open in common household areas.

Vitamin Protection from Stroke Damage?

Surely, by now, people are well-aware that when it comes to surviving stroke, time is of the essence. The sooner patients get treated, the more likely their brains will be spared significant damage. Is it worth considering, then, if you’re at high risk for stroke, that taking a supplement might offer your brain protection ahead of time? According to the results of a new study from The Ohio State University in Columbus, such a day may be close at hand.Not Just Any Vitamin EThe study, funded by the National Institutes of Health, looked into whether taking a natural form of vitamin E called tocotrienols (more on that in a minute) might prepare the brain to react better after an ischemic stroke (a stroke caused by a blood clot). Most strokes (87%) are ischemic. The report, an animal study, used dogs because their brains more closely resemble those of humans than do those of commonly studied animals, such as mice and rats. For 10 weeks, researchers gave one group of dogs 200 mg a day of tocotrienols and the other group a placebo. Then, while the animals were under anesthesia, they induced strokes in both groups by blocking the middle cerebral artery in their brains for one hour. Next, researchers conducted imaging studies of the animals’ brains both one hour after and 24 hours after the induced strokes to learn what changes had taken place. What they discovered...More blood flow. In the vitamin E group, minor blood vessels (collateral vessels) that are a normal part of the brain’s circulatory system became larger in the area of the blockage, enabling more blood flow to continue in that area and protect the brain. This did not happen to the same extent in the control group.Less tissue damage. Twenty-four hours after the strokes, brain lesions that indicate tissue damage were 80% smaller in the vitamin E group compared with those in the placebo group.Less nerve damage. In the vitamin E group after 24 hours, the brain’s internal communication network -- a crisscrossing of nerves -- remained relatively intact at the location of the stroke... while in the placebo group, the network showed major disruptions. Waking Up Sleeping Arteries The senior author of the study, Chandan K. Sen, PhD, professor in the department of surgery, has been researching tocotrienol vitamin E and its effect on the brain for more than a decade. He says that the brain contains collateral vessels that normally remain dormant. When a person has a stroke, those collateral vessels enlarge and join together to improve blood flow in the affected part of the brain. Researchers found that the tocotrienol vitamin E essentially helped "wake up" these previously inactive arteries more effectively at the time of the trauma to the brain. Dr. Sen and his group are now preparing a clinical trial using tocotrienol vitamin E in a group of people who are at an increased risk for stroke. The participants in this trial will take 400 mg daily of tocotrienol E to see if the vitamin helps prevent stroke or, in cases when stroke occurs, if it helps reduce damage to the brain, as it did with the dogs. When asked for a recommendation for readers of Daily Health News, Dr. Sen said that he would like to wait for the outcomes of the clinical trial. But people at a high risk for stroke may want to talk to their doctors about taking natural Vitamin E as we wait for the clinical trial to be completed. Read the LabelThere are two categories of natural vitamin E -- tocotrienols (which were used in the study) and tocopherols. Both types include four subtypes called alpha, beta, delta and gamma. In his research, Dr. Sen uses a mix of natural vitamin E that’s rich in alpha-tocotrienol. If your doctor advises you to start taking natural vitamin E to help prevent stroke, Dr. Sen says to look for a supplement in a health-food store or online that contains a high percentage of alpha-tocotrienol. You may notice that many vitamin E supplements contain some tocopherols, as well. This is fine, so long as the tocopherol is natural and not synthetic. Check the label, and if it includes the words synthetic alpha tocopherol -- or "dl" instead of "d," the natural form -- return the bottle to the shelf and look for all-natural E. Source: Chandan K. Sen, PhD, professor in the department of surgery and associate dean, College of Medicine, The Ohio State University, Columbus.

Synthesis and Characterization of

Synthesis and Characterization of
Iron Composite Nanoparticles for Cancer Therapy
Amanda Huey
Dr. Guandong Zhang
Dr. Daniel Cullen
PI: Dr. Ian Baker
Center for Nanomaterials Research at Dartmouth
Research Experience for Undergraduates Program
August 15, 2008
1
1. Introduction
Magnetic nanoparticles can be used for many applications, including magnetic recording
media, catalysis, MRI contrast enhancement, drug delivery and hyperthermia.1,2 Hyperthermia is
a promising form of cancer therapy that locally heats tissue to greater than 42ºC for to destroy
tissue, particularly tumors.1 The difficulty of this therapy lies in selectively heating cancer
tumors.1 By localizing magnetic nanoparticles to the desired area and applying an alternating
magnetic field (AMF), the particles can generate heat by hysteresis loss, thus killing cancer cells
with minimal injury to normal tissues. Highly hysteretic particles are desirable because they will
give off the most heat with the lowest dose to a patient.1,2 Iron, which has a very high magnetic
saturation value (220 emu/g), is ideal for hyperthermia application.
Other criteria necessary for magnetic nanoparticle hyperthermia application include:
biocompatibility, non-toxicity, high accumulation in target tissue and good dispersion in aqueous
solution.1,3 Yet iron is reactive to both oxygen and water and can eventually be oxidized into
weakly ferromagnetic and antiferromagnetic species respectively, rendering particles less useful
to hyperthermia.1 Therefore, it is critical to utilize protection strategies that chemically stabilize
naked nanoparticles against oxidation.2 Such strategies include: passivation to form a strongly
adherent magnetite (Fe3O4) shell around the iron core and coating particles with coatings that
protect the iron core.1,2 It is hypothesized that hydrophobic coatings and bilayer coatings protect
the iron core of nanoparticles better than hydrophilic and monolayer coatings. Coatings can be
attached to nanoparticles via van der Waals forces, charge attraction, or covalent bonding
directly to the nanoparticle surface. Protective layers can then be used for further
functionalization, depending on the desired application for the nanoparticles.2 For hyperthermia,
biocompatible outermost coatings must be used to help particles avoid opsonization and
phagocytosis by the reticulendothelial system (the body’s mechanism to destroy foreign
substances) so that can be implemented.3
This project focuses on synthesizing iron nanoparticles and then using surface
modification to protect the iron core and achieve a biocompatible coating. The particles’
magnetic properties, surface coating and heating properties were then characterized using:
Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD), Vibrating Sample
Magnetometry (VSM), Hyperthermia/Specific Absorption Rate (SAR), Infrared Spectroscopy
(IR) and Thermogravimetry/Differential Thermal Analysis (TG/DTA). The long-range goal of
this research is to be able to synthesize biocompatible iron composite nanoparticles for cancer
therapy.
2. Experimental
2.1 Synthesis of Fe/Fe3O4 Nanoparticles and Surface Modification
2.1.1 Synthesis of Fe/Fe3O4 Nanoparticles
Fifteen nm Fe/ Fe3O4 core-shell nanoparticles were synthesized using the microemulsion
method, in which NaBH4 reduces aqueous FeCl3. Two microemulsion solutions were prepared.
Each microemulsion solution contained the same oil phase (n-octane), surfactant (cetyl trimethyl
ammonium bromide [CTAB]) and co-surfactant (n-butanol), but the water phase differed, with
one microemulsion containing FeCl3 and the other NaBH4.
To prepare a microemulsion, 75 mL n-octane, 14.3g CTAB and 12.8 mL n-butanol were
agitated in an Erlenmeyer flask. Separately, 30 mL of 0.20M FeCl3 solution and 30 mL of 0.85M
2
NaBH4 solution were prepared and then each was added to a separate oil phase containing flask.
The microemulsions were then agitated under Argon gas protection for 45 minutes. Then, the
NaBH4 microemulsion was added drop-by-drop to the FeCl3 microemulsion over the course of
about 10 minutes under high speed agitation and the mixture was left to react for 10 more
minutes. Afterwards, under Ar protection, the fresh nanoparticles were separated on a magnetic
field and then washed with de-gassed deionized (DI) water, then methanol, then acetone as
quickly as possible to prevent oxidation. The particles were then passivated in an Ar-filled
desiccator for two days to form the protective magnetite (Fe3O4) shell.
2.1.2 Fe/Fe3O4 Nanoparticles Stabilized with Trimethylamine N-oxide
To best preserve the iron core, directly after nanoparticle synthesis130 mg freshly
synthesized particles were added to a solution of 15 mg trimethylamine N-oxide in 2 mL of
isopropyl alcohol (IPA). The mixture was sonicate and left to react for 40 minutes, then rinsed
twice with methanol and dried with Ar.
2.1.3 Phase Transfer of Fe/Fe3O4 Nanoparticles Using Tetramethyl Ammonium Hydroxide
(TMAH)
Fifteen mg Fe/Fe3O4/CTAB (original) nanoparticles were dispersed in 5 mL hexane and
then the solution was added to 5 mL 12% tetramethyl ammonium hydroxide (TMAH) solution.
The mixture was sonicated and shaken until the particles transferred to the bottom (aqueous)
layer.
2.1.4 Fe/Fe3O4 Nanoparticles Modified by Hydrophobic Silanes: Hexamethyldisilazane
(HMDS) and Octadecyltrichlorosilane (OTS)
HMDS modification: Directly following synthesis, after the methanol wash, about 50 mg
of Fe/Fe3O4 nanoparticles were washed with TMAH, then methanol again, then with 9 mL of 2:1
toluene: methanol solution, then with toluene. Then particles were dispersed in 9 mL 3.0 %
hexamethyldisilazane (HMDS) toluene solution in a glass vial filled with Ar. The sample was
then discontinuously sonicated at 50 °C for 4 hours. Finally, nanoparticles were separated and
dried at 100 °C for 2 minutes under Ar protection.
OTS modification: 30 mg Fe/Fe3O4/CTAB particles were added to a solution of 3 mL
IPA and .3 mL TMAH. The solution was sonicated 25 minutes, then rinsed twice with IPA and
once with toluene. The particles were then dispersed in 3 mL toluene plus 30 μL OTS and
sonicated 20 minutes. The particles were then rinsed once with toluene and dried on a hot plate at
50ºC under Ar protection.
2.1.5 Fe/Fe3O4 Nanoparticles Modified by Phosphatidylcholine (PC)
For a 30% PC coating, 80 mg Fe/Fe3O4/HMDS (or Fe/Fe3O4/OTS) modified particles
were added to a solution of 0.24 mL PC and 0.12 mL chloroform. The solution was sonicated 25
minutes at room temperature and then the chloroform was evaporated under Ar protection.
2.1.6 Fe/Fe3O4 Nanoparticles Modified by Pluronic® Copolymers
Pluronic® F-127: 26.1 mg Fe/Fe3O4/CTAB particles were dispersed in a solution of 7.9
mg F-127 in .15 mL chloroform and sonicated for 50 minutes, then dried under Ar protection.
Pluronic® P-123: 15.4 mg Fe/Fe3O4/CTAB particles were dispersed in a solution of 5.3
mg P-123 in .1 mL chloroform, sonicated for 50 minutes, and dried under Ar protection.
3
2.1.7 Fe/Fe3O4 Nanoparticles Modified by Covalent Bonding: Dopamine-Dicarboxylterminated
Polyethylene Glycol
First, 6 mL PBS and 14 mL DI water were mixed and sonicated, then 0.30g N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 0.30g NHydroxysuccinimide
(NHS) were added to the solution and sonicated. Then 0.30g dopamine
(DA) hydrochloride was added and the solution was sonicated. Finally, 0.2 mL dicarboxylterminated
polyethylene glycol (PEG) was added and the solution was sonicated 20 minutes and
left to react overnight (about 18 hours) to ensure amide bond formation due to the EDC reaction.
The next day, a solution of 0.1g KOH in 2 mL DI water was used to change the pH of 5.5
mL EDC reaction solution to about 6.5. Then, 23.8 mg Fe/Fe3O4/CTAB (or Fe/Fe3O4/oxidizer)
particles were added to solution, sonicated 25 minutes, then rinsed twice with methanol,
centrifuged for 5 minutes, then dried with Ar.
2.2 Characterization of Nanoparticles
The crystallinity and structure of iron composite nanoparticles were determined by using
X-ray diffraction (XRD). Transmission electron microscope (TEM) was used to verify the shape
and size of particles, as well as the presence of an iron core. The magnetic saturation and
coercivity of the nanoparticles were determined by examining the hysteresis loop produced by
the vibrating sample magnetometer (VSM). The surface coating on particles was examined by
using infrared spectroscopy (IR) and Thermogravimetry/Differential Thermal Analysis
(TG/DTA). Finally, the heating effect of the Fe/Fe3O4 nanoparticles was examined under an
alternating magnetic field measured by a heating setup in the lab.
3. Results & Discussion
Iron nanoparticles were synthesized using the microemulsion method because this
method yields monodisperse iron nanoparticles without requiring high temperature or high
pressure conditions. It is easy to control particle size and thus the magnetic properties of the
nanoparticles by changing the oil to water ratio of the solution. In the microemulsion, the water
droplets act as a mini-reactor site for equation (1) to occur:
2FeCl3 + 6NaBH4 + 18H2O → 2Fe0 ↓ + 21H2 ↑ + 6B(OH)3 + 6NaCl (1)
The iron core of freshly synthesized particles must be preserved by formation of a thin
magnetite (Fe3O4) shell. One method of Fe3O4 shell formation is passivation using Argon gas;
another uses an oxidizer, such as Trimethylamine N-oxide, which helps transfer oxygen to the
iron core to form magnetite, rather than a less magnetic iron oxide, such as hematite (α-Fe2O3) or
goethite (α-FeOOH). Figure 1 shows VSM results demonstrating the benefit of using oxidizer
directly after particle synthesis in order to preserve the iron core and thus the magnetic properties
of the particles. Particles preserved with oxidizer have a magnetic saturation (Ms) of 90 emu/g,
while the original particles only have an Ms of 58 emu/g.
4
Figure 1: VSM results showing the effect of oxidizer on iron core and magnetic property
preservation in Fe/Fe3O4 nanoparticles
Freshly synthesized particles also have a layer of surfactant (CTAB) coating, which must
be modified in order to better preserve the iron core. New coatings can replace old coatings due
to stronger bonds with nanoparticles. In addition, new coatings can be used to achieve a phase
transfer of the nanoparticles from the oil phase to the water phase, which is necessary for
biocompatibility. Phase transfer is important because even though CTAB is an amphiphilic
compound, Fe/Fe3O4/CTAB particles disperse in the oil phase. Figure 2 shows a schematic of
surfactant replacement, with tetramethyl ammonium hydroxide (TMAH) replacing CTAB and
Figure 3 shows the resulting phase transfer of the nanoparticles from oil to water phase.
Figure 2: Schematic of surfactant replacement with TMAH replacing CTAB on Fe/Fe3O4
nanoparticle surface5
Figure 3: Phase transfer of Fe/Fe3O4 particles from oil to water phase
5
One type of coating that helps preserve the iron core is a hydrophobic silane coating, such
as hexamethyldisilazane (HMDS) or octadecyltrichlorosilane (OTS), since hydrophobic coatings
impede oxidation better than hydrophilic coatings. Figure 4 shows the schematic of HMDS
modification.
Figure 4: HMDS silanization on the Fe/Fe3O4 nanoparticle surface5
Figure 5 shows the benefit of using a hydrophobic coating, rather than a hydrophilic one,
to preserve the iron core of nanoparticles. The original particles (CTAB coated) have an Ms
value of 104 emu/g and the hydrophobic HMDS coated particles have a Ms of 83 emu/g. The
decrease in the Ms value is due to the oxidation of iron that occurs during the modification
process. However, the hydrophilic TMAH coated particles only have an Ms value of 73 emu/g,
showing that nanoparticles with a hydrophilic coating are more easily oxidized.
Figure 5: VSM results for original (CTAB), HMDS, and TMAH coated Fe/Fe3O4
nanoparticles
OTS silanization works analogously to HMDS silanization, but the longer hydrophobic
tail of OTS (18 carbons) functions to better protect the iron core of the particles. Figure 6 shows
the heating results showing the heating effects of particles coated with OTS and
phosphatidylcholine (PC, a biocompatible coating, to be discussed later) versus particles coated
with HMDS and PC. Since both solutions consist of 0.6% of some iron-containing compounds,
the higher heating effect of the OTS/PC particles must be due to better preservation of the iron
core thanks to the longer hydrophobic tail on OTS.
6
Figure 6: Heating effect results comparing hydrophobic silane coatings of
Fe/Fe3O4/OTS/PC vs. Fe/Fe3O4/HMDS/PC particles
Since silanes are not biocompatible, a biocompatible coating must be added over the
silane, usually via van der Waals forces. Figure 7 shows a mechanism of the addition of a
biocompatible phospholipid, phosphatidylcholine (PC), to a silanized nanoparticle.
Figure 7: Mechanism of PC assembly via van der Waals forces to form a biocompatible
outermost layer5
Pluronic® copolymers, which are also added via van der Waals forces (Figure 9), provide
a unique way of preserving the iron core and providing a biocompatible coating due to their
hydrophobic and hydrophilic chain sections (Figure 8) and temperature dependent properties.
7
8. a) b)
Figure 8: a) General structure of Pluronic® copolymers b) Structure of the two
copolymers used to modify nanoparticles5
Figure 9: Schematic of Fe/Fe3O4 nanoparticle modification with Pluronic® copolymer5
Below critical micelle temperature (CMT), Pluronic® is hydrophilic; above it, the PPO
chain dehydrates and becomes hydrophobic and micelles self-assemble. At even higher
temperatures, the entire chain becomes hydrophobic and micelles self-associate.4 It is
hypothesized that these properties might help produce better hyperthermia heating effects.
Another way to preserve the iron core and provide a biocompatible coating is through
covalent bonds to the nanoparticle and between coating layers; these tighter bonds better
preserve the iron core and hold coatings in place. According to Yunyu Shi, 2-(3,4-
dihydroxyphenyl)ethylamine (dopamine, or DA) provides good, strong covalent bonds to Fe3O4
in nanoparticles.3 The NH2 group of dopamine can then be used for further functionalization,
such as formation of an amide bond with biocompatible carboxyl-group terminated polyethylene
glycol (PEG), yet with two carboxyl groups on PEG, cross-linking between particles becomes a
problem.3 Cross-linking was avoided by maintaining a low concentration of particles in solution,
thus linking both carboxyl groups of PEG to a single particle. Dopamine and PEG were bonded
together first via amide bond formation by the EDC reaction, as seen in Figure 10a. Particles
were then added to solution to form a bond between the hydroxyl group of dopamine and the
nanoparticle (Figure 10b). The particles were added second in order to avoid prolonged exposure
to aqueous conditions, which leads to oxidation of the iron core.
8
10. a)
10. b)
Figure 10: Schematic of Dopamine-PEG coating onto Fe/Fe3O4 nanoparticles a)
Formation of covalent amide bond between dopamine and carboxyl groups on PEG b) Formation
of covalent bonds between dopamine and Fe/Fe3O4 nanoparticle
These coating methods were successful at preserving the iron core, as seen in TEM
images of the nanoparticles (Figure 11). All three images show a good dispersion of particles
with the iron core successfully preserved and visible in the images.
11. a) b) c)
Figure 11: TEM image of a) Fe/Fe3O4/HMDC/PC b) Fe/Fe3O4/Pluronic® P-123 c)
Fe/Fe3O4/oxidizer/dopamine/PEG coated nanoparticles
Over time, the iron core and magnetite shell can still undergo oxidation to form less
magnetic or non-magnetic iron oxides. X-ray diffraction can be used to determine which iron
oxide compounds are present in nanoparticles by calculating the lattice parameter from the (311)
and (440) plane peaks and comparing these values to the known lattice parameters of compounds
such as magnetite (Fe3O4, a= 0.83967 nm) and maghemite (γ-Fe2O3, a= 0.8346 nm). To
9
determine the sequence of oxidation Fe/Fe3O4/HMDS/PC particles were dispersed in DI water
with a concentration of 0.2% for various amounts of time. Figure 12 shows the X-ray diffraction
results of Fe/Fe3O4/HMDS/PC particles in water for 4 hours.
Figure 12: XRD results of Fe/Fe3O4/HMDS/PC particles dispersed in water for 4 hours
Particles in water for longer amounts of time showed a decrease in iron oxide peak
intensities and a shift in 2θ values corresponding to the formation of less magnetic compounds.
From these results, the lattice parameters were calculated and calculations are summarized in
Table 1. (Note: Lattice parameter a (nm) is the average value of the lattice parameter calculated
from (311) and (440) plane peaks using the following formulas:
2 2 2 h k l
a
d
+ +
= ,
d
n
2
sin
!
" = )
Table 1: Oxidation of Fe/Fe3O4/HMDS/PC nanoparticles over time
Time in water (hr) 4 20 36
Lattice parameter a (nm) 0.837481 0.836522 0.834912
Compounds Close to Fe3O4 Close to γ-Fe2O3 Close to γ-Fe2O3
From observation and lattice parameter calculations, the proposed oxidation sequence of
nanoparticles is as follows:
Step 1: 3 Fe0 + 2O2 → Fe3O4
Step 2: 4 Fe3O4 + O2 → 6 γ-Fe2O3
4 Fe3O4 + O2 + 2 H2O → 4 γ-Fe2O3 + 4 α-FeOOH
Maghemite (γ-Fe2O3) is still ferromagnetic, though less so than magnetite; however,
goethite (α-FeOOH) is antiferromagnetic, which demonstrates the need to impede oxidation as
much as possible, so that particles are still usable for hyperthermia treatment.
The heating effect of the iron composite nanoparticles was measured using a heating
setup in the laboratory. Figure 13 shows the much greater heating effect achieved over the course
Fe3O4
(311)
Fe3O4
(440)
10
of a minute using Fe/Fe3O4 particles with a biocompatible coating versus just Fe3O4 particles
(also with a biocompatible coating). The high magnetic saturation of the iron core increases the
size of the hysteresis loop and thus the amount of heat given off by particles, demonstrating the
importance of the work done to preserve the iron core.
Figure 13: Heating results showing the heating effect of Fe/Fe3O4 vs. Fe/Fe3O4 particles
4. Conclusion
Fe/Fe3O4 nanoparticles were successfully synthesized using the microemulsion method.
The iron core of particles was preserved using an oxidizer and a variety of coatings. Outermost
biocompatible layers were also achieved. The magnetic properties and heating effects of particles
were examined and TEM and XRD characterizations were used to determine the appearance and
structure of particles and the presence of the iron core.
5. References
1. Dale L.. Huber. Small. 2005, No. 5, 482-501.
2. An-Hui Lu, E. L. Salabas, Ferdi Schüth. Angew. Chem. Int. Ed. 2007, 46, 1222-1244.
3. Yunyu Shi. Superparamegnetic Nanoparticles for Magnetic Resonance Imaging (MRI)
Diagnosis. Master Dissertation, University of Adelaide/University of South Australia,
2006.
4. Marcela Gonzales, Kannan M. Krishnan. Journal of Magnetism and Magnetic Materials
311. 2007, 59-62
5. Figures courtesy of Dr. Guandong Zhang
6. Acknowledgements
Thank you very much to Dr. Baker for advising, Dr. Cullen for help with the XRD,
Yifeng Liao for TEM imaging and especially Dr. Guandong Zhang for his everyday guidance.
Thank you also to the 2008 CNR@D REU program and NSF, DoD-ASSURE and SBS for
funding the program.

Broad-spectrum amino acid-sensing class C G-protein coupled receptors

Pharmacology
& Therapeutics

Volume 127, Issue 3,
September 2010,
Pages 252-260
doi:10.1016/j.pharmthera.2010.04.007 | How to Cite or Link Using DOI
Copyright © 2010 Elsevier Inc. All rights reserved.
Cited By in Scopus (0)
Permissions & Reprints
Associate Editor: Peter Molenaar

Broad-spectrum amino acid-sensing
class C G-protein coupled receptors:
Molecular mechanisms, physiological significance and options for drug
development
Arthur D. Conigravea,
,
and David R. Hampsonb

a School of Molecular Bioscience (G08),
University of Sydney, NSW 2006, Australiab
Department of Pharmaceutical Sciences and Department of Pharmacology,
University of Toronto, 144 College St. Toronto, Ontario, Canada M5S 3M2

Available online 6 May 2010.
AbstractIn this article, we consider the
molecular mechanisms that underlie broad-spectrum amino acid sensing by a discrete subgroup
of class C G-protein-coupled receptors
that includes the calcium-sensing receptor,
GPRC6A and heterodimers composed of two closely related receptor
subunits, T1R1 and T1R3. We consider their
physiological significance highlighting their diverse spectrum of
cellular responses and the phenotypes of global and conditional knock-out mice. In addition, we consider
strategies for the development of new drugs that target these receptors.

Keywords: Allosteric modulators; Amino acid-sensing; Calcium-sensing receptor; Dimerisation;
GPRC6A; G-protein coupled receptor;
Molecular modelling; Taste receptors; Transgenic
mouse; Venus FlyTrap
Abbreviations: AMPA,
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid; CaSR, calcium-sensing receptor; CR,
Cysteine-rich; ERK, extracellular regulated kinase; GABA, Gamma-amino-butyric
acid; GABAB, GABA receptor Type B; GPCR, G-protein
coupled receptor; GPRC6A, G-protein
coupled receptor class C member 6A; HH, heptahelical; mGlu, metabotropic
glutamate receptor; NMDA,
N-methyl d-aspartate; T1R, Taste Receptor
Family-1; VFT domain, Venus Fly Trap
domain
Article Outline1. Introduction2.
Domain-based organization of class C G-protein coupled
receptors3. Dimers and higher order
oligomers4. Modes of signaling by class C
GPCRs4.1. Molecular basis of signal
transmission: a physical link between the VFT and CR domains5.
Ligand binding surfaces for class C GPCRs6.
A conserved amino acid binding site in class C GPCRs:
origins of promiscuity in broad-spectrum amino acid sensors7.
mGlu subgroup-selective compounds and design options
for agents that target the VFT domains of broad-spectrum amino
acid-sensing receptors7.1. Development
of agents that target the VFT domains of broad-spectrum amino
acid-sensing receptors8. Physiological
roles of broad-spectrum amino acid-sensing class C GPCRs8.1.
Tissue expression patterns and proposed roles of
broad-spectrum amino acid-sensing receptors8.1.1. CaSR8.1.2. C6A8.1.3.
T1R1/T1R38.2.
Insights from global and tissue-specific transgenic
mice8.2.1. CaSR8.2.2. C6A8.2.3. T1R1/T1R39.
ConclusionsAcknowledgementsReferences
1. IntroductionMammalian
class C G-protein coupled receptors
include metabotropic receptors for the amino
acid neurotransmitter glutamate (the so-called metabotropic glutamate receptors; mGlus); the α-carbon decarboxylated analog
of glutamate, GABA (the GABAB receptors); several
orphans; and a recently defined subgroup of broad-spectrum amino acid-sensing receptors including the
calcium-sensing receptor (CaSR) and GPRC6A (hereafter referred to as
C6A), both of which appear to
operate as homodimers, as well as T1R1/T1R3 taste
receptor heterodimers (Fig. 1). Receptors in this
latter subgroup are promiscuous in their amino
acid-sensing properties, being ‘tuned’ to respond to two or more
classes of amino acids. Collectively, they appear to operate as sensors
of protein nutrition and exhibit potencies for individual amino acids
that are appropriate for providing an integrated receptor response to
complex amino acid mixtures. In this
article we consider the molecular basis of broad-spectrum amino acid sensing by this subgroup of
class C G-protein-coupled receptors
together with their physiological significance along with potential
strategies for the design and development of new therapeutic agents that
target them.Full-size image
(22K)
High-quality image
(60K)Fig. 1. Schematic
representation of phylogenetic relationships between different members
of GPCR Class C. The analyses were
based on a protein sequence alignment that included both the VFT and HH domains but excluded the
receptors' C-termini. The red boxes indicate broad-spectrum amino acid-sensing receptors; the green
boxes indicate narrow-spectrum receptors. The ligand selectivities of
the putative pheromone receptors are currently unknown. The CaSR is most closely related to other
broad-spectrum amino acid-activated
receptors including GPRC6A, the 5.24 receptor and the T1R taste
receptors. The figure has been reproduced (Conigrave & Hampson, 2006). 2.
Domain-based organization of class C G-protein
coupled receptorsIn general, Class C GPCRs have a large extracellular region of around 500–600
amino acids that is organized into two separate sub-domains: a
neurotransmitter or nutrient-binding, N-terminal Venus FlyTrap (VFT) domain of around 450–500 amino acids
and a Cysteine-rich (CR) domain of around 60–70 amino acids (reviews: ([Blasi et al., 2001], [Conigrave & Hampson, 2006]
and [Wellendorph & Bräuner-Osborne,
2009])). Atypically, the CR domain is absent in GABAB receptors (Hu et al., 2000). In keeping with
all known members of the GPCR
superfamily, class C GPCRs exhibit a
heptahelical (HH) domain that is required for the docking and
activation of G-proteins ([Chang et al., 2000] and [Havlickova et al., 2003]) as well
as a large C-terminus (50–200 amino acids) that binds protein partners
to direct the receptors to specific cellular compartments for the
assembly of signaling networks, temporary storage or proteolysis. The four major domains of
class C GPCRs are organized in such a
way that the CR domain acts in series to couple the nutrient-sensing VFT domain to the HH domain-C-terminal
domain signaling unit (Fig. 2). The C-terminus of
the CR domain is connected to the first transmembrane helix of the HH
domain by a short peptide linker. In the CaSR
this peptide corresponds to residues 599–613 (Ray et al., 2007).Full-size image
(38K)
High-quality image
(367K)Fig. 2. Depiction of an mGlu-1 homodimer. The Venus FlyTrap (VFT), Cys-rich (CR) and heptahelical (HH)
domains are shown. The representation of the nutrient-binding VFT domain is based on the crystal
structure of mGlu-1 ([Kunishima et al., 2000] and [Tsuchiya et al., 2002]). The CR
and HH domains are homology models based on the crystal structures of
the extracellular domain of the type I tumor
necrosis factor receptor (Naismith et al., 1996) and bovine rhodopsin
(Palczewski et al., 2000)
respectively. With the exception of the relationships between the VFT domain and CR domains in mGlu-3 (Muto et al., 2007), the relative
orientations of the three domains are unknown. Several potential binding
surfaces are shown including the cavities of the bilobed VFT domains (a and a'), the
interprotomeric interface (b), the niches between the VFT domains and CR domains (c and c'), and
pockets within the HH domains that are recognized binding sites for
allosteric modulators (d and d'). Crystal structure analysis of the VFT domains of mGlu-1 ([Kunishima et al., 2000] and [Tsuchiya et al., 2002]), as well
as mGlu-3 and mGlu-7 (Muto et al., 2007) demonstrate
that the N-terminal VFT domain is
the site of glutamate binding in mGlus ([Kunishima et al., 2000], [Tsuchiya et al., 2002] and [Muto et al., 2007]) and analyses
of mutant and chimeric receptors indicates that it is also the site of l-amino acid binding
in the CaSR ([Zhang et al., 2002], [Mun et al., 2004] and [Mun et al., 2005]) and C6A ([Kuang et al., 2005] and [Wellendorph et al., 2005]).
Sequence alignments also suggest that the l-amino acid binding site is conserved in
the VFT domain(s) of T1R1/T1R3
heterodimers (review: (Conigrave & Hampson, 2006)).
Four primary structural states are recognized, irrespective of whether
the ligand is bound. These include an open–open (inactive) state,
closed–open and open–closed (partially active) states and a
closed–closed (fully active) state. Ligand binding stabilizes the closed
states and ligand binding can occur in both VFT domains simultaneously ([Kunishima et al., 2000] and [Tsuchiya et al., 2002]). The
closed–open/open–closed and closed–closed forms of the mGlus may define distinct G-protein
selectivities (Rondard et al., 2006).3. Dimers and higher order oligomersUnlike
GPCRs of Class A which, based on
recent crystal structure analyses, may be functionally active as
monomers ([Cherezov et al., 2007], [Rasmussen et al., 2007] and [Warne et al., 2008]) or Class B
which function as heterodimers with receptor
activity modifying proteins (RAMPs; review: (Hay et al., 2006)), class C GPCRs appear to be functionally active
only as homodimers (e.g., the CaSR, mGlus) or heterodimers with closely
related GPCR subunits (e.g., the T1R
and GABAB receptors).
The dimers are stabilized by disulfide bonds in the proximal VFT domain and by additional non-covalent
interactions ([Zhang et al., 2001] and [Hampson et al., 2008]). In the CaSR, for example, intermolecular
disulfides act to asymmetrically crosslink residues C129 and C131 (Ray et al., 1999) and non-covalent
interactions involving residues L112 and L156 also participate in dimer
formation (Jiang et al., 2004b). In addition,
the crystal structure of the entire mGlu-3
extracellular domain demonstrates that intramolecular disulfides are
required for normal receptor function. Four intramolecular disulfides
stabilize the CR domain into a rigid structure to permit the application
of rotational forces from the VFT
to the HH domains. An additional intramolecular disulfide appears to
provide a lever between lobe-2 of the VFT
domain (Cys-240) and the Cys-rich domain (Cys-527; (Muto et al., 2007)). A model of
the dimer arrangements between mGlu
subunits is provided in Fig. 2.Analyses of
heterodimers, including the GABAB
and T1R1/T1R3 receptors, demonstrate that one
subunit is specialized for ligand binding in its VFT domain (e.g., T1R1 and GABAB1) whereas the other
subunit (e.g., T1R3 and GABAB2)
is required for expression and intracellular signaling responses ([Jones et al., 1998], [Kaupmann et al., 1998], [White et al., 1998], [Jiang et al., 2004a] and [Zhang et al., 2008]). Whether
subunit specialization might operate in a cyclical manner in class C
homodimers (e.g., involving mGlus, CaSR or C6A)
under certain conditions is currently unclear. In one cyclical scheme,
the VFT domain of subunit A might
mediate nutrient binding to induce signaling via the HH domain of its
protomeric partner subunit A' followed by nutrient binding in subunit A'
and attendant signaling via subunit A. Recent evidence suggesting that
only one active HH domain is required for mGlu-1
signaling (Hlavackova et al., 2005) is
consistent with such a possibility.Recent analyses in HEK293
cells and COS7 cells indicate that
RAMPs 1 and/or 3 are required for CaSR
trafficking to the plasma membrane
in certain cell contexts ([Bouschet et al., 2005] and [Bouschet et al., 2008]) raising
the possibility that the CaSR might
form heterodimers or higher order oligomers with non GPCR chaperones. Whether RAMPs are
required for CaSR trafficking in
endogenous cells or facilitate plasma
membrane targeting in cells in which the CaSR is over-expressed heterologously is
currently uncertain. If RAMPs are indeed required for normal endogenous
expression, it seems possible that they may also be required for the
expression of other members of GPCR
class C.Why are dimers formed from closely related subunits
required for the function of class C receptors and not other GPCR classes? As noted above, class C
receptors are unusual in binding nutrient ligands in their extended
extracellular VFT domains.
Therefore, a mechanism is required to transmit the molecular signal
associated with VFT domain closure
to the transmembrane domains where
intracellular signaling is initiated, requiring the transmission of
turning moments from the raised FlyTraps
to the HH domain signaling units (review: (Pin et al., 2004)). Consistent
with this notion, receptor activation appears to arise from the
approximation of neighbouring lobes 2 in receptor dimers and associated
adjustment of the spatial relationship between adjacent HH domains
(review: (Pin et al., 2009)). In one
scenario the heptahelical partners exchange domains to generate an
active G-protein binding site (Brock et al., 2007).4. Modes of signaling by class C GPCRsSeveral subclasses of metabotropic glutamate receptors are
recognized based on sequence homologies, signaling properties and
pharmacological behaviour (review: (Conn, 2003)). Upon receptor
activation, Group 1 mGlus (mGlu-1 and mGlu-5)
elicit Gq/11 dependent activation of PI-PLC associated with intracellular Ca2+
mobilization. mGlus belonging to
Groups II (mGlu-2 and -3) and III (mGlu-4, -6, -7 and -8), on the other
hand, elicit Gi/G0 dependent inhibition of adenylyl cyclase (Ferraguti & Shigemoto, 2006).
Among the broad-spectrum amino acid-sensing
subgroup of class C GPCRs, the CaSR elicits both Gq/11-dependent
activation of PI-PLC (review: (Hofer & Brown, 2003)) and
intracellular Ca2+ mobilization as well as Gi/0-dependent
inhibition of adenylyl cyclase (Gerbino et al., 2005), thereby
combining properties of both group I and group II/III receptors (Table 1). In addition, both C6A ([Kuang et al., 2005], [Wellendorph et al., 2005] and [Wellendorph et al., 2007]) and
the T1R1/T1R3 heterodimeric receptors (Nelson et al., 2002) activate PI-PLC, although their impacts on adenylyl cyclase activity are less
certain. It is not currently known whether the CaSR recruits G-proteins of both classes
simultaneously to separate G-protein docking sites, switches
repetitively between different classes of G-proteins, or is specialized
for G-protein binding according to
its protein binding partners and
membrane localization.Table 1. G-protein
selectivities of class C GPCRs.
Well-characterized members of GPCR
class C are shown. The mGlus have
been organized according to their three main groups. The broad-spectrum amino acid-sensing receptors (CaSR, C6A
and T1R1/T1R3 heterodimers) are collected
together as a subgroup at the bottom of the table. The CaSR appears to be unusual in its high
degree of promiscuity for different G-proteins.
ReceptorG-protein selectivities
mGlu Group I (1, 5)Gq/11
mGlu Group II (2, 3)Gi/o
mGlu Group III (4, 6, 7, 8)Gi/o
GABABGi/o
CaSRGq/11; Gi/o; G12/13
C6AGq/11
T1R1/T1R3α-gustducin
T1R2/T1R3α-gustducin; α-transducin
4.1.
Molecular basis of signal transmission: a physical link between the VFT and CR domainsThe mechanism
by which ligand binding in the VFT
domain induces the transmission of signals from the VFT domain to the HH domain and, in turn,
controls coupling to one or more G-proteins is critical to an
understanding of the mechanism of receptor activation. Noting the
presence of nine cysteines in the CaSR
CR domains Hu et al. investigated the possibility of a disulfide link
between the CaSR VFT and CR domains by introducing a
selective tobacco etch virus protease cleavage site (NLYFQG) between residue
E536, the last residue encoded by exon-5 at the C-terminus of the VFT domain and V537, the first residue
encoded by exon-6 (Hu et al., 2001). Following proteolytic cleavage, the VFT domains were released as C129–C131
disulfide-linked dimers from the remainder of the dimeric protein,
apparently disproving the hypothesis that a disulfide directly links the
VFT and CR domains (Hu et al., 2001).Surprisingly,
evidence for a disulfide link between the VFT
and CR domains was subsequently identified for rat mGlu-2
raising the possibility of a class-wide mode of operation (Rondard et al., 2006).
Particularly compelling was the observation that mutations of mGlu-2 Cys-234 disabled receptor function
without impairing surface expression and abolished the positive impact
of the HH domain-binding positive allosteric modulator 4-MPPTS on glutamate binding (Rondard et al., 2006). Tight
conservation of mGlu-2 Cys-234 and
its CR domain partner Cys-518, together with the demonstration of an
interdomain disulfide in the crystal structure of the entire
extracellular domain of mGlu-3 (Muto et al., 2007), strengthen the
case that a highly conserved disulfide between the VFT and CR domains is critical for signal
transmission in all class C GPCRs.
As noted recently by Hu and Spiegel (2007), the failure
to demonstrate an intramolecular disulfide between the CaSR's VFT
and CR domains in their earlier study (Hu et al., 2001) may have arisen
from its relative instability under the conditions used to process and
separate the protein bands.5.
Ligand binding surfaces for class C GPCRsSeveral
ligand binding surfaces are recognized for class C GPCRs. Given that all these receptors
appear to operate as dimers formed from closely related subunits,
potential binding surfaces include both VFT
interlobar fissures ([Kunishima et al., 2000] and [Tsuchiya et al., 2002]), the
interprotomeric interface between VFT
domains ([Tsuchiya et al., 2002] and [Abe et al., 2003]), the niches
between the VFT domains and CR
domains, and pockets within the HH domains (Miedlich et al., 2004). The
locations of these sites are shown schematically in Fig. 2. It is not yet
certain, however, whether all these potential sites are used
physiologically. The best recognized binding site in the broad-spectrum amino acid-sensing subgroup is the
conserved VFT interlobar crevice for
amino acid binding. Based on the
crystal structure of the mGlu-1 VFT domain, there is also a multivalent cation binding site in the
interprotomeric interface, which supports the agonist activity of Gd3+
(Tsuchiya et al., 2002). It seems
doubtful, however, that this site supports multivalent cation sensing in
the CaSR since the negatively
charged interface is interrupted by local positive charge. Instead the CaSR appears to provide Ca2+
binding sites within the interlobar crevice of the VFT domain ([Huang et al., 2007] and [Huang et al., 2009]) and
associated with the extracellular surface of the HH domains ([Hauache et al., 2000], [Ray & Northup, 2002] and [Mun et al., 2004]).The VFT domain interlobar crevice and HH
domain-binding pockets have proven to be suitable target sites for drug
development. In general, whereas activators that bind in the VFT domains are agonists, activators that
bind in the HH domains are positive allosteric modulators (PAMs).
Similarly whereas, in general, inhibitors that bind in the VFT domains are antagonists, inhibitors
that bind in the HH domains are negative allosteric modulators. The VFT domain-associated amino acid binding site of the CaSR appears to be an exception since it
operates as a PAM with respect to intracellular Ca2+
mobilization ([Conigrave et al., 2000a], [Conigrave et al., 2000b] and [Conigrave & Hampson, 2006]).
Successful development of high potency, high efficacy, positive and
negative allosteric modulators of class C GPCRs
including mGlus and the CaSR offer new approaches to drug
development (review: (Conn et al., 2009)).6. A conserved amino acid binding site in class C GPCRs: origins of promiscuity in
broad-spectrum amino acid sensorsIn
addition to allosteric sites in the HH domains, all deorphanized
members of Class C GPCRs possess an amino acid-, or modified amino acid binding pocket in their VFT domains. In the mGlus, the glutamate-binding
pocket occupies a relatively small cavity within the crevice of the VFT domain compared to other Class C
receptors ([Wang & Hampson, 2006] and [Wang et al., 2006]) and is highly
selective for l-glutamate ([Frauli et al., 2006] and [Kuang et al., 2006]). From the
crystal structures it appears that as many as 12 residues establish
H-bonds or salt bridges with the bound glutamate ([Kunishima et al., 2000], [Tsuchiya et al., 2002] and [Muto et al., 2007]) and
additional bonding interactions occur with more complex ligands, thereby
increasing affinity, potency and subgroup selectivity (reviews: ([Conigrave & Hampson, 2006]
and [Wellendorph & Bräuner-Osborne,
2009])). In rat mGlu-1, five residues support normal
binding of the α-amino and αcarboxylatemoieties of glutamate: S165,
T188, D208, Y236, and D318 (Kunishima et al., 2000) and these
residues are highly conserved across the class (review: (Conigrave & Hampson, 2006)).In
contrast, residues that ligate the amino
acid side chains are not conserved across class C receptors and
variation in the side-chain binding pockets is responsible for the
diversity and promiscuity of l-amino acid sensing in the CaSR, C6A
and T1R1/T1R3 heterodimers ([Wang & Hampson, 2006] and [Wang et al., 2006]). In mGlu-1, R78 and K409 ligate the
γ-carboxylate side-chain of the bound glutamate, and R78 is tightly
conserved across all three mGlu
subgroups ([Hampson et al., 1999] and [Kuang et al., 2006]). The
side-chain binding pockets of other Class C receptors are less well
defined and mediate broad-spectrum amino
acid selectivities for basic and aliphatic amino acids in 5.24
and C6A (([Speca et al., 1999], [Kuang et al., 2005] and [Wellendorph et al., 2005]) as
well as aromatic, aliphatic and polar amino acids in the CaSR (([Conigrave et al., 2000b] and [Conigrave & Hampson, 2006]);
see Fig. 3).Full-size image
(89K)
High-quality image
(1273K)Fig. 3. Comparisons of
the binding pockets of mGlu-1 C6A, 5.24 and the CaSR. The Connolly surfaces that define
the binding pockets were constructed in the absence of ligand using the
MOLCAD module in Sybyl software. Ligands are displayed as ball and stick
representations (glutamate in the case of mGlu-1;
arginine in the cases of C6A and
5.24 and l-Phe in the case of the CaSR). The figure has been adapted from
Ref. (Wang & Hampson, 2006). 7. mGlu subgroup-selective compounds and
design options for agents that target the VFT
domains of broad-spectrum amino acid-sensing
receptorsConsiderable effort has gone into the development of
high potency agonists and antagonists of the orthosteric sites in the mGlu VFT
domains. In general, these compounds exhibit either: (i) broad-spectrum
activity across all eight mGlu
subtypes or (ii) selectivity for an mGlu
subgroup (review: (Conn, 2003)) although some
receptor specific agents are also recognized. Examples of
group-selective compounds include the potent Group I-selective agonist quisqualate which binds to either mGlu-1 or mGlu-5
in an orientation that permits the formation of additional H-bonds (Sato et al., 2003), the Group
II-selective agonist DCG-IV, in
which an additional side-chain carboxylate interacts with a
group-specific Tyr residue (Y150 in mGlu-2;
([Muto et al., 2007] and [Yao et al., 2003])) and Group
III-selective agonists (e.g., l-amino-4-phosphonobutyric
acid and l-serine-O-phosphate) in which negatively charged
phosphonate side-chains bind in a positively charged microenvironment ([Hermit et al., 2004] and [Rosemond et al., 2004]).7.1. Development of agents that target
the VFT domains of broad-spectrum amino acid-sensing receptorsAlthough
potent and selective positive and negative allosteric modulators of the
CaSR's HH domains, known
respectively as calcimimetics and calcilytics, have been developed
successfully ([Nemeth, 2006] and [Marquis et al., 2009]), the
design and/or development of compounds that target the VFT domain ligand binding sites of
broad-spectrum amino acid-sensing
receptors is in its infancy. Recent molecular modelling, successfully
predicting that the VFT ligand
binding domains of the CaSR, C6A and 5.24 receptors can accommodate the
peptide glutathione (Wang et al., 2006), however,
suggests an approach to the design of novel activators and inhibitors of
all members of broad-spectrum amino acid-sensing
receptors based on γ-glutamyl or β-aspartyl nuclei. Consistent with this
notion, γ-glutamyl peptides have been reported recently to
act as CaSR-mediated taste-enhancing
agents (Ohsu et al., 2010).8. Physiological roles of
broad-spectrum amino acid-sensing
class C GPCRsThe
physiological significance of class C GPCRs
can be assessed by a consideration of tissue expression patterns and
the impact of receptor activation on cell and tissue responses.
Alternatively, their significance can be assessed in transgenic mice derived, for example, by
global or conditional deletion strategies. In the subsequent sections,
the physiological significance of the broad-spectrum amino acid-sensing class C GPCRs is considered from both these
perspectives.8.1. Tissue
expression patterns and proposed roles of broad-spectrum amino acid-sensing receptors8.1.1. CaSRThe
CaSR is widely expressed in
mammalian tissues (Brown & MacLeod, 2001). In endocrine cells including parathyroid chief cells and thyroid C-cells, it respectively inhibits
the secretion of parathyroid hormone
and promotes the secretion of calcitonin,
thereby reciprocally modulating two opposing regulators of whole body
calcium metabolism (Brown, 2007). In renal tubular
segments it modulates calcium, phosphate,
salt and water transport ([Ward & Riccardi, 2002] and [Vezzoli et al., 2009]). In the gastrointestinal tract, it is expressed
in epithelial cells of the stomach, small and large intestine, thereby promoting
digestion along with the absorption of macronutrients, micronutrients
and water (reviews: ([Conigrave & Brown, 2006] and [Geibel & Hebert, 2009])). It
is also expressed in enteroendocrine cells
in which it promotes the secretion of gut hormones including gastrin (Buchan et al., 2001) and possibly cholecystokinin (Hira et al., 2008), as well as neurones of the enteric nervous system (Chattopadhyay et al., 1998), thus
contributing to the control of intestinal motility (Geibel & Hebert, 2009). In
addition, the CaSR is expressed in
mesenchymal tissues and recent evidence suggests that it plays a key
role in the development of cartilage
and in bone homeostasis (Chang et al., 2008) (see below).The
CaSR is also expressed in the central nervous system (CNS) ([Ruat et al., 1995] and [Rogers et al., 1997]). Although
its roles in the CNS are not
well-defined, its expression is developmentally regulated and it
contributes to: (i) the central control of salt and water metabolism via
its expression in the subfornical organ
(Rogers et al., 1997); (ii) the
modulation of neurotransmission via adjustments in the activity of
non-selective cation channels (Phillips et al., 2008); and (iii)
normal CNS development including myelination ([Ferry et al., 2000] and [Chattopadhyay et al., 2008]) and
the generation of sympathetic nerves
(Vizard et al., 2008). During brain development and prior to the
formation of the blood brain barrier,
activation of the CaSR by
physiological levels of Ca2+, amino acids and/or other
activators may suppress excitatory postsynaptic currents to promote
neuronal growth and neurite
arborization.8.1.2. C6AThe recently cloned basic amino acid-sensing receptor C6A responds to arginine, lysine,
ornithine and various aliphatic amino acids as well as divalent cations
(reviews: ([Conigrave & Hampson, 2006]
and [Wellendorph et al., 2009a]). Like
the CaSR, C6A mRNA and protein are widely expressed
in tissues including the lung, kidney, liver,
spleen, heart, skeletal
muscle, brain and taste buds ([Kuang et al., 2005] and [Wellendorph et al., 2007]). In
consequence, its proposed roles are diverse and include regulation of
nutrient disposition and/or amino acid
metabolism (Wellendorph et al., 2005),
nutrient-dependent control of neurotransmission ([Kuang et al., 2005] and [Wellendorph et al., 2007]),
regulation of mesenteric blood flow (Harno et al., 2008), specialized
calcium- and amino acid-sensing in bone ([Pi et al., 2005] and [Pi et al., 2006]), and even the
detection of cell death (Civelli, 2005). C6A may also mediate some of the
long-recognized effects of basic amino acids on hormone secretion. Surprisingly, it has
proven difficult to express C6A
heterologously in cultured cells ([Kuang et al., 2005] and [Wellendorph et al., 2005])
raising the possibility that it forms heterodimers with a currently
unidentified partner(s).8.1.3.
T1R1/T1R3Three T1R taste receptors (T1R1,
T1R2, and T1R3) represent a well-defined subset
of receptors within the broad-spectrum amino
acid-sensing group. They form heterodimers in which T1R3
specializes as the signaling subunit to activate, respectively, PI-PLC and adenylyl
cyclase via the heterotrimeric
G-proteins α-gustducin and
α-transducin (reviews: ([Chandrashekar et al., 2006] and [Palmer, 2007]). T1R1
combines with T1R3 to form broad-spectrum l-amino acid
sensors, and T1R2 combines with T1R3 to form
sweet-taste sensors for simple sugars and d-amino
acids. T1R1/T1R3 amino
acid-sensing heterodimers contribute to the perception of
savoury taste (umami) in combination with purine
nucleotide enhancers including IMP
and GMP ([Li et al., 2002] and [Nelson et al., 2002]) and recent
mutational analysis and molecular modelling indicates that amino acids
and IMP bind at different sites in the extracellular domain of T1R1
(Zhang et al., 2008).Studies
of cellular expression in mouse
taste tissue indicate that T1R1 is expressed alone (i.e.,
without T1R3) in a significant number of cells in both
circumvallate and fungiform papillae (Kim et al., 2003) suggesting that
in some cell contexts it may operate either as homodimers or in
heterodimers with other members of GPCR
class C (e.g., with one or more mGlus).
T1R receptors also contribute to the coordination of nutrient digestion
and absorption in the small intestine,
operating in networks with other nutrient-sensing receptors as well as
transporters for peptides, amino
acids and monosaccharides (Mace et al., 2009). T1R2/T1R3
heterodimers, for example, promote the secretion of the
glucose-regulating hormone GLP-1
from enteroendocrine L-cells (Jang et al., 2007).8.2. Insights from global and
tissue-specific transgenic miceGlobal
and conditional null mice have been
described for several members of the broad-spectrum amino acid-sensing subgroup within GPCR class C. These mice are providing important insights into
the physiological significance of the receptors. However, mouse models need to be approached with
caution. In addition to differences between the mouse and other mammals, so-called ‘background’ genetic
differences between strains of M. musculus can modify phenotypic
outcomes. Furthermore, phenotypic differences can arise from the
strategy chosen to disrupt gene function. It is now apparent, for
example, that partial gene deletion strategies based on the targeting of
individual exons can lead to substantial diversity in the resulting
phenotype depending on the exon selected. This appears to be especially
true for the CaSR and, possibly, C6A (see below). Interestingly, the gene
structures of the cluster of broad-spectrum amino
acid-sensing receptors are strikingly similar (Fig. 4).Full-size image
(45K)
High-quality image
(336K)Fig. 4. Structural
similarities between the genes encoding the CaSR,
C6A, and T1R1 taste
receptors. The number of residues encoded by each exon is indicated
above the corresponding rectangles. Introns are depicted as lines
(sequence lengths shown in kilobases). Isoform 1 of C6A is shown together with transcript
variant 2 of T1R1. The exons that define the specific forms
of C6A and T1R1 shown are
depicted as grey boxes. 8.2.1.
CaSRMice homozygous for global disruption of
exon-5 of the CaSR gene, which
encodes residues 460–536 in the C-terminus of the human VFT
domain (www.casrdb.mcgill.ca), exhibit a phenotype
characterized by growth retardation, skeletal abnormalities, parathyroid hyperplasia,
markedly elevated serum Ca2+
and parathyroid hormone
concentrations as well as early death — features typical of untreated
neonatal severe hyperparathyroidism
in humans (Ho et al., 1995). Thus, the first
global CaSR-null mouse to be described, exhibited a
phenotype consistent with the effect of disabling a gene with a key,
non-redundant role in parathyroid Ca2+-sensing
and appeared to exclude other non-redundant roles for the CaSR. Consistent with this notion,
heterozygous CaSR-ex5+/− mice exhibited features consistent with familial hypocalciuric hypercalcemia in humans, which arises from the impact of
inactivating CaSR mutations on
calcium-sensing in the parathyroid
and kidney (Ho et al., 1995).More
recent work has led to a re-evaluation of CaSR
exon-5 as a target in transgenic mouse
studies. As a result of alternative splicing that induces in-frame
deletion of exon-5, various tissues express a shortened form of the CaSR with a modified glycosylation pattern ([Oda et al., 1998] and [Oda et al., 2000]). The exon-5
minus CaSR traffics normally to the cell membrane in growth plate chondrocytes
prepared from CaSR-ex5−/− mice and retains receptor function as
revealed by high extracellular Ca2+-stimulated inositol phosphate turnover (Rodriguez et al., 2005). These
surprising findings indicate that the exon-5 null mouse is an “incomplete knock-out”, which
retains significant residual CaSR
function, providing a theoretical basis for the development and
investigation of alternative CaSR-null
mice in which other exons are
targeted. These include exons required for the formation of the
nutrient-binding, bilobed VFT domain
(exons 2–4), the Cys-rich domain (exon-6) or the entire HH domain and
C-terminal domain (exon-7). In response to this need, several new transgenic mouse strains have been
developed from tissue-selective ablation of exon-7 in the following
tissues (promoter in parentheses): parathyroid
(PTH), osteoblasts
(Col(I) α1-subunit or Osterix) and growth plate chondrocytes
(Col(II) α1-subunit) (Chang et al., 2008). These new transgenic mice have transformed our
understanding of the physiological breadth of the non-redundant roles
played by the CaSR.As
expected, parathyroid-specific
ablation of CaSR exon-7 resulted in a
phenotype consistent with neonatal primary
hyperparathyroidism with no effect on birth length or weight but
marked postnatal growth retardation associated with skeletal
under-mineralization and hypercalcemia
(Chang et al., 2008). The phenotype
observed in these mice resembled
that of the global exon-5 null mouse
(Ho et al., 1995). Surprisingly, mice homozygous for growth plate chondrocyte-specific
ablation of CaSR exon-7, exhibited a
much more severe phenotype in which all progeny died in utero,
typically prior to embryonic day 12 (Chang et al., 2008). In addition, osteoblast-specific ablation resulted in a
major skeletal phenotype exhibiting postnatal growth retardation and
under-mineralization of the skull
and long bones. On micro-CT analysis
these mice exhibited markedly
reduced femoral trabecular number and volume and analysis of gene
expression revealed impaired expression of early and late markers of osteoblast differentiation, as well as
marked alterations in the expression of key growth and survival factors (Chang et al., 2008). In
particular, in osteoblast-specific, CaSR exon-7 null mice there was impaired expression of insulin-like growth factor-1 (IGF-1) and prosurvival factors including Bcl2.The phenotype of the osteoblast-specific CaSR-null mouse
is at variance with predictions based on previous studies of Gcm2/CaSR-ex5
(Tu et al., 2003) and PTH/CaSR-ex5
(Kos et al., 2003) double null mice in which the skeletal effects of
global exon-5 ablation were found to depend on PTH gene expression and associated primary hyperparathyroidism secondary to
defective Ca2+o-mediated feedback. These
discrepancies underscore the incomplete nature of the exon-5 knock-out
strategy. The results from the Col (I) and Osterix-directed
exon-7 null mice suggest that the CaSR plays a key role in skeletal development by up-regulating the osteoblast-dependent expression of
autocrine and paracrine growth and survival factors (Chang et al., 2008) and, together
with the findings for the Col (II)-directed exon-7 null mouse, demonstrate the existence of
physiologically critical, PTH-independent
roles for the CaSR in the
development and homeostasis of cartilage and bone.Are the multi-metabolic
sensing functions of the CaSR
including sensitivities to amino acids, ionic strength, pH and
temperature ([Conigrave et al., 2000a] and [Breitwieser et al., 2004]) or the
demonstrations of roles for the CaSR
in neurotransmission and neuronal development ([Phillips et al., 2008] and [Vizard et al., 2008])
physiologically significant or redundant i.e., largely supported by
other mechanisms? Evaluation of the wider physiological significance of CaSR expression in the kidney, gastrointestinal
tract, brain and other sites
awaits the generation of new tissue-selective CaSR-null mice.
Furthermore, recent work suggesting that the CaSR suppresses branching morphogenesis in
the lung prior to the later stages
of fetal development (Finney et al., 2008) indicates a
need for transgenic mice in which
there is developmental as well as tissue-specific manipulation of CaSR expression.8.2.2. C6AThe
recent generation of global exon-2 and exon-6 null mice in two separate studies has provided
opportunities to evaluate the non-redundant functions of C6A ([Pi et al., 2008], [Wellendorph et al., 2009b] and [Wellendorph et al., 2009a]).
Exon-2 encodes residues 66–167 of the mouse
ortholog of C6A (Kuang et al., 2005) and should
disable nutrient sensing by the VFT
domain. Exon-6, on the other hand, encodes the entire HH domain and
C-terminal domain and should eliminate the receptor's signaling function
([Kuang et al., 2005] and [Wellendorph et al., 2007]).
Surprisingly, two very different outcomes have been reported ([Pi et al., 2008] and [Wellendorph et al., 2009b]). In
the case of the exon-2 null mouse, a
multi-system phenotype was reported featuring normal body weight but
increased fat mass, decreased lean body mass, hyperglycemia and insulin resistance, proteinuria, renal calcium and phosphate wasting, impaired bone mineral density and defective
testicular function resulting in low serum
testosterone levels (Pi et al., 2008). Remarkably, in
the case of the C6A exon-6 null mouse, however, no phenotypic disturbance
was observed (Wellendorph et al., 2009b). If the
disparity is real, the results suggest that the nutrient-sensing
function of exon-2 is critical to the processing of normal metabolic
signals arising from basic amino acids and/or divalent cations but the
signaling function of exon-6 is redundant. Such a situation might arise
if, as suggested above, C6A normally
operates in heterodimers with an, as yet unidentified, signaling
partner. If this is the case, the cloned C6A
subunit might be analogous to the T1R1 and GABAB1 subunits whose
heterodimeric partners are T1R3 and GABAB2 respectively.8.2.3. T1R1/T1R3Studies
on T1R1 global null mice
generated by targeted deletion of the HH and C-terminal domains (Zhao et al., 2003), or T1R3
global null mice generated by
deletion of the N-terminal extracellular domain (Zhao et al., 2003) or entire gene
locus (Damak et al., 2003) support the
hypothesis that T1R1/T1R3 heterodimers form
broad-spectrum l-amino acid receptors, which contribute to
umami taste. Importantly, responses to l-glutamate
and other amino acids were impaired in both the absence and presence of
the umami enhancing agents IMP and GMP
([Damak et al., 2003] and [Zhao et al., 2003]). On the other
hand, evidence for residual glutamate sensing in T1R3 null mice (Damak et al., 2003), points to
roles for additional receptors (e.g., mGlu-4)
in umami sensing (Maruyama et al., 2006). The
potential physiological significance of T1Rs in coordinating intestinal
responses to nutrients has not yet been addressed in transgenic mouse models.9. ConclusionsAs we get to know
them better, Class C GPCRs are
presenting no shortage of surprises. For these receptors, multi-ligand
sensing and multi-modal signaling is the norm. Nutrient molecules, once
the preserve of biochemists and nutrition scientists, are also now of
great interest to molecular pharmacologists as well as cell and systems
physiologists. The design of the receptors is deceptively simple and
highly conserved but, depending on the cell in which they are expressed,
their preferences for nutrients, binding partners, modes of operation
and physiological roles are impressively plastic. The subgroup of
broad-spectrum amino acid sensors
including the CaSR, C6A and T1R1/T1R3
receptors have roles in nutrient sensing that contribute to taste,
control of appetite and satiety, as well as coordination of intestinal
digestion, absorption and nutrient disposition. These receptors are also
potentially important drug targets with strategies now defined for
their development. Stay tuned!
AcknowledgmentsThe authors
thank Mr Sujeenthar Tharmalingam for his assistance in preparing the
diagrams and also wish to thank the National Health and Medical Research
Council of Australia (ADC and DRH) and the Canadian Institutes for
Health Research and the Canadian Natural Sciences and Engineering
Research Council (DRH) for research support.
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