NUTRITIONAL GENOMICS
IN PROTEIN RESEARCH
2009

INVESTIGATIVE REVIEW
HUMAN IN VIVO CLINICAL TRIALS

THE SCIENCE OF TRANSCRIPTION
PATHWAYS IN PROTEIN METABOLISM

DIRECTOR OF CLINICAL INVESTIGATIONS
PROTEIN RESEARCH PROJECT
Dr. Ann de Wees Allen
Chief of Biomedical Research
Glycemic Research Institute
Washington, D.C.


ABSTRACT

In August 2009, a grant was provided, and clinical investigation was initiated in order to determine the Mechanisms of Action and Clinical Perimeter as well as the Pathobiochemical and Clinical Chemical Aspects of a recently developed form of protein designed to encompass the principles of Nutritional Genomics.

Wherein said composition was submitted to the Glycemic Research Institute® for independent clinical trial analysis, under the government Certification program, and identified as a Protein-Glycoside-Matrix (herein the “Test Food”);

The final Investigative Trials Report will summarize the clinical findings related to the Test Food, which include Glycemic Index and Load, Diabetic Index, Adipose Fat-Storage Index, LPL, protein synthesis and metabolism, bioactivity, contractile proteins, anti-ketogenic mechanism, postprandial utilization of dietary nitrogen, protein availability, protein signaling, and transcription and translation pathways.

Board Approved Human In Vivo Clinical Trials, as well as Analytical Laboratory Trials, including High-Performance Anion Exchange Chromatography, will be conducted by the Glycemic Research Institute® and provided as a Addendum to the report.

DESCRIPTION OF THE TEST FOOD

The Test Food submitted is defined as “A Protein-Glycoside-Matrix comprised of complete biological molecules in a stable conformation that are assembled from amino acids using information encoded in genes. Each protein has its own unique amino acid sequence that is specified by the nucleotide sequence of the gene encoding this protein. The Protein-Glycoside-Matrix (PGM) was further identified as “A molecular network comprised of glycoside molecules bound to specific proteins” that is protected by a United States Patent.

Upon analytical assay of the ingredients and compound, this description is determined to be technically and scientifically accurate, and the Test Food is determined to be safe for use in humans, which qualifies for participation in Human In Vivo Clinical Trials in Diabetics and Non-Diabetics.

METABOLIC DYNAMIC & PROPERTIES

The Test Food, Protein-Glycoside-Matrix, contains biologically active components bound to protein molecules. Biologically active refers to natural compounds that affect the life processes of a human organism in a beneficial manner. The metabolic dynamic of the Protein-Glycoside-Matrix (PGM) composition appears to be multifaceted, and includes the stimulation of muscle protein synthesis (MPS) which provides positive net protein balance, resulting in hypertrophy, in an Anti-Ketogenic Matrix.

The glycosides in Protein-Glycoside-Matrix (PGM) lengthen the biological life of a protein molecule by decreasing the protein's rate of clearance from the blood. Additionally, the Protein-Glycoside-Matrix (PGM) compound helps the protein to fold properly, to stabilize the protein molecules, and to provide bioactivity.

The hypertrophic advantages in muscle-mass are evidenced in response to acute administration (short-term), and is particularly evidenced when administered orally over a chronic term (long-term).

Hypertrophy is defined as the increase in the volume of an organ or tissue due to the enlargement of its component cells. Muscle Hypertrophy is the increase in muscle tissue evidenced in response to stimuli.

One of the two most common and visible forms of organ hypertrophy occurs in skeletal muscles in response to strength training (known as muscle hypertrophy), and in response to oral ingestion of contractile proteins.

Contractile proteins participate in contractile processes, and include muscle proteins as well as those found in other tissues and cells in the body. These proteins partipate in localized contractile events in the cytoplasm, in motile activity, and in cell aggregation.

Oral administration of the Protein-Glycoside-Matrix results in significant gains in contractile protein content and muscle strength in humans. Additionally, a dose of 10 grams of protein enhances the inhibition of protein breakdown.

IMPROVED POST-PRANDIAL UTILIZATION OF DIETARY NITROGEN

Whey proteins, including isolates, as well as most anabolic proteins, are considered “Fast Proteins.” Fast Proteins are contraindicated in the anabolic state, as they induce over-rapid delivery of amino acids, which cannot support the anabolic requirement throughout the post-prandial period.

Slowly digested proteins (Slow Proteins) induce superior post-prandial utilization of dietary nitrogen than rapidly digested protein (Fast Proteins), despite the high chemical score of MSPI. The Test Food (Protein-Glycoside-Matrix) appears to metabolically switch whey proteins from acting like Fast Proteins to acting like Slow Proteins.

TRANSCRIPTION PATHWAY

Protein-Glycoside-Matrix (PGM) contains Patented anabolic amino acids and proteins, including natural Branched Chain Amino Acids (BCAAs), which are used to fuel working muscles and stimulate protein synthesis. The essential amino acids, found in Protein-Glycoside-Matrix (PGM), combined with glucose availability, is sensed by both AMPK and mTOR in human muscle, which enhances AKT/mTOR signalling to key regulators of translation initiation and elongation, thus inducing a potent and rapid increase in the rate of muscle protein synthesis.

In humans, rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are important nutrient and energy-sensing and signalling proteins in skeletal muscle. AMPK activation decreases muscle protein synthesis by inhibiting mTOR signalling to regulatory proteins associated with translation initiation and elongation.

The specific amino acids found in Protein-Glycoside-Matrix (PGM) stimulate mTOR signalling and protein synthesis. These anabolic nutrients are sensed by both AMPK and mTOR, resulting in an acute and potent stimulation of human skeletal muscle protein synthesis via enhanced translation initiation and elongation. The Protein-Glycoside-Matrix (PGM) compound plays a key role in initiating the transcription pathway that fires up protein synthesis, which stimulates protein synthesis and aids in speeding recovery and adaptation to stress (exercise).


KETOSIS & CORTISOL: NEGATIVE METABOLIC STATES

Ketosis (from the root word ketones) is produced in the liver through the incomplete breakdown of fat. Unlike other proteins, the Test Food, Protein-Glycoside-Matrix (PGM) exhibits Non-Ketogenic properties. This is an extremely important facet in protein formulating, due to the negative physical events triggered by both Ketosis and Cortisol.

Ketosis is a buildup of partially broken-down fats (ketones) in the bloodstream and may occur if less than 50-100 grams of carbohydrates are consumed each day, or if protein is consumed without carbohydrates. As a result of ketone build-up in the blood, the body may produce high levels of uric acid, which is a risk factor for developing gout and kidney stones.

For pregnant women and people with diabetes or kidney disease, ketosis is especially dangerous.

SPARING PROTEINS & PREVENTING KETOSIS

Research has demonstrated that proteins and protein products that do not contain any carbohydrates, just protein, are ketogenic. This is metabolic suicide for protein metabolism and homeostasis. Proteins without carbohydrates instigate a ketogenic state in humans.

Carbohydrates are mandatory in protein synthesis

Maintaining a regular intake of carbohydrates will prevent protein from being used as an energy source. Further, an adequate amount of carbohydrates prevents the degradation of skeletal muscle and other tissues such as the heart, liver, and kidneys. The Test Food, Protein-Glycoside-Matrix, allows gluconeogenesis to slow down, allowing amino acids to be freed for the biosyntheses of enzymes, antibodies, receptors and other important proteins. More importantly, it prevents ketosis.

Although the brain will adapt to using ketones as a fuel, it preferentially uses carbohydrates and requires a minimum level of glucose circulating in the blood in order to function properly. Before the adaptation process occurs, sub-optimal hypoglycemic blood glucose levels typically cause headaches in response to a ketogenic state.

Although the processes of protein degradation and ketosis can create problems of their own during prolonged fasting, they are adaptive mechanisms during glucose shortages. The first priority of metabolism during a prolonged fast is to provide enough glucose for the brain and other organs that dependent upon it for energy in order to spare proteins for other cellular functions.

The second priority of the body is to shift the use of fuel from glucose to fatty acids and ketone bodies. From that point on, ketones significantly become more important as a source of fuel, while fatty acids and glucose become less important.

To prevent these ketotic symptoms, it is recommended that the average person consume at least 50 to 100 g of carbohydrates per day, and when ingesting dietary protein, include an appropriate ratio of protein-to-carbs symbiotic to protein synthesis.

KETOSIS CAUSES BODY FAT GAINS & SLOWS METABOLISM

High protein and/or low carbohydrate diets slow down metabolism. During high protein and/or low carbohydrate diets, weight loss may be realized but the type of weight that is actually lost includes muscle mass as well as fat. When muscle mass is reduced in humans in response to high protein and/or low carbohydrate diets, fat-burning slows down and metabolic rates decrease. This results in increased body fat and decreased lean muscle mass.

With reduced lean body mass, resting metabolic rates decrease since skeletal muscle requires more energy at rest and during exercise than adipose fat tissue. Diets and protein supplements that contain adequate Low Glycemic carbohydrates not only increase physical well being, but also contribute to body fat loss and maintenance of lean muscle mass.

KETOSIS & SPORTS PERFORMANCE

The Test Food, Protein-Glycoside-Matrix, was specifically formulated to provide a Metabolic Advantage to athletes. One of the components relative to this claim is the Anti-Ketogenic properties of the Test Food.
In athletes, Ketosis has been proven to:
• Reduce brain capacity and function
• Increase body fat
• Decrease lean muscle mass


Ketosis is one of the most severe and negative states in protein synthesis and utilization, brain function, and fat-burning. Some classes of dietary amino acids are gluconeogenic, meaning glucose producing, while many amino acids are ketogenic and cannot enter the citric acid cycle.

Thus, the level of ketones in the body rises while levels of glucose fall, and many organs begin to function less efficiently, including the brain, which relies heavily on glucose. Proteins that do not contain carbohydrates exacerbate brain-focus-reduction, reducing mental focus and sports performance.

As an athlete’s performance and career ultimately depend on fast-brain-response, this athletes cannot afford reduced-sports-performance due to ketogenic proteins or diets. Neither the athlete nor the non-athlete can afford reduced-brain-function, or disrupted protein and fat metabolism.

In terms of added body fat and energy-synthesis, fats can only be metabolized (burned) when there is a adequate amount of glucose from carbohydrates present to produce oxaloacetate, which condenses with acetyl CoA in the citric acid cycle. Since fatty acids are degraded directly to acetyl CoA, they cannot be used as an energy source, and can be transformed in ketones.

High levels of ketones in the blood stream are dangerous, and low amounts of glucose (from dietary carbohydrates) in the blood can be detrimental to the brain and to sports performance.

PROTEIN THRESHOLD:
STORAGE CAPACITY OF PROTEINS

The storage capacity of protein in humans has been well established. Storage capacity relates to the maximum amount of protein the human body can process without negative consequences. In World-Class powerlifters, who weigh up to 400 pounds, the storage capacity of proteins does not exceed 30 grams within a 2-3 hour period.

In World-Class bodybuilders, such as Mr. Universe, who hold huge amounts of muscle mass, and low amounts of body fat, regardless of weight or size, or calories burned, or degree of muscle mass, the 30-gram protein rule does not change. The average, non-athlete does not require an intake of 30 grams of protein at one time, and can achieve protein homeostasis by ingesting specific forms and amounts of elemental protein throughout the day.

In large-size competing athletes, the homeostasis-dose of protein is 30 elemental grams. The 30-gram protocol should not be used in non-athletes, as this dose of protein cannot be metabolized or utilized in the body without benefit of high-muscle mass, as well as high-energy-output and high-caloric-burning. Using proteins at the 30-gram dose in an inappropriate ketogenic formula/product can cause serious medical problems, including ketosis, elevated liver enzymes, and liver damage.

In humans, ingestion of ketogenic proteins and meal replacement products results in increased body fat levels via elevation of insulin and LPL fat-storage in adipose tissue fat cells. Ketogenic protein drinks and meal replacements are contraindicated.

Additionally, protein drinks and products that only contain sugar alcohols, synthetic sweeteners, and other non-carbohydrate ingredients, do not achieve health protein storage capacity, and can cause ketosis and liver problems.

In terms of timing, protein drinks should not be consumed near bedtime, as this causes lowered Delta-stimulated GH and testosterone production.

PATHOLOGY OF CORTISOL’s MEDICAL IMPACT
IN HUMAN HEALTH

Cortisol is a corticosteroid hormone. The synthesis of Cortisol in the adrenal gland is stimulated by the anterior lobe of the pituitary gland. The highest levels of Cortisol in humans occur in early morning, with lowest levels in the evening and 2-3 hours following the onset of the sleep cycle.

Over-expression and over-elevation of Cortisol in humans is triggered by a variety of external biochemical events resulting in mild-to-serious medical problems, including reduction of sports performance in athletes, and excess adipose tissue body fat.

The down-regulation of Cortisol is an obvious advantage in protein metabolism, and the Test Food under investigation will provide substantiation of Cortisol-down-regulation. As of September 2009, no prior substantiated evidence of proteins that down-regulate Cortisol have been introduced.

CLAIMS SUBSTANTIATION

The Test Food duly submitted to the Glycemic Research Institute®, Protein-Glycoside-Matrix (PMG), is described as an advanced protein delivery system, that is undergoing independent government Certification Human In Vivo Clinical Trials, with the goal of clinical substantiation of metabolic properties and legal claims.

The proprietary PMG formula was designed to mitigate and/or eliminate blood glucose and insulin excursions in humans caused by ingestion of High Glycemic proteins, thus blunting Lipoprotein Lipase (LPL) fat-storage, adipose tissue fat-storage, body fat weight gains, and stimulation of fat cell replication.

In addition, PMG was designed to replace ketogenic protein supplements, and meal replacements that activate Cortisol and GLP-1.

The Test Food will be recommended in the following human health areas:
• To achieve protein homeostasis
• As a protein supplement and/or meal replacement
• As an adjunct to promote lean muscle mass in active persons
• As part of a healthy weight management program
• For professional and non-professional athletes
• In post-gastric-surgery patients
• In catabolic medical conditions
• In type 2 diabetics for glucose control
• In insulin-resistant individuals
• As an adjunct to anexoria medical treatment to prevent catabolism







Protein Synthesis
Transcription & Translation

Illustrations and Data Provided by
www.ChemistryExplained.com
2009





Protein Synthesis
Transcription & Translation

The most important facet of the function of living cells is the synthesis of proteins. Because proteins carry out multiple tasks in the body, the mechanism to synthesize them is highly intricate.

Despite the overall complexity of this process, it occurs with remarkable accuracy. The rate of error is roughly one in every 10,000 amino acids. Using the processes of transcription and translation, the body makes an amazing number and variety of proteins.

The transcription and translation processes provide
the correct primary structure of the protein

The protein must fold to obtain the correct secondary and tertiary structures. Protein folding remains an active research area.

There are several stages involved in the synthesis process, including transcription and translation.

The illustration above (Protein Synthesis) shows the process whereby DNA encodes for the production of amino acids and proteins.

This process can be divided into two parts:

1. Transcription

Before the synthesis of a protein begins, the corresponding RNA molecule is produced by RNA transcription. One strand of the DNA double helix is used as a template by the RNA polymerase to synthesize a messenger RNA (mRNA). This mRNA migrates from the nucleus to the cytoplasm. During this step, mRNA goes through different types of maturation including one called splicing when the non-coding sequences are eliminated. The coding mRNA sequence can be described as a unit of three nucleotides called a codon.

The primary role of deoxyribonucleic acid (DNA) is to direct the synthesis of proteins. DNA, however, is located in the nucleus of the cell, and protein synthesis occurs in cellular structures called ribosomes, found out-side the nucleus. The process by which genetic information is transferred from the nucleus to the ribosomes is called transcription. During transcription, a strand of ribonucleic acid (RNA) is synthesized. This messenger RNA (mRNA) is complementary to the portion of DNA that directed it: as it has a complementary nucleotide at each point in the chain.

A specialized protein called an enzyme controls when transcription occurs. The enzyme called RNA polymerase is present in all cells; eukaryotic cells have three types of this enzyme. DNA has a section called the promoter region that identifies the sites where transcription starts and must be recognized by one subunit of the RNA polymerase called the sigma (s) factor. Recognition between the promoter and the s-factor helps to regulate how often a particular gene is transcribed. Once bound, the polymerase initiates the construction of mRNA (or other RNA molecules).

Initiation of the synthesis of a new RNA molecule does not always lead to a complete synthesis. After roughly ten nucleotides have been strung together, the continued addition of complementary base pairs takes place more readily in a process called elongation. The speed of addition of new nucleotides is remarkable—between twenty and fifty nucleotides per second can be added at body temperature.

Eventually the elongation process must stop. There are certain sequences of nucleotides that stop elongation, a process called termination. Often, termination occurs when the newly formed section of RNA loops back on itself in a tight formation called a hairpin. Once the hairpin structure has formed, the last component is then a string of uracil residues.

After transcription has taken place, the mRNA produced is not necessarily ready to direct the subsequent protein synthesis. Depending on the type of cell, segments of nucleotides may be removed or appended before the actual synthesis process takes place. This type of post-transcriptional processing often occurs in human cells.

2. Translation

The ribosome binds to the mRNA at the start codon (AUG) that is recognized only by the initiator tRNA. The ribosome proceeds to the elongation phase of protein synthesis. During this stage, complexes, composed of an amino acid linked to tRNA, sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the tRNA anticodon. The ribosome moves from codon to codon along the mRNA. Amino acids are added one by one, translated into polypeptidic sequences dictated by DNA and represented by mRNA. At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome. One specific amino acid can correspond to more than one codon. The genetic code is said to be degenerate.

Once the mRNA has been synthesized, and perhaps modified, the next step of protein synthesis, translation, takes place. For this stage, additional forms of RNA are needed.

Transfer RNA (tRNA) plays the role of carrying an amino acid to the synthesis site at the ribosome. tRNA molecules are relatively small, with around seventy-five nucleotides in a single strand. They form several loops, one of which is an anti-codon, a three-residue series that is complementary to the codon present in the mRNA.

The opposite end of the tRNA is where an amino acid is bound. The correct binding of an amino acid to a specific tRNA is every bit as important as the anti-codon in ensuring that the correct amino acid is incorporated in the polypeptide that is synthesized.

There are different tRNA molecules for each of the twenty amino acids that are present in living systems; some amino acids have more than one tRNA that carry them to the synthesis site.

When translation begins, mRNA forms a complex with a ribosome to form an assembly site. This complex requires the assistance of proteins called initiation factors, so the existence of an mRNA does not mean that a protein will always be synthesized. The first tRNA that takes part in the initiation always carries the same amino acid, methionine. When the protein is completely synthesized, this initial methionine is often removed.

With the initial methionine in place, another tRNA with its amino acid joins the assembly site as dictated by the codon on the mRNA. With two amino acids present, a peptide bond can be formed and the polypeptide can begin forming.

The new amino acid is added to the carbon end of the polypeptide (the C-terminus) with the peptide bond forming between the C-O of the polypeptide and the amine of the new amino acid. This structural specificity is enforced by the nature of the binding between the amino acid and the tRNA. The portion of the amino acid that is unbound in the tRNA complex is the amine.

Elongation ultimately requires the repetition of several steps:

(1)
The tRNA–amino acid complexes must be made.
(2)
This complex must bind to the mRNA-ribosome assembly site. The correct amino acid is assured by the matching of the anti-codon on the tRNA to the codon on the mRNA.
(3)
A peptide bond is formed between the new amino acid and the growing polypeptide chain.
(4)
The amino acid is cleaved from the tRNA, which can be cycled back to form another complex with an amino acid for a later synthesis.
(5)
The growing polypeptide forms a fiber-like tendril.
(6)
The ribosome essentially moves along the mRNA, reopening the initiation site for additional protein synthesis. In this way, proteins are synthesized by several ribosomes acting on the same mRNA molecule.


The structure of the ribosome plays an important role in this elongation process. There must be two sites available for synthesis to occur. One site, called the P site (for peptide), is where the growing (or nascent) polypeptide is located. Adjacent to this location is another site where the tRNA with its new amino acid can bind.

This site is called the A site (for the amino acid that is delivered there along with the tRNA).

As was the case in the elongation of mRNA noted earlier, somehow the emerging polypeptide must stop adding amino acids. The termination is actually part of the coding present in the codons. Three specific codons are known as stop codes, and when they are present in mRNA, the elongation is stopped.





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