Trait: GNB3 and cardiometabolic health
Dr Haran Sivapalan
November 22, 2021
What are G-protein coupled receptors (GPCRs)?
Cells need a way of responding to a variety of signals from their external environment, such as from hormones, neurotransmitters, nutrients, and light.
In order to do this, several living organisms (including animals, plants, and fungi) have evolved specialised receptors on their outer cell membranes, known as G-protein coupled receptors (GCPRs).
As their name suggests, GCPRs are linked or coupled to specialised proteins in the cell membrane called G-proteins.
When an external signal molecule (say, a hormone) binds to a GCPR, it activates the G-protein. The activated G-protein then goes on activate various other molecules (e.g. enzymes) inside the cell, triggering a cascade of chemical reactions known as an intracellular signalling cascade.
The end point of the signalling cascade are target proteins or genes that bring about a cell response. For instance, certain genes may be switched on or an ion channel will be opened. The overall process by which an external signal is transmitted to the interior of a cell to cause a response is known as signal transduction.
Source: Fu, J., Lee, T., & Qi, X. (2014). The identification of high-affinity G protein-coupled receptor ligands from large combinatorial libraries using multicolor quantum dot-labeled cell-based screening. Future medicinal chemistry, 6(7), 809-823.
A good illustrative example of signal transduction by GCPRs is how the hormone adrenaline / epinephrine (a signal) causes our heart rate to increase (a response).
Heart muscle cells have β-adrenergic receptors on their surface, which are a type of GPCR. When adrenaline binds to a β-adrenergic receptor, it activates a G-protein. The activated G-protein then triggers a signalling cascade, in which different enzymes are stimulated, resulting in the opening of calcium ion channels. The influx of calcium ions into heart muscle cells then causes them to contract more quickly, leading to an increase in heart rate.
Source: Klabunde, R. (2011). Cardiovascular physiology concepts. Lippincott Williams & Wilkins.
We have almost a thousand different GPCRs, with each one being specific to a particular signal. Colletively, as GPCRs can bind to variety of ligands/signals and activated G-proteins can stimulate many different types of molecules in signalling cascades, several different processes in the body rely on GCPR signalling.
- Sensation: for example, our sense of smell relies upon odorant particles binding to olfactory receptors in our nose. These olfactory receptors are GCPRs.
- Immune responses and inflammation: Various immune cells have GCPRs that are activated by signalling molecules called cytokines. This allows for the coordination of inflammatory and immune responses.
- Control of metabolism: GPCRs play key roles in various metabolic processes. For example, glucagon, which acts to increase blood glucose levels, acts via the glucagon receptor (a GPCR) on the surface of liver cells.
- Hormonal responses: Lots of different hormones e.g. FSH (follicle-stimulating hormone), TSH (thyroid-stimulating hormone), oxytocin, all exert their effects via GPCRs.
- Cell growth: GPCRs are involved in transmitting signals causing cells to proliferate.
- Control of blood pressure: Hormones that regulate blood pressure by altering kidney function (e.g. angiotensin) and altering the dilation/constriction (e.g. adrenaline) exert their effects using GCPRs.
Source: Gutkind, J. S. (1998). The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. Journal of Biological Chemistry, 273(4), 1839-1842.
G-protein coupled receptors (GPCRs) in closer detail
G-proteins are made of three different subunits: α, β and γ. The function of these three subunits changes depending on whether the GPCR and G-protein are activated by a signal or inactive.
When the G-protein is inactive, the three subunits are joined together. The inactive α subunit binds a molecule called GDP (an important energy-rich molecule similar to ADP). In its inactive state and without a signal, the whole G-protein complex is anchored to the G-protein coupled receptor (GPCR).
Source: Wiseman, D. N., Otchere, A., Patel, J. H., Uddin, R., Pollock, N. L., Routledge, S. J., ... & Goddard, A. D. (2020). Expression and purification of recombinant G protein-coupled receptors: A review. Protein expression and purification, 167, 105524.
When a signal molecule (ligand), such as a hormone, binds to the GPCR, it triggers a change in the shape of the receptor. This activates the G-protein.
On activation, the α subunit of the G-protein exchanges GDP for GTP. GTP is similar molecule to ATP in that it powers chemical reactions in cells. When the α subunit binds GTP, it causes the entire G-protein to dissociate from the GPCR.
At the same time, the α subunit dissociates from the β and γ subunits. This frees up the α subunit, allowing it to activate other molecules (e.g. enzymes, ion channels) in an intracellular signalling cascade. Similarly, the β and γ subunits, although remaining together as a complex, are also free to stimulate other molecules in a signalling cascade. These signalling cascades will eventually lead to and cause cell responses e.g. the switching on of certain genes, muscle contraction, transmission of an electrical signal.
To “switch off” the G-protein, GTP is converted back to GDP. This causes the α, β, and γ subunits to reassociate, and the entire G-protein to reconnect with the GPCR.
How are GPCRs involved in the regulation of cardiometabolic health?
Cardiometabolic health is a term that encompasses having healthy:
- Blood pressure.
- Blood sugar levels and insulin sensitivity.
- Blood lipid levels (including blood cholesterol and triglyceride levels).
- Amount of visceral fat and waist circumference.
Ensuring these parameters are within healthy ranges can protect us from cardiometabolic diseases – the name given to a spectrum of conditions including insulin resistance, obesity, heart attack, stroke, Type II diabetes, and high blood pressure.
GPCRs and GPCR signalling are heavily involved in several aspects cardiometabolic health. The regulation of blood pressure, for example, relies upon GPCRs.
As discussed in the Angiotensin II level trait article, a hormone called angiotensin II acts to increase blood pressure by causing vasoconstriction (narrowing of blood vessels) and greater reabsorption of sodium and water into the blood by our kidneys. These effects of angiotensin II (Ang II) result from the activation of angiotensin receptors (AT1 and AT2), which are G-protein coupled receptors (GCPRs).
Interestingly, drugs that block these receptors are used in the treatment of high blood pressure (hypertension).
Source: Nguyen Dinh Cat, A., Montezano, A. C., & Touyz, R. M. (2013). Renin–angiotensin–aldosterone system: new concepts. Future Medicine Ltd.
Another hormone that acts to regulate blood pressure via vasoconstriction and vasodilation is adrenaline (also known as epinephrine). When adrenaline binds to α1 receptors (which are GPCRs) on smooth muscle cells surrounding arteries, it causes them to constrict. This has the effect of increasing blood pressure.
The control of blood glucose and lipid levels is also reliant upon GPCRs. The secretion of insulin, for example, a key hormone that allows tissues to take up glucose from the bloodstream, can either be enhanced or inhibited by GPCR signalling. For instance, GLP-1 (glucagon-like peptide 1) is an incretin hormone released by the gut that acts to enhance insulin secretion. It has this effect by acting on a GPCR, the GLP-1 receptor.
By playing important roles in the regulation of blood pressure, blood glucose levels, blood lipid levels, and bodyweight, GPCR-signalling has a profound effect on our cardiometabolic health.
As we’ll discuss in the following sections, gene variants that alter GPCR-signalling can affect our risk of cardiometabolic diseases such as high blood pressure (hypertension) and obesity.
What is the GNB3 gene?
The GNB3 gene encodes the G-protein beta-3 subunit.
As explained in the G-protein coupled receptors (GPCRs) in closer detail section, G-proteins are made up of three different subunits: α, β and γ. The GNB3 gene codes for a form of the β subunit. It therefore plays an important role in G-protein activity once a GPCR is activated by a signal (e.g. a hormone).
On this note, variants of the GNB3 gene are shown to affect the activity of the β subunit, with one variant linked to greater G-protein activation and GPCR-signalling. We’ll discuss this in the following section.
How do GNB3 variants affect GPCR-signalling?
A SNP (Single Nucleotide Polymorphism), rs5443, within the GNB3 gene causes a C -->T change in the DNA code. This creates two different GNB3 gene variants or ‘alleles’: the ‘C’ allele and the ‘T’ allele.
Whereas the ‘C’ allele codes for a normal G-protein beta-3 subunit molecule, the ‘T’ allele codes for a different variant (known as a splice variant) of the beta subunit protein, one which lacks 41 amino acids and has higher activity.
More specifically, studies show that this splice variant of the G-protein beta-3 subunit (encoded by the ‘T’ allele) causes greater G-protein activation in response to signals. This enhances signalling in ‘T’ allele carriers when GPCRs are activated by signals such as hormones, leading to greater cell responses.
For example, adrenaline (a signal) binding to adrenergic receptors (a type of GPCR) on the surface of platelets triggers an intracellular signalling cascade that causes the platelets to aggregate (a cell response).
This platelet aggregation response in response to adrenaline is shown to be significantly greater in ‘T’ allele carriers compared to non-carriers.
Similarly, activation of the alpha-2-adrenergic receptor (another GPCR) causes vasoconstriction (narrowing) of the coronary arteries (the blood vessels supplying the heart).
In one study, researchers injected a compound (called BHT-933) that activates these alpha-2-adrenergic receptors and then assessed blood flow through the coronary arteries. They found that, compared to non-carriers, ‘T’ allele carriers had a greater degree of vasoconstriction, suggestive of enhanced GPCR-signalling. This is illustrated in the scatter plot below (‘T’ allele carriers are those with TC or TT genotypes).
Source: Baumgart, D., Naber, C., Haude, M., Oldenburg, O., Erbel, R., Heusch, G., & Siffert, W. (1999). G protein β3 subunit 825T allele and enhanced coronary vasoconstriction on α2-adrenoceptor activation. Circulation research, 85(10), 965-969.
On a related note, ‘T’ allele carriers are also shown to have enhanced GPCR-signalling and vasoconstriction in response to endothelin, angiotensin II and noradrenaline binding to their respective G-protein coupled receptors. All these hormones are involved in the regulation of blood pressure.
Given that GPCRs play key roles in the regulation of cardiometabolic health, we might expect enhanced GPCR-signalling to affect cardiometabolic health outcomes in ‘T’ allele carriers. Consonant with this, and as we’ll describe in the following sections, studies have found ‘T’ allele carriers to have a higher risk of hypertension (high blood pressure), obesity, and insulin resistance compared to non-carriers.
How do GNB3 variants affect risk of hypertension?
Hypertension is the medical term for high blood pressure. According to American Heart Association criteria, it is defined as having a systolic blood pressure ≥ 130 mm Hg or a diastolic blood pressure ≥ 90 mm Hg. You can read more about blood pressure, hypertension and why it’s bad for our health in the MTHFR and blood pressure trait article.
Several studies have linked the ‘T’ allele (rs5443) of the GNB3 gene to a higher risk of hypertension in Caucasian populations.
A 2017 meta-analysis that encompassed 63,729 subjects of various ethnicities found that ‘T’ allele carriers (i.e. those with ‘CT’ or ‘TT’ genotypes) had a 11% higher risk of hypertension compared to non-carriers (i.e. those with the CC genotype).
More specifically, those with one copy of the ‘T’ allele (i.e. the CT genotype) had a 9% higher risk of hypertension than non-carriers. Those with two copies of the ‘T’ allele (TT genotype) had a 16% higher risk of hypertension.
When the data was broken down by ethnicity, however, the link between the ‘T’ allele and hypertension only remained statistically significant in Caucasian subjects. ‘T’ allele carriers from Asian and African populations were not shown to have an elevated risk of hypertension.
Other studies have reported similar findings, with a 2007 meta-analysis reporting that the ‘T’ allele was associated with increased hypertension risk in Caucasian subjects but not in Asian subjects.
The reasons for this difference between ethnicities is poorly understood. It is likely due to both the impact of other gene variants that may differ between ethnicities, as well as environmental / cultural differences (such as differences in diet). On this note, it has been suggested that Caucasians consume less salt compared to Asian populations, and there is evidence that salt intake may interact with the GNB3 gene to affect risk of hypertension.
As well as having a higher baseline risk of hypertension, Caucasian ‘T’ allele carriers who already have hypertension are shown to develop more severe hypertension more quickly.
The HARVEST (Hypertension and Ambulatory Recording Venetia Study) study followed 461 people with Grade I hypertension (systolic blood pressure between 140 – 159 mm Hg and/or diastolic blood pressure of 90-99 mm Hg) over an average period of 4.7 years.
The researchers then conducted a survival analysis, whereby they assessed how many of the subjects reached a defined endpoint – in this case, whether they met the eligibility criteria for antihypertensive medication, which included having Grade II hypertension (a systolic blood pressure ≥ 160 mm Hg and/or a diastolic blood pressure ≥ 100 mm Hg).
Source: Sartori, M., Semplicini, A., Siffert, W., Mormino, P., Mazzer, A., Pegoraro, F., ... & Palatini, P. (2003). G-protein β3-subunit gene 825T allele and hypertension: a longitudinal study in young grade I hypertensives. Hypertension, 42(5), 909-914.
As illustrated in the Kaplan-Meier curves above show, a greater proportion of ‘T’ allele carriers (the top, solid line) progressed to Grade II hypertension and did so more quickly than non-carriers (the bottom, dotted line).
The impact of the ‘T’ allele on hypertension risk is likely due to enhanced GPCR-signalling. As discussed previously, greater G-protein activation and GPCR-signalling can cause greater vasoconstriction and other physiological changes that increase blood pressure.
How do GNB3 variants affect risk of pre-eclampsia?
Pre-eclampsia is a condition that affects some pregnant women and is characterised by high blood pressure and protein in urine. It typically occurs from 20 weeks gestation or soon after delivery.
A recent meta-analysis of 8 studies found that ‘T’ allele was linked to a greater risk of preeclampsia. Those with two copies of the ‘T’ allele (i.e. the TT genotype) were found to have a 21% higher risk of pre-eclampsia compared to those with the CC genotype.
The mechanisms by which the ‘T’ allele increase the risk of pre-eclampsia in pregnant women may be the same as those which increase the risk of hypertension in the general population.
How do GNB3 variants affect risk of weight gain and obesity?
The ‘T’ allele (rs5443) of the GNB3 has been linked to an increased risk of being overweight and obese across multiple ethnic groups.
For example, a study of German, Chinese and black South African men found those with one copy of the ‘T’ allele (i.e. CT genotype) had a 1.5 – 1.7 times higher risk of being overweight (BMI ≥ 25 kg/m2). This risk was greater (1.8 – 2.5 times higher) in those with two copies of the ‘T’ allele (TT genotype).
Another study of Saudi subjects, which compared the genotypes of obese and non-obese individuals, found that the TT genotype was overrepresented in the obese group. In obese females, for example, 83.6% had the TT genotype, which was significantly greater than the corresponding figure of 20% for non-obese females.
The link between the ‘T’ allele of the GNB3 gene and obesity is again likely to be due to enhanced GPCR-signalling.
Source: Zhai, M., Yang, D., Yi, W., & Sun, W. (2020). Involvement of calcium channels in the regulation of adipogenesis. Adipocyte, 9(1), 132-141.
GPCRs are highly involved in fat metabolism and animal studies have shown that enhanced GPCR-signalling can stimulate adipogenesis – the process by which stem cells become adipocytes (fat cells), which then grow in size and store higher amounts of fat. An increase in the size and amount of adipocytes in adipose tissue can then lead to obesity.
GNB3 variants and gestational weight gain
In addition to being linked to weight gain and obesity more broadly, the ‘T’ allele has been linked in some studies to excessive weight gain during pregnancy.
A degree of weight gain throughout pregnancy (known as gestational weight gain) is, of course, to be expected. Excessive weight gain, however, can have negative health effects for both mother and infant, including gestational diabetes, preeclampsia, dystocia (difficulty during labour), neonatal distress, and childhood obesity.
According to the 2009 Institute of Medicine (IoM) guidelines, the amount of gestational weight gain that is deemed healthy is inversely related to a person’s pre-pregnancy BMI (see table below). A woman with a pre-pregnancy BMI > 30 kg/m2 is expected to gain between 5 and 9 kg over the course of pregnancy. For a woman with a lower pre-pregnancy BMI <18.5 kg/m2, the recommended weight gain is between 12 and 18 kg. Weight gain above these recommended IoM levels is considered excessive.
Studies linking the ‘T’ allele to gestational weight gain have mixed findings. A study of 294 women from Caucasian, Hispanic and African-American backgrounds found that those with two copies of the ‘T’ allele (i.e. the TT genotype) gained significantly more weight than those with CT and CC genotypes.
In a Romanian study of 158 women, the ‘T’ allele was found be more common in subjects with excessive gestational weight gain compared to those with healthy weight gain, although this difference was not statistically significant.
A study of African-American women found that obese subjects with the CC genotype had lower gestational weight gain than ‘T-allele carriers, although this association did not reach statistical significance.
How do GNB3 variants affect response to sildenafil (Viagra)?
Sildenafil (Viagra) is a medicine used to treat erectile dysfunction. It works by causing relaxation of the smooth muscle surrounding blood vessels to the penis. This increases blood flow to erectile tissue and allows men to achieve and maintain an erection.
In terms of GPCR-signalling, sildenafil acts to increase levels of cGMP, a second messenger molecule involved in intracellular signalling that is produced when certain G-proteins are activated. Sildenafil inhibits an enzyme called phosphodiesterase 5 (PDE5), which is responsible for the degradation of cGMP. By inhibiting PDE5, sildenafil causes cGMP levels to remain elevated, leading to relaxation of smooth muscle and increased blood flow to erectile tissue.
Source: Cruz-Burgos, M., Losada-Garcia, A., Cruz-Hernández, C. D., Cortés-Ramírez, S. A., Camacho-Arroyo, I., Gonzalez-Covarrubias, V., ... & Rodríguez-Dorantes, M. (2021). New Approaches in Oncology for Repositioning Drugs: The Case of PDE5 Inhibitor Sildenafil. Frontiers in Oncology, 11, 208.
One study has shown that people with two copies of the ‘T’ allele (rs5443) of the GNB3 gene (i.e. the TT genotype) have a much greater response to sildenafil. The study looked at the genotypes of patients with erectile dysfunction receiving between 25 – 100 mg of sildenafil. Patients were then asked to assess their erectile response on a scale from 0 - 5 as follows:
- 0: no tumescence or rigidity,
- 1: little tumescence and no rigidity,
- 2: some tumescence and no rigidity,
- 3: full tumescence and no rigidity,
- 4: full tumescence with rigidity firm enough for intercourse
- 5: complete erection.
A much greater proportion (90.9%) of men with the TT genotype ranked their erectile response to sildenafil as 4 or 5 compared to those with the CT (48.9) or CC (50.9). Furthermore, those with a positive erectile response were 10 times more likely to have the TT genotype.
Your GNB3 and cardiometabolic health trait
Your GNB3 and cardiometabolic health trait looks at the rs5443 SNP within the GNB3 gene. Depending on your DNA results, sex and ethnicity, you will be classified into one of the following groups:
- Average risk of hypertension - you do not carry the risk 'T' allele associated with increased risk of hypertension and obesity.
- Average risk of hypertension, risk of obesity - you carry one copy of the risk 'T' allele associated with a higher risk of overweight and obesity. As the 'T' allele is not linked to hypertension in non-Caucasian populations, you are considered to have an average risk of hypertension based on GNB3 variants alone.
- Average risk of hypertension, weight gain in pregnancy - you carry two copies of the risk 'T' allele associated with a higher risk of overweight and obesity, as well as a higher risk of gestational weight gain in women. As the 'T' allele is not linked to hypertension in non-Caucasian populations, you are considered to have an average risk of hypertension based on GNB3 variants alone.
- Average risk of hypertension, responsive to Viagra - you carry two copies of the risk 'T' allele associated with a higher risk of overweight and obesity. Your genotype (TT) is also shown to have a greater response to Viagra (sildenafil) in men. As the 'T' allele is not linked to hypertension in non-Caucasian populations, you are considered to have an average risk of hypertension based on GNB3 variants alone.
- Increased risk of hypertension and obesity - you carry one copy of the risk 'T' allele associated with increased risk of hypertension (in Caucasian populations) and obesity.
- High risk of hypertension and weight gain in pregnancy - you carry two copies of the risk 'T' allele associated with a higher risk of overweight and obesity, hypertension, as well as a higher risk of gestational weight gain in women.
- High risk of hypertension, responsive to Viagra - you carry two copies of the risk 'T' allele associated with a higher risk of hypertension, overweight and obesity. Your genotype (TT) is also shown to have a greater response to Viagra (sildenafil) in men.
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