Hormone replacement therapy and coronary heart disease

As I’ve written in a previous post, the Women’s Health Initiative (WHI) examined the risks of heart disease, breast cancer, and osteoporosis in post-menopausal women. One aspect of the WHI was randomized trials investigated the effects of hormone therapy (either estrogen alone or estrogen plus progestin) on the health of post-menopausal women. The study was shut down early due to a high number of adverse effects. From the data that was collected while the study was ongoing came the somewhat surprising result that women receiving the hormone therapy were not at any reduced risk for non-fatal myocardial infarction (heart attacks) or coronary artery disease. This was surprising because it had long been thought that estrogen supplied some sort of cardioprotective function (this was the explanation for why pre-menopausal women have lower risk of heart attacks than men of similar age). Follow-up analyses after the WHI trial ended showed that the effect of estrogen on the heart seemed to be time dependent – the earlier the estrogen was administered, the more cardioprotective the result. In women aged 50-59, there was a fairly notable beneficial effect of hormone therapy. In older women, there was either no benefit, or, in the 70-79 cohort, perhaps some detriment to the hormone therapy.

A recent study in the New England Journal of Medicine looked at atherosclerotic calcification of women in the 50-59 year-old cohort using computed tomography, comparing women who had received hormones and those that hadn’t. The women studied had been part of the initial WHI study (estrogen only trial - so all the women lacked a uterus). Technicians at a central lab scored all the scans for calcification. In the end, women that had received estrogen during the WHI trial had less calcification than women that received a placebo.

The authors conclude:

The new findings from WHI-CACS [CACS = Coronary Artery Calcium Study; the name of the follow-up analysis] indicate that estrogen therapy initiated in women at 50 to 59 years of age is related to a reduced plaque burden in the coronary arteries and a reduced prevalence of subclinical coronary artery disease, providing support for the hypothesis that estrogen therapy may have cardioprotective effects in younger women.

The authors also provide a potential explanation for why estrogen might be cardioprotective in recently post-menopausal women (50-59 years old), but have negative effects on older women (70-79):

It is possible that estrogen could reduce coronary-artery calcium scores but still increase the risk of clinical CHD [coronary heart disease] events, owing to adverse effects on thrombosis and plaque rupture, which are more likely in older women with advanced stages of atherosclerosis. Such a duality of effects would not necessarily apply to younger women with lower burdens of atherosclerosis.

But nobody should get carried away with hormone replacement therapy; it still has significant risks. Any decision to initiate hormone therapy is still a balanace between those risks and the benefits:

In the meantime, hormone therapy should not be initiated (or continued) for the express purpose of preventing cardiovascular disease in either younger or older postmenopausal women. The current recommendations from many organizations that hormone therapy be limited to the treatment of moderate-to-severe menopausal symptoms, with the lowest effective dose used for the shortest duration necessary, remain appropriate.

References:

1. Manson, J.E. et al. (2007). "Estrogen Therapy and Coronary Artery Calcification." New England Journal of Medicine 365: 2591-25602. (Available for free after 6 months)

2. WHI steering committee (2004). "Effects of Conjugated Equine Estrogen in Post-menopausal Women with Hysterectomy." Journal of the American Medical Association 291(14): 1701-1712. (Free with registration)

Glucagon regulation, part 2

Getting back to glucagon regulation. The paper I mentioned at the end of my last post on this topic was written by MacDonald and colleagues, and published online in PLoS Biology: “A K_ATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans”.

The title seems to imply that the article is just about what ion channel is involved in the regulation of glucagon release. It goes beyond this however, suggesting that blood glucose alone is enough to regulate glucagon (as the thing that influences the ion channel’s activity).

“We have now compared insulin and glucagon release and α- and β-cell Ca²⁺ responses in intact mouse, rat, and human pancreatic islets. We show that glucose retained the ability to suppress glucagon release from isolated islets during blockade of the Zn²⁺ and GABA paracrine pathways, and in the absence of stimulated insulin secretion or β-cell Ca²⁺ responses. Thus we now provide evidence in both rodent and human islets supporting the direct (intrinsic) glucose regulation of glucagon release from pancreatic α-cells.”

Recall that the review I discussed previously favored the ‘paracrine/endocrine’ hypothesis – specifically emphasizing the possible role of insulin (the β-cell ‘switch-off’ hypothesis). In fact, that review repeatedly downplayed any direct role for glucose in the regulation of glucagon:

“A direct inhibitory action of glucose on α-cell secretion seems to be of little physiological significance…”

In brief, MacDonald and colleagues systematically set out to (1) test whether glucose levels were sufficient to regulate glucagon secretion and (2) examine the electrophysiology of a-cells under different experimental conditions.

To demonstrate that glucose can directly regulate glucagon (without paracrine intermediaries), MacDonald measured glucagon secretion by mouse and rat islets at two different concentrations of glucose. In addition, they selectively blocked two known paracrines: GABA and Zn²⁺ ions. (Both of these chemicals inhibit glucagon release.) The results of these experiments are shown in the figure below.

To understand this figure, you need to know that Ca²⁺-EDTA removes Zn²⁺ from solution. Actually, the Zn²⁺ is still there, but it’s bound to the EDTA, so we’d say it is not biologically available. Similarly, SR-95531 blocks the activity of GABA. The pluses and minuses below the x-axis of each graph indicate the presence (+) or absence (-) of the EDTA and the SR-95531.

We can work through the graph in the following way (taking only the mouse data for simplicity). The first two bars basically show data from islets at low (left bar) and high (right bar) glucose. Based on normal physiology, we’d expect glucagon to be high when glucose is low and vice versa. Since neither EDTA nor SR-95531 are present, the reduction in glucagon secretion could be due to either glucose, GABA, or Zn²⁺. The next pair of bars again shows the response at two different concentrations of glucose, but now we’ve prevented Zn²⁺ from acting by adding EDTA to the mix. Even without Zn²⁺, however, we still see a reduction in glucagon production at high glucose concentrations. So, Zn²⁺, isn’t what’s causing the reduction. In the third pair of bars, the only difference is a change in glucose concentration – both Zn²⁺ and GABA have been blocked. What can we conclude? Glucose alone is sufficient to reduce glucagon secretion. Notice that the drop in glucagon secretion isn’t as great this time. This might signal that the presence of GABA magnifies the effect of glucose on glucagon secretion.

These data contradict the conclusions of the review paper, but they don’t deal with the β-cell ‘switch-off’ hypothesis. However, a subsequent experiment (panel A and B of Figure 2) shows that glucagon secretion (filled circles) increases well before insulin drops (open circles) to any significant degree (this is particularly apparent in the mouse data). Conclusion: it isn’t a drop in insulin that switches on the secretion of glucagon.

A similar effect can be seen in human islets (presented in Figure 5B in a slightly different format).

The evidence for the central role of K_ATP channels can be seen in Figure 2 above. Diazoxide is a chemical that opens the channels, and tolbutamide blocks them. Panels 2A and 2B show that glucagon secretion rises as the amount of diazoxide rises (and as more K_ATP channels open). Beyond a certain point, however, increased activity of the K_ATP channels actually leads to a reduction in glucagon secretion. (I’ll try to explain the authors’ interpretation of this in a second.)

For brevity and simplicity, I’ll skip the experiments that investigated ion channels for Na⁺ and Ca²⁺, and just say that the authors conclude that both channels involved are active at intermediate membrane potentials. As the membrane becomes either hyperpolarized (more negative than usual) or too depolarized (more positive than usual) these channels become inactive and close). The effect of this is inhibition of glucagon secretion.

Having said that, I’ll jump to the conceptual model that MacDonald and crew devise to explain what they think is going on. (As an aside, this is one of the things I really liked about this paper. Although the authors might be wrong in their interpretation of what the results of these experiments mean, the model will provide other researchers with some specific things to test.) The model is summarized graphically in Figure 10.

Panel A basically depicts what they think is going on in normal α-cells: at low glucose, the K_ATP channels are mostly open (as are the Na⁺ and Ca²⁺ channels - shown in the red and blue lines) and glucagon secretion is high (solid line at bottom of panel). As glucose levels rise, the K_ATP channels begin to close (due to the increase of ATP in the cell from glucose metabolism), and the cell membrane begins to depolarize (i.e., becomes less negative because fewer K⁺ ions are leaving the cell – keeping more positive charges in the cell makes the inside less negative). At some point the membrane is sufficiently depolarized that the Na⁺ and Ca²⁺ channels close, inhibiting glucagon release.

The other three panels basically explain what they think is happening to produce some of the results they saw in their other experiments. For example, if diazoxide opens more K_ATP channels, then why does glucagon secretion decline at high levels of diazoxide? The idea is that as more and more K_ATP channels open, the cell membrane becomes hyperpolarized (more negative because more K⁺ ions are leaving the cell). This hyperpolarized state causes the Na⁺ and Ca²⁺ channels to close, reducing glucagon secretion.

I have to say that the one thing that I don’t really understand about all this is the connection between the Na⁺ and Ca²⁺ channels and action potentials of the α-cell membrane that ultimately trigger the release of glucagon (by exocytosis). I would assume that other channels that are either not voltage sensitive or sensitive to different voltages (different from the particular Na⁺ and Ca²⁺ studied in these experiments) are also active. But experience suggests that I’m probably wrong.

References:

1. 1. MacDonald, P.E., et al. (2007). "A K_ATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans” PLoS Biology. Published May 15, 2007.
2. Gromada, J., Franklin, I., and Wollheim, C.B. (2007) “α-Cells of the Endocrine Pancreas: 35 Years of Research but the Enigma Remains.” Endocrine Reviews 28(1):84-116. (Author manuscript pdf free here.)
   

Devilish details of glucagon regulation

One of the difficult things about teaching science (or any subject, I imagine) is deciding which details to leave out. On one hand, it’s often the details that make things interesting. On the other hand, each layer of complexity often requires more background for it to make sense, and the amount of information necessary for comprehension expands exponentially. As a result, we often present things as cut and dry when they are anything but. I imagine that this often gives students the impression that everything is known. Given that I’m teaching subjects that are a bit outside what I studied in graduate school, I sometimes get to experience this myself as I delve into the research literature. I’m often surprised to find out what we don’t know. As an example, take the regulation of the hormone glucagon.

Glucagon is a hormone produced and secreted by α-cells of the pancreas. The basic role of glucagon is to prevent low blood sugar – it helps maintain adequate levels of glucose in the blood. In effect, glucagon opposes the action of insulin. Insulin decreases blood glucose, glucagon raises it. It does this by triggering the release of glucose from the liver.

According to a common anatomy and physiology text (Tortora & Derrickson):

Decreased blood level of glucose, exercise and mainly protein meals stimulate [glucagon] secretion; somatostatin and insulin inhibit secretion.

From reading that, you might think the regulation of glucagon seems pretty straightforward. But if that’s true, then why did the Endocrine Society publish an article earlier this year by Jesper Gromada and colleagues titled “α-Cells of the Endocrine Pancreas: 35 Years of Research but the Enigma Remains.” (The paper is available online for free as an “author manuscript pdf” here.) Clearly, things aren’t as cut and dry as the textbook leads one to believe.

So, what do we know about glucagon regulation? Before I answer that, perhaps I should mention why this is of anything more than academic interest. If you follow the news, you’ve probably heard discussions of a diabetes epidemic. NPR recently broadcast a story about type 2 diabetes showing up in people in their teens and twenties, much younger than once was common. Traditionally, diabetes is portrayed as a problem with the hormone insulin, but actually, high levels of glucagon also play a role. Moreover, problems with glucagon regulation in people with type 1 or advanced type 2 diabetes lead to problems with low blood sugar (hypoglycemia). In short, being able to control glucagon could help diabetics maintain normal blood glucose levels. (This is important because many of the complications associated with diabetes are a result of chronic high blood glucose.)

Back to what we know about glucagon regulation. From the article in Endocrine Reviews:

The control of glucagon secretion is multifactorial and involves direct effects of nutrients [like glucose and amino acids] on α-cell stimulus-secretion coupling as well as paracrine regulation by insulin and zinc as well as other factors secreted from neighboring β- and δ-cells within the islet of Langerhans. Glucagon secretion is also regulated by circulating hormones and the autonomic nervous system.

Some of that bears explaining. Paracrines can be thought of as local hormones that affect cell types different from the type of cell that made and secreted the paracrine. A regular hormone (as opposed to a paracrine) can be thought of as a circulating hormone – meaning that it circulates throughout the body, not just a local area. β- and δ-cells are other cell types in the pancreas. β-cells release insulin and δ-cells release somatostatin (somatostatin was mentioned in the textbook description of glucagon regulation). Lastly, the islets of Langerhans are clusters of cells including α-, β-, and δ-cells that make up the endocrine pancreas. It’s referred to as the endocrine pancreas because it’s the part of the pancreas that releases hormones (as part of the endocrine system). The rest of the pancreas (the exocrine pancreas) releases digestive enzymes into the small intestine.

If we know all of these things influence glucagon regulation, then what’s the enigma referred to in the article’s title? The uncertainty lies in the relative influence of the factors:

Since the early 1970’s, the mechanism underlying the regulation of glucagon secretion by glycemia has puzzled scientists. The debate continues whether α-cells directly sense and respond to fluctuations in plasma glucose or whether the response is mediated by the autonomic nervous system and/or the paracrine/endocrine effects of secretory products from other islet cell types. Currently, a large body of research favors the latter ‘paracrine/endocrine’ hypothesis.

The ‘paracrine/endocrine’ hypothesis can be summarized briefly as the idea that a drop in insulin triggers the release of glucagon. This portion of the broader paracrine/endocrine hypothesis is often called the β-cell ‘switch-off’ hypothesis because insulin is secreted by β-cells. High levels of insulin inhibit glucagon, so the decline of insulin in the blood will free α-cells from inhibition.

The rest of the paper goes over the evidence for the various controlling factors, explaining why the authors think the ‘paracrine/endocrine’ hypothesis is most likely the major factor regulating glucagon release. One complication seems to be that research conducted on different species can be difficult to compare. Apparently, there are subtle species-specific differences in glucagon regulation. Just because α-cells of mice react a certain way doesn’t necessarily mean that rats (or humans) will too. So, research on one organism doesn’t necessarily translate perfectly to other organisms.

Having written all this, however, I haven’t really gotten much beyond what the textbook said. (Perhaps a good indication that the extra detail isn’t worth going into at the level of class I teach.) But there is something that an inquiring mind still might be wondering: How does insulin prevent the α-cells from secreting glucagon?

This post is already far too long to delve into that in much detail, but the CliffsNotes version is this. The membranes around cells are impermeable to ions like potassium (K⁺), sodium (Na⁺), and calcium (Ca²⁺). It’s still possible for these ions (and others that I haven’t named) to enter cells, but they need passages (called ion channels) through the membrane. Using these channels, cells can control which ions get in and which ions get out. In doing so, the cells set up concentration gradients. For example, K⁺ ions are generally more abundant inside cells than outside them. The reverse is true for Na⁺. A result of this is the generation of an electrical potential across the cell membrane. Changes in the electrical potential can trigger changes in the activities of a cell. (A nerve impulse – the transmission of an action potential – is an example of what can happen when the electrical potential of a cell changes.)

So, to get to the point, insulin opens an ion channel in the membrane of α-cells. The opening of this ion channel changes the electrical potential of the cell, and this indirectly prevents the vessels containing glucagon inside α-cells from releasing the hormone. When insulin levels in the blood decrease, those ion channels close. This changes the electrical potential of the α-cells, and allows the vessels containing glucagon to release the hormone from the cell.

Next week, when I get some time, I’ll take up a new research paper that deals with this issue and comes to conclusions that differ from the ‘paracrine/endocrine’ hypothesis.