Tuesday, June 5, 2007

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 KATP 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 KATP 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 KATP channels open). Beyond a certain point, however, increased activity of the KATP 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 KATP 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 KATP 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 KATP channels, then why does glucagon secretion decline at high levels of diazoxide? The idea is that as more and more KATP 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.


  1. 1. MacDonald, P.E., et al. (2007). "A KATP 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.)

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