Glucagon secretion -
B, Response of Sur1KO islets. The perifusion protocol is the same as shown in Fig. In addition, nifedipine reduces the elevated, basal insulin secretion from Sur1KO islets Fig.
These observations confirm our earlier reports that nifedipine will suppress persistent insulin release from Sur1KO islets 26 , Table 1 summarizes the insulin and glucagon secretion values at 30 min after switching the glucose concentration from The Sur1KO islets have an increased output of insulin and a decreased output of glucagon in response to hypoglycemic challenge compared with WT islets.
Glibenclamide does not affect hormone secretion from Sur1KO islets after 30 min of incubation, whereas blocking L-type calcium channels with nifedipine effectively inhibits insulin secretion in both WT and Sur1KO islets.
Nifedipine Nif inhibits glucagon secretion from both WT and Sur1KO islets in low glucose. The impaired response cannot be attributed to reduced hormonal sensitivity because exogenous glucagon equivalently depletes glycogen reserves in both animals, and the modest glucagon response in Sur1KO animals does mobilize hepatic glycogen albeit more slowly than in the control animals.
Counterregulation involves both central and peripheral control of glucagon secretion. The results extend the analysis reported for K IR 6. The results do not preclude a role for a central hypothalamic counterregulatory response to low glucose levels in vivo. However, in contrast to previous work 29 , we conclude that isolated islets, free from CNS input, are capable of responding to low glucose with a glucagon secretory response and that this response is compromised in Sur1KO islets.
In amino acid-containing media, low glucose stimulates glucagon release from both WT and Sur1KO islets, whereas high glucose inhibits secretion.
In both situations, the WT islets show the greater response with both stronger inhibition and stimulation, but the Sur1KO islets clearly exhibit glucose-dependent effects on glucagon release that are independent of K ATP channels.
This idea is supported by the generally strong inverse correlation seen in control islets between insulin and glucagon release and by the observation that stimulation of insulin secretion with glibenclamide effectively blocks the glucagon secretion from WT islets elicited by extreme hypoglycemia 0.
Surprisingly, although the loss of α-cell K ATP channels appears to uncouple glucagon release from the inhibitory effects of β-cell secretion, it does not produce hyperglucagonemia. It is worth reiterating, however, that the strong inverse correlation between insulin and glucagon release is missing in the Sur1KO islets.
This can be seen clearly, for example, in Fig. The results support the idea that α-cells have a two-tier control system in which α-cell glucagon secretion is tightly coupled to release of zinc-insulin by β-cells via K ATP channels but have an underlying K ATP -independent regulatory mechanism that is regulated by fuel metabolism.
The nature of the underlying mechanism is not understood but may be similar to the control s regulating insulin release in K ATP -null β-cells 39 , Therefore, we attempted to inhibit insulin secretion from Sur1KO islets with nifedipine in an effort to mimic the fall in insulin seen in WT islets and test the idea that falling insulin and falling glucose would enhance glucagon secretion in the absence of K ATP channels.
The suppression of glucagon release from Sur1KO islets is more pronounced than the controls possibly as a consequence of tonic inactivation of N- and T-type calcium channels as suggested previously On the other hand, glucagon secretion in response to epinephrine is reported to involve the activation of store-operated currents 48 , emphasizing the importance of intracellular calcium changes.
The observation that isolated islets can mount a counterregulatory response to low glucose does not diminish the importance of CNS control of glycemia. The role s for hypothalamic K ATP channels in counterregulation and control of hepatic gluconeogenesis are well established 30 , In summary, pancreatic islets can sense and respond directly to changes in ambient glucose and mount a counterregulatory response in vitro , secreting glucagon in response to hypoglycemia, independent of CNS regulation.
Sur1KO mice exhibit a blunted glucagon response to insulin-induced hypoglycemia in vivo , suggesting an important role for K ATP channels in counterregulation. Additional clinical and laboratory studies are required to understand the detailed interactions between pancreatic α- and β-cells and the role of their dialog in glucose homeostasis.
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Stagner JI , Samols E The vascular order of islet cellular perfusion in the human pancreas. Diabetes 41 : 93 — Diabetologia 47 : — This led to the stunning discovery, that a pancreatic hormone can be produced in large amounts outside its original endocrine gland.
Over the next 10 years, using tracer methods to measure glucose fluxes, biochemical, histological, immunological methods, electron microscopy, and purifying gastric glucagon to homogeneity, it was determined beyond all doubt that the parietal mucosa of the dog stomach can synthesize and secrete true glucagon Morita et al.
When stomach glucagon was purified to homogeneity, its effect on isolated liver cells in-vitro was quantified. The effects of the extracts were identical to those of pancreatic glucagon. Now, it was not surprising by measuring glycogenolysis, gluconeogenesis, production of lactate and pyruvate, and concentration of cAMP, that following pancreatectomy in dogs, diabetes is as severe as with the selective destruction of the β-cells Doi et al.
Another stunning finding was that in the gastric mucosa of a depancreatized dog that was maintained on insulin by for 5 years, there was a large hyperplasia of α-cells, and a large amount of glucagon in the dog's stomach.
By electron microscopy of the parietal mucosa of the stomach looked like a glucagon-producing endocrine gland Ravazzola et al. It was demonstrated with labeled tryptophan, leucine, and s-methionine, the specific biosynthesis of glucagon in mucosa pieces of the stomach Hatton et al.
These findings challenged classical views of endocrinology and provided further proof that one hormone is not necessarily produced in only one endocrine gland. Furthermore, the amount of glucagon-like peptides that are secreted exclusively from the gastro-intestinal tract was quantified Mojsov et al.
High glucagon plasma levels in the depancreatized dogs were also confirmed by others Matsuyama and Foa, Their regulation of extrapancreatic glucagon release was different than that from the pancreas Luyckx and Lefebvre, True glucagon was localized exclusively in the stomach because pancreatectomy plus gastrectomy virtually removed glucagon from plasma Muller et al.
The most extensive factors that control gastric glucagon release were ascertained by using a unique model of isolated-perfused dog stomach Lefebvre and Luyckx, Arginine elicited rapid gastric glucagon release.
This glucagon release was almost completely abolished by somatostatin. Thus, insulin is needed for hyperglycemia to inhibit gastric glucagon secretion. Perfused dog stomach provides a unique tool for investigating α-cell function in absence of endogenously released insulin.
In addition, they also reported that immune-neutralization of insulin in the blood perfusing the stomach doubled the glucagon release, and thus further confirmed the role of insulin in controlling α-cell secretion Lefebvre and Luyckx, These early observations in the dog stomach are relevant in the studies of pancreatic slices, of streptozotocin STZ and BioBreeding BB diabetic rats, which will be reported later in this review.
In contrast to dogs, in totally depancreatized humans, there is only a negligible amount of plasma glucagon, and in contrast to depancreatized dogs, in depancreatized humans, diabetes is very mild Barns et al. Thus, the discovery of extra-pancreatic glucagon led to a much better understanding of the role of glucagon in physiology and diabetes.
Glucagon-like peptides are detected in the brain Tager et al. The discovery of extra-pancreatic glucagon and quantification of release of glucagon-like peptides from the intestine, also stimulated research in the field of GLP-1 that is co-encoded in the glucagon gene as a potent stimulator of insulin release Mojsov et al.
In these mice, which exhibit no response to glucagon at any concentration, destruction of β-cells did not result in any of the diabetic abnormalities thought to be caused by insulin deficiency.
Unquestionably, this exciting new finding indicates an important role of glucagon in diabetes. The interesting question is whether there are compensatory mechanisms that occur in knock-out rodents that replace the action of insulin, such as increased insulin-like growth factor IGF -1 or increased sensitivity of insulin receptors to IGF It is also difficult with the methods presently used to ascertain that insulin has been completely removed.
One could speculate that some knock-outs procedures may alter the physiology of insulin-glucagon interactions, and may reflect a metabolic system not seen in physiology or in diabetes.
The cellular machinery that controls glucagon secretion from α-cells is perhaps surprisingly similar to that which regulates insulin secretion from β-cells Figure 1. The compliment of ion channels expressed in α-cells mirrors those found in β-cells.
Figure 1. Pancreatic endocrine cells are regulated by intrinsic and paracrine signals in response to glucose. When plasma glucose is increased B , glucose enters pancreatic islet cells through plasma membrane glucose transporters GLUT where it is metabolized through glycolysis and mitochondrial oxidative metabolism.
In the β-cell B , top left this results in membrane depolarization and firing of action potentials that, in combination with additional mitochondrial signals, results in the exocytosis of insulin-containing granules. Glucagon secretion is also inhibited by paracrine factors secreted from β-cells and pancreatic δ-cells.
These signals may interact with putative α-cell metabolic sensors i. It is not surprising then that pancreatic α-cells are electrically excitable and, like β-cells, use their electrical activity to couple changes in glucose to the regulation of glucagon release Rorsman and Hellman, ; Gromada et al.
Looking at this excitatory and exocytotic machinery alone however, is becomes difficult to explain how glucose inhibits, rather than stimulates, α-cell glucagon secretion. Understanding how the glucagon secretory machinery is regulated by signals both intrinsic and extrinsic to the α-cell will be necessary to elucidate the exact mechanism of glucose-regulated glucagon secretion.
Indeed, there are already hints that the excitatory machinery in α-cells is regulated in a manner opposite to that of β-cells: for example membrane depolarization is capable of turning off a number of the ion channels involved in α-cell electrical activity that are activated under similar conditions in β-cells Ramracheya et al.
Thus, elucidating not only the pieces of machinery that control glucagon secretion, but how these are regulated will provide novel insight into the physiological mechanism for glucose-regulated glucagon release.
This question has been a matter of debate for many years. Based solely on studies of dispersed or purified α-cells Pipeleers et al. One must be quite careful in the interpretation of such studies however, since properties of both dispersed α- and β-cells are quite different than those in intact islets.
For example, the presence of functional gap junction connections is recently proposed as necessary for the efficient suppression of glucagon secretion Ito et al. These of course would be lost upon dispersion and purification of α-cells. Within α-cells, glucose certainly has metabolic effects Detimary et al.
Recent evidence has implicated α-cell resident metabolic sensing in the control of glucagon secretion, and in the pathophysiology of glucagon secretion in diabetes through AMP-activated protein kinase AMPK Leclerc et al. Glucose-dependent inhibition of glucagon secretion was associated with an inhibition of AMPK activity, while forced activation of AMPK stimulated glucagon secretion.
This study Leclerc et al. While the idea that AMPK activation may be beneficial in diabetes may seem at odds with the glucagon-stimulating effects of AMPK activation, this must be considered in the context of the activity of upstream AMPK regulators which themselves may be regulated by glucose in α-cells.
At this time there is little or no information about the up- or down-stream regulators of AMPK in α-cells, although, this is currently an area of growing interest in the context of insulin secretion [reviewed in ref.
Fu et al. Thus, the α-cell may indeed be capable of responding to intrinsic metabolic signals, and integrating these inputs with extrinsic paracrine signals in the physiologic control of glucagon secretion. The activity of α-cell K ATP -channels is thought to contribute to the control of glucagon secretion Ronner et al.
A role for K ATP channels in the regulation of glucagon secretion is supported by the reduced glucagon secretion under low-glucose conditions seen in mice lacking functional K ATP -channels Gromada et al. However, it should be recognized that all K ATP -channel measurements in α-cells suggest that the effect of glucose on overall channel activity may be smaller than that seen with pharmacologic agents Gromada et al.
Nonetheless, given the low input resistance of α-cells, small changes in K ATP channel activity may be functionally relevant. Interestingly, work in mice expressing GFP under the control of the mouse insulin promoter MIP-GFP mice showed that α-cell K ATP channels are more sensitive to ATP than are those in β-cells Leung et al.
In those reports, we showed that insulin reduces the sensitivity of α-cell K ATP channels to ATP relatively more so than β-cell K ATP channels, and this was by its actions on the insulin receptor-phosphatidylinositol 3-kinase signaling pathway.
The factors and signaling mechanisms that control α-cell K ATP channel sensitivity to ATP are not well understood, although, regulation by paracrine factors may represent one such mechanism that could bridge the divide in understanding the interplay between paracrine and intrinsic factors controlling glucagon secretion.
Release of islet hormones is regulated not only by direct actions of glucose and other nutrients, but also indirectly and potently by paracrine factors secreted by adjacent islet cells.
The current body of knowledge shows that islet cells profoundly modulate each other's secretory functions by very complex paracrine and even autocrine pathways Gaisano and Leung, High glucose stimulates β- ad δ-cell secretion while inhibiting α-cell secretion, whereas low glucose stimulates α-cell secretion directly or indirectly, but inhibits other islet cells Dunning and Gerich, ; Quesada et al.
Since insulin reduces K ATP channel sensitivity to ATP in α-cells more so than β-cells Leung et al. Indeed, glucagon secretion has long been known to be inhibited by insulin Le Marchand and Piston, ; Andersson et al. L-glutamate released from α- Cabrera et al. Glucagon secreted by α-cells exhibits paracrine stimulatory action on β-cells and autocrine stimulation of α-cell glucagon secretion Ma et al.
It is particularly important that some diabetes patients have increased risk of hypoglycemia during insulin treatment therapy White et al. The threat of hypoglycemia has increased since the treatment for diabetes has aimed for tight blood glucose control to decrease the risk of diabetic complications.
In order to avoid hypoglycemia, many diabetic patients reduce their blood glucose control. Thus, hypoglycemia is a limiting factor for proper control of glycemia.
Therefore, it is important to develop a treatment strategy that would decrease the risk of hypoglycemia. The defect of glucagon and epinephrine responses to hypoglycemia in diabetes is puzzling because both counterregulatory responses are normal or even excessive during some stresses, such as moderate and strenuous exercise, both in dogs and humans Orci et al.
We showed that although in each islet the number of glucagon cells is greatly increased, the total amount of glucagon in the pancreas remains unchanged because of the reduction in the number of islet cells.
Clearly, alloxan or STZ destroys not only β-cells, but they also reduce the total number of islet cells. It is well known that the release of glucagon by the pancreas is inhibited by both insulin and somatostatin; and in diabetes, defects in the release of these islet paracrine hormones contribute to the perturbation of glucagon release from α-cells.
Thus, the physiological regulation of glucagon secretion is complex Figure 1. At high glucose, paracrine inputs from both β- and δ-cells are crucial physiological suppressors of glucagon release through actions on the α-cell electrical and secretory machinery. Although controversial, metabolic sensing pathways intrinsic to the α-cell likely contribute to the suppression of glucagon release either directly, by inhibiting the α-cell ion channels and exocytotic machinery, or indirectly by modulating the cellular response to paracrine signals.
As such, glucagon release is the result of an integrated α-cell response to external and internal cues. A breakdown in these mechanisms in diabetes likely contributes to hyperglucagonemia and impaired counterregulatory responses.
In T1D, there is a lack of decrement changes in intraislet insulin occurring which has been postulated to account for the defective glucagon counter-regulation to hypoglycemia.
α-cell sensitivity during hypoglycemia improves when normoglycemia is achieved by chronic phloridzin treatment, but not by insulin treatment in diabetic rats Shi et al. This is partly due to insulin inhibition of glucagon synthesis and release Liu et al.
These reports showed the ability of low glucose to stimulate α-cell secretion requires initial increase in insulin levels switch on followed by insulin deprivation switch off in presence of low glucose. Plasma somatostatin, pancreatic prosomatostatin mRNA and somatostatin protein levels are increased in diabetic humans Orci et al.
In T1D, upper-gut somatostatin, the major source of circulating somatostatin, is also increased Papachristou et al. It is generally believed that somatostatin only plays a minor role in inhibiting the α-cell in non-diabetic animals or humans.
Global somatostatin knock-out increased nutrient stimulated, but not basal glucagon secretion, compared with wild-type mice, in-vivo and in isolated islets, suggesting a role of locally released somatostatin on stimulated, but not basal insulin secretion Hauge-Evans et al.
Similarly, isolated islets from somatostatin receptor type-2 SSTR2 knock-out mice showed 2-fold greater stimulated glucagon secretion than wild type mice Strowski et al. In human isolated islets a dose-dependent reversal of SSTR2 antagonist induced suppression of glucagon secretion was achieved by using the same SSTR2 as the current study Singh et al.
Thus, using the SSTR2 antagonists may appear to also be relevant to humans. Since most β-cells have been destroyed, somatostatin becomes the main paracrine inhibitor of the α-cell in diabetes. That is why it was of particular interest that in diabetic dog islets, the ratio of somatostatin to glucagon is markedly increased.
An acute insulin injection increased this ratio further. This was the first demonstration that part of the defective mechanism in hypoglycemia may reflect alterations of this ratio in diabetes Rastogi et al. One could hypothesize that in diabetes, in absence of the tonic effect of insulin, islet α-cells are oversensitive to insulin and are exposed to increased somatostatin Papachristou et al.
Somatostatin is increased in the pancreas and also in blood. The major part of the concentration of somatostatin in blood is due to somatostatin release from the gut. Thus, the increase in local somatostatin and release of somatostatin delivery to the pancreas may both play a role in diabetes Figure 2.
It was previously demonstrated that in perifused islets and in infused isolated pancreas that the SSTR antagonist can greatly increase the response of α-cells to arginine. However, responses to insulin-induced hypoglycemia have not been tested.
In order to test the hypothesis about the importance of somatostatin in diabetic rats, a specific antagonist SSTR2 of the somatostatin receptor of α-cells was injected.
It was demonstrated that infusion of this antagonist can fully normalize glucagon responses to insulin-induced hypoglycemia in diabetic rats [Figure 3 , from ref. Yue et al. A patent Vranic et al. This could permit diabetic patients to adhere more strictly to an intensive insulin treatment and lessen the risk of diabetic complications.
Figure 2. In the normal physiology, the α-cell is under the tonic inhibitory influence of insulin and therefore somatostatin inhibition of α-cell may be of minor or no importance Singh et al. This is in contrast to diabetic islets in diabetes, where α-cell may be more sensitive to insulin and in addition, both circulating and pancreatic somatostatin, are increased.
It is generally believed that hypoglycemia is a strong stimulator of glucagon release from the α-cell. However, in islets in-vitro the effect of hypoglycemia is not consistent.
This difference may reflect the fact that between in-vitro and in-vivo systems, in-vivo the islets have abundant blood flow, which brings to the islet other factors such as amino acids i. We hypothesize therefore, that hypoglycemia has an effect only when amino acids or other substances found in blood, are present.
In absence of the tonic effect of insulin, somatostatin is the only endogenous inhibitor of glucagon release and insulin exerts a strong inhibitory effect on the α-cell. Therefore, when an antagonist blocks the α-cell receptors, despite the inhibitory effect of injected insulin, the α-cell can release normal amounts of glucagon Vranic, The figure is modified from that we previously reported Vranic, Figure 3.
In diabetic D rats, plasma glucagon increases only marginally during a glucose clamp at 2. B The response of glucagon to hypoglycemia was the same as in normal N rats.
C The data is also shown as area under the curve AUC analysis. The data is modified from that we reported in ref. The SSTR2a is highly specific for glucagon and only marginally for insulin, and it's structure is H-Fpa-cyclo[DCys-PAL-DTrp-Lys-Tle-Cys]-Nal-NH2 Yue et al.
Most importantly, infusion of the SSTR2 antagonist in absence of insulin did not affect the blood concentration of insulin, glucagon, epinephrine, or blood-sugar Yue et al. The efficacy of the SSTR2 antagonist with two different doses of the antagonist and of insulin was tested.
It is particularly interesting that in normal rats the antagonist did not improve or even decrease the response of glucagon to insulin-induced hypoglycemia. One could speculate that in normal rats, the high doses of antagonist even have some agonist properties, and confirmed that in normal rats, somatostatin is not a major inhibitor of hypoglycemia-induced glucagon release.
The response of corticosterone was also normalized. Corticosterone in contrast to glucagon is important for hypoglycemias of longer duration, since the effects of cortisol are mainly exerted through genetic mechanisms.
This could also be of importance for glucagon release because cortisol has some effect on the α-cells' control. Interestingly, delivery of the SSTR2 antagonist did not further increase pancreatic glucagon and somatostatin, or plasma somatostatin. One of the key questions was whether the SSTR2 antagonist can actually prevent hypoglycemia.
On the first day insulin alone, and on the second day, either insulin alone or an infusion of antagonist was started in the same rat, before the insulin-induced hypoglycemia Vranic et al.
The reason for such designs is that even one episode of hypoglycemia sensitizes the endocrine and metabolic system so that you would expect that on the second day the rats would need a different amount of insulin.
In order to avoid this problem, diabetic rats were injected for 3 days with insulin, in order to avoid further effect of antecedent hypoglycemia. After the injection of insulin, rats became hypoglycemic, but with the SSTR2 antagonist, hypoglycemia was avoided.
Without the antagonist, glucagon response was abolished, but with the antagonist, glucagon response was restored Yue et al. These STZ-induced diabetic rats were not treated with insulin since they still have some residual insulin in the blood and in the pancreas.
In contrast, BB rats are totally insulin-deprived, thus requiring insulin treatment, and therefore this model is more similar to human T1D; both caused by immune destruction of the β-cells.
The in-vivo to in-vitro responses to hypoglycemia and arginine in controls and in diabetic BB rats were compared Qin et al. In the in-vivo experiments, the glucose was clamped at 2. In contrast to the controls, the glucagon response was greatly diminished, but it was normalized during the infusion of the SSTR2 antagonist.
With glucagon response normalized, the BB rats did not need glucose infusion to maintain the clamp, while without the antagonist they needed a large amount of glucose infused because of the glucagon deficiency. Interestingly, we used for the first time pancreatic slices to assess the effect of hypoglycemia and arginine.
Surprisingly, hypoglycemia per se did not increase glucagon release. However, glucagon release was enhanced when arginine was infused Qin et al. The difference between in-situ and in-vitro experiments is that pancreatic slices are not controlled by the nervous system and are not exposed to hormones or metabolites such as, arginine that stimulate glucagon release.
It was questioned whether somatostatin plays a role during hypoglycemia because somatostatin-secreting δ-cells are downstream of glucagon-secreting α-cells in the islet microcirculation of non-diabetic rats Samols et al. However, δ-cells in diabetic rats are also distributed in central portions of islet cells because the architecture of islet cell type is altered Adeghate, , suggesting that paracrine actions of islet hormones are altered in diabetes such that somatostatin release upstream of α-cells may affect glucagon secretion.
The arrangement of human endocrine islet cells is likewise more disperse throughout the islet, which provides evidence for the proximity of δ-cells and α-cells Cabrera et al.
Furthermore, paracrine signaling may also occur via diffusion within the islet interstitium, independent of blood flow. The remaining question to be answered is to explore factors in blood that are necessary to sensitize the responses of α-cell to hypoglycemia and the mechanism of the potential sensitization of α-cells to insulin in diabetes.
These results indicate that SSTR2 blockade Rossowski et al. This strategy could lead to prevention of hypoglycemia in insulin-treated diabetics. Considerable work investigating glucagon secretion and α-cell signaling in healthy islets have been done as discussed above.
However, there has been relatively little progress in assessing the perturbation of α-cellular physiology and paracrine dysregulation during diabetes, which will require more innovative approaches.
One approach is the pancreatic slice preparation Huang et al. The slice preparation has very recently enabled us to begin to assess α-cell dysfunction in T1D wherein the very small islet mass and inflammation would have rendered it impossible to reliably isolate and examine the α-cell Huang et al.
This, along with the larger glucagon granules found on E. carrying larger amount of glucagon cargo, would trigger more glucagon release, thus explaining the basis of hyperglucagonemia in T1D Huang et al. Future studies employing the pancreas slice preparation will enable the elucidation of paracrine regulation within normal and diabetic islets.
Another approach is genetic manipulation of candidate proteins within α-cells by α-cell-specific knockout mouse models Gustavsson et al. Ideally, these clever approaches could be combined. From a clinical point of view, the mechanism whereby in T2D there is excessive response to glucagon during meals, and whether pharmacological intervention can prevent this problem.
A key question is also whether it is possible to prevent hypoglycemia in insulin-treated diabetics. So far, the evidence was obtained only in STZ-treated and BB rats. Patrick E. MacDonald receives research funding for his work on α-cells from Merck.
Herbert Y. Gaisano and Mladen Vranic have no financial or commercial relationships. This work was supported by a grant to Herbert Y. Gaisano from the Canadian Diabetes Association OGHG.
MacDonald holds an Alberta Innovates-Health Sciences Scholarship and the Canada Research Chair in Islet Biology. Adeghate, E.
Distribution of calcitonin-gene-related peptide, neuropeptide-Y, vasoactive intestinal polypeptide, cholecystokinin-8, substance P and islet peptides in the pancreas of normal and diabetic rats.
Neuropeptides 33, — Pubmed Abstract Pubmed Full Text CrossRef Full Text. A : Schematic of experimental approach. D : Proposed model for glucose metabolism in α-cells. All data presented are mean ± SEM. Statistics were performed with the Student t test B and C. The similar citrate content and small increase in 13 C enrichment could suggest that citrate is being used to generate other metabolites not measured here.
The increased percentage of 13 C enrichment in α-KG could suggest that citrate may be converted to α-KG. Alternatively, cataplerosis of citrate could explain why there is no increase in the total content of the metabolite Fig.
The combination of increased α-KG and decreased succinate enrichment suggests that the increases in total concentrations of fumarate, malate, and succinate content may be derived from anaplerotic metabolism.
On the basis of the findings in this experimental setup, it is likely that a proportion of citrate is used in the glucose FA cycle or for FA synthesis Fig. For glucose to contribute to the glucose FA cycle, pyruvate derived from glucose must enter the TCA cycle as oxaloacetate through pyruvate carboxylase or as acetyl-CoA through PDH.
The two metabolites can then be condensed by citrate synthase to form citrate. The activity of PDH is determined partly through inhibitory phosphorylation by pyruvate dehydrogenase kinases PDK Fig.
Mouse and human α-cells both express high levels of Pdk4 mRNA compared with other islet cell types 47 , PDK4 expression is regulated by changes in substrate availability, and consequently, expression is increased in both skeletal muscle 49 and islets 50 during fasting.
To determine whether the glucose-FA cycle is important for the effect of glucose on intracellular ATP and glucagon secretion, we generated a mouse model in which Pdk4 was specifically overexpressed in α-cells Pdk4αKI. In these mice, the higher PDH phosphorylation would lead to lower influx of pyruvate into the TCA cycle through PDH.
PDK4 staining intensity was observed exclusively in α-cells and was twofold higher in Pdk4αKI mice than in controls Fig. In these mice, the glucose-induced reduction in intracellular ATP Fig.
This suggests that PDK4 plays a key role in the regulation of glucagon secretion and that entry of pyruvate as acetyl-CoA into the TCA cycle is important for the reduction of glucagon secretion and intracellular ATP in α-cells. Regulation of FAO is vital for glucose-induced glucagon repression.
A : A depiction of the interaction between glucose and FAO pathways. PC, pyruvate carboxylase. B : From left to right: glucagon cyan , Pdk4 yellow , and composite staining in pancreatic sections from control and Pdk4αKI mice. E : Dot plots of the last three frames of each condition in D. G : Glucagon content for F.
I : Glucagon content for H. K : Dot plots of the last three frames of each condition in J. See also Supplementary Fig. Statistics performed were unpaired t test C , G , and I , two-way ANOVA with the Tukey E and K or Šidák F and H post hoc test, and mixed-effects analysis with the Šidák post hoc test L.
Cpt1a KO in α-cells leads to decreased long-chain FAO and reduced glucagon secretion at low glucose 14 , identical to the effects observed when islets are incubated with low substrate levels Fig.
We therefore investigated the effect of glucose on ATP levels in islets from mice with Cpt1a KO specifically in α-cells αCPT1aKO.
Intracellular ATP did not change in α-cells from these mice when exposed to 0. Some studies have suggested that inhibition of mitochondrial FAO can activate peroxisomal FAO 52 — These findings indicate that the glucose-FA cycle is active in α-cells and that glucose inhibits glucagon secretion by lowering FAO and intracellular ATP.
To test whether PDK4 expression in α-cells contributes to the regulation of glucagon secretion in vivo, we measured blood glucose and plasma glucagon in Pdk4αKI mice Fig. Glucose tolerance and plasma glucagon levels were unaffected in male and female Pdk4αKI mice Fig. However, unlike controls, glucagon levels failed to reduce in female Pdk4αKI mice after glucose administration Fig.
This was also the case when sexes were combined to increase statistical power Fig. No changes in body weight, blood glucose, or ketones were observed between genotypes Fig. This suggests that, while PDK4 expression in α-cells is an important regulator of glucose-regulated glucagon secretion, it is not enough to drive the development of hyperglycemia or hyperketonemia in vivo.
Overexpression of Pdk4 in α-cells affects glucose-regulated glucagon secretion in vivo. A : Intraperitoneal i. B : Area under the curve AUC for A.
C : Plasma glucagon at 0 and 30 min after i. Statistics performed were unpaired t test B , E , H , J , K , and L and two-way ANOVA with the Šidák post hoc test C , F , and I. Pancreatic α-cells express K ATP channels 48 , The observation that glucose decreased intracellular ATP in the presence of NEFA therefore prompted us to investigate the electrical activity in α-cells under these experimental conditions.
The addition of 0. Unlike in control recordings without NEFA and BSA Fig. This could indicate that the reduction in ATP caused by increasing glucose levels leads to opening of K ATP channels and suggests that membrane potential in α-cells is more negative than previously reported Electrical activity 11 and FAs 40 have both been suggested to regulate glucagon secretion through changes in cytosolic calcium.
To investigate whether the addition of 0. This suggests that at low glucose, the membrane potential is depolarized and that increasing glucose repolarizes the plasma membrane to inhibit electrical activity, calcium entry, and glucagon secretion.
Glucose-induced reduction in ATP repolarizes the plasma membrane in α-cells. E : Representative trace of calcium oscillations in an α-cells in intact WT islets. Statistics performed were one-way ANOVA with the Tukey C , D , and F post hoc test. Here we propose that the regulation of ATP production in α-cells is highly dependent on enzymes that promote FAO, such as PDK4 and CPT1a.
We find that inhibition of pyruvate entry into the TCA cycle as acetyl-CoA, or FA transport into the mitochondria, disconnects changes in glucose levels from changes in ATP production and glucagon secretion.
Based on the observations made here, we suggest that glucose regulates glucagon secretion, not by increasing intracellular ATP, but by inhibiting FAO to lower intracellular ATP. Our findings suggest that in α-cells, FAO is subject to suppression from glucose, as suggested by the glucose-FA cycle.
Despite this, α-cells do oxidize glucose to some extent 56 , 57 , at least in the absence of other substrates and BSA, where increases in extracellular glucose results in ATP production 11 , 19 , 21 , 24 , However, we show here that in the absence of BSA, α-cells do not secrete much glucagon and do not respond to glucose.
While it is not clear why BSA is important for α-cell function, albumin has previously been shown to impact both intracellular lipid, pH, and redox homeostasis 59 , Whether this discrepancy is due to differences in the experimental paradigm or the two cell types is unclear. Previous hypotheses of how glucose regulates glucagon secretion suggest that increased ATP from glucose oxidation leads to membrane depolarization in α-cells 11 , However, as with the previous measurements of ATP, these experiments were performed with glucose as the only substrate.
The findings we present here show that glucose repolarizes the plasma membrane in α-cells when applied in the presence of NEFA, consistent with the observed reduction in ATP under the same experimental conditions.
The finding that this effect was reversed by tolbutamide suggests it reflects activation of K ATP channels. It is, therefore, more likely that activation of K ATP channels drives the change in membrane potential in α-cells in response to increased glucose levels. In addition, the current observation that ATP is reduced may also be aligned with the proposed reduction in intracellular cAMP in α-cells The lower cAMP could also be caused by increases in intracellular FAs as a consequence of the lower FAO at higher glucose, as adenylate cyclase in other tissues has been suggested to be inhibited by increases in intracellular FA levels Under conditions with 0.
This suggests that the lowering of plasma levels of glucagon in response to a glucose tolerance test may also be driven by changes in FA availability.
This is supported by the ex vivo experiments presented here. However, overexpression of PDK4 in α-cells results in a rather mild phenotype. That PDK4 overexpression in α-cells alone is not enough to drive the development of hyperglycemia or hyperketonemia in vivo is not surprising.
Other models of impaired glucagon secretion also have relatively mild phenotypes 7 , 14 , 21 , In the case of this model, this may reflect that paracrine factors also contribute to the inhibition glucagon secretion.
Despite this, our data indicate that changing PDH activity in α-cells can affect circulating glucagon levels. Thus, α-cells may rely on sensing circulating levels of FA as well as glucose. However, it should be considered that other substrates, such as amino acids, could also contribute and thereby regulate glucagon secretion in α-cells In conclusion, we propose a framework for α-cell metabolism and glucose-regulated glucagon secretion, reciprocal to that observed in β-cells 65 , in which the metabolic phenotype of α-cells enables a specialized glucose response, which lowers intracellular ATP and leads to reduced glucagon secretion through activation of K ATP channels and repolarization of the plasma membrane.
This model of α-cell metabolism and glucagon secretion suggests that α-cells can act as sensors of changes in both circulating glucose and NEFA concentrations.
The authors thank Dorthe Nielsen University of Copenhagen for technical assistance during data collection, Professor Leanne Hodson and Dr. Katherine Pinnick University of Oxford for scientific advice, and Professor Seung Kim Stanford University for providing the GUTR2 construct used for live cell imaging experiments.
Imaging experiments were performed at the Centre for Advanced Bioimaging CAB at the University of Copenhagen. is supported by a fellowship from Svenska Sällskapet for Medicinsk Forskning SSMF.
is supported by long term structural funding - Methusalem funding by the Flemish government, the Fund for Scientific Research-Flanders FWO-Vlaanderen , European Research Council Advanced Research Grant EU- ERC , and a Novo Nordisk Foundation Denmark NNF Laureate Research Grant.
is supported by the Novo Nordisk Foundation grant no. NNF18CC L. is supported by the Swedish Research grant SRA-Exodiab and project grant , the Swedish Foundation for Strategic Research IRC-LUDC , and The Swedish Diabetes Foundation.
is supported by the Swedish Research Council, the Helmsley Trust, and the Medical Research Council MRC , J. Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. contributed to the investigation. reviewed and edited the manuscript. and J. conceptualized the study. conceived the experimental design. wrote the original draft. contributed materials. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Prior Presentation. Parts of this study were presented as an abstract at the 56th Annual Meeting of the European Association for the Study of Diabetes, virtual meeting, 21—25 September , and at the 81st Scientific Sessions of the American Diabetes Association, virtual meeting, 25—29 June Sign In or Create an Account.
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Patients Glucagon secretion diabetes Immune system strength exhibit hyperglucagonemia, or Glucagon secretion glucagon secretion, which may Glucagon secretion the underlying cause of the secretoin Glucagon secretion diabetes. Defective alpha cell secretory responses to glucose and paracrine effectors in Performance nutrition coach Type 1 Glucaon Glucagon secretion 2 secretiom may drive the development of hyperglucagonemia. Therefore, uncovering Glucaogn mechanisms that regulate glucagon secretion from the pancreatic alpha cell is critical for developing improved treatments for diabetes. In this review, we focus on aspects of alpha cell biology for possible mechanisms for alpha cell dysfunction in diabetes: proglucagon processing, intrinsic and paracrine control of glucagon secretion, secretory granule dynamics, and alterations in intracellular trafficking. We explore possible clues gleaned from these studies in how inhibition of glucagon secretion can be targeted as a treatment for diabetes mellitus. Glucagon is a amino acid peptide hormone produced by the alpha α cells of the pancreatic islet. Sarah L. ArmourSecretioon FruehBCAA and muscle performance improvement V. ChibalinaHaiqiang DouLidia Glucagon secretion secretioj, Alexander Hamilton Glucagon secretion, Georgios Katzilieris-PetrasGlucagon secretion CarmelietBenjamin DaviesThomas MoritzLena EliassonPatrik RorsmanJakob G. Knudsen; Glucose Controls Glucagon Secretion by Regulating Fatty Acid Oxidation in Pancreatic α-Cells. Diabetes 1 October ; 72 10 : — Whole-body glucose homeostasis is coordinated through secretion of glucagon and insulin from pancreatic islets.
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