Previous studies have shown that the α cell is critical for a normal counterregulatory response to insulin-induced hypoglycemia (4
). In fact, glucagon is widely thought to provide the primary defense against a low blood glucose level. On the other hand, insulin is known to exert a powerful restraining effect on glucagon’s action (3
). This raises the question of how glucagon can have such a prominent role in counterregulation if it is so easily subject to insulin’s inhibitory action. The aim of the present study, therefore, was to determine the extent to which hypoglycemia enhances glucagon’s ability to overcome insulin’s inhibitory action on the liver and to shed light on the mechanism by which this occurs. The present results indicate that hypoglycemia (~50 mg/dl), or some factor associated with it, enhanced glucagon’s ability to increase glucose production almost 3-fold, even in the presence of extremely high insulin levels. Furthermore, they showed that this change reflected a marked increase in glycogenolysis, which was associated with hypoglycemia-induced inhibition of insulin signaling and increased AMPK activity caused by the combined effects of the fall in glucose and the rise in glucagon.
In the current study, in the presence of hyperinsulinemia and euglycemia, a physiologic rise in glucagon mimicking that occurs in response to insulin-induced hypoglycemia, caused an increase in NHGO that had a ΔAUC of 85 mg/kg/120 min. When the same rise in glucagon was brought about under hypoglycemic conditions, it produced a 3-fold greater increase (251 mg/kg/120 min) in NHGO. This correlated with a 2.3-fold greater increase in glycogenolysis and little or no difference in gluconeogenesis. It should be noted that the changes in plasma epinephrine, norepinephrine, and cortisol that occurred in response to hypoglycemia were the same whether glucagon was increased or not; thus, a differential response in their secretion or subsequent effects on the liver cannot explain the difference in glucose production in the 2 hypoglycemic groups.
Although differences in gluconeogenesis were not part of the explanation for the increase in glucagon action, hypoglycemia with or without glucagon (SH and GH) caused a marked 1.7-mg/kg/min increase in NHGNG flux. This leads to the question of how the increase in gluconeogenesis came about, since the cellular indices of the gluconeogenic program were either at or below values evident in the control animals. The answer lies in the fact that hypoglycemia caused marked increases in the release of lactate from muscle and glycerol from fat. This resulted in large increments in their delivery to and uptake by the liver, which in turn caused a substrate-driven increase in gluconeogenic flux. The increase in muscle glycogenolysis undoubtedly resulted from the hypoglycemia-driven rise in plasma epinephrine, whereas the increase in lipolysis probably resulted from the hypoglycemia-driven increase in neural input (norepinephrine) to fat (10
The question then arises as to the mechanism by which the liver’s glycogenolytic responsiveness to glucagon was enhanced by hypoglycemia. It is unclear which physiologic signal — increased cortisol; increased epinephrine; increased norepinephrine; increased neural input to the liver, fat, and muscle; hypoglycemia per se; or some combination of these — explains this important adaptive response. It is clear that the liver is capable of adjusting its glucose output in response to variations in the blood glucose concentration, independent of hormonal changes (12
). This phenomenon of hepatic autoregulation has been demonstrated to occur in various in vitro preparations (14
) and in vivo (12
). It is therefore possible that hypoglycemia per se sensitized the liver to glucagon.
Another possibility is that the enhanced response of the liver to glucagon during insulin-induced hypoglycemia is related to the interaction of glucagon with one or other of the counterregulatory hormones. Several studies have looked at the acute interaction of the counterregulatory hormones (17
). Lecavalier et al. (19
) performed studies in humans to assess the interaction between glucagon and cortisol on gluconeogenesis from 14
C lactate. In their studies, glucose was clamped, and they used a pancreatic pituitary clamp to control for changes in insulin, glucagon, and growth hormone. They found that cortisol and glucagon stimulated gluconeogenesis in an additive manner. However, the increment in glucagon that they used was very high, so it is conceivable that a synergistic effect of the hormones could have been missed. Gustavson et al. (20
) studied the interaction of epinephrine and glucagon in the regulation of hepatic glucose production at a time when plasma insulin and hyperglycemia were fixed in overnight-fasted conscious dogs. They observed that the effects of glucagon and epinephrine on hepatic glucose production were additive, but not synergistic. Thus, the data currently available do not support a synergistic interaction between glucagon and the other counterregulatory hormones. It must be remembered, however, that none of the above studies were carried out under hypoglycemic conditions. It is possible that the combination of hypoglycemia and the rise in counterregulatory hormones is critical to the enhanced response.
It is also of interest to examine the cellular changes associated with the increased glycogenolytic effect of glucagon. Berglund et al. (21
) recently demonstrated that a physiological rise in glucagon or hypoglycemia can activate AMPK in mouse liver. In addition, previous studies have shown that portal infusion of the AMPK activator AICAR stimulated hepatic glucose production (through an increase in glycogenolysis) despite substantial hyperinsulinemia and fixed glucagon levels (22
). In light of these data, we evaluated hepatic AMPK activation in the present studies. In the presence of high insulin, low glucagon, and euglycemia, P-AMPK (Thr172) was not different from that in control animals. On the other hand, the addition of glucagon (GE) or hypoglycemia (SH) led to a similar increase in AMPK (Thr172) phosphorylation. Furthermore, the combination of glucagon and hypoglycemia had an additive effect on AMPK phosphorylation. Since AMPK activation correlated with NHGLY flux in all groups, one can speculate that AMPK played a key regulatory role in promoting glycogenolysis in response to glucagon and/or hypoglycemia.
It is possible that AMPK regulates glycogen synthase and glycogen phosphorylase within the liver, although to date, this regulation has not been clearly established in vivo. To our knowledge, there is no evidence that AMPK can directly phosphorylate GP. However, a recent in vitro study suggested that AMPK may indirectly reduce PP1-mediated dephosphorylation of GS and GP, the net effect of which would favor glycogenolysis (23
). In addition, AMPK can directly phosphorylate GS at Ser7 (24
). Furthermore, Ser7 has been shown to be the dominant phosphorylation site responsible for GS inhibition, at least in isolated hepatocytes (25
). Unfortunately, we were unable to evaluate GS (Ser7) phosphorylation because of the unavailability of a commercial antibody. However, the alterations (increased GP, decreased GS) observed with either hypoglycemia or glucagon correlated with AMPK activation. That the most marked change in the activity ratios of GP and GS (favoring glycogenolytic flux) occurred with combination of glucagon and hypoglycemia (and the greatest increase in AMPK activation) is consistent with the suggestion that AMPK may be one of the prominent regulators of glycogen metabolism in this context.
It is also of interest to note that AMPK phosphorylation at Ser485 was markedly enhanced by the presence of high insulin (SE). Akt is known to phosphorylate hepatic AMPK at Ser485, which prevents phosphorylation of AMPK at Thr172 and activation of the enzyme (26
). Thus, Ser485 phosphorylation can be considered an index of AMPK inhibition by insulin. Ser485 phosphorylation was reduced equivalently by glucagon and/or hypoglycemia in the present study, whereas previous studies in isolated cells indicated that cAMP-elevating agents increased Ser485 phosphorylation of AMPK (27
). This discrepancy may be explained, at lease in part, by the fact that the former studies were performed in vitro and thus lacked important inputs for hepatic glucose regulation that were present in our studies. In any case, we believe our data are the first to suggest that AMPK dephosphorylation at Ser485, mediated by either glucagon or hypoglycemia, may be relevant to the ability of the liver to overcome the effects of insulin on glucose production.
In the presence of high insulin, low glucagon, and euglycemia (SE), the insulin signaling pathway was strongly activated (as evidenced by marked increases in phosphorylated Akt and GSK3β and decreased phosphorylation of GS at Ser641), which was correlated with inhibited glycogenolysis and stimulated glycogen synthesis. The addition of glucagon under euglycemic conditions (GE) led to an increase in glycogenolytic flux, despite no attenuation of insulin signaling. When hypoglycemia was allowed to occur in the absence of glucagon (SH), insulin signaling was blunted (decreased phosphorylation of Akt, GSK3β, and increased Ser641 phosphorylation of GS), and this resulted in increased glycogenolysis. The addition of glucagon to hypoglycemia (GH) did not cause a further reduction in insulin signaling, yet it caused the greatest increase in the glycogenolytic rate. Thus, our data suggest that hypoglycemia, or some aspect associated with it, can interfere with insulin-mediated regulation of Akt, GSK3β, and GS (Ser641), which undoubtedly facilitates hepatic glycogenolysis. Moreover, we posit that the addition of glucagon (which does not impair insulin signaling) stimulated glycogenolysis, possibly through AMPK-mediated alteration of GS and GP activities. Finally, this glycogenolytic effect was magnified when glucagon and hypoglycemia were combined, leading to an additive effect on AMPK activation, in the presence of hypoglycemia-mediated impairment of insulin signaling.
Although gluconeogenic flux was not a major factor in increased glucose production in the acute response to glucagon and/or hypoglycemia, it is interesting to note that the substantial suppression of the gluconeogenic program at the genetic level was only modestly lessened by addition of glucagon and/or the hypoglycemic condition. Insulin (SE) predictably led to increased FOXO1, STAT3, and CRTC2 phosphorylation and decreased CREB phosphorylation, all of which likely factored into the substantial decrease observed in gluconeogenic gene expression. Addition of glucagon and/or hypoglycemia (GE, SH, and GH) led to slight increases in gluconeogenic mRNA that correlated with alterations in CREB and FOXO1 phosphorylation. However, STAT3 and CRTC2 phosphorylation were maintained at elevated levels, and since gluconeogenic gene expression was still largely suppressed, it appears that these factors may dominate the control of PCK1
gene expression, at least during hyperinsulinemia. In addition, AMPK, much like insulin, can phosphorylate CRTC2, resulting in the cytoplasmic sequestration of this protein and a consequent reduction in gluconeogenic gene expression (28
). However, our results indicate that the level of CRTC2 phosphorylation in response to insulin (SE) was not further increased in the GE, SH or GH groups despite the activation of AMPK. It therefore appears that in the presence of marked hyperinsulinemia, AMPK activation by glucagon and/or hypoglycemia does not cause further CRTC2 phosphorylation.
In summary, hypoglycemia increased glucagon’s ability to overcome insulin’s inhibitory effect on hepatic glucose production. This effect was attributable to marked enhancement of net glycogen breakdown with no effect on gluconeogenesis. At the cellular level, it was associated with reduced insulin signaling caused by some aspect of hypoglycemia and by activation of AMPK resulting from the combination of increased glucagon and hypoglycemia. Which aspect of hypoglycemia causes these changes (hypoglycemia per se, or some other component of the counterregulatory response) remains to be determined.