Glucagon Receptor Signaling and Lipid Metabolism

1Glucagon is processed from its precursor, proglucagon, by prohormone convertase 2 and secreted from pancreatic alpha cells. The role of glucagon in glucose metabolism has been intensively studied, and comprehensive reviews are found elsewhere. In addition to regulating glucose metabolism, glucagon also seems important for minute-to-minute regulation of amino acid metabolism as part of the recently described liver-alpha cell axis, in which amino acids stimulate glucagon secretion and glucagon in turn stimulates hepatic amino acid uptake and metabolism (ureagenesis) and, thus, circulating amino acid concentrations as well as increased hepatic NADH/NAD+ ratio. The actions of glucagon are mediated via the glucagon receptor, a seven transmembrane receptor coupled to Gαs- and Gq-proteins, which regulate adenylate cyclase (AC) and phospholipase C activities when activated. The glucagon receptor is primarily expressed in the liver, but it is also expressed in varying amounts in the central nervous system, kidneys, gastro-intestinal tract, heart (controversial), and pancreas.

2Glucagon receptor expression has been reported in rat adipocytes, where a lipolytic effect of glucagon may be of physiological relevance. As type 2 diabetic hyperglucagonaemia contributes to the hyperglycemic state of patients with type 2 diabetes (T2D), inhibition of glucagon receptor signaling has been investigated as glucose-lowering therapy in T2D patients. Interestingly, potential adverse effects of this therapeutic approach include increased low-density lipoprotein (LDL) plasma concentrations and increased hepatic fat accumulation. Furthermore, hepatocyte studies have shown that glucagon stimulates beta-oxidation, inhibits lipogenesis and decrease triglyceride (TG) and very-low-density lipoprotein (VLDL) secretion emphasizing a potentially important role of glucagon in lipid metabolism.

3Lipolysis in adipocytes depends on activation of AC and thereby increased protein kinase A (PKA) activity. PKA phosphorylates (hence activates) perilipins and hormone-sensitive lipase (HSL), and two additional lipases, resulting in hydrolysis of TGs and release of glycerol and free fatty acids (FFAs), e.g., palmitate (;;;;; Figure 1). Circulating levels of FFAs and glycerol therefore reflect the rate of lipolysis. For glucagon to directly influence adipocyte function, its cognate receptor must be expressed. Glucagon receptor mRNA has been detected in rat adipocytes, but to determine the physiological relevance of glucagon receptor mRNA expression, it is necessary to investigate whether the mRNA is actually translated into a functional receptor. Specific antibodies directed against the glucagon receptor are necessary in addressing this question, but development of specific antibodies against glucagon receptors has been challenging and the antibodies available are unspecific and therefore not suitable for receptor localization. As an example, one study reported localization of the glucagon receptor in rat adipocytes using a monoclonal antibody whereas another using autoradiography, glucagon receptors were not found to be expressed, and no studies have demonstrated presence of glucagon receptors on human adipocytes. Clearly, future studies should investigate glucagon receptor expression using antibody and antibody-independent methods.

4Glucagon has been reported to activate HSL and lipolysis in rat adipocytes within minutes at concentrations as low as 6 × 10−10 M and 10−11 M. Glucagon has also been shown to stimulate lipolysis in birds, rabbits, and human adipocytes in vitro at concentrations near 10−8 M. At physiological plasma concentrations (1–40 pM), a lipolytic effect of glucagon in human adipocytes has been difficult to demonstrate. One of the first human studies reporting a lipolytic effect of glucagon, demonstrated that an injection of 7.5 μg glucagon into the branchial artery resulted in a rapid increase in FFA plasma concentrations in the corresponding vein but this was not replicated in a similar study with mean increases of glucagon plasma concentrations by 237 pM in overnight fasted subjects. An increase in FFA plasma concentrations has been demonstrated upon glucagon infusion (mean glucagon increment 209 ± 15 pM) and intravenous injection of glucagon [reaching plasma concentrations of >1,000 pM]. Since supra-physiological glucagon concentrations were applied, these studies may lack specificity because of interaction of glucagon with other related G protein-coupled receptors (e.g., the glucagon-like peptide 1 (GLP-1) receptor). Pharmacological concentrations of glucagon also stimulate secretion of catecholamines and growth hormone, both of which have powerful lipolytic effects, possibly as part of a generalized sympathetic nervous system discharge. Glucagon was not found to have any lipolytic effects in clinical studies using glucagon concentrations ranging from 19 to 64 pM. In some clinical studies investigating the lipolytic effect of supra-physiological glucagon concentrations, the lipolytic effect of glucagon could be abolished by insulin, and in rat adipocytes insulin is a potent inhibitor of lipolysis. A lipolytic effect of glucagon, if any, on human adipocytes may therefore only be physiologically relevant when insulin secretion is low. Supporting this, a 2-h infusion of 1 ng/kg × min glucagon (presumably resulting in physiologically relevant elevations) and somatostatin in insulin-deficient diabetic subjects caused a two to three-fold increase in FFA and glycerol plasma concentrations, compared to infusion of somatostatin alone. However, when insulin, somatostatin, and glucagon were infused together, glucagon had no lipolytic effect. Furthermore, infusion with saline only gave the same increase in FFA as compared to glucagon infusion. In another study glucagon was infused at 1.2 ng/kg × min (high but also relevant) together with somatostatin for 2 h, but there was no lipolytic effect of glucagon at insulin concentrations of 38 pM. In contrast, a 2-h glucagon infusion at 1.3 ng/kg × min, during a mean insulin plasma concentration of 65 pM, increased the rate of appearance of labeled FFA and glycerol by 40 and 36%, respectively. As glucagon receptors are expressed on beta cells and may stimulate insulin secretion through both GLP-1 and glucagon receptors it may be speculated that intraislet regulation of insulin through glucagon may contribute to its effect on lipid metabolism.

5It is important to note that FFA and glycerol in plasma are not only determined by release from adipocytes, but also by rate of uptake and re-esterification in other tissues. A lack of effect of glucagon on the free plasma pool of FFA and glycerol, does therefore not rule out that glucagon has a direct effect on lipid metabolism in adipocytes and hepatocytes (Figure 1).

6In hepatocytes, glucagon action increases the transcription factor cAMP responsive element binding (CREB) protein, which induces the transcription of carnitine acyl transferase 1 (CPT-1). CPT-1 enables catabolism of long-chain fatty acids by converting fatty acids to acyl-carnitines, which are transported into the mitochondria and subjected to beta-oxidation. During beta-oxidation the fatty acids are degraded into acetate, which ultimately enters the citric acid cycle. Furthermore, through PKA-dependent phosphorylation, glucagon receptor signaling inactivates acetyl-CoA carboxylase, the enzyme catalyzing the formation of malonyl-CoA. Malonyl-CoA is the first intermediate in fatty acid synthesis and inhibits CPT-1 (i.e., inhibits beta-oxidation). By inhibiting the formation of malonyl-CoA, glucagon diverts FFAs to beta-oxidation rather than re-esterification into TGs (Figure 2). Periportal and perivenous hepatocytes receive different concentrations of substrates and oxygen and as a consequence periportal hepatocytes primarily mediate oxidative processes, including beta-oxidation, whereas perivenous hepatocytes preferentially mediate glucose uptake and lipogenesis.

7In hepatocytes, glucagon may bring about an energy-depleted state (increasing the AMP/ATP ratio) sufficient to activate AMP-activated kinase, which phosphorylates acetyl-CoA carboxylase and p38 mitogen-activated protein kinase, leading to transcriptional activation of peroxisome proliferator-activated receptor-α (PPARα). PPARα stimulates the transcription of genes involved in beta-oxidation including CPT-1, CPT-2, and acetyl-CoA oxidase, and the transcription of fibroblast growth factor 21, which is produced in the liver in response to glucagon. Glucagon also stimulates forkhead transcription factor A2 activity (FoxA2), which induces transcription of genes involved in beta-oxidation, such as CPT-1, very-, and medium- long-chain acyl-CoA dehydrogenase. Subsequent to activating its receptors on hepatocytes, insulin suppresses most of these pathways, and the metabolic state in the hepatocytes may therefore be determined by the insulin-glucagon ratio, rather than by the hormone concentrations per se. Insulin inhibits lipolysis in adipocytes and by reducing the amount of substrate (FFA and glycerol) reaching the liver may reduce hepatic gluconeogenesis.

8To investigate the physiological effects of glucagon in lipid metabolism, several studies have relied on glucagon receptor knockout (Gcgr−/−) mice or animals treated with GRA. In the livers of Gcgr−/− mice there is an increase in glycolysis and a decrease in gluconeogenesis and citric acid cycle activity, which results in decreased acetyl-CoA oxidation and acetyl-CoA accumulation. The accumulation of acetyl-CoA in the cytosol of hepatocytes results in increased lipogenesis. Supporting this, genes involved in lipogenesis, e.g., ATP citrate lyase and fatty acid synthase, were found to be upregulated in livers of Gcgr−/− mice at both the mRNA and protein level, while CPT-1 and -2 levels, and other enzymes necessary for beta-oxidation, were downregulated. Hepatic beta-oxidation is essential for the production of both glucose and ketones since it provides the substrates acetyl-CoA and acetate and mitochondrial energy supply (ATP/NADH) needed for gluconeogenesis. The hepatic gene expression profile changes markedly in response to fasting, and major differences have been reported in expression levels of genes involved in lipid metabolism between the fed and fasted state. Following a prolonged fast (16 h), wild-type mice had an increased hepatic expression of genes involved in beta-oxidation, such as CPT-1, CPT-2, and acyl-CoA dehydrogenase, but this was not observed in Gcgr−/− mice, which displayed an impaired beta-oxidation in both the fasted and fed state and Gcgr−/− mice failed to change the hepatic energy state in response to fasting. Furthermore, Gcgr−/− mice showed increased hepatic TG secretion and increased plasma concentrations of TG and FFA after a 16 h fasting period, but not after 5 h of fasting. Others also found similar TG and FFA plasma concentrations in Gcgr−/− and wild-type mice after a short-term fast; they did, however, find increased plasma concentrations of LDL in Gcgr−/− mice. Glucagon thus seems to regulate hepatic metabolism in response to fasting by stimulating glucose-producing processes, including beta-oxidation. When challenged with a high fat diet (HFD) for 8 weeks, Gcgr−/− mice did not increase the amount of inguinal and epididymal fat, whereas the amount of both doubled in wild-type mice. In line with this, others showed a decrease in white adipose tissue mass and an increase in lean body mass in Gcgr−/− compared to wild-type mice, without changes in bodyweight, food consumption, or energy expenditure and one group found that Gcgr−/− mice had lower amounts of white adipose tissue when fed both a HFD and a low fat diet compared to wild-type mice, and thus seemed to be resistant to diet-induced obesity. This could reflect an inability of Gcgr−/− mice to mobilize the hepatic lipid storage; instead adipocyte lipolysis (by catecholamines) maintain the energy supply to other metabolically active tissues.

9Administration of GRAs has been associated with increased hepatic fat content (assessed as hepatic fat fraction measured by magnetic resonance imaging) and increased plasma concentrations of LDL. Furthermore, subjects with endogenous glucagon deficiency (pancreatectomized subjects) and rats and diabetic (db/db) mice treated with glucagon antisense oligonucleotide have increased hepatic fat. These data suggest that inhibition of glucagon receptor signaling results in hepatic lipid accumulation. In addition, Gcgr−/− mice may be prone to steatosis when challenged with a high fat diet (HFD) for 8 weeks. However, a study involving a similar HFD diet for 12 weeks and mice with the same sex, gene modification, and background (C57BL/6J), showed that Gcgr−/− mice were protected from steatosis. Of notice, C57BL/6J mice do not consistently develop steatosis upon HFD feeding, and this might have influenced the results. In rats, impaired glucagon action also associates with development of hepatic steatosis. Interestingly, HFD feeding has been reported to decrease glucagon receptor expression at the plasma membrane of rat hepatocytes. These data suggest that hepatic lipid accumulation may cause impaired glucagon receptor signaling, and that this (as demonstrated using GRAs) may contribute to and accelerate hepatic lipid accumulation.

10Acute administration of 30 μg/kg glucagon decreased FFA and TG plasma concentrations and reduced hepatic TG content and secretion in mice. Chronic hyperglucagonemia (injection of 10 μg glucagon every 8 h for 21 days) had hypolipidemic effects in rats, evident by a 70 and 38% decrease in plasma concentrations of TGs and phospholipids, respectively. Consistent with this, glucagon inhibited synthesis and secretion of TGs in cultured hepatocytes, in perfused rat livers, and decreased the synthesis of hepatic VLDL in rats. In humans, hyperglucagonemia (56 ± 20 pM), during a pancreatic clamp, reduced hepatic lipoprotein particle turnover, and glucagon administration increased hepatic beta-oxidation in humans. In diet-induced obese (DIO) mice, a once-weekly treatment with 70 nmol/kg glucagon/GLP-1 receptor co-agonist resulted in loss of fat mass, which in the same study was also found, although less pronounced in GLP-1 receptor knockout mice, and improved hepatic lipid metabolism and steatosis within 4 weeks. Another glucagon/GLP-1 co-agonist (1.9 μmol/kg daily for 14 days) decreased acetyl-CoA and malonyl-CoA concentrations and increased CPT-1 mRNA in the livers of DIO mice, whereas a selective GLP-1 receptor agonist had no effect. Both of these dual agonists reduced hepatic steatosis, increased HSL activity in adipocytes, and improved dyslipidemia in DIO mice. Supporting these data, other glucagon/GLP-1 receptor co-agonists have been reported to lower plasma concentrations of TG and cholesterol, decrease hepatic fat content, and reduce adipose mass in rodent models of T2D and obesity. Importantly, acute administration of 25 nmol/kg glucagon/GLP-1 co-agonist decreased plasma concentration of TGs, cholesterol, and LDL in DIO mice within 1 h, whereas liraglutide (a pure GLP-1 receptor agonist) administration had no effect. In addition, hepatic synthesis of VLDL and palmitate, and fatty acid esterification decreased, while beta-oxidation and LDL receptors expression increased upon co-agonist, but not liraglutide, administration. The inhibitory effect on hepatic lipogenesis and stimulatory effect on beta-oxidation therefore seems to be mediated by glucagon receptor signaling. Several clinical studies are currently investigating the potential treatment of obesity and T2D using glucagon/GLP-1 co-agonists.

11FFAs are under certain circumstances insulin secretagogs but their ability to stimulate glucagon secretion remains debated. Some clinical studies found a suppression of glucagon secretion at increased FFA concentrations whereas isolated alpha cells were shown to secrete glucagon in response to FFA stimulation. In isolated rat pancreatic islets, palmitate stimulated glucagon secretion. Others found palmitate to stimulate glucagon secretion in a glucose-dependent manner using isolated pancreatic islets; increasing at glucose concentrations of 2.8, 5.6, and 10 mM but not at 16.7 mM. Medium and long-chain fatty acids (>C5) have been reported to stimulate glucagon secretion by activation of FFA receptor G protein-coupled receptor 40 (GPR40) and GPR119, both present in the pancreatic islets. FFAs may also function as metabolic substrate and stimulate alpha cell secretion through beta-oxidation. FFAs decrease secretion of somatostatin, and may lower the tonic inhibition of somatostatin on alpha cells. A clinical study investigating the effects of ingestion of lipids on hormone secretion, found no change in glucagon secretion after intravenous or oral administration of a lipid emulsion (3 ml/kg), neither did glucagon plasma concentrations change upon a 300 min lipid infusion raising FFA plasma concentrations from 0.4 to 0.8 mM. No difference in glucagon secretion was observed between subjects consuming a HFD or a low-fat diet for 2 weeks. In contrast to this, ingestion of long–chain fatty acids (olive oil and C8 fatty acids) lead to increased plasma concentrations of glucagon 40 min after, whereas no increase was observed after ingestion of short-chain fatty acids (C4), however, glucose-dependent insulinotropic polypeptide (GIP) concentrations also increased upon ingestion of long-chain fatty acids and this may have caused an increase in glucagon secretion. Another study observed that a meal rich in mono-unsaturated fatty acids resulted in a larger glucagon response when compared to a control meal. Others also observed an increase in glucagon concentrations upon fat-enriched meals. The glucagon response observed upon a 90 min intraduodenal infusion of linoleic, oleic, and palmitic acids were significant lower than observed upon protein infusion. Studies of ability of FFAs to stimulate glucagon secretion are complex, since FFAs are found in many forms and their stimulatory effect may vary [as is the case for incretin secretion]. Furthermore, the increased glucagon concentrations reported in some studies may result from other proglucagon products (e.g., glicentin or oxyntomodulin), since measurements of plasma glucagon concentrations have been marred with problems regarding sensitivity and specificity, and further studies investigating the regulation of glucagon secretion by FFAs are needed.

12Glucagon may, aside from its physiological actions on glucose and amino acid metabolism, also be important for lipid metabolism via effects on hepatic beta-oxidation and lipogenesis, and potentially increased lipolysis in adipocytes. A direct role of glucagon on adipocytes may be of importance in rodents, as glucagon stimulates lipolysis, whereas in humans an adipocyte-dependent lipolysis of glucagon is more complex. In both rodents and humans, glucagon is a powerful regulator of hepatic lipid metabolism as highlighted in studies using GRAs. The clinical use of GRAs is further challenged by glucagon’s role in amino acid metabolism, and blocking the glucagon receptor results in hyperaminoacidemia and eventually alpha cell hyperplasia. Treatment of diabetes using the current GRAs may therefore not be feasible, however, one may speculate that targeted antagonism of glucagon signaling may circumvent these unwarranted side-effects. Currently glucagon receptor agonists, combined with GLP-1 and GIP receptor agonists, are investigated as possible therapeutic agents. In preclinical studies, these agents improve steatosis and dyslipidemia, possibly as a consequence of regulation of hepatic lipid metabolism by glucagon agonism.

13Taken together, glucagon seems to play an important physiological role in the acute regulation of lipid metabolism but clearly further studies particularly in humans are warranted.

14All authors wrote and approved the final edition of the manuscript.

15The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.