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AIMS/HYPOTHESES: Glucagon and glucagon-like peptide-1 (GLP-1) are derived from the same precursor; proglucagon, and dual agonists of their receptors are currently being explored for the treatment of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Elevated levels of endogenous glucagon (hyperglucagonaemia) have been linked with hyperglycaemia in individuals with type 2 diabetes but are also observed in individuals with obesity and MASLD. GLP-1 levels have been reported to be largely unaffected or even reduced in similar conditions. We investigated potential determinants of plasma proglucagon and associations of glucagon receptor signalling with metabolic diseases based on data from the UK Biobank. METHODS: We used exome sequencing data from the UK Biobank for ~410,000 white participants to identify glucagon receptor variants and grouped them based on their known or predicted signalling. Data on plasma levels of proglucagon estimated using Olink technology were available for a subset of the cohort (~40,000). We determined associations of glucagon receptor variants and proglucagon with BMI, type 2 diabetes and liver fat (quantified by liver MRI) and performed survival analyses to investigate if elevated proglucagon predicts type 2 diabetes development. RESULTS: Obesity, MASLD and type 2 diabetes were associated with elevated plasma levels of proglucagon independently of each other. Baseline proglucagon levels were associated with the risk of type 2 diabetes development over a 14 year follow-up period (HR 1.13; 95% CI 1.09, 1.17; n=1562; p=1.3×10-12). This association was of the same magnitude across strata of BMI. Carriers of glucagon receptor variants with reduced cAMP signalling had elevated levels of proglucagon (ß 0.847; 95% CI 0.04, 1.66; n=17; p=0.04), and carriers of variants with a predicted frameshift mutation had higher levels of liver fat compared with the wild-type reference group (ß 0.504; 95% CI 0.03, 0.98; n=11; p=0.04). CONCLUSIONS/INTERPRETATION: Our findings support the suggestion that glucagon receptor signalling is involved in MASLD, that plasma levels of proglucagon are linked to the risk of type 2 diabetes development, and that proglucagon levels are influenced by genetic variation in the glucagon receptor, obesity, type 2 diabetes and MASLD. Determining the molecular signalling pathways downstream of glucagon receptor activation may guide the development of biased GLP-1/glucagon co-agonist with improved metabolic benefits. DATA AVAILABILITY: All coding is available through https://github.com/nicwin98/UK-Biobank-GCG.
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Increased plasma concentrations of glucagon (hyperglucagonemia) are reported in patients with type 2 diabetes (T2D) and act as a prediabetogenic risk factor. Emerging evidence suggests that hepatic steatosis in obesity is causing a condition of glucagon resistance towards amino acid catabolism, resulting in a compensatory hyperglucagonemia. We investigated the presence of hyperglucagonemia in individuals with biopsy-verified metabolic dysfunction-associated steatotic liver disease (MASLD), and whether body mass index (BMI), T2D, hepatic steatosis and/or fibrosis contribute to this relationship. To dissect potential mechanisms, we determined hepatic gene expression related to amino acid transport and catabolism. Individuals with MASLD had hyperglucagonemia (controls (n=74) versus MASLD (n=106); median [Q1, Q3]; 4 [3, 7] versus 8 [6, 13] pM), p<.0001) and were glucagon resistant (assessed by the glucagon-alanine index) (1.3 [0.9, 2.1] versus 3.3 [2.1, 5.3] pM*mM, p<.0001). These changes associated with hepatic steatosis (p<.001, R2>.25) independently of BMI, sex, age, and T2D. Plasma levels of glucagon were similar in individuals with MASLD when stratified on T2D status (MASLD-T2D (n=52) versus MASLD+T2D (n=54); 8 [6, 11] versus 8 [6, 13] pM, p=.34) and hepatic fibrosis (MASLD+F0 (n=25) versus MASLD+F1-F3 (n=67); 8.4 [7.0, 13.3] versus 7.9 [5.2, 11.6] pM, p=.43). Obesity (BMI=30kg/m2) did not alter glucagon levels (p=.65) within groups (control/MASLD). The mRNA expression of proteins involved in amino acid transport and catabolism were downregulated in MASLD. Thus, prediabetogenic hyperglucagonemia is present in individuals with biopsy-verified MASLD, and hepatic steatosis partially drives hyperglucagonemia and glucagon resistance, irrespective of T2D, BMI, and hepatic fibrosis.
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PURPOSE OF REVIEW: Glucagon increases hepatic glucose production and in patients with metabolic diseases, glucagon secretion is increased contributing to diabetic hyperglycemia. This review explores the role of amino acids and lipids in the regulation of glucagon secretion and how it may be disturbed in metabolic diseases such as obesity and metabolic associated fatty liver disease (MAFLD). RECENT FINDINGS: Human and animal studies have shown that MAFLD is associated with glucagon resistance towards amino acid catabolism, resulting in elevated plasma levels of amino acids. A recent clinical study showed that MAFLD is also associated with glucagon resistance towards lipid metabolism. In contrast, MAFLD may not decrease hepatic sensitivity to the stimulatory effects of glucagon on glucose production. SUMMARY: Elevated plasma levels of amino acids and lipids associated with MAFLD may cause diabetogenic hyperglucagonemia. MAFLD and glucagon resistance may therefore be causally linked to hyperglycemia and the development of type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Hiperglicemia , Hepatopatia Gordurosa não Alcoólica , Animais , Humanos , Glucose/metabolismo , Glucagon/metabolismo , Aminoácidos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Retroalimentação , LipídeosRESUMO
BACKGROUND AND AIMS: Patients with some chronic liver diseases have increased risk of diabetes. Whether this is also the case for patients with autoimmune liver diseases is unknown. The study aimed to calculate risk and worldwide prevalence of diabetes in patients with autoimmune hepatitis (AIH), primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). METHODS: We performed a case-control study using data from the United Kingdom Biobank (UKB) and compared frequency of type 1 diabetes (T1D) and type 2 diabetes (T2D) in AIH and PBC with age-, sex-, BMI- and ethnicity-matched controls. Next, we performed a systematic review and proportional meta-analysis searching PubMed, Embase, Cochrane Library and Web of Science (inception to 1 May 2022 [AIH]; 20 August 2022 [PBC]; 11 November 2022 [PSC]). The pooled prevalence of diabetes was calculated using an inverse method random effects model. RESULTS: Three hundred twenty-eight AIH patients and 345 PBC patients were identified in UKB and risk of T1D and T2D significantly increased compared with matched controls. Our systematic search identified 6914 records including the UKB study. Of these, 77 studies were eligible for inclusion comprising 36 467, 39 924 and 4877 individuals with AIH, PBC and PSC, respectively. The pooled prevalence of T1D was 3.8% (2.6%-5.7%), 1.7% (0.9%-3.1%), 3.1% (1.9%-4.8%) and of T2D 14.8% (11.1%-19.5%), 18.1% (14.6%-22.2%), 6.3% (2.8%-13.3%) in patients with AIH, PBC and PSC, respectively. CONCLUSIONS: Patients with autoimmune liver diseases have increased risk of diabetes. Increased awareness of diabetes risk in patients with autoimmune liver diseases is warranted.
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Doenças Autoimunes , Colangite Esclerosante , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Hepatite Autoimune , Cirrose Hepática Biliar , Hepatopatias , Humanos , Cirrose Hepática Biliar/epidemiologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Estudos de Casos e Controles , Pontuação de Propensão , Hepatopatias/epidemiologia , Doenças Autoimunes/complicações , Doenças Autoimunes/epidemiologia , Hepatite Autoimune/complicações , Hepatite Autoimune/epidemiologia , Colangite Esclerosante/complicações , Colangite Esclerosante/epidemiologiaRESUMO
Amino acids stimulate the secretion of glucagon, and glucagon receptor signaling regulates amino acid catabolism via ureagenesis, together constituting the liver-α cell axis. Impairment of the liver-α cell axis is observed in metabolic diseases such as diabetes. It is, however, unknown whether glucose affects the liver-α cell axis. We investigated the role of glucose on the liver-α cell axis in vivo and ex vivo. The isolated perfused mouse pancreas was used to evaluate the direct effect of low (3.5 mmol/L) and high (15 mmol/L) glucose levels on amino acid (10 mmol/L arginine)-induced glucagon secretion. High glucose levels alone lowered glucagon secretion, but the amino acid-induced glucagon responses were similar in high and low glucose conditions (P = 0.38). The direct effect of glucose on glucagon and amino acid-induced ureagenesis was assessed using isolated perfused mouse livers stimulated with a mixture of amino acids (VaminR, 10 mmol/L) and glucagon (10 nmol/L) during high and low glucose conditions. Urea production increased robustly but was independent of glucose levels (P = 0.95). To investigate the whole body effects of glucose on the liver-α cell axis, four groups of mice received intraperitoneal injections of glucose-Vamin (2 g/kg, + 3.5 µmol/g, respectively, G/V), saline-Vamin (S/V), glucose-saline (G/S), or saline-saline (S/S). Blood glucose did not differ significantly between G/S and G/V groups. Levels of glucagon and amino acids were similar in the G/V and S/V groups (P = 0.28). Amino acids may overrule the inhibitory effect of glucose on glucagon secretion and the liver-α cell axis may operate independently of glucose in mice.NEW & NOTEWORTHY Glucagon is an essential regulator of our metabolism. Recent evidence suggests that the physiological actions of glucagon reside in amino acid catabolism in the so-called liver-α cell axis, in which amino acids stimulate glucagon secretion and glucagon enhances hepatic amino acid catabolism. Here, it is demonstrated that this feedback system is independent of glycemia possibly explaining why hyperglycemia in diabetes may not suppress α cell secretion.
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Arginina , Glicemia , Células Secretoras de Glucagon , Glucagon , Aminoácidos/biossíntese , Animais , Arginina/metabolismo , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Insulina , Camundongos , UreiaRESUMO
Glucagon regulates the hepatic amino acid metabolism and increases ureagenesis. Ureagenesis is activated by N-acetylglutamate (NAG), formed via activation of N-acetylglutamate synthase (NAGS). With the aim to identify the steps whereby glucagon both acutely and chronically regulates ureagenesis, we investigated whether glucagon receptor-mediated activation of ureagenesis is required in a situation where NAGS activity and/or NAG levels are sufficient to activate the first step of the urea cycle in vivo. Female C57BL/6JRj mice treated with a glucagon receptor antagonist (GRA), glucagon receptor knockout (Gcgr-/-) mice, and wild-type (Gcgr+/+) littermates received an intraperitoneal injection of N-carbamoyl glutamate (Car; a stable variant of NAG), l-citrulline (Cit), Car and Cit (Car + Cit), or PBS. In separate experiments, Gcgr-/- and Gcgr+/+ mice were administered N-carbamoyl glutamate and l-citrulline (wCar + wCit) in the drinking water for 8 wk. Car, Cit, and Car + Cit significantly (P < 0.05) increased plasma urea concentrations, independently of pharmacological and genetic disruption of glucagon receptor signaling (P = 0.9). Car increased blood glucose concentrations equally in GRA- and vehicle-treated mice (P = 0.9), whereas the increase upon Car + Cit was impaired in GRA-treated mice (P = 0.008). Blood glucose concentrations remained unchanged in Gcgr-/- mice upon Car (P = 0.2) and Car + Cit (P = 0.9). Eight weeks administration of wCar + wCit did not change blood glucose (P > 0.2), plasma amino acid (P > 0.4), and urea concentrations (P > 0.3) or the area of glucagon-positive cells (P > 0.3) in Gcgr-/- and Gcgr+/+ mice. Our data suggest that glucagon-mediated activation of ureagenesis is not required when NAGS activity and/or NAG levels are sufficient to activate the first step of the urea cycle.NEW & NOTEWORTHY Hepatic ureagenesis is essential in amino acid metabolism and is importantly regulated by glucagon, but the exact mechanism is unclear. With the aim to identify the steps whereby glucagon both acutely and chronically regulates ureagenesis, we here show, contrary to our hypothesis, that glucagon receptor-mediated activation of ureagenesis is not required when N-acetylglutamate synthase activity and/or N-acetylglutamate levels are sufficient to activate the first step of the urea cycle in vivo.
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Citrulina/administração & dosagem , Glucagon/metabolismo , Glutamatos/administração & dosagem , Fígado/efeitos dos fármacos , Receptores de Glucagon/deficiência , Receptores de Glucagon/metabolismo , Ureia/sangue , Aminoácido N-Acetiltransferase/metabolismo , Animais , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Feminino , Glutamatos/metabolismo , Antagonistas de Hormônios/administração & dosagem , Fígado/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/genéticaRESUMO
Glucagon and insulin are important regulators of blood glucose. The importance of insulin receptor signaling for alpha-cell secretion and of glucagon receptor signaling for beta-cell secretion is widely discussed and of clinical interest. Amino acids are powerful secretagogues for both hormones, and glucagon controls amino acid metabolism through ureagenesis. The role of insulin in amino acid metabolism is less clear. Female C57BL/6JRj mice received an insulin receptor antagonist (IRA) (S961; 30 nmol/kg), a glucagon receptor antagonist (GRA) (25-2648; 100 mg/kg), or both GRA and IRA (GRA + IRA) 3 h before intravenous administration of similar volumes of saline, glucose (0.5 g/kg), or amino acids (1 µmol/g) while anesthetized with isoflurane. IRA caused basal hyperglycemia, hyperinsulinemia, and hyperglucagonemia. Unexpectedly, IRA lowered basal plasma concentrations of amino acids, whereas GRA increased amino acids, lowered glycemia, and increased glucagon but did not influence insulin concentrations. After administration of GRA + IRA, insulin secretion was significantly reduced compared with IRA administration alone. Blood glucose responses to a glucose and amino acid challenge were similar after vehicle and GRA + IRA administration but greater after IRA and lower after GRA. Anesthesia may have influenced the results, which otherwise strongly suggest that both hormones are essential for the maintenance of glucose homeostasis and that the secretion of both is regulated by powerful negative feedback mechanisms. In addition, insulin limits glucagon secretion, while endogenous glucagon stimulates insulin secretion, revealed during lack of insulin autocrine feedback. Finally, glucagon receptor signaling seems to be of greater importance for amino acid metabolism than insulin receptor signaling.
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Aminoácidos/metabolismo , Glicemia/metabolismo , Glucagon/metabolismo , Receptor de Insulina/metabolismo , Receptores de Glucagon/metabolismo , Aminoácidos/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Glucagon/efeitos dos fármacos , Glucose/metabolismo , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Camundongos , Peptídeos/farmacologia , Receptor de Insulina/antagonistas & inibidores , Receptores de Glucagon/antagonistas & inibidoresRESUMO
Hundred years after the discovery of glucagon, its biology remains enigmatic. Accurate measurement of glucagon has been essential for uncovering its pathological hypersecretion that underlies various metabolic diseases including not only diabetes and liver diseases but also cancers (glucagonomas). The suggested key role of glucagon in the development of diabetes has been termed the bihormonal hypothesis. However, studying tissue-specific knockout of the glucagon receptor has revealed that the physiological role of glucagon may extend beyond blood-glucose regulation. Decades ago, animal and human studies reported an important role of glucagon in amino acid metabolism through ureagenesis. Using modern technologies such as metabolomic profiling, knowledge about the effects of glucagon on amino acid metabolism has been expanded and the mechanisms involved further delineated. Glucagon receptor antagonists have indirectly put focus on glucagon's potential role in lipid metabolism, as individuals treated with these antagonists showed dyslipidemia and increased hepatic fat. One emerging field in glucagon biology now seems to include the concept of hepatic glucagon resistance. Here, we discuss the roles of glucagon in glucose homeostasis, amino acid metabolism, and lipid metabolism and present speculations on the molecular pathways causing and associating with postulated hepatic glucagon resistance.
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Glucagon/metabolismo , Receptores de Glucagon/metabolismo , Aminoácidos/metabolismo , Animais , Biomarcadores/metabolismo , Humanos , Metabolismo dos Lipídeos , Transdução de SinaisRESUMO
AIMS/HYPOTHESIS: The secretion of glucagon is controlled by blood glucose and inappropriate secretion of glucagon contributes to hyperglycaemia in diabetes. Besides its role in glucose regulation, glucagon regulates amino acid metabolism in hepatocytes by increasing ureagenesis. Disruption of this mechanism causes hyperaminoacidaemia, which in turn increases glucagon secretion. We hypothesised that hepatic insulin resistance (secondary to hepatic steatosis) via defective glucagon signalling/glucagon resistance would lead to impaired ureagenesis and, hence, increased plasma concentrations of glucagonotropic amino acids and, subsequently, glucagon. METHODS: To examine the association between glucagon and amino acids, and to explore whether this relationship was modified by hepatic insulin resistance, we studied a well-characterised cohort of 1408 individuals with normal and impaired glucose regulation. In this cohort, we have previously reported insulin resistance to be accompanied by increased plasma concentrations of glucagon. We now measure plasma levels of amino acids in the same cohort. HOMA-IR was calculated as a marker of hepatic insulin resistance. RESULTS: Fasting levels of glucagonotropic amino acids and glucagon were significantly and inversely associated in linear regression models (persisting after adjustment for age, sex and BMI). Increasing levels of hepatic, but not peripheral insulin resistance (p > 0.166) attenuated the association between glucagon and circulating levels of alanine, glutamine and tyrosine, and was significantly associated with hyperaminoacidaemia and hyperglucagonaemia. A doubling of the calculated glucagon-alanine index was significantly associated with a 30% increase in hepatic insulin resistance, a 7% increase in plasma alanine aminotransferase levels, and a 14% increase in plasma γ-glutamyltransferase levels. CONCLUSIONS/INTERPRETATION: This cross-sectional study supports the existence of a liver-alpha cell axis in humans: glucagon regulates plasma levels of amino acids, which in turn feedback to regulate the secretion of glucagon. With hepatic insulin resistance, reflecting hepatic steatosis, the feedback cycle is disrupted, leading to hyperaminoacidaemia and hyperglucagonaemia. The glucagon-alanine index is suggested as a relevant marker for hepatic glucagon signalling.
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Aminoácidos/sangue , Glucagon/sangue , Resistência à Insulina/fisiologia , Fígado/citologia , Fígado/metabolismo , Idoso , Alanina/sangue , Estudos Transversais , Feminino , Teste de Tolerância a Glucose , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-IdadeRESUMO
Glucagon secreted from the pancreatic alpha-cells is essential for regulation of blood glucose levels. However, glucagon may play an equally important role in the regulation of amino acid metabolism by promoting ureagenesis. We hypothesized that disruption of glucagon receptor signaling would lead to an increased plasma concentration of amino acids, which in a feedback manner stimulates the secretion of glucagon, eventually associated with compensatory proliferation of the pancreatic alpha-cells. To address this, we performed plasma profiling of glucagon receptor knockout ( Gcgr-/-) mice and wild-type (WT) littermates using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, and tissue biopsies from the pancreas were analyzed for islet hormones and by histology. A principal component analysis of the plasma metabolome from Gcgr-/- and WT littermates indicated amino acids as the primary metabolic component distinguishing the two groups of mice. Apart from their hyperaminoacidemia, Gcgr-/- mice display hyperglucagonemia, increased pancreatic content of glucagon and somatostatin (but not insulin), and alpha-cell hyperplasia and hypertrophy compared with WT littermates. Incubating cultured α-TC1.9 cells with a mixture of amino acids (Vamin 1%) for 30 min and for up to 48 h led to increased glucagon concentrations (~6-fold) in the media and cell proliferation (~2-fold), respectively. In anesthetized mice, a glucagon receptor-specific antagonist (Novo Nordisk 25-2648, 100 mg/kg) reduced amino acid clearance. Our data support the notion that glucagon secretion and hepatic amino acid metabolism are linked in a close feedback loop, which operates independently of normal variations in glucose metabolism.
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Aminoácidos/efeitos adversos , Aminoácidos/sangue , Comunicação Celular , Células Secretoras de Glucagon/fisiologia , Hepatócitos/fisiologia , Receptores de Glucagon/genética , Animais , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Eletrólitos/efeitos adversos , Eletrólitos/sangue , Feminino , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/patologia , Glucose/efeitos adversos , Hepatócitos/efeitos dos fármacos , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Soluções/efeitos adversosRESUMO
AIMS/HYPOTHESIS: In humans, glucagon-like peptide-1 (GLP-1) is rapidly degraded by dipeptidyl peptidase-4 to a relatively stable metabolite, GLP-1(9-36)NH2, which allows measurement of GLP-1 secretion. However, little is known about the kinetics of the GLP-1 metabolite in mice. We hypothesised that the GLP-1 metabolite is rapidly degraded in this species by neutral endopeptidase(s) (NEP[s]). METHODS: We administered glucose, mixed meal or water orally to 256 mice, and took blood samples before and 2, 6, 10, 20, 30, 60 or 90 min after stimulation. To study the metabolism of the GLP-1 metabolite, i.v. GLP-1(9-36)NH2 (800 fmol) or saline (154 mmol/l NaCl) was administered to 160 mice, some of which had a prior injection of a selective NEP 24.11 ± inhibitor (candoxatril, 5 mg/kg) or saline. Blood was collected before and 1, 2, 4 and 12 min after GLP-1/saline injection. Plasma GLP-1 levels were analysed using a customised single-site C-terminal ELISA, two different two-site ELISAs and MS. RESULTS: GLP-1 secretion profiles after oral glucose administration differed markedly when assayed by C-terminal ELISA compared with sandwich ELISAs, with the former showing a far higher peak value and AUC. In mice injected with GLP-1(9-36)NH2, immunoreactive GLP-1 plasma levels peaked at approximately 75 pmol/l at 1 min when measured with sandwich ELISAs, returning to baseline (~20 pmol/l) after 12 min, but remained elevated using the C-terminal ELISA (~90 pmol/l at 12 min). NEP 24.11 inhibition by candoxatril significantly attenuated GLP-1(9-36)NH2 degradation in vivo and in vitro. MS identified GLP-1 fragments consistent with NEP 24.11 degradation. CONCLUSIONS/INTERPRETATION: In mice, the GLP-1 metabolite is eliminated within a few minutes owing to endoproteolytic cleavage by NEP 24.11. Therefore, accurate measurement of GLP-1 secretion in mice requires assays for NEP 24.11 metabolites. Conventional sandwich ELISAs are inadequate because of endoproteolytic cleavage of the dipeptidyl peptidase-4-generated metabolite.
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Peptídeo 1 Semelhante ao Glucagon/sangue , Período Pós-Prandial/fisiologia , Animais , Feminino , Glucose/farmacologia , Indanos/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Neprilisina/antagonistas & inibidores , Período Pós-Prandial/efeitos dos fármacos , Propionatos/farmacologia , Inibidores de Proteases/farmacologiaRESUMO
INTRODUCTION: Glucagon receptor agonism is currently explored for the treatment of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). The metabolic effects of glucagon receptor agonism may in part be mediated by increases in circulating levels of Fibroblast Growth Factor 21 (FGF21) and Growth Differentiation Factor 15 (GDF15). The effect of glucagon agonism on FGF21 and GDF15 levels remains uncertain, especially in the context of elevated insulin levels commonly observed in metabolic diseases. METHODS: We investigated the effect of a single bolus of glucagon and a continuous infusion of glucagon on plasma concentrations of FGF21 and GDF15 in conditions of endogenous low or high insulin levels. The studies included individuals with overweight with and without MASLD, healthy controls (CON) and individuals with type 1 diabetes (T1D). The direct effect of glucagon on FGF21 and GDF15 was evaluated using our in-house developed isolated perfused mouse liver model. RESULTS: FGF21 and GDF15 correlated with plasma levels of insulin, but not glucagon, and their secretion was highly increased in MASLD compared with CON and T1D. Furthermore, FGF21 levels in individuals with overweight with or without MASLD did not increase after glucagon stimulation when insulin levels were kept constant. FGF21 and GDF15 levels were unaffected by direct stimulation with glucagon in the isolated perfused mouse liver. CONCLUSION: The glucagon-induced secretion of FGF21 and GDF15 is augmented in MASLD and may depend on insulin. Thus, glucagon receptor agonism may augment its metabolic benefits in patients with MASLD through enhanced secretion of FGF21 and GDF15.
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Fatores de Crescimento de Fibroblastos , Glucagon , Fator 15 de Diferenciação de Crescimento , Fator 15 de Diferenciação de Crescimento/metabolismo , Fator 15 de Diferenciação de Crescimento/sangue , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/sangue , Glucagon/sangue , Glucagon/metabolismo , Animais , Humanos , Camundongos , Masculino , Feminino , Adulto , Insulina/farmacologia , Insulina/sangue , Insulina/metabolismo , Pessoa de Meia-Idade , Fígado/metabolismo , Fígado/efeitos dos fármacos , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/sangue , Obesidade/metabolismo , Camundongos Endogâmicos C57BL , Fígado Gorduroso/metabolismo , Sobrepeso/metabolismoRESUMO
The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.
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Lipólise , Ureia , Ratos , Camundongos , Animais , Ureia/metabolismo , Fígado/metabolismo , Veia Cava Inferior , Glucose/metabolismo , Modelos Animais de Doenças , Perfusão/métodosRESUMO
Autoimmune liver diseases include autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis. They are chronic, heterogenous diseases affecting the liver which is a key metabolic organ that ensures glucose homeostasis. It is well known that patients with other chronic liver diseases such as cirrhosis and nonalcoholic fatty liver disease (NAFLD) display glucose disturbances like insulin resistance and have an increased risk of diabetes. Previous evidence on glucose disturbances in patients with autoimmune liver disease is scarce but does point towards a potentially increased risk of type 1 diabetes and type 2 diabetes. The underlying mechanisms are unknown but may reflect genetic predisposition, concurrent NAFLD and or cirrhosis development, and treatment (steroid) related impairment of glucose homeostasis. Therefore, increased awareness and surveillance of diabetes development in patients with autoimmune liver disease may be important. Overall, detection and treatment of diabetes generally follow the usual diabetes guidelines; however, in patients with advanced liver cirrhosis, HbA1c may not be a reliable marker of average glucose levels, and treatment with insulin is generally recommended. In addition, it has recently been suggested that sodium-glucose cotransporter 2 inhibitors may be beneficial in treating refractory ascites. Further research on diabetes risk in autoimmune liver disease is warranted.
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Doenças Autoimunes , Colangite Esclerosante , Diabetes Mellitus Tipo 2 , Hepatite Autoimune , Cirrose Hepática Biliar , Hepatopatias , Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/complicações , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Cirrose Hepática Biliar/etiologia , Colangite Esclerosante/complicações , Hepatopatias/diagnóstico , Hepatopatias/epidemiologia , Hepatopatias/etiologia , Doenças Autoimunes/complicações , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/tratamento farmacológico , Hepatite Autoimune/complicações , Hepatite Autoimune/diagnóstico , Hepatite Autoimune/tratamento farmacológico , Doença Crônica , Cirrose Hepática/complicações , Glucose , FígadoRESUMO
Glucagon and insulin are the main regulators of blood glucose. While the actions of insulin are extensively mapped, less is known about glucagon. Besides glucagon's role in glucose homeostasis, there are additional links between the pancreatic α-cells and the hepatocytes, often collectively referred to as the liver-α-cell axis, that may be of importance for health and disease. Thus, glucagon receptor antagonism (pharmacological or genetic), which disrupts the liver-α-cell axis, results not only in lower fasting glucose but also in reduced amino acid turnover and dyslipidemia. Here, we review the actions of glucagon on glucose homeostasis, amino acid catabolism, and lipid metabolism in the context of the liver-α-cell axis. The concept of glucagon resistance is also discussed, and we argue that the various elements of the liver-α-cell axis may be differentially affected in metabolic diseases such as diabetes, obesity, and nonalcoholic fatty liver disease (NAFLD). This conceptual rethinking of glucagon biology may explain why patients with type 2 diabetes have hyperglucagonemia and how NAFLD disrupts the liver-α-cell axis, compromising the normal glucagon-mediated enhancement of substrate-induced amino acid turnover and possibly fatty acid ß-oxidation. In contrast to amino acid catabolism, glucagon-induced glucose production may not be affected by NAFLD, explaining the diabetogenic effect of NAFLD-associated hyperglucagonemia. Consideration of the liver-α-cell axis is essential to understanding the complex pathophysiology underlying diabetes and other metabolic diseases.
Assuntos
Diabetes Mellitus Tipo 2 , Hepatopatia Gordurosa não Alcoólica , Aminoácidos/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucagon/metabolismo , Glucose , Hepatócitos/metabolismo , Humanos , Insulina/metabolismoRESUMO
The pancreatic hormone, glucagon, is known to regulate hepatic glucose production, but recent studies suggest that its regulation of hepatic amino metabolism is equally important. Here, we show that chronic glucagon receptor activation with a long-acting glucagon analog increases amino acid catabolism and ureagenesis and causes alpha cell hypoplasia in female mice. Conversely, chronic glucagon receptor inhibition with a glucagon receptor antibody decreases amino acid catabolism and ureagenesis and causes alpha cell hyperplasia and beta cell loss. These effects were associated with the transcriptional regulation of hepatic genes related to amino acid uptake and catabolism and by the non-transcriptional modulation of the rate-limiting ureagenesis enzyme, carbamoyl phosphate synthetase-1. Our results support the importance of glucagon receptor signaling for amino acid homeostasis and pancreatic islet integrity in mice and provide knowledge regarding the long-term consequences of chronic glucagon receptor agonism and antagonism.
RESUMO
Glucagon is a major regulator of metabolism and drugs targeting the glucagon receptor (GCGR) are being developed. Insight into tissue and cell-specific expression of the GCGR is important to understand the biology of glucagon and to differentiate between direct and indirect actions of glucagon. However, it has been challenging to localize the GCGR in tissue due to low expression levels and lack of specific methods. Immunohistochemistry has frequently been used for GCGR localization, but antibodies targeting G-protein-coupled-receptors may be inaccurate. We evaluated all currently commercially available GCGR antibodies. The antibody, ab75240 (Antibody no. 11) was found to perform best among the twelve antibodies tested and using this antibody we found expression of the GCGR in the kidney, liver, preadipocytes, pancreas, and heart. Three antibody-independent approaches all confirmed the presence of the GCGR within the pancreas, liver and the kidneys. GCGR expression should be evaluated by both antibody and antibody-independent approaches.
Assuntos
Glucagon , Receptores de Glucagon , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Expressão Gênica , Anticorpos/metabolismo , Fígado/metabolismoRESUMO
OBJECTIVE: Treatment with glucagon receptor antagonists (GRAs) reduces blood glucose but causes dyslipidemia and accumulation of fat in the liver. We investigated the acute and chronic effects of glucagon on lipid metabolism in mice. METHODS: Chronic effects of glucagon receptor signaling on lipid metabolism were studied using oral lipid tolerance tests (OLTTs) in overnight fasted glucagon receptor knockout (Gcgr-/-) mice, and in C57Bl/6JRj mice treated with a glucagon receptor antibody (GCGR Ab) or a long-acting glucagon analogue (GCGA) for eight weeks. Following treatment, liver tissue was harvested for RNA-sequencing and triglyceride measurements. Acute effects were studied in C57Bl/6JRj mice treated with a GRA or GCGA 1 h or immediately before OLTTs, respectively. Direct effects of glucagon on hepatic lipolysis were studied using isolated perfused mouse liver preparations. To investigate potential effects of GCGA and GRA on gastric emptying, paracetamol was, in separate experiments, administered immediately before OLTTs. RESULTS: Plasma triglyceride concentrations increased 2-fold in Gcgr-/- mice compared to their wild-type littermates during the OLTT (P = 0.001). Chronic treatment with GCGR Ab increased, whereas GCGA treatment decreased, plasma triglyceride concentrations during OLTTs (P < 0.05). Genes involved in lipid metabolism were upregulated upon GCGR Ab treatment while GCGA treatment had opposite effects. Acute GRA and GCGA treatment, respectively, increased (P = 0.02) and decreased (P = 0.003) plasma triglyceride concentrations during OLTTs. Glucagon stimulated hepatic lipolysis, evident by an increase in free fatty acid concentrations in the effluent from perfused mouse livers. In line with this, GCGR Ab treatment increased, while GCGA treatment decreased, liver triglyceride concentrations. The effects of glucagon appeared independent of changes in gastric emptying of paracetamol. CONCLUSIONS: Glucagon receptor signaling regulates triglyceride metabolism, both chronically and acutely, in mice. These data expand glucagon´s biological role and implicate that intact glucagon signaling is important for lipid metabolism. Glucagon agonism may have beneficial effects on hepatic and peripheral triglyceride metabolism.
Assuntos
Glucagon , Receptores de Glucagon , Triglicerídeos , Animais , Camundongos , Acetaminofen/farmacologia , Glucagon/metabolismo , Metabolismo dos Lipídeos/fisiologia , Camundongos Endogâmicos C57BL , Receptores de Glucagon/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
CONTEXT: Inhibitors of the protease neprilysin (NEP) are used for treating heart failure, but are also linked to improvements in metabolism. NEP may cleave proglucagon-derived peptides, including the glucose and amino acid (AA)-regulating hormone glucagon. Studies investigating NEP inhibition on glucagon metabolism are warranted. OBJECTIVE: This work aims to investigate whether NEP inhibition increases glucagon levels. METHODS: Plasma concentrations of glucagon and AAs were measured in eight healthy men during a mixed meal with and without a single dose of the NEP inhibitor/angiotensin II type 1 receptor antagonist, sacubitril/valsartan (194 mg/206 mg). Long-term effects of sacubitril/valsartan (8 weeks) were investigated in individuals with obesity (nâ =â 7). Mass spectrometry was used to investigate NEP-induced glucagon degradation, and the derived glucagon fragments were tested pharmacologically in cells transfected with the glucagon receptor (GCGR). Genetic deletion or pharmacological inhibition of NEP with or without concomitant GCGR antagonism was tested in mice to evaluate effects on AA metabolism. RESULTS: In healthy men, a single dose of sacubitril/valsartan significantly increased postprandial concentrations of glucagon by 228%, concomitantly lowering concentrations of AAs including glucagonotropic AAs. Eight-week sacubitril/valsartan treatment increased fasting glucagon concentrations in individuals with obesity. NEP cleaved glucagon into 5 inactive fragments (in vitro). Pharmacological NEP inhibition protected both exogenous and endogenous glucagon in mice after an AA challenge, while NEP-deficient mice showed elevated fasting and AA-stimulated plasma concentrations of glucagon and urea compared to controls. CONCLUSION: NEP cleaves glucagon, and inhibitors of NEP result in hyperglucagonemia and may increase postprandial AA catabolism without affecting glycemia.
RESUMO
OBJECTIVE: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear. METHODS: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD). RESULTS: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis). CONCLUSIONS: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.