Glucagon-like peptide-1 analog liraglutide leads to multiple metabolic alterations in diet-induced obese mice

Liraglutide, a glucagon-like peptide-1 analog, has beneficial metabolic effects in patients with type 2 diabetes and obesity. Although the high efficacy of liraglutide as an anti-diabetic and anti-obesity drug is well known, liraglutide-induced metabolic alterations in diverse tissues remain largely unexplored. Here, we report the changes in metabolic profiles induced by a 2-week subcutaneous injection of liraglutide in diet-induced obese mice fed a high-fat diet for 8 weeks. Our comprehensive metabolomic analyses of the hypothalamus, plasma, liver, and skeletal muscle showed that liraglutide intervention led to various metabolic alterations in comparison with diet-induced obese or nonobese mice. We found that liraglutide remarkably coordinated not only fatty acid metabolism in the hypothalamus and skeletal muscle but also amino acid and carbohydrate metabolism in plasma and liver. Comparative analyses of metabolite dynamics revealed that liraglutide rewired intertissue metabolic correlations. Our study points to a previously unappreciated metabolic alteration by liraglutide in several tissues, which may underlie its therapeutic effects within and across the tissues. Liraglutide, a glucagon-like peptide-1 analog, has beneficial metabolic effects in patients with type 2 diabetes and obesity. Although the high efficacy of liraglutide as an anti-diabetic and anti-obesity drug is well known, liraglutide-induced metabolic alterations in diverse tissues remain largely unexplored. Here, we report the changes in metabolic profiles induced by a 2-week subcutaneous injection of liraglutide in diet-induced obese mice fed a high-fat diet for 8 weeks. Our comprehensive metabolomic analyses of the hypothalamus, plasma, liver, and skeletal muscle showed that liraglutide intervention led to various metabolic alterations in comparison with diet-induced obese or nonobese mice. We found that liraglutide remarkably coordinated not only fatty acid metabolism in the hypothalamus and skeletal muscle but also amino acid and carbohydrate metabolism in plasma and liver. Comparative analyses of metabolite dynamics revealed that liraglutide rewired intertissue metabolic correlations. Our study points to a previously unappreciated metabolic alteration by liraglutide in several tissues, which may underlie its therapeutic effects within and across the tissues. Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone that induces insulin secretion, glucagon suppression, and appetite suppression through the signaling cascades of GLP-1 receptor (GLP-1R), which maintains glucose homeostasis and induces weight loss (1Gribble F.M. Reimann F. Metabolic messengers: glucagon-like peptide 1.Nat. Metab. 2021; 3: 142-148Crossref PubMed Scopus (61) Google Scholar, 2Drucker D.J. 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Biotechnol. 2019; 188: 165-184Crossref PubMed Scopus (12) Google Scholar). A recent NMR spectroscopy–based urine metabolomics study has detected the effects of liraglutide treatment in diet-induced obese (DIO) mice: liraglutide decreased urine levels of nicotinamide metabolites and taurine and increased those of creatinine and creatine (33Buganova M. Pelantova H. Holubova M. Sediva B. Maletinska L. Zelezna B. et al.The effects of liraglutide in mice with diet-induced obesity studied by metabolomics.J. Endocrinol. 2017; 233: 93-104Crossref PubMed Scopus (23) Google Scholar). Although urine metabolomics has advantages such as noninvasiveness and the ease of obtaining large volumes of samples, it is difficult to interpret the data on urine metabolites, which are in the final waste products and often do not represent actual changes in various tissues (34Bouatra S. Aziat F. Mandal R. Guo A.C. Wilson M.R. Knox C. et al.The human urine metabolome.PLoS One. 2013; 8e73076Crossref PubMed Scopus (1018) Google Scholar). Therefore, the integration of metabolomics data from multiple tissues is required for better understanding the aberrant metabolism at the sites of disease pathogenesis or during clinical challenges (pre- versus post-treatment). Here, we report a comprehensive metabolomic study to understand the metabolic effects of liraglutide in the hypothalamus, plasma, liver, and skeletal muscle of DIO mice fed high-fat diet (HFD) using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and gas chromatography–tandem mass spectrometry (GC-MS/MS). The major metabolites and metabolic pathways restored by liraglutide in each tissue of DIO mice were also identified in comparison to those in nonobese mice. Our metabolomic results highlight how liraglutide contributes to the therapeutic profile of GLP-1R agonism, offering insights into the clinical efficacy of liraglutide and the understanding of the systems-level effects on metabolic profile associated with metabolic diseases such as obesity and diabetes. To investigate metabolic changes induced by liraglutide, mice were fed a normal chow diet or HFD for 8 weeks and were given daily subcutaneous injections of saline (hereafter referred to as DIO saline) or 400 μg/kg of liraglutide for 14 days (hereafter referred to as DIO liraglutide). Liraglutide has no effect on body weight, food intake, body fat, and glucose tolerance in nonobese mice, although it changes several pancreatic and adipogenic parameters in those mice (35Mondragon A. Davidsson D. Kyriakoudi S. Bertling A. Gomes-Faria R. Cohen P. et al.Divergent effects of liraglutide, exendin-4, and sitagliptin on beta-cell mass and indicators of pancreatitis in a mouse model of hyperglycaemia.PLoS One. 2014; 9e104873Crossref Scopus (25) Google Scholar, 36Zhou J. Poudel A. Chandramani-Shivalingappa P. Xu B. Welchko R. Li L. Liraglutide induces beige fat development and promotes mitochondrial function in diet induced obesity mice partially through AMPK-SIRT-1-PGC1-alpha cell signaling pathway.Endocrine. 2019; 64: 271-283Crossref PubMed Scopus (33) Google Scholar). We injected nonobese mice with saline as control (hereafter referred to as Chow saline) to investigate the beneficial effects of liraglutide on obese phenotypes and restoration of metabolism in obese animals. After the last injection, the hypothalamus, plasma, liver, and skeletal muscle were extracted and metabolites were analyzed using LC-MS/MS and GC-MS/MS (Fig. 1A). Liraglutide resulted in a 26.1 ± 4.5% weight loss (Fig. 1B) and significantly reduced caloric intake in DIO mice (Fig. 1C). To assess the effect of liraglutide on body composition, we measured fat and lean masses using dual-energy X-ray absorptiometry after the last dose. Both masses were significantly reduced by liraglutide in DIO mice and to a level similar to that of Chow saline (Fig. 1D). Eight-week HFD significantly increased bone mineral density and contents in comparison with Chow saline. Liraglutide treatment of DIO mice for 14 days reduced bone mineral contents but not density in comparison with DIO saline (Fig. 1E). Collectively, these results indicate that liraglutide ameliorates obese phenotypes in DIO mice. To assess changes in systemic metabolism induced by liraglutide in DIO mice compared to Chow saline mice, we performed targeted and nontargeted metabolomics and detected 647 annotated metabolites in total (125 in the hypothalamus, 178 in plasma, 193 in liver, and 151 in skeletal muscle). Unsupervised principal component analysis (PCA) revealed the effects of HFD and liraglutide on the metabolome. The components containing metabolomic features were clearly distinguishable and did not overlap among the three groups (Chow saline, DIO saline, and DIO liraglutide) (Fig. 2A). In a heatmap, the 647 metabolites displayed a different pattern in each group (Fig. 2B). Among these metabolites, 377 showed a significant difference in response to either HFD or liraglutide (Fig. 2C). To determine changes in the metabolome and metabolic pathways caused by HFD or liraglutide, pairwise comparisons were performed (DIO saline versus Chow saline, DIO liraglutide versus DIO saline, and DIO liraglutide versus Chow saline) (Fig. 2, D‒I). Each group was obviously distinct from the corresponding comparison group in PCA analysis (Fig. S1, A‒C). Supervised orthogonal partial least squares discriminant analysis (OPLS-DA) also showed a clear distinction from the comparison groups (Fig. S1, D‒F). We estimated the variable influence on projection (VIP) scores, which indicate the importance of metabolites to the OPLS-DA model, and listed the scores of the top 10 metabolites (Fig. S1, G‒I). Metabolites with significant differences and their metabolites are shown through volcano plots showing the fold changes and p values (Fig. 2, D, F, and H). HFD significantly increased 41 metabolites in the plasma, liver, and skeletal muscle (17, 22, and 2 metabolites, respectively), mainly long-chain fatty acids and mono/polyunsaturated fatty acids (Fig. 2D). HFD significantly decreased 219 metabolites, mainly in the hypothalamus and skeletal muscle (75 and 79 metabolites, respectively). To identify the metabolic pathways enriched by HFD, we performed a metabolite set enrichment analysis (top 5) for either increased or decreased metabolites. The increased metabolites impacted the robust enrichment of metabolic pathways involved in fatty acid biosynthesis, alpha linolenic acid/linoleic acid, taurine/hypotaurine, pentose phosphate pathway, and glucose‒alanine cycle metabolism, whereas the decreased metabolites impacted the Warburg effect, urea cycle, ammonia recycling, glutamate, and glycine/serine metabolism (Fig. 2E). Liraglutide increased 31 metabolites, mostly in plasma (25 metabolites) and more than half of the 76 decreased metabolites were detected in the liver (46 metabolites) (Fig. 2F). Notably, most of the decreased metabolites are the intermediates of the pentose phosphate pathway or Warburg effect in the liver. Consistently, the decreased metabolites were enriched in the pentose phosphate pathway, Warburg effect, galactose, amino sugar metabolism, and cysteine metabolism, while the increased metabolites were enriched in the malate‒aspartate shuttle, ammonia recycling, aspartate, phenylalanine/tyrosine metabolism, and urea cycle (Fig. 2G). Our results suggested that liraglutide decreased the pentose phosphate pathway increased by HFD but elevated the urea cycle and ammonia recycling decreased by HFD (Fig. 2, E and G). The greatest number of metabolites differed between DIO liraglutide and Chow saline. Metabolites in DIO liraglutide were increased mostly in plasma (60 metabolites) and decreased in the hypothalamus and skeletal muscle (103 metabolites in each) (Fig. 2H). The increased metabolites were enriched in spermidine/spermine biosynthesis and metabolism of amino acids including glycine, serine, arginine, proline, and methionine, whereas the decreased metabolites were highly enriched in the Warburg effect, glutamate, ammonia recycling, and amino sugar metabolism. The urea cycle overlapped with upregulated and downregulated metabolic pathways (Fig. 2I). The hypothalamus is a key brain area controlling energy balance and glucose metabolism via numerous metabolic signals such as lipids and amino acids which play a critical role in regulating food intake (37Blouet C. Jo Y.H. Li X. Schwartz G.J. Mediobasal hypothalamic leucine sensing regulates food intake through activation of a hypothalamus-brainstem circuit.J. 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All metabolites were compared in group pairs by OPLS-DA, resulting in clear distinction (Fig. S2, A, C, and E). HFD increased the metabolites of fatty acid metabolism, namely myristic, erucic, behenic, arachidic, heneicosanoic, lignoceric, palmitoleic, eicosanoic, undecanoic, tricosanoic, myristoleic, pentadecanoic, lauric, eicosatrienoic, and tridecanoic acids, which were further increased by liraglutide (Fig. 3C). These results were also consistent between the two pairs of comparison groups (Fig. S2, B, D, and F). Notably, erucic, behenic, arachidic, and lignoceric acids are products of fatty acid biosynthesis in which enzymes synthesize fatty acids from the substrates such as malonyl-CoA, acetyl-CoA, and NADPH. HFD also increased glycine, threonine, and valine, but liraglutide decreased these amino acids (Fig. 3C). Adenosine was decreased in DIO liraglutide compared to both DIO saline and Chow saline (Fig. S2, D, and F). To identify the metabolic pathways impacted by HFD or liraglutide, we performed metabolite set enrichment analysis for 30 significant metabolites in each group and identified metabolic pathways common to all three groups. Fatty acid biosynthesis and fatty acid metabolism, which includes fatty acid biosynthesis, were specifically increased in DIO saline and DIO liraglutide compared to Chow saline. Liraglutide markedly increased fatty acid metabolism, including biosynthesis, in the hypothalamus (Fig. 3D). To understand the increase or decrease of metabolite classes by HFD or liraglutide, we displayed the flow charts of metabolites. Among the 28 metabolites that showed significant differences (VIP score ≥ 1.2) in the plasma of the three groups, 88.2% of fatty acids and 80% of amino acids were increased in DIO saline compared to Chow saline; 88.2% of fatty acids were further increased and 80% amino acids were decreased in DIO liraglutide; 83.3% of carbohydrates were decreased in DIO saline compared to Chow saline but 50% of carbohydrates were increased in DIO liraglutide (Fig. 3E). Overall, liraglutide mainly increased fatty acids and decreased amino acids that were increased by HFD in the hypothalamus. Plasma metabolomics is widely used in clinical and biological studies on metabolic diseases (43Dettmer K. Aronov P.A. Hammock B.D. Mass spectrometry-based metabolomics.Mass Spectrom. Rev. 2007; 26: 51-78Crossref PubMed Scopus (1633) Google Scholar). The components were not dramatically distinct among the three groups either in PCA or in PLS-DA (Fig. 4A). Among 178 annotated metabolites, 96 metabolites showed significantly different responses among the three groups (Fig. 4B). OPLS-DA between two groups showed clear differences (Fig. S3, A, C, and E). HFD decreased but liraglutide increased the levels of essential or nonessential amino acids including branched-chain amino acids (Figs. 4C, S3, B, and D). On the contrary, glutamic acid, histidine, and tyrosine, which were decreased by HFD, were further decreased by liraglutide (Fig. 4C). Compared to Chow saline, liraglutide decreased cysteine, glycine, glutamate, serine, methionine, phenylalanine, histidine, glutamine, and tyrosine (Fig. 3F). HFD also decreased the intermediates of glycolysis, citric acid cycle, and epinephrine/norepinephrine compared to Chow saline; interestingly, liraglutide reorganized the levels of these metabolites (Fig. 4C). In addition, both of HFD and liraglutide increased arachidonic acid and taurine compared to those in Chow saline (Fig. S3, B, and F). Compared to Chow saline, DIO saline tended to decrease almost all metabolic pathways that were common among the three groups, including amino acid metabolism. Intriguingly, liraglutide restored most of the metabolic pathways severely reduced by HFD, except for amino sugar and galactose metabolism, and additionally enhanced cysteine metabolism in the plasma of DIO mice (Fig. 4D). Among the 54 metabolites that showed significant differences (VIP score ≥ 1.2) in the plasma of the three groups, 95.5% of amino acids, 90.5% of carbohydrates, and 66.7% of fatty acids were decreased in DIO saline compared to Chow saline; 81.8% of amino acids, 66.7% of carbohydrates, and 66.7% of fatty acids were increased in DIO liraglutide compared to DIO saline. The neurotransmitters epinephrine and norepinephrine were reduced in DIO saline compared to Chow saline and increased in DIO liraglutide compared to DIO saline (Fig. 4E). Taken together, these data highlight that liraglutide restores the plasma metabolism of amino acids, carbohydrates, fatty acids, and neurotransmitters that was reduced by HFD. Metabolomics analysis in the liver showed that the components of DIO saline and DIO liraglutide mostly overlapped in PCA, but not in PLS-DA (Fig. 5A). Among 193 annotated metabolites, 66 metabolites showed significantly different responses among the three groups (Fig. 5, B). OPLS-DA between two groups showed clear differences (Fig. S4, A, C, and E). HFD increased fatty acids such as erucic, eicosanoic, myristic, behenic, and heptadecanoic acids, whereas liraglutide reduced fatty acids (erucic, lauric, myristic, palmitic, arachidic, behenic, heneicosanoic, myristoleic, palmitoleic, oleic, and arachidonic acids) in comparison with DIO liraglutide and DIO saline (Figs. 5C, S4, B, and D). HFD also increased methionine, taurine, Nα-acetyl ornithine, and β-glutamic acid. Notably, these amino a