Targeted metabolomics in human and animal biofluids and tissues using liquid chromatography coupled with tandem mass spectrometry.

Here, we present a targeted polar metabolomics protocol for the analysis of biofluids and frozen tissue biopsies using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We describe steps for sample pretreatment, liquid-liquid extraction, and isolation of polar metabolites. We then detail procedures for target LC-MS/MS analysis. In this protocol, we focus on the analysis of plasma and serum samples. We also provide brief instructions on how to process other biological matrices as supplemental information. For complete details on the use and execution of this protocol, please refer to Coskun et al. (2022).1.

Effects of Tirzepatide, a Dual GIP and GLP-1 RA, on Lipid and Metabolite Profiles in Subjects With Type 2 Diabetes.

CONTEXT: Tirzepatide substantially reduced hemoglobin A1c (HbA1c) and body weight in subjects with type 2 diabetes (T2D) compared with the glucagon-like peptide 1 receptor agonist dulaglutide. Improved glycemic control was associated with lower circulating triglycerides and lipoprotein markers and improved markers of beta-cell function and insulin resistance (IR), effects only partially attributable to weight loss. OBJECTIVE: Assess plasma metabolome changes mediated by tirzepatide. DESIGN: Phase 2b trial participants were randomly assigned to receive weekly subcutaneous tirzepatide, dulaglutide, or placebo for 26 weeks. Post hoc exploratory metabolomics and lipidomics analyses were performed. SETTING: Post hoc analysis. PARTICIPANTS: 259 subjects with T2D. INTERVENTION(S): Tirzepatide (1, 5, 10, 15 mg), dulaglutide (1.5 mg), or placebo. MAIN OUTCOME MEASURE(S): Changes in metabolite levels in response to tirzepatide were assessed against baseline levels, dulaglutide, and placebo using multiplicity correction. RESULTS: At 26 weeks, a higher dose tirzepatide modulated a cluster of metabolites and lipids associated with IR, obesity, and future T2D risk. Branched-chain amino acids, direct catabolic products glutamate, 3-hydroxyisobutyrate, branched-chain ketoacids, and indirect byproducts such as 2-hydroxybutyrate decreased compared to baseline and placebo. Changes were significantly larger with tirzepatide compared with dulaglutide and directly proportional to reductions of HbA1c, homeostatic model assessment 2-IR indices, and proinsulin levels. Proportional to metabolite changes, triglycerides and diglycerides were lowered significantly compared to baseline, dulaglutide, and placebo, with a bias toward shorter and highly saturated species. CONCLUSIONS: Tirzepatide reduces body weight and improves glycemic control and uniquely modulates metabolites associated with T2D risk and metabolic dysregulation in a direction consistent with improved metabolic health.

Circulating proteins protect against renal decline and progression to end-stage renal disease in patients with diabetes.

Diabetic kidney disease (DKD) and its major clinical manifestation, progressive renal decline that leads to end-stage renal disease (ESRD), are a major health burden for individuals with diabetes. The disease process that underlies progressive renal decline comprises factors that increase risk as well as factors that protect against this outcome. Using untargeted proteomic profiling of circulating proteins from individuals in two independent cohorts with type 1 and type 2 diabetes and varying stages of DKD followed for 7 to 15 years, we identified three elevated plasma proteins-fibroblast growth factor 20 (OR, 0.69; 95% CI, 0.54 to 0.88), angiopoietin-1 (OR, 0.72; 95% CI, 0.57 to 0.91), and tumor necrosis factor ligand superfamily member 12 (OR, 0.75; 95% CI, 0.59 to 0.95)-that were associated with protection against progressive renal decline and progression to ESRD. The combined effect of these three protective proteins was demonstrated by very low cumulative risk of ESRD in those who had baseline concentrations above median for all three proteins, whereas the cumulative risk of ESRD was high in those with concentrations below median for these proteins at the beginning of follow-up. This protective effect was shown to be independent from circulating inflammatory proteins and clinical covariates and was confirmed in a third cohort of diabetic individuals with normal renal function. These three protective proteins may serve as biomarkers to stratify diabetic individuals according to risk of progression to ESRD and might also be investigated as potential therapeutics to delay or prevent the onset of ESRD.

GIPR agonism mediates weight-independent insulin sensitization by tirzepatide in obese mice.

Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor (GLP-1R) agonist, delivered superior glycemic control and weight loss compared with GLP-1R agonism in patients with type 2 diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine whether GIPR agonism contributes, we compared the effect of tirzepatide in obese WT and Glp-1r-null mice. In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support of this, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino acids (BCAAs) and ketoacids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid, and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent and -independent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual-receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.

Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms.

Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.

Integrative analysis of prognostic biomarkers derived from multiomics panels helps discrimination of chronic kidney disease trajectories in people with type 2 diabetes.

Clinical risk factors explain only a fraction of the variability of estimated glomerular filtration rate (eGFR) decline in people with type 2 diabetes. Cross-omics technologies by virtue of a wide spectrum screening of plasma samples have the potential to identify biomarkers for the refinement of prognosis in addition to clinical variables. Here we utilized proteomics, metabolomics and lipidomics panel assay measurements in baseline plasma samples from the multinational PROVALID study (PROspective cohort study in patients with type 2 diabetes mellitus for VALIDation of biomarkers) of patients with incident or early chronic kidney disease (median follow-up 35 months, median baseline eGFR 84 mL/min/1.73 m2, urine albumin-to-creatinine ratio 8.1 mg/g). In an accelerated case-control study, 258 individuals with a stable eGFR course (median eGFR change 0.1 mL/min/year) were compared to 223 individuals with a rapid eGFR decline (median eGFR decline -6.75 mL/min/year) using Bayesian multivariable logistic regression models to assess the discrimination of eGFR trajectories. The analysis included 402 candidate predictors and showed two protein markers (KIM-1, NTproBNP) to be relevant predictors of the eGFR trajectory with baseline eGFR being an important clinical covariate. The inclusion of metabolomic and lipidomic platforms did not improve discrimination substantially. Predictions using all available variables were statistically indistinguishable from predictions using only KIM-1 and baseline eGFR (area under the receiver operating characteristic curve 0.63). Thus, the discrimination of eGFR trajectories in patients with incident or early diabetic kidney disease and maintained baseline eGFR was modest and the protein marker KIM-1 was the most important predictor.

Long-Acting and Selective Oxytocin Peptide Analogs Show Antidiabetic and Antiobesity Effects in Male Mice.

Oxytocin (OXT) has been shown to suppress appetite, induce weight loss, and improve glycemic control and lipid metabolism in several species, including humans, monkeys, and rodents. However, OXT's short half-life in circulation and lack of receptor selectivity limit its application and efficacy. In this study, we report an OXT peptide analog (OXTGly) that is potent and selective for the OXT receptor (OXTR). OXT, but not OXTGly, activated vasopressin receptors in vitro and acutely increased blood pressure in vivo when administered IP. OXT suppressed food intake in mice, whereas OXTGly had a moderate effect on food intake when administered IP or intracerebroventricularly. Both OXT (IP) and OXTGly (IP) improved glycemic control in glucose tolerance tests. Additionally, both OXT (IP) and OXTGly (IP) stimulated insulin, glucagon-like peptide 1, and glucagon secretion in mice. We generated lipid-conjugated OXT (acylated-OXT) and OXTGly (acylated-OXTGly) and demonstrated that these molecules have significantly extended half-lives in vivo. Compared with OXT, 2-week treatment of diet-induced obese mice with acylated-OXT [subcutaneous(ly) (SC)] resulted in enhanced body weight reduction, an improved lipid profile, and gene expression changes consistent with increased lipolysis and decreased gluconeogenesis. Treatment with acylated-OXTGly (SC) also resulted in a statistically significant weight loss, albeit to a lesser degree compared with acylated-OXT treatment. In conclusion, we demonstrate that selective activation of the OXTR pathway results in both acute and chronic metabolic benefits, whereas potential activation of vasopressin receptors by nonselective OXT analogs causes physiological stress that contributes to additional weight loss.

The effects of vertical sleeve gastrectomy in rodents are ghrelin independent.

Reductions in levels of the hunger-stimulating hormone ghrelin have been proposed to mediate part of the effects of vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass surgeries for obesity. We studied circulating levels of acyl and desacyl ghrelin in rats after these surgeries. We found that blood levels of ghrelin were reduced after VSG, but not after Roux-en-Y gastric bypass, based on enzyme-linked immunosorbent assay and mass-spectrometry analyses. We compared the effects of VSG in ghrelin-deficient mice and wild-type mice on food intake, body weight, dietary fat preference, and glucose tolerance. We found that VSG produced comparable outcomes in each strain. Reduced ghrelin signaling therefore does not appear to be required for these effects of VSG.

From ghrelin to ghrelin's O-acyl transferase.

The hormone ghrelin is a unique signaling peptide with powerful metabolic effects, mediated by its acylated forms. The acyl modification of ghrelin is unique in that it takes place via a susceptible ester linkage in the conserved serine-3 of ghrelin and is composed principally of octanoyl and, to lesser extent, decanoyl fatty acids. The nature of this ester linkage makes it susceptible to esterases, which convert it to its des-acyl forms, and, if not adequately inhibited, the conversion to des-acyl ghrelin, particularly post sample collection, can lead to artifactual and misleading results. Here, we describe sample processing and mass spectrometric methodologies for the accurate and simultaneous quantification of acylated and des-acylated forms of ghrelin. We exploited these methodologies (1) to characterize circulating and tissue-specific forms of acyl and des-acyl ghrelin, (2) to optimize a cell system for acyl ghrelin production and search for the enzyme responsible for ghrelin's acylation, and (3) to demonstrate that GOAT is ghrelin's O-acyl transferase.

GOAT links dietary lipids with the endocrine control of energy balance.

Central nervous system nutrient sensing and afferent endocrine signaling have been established as parallel systems communicating metabolic status and energy availability in vertebrates. The only afferent endocrine signal known to require modification with a fatty acid side chain is the orexigenic hormone ghrelin. We find that the ghrelin O-acyl transferase (GOAT), which is essential for ghrelin acylation, is regulated by nutrient availability, depends on specific dietary lipids as acylation substrates and links ingested lipids to energy expenditure and body fat mass. These data implicate the ghrelin-GOAT system as a signaling pathway that alerts the central nervous system to the presence of dietary calories, rather than to their absence as is commonly accepted.

Ghrelin octanoylation mediated by an orphan lipid transferase.

The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelin's physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.