Comprehensive characterization of post-translationally modified histone proteoforms is challenging due to their high isobaric and isomeric content. Trapped ion mobility spectrometry (TIMS), ...implemented on a quadrupole/time-of-flight (Q-ToF) mass spectrometer, has shown great promise in discriminating isomeric complete histone tails. The absence of electron activated dissociation (ExD) in the current platform prevents the comprehensive characterization of unknown histone proteoforms. In the present work, we report for the first time the use of an electromagnetostatic (EMS) cell devised for nonergodic dissociation based on electron capture dissociation (ECD), implemented within a nESI-TIMS-Q-ToF mass spectrometer for the characterization of acetylated (AcK18 and AcK27) and trimethylated (TriMetK4, TriMetK9 and TriMetK27) complete histone tails. The integration of the EMS cell in a TIMS-q-TOF MS permitted fast mobility-selected top-down ECD fragmentation with near 10% efficiency overall. The potential of this coupling was illustrated using isobaric (AcK18/TriMetK4) and isomeric (AcK18/AcK27 and TriMetK4/TriMetK9) binary H3 histone tail mixtures, and the H3.1 TriMetK27 histone tail structural diversity (e.g., three IMS bands at z = 7+). The binary isobaric and isomeric mixtures can be separated in the mobility domain with R IMS > 100 and the nonergodic ECD fragmentation permitted the PTM localization (sequence coverage of ∼86%). Differences in the ECD patterns per mobility band of the z = 7+ H3 TriMetK27 molecular ions suggested that the charge location is responsible for the structural differences observed in the mobility domain. This coupling further enhances the structural toolbox with fast, high resolution mobility separations in tandem with nonergodic fragmentation for effective proteoform differentiation.
Central obesity is a major risk factor for heart failure with preserved ejection fraction (HFpEF), particularly in women, but the mechanisms remain unclear. We hypothesized that sex-specific ...differences in visceral adipose tissue (VAT) content would differentially relate to haemodynamic severity of HFpEF in women and men.
Abdominal computed tomography (CT) and invasive haemodynamic exercise testing were performed in 105 subjects with HFpEF (63 women) and 105 age-, sex-, and body mass index-matched controls. Visceral adipose tissue area was quantified by CT. As compared with control women, VAT area was 34% higher in women with HFpEF (186 ± 112 vs. 139 ± 72 cm2, P = 0.006), while VAT area was not significantly different in men with or without HFpEF (294 ± 158 vs. 252 ± 92 cm2, P = 0.1). During exercise, pulmonary capillary wedge pressure (PCWP) increased markedly and to similar extent in both men and women with HFpEF. Women with increased VAT area displayed 33% higher PCWP during exercise compared with women with normal VAT area (28 ± 10 vs. 21 ± 10 mmHg, P = 0.001), whereas exercise PCWP was similar in men with or without excess VAT (24 ± 9 vs. 25 ± 6, P = 0.89). In women, each 100 cm2 increase in VAT area was associated with a 4.0 mmHg higher PCWP (95% CI 2.1, 6.0 mmHg; P < 0.0001), but there was no such relationship in men (interaction P = 0.009).
These data suggest that accumulation of excess VAT plays a distinct and important role in the pathophysiology of HFpEF preferentially in women. Further research is needed to better understand the mechanisms and treatment implications for visceral fat in HFpEF.
Unambiguous identification of distinct proteoforms and their biological functions is a significant analytical challenge due to the many combinations of post-translational modifications (PTM) that ...generate isomeric proteoforms. Resulting chimeric tandem mass spectra hinder detailed structural characterization of individual proteoforms for mixtures with more than two isomers. Large isomeric peptides and intact isomeric proteins are extremely difficult to distinguish with traditional chromatographic separation methods. Gas-phase ion separation techniques such as ion mobility spectrometry (IMS) methods now offer high resolving power that may enable separation of isomeric biomolecules, such as peptides and proteins. We explored novel high-resolution cyclic ion mobility spectrometry (cIM) combined with an electro-magnetostatic cell for “on-the-fly” electron capture dissociation (ECD) for separation and sequencing of large isomeric peptides. We demonstrate the effectiveness of this approach on ternary mixtures of mono- and trimethylated isomers of histone H3 N-tails (∼5.4 kDa), achieving a complete separation of these isomers with an average resolving power of ∼400 and a resolution of 1.5 and with nearly 100% amino acid sequence coverage. Our results demonstrate the potential of the cIM-MS/MS(ECD) technology to enhance middle-down and top-down proteomics workflows, thereby facilitating the identification of near-identical proteoforms with essential biological functions in complex mixtures.
Aims
Obesity is a risk factor for heart failure with preserved ejection fraction (HFpEF), particularly in women, but the mechanisms remain unclear. The present study aimed to investigate the impact ...of central adiposity in patients with HFpEF and explore potential sex differences.
Methods and results
A total of 124 women and 105 men with HFpEF underwent invasive haemodynamic exercise testing and rest echocardiography. Central obesity was defined as a waist circumference (WC) ≥88 cm for women and ≥102 cm for men. Exercise‐normalized pulmonary capillary wedge pressure (PCWP) responses were evaluated by the ratio of PCWP to workload (PCWP/W) and after normalizing to body weight (PCWL). The prevalence of central obesity (77%) exceeded that of general obesity (62%) defined by body mass index ≥30 kg/m2. Compared to patients without central adiposity, patients with HFpEF and central obesity displayed greater prevalence of diabetes and dyslipidaemia, higher right and left heart filling pressures and pulmonary artery pressures during exertion, and more severely reduced aerobic capacity. Associations between WC and fasting glucose, low‐density lipoprotein (LDL) cholesterol, peak workload, and pulmonary artery pressures were observed in women but not in men with HFpEF. Although increased WC was associated with elevated PCWP in both sexes, the association with PCWP/W was observed in women but not in men. The strength of correlation between PCWP/W and WC was more robust in women with HFpEF as compared to men (Meng's test p = 0.0008), and a significant sex interaction was observed in the relationship between PCWL and WC (p for interaction = 0.02).
Conclusions
Central obesity is even more common than general obesity in HFpEF, and there appear to be important sexual dimorphisms in its relationships with metabolic abnormalities and haemodynamic perturbations, with greater impact in women.
Increased plasma levels of glucagon (hyperglucagonaemia) promote diabetes development but is also observed in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). This may ...reflect hepatic glucagon resistance towards amino acid catabolism. A clinical test for measuring glucagon resistance has not been validated. We evaluated our glucagon sensitivity (GLUSENTIC) test, consisting of two study days: a glucagon injection and measurements of plasma amino acids, and an infusion of mixed amino acids and subsequent calculation of the GLUSENTIC index (primary outcome measure) from measurements of glucagon and amino acids. To distinguish glucagon-dependent from insulin-dependent actions on amino acid metabolism, we also studied patients with type 1 diabetes (T1D). The delta-decline in total amino acids was 49% lower in MASLD following exogenous glucagon (p=0.01), and the calculated GLUSENTIC index was 34% lower in MASLD (p<0.0001), but not T1D (p>0.99). In contrast, glucagon-induced glucose increments were similar in controls and MASLD (p=0.41). The GLUSENTIC test and index may be used to measure glucagon resistance in individuals with obesity and MASLD.
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.
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.
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.
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.
Display omitted
•FGF21 and GDF15 were highly increased in MASLD following glucagon stimulation.•Plasma FGF21 and GDF15 levels correlated with insulin, but not glucagon.•FGF21 was unaffected by glucagon stimulation when insulin levels were constant.•No direct effect of glucagon on FGF21 and GDF15 secretion•The increase of FGF21 and GDF15 by glucagon in MASLD may depend on insulin.
Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in genes coding for proteins involved in sarcomere function. The disease is associated with mitochondrial dysfunction. ...Evolutionarily developed variation in mitochondrial DNA (mtDNA), defining mtDNA haplogroups and haplogroup clusters, is associated with functional differences in mitochondrial function and susceptibility to various diseases, including ischemic cardiomyopathy. We hypothesized that mtDNA haplogroups, in particular H, J and K, might modify disease susceptibility to HCM. Mitochondrial DNA, isolated from blood, was sequenced and haplogroups identified in 91 probands with HCM. The association with HCM was ascertained using two Danish control populations. Haplogroup H was more prevalent in HCM patients, 60% versus 46% (p = 0.006) and 41% (p = 0.003), in the two control populations. Haplogroup J was less prevalent, 3% vs. 12.4% (p = 0.017) and 9.1%, (p = 0.06). Likewise, the UK haplogroup cluster was less prevalent in HCM, 11% vs. 22.1% (p = 0.02) and 22.8% (p = 0.04). These results indicate that haplogroup H constitutes a susceptibility factor and that haplogroup J and haplogroup cluster UK are protective factors in the development of HCM. Thus, constitutive differences in mitochondrial function may influence the occurrence and clinical presentation of HCM. This could explain some of the phenotypic variability in HCM. The fact that haplogroup H and J are also modifying factors in ischemic cardiomyopathy suggests that mtDNA haplotypes may be of significance in determining whether a physiological hypertrophy develops into myopathy. mtDNA haplotypes may have the potential of becoming significant biomarkers in cardiomyopathy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Glucagon is essential for glucose control and increased levels of glucagon (hyperglucagonemia) observed in patients with type 2 diabetes contribute to their hyperglycemia. Recently, hyperglucagonemia ...has also been found in individuals with non-alcoholic fatty liver disease (NAFLD) as well as impaired actions of glucagon on amino acid catabolism. Whether glucagon actions on hepatic glucose production are impaired is unknown. We investigated the acute effects of a single bolus of glucagon (0.2mg) on glucose dynamics in 18 normoglycemic individuals (age: 51±3 years, BMI; 31± 0.8kg/m2, hepatic fat content: 20±2%, fasting glucose: 5.5±0.1mM) with magnetic resonance imaging verified NAFLD and 22 controls (age: 38±3 years, BMI; 24± 0.8kg/m2, hepatic fat content: 4±0.1%, fasting glucose: 5.0±0.1mM) . On a separate day, a mixture of amino acids (14 g/L; 331 mg/min/kg body weight) was infused intravenously for 45min to evaluate the actions of endogenous glucagon on glucose dynamics. Glucose levels (see figure) were significantly increased in individuals with NAFLD 60min after the glucagon bolus and during the amino acid infusion with a maximal difference of 0.5mM 30min into the infusion. These data suggest that the actions of glucagon on hepatic glucose production are not impaired by NAFLD. Therefore, the hyperglucagonemia in patients with NAFLD may constitute a diabetogenic risk factor.
Disclosure
S.Kjeldsen: None. H.Vilstrup: None. F.V.Schiødt: Advisory Panel; Novo Nordisk. A.Møller: None. E.B.Rashu: None. L.Gluud: Advisory Panel; Novo Nordisk, Consultant; Pfizer Inc., Research Support; Alexion Pharmaceuticals, Inc., Gilead Sciences, Inc., Novo Nordisk, Sobi. S.B.Haugaard: None. J.J.Holst: Advisory Panel; Novo Nordisk, Board Member; Antag Therapeutics, Bainan Biotech. J.Rungby: Advisory Panel; Abbott, Boehringer Ingelheim International GmbH, Speaker’s Bureau; AstraZeneca, Bayer AG, Novo Nordisk, Pfizer Inc. N.J.Wewer albrechtsen: Research Support; Mercodia AB, Novo Nordisk, Regeneron Pharmaceuticals Inc., Speaker’s Bureau; Merck & Co., Inc., Mercodia AB. N.J.Jensen: None. M.Nilsson: None. N.Heinz: None. J.D.Nybing: None. F.H.Linden: None. E.Høgh-schmidt: n/a. M.P.Boesen: None. S.Madsbad: None.
Funding
NNF Excellence Emerging Investigator Grant – Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude.
Glucagon regulates hepatic glucose production and hyperglucagonemia contributes to diabetes. Equally important, glucagon may regulate amino acid (AA) levels that in turn control glucagon secretion. ...Hepatic steatosis may uncouple glucagon's effect on AA metabolism causing impaired actions of glucagon (resistance) on AA metabolism but not glucose production, thereby creating a diabetogenic circle. In order to quantify glucagon's effect on AA metabolism, we developed and evaluated a glucagon sensitivity test. The test consists of a bolus-infusion of glucagon (200 μg) and an AA infusion (330 mg/min/kg body weight for 45 min) on two separate days following an overnight fast. Liver fat was measured using magnetic resonance imaging. Preliminary data from six individuals without diabetes (HbA1c < 48mmol/mol) including three lean controls (CON) (mean ± SD; Age: 32 ± 7 years, liver fat: 4.1 ± 1 %, BMI; 22 ± 2 kg/m2) and three individuals with obesity (OBE) (47 ± 12 years, 12 ± 6 %, 30 ± 4 kg/m2) are presented. A glucagon injection reduced AA levels 29% less in OBE compared to CON (dAUC0-120min; 41 ± 6 vs. 29 ± 10 mmol/L x min) during the fasted state. AA levels increased 33% more in OBE compared to CON during an AA infusion (iAUC0-45min; 118 ± 28 vs. 89 ± 12 mmol/L x min). We conclude that glucagon sensitivity towards AA metabolism may be evaluated by a bolus-infusion of glucagon and an AA infusion, and that hepatic steatosis may cause glucagon resistance.
PDF
