Objective and Methods

Pancreatic β cell dysfunction is critical to the development of type 2 diabetes (T2D). Our previous studies suggested that C3aR1 on β cells promotes insulin secretion and cell survival. However, as C3aR1 is expressed on many other cell types including within the islets, whole-body C3aR1 knockout models confound the analyses of direct impacts on β cells. To clarify the role of C3aR1 in β cells under T2D conditions, we generated β cell-specific C3aR1 knockout mice. We assessed glucose homeostasis, focusing on β cell function and mass under metabolic stress conditions, to interrogate the effects of C3aR1 on β cells in a mouse model of T2D. We performed proteomic analyses on islets from control and β cell-specific C3aR1 knockout mice. To determine potential translational relevance, C3AR1 was assessed alongside glucose-stimulated insulin secretion in human islets.

Results

We show that the complement receptor C3aR1 on β cells plays an essential role in maintaining β cell homeostasis, especially under the metabolic duress of obesity and T2D. Male mice with β cell specific deletion of C3ar1 (β-C3aR1 KO) exhibit worse glucose tolerance and lower insulin levels when fed regular or high fat diet. Under high fat diet, β-C3aR1 KO also have diminished β cell mass. Islets from β-C3aR1 KO mice demonstrate impaired insulin secretion. β cells lacking C3aR1 display increased susceptibility to lipotoxicity-mediated cell death. Markers of β cell identity are decreased in β-C3aR1 KO mice while stress markers are elevated. Disruption of C3ar1 on β cells ablates the insulin secretory response to C3a, establishing a signaling axis between C3a and β cell-derived C3aR1. Islet proteomic analyses highlight the MAPK pathway and mitochondrial dysfunction with C3aR1 loss in β cells. Finally, we show that C3AR1 is positively correlated with insulin secretion in human islets.

Conclusions

These findings indicate that C3aR1 expression on β cells is necessary to maintain optimal β cell function and preserve β cell mass in T2D.

Purpose

Metabolic dysfunction-associated fatty liver disease (MAFLD) has emerged as an important public health concern that poses a significant threat to human health and imposes a substantial economic burden. Research has demonstrated that ubiquitin ligase-mediated substrate protein ubiquitination is a pivotal factor influencing liver lipid homeostasis and metabolic abnormalities in MAFLD. Nevertheless, the specific enzyme molecules implicated in this regulatory process remain to be elucidated. We have published a transcriptome-overexpressing ubiquitin ligase, membrane-associated ring–CH–type finger 2 (MARCH2), in HepG2 cells, and subsequent reanalysis of these transcriptome data revealed a close association between MARCH2 and lipid metabolism.

Methods

By employing a range of methodologies, including recombinant adeno-associated virus (rAAV) transduction, lentiviral transduction, immunoblotting, quantitative PCR, tissue section staining, ubiquitination assays, serum biochemical analysis, immunoprecipitation, and mass spectrometry, this study investigated the functions and mechanisms of MARCH2 in the progression of MAFLD at the molecular, cellular, and organismal levels.

Results

Overexpression of MARCH2, but not its catalytically inactive ligase variant, inhibited lipid accumulation in HepG2 cells. Additionally, MARCH2 undergoes K48-linked self-polyubiquitination and subsequent proteasomal degradation in response to oleic acid/palmitic acid stimulation. Furthermore, knockout of MARCH2 exacerbates the progression of MAFLD-related phenotypes, including increased body weight, impaired glucose tolerance, reduced insulin sensitivity, hypercholesterolemia, hepatic lipid accumulation, and steatosis, in high-fat diet-fed mice, irrespective of sex. Mechanistically, MARCH2 facilitates the polyubiquitination and degradation of fatty acid synthase (FASN) in the de novo lipogenesis pathway. And liver-specific overexpression of MARCH2 by rAAV effectively reduces FASN levels and further ameliorates MAFLD in ob/ob mice.

Conclusion

MARCH2 undergoes self-ubiquitination and plays an important role in maintaining the liver lipid homeostasis of MAFLD, and drug intervention in the MARCH2-FASN axis is a promising approach for treating systemic metabolic abnormalities in MAFLD.

In addition to sustained glucose lowering, centrally administered fibroblast growth factor 1 (FGF1) induces a potent but transient anorexia in animal models of type 2 diabetes. To investigate the mechanism(s) underlying this anorexic response, the current work focused on a specific neuronal subset located in the external lateral subdivision of the parabrachial nucleus marked by the expression of calcitonin gene-related peptide (elPBN^CGRP neurons). These neurons can be activated by withdrawal of upstream GABAergic inhibitory input and are implicated as mediators of the adaptive response (including anorexia) to a wide range of aversive stimuli. To determine if FGF1-induced anorexia is associated with elPBN^CGRP neuron activation, we employed adult male Calca^Cre:GFP/+ transgenic mice in which GFP is fused to Cre recombinase driven by the CGRP-encoding gene Calca. Here, we show that FGF1 activates elPBN^CGRP neurons, both after intracerebroventricular (icv) injection in vivo and when applied ex vivo in a slice preparation, and that the mechanism underlying this effect depends upon reduced GABAergic input from neurons lying upstream. Consistent with this interpretation, we report that the anorexic response to icv FGF1 is reduced by ∼70% when elPBN^CGRP neurons are silenced using chemogenetics. Last, we report that effects of icv FGF1 injection on both elPBN^CGRP neuron activity and food intake are strongly attenuated by systemic administration of the GABA_A receptor agonist Bretazenil. We conclude that in adult male mice, elPBN^CGRP neuron activation is a key mediator of FGF1-induced anorexia, and that this activation response is mediated at least in part by withdrawal of GABAergic inhibition.

Objective

The activation of brown adipose tissue (BAT) promotes energy expenditure is recognized as a promising therapeutic strategy for combating obesity. The deubiquitinating enzyme family members are widely involved in the process of energy metabolism. However, the specific deubiquitinating enzyme member that affects the BAT thermogenesis remains largely unexplored.

Methods

Adeno-associated virus, lentivirus and small molecule inhibitor were applied to generate USP2 gain- or loss-of-function both in vivo and in vitro. OxyMax comprehensive laboratory animal monitoring system, seahorse and transmission electron microscopy were used to determine the energy metabolism. Quantitative proteomics, immunofluorescence staining and co-immunoprecipitation were performed to reveal the potential substrates of USP2.

Results

USP2 is upregulated upon thermogenic activation in adipose, and has a close correlation with UCP1 mRNA levels in human adipose tissue. BAT-specific Usp2 knockdown or systemic USP2 inhibition resulted in impaired thermogenic programs both in vivo and in vitro. Conversely, overexpression of Usp2 in BAT conferred protection against high-fat diet-induced obesity and associated metabolic disorders. Proteome-wide analysis identified EBF2 as the substrate of USP2 that mediates the thermogenic function of USP2 in BAT.

Conclusions

Our data demonstrated the vital role of USP2 in regulating BAT activation and systemic energy homeostasis. Activation of USP2-EBF2 interaction could be a potential therapeutic strategy against obesity.

Excessive exercise combined with inadequate recovery time may trigger fatigue, performance impairment, and ultimately the overtraining syndrome. The intramyocellular mechanisms involved in the overtraining syndrome remain only partially known. Here, we combined multi-omics analyses from isogenic BXD mouse strains with a mouse model of overtraining and excessive exercise protocol in mice and humans to evaluate the molecular mechanism involved in the performance impairment induced by excessive exercise. We identified that BXD mouse strains with elevated levels of Parp1 gene expression in the skeletal muscle displayed features like overtraining syndrome and abnormal muscle genetic signature. High PARP1 protein content and aberrant PARylation of proteins were detected in the skeletal muscle of overtrained, but not in trained mice. Overtraining syndrome reduced mitochondrial function promoted by exercise training, induced muscle hyperalgesia, reduced muscle fiber size and promoted a similar gene signature of myopathy and atrophy models. Short periods of excessive exercise also increased PARylation in the skeletal muscle of mice and healthy subjects. The pharmacological inhibition of PARP1, using Olaparib, and genetic Parp1 ablation, preserved muscle anatomy and protected against physical performance impairment and other symptoms of the overtraining syndrome in mice. In conclusion, PARP1 excessive activation is related to muscle abnormalities led by long or short periods of excessive exercise, and here we suggest that PARP1 is a potential target in the treatment and prevention of overtraining syndrome.

Lipolysis in white adipose tissue (WAT) provides fatty acids as energy substrates for thermogenesis to increase energy expenditure. Syndecan-4 (Sdc4) is a transmembrane proteoglycan bearing heparan sulfate chains. Although single nucleotide polymorphisms (SNPs) of the Sdc4 gene have been identified linking to metabolic syndromes, its specific function in adipose tissue remains obscure. Here, we show that Sdc4 serves as a regulator of lipid metabolism and adaptive thermogenesis. Sdc4 expression and shedding are elevated in the WAT of diet-induced obese mice. Adipocyte-specific deletion of Sdc4 promotes lipolysis and WAT browning, thereby raising whole-body energy expenditure to protect against diet-induced obesity. Mechanistically, fibroblast growth factor 2 (FGF2) is a paracrine factor that maintains energy homeostasis. Elevated shed Sdc4 concentrates and delivers FGF2 to fibroblast growth factor receptor 1 (FGFR1) on adipocytes, which in turn suppresses lipolysis by reducing hormone-sensitive lipase (HSL) activity, thus exaggerating adipose tissue dysfunction upon high-fat diet induction. Sdc4-deficient adipocytes show higher lipolytic and thermogenic capacity by enhancing HSL phosphorylation and UCP1 expression. Overall, our study reveals that adipocyte-derived shed Sdc4 is a novel suppressor of lipolysis, contributing to decreased energy expenditure, thus exaggerating obesity. Targeting shed Sdc4 is a potential therapeutic strategy for obesity.

Objective

Liver fibrosis is a crucial condition for evaluating the prognosis of chronic liver disease. Lectin-1ike oxidized low density lipoprotein receptor-1 (LOX-1) has been shown potential research value and therapeutic targeting possibilities in different fibrotic diseases. However, the role of LOX-1 and the underlying mechanisms in liver fibrosis progression remain unclear.

Methods

LOX-1 expression was detected in liver tissues from patients and rodents with liver fibrosis. LOX-1 knockout rats were subjected to CCl₄ or methionine and choline-deficient diet (MCD) to induce liver fibrosis. Transcriptomic and metabolomics analysis were used to investigate the involvement and mechanism of LOX-1 on liver fibrosis.

Results

We found that LOX-1 exacerbated liver fibrosis by promoting hepatic stellate cells (HSCs) activation. LOX-1 deletion reversed the development of liver fibrosis. We further verified that LOX-1 drove liver fibrosis by reprogramming glutamine metabolism through mediating isoform switching of glutaminase (GLS). Mechanistically, we revealed the crucial role of the LOX-1/OCT1/GLS1 axis in the pathogenesis of liver fibrosis. Moreover, LOX-1 rewired ammonia metabolism by regulating glutamine metabolism-urea cycle to drive the progression of liver fibrosis.

Conclusions

Our findings uncover the pivotal role of LOX-1 in the progression of liver fibrosis, enrich the pathological significance of LOX-1 regulation of hepatic ammonia metabolism, and provide an insight into promising targets for the therapeutic strategy of liver fibrosis, demonstrating the potential clinical value of targeting LOX-1 in antifibrotic therapy.

Objective

Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonism is foundational to modern obesity pharmacotherapies. These compounds were engineered for maximal G protein alpha(s) (Gsα) signaling potency and downstream cAMP production. However, this strategy requires reconsideration as partial, biased GLP-1R agonists characterized by decreased Gsα signaling and disproportionate reductions in β-arrestin recruitment relative to the native ligand provide greater weight loss than full, balanced agonists in preclinical models.

Methods

We tested the hypothesis that in vitro signaling bias, which considers both cAMP signaling and β-arrestin recruitment, better predicts weight loss efficacy in diet induced obese (DIO) rodents than cAMP potency alone.

Results

Our data demonstrate that signaling bias significantly correlates to GLP-1R agonist mediated weight loss in diet-induced obese mice. We further characterized a protracted GLP-1 analogue (NNC5840) which exhibits a partial-Gsα, cAMP-biased GLP-1R signaling profile in vitro and demonstrates superior maximal body weight reduction compared to semaglutide in DIO mice. The NNC5840 weight loss profile is characterized by reduced in vivo potency but increased maximal efficacy.

Conclusion

The data demonstrate that biased agonism is a strong predictor of in vivo efficacy for GLP-1R agonists independent of factors like intrinsic cAMP potency or pharmacokinetics. These data suggest that drug discovery screening strategies which take a holistic approach to target receptor signaling may provide more efficacious candidate molecules. The interpretations of these studies are limited by unknowns including how structural modifications to the biased GLP-1R agonist effect physiochemical properties of the molecules.

Background

Cushing's Syndrome (CS) results from prolonged exposure to excessive glucocorticoids (GCs), leading to metabolic disturbances and adrenal insufficiency (AI). Fibroblast growth factor 21 (FGF21) has shown promise as a potential therapeutic target for metabolic disorders. This study explores the effects of FGF21 on adrenal gland function in a mouse model of AI following chronic hypercortisolism and investigates sex-dependent differences in the hypothalamic-pituitary-adrenal (HPA) axis response.

Methods

We employed a mouse model of AI after chronic corticosterone (CORT) treatment. We studied the effects of recombinant human FGF21 (hFGF21) administration on adrenal function in AI mice. We then investigated male and female wild-type (WT) and FGF21-overexpressing transgenic (Tg) mice subjected to 5 weeks of CORT treatment reaching CS phenotype, followed by immediate analysis or a 10-week recovery period. We evaluated metabolic parameters, HPA axis function, and adrenal gland morphology and gene expression.

Results

Prolonged CORT exposure resulted in metabolic disturbances and HPA axis dysregulation. hFGF21 treatment increased CORT and ACTH secretion in AI mice. FGF21 overexpression influenced glucose homeostasis and insulin regulation during CORT treatment and recovery, with sex-specific effects. Tissue-specific regulation of Klb expression was observed across the HPA axis, with distinct patterns between males and females. Tg mice displayed altered adrenal progenitor cell activation and steroidogenic gene expression. Sex-specific differences were observed in adrenal capsule remodeling and gene expression patterns during recovery.

Conclusions

This study reveals the complex interplay between FGF21 signaling and GC-induced metabolic and endocrine changes, suggesting a potential sex-specific role of FGF21 in the metabolic and HPA after CS.

Single molecules that combine complementary modes of action with glucagon-like peptide-1 receptor (GLP-1R) agonism are best-in-class therapeutics for obesity treatment. NN1706 (MAR423, RO6883746) is a fatty-acylated tri-agonist designed for balanced activity at GLP-1R and glucose-dependent insulinotropic polypeptide receptor (GIPR) with lower relative potency at the glucagon receptor (GcgR). Obese mice, rats and non-human primates dosed with NN1706 showed significant body weight reductions and improved glycemic control. In human participants with overweight or obesity, daily subcutaneous NN1706 treatment resulted in substantial body weight loss in a dose-dependent manner without impairing glycemic control (NCT03095807, NCT03661879). However, increased heart rate was observed across NN1706 treatment cohorts, which challenges further clinical development of NN1706.

Objective

Carriers of PAM (peptidylglycine alpha-amidating monooxygenase) coding variant alleles have reduced insulinogenic index, higher risk of developing type 2 diabetes (T2D), and islets from heterozygous carriers of the PAM p.Asp563Gly variant display reduced insulin secretion. Exactly how global PAM deficiency contributes to hyperglycemia remains unclear. PAM is the only enzyme capable of converting glycine-extended peptide hormones into amidated products. Like neuropeptide Y (NPY), α-melanocyte stimulating hormone (αMSH), and glucagon-like peptide 1 (GLP-1), islet amyloid polypeptide (IAPP), a beta cell peptide that forms islet amyloid in type 2 diabetes, is a PAM substrate. We hypothesized that Pam deficiency limited to beta cells would lead to reduced insulin secretion, prevent the production of amidated IAPP, and reveal the extent to which loss of Pam in β-cells could accelerate the onset of hyperglycemia in mice.

Methods

PAM activity was assessed in human islets from donors based on their PAM genotype. We generated beta cell-specific Pam knockout (Ins1^Cre/+, Pam^fl/fl; βPamKO) mice and performed islet culture, histological, and metabolic assays to evaluate the physiological roles of Pam in beta cells. We analyzed human IAPP (hIAPP) amyloid fibril forming kinetics using synthetic amidated and non-amidated hIAPP peptides, and generated hIAPP knock-in beta cell-specific Pam knockout (hIAPP^w/w βPamKO) mice to determine the impact of hIAPP amidation on islet amyloid burden, islet graft survival, and glucose tolerance.

Results

PAM enzyme activity was significantly reduced in islets from donors with the PAM p.Asp563Gly T2D-risk allele. Islets from βPamKO mice had impaired second-phase glucose- and KCl- induced insulin secretion. Beta cells from βPamKO mice had larger dense-core granules and fewer and shorter cilia. Interestingly, non-amidated hIAPP was less fibrillogenic in vitro, and high glucose-treated hIAPP^w/w βPamKO islets had reduced amyloid burden. Despite these changes in beta cell function, βPamKO mice were not more susceptible to diet-induced hyperglycemia. In vitro beta cell death and in vivo islet graft survival remained comparable between hIAPP^w/w βPamKO and hIAPP^w/w islets. Surprisingly, aged hIAPP^w/w βPamKO mice had improved insulin secretion and glucose tolerance.

Conclusions

Eliminating Pam expression only in beta cells leads to morphological changes in insulin granules, reduced insulin secretion, reduced hIAPP amyloid burden and altered ciliogenesis. However, in mice beta-cell Pam deficiency has no impact on the development of diet- or hIAPP-induced hyperglycemia. Our data are consistent with current studies revealing ancient, highly conserved roles for peptidergic signaling in the coordination of the diverse signals needed to regulate fundamental processes such as glucose homeostasis.