Cover Story
Glucagon-like peptide 1 (GLP-1) is an important gut derived hormone that enables nutrient assimilation. There are several well-established actions of GLP-1, including stimulation of postprandial insulin secretion from β cells supporting development of GLP-1 medicines for type 2 diabetes (T2D), and reduction of appetite, enabling weight loss and approval of GLP-1-based medicines for the treatment of people with obesity. The expanding actions and improved efficacy of modern GLP-1 receptor agonists (GLP-1RAs) have led to increasing utilization of these medicines for people with T2D and obesity. Moreover, the safety of these medicines has been reinforced by data from cardiovascular outcome trials demonstrating reductions in rates of non-fatal myocardial infarction, non-fatal stroke, cardiovascular death and all-cause mortality.
All Articles
- Abstract
GLP-1R signaling modulates colonic energy metabolism, goblet cell number and survival in the absence of gut microbiota
Objectives
Gut microbiota increases energy availability through fermentation of dietary fibers to short-chain fatty acids in conventionally raised mice. Energy deficiency in germ-free (GF) mice increases glucagon-like peptide-1 (GLP-1) levels, which slows intestinal transit. To further analyze the role of GLP-1-mediated signaling in this model of energy deficiency, we re-derived mice lacking GLP-1 receptor (GLP-1R KO) as GF.
Methods
GLP-1R KO mice were rederived as GF through hysterectomy and monitored for 30 weeks. Mice were subjected to rescue experiments either through feeding an energy-rich diet or colonization with a normal cecal microbiota. Histology and intestinal function were assessed at different ages. Intestinal organoids were assessed to investigate stemness.
Results
Unexpectedly, 25% of GF GLP-1R KO mice died before 20 weeks of age, associated with enlarged ceca, increased cecal water content, increased colonic expression of apical ion transporters, reduced number of goblet cells and loss of colonic epithelial integrity. Colonocytes from GLP-1R KO mice were energy-deprived and exhibited increased ER-stress; mitochondrial fragmentation, increased oxygen levels and loss of stemness. Restoring colonic energy levels either by feeding a Western-style diet or colonization with a normal gut microbiota normalized gut phenotypes and prevented lethality.
Conclusions
Our findings reveal a heretofore unrecognized role for GLP-1R signaling in the maintenance of colonic physiology and survival during energy deprivation.
- Abstract
Tyrosine-phosphorylated DNER sensitizes insulin signaling in hepatic gluconeogenesis by inducing proteasomal degradation of TRB3
Objective
Hepatic insulin resistance, which leads to increased hepatic gluconeogenesis, is a major contributor to fasting hyperglycemia in type 2 diabetes mellitus (T2DM). However, the mechanism of impaired insulin-dependent suppression of hepatic gluconeogenesis remains elusive. Delta/Notch-like epidermal growth factor (EGF)-related receptor (DNER), firstly described as a neuron-specific Notch ligand, has been recently identified as a susceptibility gene for T2DM through genome-wide association studies. We herein investigated whether DNER regulates hepatic gluconeogenesis and whether this is mediated by enhanced insulin signaling.
Methods
The association between DNER, tribbles homolog 3 (TRB3) and Akt signaling was evaluated in C57BL/6J, ob/ob and db/db mice by western blot analysis. DNER loss-of-function and gain-of-function in hepatic gluconeogenesis were analyzed by western blot analysis, quantitative real-time PCR, glucose uptake and output assay in AML-12 cells and partially validated in primary mouse hepatocytes. Hepatic DNER knockdown mice were generated by tail vein injection of adenovirus to confirm the effects of DNER in vivo. The interaction between DNER and TRB3 was investigated by rescue experiments, cycloheximide chase analysis, co-immunoprecipitation and immunofluorescence. The potential insulin-stimulated phosphorylation sites of DNER were determined by co-immunoprecipitation, LC-MS/MS analysis and site-specific mutagenesis.
Results
Here we show that DNER enhanced hepatic insulin signaling in gluconeogenesis by inhibiting TRB3, an endogenous Akt inhibitor, through the ubiquitin-proteasome degradation pathway. In AML-12 hepatocytes, insulin-stimulated activation of Akt and suppression of gluconeogenesis are attenuated by DNER knockdown, but potentiated by DNER over-expression. In C57BL/6J mice, hepatic DNER knockdown is accompanied by impaired glucose and pyruvate tolerance. Furthermore, the in vitro effects of DNER knockdown or over-expression on both Akt activity and hepatic gluconeogenesis can be rescued by TRB3 knockdown or over-expression, respectively. In response to insulin stimulation, DNER interacted directly with insulin receptor and was phosphorylated at Tyr677. This site-specific phosphorylation is essential for DNER to upregulate Akt activity and then downregulate G6Pase and PEPCK expression, by interacting with TRB3 directly and inducing TRB3 proteasome-dependent degradation.
Conclusions
Taken together, the crosstalk between insulin-Akt and DNER-TRB3 pathways represents a previously unrecognized mechanism by which insulin regulates hepatic gluconeogenesis.
- Abstract
Elevation of hypothalamic ketone bodies induces a decrease in energy expenditures and an increase risk of metabolic disorder
Objective
Ketone bodies (such as β-hydroxybutyrate or BHB) have been recently proposed as signals involved in brain regulation of energy homeostasis and obesity development. However, the precise role of ketone bodies sensing by the brain, and its impact on metabolic disorder development remains unclear. Nevertheless, partial deletion of the ubiquitous ketone bodies transporter MCT1 in mice (HE mice) results in diet-induced obesity resistance, while there is no alteration under normal chow diet. These results suggest that ketone bodies produced during the high fat diet would be important signals involved in obesity onset.
Methods
In the present study we used a specific BHB infusion of the hypothalamus and analyzed the energy homeostasis of WT or HE mice fed a normal chow diet.
Results
Our results indicate that high BHB levels sensed by the hypothalamus disrupt the brain regulation of energy homeostasis. This brain control dysregulation leads to peripheral alterations of energy expenditure mechanisms.
Conclusions
Altogether, the changes induced by high ketone bodies levels sensed by the brain increase the risk of obesity onset in mice.
- Abstract
Salubrinal promotes phospho-eIF2α-dependent activation of UPR leading to autophagy-mediated attenuation of iron-induced insulin resistance
Identification of new mechanisms mediating insulin sensitivity is important to allow validation of corresponding therapeutic targets. In this study, we first used a cellular model of skeletal muscle cell iron overload and found that endoplasmic reticulum (ER) stress and insulin resistance occurred after iron treatment. Insulin sensitivity was assessed using cells engineered to express an Akt biosensor, based on nuclear FoxO localization, as well as western blotting for insulin signaling proteins. Use of salubrinal to elevate eIF2α phosphorylation and promote the unfolded protein response (UPR) attenuated iron-induced insulin resistance. Salubrinal induced autophagy flux and its beneficial effects on insulin sensitivity were not observed in autophagy-deficient cells generated by overexpressing a dominant-negative ATG5 mutant or via knockout of ATG7. This indicated the beneficial effect of salubrinal-induced UPR activation was autophagy-dependent. We translated these observations to an animal model of systemic iron overload-induced skeletal muscle insulin resistance where administration of salubrinal as pretreatment promoted eIF2α phosphorylation, enhanced autophagic flux in skeletal muscle and improved insulin responsiveness. Together, our results show that salubrinal elicited an eIF2α-autophagy axis leading to improved skeletal muscle insulin sensitivity both in vitro and in mice.
- Abstract
Tumoral acidosis promotes adipose tissue depletion by fostering adipocyte lipolysis
Objective
Tumour progression drives profound alterations in host metabolism, such as adipose tissue depletion, an early event of cancer cachexia. As fatty acid consumption by cancer cells increases upon acidosis of the tumour microenvironment, we reasoned that fatty acids derived from distant adipose lipolysis may sustain tumour fatty acid craving, leading to the adipose tissue loss observed in cancer cachexia.
Methods
To evaluate the pro-lipolytic capacities of acid-exposed cancer cells, primary mouse adipocytes from subcutaneous and visceral adipose tissue were exposed to pH-matched conditioned medium from human and murine acid-exposed cancer cells (pH 6.5), compared to naive cancer cells (pH 7.4). To further address the role of tumoral acidosis on adipose tissue loss, a pH-low insertion peptide was injected into tumour-bearing mice, and tumoral acidosis was neutralised with a sodium bicarbonate buffer. Prolipolytic mediators were identified by transcriptomic approaches and validated on murine and human adipocytes.
Results
Here, we reveal that acid-exposed cancer cells promote lipolysis from subcutaneous and visceral adipocytes and that dampening acidosis in vivo inhibits adipose tissue depletion. We further found a set of well-known prolipolytic factors enhanced upon acidosis adaptation and unravelled a role for β-glucuronidase (GUSB) as a promising new actor in adipocyte lipolysis.
Conclusions
Tumoral acidosis promotes the mobilization of fatty acids derived from adipocytes via the release of soluble factors by cancer cells. Our work paves the way for therapeutic approaches aimed at tackling cachexia by targeting the tumour acidic compartment.
- Abstract
Ablation of IFNγ in myeloid cells suppresses liver inflammation and fibrogenesis in mice with hepatic small heterodimer partner (SHP) deletion
Background
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a common complication of obesity and, in severe cases, progresses to metabolic dysfunction-associated steatohepatitis (MASH). Small heterodimer partner (SHP) is an orphan member of the nuclear receptor superfamily and regulates metabolism and inflammation in the liver via a variety of pathways. In this study, we investigate the molecular foundation of MASH progression in mice with hepatic SHP deletion and explore possible therapeutic means to reduce MASH.
Methods
Hepatic SHP knockout mice (SHPΔhep) and their wild-type littermates (SHPfl/fl) of both sexes were fed a fructose diet for 14 weeks and subjected to an oral glucose tolerance test. Then, plasma lipids were determined, and liver lipid metabolism and inflammation pathways were analyzed with immunoblotting, RNAseq, and qPCR assays. To explore possible therapeutic intersections of SHP and inflammatory pathways, SHPΔhep mice were reconstituted with bone marrow lacking interferon γ (IFNγ−/−) to suppress inflammation.
Results
Hepatic deletion of SHP in mice fed a fructose diet decreased liver fat and increased proteins for fatty acid oxidation and liver lipid uptake, including UCP1, CPT1α, ACDAM, and SRBI. Despite lower liver fat, hepatic SHP deletion increased liver inflammatory F4/80+ cells and mRNA levels of inflammatory cytokines (IL-12, IL-6, Ccl2, and IFNγ) in both sexes and elevated endoplasmic reticulum stress markers of Cox2 and CHOP in female mice. Liver bulk RNAseq data showed upregulation of genes whose protein products regulate lipid transport, fatty acid oxidation, and inflammation in SHPΔhep mice. The increased inflammation and fibrosis in SHPΔhep mice were corrected with bone marrow-derived IFNγ−/− myeloid cell transplantation.
Conclusion
Hepatic deletion of SHP improves fatty liver but worsens hepatic inflammation possibly by driving excess fatty acid oxidation, which is corrected by deletion of IFNγ specifically in myeloid cells. This suggests that hepatic SHP limits fatty acid oxidation during fructose diet feeding but, in doing so, prevents pro-MASH pathways. The IFNγ-mediated inflammation in myeloid cells appears to be a potential therapeutic target to suppress MASH.
- Abstract
Letter-to-the-editor on “Acetyl-CoA synthetase (ACSS2) does not generate butyryl- and crotonyl-CoA”
Dear editors of Molecular Metabolism,
We read with great interest the article by Zeaiter et al. concerning “Acetyl-CoA synthetase (ACSS2) does not generate butyryl- and crotonyl-CoA” in Molecular Metabolism. The authors conducted LC-MS/MS for coenzyme A (CoAs) measurement with short-chain fatty acids (SCFAs) and purified or recombinant acetyl-CoA synthetase 2 (ACSS2) enzymes in vitro to test the ability of ACSS2 to generate SCFA-CoAs from corresponding 3- and 4-carbon SCFAs, such as propionate, butyrate and crotonate. ACSS2 was unable to generate SCFA-CoAs from butyrate and crotonate and had very low activity with propionate. Additionally, structural modeling also indicated that the ACCS2 active site was poorly compatible with crotonyl-AMP. This topic is interesting, and I believe the methods in this article are clear. However, in view of the CoAs testing results, I would like to point out some concerns that may make the conclusions more convincing.
Firstly, the use of commercially available purified acetyl-CoA synthetase enzymes for the catalysis of SCFAs in vitro reaction systems need to be discussed. Enzyme activity may be affected in vitro and could lead to abnormal function of ACSS2, which differs from physiological conditions. Thus, interfering with ACSS2 expression (overexpressed or silenced) in cells to detect CoAs may be a better approach, as described in other articles.
Secondly, in my opinion, one of the greatest limitations of this study is that it may not be appropriate to optimize the reaction conditions with acetate as the substrate. It is worth noting that the unit of MS intensity is “104 AU”, as shown in Table 1; therefore, we speculate that the MS intensity of crotonyl-CoA and butyryl-CoA may be much less than “104 AU”, rather than completely “zero”. We believe that the amounts of crotonyl-CoA and butyryl-CoA are small but measurable, while using the highest amount of acetyl-CoA as a control would lead to smaller values for crotonyl-CoA and butyryl-CoA. Therefore, ACSS2 is much more capable of using acetate than butyrate and crotonate, but this finding does not indicate that ACSS2 cannot catalyze butyrate or crotonate. Additionally, for the catalytic effect of ACSS2, 0.5 mM acetate is sufficient but may not be sufficient for crotonate or butyrate. Previous study reported that intracellular crotonyl-CoA concentration is about 600- to 1,000-fold lower than that of acetyl-CoA, and the doses of crotonate used for crotonyl-CoA detection were usually 2.5 mM, 5 mM, or 10 mM. Sabari et al. also reported that knockdown of ACSS2 reduced the amount of crotonyl-CoA produced in the presence of 10 mM crotonate in HeLa S3 cells. Thus, we believe that increasing the content of crotonate may promote the generation of crotonyl-CoA by ACSS2.
In summary, we obtained a new method to investigate the ability of ACSS2 to generate other CoAs, such as structural modeling. However, for these CoAs detection, we suppose that regulating ACSS2 expression in living cells may be more credible. Using the highest amount of acetyl-CoA as a contrast for other CoAs may not be appropriate. Additionally, increasing the concentration of butyrate and crotonate to measure the CoAs content might be more convincing.
- Abstract
Roles and therapeutic targeting of ceramide metabolism in cancer
Background
Ceramides are sphingolipids that act as signaling molecules involved in regulating cellular processes including apoptosis, proliferation, and metabolism. Deregulation of ceramide metabolism contributes to cancer development and progression. Therefore, regulation of ceramide levels in cancer cells is being explored as a new approach for cancer therapy.
Scope of the review
This review discusses the multiple roles of ceramides in cancer cells and strategies to modulate ceramide levels for cancer therapy. Ceramides attenuate cell survival signaling and metabolic pathways, while activating apoptotic mechanisms, making them tumor-suppressive. Approaches to increase ceramide levels in cancer cells include using synthetic analogs, inhibiting ceramide degradation, and activating ceramide synthesis. We also highlight combination therapies such as use of ceramide modulators with chemotherapies, immunotherapies, apoptosis inducers, and anti-angiogenics, which offer synergistic antitumor effects. Additionally, we also describe ongoing clinical trials evaluating ceramide nanoliposomes and analogs. Finally, we discuss the challenges of these therapeutic approaches including the complexity of ceramide metabolism, targeted delivery, cancer heterogeneity, resistance mechanisms, and long-term safety.
Major conclusions
Ceramide-based therapy is a potentially promising approach for cancer therapy. However, overcoming hurdles in pharmacokinetics, specificity, and resistance is needed to optimize its efficacy and safety. This requires comprehensive preclinical/clinical studies into ceramide signaling, formulations, and combination therapies. Ceramide modulation offers opportunities for developing novel cancer treatments, but a deeper understanding of ceramide biology is vital to advance its clinical applications.
- Abstract
Loss of GIPR in LEPR cells impairs glucose control by GIP and GIP:GLP-1 co-agonism without affecting body weight and food intake in mice
Objective
The glucose-dependent insulinotropic polypeptide (GIP) decreases body weight via central GIP receptor (GIPR) signaling, but the underlying mechanisms remain largely unknown. Here, we assessed whether GIP regulates body weight and glucose control via GIPR signaling in cells that express the leptin receptor (Lepr).
Methods
Hypothalamic, hindbrain, and pancreatic co-expression of Gipr and Lepr was assessed using single cell RNAseq analysis. Mice with deletion of Gipr in Lepr cells were generated and metabolically characterized for alterations in diet-induced obesity (DIO), glucose control and leptin sensitivity. Long-acting single- and dual-agonists at GIPR and GLP-1R were further used to assess drug effects on energy and glucose metabolism in DIO wildtype (WT) and Lepr-Gipr knock-out (KO) mice.
Results
Gipr and Lepr show strong co-expression in the pancreas, but not in the hypothalamus and hindbrain. DIO Lepr-Gipr KO mice are indistinguishable from WT controls related to body weight, food intake and diet-induced leptin resistance. Acyl-GIP and the GIPR:GLP-1R co-agonist MAR709 remain fully efficacious to decrease body weight and food intake in DIO Lepr-Gipr KO mice. Consistent with the demonstration that Gipr and Lepr highly co-localize in the endocrine pancreas, including the β-cells, we find the superior glycemic effect of GIPR:GLP-1R co-agonism over single GLP-1R agonism to vanish in Lepr-Gipr KO mice.
Conclusions
GIPR signaling in cells/neurons that express the leptin receptor is not implicated in the control of body weight or food intake, but is of crucial importance for the superior glycemic effects of GIPR:GLP-1R co-agonism relative to single GLP-1R agonism.
- Abstract
Response to letter-to-the-editor: “Acetyl-CoA synthetase (ACSS2) does not generate butyryl- and crotonyl-CoA”
Dear Editors of Molecular Metabolism,
Thank you for the opportunity to respond to the Letter-to-the-Editor regarding our recent publication on the substrate specificity of acetyl-CoA synthetase short chain 2 (ACSS2) and to provide clarification on certain aspects of our study.
The authors of this comment suggest that detecting short-chain acyl-CoAs (SCA-CoAs) through the regulation of ACSS2 expression in living cells might offer a more reliable approach to evaluating its role in SCA-CoA production. We respectfully disagree. ACSS2 depletion and the resulting drop in acetyl-CoA concentration would indirectly affect the cellular concentration of several SCA-CoAs, including crotonyl-CoA. Indeed, acetyl-CoA produced by ACSS2 feeds acetyl-CoA carboxylase 1 (ACC1), whose activity is required for histone butyrylation and crotonylation. Furthermore, ACSS2 regulates the expression of genes involved in gluconeogenesis and fat metabolism, thereby potentially impacting SCA-CoA production significantly. Thus, in our opinion, altering ACSS2 levels in cells would not provide conclusive evidence for its substrate specificity, which was the primary focus of our study.
By far the most reliable way to assess the ability of ACSS2 to generate SCA-CoAs from the corresponding short-chain fatty acids (SCFA) is through in vitro assays with purified components. Although it is acknowledged that in vitro conditions, despite closely mimicking in vivo environments as in our study, might impact the specific activity of an enzyme, it is highly improbable that they would affect its substrate specificity, which constituted the main objective of our research. To further minimize any issue with individual acetyl-CoA synthetase preparations, we used human and yeast enzymes from different sources.
The authors of the comment further raise doubts regarding our study's conclusion that ACSS2 cannot generate butyryl- or crotonyl-CoA, speculating that a low concentration of these molecules in our assay may have escaped detection. Importantly, our mass spectrometry covers an SCA-CoA concentration range that is linear for at least 4 orders of magnitude and has a detection limit for SCA-CoAs of <0.5 to <10 nM, depending on the species. Since our assay converted up to half of 0.5 mM acetate into acetyl-CoA, a 1000-fold lower efficiency for crotonyl-CoA as suggested by the authors of the comment would yield 0.5 μM, still largely above the detection limits. Thus, at the analyzed SCFA concentrations of 0.5 mM, we can exclude production of butyryl- and crotonyl-CoA. This concentration was deliberately chosen to be much higher than the intracellular levels of SCFA. The concentration of acetate, by far the most abundant species, is 0.05–0.2 mM in the plasma and is unlikely to be higher within cells, consistent with the reported Km value of ACSS2 for acetate of about 0.05–0.11 mM. If trace amounts of other SCA-CoAs were to be generated using excessively higher SCFA concentrations, for which we have no data, this result would not be physiologically relevant. Conclusions similar to ours were already reached previously with commercially available or self-purified enzyme, albeit using less sensitive methodology. For instance, Frenkel and Kitchens reported that acetyl-CoA synthetase purified from Baker's yeast had high substrate specificity for acetate and propionate. Patel and Walt found that commercially purchased enzymes accepted 3-chloropropionic acid, similar in size to butyric acid, as a substrate, but not butyric acid itself. This finding provides evidence that these enzymes can maintain their activity on substrates other than acetate, but are highly selective in their choice of longer-chain fatty acid molecules.
Mindful of the potential limitations of individual methodologies, we combined three independent approaches – cellular, in vitro and in silico – which consistently support the conclusions of our study. We feel that it is crucial to convey this information to the community. We suggest that the indirect effects of ACSS2 knockdown should be reevaluated based on our findings and those reported in the existing literature, rather than accepting the hypothesis of its broad substrate specificity as an established fact.
- Abstract
Scd1 and monounsaturated lipids are required for autophagy and survival of adipocytes
Objective
Exposure of adipocytes to ‘cool’ temperatures often found in the periphery of the body induces expression of Stearoyl-CoA Desaturase-1 (Scd1), an enzyme that converts saturated fatty acids to monounsaturated fatty acids. The goal of this study is to further investigate the roles of Scd in adipocytes.
Method
In this study, we employed Scd1 knockout cells and mouse models, along with pharmacological Scd1 inhibition to dissect the enzyme's function in adipocyte physiology.
Results
Our study reveals that production of monounsaturated lipids by Scd1 is necessary for fusion of autophagosomes to lysosomes and that with a Scd1-deficiency, autophagosomes accumulate. In addition, Scd1-deficiency impairs lysosomal and autolysosomal acidification resulting in vacuole accumulation and eventual cell death. Blocking autophagosome formation or supplementation with monounsaturated fatty acids maintains vitality of Scd1-deficient adipocytes.
Conclusion
This study demonstrates the indispensable role of Scd1 in adipocyte survival, with its inhibition in vivo triggering autophagy-dependent cell death and its depletion in vivo leading to the loss of bone marrow adipocytes.
- Abstract
Intestinal Acyl-CoA synthetase 5 (ACSL5) deficiency potentiates postprandial GLP-1 & PYY secretion, reduces food intake, and protects against diet-induced obesity
Objective
In the small intestine, the products of digestion of dietary triacylglycerol (TAG), fatty acids (FA) and monoacylglycerol, are taken up by absorptive cells, enterocytes, for systemic energy delivery. These digestion products can also bind receptors on endocrine cells to stimulate the release of hormones capable of influencing systemic energy metabolism. The initial phase of intestinal FA absorption involves the acylation of FAs to acyl-CoA by the acyl-CoA long chain synthetase (ACSL) enzymes. ACSL5 is abundantly expressed in the small intestinal epithelium where it is the major ACSL isoform, contributing approximately 80% of total ACSL activity. In mice with whole body deficiency of ACSL5, the rate of dietary fat absorption is reduced and energy expenditure is increased. However, the mechanisms by which intestinal ACSL5 contributes to intestinal FA metabolism, enteroendocrine signaling, and regulation of energy expenditure remain undefined. Here, we test the hypothesis that intestinal ACSL5 regulates energy metabolism by influencing dietary fat absorption and enteroendocrine signaling.
Methods
To explore the role of intestinal ACSL5 in energy balance and intestinal dietary fat absorption, a novel mouse model of intestine specific ACSL5 deficiency (ACSL5IKO) was generated by breeding ACSL5 floxed (ACSL5loxP/loxP) to mice harboring the tamoxifen inducible, villin-Cre recombinase. ACSL5IKO and control, ACSL5loxP/loxP mice were fed chow (low in fat) or a 60% high fat diet (HFD), and metabolic phenotyping was performed including, body weight, body composition, insulin and glucose tolerance tests, energy expenditure, physical activity, and food intake studies. Pair-feeding studies were performed to determine the role of food intake in regulating development of obesity. Studies of dietary fat absorption, fecal lipid excretion, intestinal mucosal FA content, and circulating levels of glucagon like peptide 1 (GLP-1) and peptide YY (PYY) in response to a TAG challenge were performed. Treatment with a GLP-1 receptor antagonist was performed to determine the contribution of GLP-1 to acute regulation of food intake.
Results
We found that ACSL5IKO mice experienced rapid and sustained protection from body weight and fat mass accumulation during HFD feeding. While intestine specific deficiency of ACSL5 delayed gastric emptying and reduced dietary fat secretion, it did not result in increased excretion of dietary lipid in feces. Energy expenditure and physical activity were not increased in ACSL5IKO mice. Mice deficient in intestinal ACSL5 display significantly reduced energy intake during HFD, but not chow feeding. When HFD intake of control mice was matched to ACSL5IKO during pair-feeding studies, no differences in body weight or fat mass gain were observed between groups. Postprandial GLP-1 and PYY were significantly elevated in ACSL5IKO mice secondary to increased FA content in the distal small intestine. Blockade of GLP-1 signaling by administration of a long-acting GLP-1 receptor antagonist partially restored HFD intake of ACSL5IKO.
Conclusions
These data indicate that intestinal ACSL5 serves as a critical regulator of energy balance, protecting mice from diet-induced obesity exclusively by increasing satiety and reducing food intake during HFD feeding. The reduction in food intake observed in ACSL5IKO mice is driven, in part, by increased postprandial GLP-1 and PYY secretion. These effects are only observed during HFD feeding, suggesting that altered processing of dietary fat following intestinal ACSL5 ablation contributes to GLP-1 and PYY mediated increases in satiety.
- Abstract
Histone lactylation in macrophages is predictive for gene expression changes during ischemia induced-muscle regeneration
Objectives
We have previously shown that lactate is an essential metabolite for macrophage polarisation during ischemia-induced muscle regeneration. Recent in vitro work has implicated histone lactylation, a direct derivative of lactate, in macrophage polarisation. Here, we explore the in vivo relevance of histone lactylation for macrophage polarisation after muscle injury.
Methods
To evaluate macrophage dynamics during muscle regeneration, we subjected mice to ischemia-induced muscle damage by ligating the femoral artery. Muscle samples were harvested at 1, 2, 4, and 7 days post injury (dpi). CD45+CD11b+F4/80+CD64+ macrophages were isolated and processed for RNA sequencing, Western Blotting, and CUT&Tag-sequencing to investigate gene expression, histone lactylation levels, and histone lactylation genomic localisation and enrichment, respectively.
Results
We show that, over time, macrophages in the injured muscle undergo extensive gene expression changes, which are similar in nature and in timing to those seen after other types of muscle-injuries. We find that the macrophage histone lactylome is modified between 2 and 4 dpi, which is a crucial window for macrophage polarisation. Absolute histone lactylation levels increase, and, although subtly, the genomic enrichment of H3K18la changes. Overall, we find that histone lactylation is important at both promoter and enhancer elements. Lastly, H3K18la genomic profile changes from 2 to 4 dpi were predictive for gene expression changes later in time, rather than being a reflection of prior gene expression changes.
Conclusions
Our results suggest that histone lactylation dynamics are functionally important for the function of macrophages during muscle regeneration.
- Abstract
Identification of a chromatin-bound ERRα interactome network in mouse liver
Objectives
Estrogen-related-receptor α (ERRα) plays a critical role in the transcriptional regulation of cellular bioenergetics and metabolism, and perturbations in its activity have been associated with metabolic diseases. While several coactivators and corepressors of ERRα have been identified to date, a knowledge gap remains in understanding the extent to which ERRα cooperates with coregulators in the control of gene expression. Herein, we mapped the primary chromatin-bound ERRα interactome in mouse liver.
Methods
RIME (Rapid Immuno-precipitation Mass spectrometry of Endogenous proteins) analysis using mouse liver samples from two circadian time points was used to catalog ERRα-interacting proteins on chromatin. The genomic crosstalk between ERRα and its identified cofactors in the transcriptional control of precise gene programs was explored through cross-examination of genome-wide binding profiles from chromatin immunoprecipitation-sequencing (ChIP-seq) studies. The dynamic interplay between ERRα and its newly uncovered cofactor Host cell factor C1 (HCFC1) was further investigated by loss-of-function studies in hepatocytes.
Results
Characterization of the hepatic ERRα chromatin interactome led to the identification of 48 transcriptional interactors of which 42 were previously unknown including HCFC1. Interrogation of available ChIP-seq binding profiles highlighted oxidative phosphorylation (OXPHOS) under the control of a complex regulatory network between ERRα and multiple cofactors. While ERRα and HCFC1 were found to bind to a large set of common genes, only a small fraction showed their colocalization, found predominately near the transcriptional start sites of genes particularly enriched for components of the mitochondrial respiratory chain. Knockdown studies demonstrated inverse regulatory actions of ERRα and HCFC1 on OXPHOS gene expression ultimately dictating the impact of their loss-of-function on mitochondrial respiration.
Conclusions
Our work unveils a repertoire of previously unknown transcriptional partners of ERRα comprised of chromatin modifiers and transcription factors thus advancing our knowledge of how ERRα regulates metabolic transcriptional programs.
- Abstract
A practical and robust method to evaluate metabolic fluxes in primary pancreatic islets
Objective
Evaluation of mitochondrial oxygen consumption and ATP production is important to investigate pancreatic islet pathophysiology. Most studies use cell lines due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables, is expensive and poorly reproducible. Our aim was to establish a practical method to assess primary islet metabolic fluxes using standard commercial consumables.
Methods
Pancreatic islets were isolated from mice/rats, dispersed with trypsin, and adhered to pre-coated standard Seahorse or Resipher microplates. Oxygen consumption was evaluated using a Seahorse Extracellular Flux Analyzer or a Resipher Real-time Cell Analyzer.
Results
We provide a detailed protocol with all steps to optimize islet isolation with high yield and functionality. Our method requires a few islets per replicate; both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators and glucose. The technique was validated by other functional assays, which show these cells present conserved calcium influx and insulin secretion in response to glucose. We also show that our dispersed islets maintain robust basal respiration levels, in addition to maintaining up to 89% viability after five days in dispersed cultures. Furthermore, OCRs can be measured in Seahorse analyzers and in other plate respirometry systems, using standard materials.
Conclusions
Overall, we established a practical and robust method to assess islet metabolic fluxes and oxidative phosphorylation, a valuable tool to uncover basic β-cell metabolic mechanisms as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols.