Volume 61 | July 2022

Background

Ferroptosis, as a new form of cell death, is different from other cell deaths such as autophagy or senescence. Ferroptosis involves in the pathophysiological progress of several diseases, including cancers, cardiovascular diseases, nervous system diseases, and kidney damage. Since oxidative stress and iron deposition are the broad pathological features of neurological diseases, the role of ferroptosis in neurological diseases has been widely explored.

Scope of review

Ferroptosis is mainly characterized by changes in iron homeostasis, iron-dependent lipid peroxidation, and glutamate toxicity accumulation, of which can be specifically reversed by ferroptosis inducers or inhibitors. The ferroptosis is mainly regulated by the metabolism of iron, lipids and amino acids through System Xc⁻, voltage-dependent anion channels, p53, p62-Keap1-Nrf2, mevalonate and other pathways. This review also focus on the regulatory pathways of ferroptosis and its research progress in neurological diseases.

Major conclusions

The current researches of ferroptosis in neurological diseases mostly focus on the key pathways of ferroptosis. At the same time, ferroptosis was found playing a bidirectional regulation role in neurological diseases. Therefore, the specific regulatory mechanisms of ferroptosis in neurological diseases still need to be further explored to provide new perspectives for the application of ferroptosis in the treatment of neurological diseases.

Objective

Aberrant ketogenesis is correlated with the degree of steatosis in non-alcoholic fatty liver disease (NAFLD) patients, and an inborn error of ketogenesis (mitochondrial HMG-CoA synthase deficiency) is commonly associated with the development of the fatty liver. Here we aimed to determine the impact of Hmgcs2-mediated ketogenesis and its modulations on the development and treatment of fatty liver disease.

Methods

Loss- and gain-of-ketogenic function models, achieved by Hmgcs2 knockout and overexpression, respectively, were utilized to investigate the role of ketogenesis in the hepatic lipid accumulation during postnatal development and in a high-fat diet-induced NAFLD mouse model.

Results

Ketogenic function was decreased in NAFLD mice with a reduction in Hmgcs2 expression. Mice lacking Hmgcs2developed spontaneous fatty liver phenotype during postnatal development, which was rescued by a shift to a low-fat dietary composition via early weaning. Hmgcs2 heterozygous adult mice, which exhibited lower ketogenic activity, were more susceptible to diet-induced NAFLD development, whereas HMGCS2 overexpression in NAFLD mice improved hepatosteatosis and glucose homeostasis.

Conclusions

Our study adds new knowledge to the field of ketone body metabolism and shows that Hmgcs2-mediated ketogenesis modulates hepatic lipid regulation under a fat-enriched nutritional environment. The regulation of hepatic ketogenesis may be a viable therapeutic strategy in the prevention and treatment of hepatosteatosis.

Objectives

Glucagon secretion to stimulate hepatic glucose production is the first line of defense against hypoglycemia. This response is triggered by so far incompletely characterized central hypoglycemia-sensing mechanisms, which control autonomous nervous activity and hormone secretion. The objective of this study was to identify novel hypothalamic genes controlling insulin-induced glucagon secretion.

Methods

To obtain new information on the mechanisms of hypothalamic hypoglycemia sensing, we combined genetic and transcriptomic analysis of glucagon response to insulin-induced hypoglycemia in a panel of BXD recombinant inbred mice.

Results

We identified two QTLs on chromosome 8 and chromosome 15. We further investigated the role of Irak4 and Cpne8, both located in the QTL on chromosome 15, in C57BL/6J and DBA/2J mice, the BXD mouse parental strains. We found that the poor glucagon response of DBA/2J mice was associated with higher hypothalamic expression of Irak4, which encodes a kinase acting downstream of the interleukin-1 receptor (Il-1R), and of Il-ß when compared with C57BL/6J mice. We showed that intracerebroventricular administration of an Il-1R antagonist in DBA/2J mice restored insulin-induced glucagon secretion; this was associated with increased c-fos expression in the arcuate and paraventricular nuclei of the hypothalamus and with higher activation of both branches of the autonomous nervous system. Whole body inactivation of Cpne8, which encodes a Ca⁺⁺-dependent regulator of membrane trafficking and exocytosis, however, had no impact on insulin-induced glucagon secretion.

Conclusions

Collectively, our data identify Irak4 as a genetically controlled regulator of hypoglycemia-activated hypothalamic neurons and glucagon secretion.

Objective

The mechanisms by which glucose stimulates insulin secretion from β-cells are well established and involve inhibition of ATP-sensitive K⁺ (K_ATP) channels, followed by a rise in [Ca²⁺]_c that triggers exocytosis. However, the mechanisms by which glucose controls glucagon release from α-cells are much less known. In particular, it is debated whether the sugar controls glucagon secretion by changing α-cell [Ca²⁺]_c, and whether K_ATP channels or paracrine factors are involved. The present study addresses these issues.

Methods

We tested the effect of a decrease or an increase of glucose concentration (Gx, with x = concentration in mM) on α-cell [Ca²⁺]_c and glucagon secretion. α-cell [Ca²⁺]_c was monitored using GluCreGCaMP6f mice expressing the Ca²⁺-sensitive fluorescent protein, GCaMP6f, specifically in α-cells. [Ca²⁺]_c was compared between dispersed α-cells and α-cells within islets to evaluate the potential contribution of an indirect effect of glucose. The same protocols were used for experiments of glucagon secretion from whole islets and [Ca²⁺]_c measurements to test if changes in glucagon release mirror those in α-cell [Ca²⁺]_c.

Results

Blockade of K_ATP channels by sulfonylureas (tolbutamide 100 μM or gliclazide 25 μM) strongly increased [Ca²⁺]_c in both dispersed α-cells and α-cells within islets. By contrast, glucose had no effect on [Ca²⁺]_c in dispersed α-cells, whereas it affected it in α-cells within islets. The effect of glucose was however different in islets expressing (Sst^+/+) or not somatostatin (SST) (Sst^−/−). Decreasing glucose concentration from G7 to G1 modestly increased α-cell [Ca²⁺]_c, but to a slightly larger extent in Sst^+/+ islets than in Sst^−/− islets. This G1-induced [Ca²⁺]_c rise was also observed in the continuous presence of sulfonylureas in both Sst^+/+ and Sst^−/− islets. Increasing glucose concentration from G7 to G20 did not affect α-cell [Ca²⁺]_c in Sst^+/+ islets which remained low, whereas it strongly increased it in Sst^−/− islets. The observations that this increase was seen only in α-cells within islets but never in dispersed α-cells and that it was abrogated by the gap junction inhibitor, carbenoxolone, point to an indirect effect of G20 and suggest that, in Sst^−/− islets, G20-stimulated β-cells entrain α-cells whereas, in Sst^+/+ islets, the concomitant release of SST keeps α-cell [Ca²⁺]_c at low levels. The [Ca²⁺]_c lowering effect of endogenous SST is also supported by the observation that SST receptor antagonists (SSTR2/3) increased [Ca²⁺]_c in α-cells from Sst^+/+ islets. All these [Ca²⁺]_c changes induced parallel changes in glucagon release. To test if glucose also controls glucagon release independently of [Ca²⁺]_c changes, additional experiments were performed in the continuous presence of 30 mM K⁺ and the K_ATP channel opener diazoxide (250 μM). In these conditions, α-cell [Ca²⁺]_c within islets was elevated and its steady-state level was unaffected by glucose. However, decreasing the glucose concentration from G7 to G1 stimulated glucagon release whereas increasing it from G1 to G15 inhibited it. These effects were also evident in Sst^−/− islets, and opposite to those on insulin secretion.

Conclusions

We propose a model according to which glucose controls α-cell [Ca²⁺]_c and glucagon secretion through multiple mechanisms. Increasing the glucose concentration modestly decreases [Ca²⁺]_c in α-cells independently of their K_ATPchannels and partly via SST. The involvement of SST increases with the glucose concentration, and one major effect of SST is to keep α-cell [Ca²⁺]_c at low levels by counteracting the effect of an entrainment of α-cells by β-cells when β-cells become stimulated by glucose. All these [Ca²⁺]_c changes induce parallel changes in glucagon release. Glucose also decreases the efficacy of Ca²⁺ on exocytosis by an attenuating pathway that is opposite to the well-established amplifying pathway controlling insulin release in β-cells.

Objective

Mitochondrial “retrograde” signaling may stimulate organelle biogenesis as a compensatory adaptation to aberrant activity of the oxidative phosphorylation (OXPHOS) system. To maintain energy-consuming processes in OXPHOS deficient cells, alternative metabolic pathways are functionally coupled to the degradation, recycling and redistribution of biomolecules across distinct intracellular compartments. While transcriptional regulation of mitochondrial network expansion has been the focus of many studies, the molecular mechanisms promoting mitochondrial maintenance in energy-deprived cells remain poorly investigated.

Methods

We performed transcriptomics, quantitative proteomics and lifespan assays to identify pathways that are mechanistically linked to mitochondrial network expansion and homeostasis in Caenorhabditis elegans lacking the mitochondrial calcium uptake protein 1 (MICU-1/MICU1). To support our findings, we carried out biochemical and image analyses in mammalian cells and mouse-derived tissues.

Results

We report that micu-1(null) mutations impair the OXPHOS system and promote C. elegans longevity through a transcriptional program that is independent of the mitochondrial calcium uniporter MCU-1/MCU and the essential MCU regulator EMRE-1/EMRE. We identify sphingosine phosphate lyase SPL-1/SGPL1 and the ATFS-1-target HOPS complex subunit VPS-39/VPS39 as critical lifespan modulators of micu-1(null) mutant animals. Cross-species investigation indicates that SGPL1 upregulation stimulates VPS39 recruitment to the mitochondria, thereby enhancing mitochondria-lysosome contacts. Consistently, VPS39 downregulation compromises mitochondrial network maintenance and basal autophagic flux in MICU1 deficient cells. In mouse-derived muscles, we show that VPS39 recruitment to the mitochondria may represent a common signature associated with altered OXPHOS system.

Conclusions

Our findings reveal a previously unrecognized SGPL1/VPS39 axis that stimulates intracellular organelle interactions and sustains autophagy and mitochondrial homeostasis in OXPHOS deficient cells.

Objective

Due to the increasing prevalence of obesity and insulin resistance, there is an urgent need for better treatment of obesity and its related metabolic disorders. This study aimed to elucidate the role of SERPINA3C, an adipocyte secreted protein, in obesity and related metabolic disorders.

Methods

Male wild type (WT) and knockout (KO) mice were fed with high-fat diet (HFD) for 16 weeks, adiposity, insulin resistance, and inflammation were assessed. AAV-mediated overexpression of SERPINA3C was injected locally in inguinal white adipose tissue (iWAT) to examine the effect of SERPINA3C. In vitro analyses were conducted in 3T3-L1 adipocytes to explore the molecular pathways underlying the function of SERPINA3C.

Results

Functional exploration of the SERPINA3C knockout mice revealed that SERPINA3C deficiency led to an impaired metabolic phenotype (more severe obesity, lower metabolic rates, worse glucose intolerance and insulin insensitivity), which was associated with anabatic inflammation and apoptosis of white adipose tissues. Consistent with these results, overexpression of SERPINA3C in inguinal adipose tissue protected mice against diet-induced obesity and metabolic disorders with less inflammation and apoptosis in adipose tissue. Mechanistically, SERPINA3C inhibited Cathepsin Gactivity, acting as a serine protease inhibitor, which blocked Cathepsin G-mediated turnover of α5/β1 Integrin protein. Then, the preserved integrity (increase) of α5/β1 Integrin signaling activated AKT to decrease JNK phosphorylation, thereby inhibiting inflammation and promoting insulin sensitivity in adipocytes.

Conclusions/interpretation

These findings demonstrate a previously unknown SERPINA3C/Cathepsin G/Integrin/AKT pathway in regulating adipose tissue inflammation, and suggest the therapeutic potential of targeting SERPINA3C/Cathepsin G axis in adipose tissue for the treatment of obesity and metabolic diseases.

Objective

IL-6 is an important contributor to glucose and energy homeostasis through changes in whole-body glucose disposal, insulin sensitivity, food intake and energy expenditure. However, the relative contributions of peripheral versus central IL-6 signaling to these metabolic actions are presently unclear. A conditional mouse model with reduced brain IL-6Ra expression was used to explore how blunted central IL-6 signaling alters metabolic status in lean and obese mice.

Methods

Transgenic mice with reduced levels of central IL-6 receptor alpha (IL-6Ra) (IL-6Ra KD mice) and Nestin Cre controls (Cre^+/- mice) were fed standard chow or high-fat diet for 20 weeks. Obese and lean mouse cohorts underwent metabolic phenotyping with various measures of energy and glucose homeostasis determined. Glucose-stimulated insulin secretionwas assessed in vivo and ex vivo in both mouse groups.

Results

IL-6Ra KD mice exhibited altered body fat mass, liver steatosis, plasma insulin, IL-6 and NEFA levels versus Cre^+/- mice in a diet-dependent manner. IL-6Ra KD mice had increased food intake, higher RER, decreased energy expenditure with diminished cold tolerance compared to Cre^+/- controls. Standard chow-fed IL-6Ra KD mice displayed reduced plasma insulin and glucose-stimulated insulin secretion with impaired glucose disposal and unchanged insulin sensitivity. Isolated pancreatic islets from standard chow-fed IL-6Ra KD mice showed comparable morphology and glucose-stimulated insulin secretion to Cre^+/- controls. The diminished in vivo insulin secretion exhibited by IL-6Ra KD mice was recovered by blockade of autonomic ganglia.

Conclusions

This study shows that central IL-6Ra signaling contributes to glucose and energy control mechanisms by regulating food intake, energy expenditure, fuel flexibility and insulin secretion. A plausible mechanism linking central IL-6Ra signaling and pancreatic insulin secretion is through the modulation of autonomic output activity. Thus, brain IL-6 signaling may contribute to the central adaptive mechanisms engaged in response to metabolic stress.

Objective

Exercise is a critical component of a healthy lifestyle and a key strategy for the prevention and management of metabolic disease. Identifying molecular mechanisms underlying adaptation in response to chronic physical activity is of critical interest in metabolic physiology. Circadian rhythms broadly modulate metabolism, including muscle substrate utilizationand exercise capacity. Here, we define the molecular and physiological changes induced across the daily cycle by voluntary low intensity daily exercise.

Methods

Wildtype C57BL6/J male and female mice were housed with or without access to a running wheel for six weeks. Maximum running speed was measured at four different zeitgeber times (ZTs, hours after lights on) using either electrical or manual stimulation to motivate continued running on a motorized treadmill. RNA isolated from plantaris muscles at six ZTs was sequenced to establish the impact of daily activity on genome-wide transcription. Patterns of gene expression were analyzed using Gene Set Enrichment Analysis (GSEA) and Detection of Differential Rhythmicity (DODR). Blood glucose, lactate, and ketones, and muscle and liver glycogen were measured before and after exercise.

Results

We demonstrate that the use of mild electrical shocks to motivate running negatively impacts maximum running speed in mice, and describe a manual method to motivate running in rodent exercise studies. Using this method, we show that time of day influences the increase in exercise capacity afforded by six weeks of voluntary wheel running: when maximum running speed is measured at the beginning of the nighttime active period in mice, there is no measurable benefit from a history of daily voluntary running, while maximum increase in performance occurs at the end of the night. We show that daily voluntary exercise dramatically remodels the murine muscle circadian transcriptome. Finally, we describe daily rhythms in carbohydrate metabolism associated with the time-dependent response to moderate daily exercise in mice.

Conclusions

Collectively, these data indicate that chronic nighttime physical activity dramatically remodels daily rhythms of murine muscle gene expression, which in turn support daily fluctuations in exercise performance.

Objective

Classical ATP-independent non-shivering thermogenesis enabled by uncoupling protein 1 (UCP1) in brown adipose tissue(BAT) is activated, but not essential for survival, in the cold. It has long been suspected that futile ATP-consuming substrate cycles also contribute to thermogenesis and can partially compensate for the genetic ablation of UCP1 in mouse models. Futile ATP-dependent thermogenesis could thereby enable survival in the cold even when brown fat is less abundant or missing.

Methods

In this study, we explore different potential sources of UCP1-independent thermogenesis and identify a futile ATP-consuming triglyceride/fatty acid cycle as the main contributor to cellular heat production in brown adipocytes lacking UCP1. We uncover the mechanism on a molecular level and pinpoint the key enzymes involved using pharmacological and genetic interference.

Results

ATGL is the most important lipase in terms of releasing fatty acids from lipid droplets, while DGAT1 accounts for the majority of fatty acid re-esterification in UCP1-ablated brown adipocytes. Furthermore, we demonstrate that chronic cold exposure causes a pronounced remodeling of adipose tissues and leads to the recruitment of lipid cycling capacity specifically in BAT of UCP1-knockout mice, possibly fueled by fatty acids from white fat. Quantification of triglyceride/fatty acid cycling clearly shows that UCP1-ablated animals significantly increase turnover rates at room temperature and below.

Conclusion

Our results suggest an important role for futile lipid cycling in adaptive thermogenesis and total energy expenditure.

Objective

Obesity, a growing threat to the modern society, represents an imbalance of metabolic queues that normally signal to the arcuate hypothalamic nucleus, a critical brain region sensing and regulating energy homeostasis. This is achieved by various neurons many of which developmentally originate from the proopiomelanocortin (POMC)-expressing lineage. Within the mature neurons originating from this lineage, we aimed to identify non-coding genes in control of metabolic function in the adulthood.

Methods

In this work, we used microRNA mimic delivery and POMC^Cre-dependent CRISPR-Cas9 knock-out strategies in young or aged mice. Importantly, we also used CRISPR guides directing suicide cleavage of Cas9 to limit the off-target effects.

Results

Here we found that mature neurons originating from the POMC lineage employ miR-29a to protect against insulin resistance obesity, hyperphagia, decreased energy expenditure and obesity. Moreover, we validated the miR-29 family as a prominent regulator of the PI3K-Akt-mTOR pathway. Within the latter, we identified a direct target of miR-29a-3p, Nras, which was up-regulated in those and only those mature POMC^CreCas9 neurons that were effectively transduced by anti-miR-29 CRISPR-equipped construct. Moreover, POMC^Cre-dependent co-deletion of Nras in mature neurons attenuated miR-29 depletion-induced obesity.

Conclusions

Thus, the first to our knowledge case of in situ Cre-dependent CRISPR-Cas9-mediated knock-out of microRNAs in a specific hypothalamic neuronal population helped us to decipher a critical metabolic circuit in adult mice. This work significantly extends our understanding about the involvement of neuronal microRNAs in homeostatic regulation.

Objective

Lipids exert dynamic biological functions which are determined both by their fatty acyl compositions and spatiotemporal distributions inside the cell. However, it remains a daunting task to investigate any of these features for each of the more than 1000 lipid species due to a lack of a universal labeling method for individual lipid moieties in live cells. Here we report a de novo lipid labeling method for individual lipid species with precise acyl compositions in live cells. The method is based on the principle of de novo lipid remodeling of exogenously added lysolipids with fluorescent acyl-CoA, leading to the re-synthesis of fluorescence-labeled lipids which can be imaged by confocal microscopy.

Methods

The cells were incubated with lysolipids and a nitro-benzoxadiazolyl (NBD) labeled acyl-CoA. The newly remodeled NBD-labeled lipids and their subcellular localization were analyzed by confocal imaging in live cells. Thin layer chromatography was carried out to verify the synthesis of NBD-labeled lipids. The mitochondrial trafficking of NBD-labeled lipids was validated in live cells with targeted deletion of phospholipids transporters, including TRIAP1/PRELI protein complex and StarD7.

Results

Incubation cells with lysolipids and NBD-acyl-CoA successfully labeled major lipid species with precise acyl compositions, including phospholipids, cholesterol esters, and neutral lipids, which can be analyzed by confocal imaging in live cells. In contrast to exogenously labeled lipids, the de novo labeled lipids retained full biological properties of their endogenous counterparts, including subcellular localization, trafficking, and recognition by lipid transporters. This method also uncovered some unexpected features of newly remodeled lipids and their transporters.

Conclusions

The de novo lipid labeling method not only provides a powerful tool for functional analysis of individual lipid species and lipid transporters, but also calls for re-evaluation of previously published results using exogenously labeled lipids.

Objective

Deep sequencing offers unparalleled access to rare variants in human populations. Understanding their role in disease is a priority, yet prohibitive sequencing costs mean that many cohorts lack the sample size to discover these effects on their own. Meta-analysis of individual variant scores allows the combination of rare variants across cohorts and study of their aggregated effect at the gene level, boosting discovery power. However, the methods involved have largely not been field-tested. In this study, we aim to perform the first meta-analysis of gene-based rare variant aggregation optimal tests, applied to the human cardiometabolic proteome.

Methods

Here, we carry out this analysis across MANOLIS, Pomak and ORCADES, three isolated European cohorts with whole-genome sequencing (total N = 4,422). We examine the genetic architecture of 250 proteomic traits of cardiometabolic relevance. We use a containerised pipeline to harmonise variant lists across cohorts and define four sets of qualifying variants. For every gene, we interrogate protein-damaging variants, exonic variants, exonic and regulatory variants, and regulatory only variants, using the CADD and Eigen scores to weigh variants according to their predicted functional consequence. We perform single-cohort rare variant analysis and meta-analyse variant scores using the SMMAT package.

Results

We describe 5 rare variant pQTLs (RV-pQTL) which pass our stringent significance threshold (7.45 × 10⁻¹¹) and quality control procedure. These were split between four cis signals for MARCO, TEK, MMP2 and MPO, and one transassociation for GDF2 in the SERPINA11 gene. We show that the cis-MPO association, which was not detectable using the single-point data alone, is driven by 5 missense and frameshift variants. These include rs140636390 and rs119468010, which are specific to MANOLIS and ORCADES, respectively. We show how this kind of signal could improve the predictive accuracy of genetic factors in common complex disease such as stroke and cardiovascular disease.

Conclusions

Our proof-of-concept study demonstrates the power of gene-based meta-analyses for discovering disease-relevant associations complementing common-variant signals by incorporating population-specific rare variation.

Objectives

Lysosomal acid lipase (LAL) is the key enzyme, which degrades neutral lipids at an acidic pH in lysosomes. The role of LAL in various cellular processes has mostly been studied in LAL-knockout mice, which share phenotypical characteristics with humans suffering from LAL deficiency. In vitro, the cell-specific functions of LAL have been commonly investigated by using the LAL inhibitors Lalistat-1 and Lalistat-2.

Methods

We performed lipid hydrolase activity assays and serine hydrolase-specific activity-based labeling combined with quantitative proteomics to investigate potential off-target effects of Lalistat-1 and -2.

Results

Pharmacological LAL inhibition but not genetic loss of LAL impairs isoproterenol-stimulated lipolysis as well as neutral triglyceride and cholesteryl ester hydrolase activities. Apart from LAL, Lalistat-1 and -2 also inhibit major cytosolic lipid hydrolases responsible for lipid degradation in primary cells at neutral pH through off-target effects. Their binding to the active center of the enzymes leads to a decrease in neutral lipid hydrolase activities in cells overexpressing the respective enzymes.

Conclusions

Our findings are critically important since they demonstrate that commonly used concentrations of these inhibitors are not suitable to investigate the role of LAL-specific lipolysis in lysosomal function, signaling pathways, and autophagy. The interpretation of their effects on lipid metabolism should be taken with caution and the applied inhibitor concentrations in cell culture studies should not exceed 1 μM.

Objective

Tamoxifen is widely used for inducible Cre-LoxP systems but has several undesirable side effects for researchers investigating metabolism or energy balance, including weight loss, lipoatrophy, and drug incorporation into lipid stores. For this reason, we sought to determine whether a doxycycline-inducible system would be more advantageous for adipocyte-specific Cre mouse models, but serendipitously discovered widespread ectopic tetracycline response elementCre (TRE-Cre) recombinase activity.

Methods

Adipocyte-specific tamoxifen- and doxycycline-inducible Cre mice were crossed to fluorescent Cre reporter mice and visualized by confocal microscopy to assess efficiency and background activity. TRE-Cre mice were crossed to stop-floxed diphtheria toxin mice to selectively ablate cells with background Cre activity.

Results

Tamoxifen- and doxycycline-inducible systems performed similarly in adipose tissues, but ectopic Cre recombination was evident in numerous other cell types of the latter, most notably neurons. The source of ectopic Cre activity was isolated to the TRE-Cre transgene, driven by the pTet (tetO7) tetracycline-inducible promoter. Ablation of cells with ectopic recombination in mice led to stunted growth, diminished survival, and reduced brain mass.

Conclusions

These results indicate that tamoxifen- and doxycycline-inducible adipocyte-specific Cre mouse models are similarly efficient, but the TRE-Cre component of the latter is inherently leaky. TRE-Cre background activity is especially pronounced in the brain and peripheral nerve fibers, and selective ablation of these cells impairs mouse development and survival. Caution should be taken when pairing TRE-Cre with floxed alleles that have defined roles in neural function, and additional controls should be included when using this model system.

Background/Purpose

Type 2 diabetes and obesity increase the risk of developing colorectal cancer. Metformin may reduce colorectal cancer but the mechanisms mediating this effect remain unclear. In mice and humans, a high-fat diet (HFD), obesity and metformin are known to alter the gut microbiome but whether this is important for influencing tumor growth is not known.

Methods

Mice with syngeneic MC38 colon adenocarcinomas were treated with metformin or feces obtained from control or metformin treated mice.

Results

We find that compared to chow-fed controls, tumor growth is increased when mice are fed a HFD and that this acceleration of tumor growth can be partially recapitulated through transfer of the fecal microbiome or in vitro treatment of cells with fecal filtrates from HFD-fed animals. Treatment of HFD-fed mice with orally ingested, but not intraperitoneally injected, metformin suppresses tumor growth and increases the expression of short-chain fatty acid (SCFA)-producing microbes Alistipes, Lachnospiraceae and Ruminococcaceae. The transfer of the gut microbiome from mice treated orally with metformin to drug naïve, conventionalized HFD-fed mice increases circulating propionate and butyrate, reduces tumor proliferation, and suppresses the expression of sterol response element binding protein (SREBP) gene targets in the tumor.

Conclusion

These data indicate that in obese mice fed a HFD, metformin reduces tumor burden through changes in the gut microbiome.

Objective

Brown adipocytes play a key role in maintaining body temperature as well as glucose and lipid homeostasis. However, brown adipocytes need to adapt their thermogenic activity and substrate utilization to changes in nutrient availability. Amongst the multiple factors influencing brown adipocyte activity, autophagy is an important regulatory element of thermogenic capacity and activity. Nevertheless, a specific sensing mechanism of extracellular amino acid availability linking autophagy to nutrient availability in brown adipocytes is unknown.

Methods

To characterize the role of the amino acid transporter PAT2/SLC36A2 in brown adipocytes, loss or gain of function of PAT2 were studied with respect to differentiation, subcellular localization, lysosomal activity and autophagy. Activity of vATPase was evaluated by quenching of EGFP fused to LC3 or FITC-dextran loaded lysosomes in brown adipocytes upon amino acid starvation, whereas the effect of PAT2 on assembly of the vATPase was investigated by Native-PAGE.

Results

We show that PAT2 translocates from the plasma membrane to the lysosome in response to amino acid withdrawal. Loss or overexpression of PAT2 impair lysosomal acidification and starvation-induced S6K re-phosphorylation, as PAT2 facilitates the assembly of the lysosomal vATPase, by recruitment of the cytoplasmic V1 subunit to the lysosome.

Conclusions

PAT2 is an important sensor of extracellular amino acids and regulator of lysosomal acidification in brown adipocytes.

Background/Purpose

Adipose tissue contains progenitor cells that contribute to beneficial tissue expansion when needed by de novo adipocyteformation (classical white or beige fat cells with thermogenic potential). However, in chronic obesity, they can exhibit an activated pro-fibrotic, extracellular matrix (ECM)-depositing phenotype that highly aggravates obesity-related adipose tissue dysfunction.

Methods

Given that progenitors' fibrotic activation and fat cell browning appear to be antagonistic cell fates, we have examined the anti-fibrotic potential of pro-browning agents in an obesogenic condition.

Results

In obese mice fed a high fat diet, thermoneutral housing, which induces brown fat cell dormancy, increases the expression of ECM gene programs compared to conventionally raised animals, indicating aggravation of obesity-related tissue fibrosis at thermoneutrality. In a model of primary cultured murine adipose progenitors, we found that exposure to β-hydroxybutyrate selectively reduced Tgfβ-dependent profibrotic responses of ECM genes like Ctgf, Loxl2 and Fn1. This effect is observed in both subcutaneous and visceral-derived adipose progenitors, as well as in 3T3-L1 fibroblasts. In 30 patients with obesity eligible for bariatric surgery, those with higher circulating β-hydroxybutyrate levels have lower subcutaneous adipose tissue fibrotic scores. Mechanistically, β-hydroxybutyrate limits Tgfβ-dependent collagen accumulation and reduces Smad2-3 protein expression and phosphorylation in visceral progenitors. Moreover, β-hydroxybutyrate induces the expression of the ZFP36 gene, encoding a post-transcriptional regulator that promotes the degradation of mRNA by binding to AU-rich sites within 3′UTRs. Importantly, complete ZFP36 deficiency in a mouse embryonic fibroblast line from null mice, or siRNA knock-down in primary progenitors, indicate that ZFP36 is required for β-hydroxybutyrate anti-fibrotic effects.

Conclusion

These data unravel the potential of β-hydroxybutyrate to limit adipose tissue matrix deposition, a finding that might exploited in an obesogenic context.

Objectives

Dysregulation of cholesterol metabolism in the liver and hematopoietic stem and progenitor cells (HSPCs) promotes atherosclerosis development. Previously, it has been shown that HMG-CoA-Reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway, can be phosphorylated and inactivated by the metabolic stress sensor AMP-activated protein kinase (AMPK). However, the physiological significance of AMPK regulation of HMGCR to atherogenesis has yet to be elucidated. The aim of this study was to determine the role of AMPK/HMGCR axis in the development of atherosclerosis.

Methods

We have generated a novel atherosclerotic-prone mouse model with defects in the AMPK regulation of HMGCR (Apoe^−/−/Hmgcr KI mice). Atherosclerotic lesion size, plaque composition, immune cell and lipid profiles were assessed in Apoe^−/− and Apoe^−/−/Hmgcr KI mice.

Results

In this study, we showed that both male and female atherosclerotic-prone mice with a disruption of HMGCR regulation by AMPK (Apoe^−/−/Hmgcr KI mice) display increased aortic lesion size concomitant with an increase in plaque-associated macrophages and lipid accumulation. Consistent with this, Apoe^−/−/Hmgcr KI mice exhibited an increase in total circulating cholesterol and atherogenic monocytes, Ly6-C^hi subset. Mechanistically, increased circulating atherogenic monocytes in Apoe^−/−/Hmgcr KI mice was associated with enhanced egress of bone marrow HSPCs and extramedullary myelopoiesis, driven by a combination of elevated circulating 27-hydroxycholesterol and intracellular cholesterol in HSPCs.

Conclusions

Our results uncovered a novel signalling pathway involving AMPK-HMGCR axis in the regulation of cholesterol homeostasis in HSPCs, and that inhibition of this regulatory mechanism accelerates the development and progression of atherosclerosis. These findings provide a molecular basis to support the use of AMPK activators that currently undergoing Phase II clinical trial such as O–3O4 and PXL 770 for reducing atherosclerotic cardiovascular disease risks.