Liver-specific activation of AMPK prevents steatosis on a high fructose diet

AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. We identified a mutation in the γ1 subunit (γ1D316A) that leads to activation of AMPK. We generated mice with this mutation to study the effect of chronic liver-specific activation of AMPK in vivo. Primary hepatocytes isolated from these mice have reduced gluconeogenesis and fatty acid synthesis, but there is no effect on fatty acid oxidation compared to cells from wild-type mice. Liver-specific activation of AMPK decreases lipogenesis in vivo and completely protects against hepatic steatosis when mice are fed a high-fructose diet. Our findings demonstrate that liver-specific activation of AMPK is sufficient to protect against hepatic triglyceride accumulation, a hallmark of non-alcoholic fatty liver disease (NAFLD). These results emphasize the clinical relevance of activating AMPK in the liver to combat NAFLD and potentially other associated complications (e.g., cirrhosis and hepatocellular carcinoma)

IDOL regulates systemic energy balance through control of neuronal VLDLR expression.

Liver X receptors limit cellular lipid uptake by stimulating the transcription of Inducible Degrader of the LDL Receptor (IDOL), an E3 ubiquitin ligase that targets lipoprotein receptors for degradation. The function of IDOL in systemic metabolism is incompletely understood. Here we show that loss of IDOL in mice protects against the development of diet-induced obesity and metabolic dysfunction by altering food intake and thermogenesis. Unexpectedly, analysis of tissue-specific knockout mice revealed that IDOL affects energy balance, not through its actions in peripheral metabolic tissues (liver, adipose, endothelium, intestine, skeletal muscle), but by controlling lipoprotein receptor abundance in neurons. Single-cell RNA sequencing of the hypothalamus demonstrated that IDOL deletion altered gene expression linked to control of metabolism. Finally, we identify VLDLR rather than LDLR as the primary mediator of IDOL effects on energy balance. These studies identify a role for the neuronal IDOL-VLDLR pathway in metabolic homeostasis and diet-induced obesity

AMPK activation protects against diet induced obesity through Ucp1-independent thermogenesis in subcutaneous white adipose tissue

Obesity results from a chronic imbalance between energy intake and energy output but remains difficult to prevent or treat in humans. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an important regulator of energy homeostasis1,2,3 and is a molecular target of drugs used for the treatment of metabolic diseases, including obesity4,5. Here we show that mice expressing a gain-of-function AMPK mutant6 display a change in morphology of subcutaneous white adipocytes that is reminiscent of browning. However, despite a dramatic increase in mitochondrial content, Ucp1 expression is undetectable in these adipocytes. In response to a high-fat diet (HFD), expression of skeletal muscle–associated genes is induced in subcutaneous white adipocytes from the gain-of-function AMPK mutant mice. Chronic genetic AMPK activation results in protection against diet-induced obesity due to an increase in whole-body energy expenditure, most probably because of a substantial increase in the oxygen consumption rate of white adipose tissue. These results suggest that AMPK activation enriches, or leads to the emergence of, a population of subcutaneous white adipocytes that produce heat via Ucp1-independent uncoupling of adenosine triphosphate (ATP) production on a HFD. Our findings indicate that AMPK activation specifically in adipose tissue may have therapeutic potential for the treatment of obesity

A combined human in silico and CRISPR/Cas9-mediated in vivo zebrafish based approach to provide phenotypic data for supporting early target validation

This is the final version. Available on open access from Frontiers Media via the DOI in this record. Data availability statement: The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding authors.The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts as part of the process for validating potential new drug targets. Traditional approaches (e.g., genetically-modified mice) are optimal for assessing small numbers of genes, but less practical when multiple genes are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation, by providing more confidence in target relevance and facilitating gene selection for definitive loss of function studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including changes in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports further investigation of the potential roles of API5, HSPB7, and LMO2 in human cardiovascular disease. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.Royal SocietyAstraZenec

Ablation of PGC-1β Results in Defective Mitochondrial Activity, Thermogenesis, Hepatic Function, and Cardiac Performance

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1β (PGC-1β) has been implicated in important metabolic processes. A mouse lacking PGC-1β (PGC1βKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1βKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1β ablation was partially compensated by up-regulation of PGC-1α in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1βKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1β was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1βKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1βKO mice have impaired mitochondrial function. Lack of PGC-1β also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1β plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress

Adaptive changes of the Insig1/SREBP1/SCD1 set point help adipose tissue to cope with increased storage demands of obesity.

The epidemic of obesity imposes unprecedented challenges on human adipose tissue (WAT) storage capacity that may benefit from adaptive mechanisms to maintain adipocyte functionality. Here, we demonstrate that changes in the regulatory feedback set point control of Insig1/SREBP1 represent an adaptive response that preserves WAT lipid homeostasis in obese and insulin-resistant states. In our experiments, we show that Insig1 mRNA expression decreases in WAT from mice with obesity-associated insulin resistance and from morbidly obese humans and in in vitro models of adipocyte insulin resistance. Insig1 downregulation is part of an adaptive response that promotes the maintenance of SREBP1 maturation and facilitates lipogenesis and availability of appropriate levels of fatty acid unsaturation, partially compensating the antilipogenic effect associated with insulin resistance. We describe for the first time the existence of this adaptive mechanism in WAT, which involves Insig1/SREBP1 and preserves the degree of lipid unsaturation under conditions of obesity-induced insulin resistance. These adaptive mechanisms contribute to maintain lipid desaturation through preferential SCD1 regulation and facilitate fat storage in WAT, despite on-going metabolic stress

Selecting the right therapeutic target for kidney disease

Kidney disease is a complex disease with several different etiologies and underlying associated pathophysiology. This is reflected by the lack of effective treatment therapies in chronic kidney disease (CKD) that stop disease progression. However, novel strategies, recent scientific breakthroughs, and technological advances have revealed new possibilities for finding novel disease drivers in CKD. This review describes some of the latest advances in the field and brings them together in a more holistic framework as applied to identification and validation of disease drivers in CKD. It uses high-resolution ‘patient-centric’ omics data sets, advanced in silico tools (systems biology, connectivity mapping, and machine learning) and ‘state-of-the-art‘ experimental systems (complex 3D systems in vitro, CRISPR gene editing, and various model biological systems in vivo). Application of such a framework is expected to increase the likelihood of successful identification of novel drug candidates based on strong human target validation and a better scientific understanding of underlying mechanisms

Mitochondria are required for pro-ageing features of the senescent phenotype

Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro‐inflammatory and pro‐oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent‐associated changes are dependent on mitochondria, particularly the pro‐inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC‐1β‐dependent mitochondrial biogenesis, contributing to a ROS‐mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC‐1β deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues

Clusterin Is Required for β-Amyloid Toxicity in Human iPSC-Derived Neurons

Our understanding of the molecular processes underlying Alzheimer’s disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating β-amyloid (Aβ) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in Aβ processing. Here we investigate how CLU mediates Aβ-driven neurodegeneration in human induced pluripotent stem cell (iPSC)-derived neurons. We generated a novel CLU-knockout iPSC line by CRISPR/Cas9-mediated gene editing to investigate Aβ-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to Aβ using an imaging assay and measured changes in gene expression using qPCR and RNA sequencing. In wild type neurons imaging indicated that neuronal processes degenerate following treatment with Aβ25-35 peptides and Aβ1-42 oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following Aβ treatment. However, in CLU knockout neurons Aβ exposure did not affect neurite length, suggesting that clusterin is an important component of the amyloid cascade. Transcriptomic data were analyzed to elucidate the pathways responsible for the altered response to Aβ in neurons with the CLU deletion. Four of the five genes previously identified as downstream to Aβ and Dickkopf-1 (DKK1) proteins in an Aβ-driven neurotoxic pathway in rodent cells were also dysregulated in human neurons with the CLU deletion. AD and lysosome pathways were the most significantly dysregulated pathways in the CLU knockout neurons, and pathways relating to cytoskeletal processes were most dysregulated in Aβ treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to Aβ compared to the wild type neurons supports the role of clusterin in Aβ-mediated AD pathogenesis

SCFAs Induce Mouse Neutrophil Chemotaxis through the GPR43 Receptor

Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43−/− BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3Kγ, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways