Regulatory T cells (Tregs) that express the transcription factor Foxp3 are critical for regulating intestinal inflammation. Candidate microbe approaches have identified bacterial species and strain-specific molecules that can affect intestinal immune responses, including species that modulate Treg responses. Because neither all humans nor mice harbor the same bacterial strains, we posited that more prevalent factors exist that regulate the number and function of colonic Tregs. We determined that short chain fatty acids (SCFA), gut microbiota-derived bacterial fermentation products, regulate the size and function of the colonic Treg pool and protect against colitis in a Ffar2(GPR43)-dependent manner in mice. Our study reveals that a class of abundant microbial metabolites underlies adaptive immune microbiota co-adaptation and promotes colonic homeostasis and health.

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in a variety of tissues during ageing in humans, monkeys and rodents. These mutations are unevenly distributed and can accumulate clonally in certain cells, causing a mosaic pattern of respiratory chain deficiency in tissues such as heart, skeletal muscle and brain. In terms of the ageing process, their possible causative effects have been intensely debated because of their low abundance and purely correlative connection with ageing. We have now addressed this question experimentally by creating homozygous knock-in mice that express a proof-reading-deficient version of PolgA, the nucleus-encoded catalytic subunit of mtDNA polymerase. Here we show that the knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations is associated with reduced lifespan and premature onset of ageing-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement. Our results thus provide a causative link between mtDNA mutations and ageing phenotypes in mammals.

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.

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.

We investigated whether ghrelin action at the level of the ventral tegmental area (VTA), a key node in the mesolimbic reward system, is important for the rewarding and motivational aspects of the consumption of rewarding/palatable food. Mice with a disrupted gene encoding the ghrelin receptor (GHS-R1A) and rats treated peripherally with a GHS-R1A antagonist both show suppressed intake of rewarding food in a free choice (chow/rewarding food) paradigm. Moreover, accumbal dopamine release induced by rewarding food was absent in GHS-R1A knockout mice. Acute bilateral intra-VTA administration of ghrelin increased 1-hour consumption of rewarding food but not standard chow. In comparison with sham rats, VTA-lesioned rats had normal intracerebroventricular ghrelin-induced chow intake, although both intake of and time spent exploring rewarding food was decreased. Finally, the ability of rewarding food to condition a place preference was suppressed by the GHS-R1A antagonist in rats. Our data support the hypothesis that central ghrelin signaling at the level of the VTA is important for the incentive value of rewarding food.

Free fatty acid receptor 2 (Ffar2), also known as GPR43, is activated by short-chain fatty acids (SCFA) and expressed in intestine, adipocytes, and immune cells, suggesting involvement in lipid and immune regulation. In the present study, Ffar2-deficient mice (Ffar2-KO) were given a high-fat diet (HFD) or chow diet and studied with respect to lipid and energy metabolism. On a HFD, Ffar2-KO mice had lower body fat mass and increased lean body mass. The changed body composition was accompanied by improved glucose control and lower HOMA index, indicating improved insulin sensitivity in Ffar2-KO mice. Moreover, the Ffar2-KO mice had higher energy expenditure accompanied by higher core body temperature and increased food intake. The liver weight and content of triglycerides as well as plasma levels of cholesterol were lower in the Ffar2-KO mice fed a HFD. A histological examination unveiled decreased lipid interspersed in brown adipose tissue of the Ffar2-KO mice. Interestingly, no significant differences in white adipose tissue (WAT) cell size were observed, but significantly lower macrophage content was detected in WAT from HFD-fed Ffar2-KO compared with wild-type mice. In conclusion, Ffar2 deficiency protects from HFD-induced obesity and dyslipidemia at least partly via increased energy expenditure.

The adipocyte-derived hormone adiponectin regulates glucose and lipid metabolism and influences the risk for developing obesity, type 2 diabetes, and cardiovascular disease. Adiponectin binds to two different seven-transmembrane domain receptors termed AdipoR1 and AdipoR2. To study the physiological importance of these receptors, AdipoR1 gene knockout mice (AdipoR1 Ϫ/Ϫ ) and AdipoR2 gene knockout mice (AdipoR2 Ϫ/Ϫ ) were generated. AdipoR1 Ϫ/Ϫ mice showed increased adiposity associated with decreased glucose tolerance, spontaneous locomotor activity, and energy expenditure. However, AdipoR2Ϫ/Ϫ mice were lean and resistant to high-fat diet-induced obesity associated with improved glucose tolerance and higher spontaneous locomotor activity and energy expenditure and reduced plasma cholesterol levels. Thus, AdipoR1 and AdipoR2 are clearly involved in energy metabolism but have opposing effects. Diabetes 56:583-593, 2007

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications but identifying unwanted off-target mutations is important for clinical translation. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.