Background & Aims: Expression of P21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD); however, the underlying mechanisms remain unknown. In the present study, we investigated the function of CDKN1A in SLD. Methods: CDKN1A expression levels were evaluated in different patient cohorts with SLD, fibrosis, and advanced chronic liver disease (ACLD). Cdkn1a-/- and Cdkn1a+/+ mice were fed with either a Western diet (WD), a Lieber-DeCarli (LdC) diet plus multiple EtOH (ethanol) binges, or a DuAL diet (metabolic dysfunction-associated fatty liver disease and alcohol-related liver). Primary hepatocytes were isolated and functional assays performed. Results: A significant increase in CDKN1A expression was observed in patients with steatohepatitis and fibrosis (with a positive correlation with both NAFLD Activity Score and fibrosis staging scores), cirrhosis and ACLD. Cdkn1a+/+ mice, fed a DuAL diet exhibited liver injury and cell death increased reactive oxygen species (ROS), and markers of senescence (γH2AX, β-GAL, Cdkn1a/p53) contributing to steatosis and inflammation. In contrast, Cdkn1a-/- mutant mice showed a significant decrease in senescence-associated markers as well as in markers of liver injury, hepatic steatosis and an increase in fatty acid oxidation and reduction in free fatty acid uptake as well as de novo lipogenesis. Mechanistically, activation of the AMPK-SIRT3 was observed in Cdkn1a-deleted animals. Conclusions: Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis via the AMPK-SIRT3 axis. CDKN1A expression was found to be directly correlated with increased severity of NAFLD Activity Score and fibrosis in patients with SLD. CDKN1A could be a potential theragnostic target for the treatment of metabolic dysregulation in patients with SLD, with and without alcohol consumption. Impact and implications:: Expression of p21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD), but the molecular mechanisms remain elusive. Interestingly, in this study we found that Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis, via the AMPK-SIRT3 axis. Translationally, Cdkn1a expression was found to be directly correlated with increased severity of NAFLD Activity Score (NAS) and fibrosis in SLD patients, and therefore, CDKN1A might be used potential theragnostic target for the treatment of metabolically induced SLD, with and without alcohol consumption.

Small molecules selectively inducing peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α acetylation and inhibiting glucagon-dependent gluconeogenesis causing anti-diabetic effects have been identified. However, how these small molecules selectively suppress the conversion of gluconeogenic metabolites into glucose without interfering with lipogenesis is unknown. Here, we show that a small molecule SR18292 inhibits hepatic glucose production by increasing lactate and glucose oxidation. SR18292 increases phosphoenolpyruvate carboxykinase 1 (PCK1) acetylation, which reverses its gluconeogenic reaction and favors oxaloacetate (OAA) synthesis from phosphoenolpyruvate. PCK1 reverse catalytic reaction induced by SR18292 supplies OAA to tricarboxylic acid (TCA) cycle and is required for increasing glucose and lactate oxidation and suppressing gluconeogenesis. Acetylation mimetic mutant PCK1 K91Q favors anaplerotic reaction and mimics the metabolic effects of SR18292 in hepatocytes. Liver-specific expression of PCK1 K91Q mutant ameliorates hyperglycemia in obese mice. Thus, SR18292 blocks gluconeogenesis by enhancing gluconeogenic substrate oxidation through PCK1 lysine acetylation, supporting the anti-diabetic effects of these small molecules.

Mitochondria provide essential metabolites and adenosine triphosphate (ATP) for the regulation of energy homeostasis. For instance, liver mitochondria are a vital source of gluconeogenic precursors under a fasted state. However, the regulatory mechanisms at the level of mitochondrial membrane transport are not fully understood. Here, we report that a liver-specific mitochondrial inner-membrane carrier SLC25A47 is required for hepatic gluconeogenesis and energy homeostasis. Genome-wide association studies found significant associations between SLC25A47 and fasting glucose, HbA1c, and cholesterol levels in humans. In mice, we demonstrated that liver-specific depletion of SLC25A47 impaired hepatic gluconeogenesis selectively from lactate, while significantly enhancing whole-body energy expenditure and the hepatic expression of FGF21. These metabolic changes were not a consequence of general liver dysfunction because acute SLC25A47 depletion in adult mice was sufficient to enhance hepatic FGF21 production, pyruvate tolerance, and insulin tolerance independent of liver damage and mitochondrial dysfunction. Mechanistically, SLC25A47 depletion leads to impaired hepatic pyruvate flux and malate accumulation in the mitochondria, thereby restricting hepatic gluconeogenesis. Together, the present study identified a crucial node in the liver mitochondria that regulates fasting-induced gluconeogenesis and energy homeostasis.

Liver mitochondria undergo architectural remodeling that maintains energy homeostasis in response to feeding and fasting. However, the specific components and molecular mechanisms driving these changes and their impact on energy metabolism remain unclear. Through comparative mouse proteomics, we found that fasting induces strain-specific mitochondrial cristae formation in the liver by upregulating MIC19, a subunit of the MICOS complex. Enforced MIC19 expression in the liver promotes cristae formation, mitochondrial respiration, and fatty acid oxidation while suppressing gluconeogenesis. Mice overexpressing hepatic MIC19 show resistance to diet-induced obesity and improved glucose homeostasis. Interestingly, MIC19 overexpressing mice exhibit elevated energy expenditure and increased pedestrian locomotion. Metabolite profiling revealed that uracil accumulates in the livers of these mice due to increased uridine phosphorylase UPP2 activity. Furthermore, uracil-supplemented diet increases locomotion in wild-type mice. Thus, MIC19-induced mitochondrial cristae formation in the liver increases uracil as a signal to promote locomotion, with protective effects against diet-induced obesity.

ABSTRACT Mitochondria provide essential metabolites and ATP for the regulation of energy homeostasis. For instance, liver mitochondria are a vital source of gluconeogenic precursors under a fasted state. However, the regulatory mechanisms at the level of mitochondrial membrane transport are not fully understood. Here, we report a liver-specific mitochondrial inner-membrane carrier, SLC25A47, which is required for hepatic gluconeogenesis and energy homeostasis. Genome-wide association studies found significant associations between SLC25A47 and fasting glucose, HbA1c, and cholesterol levels in humans. In mice, we demonstrated that liver-specific deletion of Slc25a47 impaired hepatic gluconeogenesis selectively from lactate, while significantly enhancing whole-body energy expenditure and the hepatic expression of FGF21. These metabolic changes were not a consequence of general liver dysfunction because acute SLC25A47 deletion in adult mice was sufficient to enhance hepatic FGF21 production, pyruvate tolerance, and insulin tolerance independent of liver damage and mitochondrial dysfunction. Mechanistically, SLC25A47 loss leads to impaired hepatic pyruvate flux and malate accumulation in the mitochondria, thereby restricting hepatic gluconeogenesis. Together, the present study identified a crucial node in the mitochondrial inner-membrane that regulates fasting-induced gluconeogenesis and energy homeostasis. SIGNIFICANCE Given the impenetrable nature of the mitochondrial inner-membrane, most of the known metabolite carrier proteins, including SLC25A family members, are ubiquitously expressed in mammalian tissues. One exception is SLC25A47 which is selectively expressed in the liver. The present study showed that depletion of SLC25A47 reduced mitochondrial pyruvate flux and hepatic gluconeogenesis under a fasted state, while activating energy expenditure. The present work offers a liver-specific target through which we can restrict hepatic gluconeogenesis, which is often in excess under hyperglycemic and diabetic conditions.

General Control Non-repressed 5 protein (GCN5), encoded by the mammalian gene Kat2a, is the first histone acetyltransferase discovered to link histone acetylation to transcriptional activation [1]. The enzymatic activity of GCN5 is linked to cellular metabolic and energetic states regulating gene expression programs. GCN5 has a major impact on energy metabolism by i) sensing acetyl-CoA, a central metabolite and substrate of the GCN5 catalytic reaction, and ii) acetylating proteins such as PGC-1α, a transcriptional coactivator that controls genes linked to energy metabolism and mitochondrial biogenesis. PGC-1α is biochemically associated with the GCN5 protein complex during active metabolic reprogramming. In the first part of the review, we examine how metabolism can change GCN5-dependent histone acetylation to regulate gene expression to adapt cells. In the second part, we summarize the GCN5 function as a nutrient sensor, focusing on non-histone protein acetylation, mainly the metabolic role of PGC-1α acetylation across different tissues.

Cannabinoid receptor 1 (CB-1) antagonists are potential candidates for treating obesity and metabolic complications. Despite clear metabolic benefits, unwanted side effects in the brain pose issues for patients. With the hope of overcoming this obstacle, CB-1 in peripheral tissues has become a potential drug target. Previous studies had suggested that liver CB-1 would be an excellent target to prevent development of nonalcoholic steatohepatitis (NAFLD). However, in this issue of the JCI , Wang et al. showed that CB-1 was barely detectable in the liver and deletion of CB-1 in hepatocytes provided no metabolic benefits against NAFLD. These contradictory results raise substantial concerns about the potential benefits of peripheral CB-1 blockers against NAFLD.

Significance Glucagon action in liver is a central response to fasting and type 2 diabetes. Glucagon action has been delineated through regulatory mechanisms involving signaling, transcription factor/coactivator-based gluconeogenic gene expression, and metabolic enzyme activity. Understanding the molecular mechanisms whereby glucagon controls energy metabolism will define new strategies and potential therapies to treat metabolic diseases. Here, we have identified a regulatory mechanism whereby PGC-1α, a known transcriptional regulator of glucagon action, binds RNAs linked to glucose energy metabolism. PGC-1α represents a class of RNA-binding proteins that act as a transcriptional coactivator through transcription factor binding, but also binds to RNA sequences to control specific mRNA transcripts encoding for metabolic and bioenergetic genes.

During the first hours of embryogenesis, formation of higher-order heterochromatin coincides with the loss of developmental potential. Here, we examine the relationship between these two events, and we probe the processes that contribute to the timing of their onset. Mutations that disrupt histone H3 lysine 9 (H3K9) methyltransferases reveal that the methyltransferase MET-2 helps terminate developmental plasticity, through mono- and di-methylation of H3K9 (me1/me2), and promotes heterochromatin formation, through H3K9me3. Although loss of H3K9me3 perturbs formation of higher-order heterochromatin, embryos are still able to terminate plasticity, indicating that the two processes can be uncoupled. Methylated H3K9 appears gradually in developing C. elegans embryos and depends on nuclear localization of MET-2. We find that the timing of H3K9me2 and nuclear MET-2 is sensitive to rapid cell cycles, but not to zygotic genome activation or cell counting. These data reveal distinct roles for different H3K9 methylation states in the generation of heterochromatin and loss of developmental plasticity by MET-2, and identify the cell cycle as a crucial parameter of MET-2 regulation.

H3K9me3 (histone H3 modified with tri-methylation at lysine 9) is a hallmark of transcriptional silencing and heterochromatin. However, its global effects on the genome, including euchromatin, are less well understood. Here we develop Formaldehyde-Assisted Identification of Regulatory Elements (FAIRE) for C. elegans to examine the chromatin configuration of mutants that lack virtually all H3K9me3, while leaving H3K9me1 and H3K9me2 intact. We find that nucleosomes are mildly disrupted, and levels of H3K9me2 and H3K27me3 rise in mutant embryos. In addition to these expected changes, the most dramatic change occurs in euchromatin: many regions encompassing transcription start sites (TSSs) gain an average of two nucleosomes in mutants. The affected regions normally lack H3K9me3, revealing a locus non-autonomous role for H3K9me3. Affected TSSs are associated with genes that are active in epithelia and muscles, and implicated in development, locomotion, morphogenesis and transcription. Mutant embryos develop normally under ideal laboratory conditions but die when challenged, with defects in morphogenesis and development. Our findings reveal that H3K9me3 protects transcription start sites within euchromatin from nucleosome deposition. These results may be relevant to mammals, where diseases that disrupt the nuclear lamina and heterochromatin can alter epithelial and muscle gene expression.

Heterochromatin formation during early embryogenesis is timed precisely, but how this process is regulated remains elusive. We report the discovery of a histone methyltransferase complex whose nuclear accumulation and activation establish the onset of heterochromatin formation in Caenorhabditis elegans embryos. We find that the inception of heterochromatin generation coincides with the accumulation of the histone H3 lysine 9 (H3K9) methyltransferase MET-2 (SETDB) into nuclear hubs. The absence of MET-2 results in delayed and disturbed heterochromatin formation, whereas accelerated nuclear localization of the methyltransferase leads to precocious H3K9 methylation. We identify two factors that bind to and function with MET-2: LIN-65, which resembles activating transcription factor 7-interacting protein (ATF7IP) and localizes MET-2 into nuclear hubs, and ARLE-14, which is orthologous to adenosine 5'-diphosphate-ribosylation factor-like 14 effector protein (ARL14EP) and promotes stable association of MET-2 with chromatin. These data reveal that nuclear accumulation of MET-2 in conjunction with LIN-65 and ARLE-14 regulates timing of heterochromatin domains during embryogenesis.

During the first hours of embryogenesis, formation of higher-order heterochromatin coincides with the loss of developmental potential. Here we examine the relationship between these two processes, and we probe the determinants that contribute to their onset. Mutations that disrupt histone H3 lysine 9 (H3K9) methyltransferases reveal that the methyltransferase MET-2 helps terminate developmental plasticity, likely through mono- and di- methylation of H3K9 (me1/me2), and promotes heterochromatin formation, likely through H3K9me3. We examine how MET-2 is regulated and find that methylated H3K9 appears gradually and depends on the accumulated time of embryogenesis. H3K9me is independent of zygotic genome activation or cell counting. These data reveal how central events are synchronized during embryogenesis and distinguish distinct roles for different H3K9 methylation states. Summary Statement During early embryogenesis, heterochromatin formation and loss of developmental plasticity are coordinately regulated by distinct Histone H3 Lysine 9 (H3K9) methylation states, by the methyltransferase MET-2.

One drug may suppress the effects of another. Although knowledge of drug suppression is vital to avoid efficacy-reducing drug interactions or discover countermeasures for chemical toxins, drug-drug suppression relationships have not been systematically mapped. Here, we analyze the growth response of Saccharomyces cerevisiae to anti-fungal compound ("drug") pairs. Among 440 ordered drug pairs, we identified 94 suppressive drug interactions. Using only pairs not selected on the basis of their suppression behavior, we provide an estimate of the prevalence of suppressive interactions between anti-fungal compounds as 17%. Analysis of the drug suppression network suggested that Bromopyruvate is a frequently suppressive drug and Staurosporine is a frequently suppressed drug. We investigated potential explanations for suppressive drug interactions, including chemogenomic analysis, coaggregation, and pH effects, allowing us to explain the interaction tendencies of Bromopyruvate.

Drug synergy allows a therapeutic effect to be achieved with lower doses of component drugs.Drug synergy can result when drugs target the products of genes that act in parallel pathways ('specific synergy').Such cases of drug synergy should tend to correspond to synergistic genetic interaction between the corresponding target genes.Alternatively, 'promiscuous synergy' can arise when one drug non-specifically increases the effects of many other drugs, for example, by increased bioavailability.To assess the relative abundance of these drug synergy types, we examined 200 pairs of antifungal drugs in S. cerevisiae.We found 38 antifungal synergies, 37 of which were novel.While 14 cases of drug synergy corresponded to genetic interaction, 92% of the synergies we discovered involved only six frequently synergistic drugs.Although promiscuity of four drugs can be explained under the bioavailability model, the promiscuity of Tacrolimus and Pentamidine was completely unexpected.While many drug synergies correspond to genetic interactions, the majority of drug synergies appear to result from non-specific promiscuous synergy.