Spatial hepatocyte plasticity of gluconeogenesis during the metabolic transitions between fed, fasted and starvation states.

The liver acts as a master regulator of metabolic homeostasis in part by performing gluconeogenesis. This process is dysregulated in type 2 diabetes, leading to elevated hepatic glucose output. The parenchymal cells of the liver (hepatocytes) are heterogeneous, existing on an axis between the portal triad and the central vein, and perform distinct functions depending on location in the lobule. Here, using single cell analysis of hepatocytes across the liver lobule, we demonstrate that gluconeogenic gene expression ( Pck1 and G6pc ) is relatively low in the fed state and gradually increases first in the periportal hepatocytes during the initial fasting period. As the time of fasting progresses, pericentral hepatocyte gluconeogenic gene expression increases, and following entry into the starvation state, the pericentral hepatocytes show similar gluconeogenic gene expression to the periportal hepatocytes. Similarly, pyruvate-dependent gluconeogenic activity is approximately 10-fold higher in the periportal hepatocytes during the initial fasting state but only 1.5-fold higher in the starvation state. In parallel, starvation suppresses canonical beta-catenin signaling and modulates expression of pericentral and periportal glutamine synthetase and glutaminase, resulting in an enhanced pericentral glutamine-dependent gluconeogenesis. These findings demonstrate that hepatocyte gluconeogenic gene expression and gluconeogenic activity are highly spatially and temporally plastic across the liver lobule, underscoring the critical importance of using well-defined feeding and fasting conditions to define the basis of hepatic insulin resistance and glucose production.

Involution of brown adipose tissue through a Syntaxin 4 dependent pyroptosis pathway.

Aging, chronic high-fat diet feeding, or housing at thermoneutrality induces brown adipose tissue (BAT) involution, a process characterized by reduction of BAT mass and function with increased lipid droplet size. Single nuclei RNA sequencing of aged mice identifies a specific brown adipocyte population of Ucp1-low cells that are pyroptotic and display a reduction in the longevity gene syntaxin 4 (Stx4a). Similar to aged brown adipocytes, Ucp1-STX4KO mice display loss of brown adipose tissue mass and thermogenic dysfunction concomitant with increased pyroptosis. Restoration of STX4 expression or suppression of pyroptosis activation protects against the decline in both mass and thermogenic activity in the aged and Ucp1-STX4KO mice. Mechanistically, STX4 deficiency reduces oxidative phosphorylation, glucose uptake, and glycolysis leading to reduced ATP levels, a known triggering signal for pyroptosis. Together, these data demonstrate an understanding of rapid brown adipocyte involution and that physiologic aging and thermogenic dysfunction result from pyroptotic signaling activation.

Complement C3 Reduces Apoptosis via Interaction with the Intrinsic Apoptotic Pathway.

Myocardial ischemia/reperfusion (I/R) elicits an acute inflammatory response involving complement factors. Recently, we reported that myocardial necrosis was decreased in complement C3-/- mice after heart I/R. The current study used the same heart model to test the effect of C3 on myocardial apoptosis and investigated if C3 regulation of apoptosis occurred in human cardiomyocytes. Comparative proteomics analyses found that cytochrome c was present in the myocardial C3 complex of WT mice following I/R. Incubation of exogenous human C3 reduced apoptosis in a cell culture system of human cardiomyocytes that did not inherently express C3. In addition, human C3 inhibited the intrinsic apoptosis pathway in a cell-free apoptosis system. Finally, human pro-C3 was found to bind with an apoptotic factor, pro-caspase 3, in a cell-free system. Thus, we present firsthand evidence showing that C3 readily reduces myocardial apoptosis via interaction with the intrinsic apoptotic pathway.

The conserved Mediator subunit cyclin C (CCNC) is required for brown adipocyte development and lipid accumulation.

OBJECTIVE: Cyclin C (CCNC) is the most conserved subunit of the Mediator complex, which is an important transcription cofactor. Recently, we have found that CCNC facilitates brown adipogenesis in vitro by activating C/EBPα-dependent transcription. However, the role of CCNC in brown adipose tissue (BAT) in vivo remains unclear. METHODS: We generated conditional knock-out mice by crossing Ccncflox/flox mice with Myf5Cre, Ucp1Cre or AdipoqCre transgenic mice to investigate the role of CCNC in BAT development and function. We applied glucose and insulin tolerance test, cold exposure and indirect calorimetry to capture the physiological phenotypes and used immunostaining, immunoblotting, qRT-PCR, RNA-seq and cell culture to elucidate the underlying mechanisms. RESULTS: Here, we show that deletion of CCNC in Myf5+ progenitor cells caused BAT paucity, despite the fact that there was significant neonatal lethality. Mechanistically different from in vitro, CCNC deficiency impaired the proliferation of embryonic brown fat progenitor cells without affecting brown adipogenesis or cell death. Interestingly, CCNC deficiency robustly reduced age-dependent lipid accumulation in differentiated brown adipocytes in all three mouse models. Mechanistically, CCNC in brown adipocytes is required for lipogenic gene expression through the activation of the C/EBPα/GLUT4/ChREBP axis. Consistent with the importance of de novo lipogenesis under carbohydrate-rich diets, high-fat diet (HFD) feeding abolished CCNC deficiency -caused defects of lipid accumulation in BAT. Although insulin sensitivity and response to acute cold exposure were not affected, CCNC deficiency in Ucp1+ cells enhanced the browning of white adipose tissue (beiging) upon prolonged cold exposure. CONCLUSIONS: Together, these data indicate an important role of CCNC-Mediator in the regulation of BAT development and lipid accumulation in brown adipocytes.

Genomic expansion of Aldh1a1 protects beavers against high metabolic aldehydes from lipid oxidation.

The North American beaver is an exceptionally long-lived and cancer-resistant rodent species. Here, we report the evolutionary changes in its gene coding sequences, copy numbers, and expression. We identify changes that likely increase its ability to detoxify aldehydes, enhance tumor suppression and DNA repair, and alter lipid metabolism, potentially contributing to its longevity and cancer resistance. Hpgd, a tumor suppressor gene, is uniquely duplicated in beavers among rodents, and several genes associated with tumor suppression and longevity are under positive selection in beavers. Lipid metabolism genes show positive selection signals, changes in copy numbers, or altered gene expression in beavers. Aldh1a1, encoding an enzyme for aldehydes detoxification, is particularly notable due to its massive expansion in beavers, which enhances their cellular resistance to ethanol and capacity to metabolize diverse aldehyde substrates from lipid oxidation and their woody diet. We hypothesize that the amplification of Aldh1a1 may contribute to the longevity of beavers.

Comparative impact of dietary carbohydrates on the liver transcriptome in two strains of mice.

Excessive long-term consumption of dietary carbohydrates, including glucose, sucrose, or fructose, has been shown to have significant impact on genome-wide gene expression, which likely results from changes in metabolic substrate flux. However, there has been no comprehensive study on the acute effects of individual sugars on the genome-wide gene expression that may reveal the genetic changes altering signaling pathways, subsequent metabolic processes, and ultimately physiological/pathological responses. Considering that gene expressions in response to acute carbohydrate ingestion might be different in nutrient sensitive and insensitive mammals, we conducted comparative studies of genome-wide gene expression by deep mRNA sequencing of the liver in nutrient sensitive C57BL/6J and nutrient insensitive BALB/cJ mice. Furthermore, to determine the temporal responses, we compared livers from mice in the fasted state and following ingestion of standard laboratory mouse chow supplemented with plain drinking water or water containing 20% glucose, sucrose, or fructose. Supplementation with these carbohydrates induced unique extents and temporal changes in gene expressions in a strain specific manner. Fructose and sucrose stimulated gene changes peaked at 3 h postprandial, whereas glucose effects peaked at 12 h and 6 h postprandial in C57BL/6J and BABL/cJ mice, respectively. Network analyses revealed that fructose changed genes were primarily involved in lipid metabolism and were more complex in C57BL/6J than in BALB/cJ mice. These data demonstrate that there are qualitative and antitative differences in the normal physiological responses of the liver between these two strains of mice and C57BL/6J is more sensitive to sugar intake than BALB/cJ.

The Mediator complex kinase module is necessary for fructose regulation of liver glycogen levels through induction of glucose-6-phosphatase catalytic subunit (G6pc).

OBJECTIVE: Liver glycogen levels are dynamic and highly regulated by nutrient availability as the levels decrease during fasting and are restored during the feeding cycle. However, feeding in the presence of fructose in water suppresses glycogen accumulation in the liver by upregulating the expression of the glucose-6-phosphatase catalytic subunit (G6pc) gene, although the exact mechanism is unknown. We generated liver-specific knockout MED13 mice that lacked the transcriptional Mediator complex kinase module to examine its effect on the transcriptional activation of inducible target gene expression, such as the ChREBP- and FOXO1-dependent control of the G6pc gene promoter. METHODS: The relative changes in liver expression of lipogenic and gluconeogenic genes as well as glycogen levels were examined in response to feeding standard low-fat laboratory chow supplemented with water or water containing sucrose or fructose in control (Med13fl/fl) and liver-specific MED13 knockout (MED13-LKO) mice. RESULTS: Although MED13 deficiency had no significant effect on constitutive gene expression, all the dietary inducible gene transcripts were significantly reduced despite the unchanged insulin sensitivity in the MED13-LKO mice compared to that in the control mice. G6pc gene transcription displayed the most significant difference between the Med13 fl/fl and MED13-LKO mice, particularly when fed fructose. Following fasting that depleted liver glycogen, feeding induced the restoration of glycogen levels except in the presence of fructose. MED13 deficiency rescued the glycogen accumulation defect in the presence of fructose. This resulted from the suppression of G6pc expression and thus G6PC enzymatic activity. Among two transcriptional factors that regulate G6pc gene expression, FOXO1 binding to the G6pc promoter was not affected, whereas ChREBP binding was dramatically reduced in MED13-LKO hepatocytes. In addition, there was a marked suppression of FOXO1 and ChREBP-ß transcriptional activities in MED13-LKO hepatocytes. CONCLUSIONS: Taken together, our data suggest that the kinase module of the Mediator complex is necessary for the transcriptional activation of metabolic genes such as G6pc and has an important role in regulating glycogen levels in the liver through altering transcription factor binding and activity at the G6pc promoter.

Regulation of gene expression during the fasting-feeding cycle of the liver displays mouse strain specificity.

The liver maintains metabolic homeostasis by integrating the regulation of nutrient status with both hormonal and neural signals. Many studies on hepatic signaling in response to nutrients have been conducted in mice. However, no in-depth study is currently available that has investigated genome-wide changes in gene expression during the normal physiological fasting-feeding cycle in nutrient-sensitive and -insensitive mice. Using two strains of mice, C57BL/6J and BALB/cJ, and deploying deep RNA-Seq complemented with quantitative RT-PCR, we found that feeding causes substantial and transient changes in gene expression in the livers of both mouse strains. The majority of significantly changed transcripts fell within the areas of biological regulation and cellular and metabolic processes. Among the metabolisms of three major types of macronutrients (i.e. carbohydrates, proteins, and lipids), feeding affected lipid metabolism the most. We also noted that the C57BL/6J and BALB/cJ mice significantly differed in gene expression and in changes in gene expression in response to feeding. In both fasted and fed states, both mouse strains shared common expression patterns for about 10,200 genes, and an additional 400-600 genes were differentially regulated in one strain but not the other. Among the shared genes, more lipogenic genes were induced upon feeding in BABL/cJ than in C57BL/6J mice. In contrast, in the population of differentially enriched genes, C57BL/6J mice expressed more genes involved in lipid metabolism than BALB/cJ mice. In summary, these results reveal that the two mouse strains used here exhibit several differences in feeding-induced hepatic responses in gene expression, especially in lipogenic genes.

Lipogenic SREBP-1a/c transcription factors activate expression of the iron regulator hepcidin, revealing cross-talk between lipid and iron metabolisms.

The sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors best known for stimulating the expression of genes encoding key lipogenic enzymes. However, SREBP functions beyond lipid metabolism are less understood. Here, we show that hepcidin antimicrobial peptide (Hamp), encoding the hormone hepcidin essential for iron homeostasis and regulated by dietary iron and inflammation, is a target gene of the two SREBP isoforms SREBP-1a/c. We found that in tissue culture, mature, active, and nuclear forms of the SREBP-1a/c proteins induce endogenous Hamp gene expression and increase the Hamp promoter activity primarily via three regulatory sequences, including an E-box. Moreover, ChIP experiments revealed that SREBP-1a binds to the Hamp gene promoter. Overexpression of nuclear SREBP-1a under the control of the phosphoenolpyruvate carboxylase-1 (Pck1) promoter in mice increased hepatic Hamp mRNA and blood hepcidin levels, and as expected, caused fatty liver. Consistent with the known effects of Hamp up-regulation, SREBP-1a-overexpressing mice displayed signs of dysregulation in iron metabolism, including reduced serum iron and increased hepatic and splenic iron storage. Conversely, liver-specific depletion of the nuclear forms of SREBPs, as in SREBP cleavage-activating protein knockout mice, impaired lipopolysaccharide-induced up-regulation of hepatic Hamp Together, these results indicate that the SREBP-1a/c transcription regulators activate hepcidin expression and thereby contribute to the control of mammalian iron metabolism.

The subunit assembly state of the Mediator complex is nutrient-regulated and is dysregulated in a genetic model of insulin resistance and obesity.

The Mediator complex plays a critical role in the regulation of transcription by linking transcription factors to RNA polymerase II. By examining mouse livers, we have found that in the fasted state, the Mediator complex exists primarily as an approximately 1.2-MDa complex, consistent with the size of the large Mediator complex, whereas following feeding, it converts to an approximately 600-kDa complex, consistent with the size of the core Mediator complex. This dynamic change is due to the dissociation and degradation of the kinase module that includes the MED13, MED12, cyclin-dependent kinase 8 (CDK8), and cyclin C (CCNC) subunits. The dissociation and degradation of the kinase module are dependent upon nutrient activation of mTORC1 that is necessary for the induction of lipogenic gene expression because pharmacological or genetic inhibition of mTORC1 in the fed state restores the kinase module. The degradation but not dissociation of the kinase module depends upon the E3 ligase, SCFFBW7 In addition, genetically insulin-resistant and obese db/db mice in the fasted state displayed elevated lipogenic gene expression and loss of the kinase module that was reversed following mTORC1 inhibition. These data demonstrate that the assembly state of the Mediator complex undergoes physiologic regulation during normal cycles of fasting and feeding in the mouse liver. Furthermore, the assembly state of the Mediator complex is dysregulated in states of obesity and insulin resistance.

Regulation and Metabolic Significance of De Novo Lipogenesis in Adipose Tissues.

De novo lipogenesis (DNL) is a complex and highly regulated process in which carbohydrates from circulation are converted into fatty acids that are then used for synthesizing either triglycerides or other lipid molecules. Dysregulation of DNL contributes to human diseases such as obesity, type 2 diabetes, and cardiovascular diseases. Thus, the lipogenic pathway may provide a new therapeutic opportunity for combating various pathological conditions that are associated with dysregulated lipid metabolism. Hepatic DNL has been well documented, but lipogenesis in adipocytes and its contribution to energy homeostasis and insulin sensitivity are less studied. Recent reports have gained significant insights into the signaling pathways that regulate lipogenic transcription factors and the role of DNL in adipose tissues. In this review, we will update the current knowledge of DNL in white and brown adipose tissues with the focus on transcriptional, post-translational, and central regulation of DNL. We will also summarize the recent findings of adipocyte DNL as a source of some signaling molecules that critically regulate energy metabolism.

Cyclin C regulates adipogenesis by stimulating transcriptional activity of CCAAT/enhancer-binding protein α.

Brown adipose tissue is important for maintaining energy homeostasis and adaptive thermogenesis in rodents and humans. As disorders arising from dysregulated energy metabolism, such as obesity and metabolic diseases, have increased, so has interest in the molecular mechanisms of adipocyte biology. Using a functional screen, we identified cyclin C (CycC), a conserved subunit of the Mediator complex, as a novel regulator for brown adipocyte formation. siRNA-mediated CycC knockdown (KD) in brown preadipocytes impaired the early transcriptional program of differentiation, and genetic KO of CycC completely blocked the differentiation process. RNA sequencing analyses of CycC-KD revealed a critical role of CycC in activating genes co-regulated by peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Overexpression of PPARγ2 or addition of the PPARγ ligand rosiglitazone rescued the defects in CycC-KO brown preadipocytes and efficiently activated the PPARγ-responsive promoters in both WT and CycC-KO cells, suggesting that CycC is not essential for PPARγ transcriptional activity. In contrast, CycC-KO significantly reduced C/EBPα-dependent gene expression. Unlike for PPARγ, overexpression of C/EBPα could not induce C/EBPα target gene expression in CycC-KO cells or rescue the CycC-KO defects in brown adipogenesis, suggesting that CycC is essential for C/EBPα-mediated gene activation. CycC physically interacted with C/EBPα, and this interaction was required for C/EBPα transactivation domain activity. Consistent with the role of C/EBPα in white adipogenesis, CycC-KD also inhibited differentiation of 3T3-L1 cells into white adipocytes. Together, these data indicate that CycC activates adipogenesis in part by stimulating the transcriptional activity of C/EBPα.

Regulation of metabolism by the Mediator complex.

The Mediator complex was originally discovered in yeast, but it is conserved in all eukaryotes. Its best-known function is to regulate RNA polymerase II-dependent gene transcription. Although the mechanisms by which the Mediator complex regulates transcription are often complicated by the context-dependent regulation, this transcription cofactor complex plays a pivotal role in numerous biological pathways. Biochemical, molecular, and physiological studies using cancer cell lines or model organisms have established the current paradigm of the Mediator functions. However, the physiological roles of the mammalian Mediator complex remain poorly defined, but have attracted a great interest in recent years. In this short review, we will summarize some of the reported functions of selective Mediator subunits in the regulation of metabolism. These intriguing findings suggest that the Mediator complex may be an important player in nutrient sensing and energy balance in mammals.

CDK8-Cyclin C Mediates Nutritional Regulation of Developmental Transitions through the Ecdysone Receptor in Drosophila.

The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition.

mTORC1 Down-Regulates Cyclin-Dependent Kinase 8 (CDK8) and Cyclin C (CycC).

In non-alcoholic fatty liver disease (NAFLD) and insulin resistance, hepatic de novo lipogenesis is often elevated, but the underlying mechanisms remain poorly understood. Recently, we show that CDK8 functions to suppress de novo lipogenesis. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a critical regulator of CDK8 and its activating partner CycC. Using pharmacologic and genetic approaches, we show that increased mTORC1 activation causes the reduction of the CDK8-CycC complex in vitro and in mouse liver in vivo. In addition, mTORC1 is more active in three mouse models of NAFLD, correlated with the lower abundance of the CDK8-CycC complex. Consistent with the inhibitory role of CDK8 on de novo lipogenesis, nuclear SREBP-1c proteins and lipogenic enzymes are accumulated in NAFLD models. Thus, our results suggest that mTORC1 activation in NAFLD and insulin resistance results in down-regulation of the CDK8-CycC complex and elevation of lipogenic protein expression.

Regulation of lipogenic gene expression by lysine-specific histone demethylase-1 (LSD1).

Dysregulation of lipid homeostasis is a common feature of several major human diseases, including type 2 diabetes and cardiovascular disease. However, because of the complex nature of lipid metabolism, the regulatory mechanisms remain poorly defined at the molecular level. As the key transcriptional activators of lipogenic genes, such as fatty acid synthase (FAS), sterol regulatory element-binding proteins (SREBPs) play a pivotal role in stimulating lipid biosynthesis. Several studies have shown that SREBPs are regulated by the NAD(+)-dependent histone deacetylase SIRT1, which forms a complex with the lysine-specific histone demethylase LSD1. Here, we show that LSD1 plays a role in regulating SREBP1-mediated gene expression. Multiple lines of evidence suggest that LSD1 is required for SREBP1-dependent activation of the FAS promoter in mammalian cells. LSD1 knockdown decreases SREBP-1a at the transcription level. Although LSD1 affects nuclear SREBP-1 abundance indirectly through SIRT1, it is also required for SREBP1 binding to the FAS promoter. As a result, LSD1 knockdown decreases triglyceride levels in hepatocytes. Taken together, these results show that LSD1 plays a role in regulating lipogenic gene expression, suggesting LSD1 as a potential target for treating dysregulation of lipid metabolism.