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Glucagon regulates the stability of REV-ERBα to modulate hepatic glucose production in a model of lung cancer–associated cachexia

Glucagon signaling governs REV-ERBα stability to modulate hepatic glucose production in a model of lung cancer cachexia.

The mechanism of how the circadian clock adjusts to environmental perturbation has been characterized in dietary and nutritional challenge paradigms (14,16,19,25), but little is known about how the clock is involved in metabolic adaption to disease states. Late-stage tumor burden is associated with cachexia in specific cancer types and is linked with marked inflammation that causes breakdown of adipose and muscle tissues, which results in severe weight loss due to tissue wasting (28,29). Unlike adipose and muscle tissue, the liver maintains its mass and even increases its metabolic rate under cachexic conditions (30). Disrupted insulin signaling and elevated glucose levels have been reported in patients with cancer (31,32), as one of the features of cachexia is activated gluconeogenesis (29,33). Historical studies point to a potential cancer-associated type of Cori cycle where tumor-secreted lactate can fuel hepatic glucose production (31,34). We and others previously demonstrated that mouse models of lung (27) and breast cancer (26), which are prone to cancer-associated cachexia (CAC), exhibited alterations in systemic inflammation that led to deregulation in circadian metabolic rhythms in peripheral tissues such as the liver. We further identified that lung adenocarcinoma tumor-bearing (TB) mice displayed disruptions in insulin/glucose signaling pathways that resulted in elevated fasting glucose levels (27), although the detailed molecular mechanism underpinning these metabolic alterations remain unknown.
Gluconeogenesis, or the de novo production of glucose from lactate, glycerol, or certain amino acids, is especially important during periods of prolonged fasting or nutritional deprivation, as a mechanism to sustain blood glucose levels (35,36). Gluconeogenesis is initiated through glucagon (GC) signaling and downstream stimulation of cyclic adenosine monophosphate (cAMP) release that activates protein kinase A (PKA)-dependent phosphorylation of cAMP response element-binding protein (CREB), resulting in an upregulation of gluconeogenic targets, Pck1 and glucose 6-phosphatase (G6pc) (35,37,38). Furthermore, the circadian clock has been reported to regulate hepatic gluconeogenesis, although different putative proteins have been proposed. The circadian repressors, cryptochromes 1 and 2 (CRY1 and CRY2), have been reported to block the GC-mediated release of cAMP, which results in the repression of the PKA/CREB axis (39). Glucocorticoids also promote gluconeogenesis through CRY-mediated repression of the glucocorticoid receptor (40,41), and several clock proteins regulate nuclear receptors to control circadian metabolic gene expression programs (42). In addition, the circadian repressor NR1D1 or REV-ERB alpha (REV-ERB), a known heme sensor that integrates circadian and metabolic rhythms (43), has been reported to primarily control not only hepatic lipid metabolism but also gluconeogenic gene expression (6,7,44,45). REV-ERB is known to repress the expression of Pck1 and G6pc and thereby suppress hepatic glucose production (44), and disruption of hepatic REV-ERB is also reported to impinge on insulin signaling, carbohydrate metabolism, and fasting ex vivo approach to confirm these observations, and PH were isolated from livers of WT and TB mice and treated with dexamethasone (DEX) to synchronize the circadian clock. Consistent with our in vivo findings, REV-ERB expression was also reduced in synchronized PH isolated from livers of TB mice after 20, 24, and 28 hours of DEX shock ( Fig. 1D and fig. S2A) with no differences in Rev-erb gene expression between both genotypes (Fig. 1E). Considering the known repressive function of REV-ERB, the expression of core clock and metabolic target genes was examined. Gene expression of Bmal1 and Cry1 and their protein levels were elevated in PH isolated from livers of TB mice ( fig. S2, A and B), as they are known targets of the transcriptional repressor REV-ERB (43). Similarly, increased gene expression of Pck1, which encodes phosphoenolpyruvate carboxylase (PEPCK), and G6pc, rate-limiting enzymes of the gluconeogenesis pathway (50), was observed from liver tissue and PH isolated from livers of TB mice ( Fig. 1F and fig. S2B). Increased Pck1 gene expression in PH is in accordance with our previous findings showing that lung TB mice displayed elevated fasting serum glucose levels (27), suggesting enhanced hepatic gluconeogenesis in lung TB mice.
Insulin and GC are the two most important hormones regulating hepatic gluconeogenesis, and we previously showed that impaired insulin secretion occurs in a time-dependent manner in lung TB mice (27). To corroborate these findings, we measured GC levels in the serum of WT and lung TB mice and found a significant elevation of GC in TB mice at ZT8 (Fig. 1G). GC levels are increased during fasting and decrease after refeeding (51). Consistent with these findings, serum GC levels are elevated in fasted WT mice and decrease upon feeding at ZT16; however, this dynamic endocrine response in GC levels is ablated in TB mice ( fig. S2C). GC signals through its receptor on the surface of hepatocytes to activate heterotrimeric G proteins, and the Gs alpha subunit activates the adenylyl cyclases to stimulate production of cAMP (52). Using mass spectrometrybased metabolomics analysis, we measured cAMP levels in livers isolated from WT and TB mice over the circadian cycle. cAMP peaked at ZT16 in livers from WT mice, while cAMP levels were significantly shifted and peaked in abundance at ZT12 in livers isolated from TB mice (Fig. 1H). Together, these data suggest that activation of the GC signaling pathway in the livers of TB mice likely impinges on cAMP release to potentially control gluconeogenesis.

The circadian protein REV-ERB is a target of the PKA signaling pathway
To investigate the link between GC signaling and clock-dependent control of gluconeogenesis, we examined a potential cross-talk between REV-ERB and hormonal control of glucose metabolism. Increased cAMP is known to activate PKA and the downstream transcription factor CREB (37,38). Given the changes in GC and cAMP levels we observed in TB mice, we investigated the pattern of PKA-dependent CREB activation by phosphorylation on its target residue, serine-133 (S133). CREB phosphorylation is rhythmic and peaks at ZT16, ZT20, and ZT0 in the livers of WT mice ( Fig. 2A and  fig. S2D), which is in accordance with the peak in cAMP observed at ZT16 (Fig. 1H). Conversely, the pattern of CREB phosphorylation was shifted in livers of TB mice where a clear activation was detected at ZT8 and ZT12 ( Fig. 2A and fig. S2D), consistent with an earlier peak in cAMP levels in liver of TB mice (Fig. 1H). Similar to in vivo findings, a phase advance of pCREB-Ser 133 was observed in PH isolated from livers of TB mice versus WT ( Fig. 2B  and fig. S2E). (G) Serum GC levels as determined by enzyme-linked immunosorbent assay (ELISA) (n = 6 mice per genotype). (H) Levels of cAMP as determined by metabolomics analysis in liver (n = 5 independent mice per genotype and time point). Data represent the means ± SEM with P value cutoff indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as determined by multiple t test. immediately after synchronization with DEX (three independent experiments). Data represent the means ± SEM with P value cutoff indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as determined by multiple t test or one-way analysis of variance (ANOVA) (with more than two groups).
Intriguingly, activation of CREB ( Fig. 2A) is antiphasic with REV-ERB protein abundance in WT livers (Fig. 1B). However, CREB activation ( Fig. 2A) is phase advanced and coincides with the down-regulation of REV-ERB protein levels in livers of TB mice (Fig. 1B). These data suggest that the GC/PKA signaling axis may impinge on REV-ERB stability. To investigate this, we injected WT mice with saline or GC and analyzed hepatic protein abundance of REV-ERB and BMAL1. Notably, a net reduction in REV-ERB protein was observed in the livers of GC-stimulated mice, while no decrease in BMAL1 was detected, and these mice displayed the expected increase in blood glucose levels (Fig. 2C). Similarly, we treated synchronized PH with GC or forskolin (FSK), an adenylyl cyclase activator, to stimulate the cAMP/PKA signaling pathway. Notably, both FSK and GC induced significant down-regulation of REV-ERB protein in DEX-synchronized PH when treated for 2 hours ( Fig. 2D and fig. S3A), while no major decrease was observed in other circadian proteins such as BMAL1 and CRY1 ( fig. S3B). We did not observe any differences in the gene expression of Bmal1 and Cry1 at 2 hours; however, gene expression was increased at later time points ( fig. S3C). REV-ERB represses gluconeogenesis and CREB is known to stimulate glucose production by binding to the CREB response elements (CRE) in the Pck1 and G6pc promoters (37,38). FSK and GC treatment induced strong activation of Pck1 and G6pc gene expression in PH (Fig. 2E).
To dissect the mechanism of REV-ERB protein destabilization by pharmacological PKA activation, we used an in vitro cell culture system using the nontumorigenic mouse hepatocyte cell line AML12 (alpha mouse liver 12), derived from livers of transgenic mice overexpressing a hepatocyte mitogen. In AML12 cells, REV-ERB consistently peaked 2 to 4 hours after DEX treatment (Fig. 3A). Next, AML12 cells were treated with FSK and GC to determine the impact of cAMP and downstream PKA signaling on REV-ERB stability. REV-ERB protein abundance was strongly reduced in the presence of FSK and GC 2 to 4 hours after DEX treatment, corresponding with activation of CREB ( Fig. 3A and fig. S4, A and B). Also, consistent with down-regulation of REV-ERB protein levels, FSK treatment of AML12 cells significantly elevated gene expression of Pck1, G6pc, Bmal1, and Cry1, direct targets of REV-ERB ( Fig. 3B  and fig. S4C). Similar trends in gene expression were observed with GC treatment of AML12 cells ( Fig. 3B and fig. S4C). FSK and GC stimulate the production of cAMP via activation of the adenylyl cyclases. To confirm that the observed effects are specific to cAMP signaling, we used dibutyryl-cAMP (dbcAMP), a cell-permeable cAMP analog, and 3-isobutyl-1-methylxanthine (IBMX), a competitive nonselective phosphodiesterase inhibitor. Both dbcAMP and IBMX led to a significant reduction in REV-ERB protein levels ( Fig. 3C and  fig. S5, A and B). Moreover, AML12 cells were treated with a cell-permeable PKA peptide inhibitor, PKI 14-22 amide (PKI), to directly block PKAdependent kinase activity. PKI promoted a significant accumulation of REV-ERB at 2 to 4 hours after DEX treatment, when the protein reaches its zenith ( Fig. 3D and fig. S5C). Together, these data demonstrate that the stability of the circadian protein REV-ERB is regulated by cAMP and downstream PKA signaling.

PKA targets serine residues on REV-ERB to regulate protein stability
Our data support the hypothesis that activated PKA signaling promotes the degradation of REV-ERB protein. To confirm that PKAdependent signaling is responsible for REV-ERB destabilization through proteasomal degradation, AML12 cells were treated with FSK, dbcAMP, GC, or IBMX with the addition of the proteasome inhibitor MG132. Degradation of REV-ERB induced by the cAMP and PKA pathway activators was blocked in the presence of MG132 (Fig. 3, E and F, and fig. S6, A to D). We also performed the opposing experiment using a cycloheximide (CHX) chase to block new protein translation and identified that REV-ERB is more rapidly degraded in the presence of FSK and GC versus PKI ( fig. S6E). These data indicate that REV-ERB protein stability is targeted by the PKA signaling pathway for proteasomal degradation.
To identify PKA-dependent phosphorylation sites on REV-ERB, we used the Scansite (https://scansite4.mit.edu) and NetPhos programs (https://services.healthtech.dtu.dk). Based on the PKA consensus motif, residue conservation among multiple species, and surface accessibility to the kinase, we selected S28, S160, and S281 as putative phosphorylation sites for further analysis. These three serine residues are well conserved among various species of vertebrates and might reflect their evolutionary importance in regulating REV-ERB stability (Fig. 4A). Also, these sites are not located in the ligand-binding domain (Fig. 4A) and therefore are unlikely to perturb heme binding. S28, S160, and S281 were mutated to the nonphosphorylatable alanine by site-directed mutagenesis. The single mutation of each of these sites did not rescue REV-ERB from degradation using IBMX, although a minor stabilization in protein level was observed with REV-ERB S160A ( Fig. 4B and fig. S7A). These findings are consistent with the fact that S160 was identified using Scansite as the strongest PKA phosphorylation motif within REV-ERB. The double mutants, REV-ERB S28A + S160A and REV-ERB S28A + S281A, almost fully rescued REV-ERB from PKA-dependent degradation, while REV-ERB S160A + S281A prevented degradation by PKA activation (Fig. 4B and fig. S7A). These data suggest that all three sites are substrates for PKA, with S160 and S281 potentially being the two most important sites. Based on these findings, we created the non-degradable triple mutant, REV-ERB S28A + S160A + S281A, which is fully stabilized in the presence of pharmacological PKA activation using IBMX ( Fig. 4B and fig. S7A). In addition, the nondegradable triple mutant displayed an increased protein stability compared to WT using a CHX chase experiment ( Fig. 4C and fig. S7B). To further pinpoint the direct contribution of these serine residues on REV-ERB stability, we compared the S160A + S281A double mutant to the S28A + S160A + S281A triple mutant using a CHX chase experiment and identified that all three serine residues of the triple mutant contribute to the increased stability of REV-ERB ( Fig. 4C and fig. S7B). These data indicate that PKA targets REV-ERB stability through three different conserved serine residues within the protein.

REV-ERB controls Pck1 expression through promoter regulation
We identified a significant up-regulation of Pck1 gene expression in the livers of TB mice and PH isolated from these animals (Fig. 1F), yet the precise transcriptional regulatory mechanism of how REV-ERB controls Pck1 expression needed further validation. To determine the binding pattern of REV-ERB to the Pck1 promoter, we analyzed published chromatin immunoprecipitation sequencing (ChIP-seq) data (45). REV-ERB binds to the proximal promoter of Pck1 in a region containing a strong and unique consensus hepatocyte nuclear factor 4 alpha (HNF4) recognition motif (Fig. 5A). In addition, the REV-ERB cistrome in liver has been shown to overlap with HNF4 . Data represent the means ± SEM with P value cutoff indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as determined by multiple t test.
motifs (53,54), where REV-ERB tethers to the liver-specific transcription factor to regulate metabolic genes. This is in contrast to the direct occupancy of REV-ERB at clock gene promoters through the RevDR2 response element (43). The protein level of HNF4 and its gene expression in the livers of WT and TB mice remained unchanged ( fig. S7C), suggesting that Pck1 gene expression could be linked to the stability of the transcriptional repressor REV-ERB.
To define the REV-ERB-dependent regulation of Pck1 in a functional transcriptional assay, we constructed the Pck1-luciferase construct containing the proximal promoter region that harbors both the HNF4 and the CREB binding sites, pGL4-Pck1-Luc HNF4; CREB . This CREB binding site was rated as the strongest CRE regulatory element, P value (2.27 × 10 −4 ), according to the genome-wide position weight matrix scanner (https://ccg.epfl.ch//pwmscan/), and has been previously reported as a robust regulatory motif in the Pck1 proximal promoter (55,56). Ectopic expression of WT and the REV-ERB triple mutant repressed Pck1 gene transcription in isolated PH, and this repression was maintained when cells were (C) Western blot analysis of FLAG-tagged WT REV-ERB or REV-ERB double (S160A + S281A) or triple (S28A + S160A + S281A) mutants in AML12 cells treated with CHX (20 g/ml) for 6 hours (two independent experiments). Data represent the means ± SEM with P value cutoff indicated as **P < 0.01 and ***P < 0.001 as determined by multiple t test.

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treated with GC (Fig. 5B). One major limitation of this pGL4-Pck1-Luc HNF4; CREB construct is its inability to discriminate between GC-stimulated CREB-dependent transcriptional activity versus REV-ERB gene regulation through HNF4 binding of the Pck1 promoter. Therefore, to isolate these two opposing transcriptional regulatory axes, we mutated the consensus CRE. This is especially relevant given the strong induction of Pck1-dependent luciferase activity upon GC treatment, which is activated by CREB (Fig. 5B). We deleted the six central nucleotides of the consensus CREB recognition motif in the Pck1 promoter (pGL4-Pck1-Luc HNF4; CREBCRE ), consistent with previous reports that these central nucleotides within the CRE are critical for CREB binding to DNA (37,38,57). Using this pGL4-Pck1-Luc HNF4; CREBCRE luciferase reporter, we observed an expected REV-ERB-mediated repression of Pck1-driven luciferase activity (Fig. 5B). In contrast, upon GC treatment, a derepression of Pck1 gene transcription was observed with WT REV-ERB, suggesting degradation of the transcriptional repressor (Fig. 5B). Conversely, the REV-ERB phosphomutant was resistant to GC-dependent degradation and therefore significantly repressed Pck1 luciferase activity through the HNF4 regulatory motif (Fig. 5B).
As a control, we confirmed the expected GC-mediated turnover of WT REV-ERB, while the REV-ERB mutant is stable in the presence of GC ( Fig. 5B and fig. S7D). Collectively, these data suggest that degradation of REV-ERB alleviates the transcriptional repression on Pck1 gene expression.

Degradation of hepatic REV-ERB alleviates repression of gluconeogenesis to enhance de novo glucose production
Our data indicate that REV-ERB represses Pck1 gene expression and this transcriptional inhibition is alleviated when REV-ERB is degraded through the cAMP/PKA signaling axis. These data suggest that down-regulation of REV-ERB protein in the livers of lung TB mice could derepress gluconeogenesis and subsequently enhance de novo glucose production. To test this hypothesis ex vivo and provide a physiological context for our findings, we performed a glucose production assay in synchronized PH isolated from livers of WT and lung TB mice. PH derived from livers of lung TB mice produced significantly more glucose than their WT counterparts after 120, 240, and 480 min, in the presence of gluconeogenic substrates lactate and pyruvate (Fig. 6A). In addition, we isolated PH from WT and TB mice and ectopically expressed REV-ERB to determine its direct role in glucose production. Consistent with Fig. 6A, de novo glucose production was elevated from PH isolated from TB mice versus WT, and glucose levels were significantly suppressed upon ectopic expression of REV-ERB, specifically in hepatocytes from TB mice (Fig. 6B). As a control, levels of REV-ERB are quantified by Western blot analysis in PH isolated from WT and TB mice (Fig. 6C). To confirm this finding in vivo, a PTT was performed to quantify de novo glucose production, as an indicator of active gluconeogenesis. Overnight fasted WT and TB mice were administered pyruvate intraperitoneally and their blood glucose was measured over time. Lung TB mice had the largest fold change in blood glucose produced 1 hour post injection of pyruvate (Fig. 6D), which is indicative of active gluconeogenesis. When this PTT was performed using 2-month post-intratracheal viral delivery old mice, where REV-ERB protein was not decreased in the liver ( fig. S7E), no difference was observed in blood glucose levels ( fig. S7F). These data demonstrate that lung TB mice have an enhanced rate of de novo glucose production through gluconeogenesis both in vivo and from PH isolated ex vivo. Lung adenocarcinomas are known to be highly glycolytic and metabolize glucose through aerobic glycolysis, or the Warburg effect, resulting in secretion of large amounts of lactate as a glycolytic by-product (58). Lactate has not only been shown to be the primary carbon source to fuel the tricarboxylic acid cycle in GEMMs of lung cancer (59, 60) but lactate is also converted to pyruvate to drive gluconeogenesis following pyruvate carboxylation to oxaloacetate (59,60). We previously demonstrated that lung TB mice have elevated expression of lactate dehydrogenases (LDH) that interconvert lactate and pyruvate (27). Therefore, to trace the metabolic fate of lactate and pyruvate, PH isolated from WT and lung TB mice were incubated with uniformly labeled 13 C (U-13 C) lactate and U-13 C pyruvate stable isotopes followed by targeted metabolomics analysis. The total abundance of U-13 C lactate and pyruvate was similar between all samples (Fig. 6E). Incubation of U-13 C lactate and pyruvate resulted in labeling of several gluconeogenic intermediates including 3-phosphoglycerate, glyceraldehyde-3-phosphate, and fructose 6-phosphate. PH isolated from TB mice had increased labeling of the gluconeogenic intermediates and glucose, which was dampened upon ectopic expression of REV-ERB (Fig. 6E). Unexpectedly, we also observed elevated labeling of glycerol 3-phosphate in hepatocytes isolated from TB mice (Fig. 6E). This finding further supports the usage of lactate and pyruvate in a parallel pathway called glyceroneogenesis, which is used to synthesize triglycerides, and this pathway shares common enzymes with gluconeogenesis, including PEPCK (61). Using metabolic fate mapping through stable isotope tracing, our data indicate that gluconeogenesis is elevated in hepatocytes isolated from lung TB mice versus WT, and that REV-ERB is a critical regulator of hepatic glucose production in a model of CAC.

DISCUSSION
Our findings define a previously unknown molecular mechanism for how the circadian protein REV-ERB integrates hormonal signaling with the downstream modulation of hepatic gluconeogenesis (Fig. 7). Our model illustrates that GC signaling, mediated through the cAMP/PKA axis, is responsible for the proteasome-mediated degradation of REV-ERB protein. Loss of REV-ERB alleviates the transcriptional repression of gluconeogenic gene expression. Using a mouse model of lung adenocarcinoma that undergoes CAC, we show that loss of hepatic REV-ERB and activation of the gluconeogenic program enhance de novo glucose production in isolated PH from lung TB mice. In addition, using stable isotope metabolic tracing, we demonstrate that lactate and pyruvate are used as gluconeogenic substrates to produce increased glucose in hepatocytes from TB mice, which is suppressed by REV-ERB. Together, our data demonstrate that the circadian protein REV-ERB is an important point of convergence that integrates GC-mediated signaling with the direct control of glucose production from the liver. These findings suggest that elevated hepatic glucose production could be beneficial to support tumor metabolism, and further studies are required to address this.
The circadian protein REV-ERB is a nuclear hormone receptor that regulates rhythmic metabolic pathways in the liver (6,7,45,62,63). Rev-erb −/− mice exhibit disrupted lipid metabolism and elevated fasting hyperglycemia (7,44). Similarly, the Rev-erb/ double knockout has been shown to modulate insulin signaling and systemic glucose levels (6,45). Specifically, REV-ERB is reported to repress the expression of gluconeogenic target genes Pck1 and G6pc (44), and the expression of Pck1 and the gluconeogenic pathway are known to be circadian. Yet, the upstream REV-ERB-dependent regulatory mechanism that governs rhythmic gluconeogenesis to control hepatic glucose production remained undefined. Our data now suggest that REV-ERB stability is regulated by GC-activated PKA signaling, and degradation of the transcriptional repressor REV-ERB activates gluconeogenic gene expression. In line with published ChIP-seq data (53), we now show through functional luciferase assays that REV-ERB controls Pck1 expression through the HNF4 binding motif, located within the proximal promoter region of Pck1. Our data do not exclude a role for CREB in regulating Pck1 expression and the gluconeogenic program, but rather, we suggest a cooperative transcriptional regulatory function of REV-ERB with the CREB pathway. Further experiments are required to define the precise cross-talk and timing of the PKA-dependent phosphorylation of REV-ERB and CREB that dictate control of gluconeogenesis. The opposing insulin and GC signaling pathways are a critical endocrine axis that control carbohydrate and lipid metabolism in the fed versus fasted states (64,65). Given that nutritional deprivation activates GC-dependent signaling and downstream activation of glucose production (64,65), these fasting-dependent endocrine and metabolic features mirror the biological consequences of CAC. Therefore, we believe that our findings regarding cachexia-induced activation of gluconeogenesis parallel fasting paradigms in terms of the role of the circadian clock. Consistent with our findings, fasting results in a marked down-regulation of REV-ERB protein abundance in liver and muscle that is linked with the activation of fastingsensing transcriptional pathways, such as CREB (19,20). These data suggest that REV-ERB could be the upstream regulatory node of the circadian clock that is coordinately controlled during nutritional deprivation and CAC. However, inherent downstream differences likely exist between fasting and CAC-dependent activation of gluconeogenesis based on substrate utilization for glucose production. For instance, glycogen stores are immediately liberated through glycogenolysis to regulate blood glucose levels during the initial stages of short-term fasting, which is subsequently followed by activation of gluconeogenesis (66). In our model of CAC, lung TB mice have depleted short-term energy storage depots (27) and instead rely primarily on gluconeogenesis for glucose production. Furthermore, our data indicate that lung TB mice use tumor-derived lactate, which is converted into pyruvate, as a substrate for hepatic glucose production based on PTT data and stable isotope tracing experiments (Fig. 6, C and D). To further support this concept, lung TB mice exhibit elevated expression of LDH and pyruvate in the liver (27), suggesting that tumor-derived lactate can readily be converted into pyruvate to fuel hepatic gluconeogenesis. Collectively, our findings indicate a potential parallel biological response between nutritional deprivation and CAC that impinges on GC-mediated activation of gluconeogenesis through the cAMP/PKA signaling axis to control REV-ERB stability. However, further studies are needed to dissect the inherent differences between these metabolic adaptation responses that likely converge on substrate preference and utilization for hepatic glucose production.
Moreover, REV-ERB is a dynamically regulated protein as evidenced by the different sites of phosphorylation that have been demonstrated to dictate its transcriptional activity and stability under fed versus fasted states. For example, the insulin axis through GSK3 has been reported to phosphorylate REV-ERB at serine residues S55 and S59 to stabilize the protein (67). Also, untargeted mapping of the circadian phosphoproteome in the liver has revealed additional sites of rhythmic phosphorylation on REV-ERB, including a known CDK1 site (68,69). Our findings now demonstrate that PKA targets REV-ERB to control its stability and regulate downstream gluconeogenic gene expression, and these results broaden the known mode of action of PKA (70,71). Given this dynamic posttranslational regulation, these findings suggest that REV-ERB may play an integral role in rewiring circadian metabolic rhythms. To support this hypothesis, our findings using a model of cachexia and recent published data looking at the fed/fasted state (72) place REV-ERB as a central player in the adaptation response to metabolic perturbation, suggesting that this circadian protein could be a sensor for changes in metabolic homeostasis. Overall, these findings suggest that several signaling pathways and downstream kinases likely impinge on REV-ERB to regulate its transcriptional GC signaling activates the cAMP/PKA axis that targets REV-ERB, on serine residues S28, S160, and S281, for proteasome-mediated degradation. Turnover of REV-ERB protein alleviates the transcriptional repression of gluconeogenic target genes and results in elevated hepatic glucose production.
activity, and further studies are required to delineate these pathways under various conditions of nutritional challenge and metabolic stress.
In addition, the circadian clock has also been reported to modulate gluconeogenesis through CRY-dependent inhibition of cAMP signaling and the CREB transcriptional pathway (39). Yet, published findings on the effects of fasting on the circadian clock reveal that CRY1 expression is actually markedly decreased in the liver (19,23), where gluconeogenesis is actually activated. Therefore, an alternative model may support a role for the CRY proteins in fine-tuning gluconeogenesis exclusively during the day/night transition, while more severe insults such as fasting, starvation, and CAC may cause a robust rewiring of transcription that is dependent on REV-ERB and its control of rhythmic metabolic pathways in the liver. Together, these data suggest that multiple pathways may regulate gluconeogenesis, and further studies are required to tease out the precise control of the circadian clock under nutritional challenge paradigms such as fasting, caloric restriction, time-restricted feeding, and starvation.
Our findings indicate that lung TB mice produce more hepatic glucose through gluconeogenesis (Fig. 6). These results are especially interesting in the context of tissue-tissue communication that is likely coordinated through the circadian clock, and specifically, it is of importance to dissect how these communication cues are altered in disease state. Mapping the metabolic fate of liver-derived glucose in GEMMs of cancer is imperative to determine whether this fuel source is shunted to the tumor to satisfy the heightened metabolic demand of rapidly proliferating cancer cells. In addition, the contribution of other peripheral tissues could be of interest to further characterize the extent of the tumor macroenvironment and how it is influenced by systemic metabolism. Although GC action is mainly liver specific, GC receptors are also expressed in human adipose tissue and can stimulate lipolysis through the cAMP/PKA signaling pathway, resulting in increased whole-body energy expenditure (73,74). Also, REV-ERB represses the gene expression of uncoupling protein 1 (Ucp1) (75). Therefore, loss of REV-ERB could alleviate Ucp1 transcriptional repression to uncouple mitochondrial respiration, leading to elevated lipid mobilization and energy expenditure. Activated lipolysis and changes in mitochondrial respiration are hallmark features of cachexia (76), suggesting that REV-ERB may play a role in cachexia-dependent changes in adipose tissue architecture and metabolism. Finally, determining whether cancer cachexia underpins activation of gluconeogenesis will be important to dissect in different GEMMs of cancer that undergo CAC, including pancreatic, gastric, and colorectal cancers. In summary, our findings define a molecular mechanism for GC-mediated activation of the cAMP/PKA axis that impinges on REV-ERB as a central player in regulating gluconeogenesis and hepatic glucose production in a mouse model of cancer cachexia.

Animal housing and experimental procedures
Kras LSL-G12D/+ ;p53 fl/fl mice have been previously described (46,47,77,78). All experiments were performed in accordance with the Institutional Animal Care and Use Committee guidelines at the University of California, Irvine. Animals were housed in a standard 12-hour light/dark paradigm and fed ad libitum. For viral infection, ad5-CMV:FlpO or ad5-CMV:Cre (University of Iowa, Viral Vector Core) were used at a titer of 3 × 10 7 plaque-forming units and administered by intratracheal delivery. Mice were euthanized between 3.5 and 4 months post infection for tissue collection.

Construction of plasmids and site-directed mutagenesis
The proximal Pck1 promoter was amplified from mouse liver genomic DNA (The Jackson Laboratory) and cloned into pGL4.10[luc2] vector using restriction enzymes Sac I and Xho I to create the luciferase reporter Pck1-Luc HNF4; CREB , containing the consensus DNA binding motifs of HNF4 and CREB. Site-directed mutagenesis of Pck1-Luc HNF4; CREB was performed to delete the six central nucleotides of the CREB binding motif, the CRE to create Pck1-Luc HNF4; CREBCRE . Also, serine residues S28, S160, and S281 were mutated to alanine by site-directed mutagenesis. Primer sequences used for Pck1 promoter amplification, deletion of the CRE sequence, and generation of the REV-ERB point mutants are listed in table S1.

Pyruvate tolerance test
WT and lung TB mice were overnight fasted for 12 hours. Blood samples were collected via tail tip excision and measured using a glucometer before the start of the experiment to measure basal glucose levels and at 15,30,45,60,90,120, and 150 min following administration of sodium pyruvate (2 g/kg body weight) (Sigma-Aldrich, St. Louis, MO) by intraperitoneal injection.
PH glucose production assay PH were isolated from WT and lung TB mice, cultured in lowglucose Dulbecco's modified Eagle's media (DMEM) (Thermo Fisher Scientific, Indianapolis, IN) for 24 hours, and synchronized with 100 nM DEX in glucose-free DMEM-F12 media (Biowest, Riverside, MO) for 1 hour. For ectopic expression of REV-ERB, PH isolated from livers of WT and TB mice were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Indianapolis, IN) and synchronized with DEX 24 hours later. After synchronization, PH were incubated 12 hours in DMEM-F12 supplemented with 10% fetal bovine serum (FBS). Then, cells were washed thoroughly with Krebs-Ringer HEPES buffer (115 mM NaCl, 5 mM KCI, 1 mM KH 2 PO 4 , 25 mM sodium HEPES, 2.5 mM MgSO 4 , and 2 mM CaCl 2 ) and incubated in Krebs-Ringer HEPES buffer supplemented with 1% BSA, 20 mM lactate, and 2 mM pyruvate at 37°C. The media were removed at the indicated times and centrifuged at 15,000g for 10 min to remove debris. The glucose content of the supernatant was measured with the Glucose (HK) Assay Kit (Sigma-Aldrich, St. Louis, MO). Cell lysates were lysed and REV-ERB protein level was determined by Western blotting.

Gene expression analysis
Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Indianapolis, IN), according to manufacturer recommendations. Equal amounts of total RNA were reverse transcribed to complementary DNA (cDNA) using Maxima H Minus cDNA Synthesis Master Mix (Thermo Fisher Scientific, Indianapolis, IN) according to the manufacturer's protocol. cDNA was used for quantitative real-time polymerase chain reaction using the PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA). Gene expression was normalized to 18S ribosomal RNA. Primer sequences used for gene expression analysis are listed in table S2.

Nuclear fractionation
Nuclear fractionation in mouse liver was performed according to a previously published protocol, with slight modifications (80). Briefly, mouse liver was resuspended and homogenized in STM buffer [250 mM sucrose, 50 mM tris-HCl (pH 7.4), and 5 mM MgCl 2 , containing proteases and phosphatases inhibitors]. The homogenate was incubated on ice for 30 min, vortexed, and centrifuged at 800g for 15 min. The pellet contains nuclei and debris, and the supernatant comprises the cytosol. The pellet was washed 2× in STM buffer, resuspended in NET buffer [20 mM HEPES (pH 7.9), 1.5 mM MgCl 2 , 0.5 M NaCl, 0.2 mM EDTA, 20% glycerol, and 1% Triton X-100, containing proteases and phosphatases inhibitors], incubated on ice for 30 min, briefly sonicated, and centrifuged at 9000g for 30 min. The resultant supernatant is the final nuclear fraction. The supernatant from the homogenate was washed 2× in STM buffer, centrifuged at 11,000g for 10 min, and the resultant supernatant was collected and constitutes the final cytosolic fraction.

Metabolite extraction for metabolomics
Metabolites were extracted from 1 million isolated PH cultured in a 6-well plate. Nine hundred microliters of ice-cold 80% MeOH (extraction solvent) containing 0.5% formic acid was added to each well and the plate was gently nutated for 10 s. NH 4 HCO 3 [15% (w/v); 80 l for 900 l of a 80% MeOH solution] was added to every well to neutralize pH. Plates were placed in −80°C for 1 hour. Cells were scraped and the entire content of the well was transferred to an Eppendorf tube. The well was rinsed with a small amount of icecold 80% MeOH and the remaining cells were added to the tube. Samples were vortexed for 10 s and centrifuged at 16,000g for 15 min at 4°C. The supernatant was transferred to a new tube (first extraction), and 100 l of extraction solvent was added to resuspend the pellet, followed by vortexing and centrifugation at 16,000g for 5 min at 4°C. The resulting supernatant that represents the second extraction was combined with the first extraction. The extract was dried down with nitrogen to concentrate metabolites 10 times. The samples were centrifuged at 16,000g for 10 min at 4°C, and 30 l of supernatant was transferred to vials for mass spectrometry analysis.

Liquid chromatography-mass spectrometry metabolomics analysis
Metabolite abundance and labeling was measured by quadrupoleorbitrap mass spectrometer (Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer, Thermo Fisher Scientific), operating in a negative ion mode via electrospray ionization and used to scan from mass/charge ratio 70 to 830 and 140,000 resolution. Liquid chromatography (LC) separation was on an Xbridge BEH Amide column (2.1 mm by 150 mm, 2.5-m particle size, 130-Å pore size; Waters) at 25°C using a gradient of solvent A (5% acetonitrile in water with 20 mM ammonium acetate and 20 mM ammonium hydroxide) and solvent B (100% acetonitrile). Flow rate was 350 l/min. The LC gradient was as follows: 0 min, 75% B; 3 min, 75% B; 4 min, 50% B; 5 min, 10% B; 7 min, 10% B; 7.5 min, 75% B; and 11 min, 75% B. The injection volume of the sample was 3 l. Data were analyzed using the MAVEN software, and natural isotope correction was performed with the AccuCor R code (https://github. com/XiaoyangSu/AccuCor).

SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/7/26/eabf3885/DC1 View/request a protocol for this paper from Bio-protocol.