Liver is the central nexus of energy metabolism and governs fate of substrates in response to nutritional and hormonal signals. Under normal physiological conditions, both hepatic glucose production and lipid synthesis display circadian rhythm20
. Our findings establish that rhythmic rerouting of metabolic intermediates between the two synthetic pathways is directly regulated by the molecular circadian clock through the epigenomic modifier Hdac3 (Supplementary Fig. 8
). During the day, when the nocturnal mouse is inactive and feeding less, Hdac3 is recruited to the genome and represses expression of genes involved in lipid synthesis and sequestration, and allows metabolic precursors to be directed towards gluconeogenesis in order to maintain normoglycemia. During the active and feeding night period, the virtual absence of Hdac3 at these genomic regions de-represses lipogenesis, and reroutes metabolic intermediates towards lipid synthesis for energy storage at the expense of glucose production. This model is supported by the following findings: i) depletion of Hdac3 in liver results in upregulation of genes in lipid synthesis and sequestration, but does not alter expression of key gluconeogenic genes; ii) the chromatin occupancy of Hdac3 is predominantly on genes in lipid metabolism and has a circadian pattern with the maximum occupancy during fasting and minimum occupancy during feeding23
; iii) hepatic glycogen is reduced upon Hdac3 deletion, contrary to what would be expected if there were a primary defect in G6pc activity and inability of generating glucose from glycogen40,43
; iv) glucose synthesis from canonical precursors pyruvate and lactate is reduced in Hdac3-depleted hepatocytes, concurrent with increased lipid synthesis from the same precursors; v) the low glucose production in Hdac3-depleted hepatocytes can be rescued by fructose, a six carbon precursors of glucose, or by inhibition of lipid synthesis, suggesting that gluconeogenesis is inherently intact, but dominated by the routing of three carbon metabolites toward lipid synthesis; vi) the low acetyl-CoA content is accompanied with enhanced lipogenesis flux in Hdac3-depleted liver, suggesting that acetyl-CoA is shunted toward lipogenesis, which may limit pyruvate carboxylase activity and thus reduce gluconeogenesis; vii) the improved glucose and insulin tolerance in Hdac3 liver-specific knockout mice is attenuated by suppression of the metabolic rerouting towards lipid synthesis and storage through knockdown of lipid sequestering LDs-coating protein Plin2; and viii) overexpression of Plin2 in liver in wild-type mice results in hepatosteatosis and improved insulin sensitivity, mimicking the metabolic phenotype in Hdac3 liver-specific knockout mice.
The present study highlights the integrated nature of metabolism. It shows that a given metabolic process can be modulated by rerouting of metabolites, without altering enzymes immediately involved in that process, as exemplified by the indirect regulation of gluconeogenesis by Hdac3. Notably, a parallel and independent metabolic rerouting may be coordinated by hepatic FoxOs, which are active in driving gluconeogenic genes expression during fasting and are inhibited by Akt-mediated phosphorylation and p300-mediated acetylation during feeding44
. Constitutively active Foxo1 increases glucose production, and seems to reduce hepatic lipid output as evidenced by hypolipidemia45,46
. Conversely, depletion of hepatic FoxOs decreases glucose production, and tends to increase lipid synthesis as evidenced by hepatosteatosis or hyperlipidemia47,48
. In both situations, the effects of FoxOs on gluconeogenesis are through well-characterized direct transcriptional activation, while the effects of FoxOs on lipid metabolism are less well-defined and somewhat controversial. Of note, acetylation of FoxOs decreases their transactivation activity44
, and it has been shown that recombinant Hdac3 protein can deacetylate Foxo1 in vitro40
. However, our findings that deletion of Hdac3 in liver does not alter Foxo1-dependent transcription, and does not cause accumulation of glycogen as predicted by the in vitro
clearly indicate that the potential deacetylation of Foxo1 is not essential for effects of Hdac3 on glucose metabolism in vivo
. We speculate that FoxOs-mediated transcriptional activation and Hdac3-mediated transcriptional repression may function collectively, but independently, to divert metabolic intermediates in between glucose production and lipid synthesis during the fasting/feeding and light/dark cycles. Although Hdac3 clearly functions as an epigenomic modifier in liver23
, it may also have non-histone protein targets as well as deacetylase-independent functions.
In contrast to the circadian shifting between hepatic lipogenesis and gluconeogenesis under normal physiological conditions, pathological overnutrition provides excess metabolic intermediates that oversaturate and constantly activate both pathways. This leads to co-existence of hepatosteatosis and insulin resistance. However, the concurrent insulin hypersensitivity and severe hepatosteatosis in mice without hepatic Hdac3 clearly demonstrates that hepatosteatosis and insulin resistance per se
do not necessarily have a cause and effect relationship. Among the most prominently upregulated genes in Hdac3-depleted liver are those encoding lipid droplets-associated proteins, including Plin2
, that negatively regulate lipolysis and subsequent release of lipids in a cooperative manner8,16,34,49,50,35,36
. Low acyl-CoA content is accompanied with reduced FAO flux in Hdac3-depleted liver, suggesting that acyl-CoAs are shunted toward glycerolipid synthesis and subsequent sequestration from mitochondrial oxidation machinery. Prkce activity and inflammation is not enhanced despite elevated total DAG content and other lipid species in Hdac3-depleted liver, suggesting that lipid is sequestered from activating cytosolic kinases. Dampening the upregulation of Plin2 masked the insulin hypersensitivity in mice without hepatic Hdac3, while hepatic overexpression of Plin2 in wild type mice results in steatosis and improves whole-body insulin sensitivity, stressing the essential role of lipid sequestration in preventing lipotoxicity and lipid-induced hepatic insulin resistance. Consistent with our finding, the insulin sensitizing effect of Plin2 was observed previously in cultured hepatocytes where depletion of Plin2 and other perilipins decreased cellular responses to insulin51
. In addition, dissociation of hepatosteatosis and insulin resistance was observed in mice or humans that are deficient in either lipolysis or hepatic triglyceride secretion8,18,52–54
. All these findings can be unified under the hypothesis that hepatic insulin resistance is determined by whether lipid droplets sequester lipid content well enough to prevent cytosolic accumulation of deleterious lipid species.
Although hepatic lipogenesis is upregulated and required for the development of steatosis in both chow-fed mice lacking hepatic Hdac3 and in wild type mice on HFD, a key difference lies in the balance between lipid sequestration capacity and the content of metabolic intermediates. In overnutrition mouse models (HFD or ob/ob), excess delivery of metabolic intermediates to the liver is the primary event, while increased hepatic lipid synthesis and storage is an adaptive response. But the lipid sequestration capacity is insufficient relative to the overwhelming lipid content influx, leading to accumulation of harmful lipid intermediates, such as DAG and ceramide that promote insulin resistance. In this situation, ablation of Plin2 reduces lipid synthesis and thus reduces generation of harmful lipid species, leading to improved insulin sensitivity42,55
. In the Hdac3-deficient model, the primary event of upregulated lipogenesis diverts metabolic precursors away from glucose production. In addition, reduced fatty acid oxidation and acetyl-CoA levels may contribute to decrease pyruvate carboxylase activity and thus limit the flux of pyruvate to glucose production. Collectively, these changes result in reduced glucose output and insulin hypersensitivity. Knockdown of Plin2 on this background reduces lipid synthesis and the metabolic rerouting, and therefore ameliorates insulin hypersensitivity. This highlights the concept that hepatic lipid accumulation have diverse pathogenesis and require specifically targeted therapies, which is critically important in light of the failure of large therapeutic trials (such as ACCORD) that lump all diabetes together56
Finally, our data establish Hdac3 as a pivotal epigenomic modifier that directly receives signal input from the molecular circadian clock and promotes hepatic glucose production via rerouting of gluconeogenic metabolites away from lipid synthesis rather than direct transcriptional regulation of gluconeogenic genes. This novel mechanism highlights the dissociation of hepatic steatosis from insulin resistance due to lipid sequestration, which may explain similar phenotypes that have been observed in other mouse models8,17,18,53,57