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Identification of a novel subset of hepatocytes that are simultaneously gluconeogenic and de novo lipogenic in the fed state and are naturally insulin resistant

ABSTRACT: It is generally accepted that hepatic gluconeogenesis, the synthesis of glucose from other carbon substrates (i.e.: glycerol, pyruvate, amino acids), is active in the fasted state and inactive in the fed state. In contrast, de novo lipogenesis (synthesis of fatty acids) is active in the fed state and is inactive in the fasted state. However, RNAPII ChIP-seq demonstrated that periportal but not pericentral hepatocytes display a basal (fed state) transcriptional activation of gluconeogenic genes that correlated with the basal level of gluconeogenic mRNA expression. Targeted single cell RNA-seq, HCR RNA-FISH, and PrimeFlow identified a subpopulation of periportal hepatocytes (approximately 10% of total hepatocytes) that simultaneously co-express both gluconeogenic and lipogenic genes in the fed state. These dual-positive hepatocytes identified in normal mouse livers are also present in human hepatocytes from humanized mouse livers. Spatial metabolic imaging coupled with stable isotope tracing analyses at the single cell level demonstrated that these individual hepatocytes simultaneously undergo both gluconeogenesis and de novo lipogenesis. Euglycemic-hyperinsulinemic clamps coupled with PrimeFlow analyses further demonstrated that this novel hepatocyte subpopulation is naturally insulin resistant. Moreover, high-fat diet-induced insulin resistance and hepatic steatosis were accompanied by an increase in the number of these dual-positive hepatocytes, suggesting a paradigm shift in our understanding of how the liver becomes insulin resistant. Taken together, these data demonstrate the existence of a unique, naturally insulin-resistant subset of mouse and human periportal hepatocytes that have the capacity to simultaneously undergo both gluconeogenesis and de novo lipogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE315776 | GEO | 2026/04/10

REPOSITORIES: GEO

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Project description:During fasting, increases in circulating pancreatic glucagon maintain glucose balance by up-regulating hepatic gluconeogenesis. Triggering of the cAMP pathway stimulates the gluconeogenic program through the phosphorylation of CREB and via the de-phosphorylation of the CREB coactivator CRTC2. Hormonal and nutrient signals are also thought to modulate gluconeogenic genes by promoting epigenetic changes that facilitate assembly of the transcriptional machinery, although the nature of these modifications is unclear. Here we show that histone H3 acetylation at Lys 9 (H3K9Ac) is elevated over gluconeogenic genes during fasting and in diabetes, where it contributes to increases in hepatic glucose production. Following its dephosphorylation, CRTC2 promoted increases in H3K9Ac by mediating the recruitment of the lysine acetyltransferase 2B (KAT2B) and WD repeat-containing protein 5 (WDR5), a core subunit of histone methyltransferase (HMT) complexes. In turn, KAT2B and WDR5 stimulated the gluconeogenic program through a self-reinforcing cycle whereby increases in H3K9Ac further potentiated CRTC2 occupancy at CREB binding sites. Breaking this cycle, by depletion of KAT2B or WDR5, decreased gluconeogenic gene expression. As administration of a small molecule KAT2B antagonist lowered circulating blood glucose concentrations in insulin resistance, our results demonstrate how this enzyme may be a useful target for diabetes treatment. A subset of cAMP responsive genes depend on specific recruitment of KAT2B (pcaf), which in concert with WDR5 acetylates H3K9. By selectively depleting hepatocytes for KAT2B or WDR5 prior to glucagon stimulation we explore, which genes rely on KAT2B and WDR5 activity. mKAT2B or mWDR5 were knocked down in primary mouse hepatocytes using adenoviral transduction with appropriate shRNAs. Control cells were transduced with a non-specific (NS) shRNA. 72 hours post transduction some cells were stimulated for 90 minutes with 100nM glucagon and others with PBS. Total RNA was purified and subjected to micro-RNA analysis. All samples are pools of RNA from three sepearate dishes. One replicate is included for most samples.

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Identification of a novel subset of hepatocytes that are simultaneously gluconeogenic and de novo lipogenic in the fed state and are naturally insulin resistant

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