Novel liver-specific TORC2 siRNA corrects hyperglycemia in rodent models of type 2 diabetes

Abstract

[transducer of regulated cAMP-responsive element-binding protein (CREB) activity 2] is a major regulator of hepatic gluconeogenesis and is increased in hyperglycemic rodent models. Because chronic hyperglycemia and increased hepatic glucose production, via increased gluconeogenesis, is a key feature of type 2 diabetes, an effective in vivo method to efficiently knock down TORC2 could provide a potential therapy for treating hyperglycemia and type 2 diabetes. To assess this, primary mouse hepatocytes, high-fat diet (HFD)-fed mice, and Zucker diabetic fatty (ZDF) rats were treated with a siRNA against TORC2 (siTORC2), which was delivered via a novel lipid nanoparticle system, or control siRNA (siCON). Compared with siCON, administration of siTORC2 resulted in highly efficient, sustained (1-3 wk) knockdown of TORC2 and its gluconeogenic target genes phosphoenolpyruvate carboxykinase and glucose-6-phophatase in primary mouse hepatocytes and in the livers of HFD-fed mice. In mice, this knockdown was specific to the liver and did not occur in kidney, skeletal muscle, or adipose tissue. In HFD-fed mice, siTORC2 reduced in vivo gluconeogenic capacity, fasting hepatic glucose production, and hyperglycemia, and led to improved hepatic and skeletal muscle insulin sensitivity. siTORC2 treatment also improved systemic hyperglycemia in ZDF rats. In conclusion, these results demonstrate the importance of TORC2 in modulating HGP in vivo and highlight a novel, liver-specific siRNA approach for the potential treatment of hyperglycemia and type 2 diabetes.transducer of regulated cAMP-responsive element-binding protein activity 2; diabetes; small interfering RNA IN PATIENTS WITH TYPE 2 DIABETES, increased glucose output from the liver is a major cause of fasting hyperglycemia (5,15,20,22,26), and, consistent with this idea, the degree of fasting hyperglycemia is positively correlated to the magnitude of the increase in hepatic glucose production (HGP) (5,15,20,33). Hepatic gluconeogenesis is a key component of HGP (30), and studies in fasted individuals using magnetic resonance spectroscopy and isotope dilution-based techniques suggest that increased gluconeogenesis is the major contributor to fasting hyperglycemia in type 2 diabetes (5,22,33). Excessive glucose output from the liver can also contribute to postprandial hyperglycemia (22, 30), which is due to a reduced ability of insulin to suppress HGP as well as impaired insulin-stimulated glucose uptake in insulin sensitive tissues (15,26).The cAMP-responsive element-binding protein (CREB) is a major positive transcriptional regulator of the gluconeogenic gene program (11). The ability of CREB to induce gluconeogenesis is dependent on the recently identified CREB coactivator transducer of regulated CREB activity 2 [TORC2, also known as CREB-regulated transcription coactivator 2 (CRTC2)] (3, 7, 8, 16). When hepatic cAMP levels are increased (e.g., by increased glucagon), TORC2 becomes dephosphorylated and translocates to the nucleus, where it coactivates CREB to induce transcripti...

Targeted disruption of the CREB coactivator Crtc2 increases insulin sensitivity

“…Mouse primary hepatocytes were isolated and cultured as previously described (15). Transient assays were performed as previously described using adenoviral (Ad-G6Pase-luc) reporters for primary hepatocytes and plasmid based reporters (EVX-luc) for HEK293T cells (4,6,10). Cells were exposed to glucagon (100 nm) or FSK (10 μM) for 4 h, and luciferase activities were normalized to β-galactosidase activity from adenoviral or plasmid-encoded RSV β-gal.…”

“…Increases in circulating glucagon are thought to trigger gluconeogenic gene expression in part through the cAMP-dependent phosphorylation of the transcription factor CREB and through the dephosphorylation of its cognate coactivator CRTC2 (4)(5)(6). In parallel, decreases in circulating insulin concentrations during fasting also stimulate gluconeogenic genes by the dephosphorylation of the forkhead activator FOXO1 (7).…”

“…The importance of CRTC2 for gluconeogenic gene expression is supported by RNAi-mediated knockdown studies, where acute depletion of CRTC2 lowered hepatic glucose production in fasted mice and by overexpression studies in which phosphorylation-defective, active CRTC2 increases circulating glucose levels under both fasting and feeding conditions (4,6,10).…”

Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2

“…The importance of the CREB:CRTC pathway in hepatic gluconeogenesis is supported by studies in which acute RNAi-me-diated knockdown of CRTC2 in liver decreases gluconeogenic gene expression as well as circulating glucose concentrations (9,14). Cultured hepatocytes from CRTC2 knock-out mice also exhibit decreases in glucose production (12,15), although these effects are more modest due to compensation by CRTC3 (16).…”

Opposing Effects of Reduced Kidney Mass on Liver and Skeletal Muscle Insulin Sensitivity in Obese Mice

“…Endogenous glucose production during the clamp was calculated by subtracting the glucose infusion rate from the glucose disposal rate (25,26). Insulin-stimulated glucose disposal rate (IS-GDR) was calculated by subtracting basal endogenous glucose production (equal to basal glucose disposal rate) from glucose disposal rate during the clamp (27). In order to assess tissue-specific glucose uptake, a bolus (10 mCi) of 2-[1- C]deoxyglucose to calculate glucose uptake, as was previously described (28).…”