Skeletal muscle-specific Cre recombinase expression, controlled by the human α-skeletal actin promoter, improves glucose tolerance in mice fed a high-fat diet

Parts used

Key findings

• Cre recombinase expressed from the human α-skeletal actin (HSA/ACTA1) promoter in the Tg(ACTA1-cre/Esr1*)2Kesr mouse is detectable by western blot in gastrocnemius and quadriceps skeletal muscle but not in heart or liver, and Cre mRNA is undetectable in pancreatic islet tissue. Cre protein is present in muscle of both vehicle- and tamoxifen-treated mice, so the promoter's activity, not tamoxifen, sets where Cre is made.
• In the tamoxifen-inducible HSA-Cre mouse, Cre recombinase is held in the cytoplasm as a complex with heat shock protein 90 (Hsp90) through a fused mutated oestrogen receptor. Tamoxifen treatment dissociates Cre from Hsp90 and Cre appears in the nuclear fraction of gastrocnemius muscle, whereas in corn-oil-treated mice Cre remains Hsp90-bound and cytosolic.
• Tamoxifen-treated mice expressing Cre recombinase from the human α-skeletal actin promoter develop improved glucose tolerance and higher glucose-stimulated circulating insulin than wild-type littermates after 12 weeks on a 60% high-fat diet, with no difference in body weight or body composition. On a 10% low-fat diet the same mice show normal glucose tolerance and insulin levels, so the phenotype requires the high-fat diet.
• The improved glucose tolerance of HSA-Cre mice requires tamoxifen-mediated nuclear liberation of Cre recombinase: corn-oil-treated HSA-Cre mice match wild-type littermates in glucose tolerance, circulating insulin and insulin sensitivity on both low- and high-fat diets, despite expressing Cre protein in skeletal muscle.
• Improved glucose tolerance in tamoxifen-treated HSA-Cre mice is not explained by insulin sensitivity: insulin tolerance tests and phosphorylation of Akt, GSK3β and mTOR in gastrocnemius muscle are unchanged versus wild-type littermates on both low- and high-fat diets. Pancreatic islet morphology by insulin and glucagon staining and mRNA for key pancreatic regulatory genes are likewise unchanged.
• The improved glucose tolerance of tamoxifen-treated HSA-Cre mice on a high-fat diet is not accompanied by changes in whole-body energy metabolism: respiratory exchange ratio, oxygen consumption, ambulatory activity, food intake and water intake match wild-type littermates, as do gastrocnemius triacylglycerol content and mRNA levels for genes of mitochondrial function, lipolysis, fatty acid oxidation and ketone body metabolism.
• Conditioned media from C2C12 myotubes overexpressing Cre recombinase does not augment glucose-stimulated insulin secretion from INS-1 832/3 beta cells, while D-Ala2 GIP robustly does, leaving the mechanism linking skeletal muscle Cre recombinase activity to raised circulating insulin unidentified.

Abstract

Cre-loxP systems are frequently used in mouse genetics as research tools for studying tissue-specific functions of numerous genes/proteins. However, the expression of Cre recombinase in a tissue-specific manner often produces undesirable changes in mouse biology that can confound data interpretation when using these tools to generate tissue-specific gene knockout mice. Our objective was to characterise the actions of Cre recombinase in skeletal muscle, and we anticipated that skeletal muscle-specific Cre recombinase expression driven by the human α-skeletal actin (HSA) promoter would influence glucose homeostasis. Eight-week-old HSA-Cre expressing mice and their wild-type littermates were fed a low- or high-fat diet for 12 weeks. Glucose homeostasis (glucose/insulin tolerance testing) and whole-body energy metabolism (indirect calorimetry) were assessed. We also measured circulating insulin levels and the muscle expression of key regulators of energy metabolism. Whereas tamoxifen-treated HSA-Cre mice fed a low-fat diet exhibited no alterations in glucose homeostasis, we observed marked improvements in glucose tolerance in tamoxifen-treated, but not corn-oil-treated, HSA-Cre mice fed a high-fat diet vs their wild-type littermates. Moreover, Cre dissociation from heat shock protein 90 and translocation to the nucleus was only seen following tamoxifen treatment. These improvements in glucose tolerance were not due to improvements in insulin sensitivity/signalling or enhanced energy metabolism, but appeared to stem from increases in circulating insulin. The intrinsic glycaemia phenotype in the HSA-Cre mouse necessitates the use of HSA-Cre controls, treated with tamoxifen, when using Cre-loxP models to investigate skeletal muscle-specific gene/protein function and glucose homeostasis.