MicroRNA-193b impairs muscle growth in mouse models of type 2 diabetes by targeting the PDK1/Akt signalling pathway

Aims/hypothesis Type 2 diabetes is associated with a reduction in skeletal muscle mass; however, how the progression of sarcopenia is induced and regulated remains largely unknown. We aimed to find out whether a specific microRNA (miR) may contribute to skeletal muscle atrophy in type 2 diabetes. Methods Adeno-associated virus (AAV)-mediated skeletal muscle miR-193b overexpression in C57BLKS/J mice, and skeletal muscle miR-193b deficiency in db/db mice were used to explore the function of miR-193b in muscle loss. In C57BL/6 J mice, tibialis anterior-specific deletion of 3-phosphoinositide-dependent protein kinase-1 (PDK1), mediated by in situ AAV injection, was used to confirm whether miR-193b regulates muscle growth through PDK1. Serum miR-193b levels were also analysed in healthy individuals (n = 20) and those with type 2 diabetes (n = 20), and correlations of miR-193b levels with HbA1c, fasting blood glucose (FBG), body composition, triacylglycerols and C-peptide were assessed. Results In this study, we found that serum miR-193b levels increased in individuals with type 2 diabetes and negatively correlated with muscle mass in these participants. Functional studies further showed that AAV-mediated overexpression of miR-193b induced muscle loss and dysfunction in healthy mice. In contrast, suppression of miR-193b attenuated muscle loss and dysfunction in db/db mice. Mechanistic analysis revealed that miR-193b could target Pdk1 expression to inactivate the Akt/mammalian target of rapamycin (mTOR)/p70S6 kinase (S6K) pathway, thereby inhibiting protein synthesis. Therefore, knockdown of PDK1 in healthy mice blocked miR-193b-induced inactivation of the Akt/mTOR/S6K pathway and impairment of muscle growth. Conclusions/interpretation Our results identified a previously unrecognised role of miR-193b in muscle function and mass that could be a potential therapeutic target for treating sarcopenia. Graphical abstract Supplementary Information The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-021-05616-y.

sections for staining were cut using a paraffin slicer. For muscle histology, the paraffin slicers were stained with Mayer's hematoxylin and eosin staining kit (Solarbio & Technology, Beijing, China).
Determination of re-fed serum glucose and serum insulin level For re-fed blood glucose, mice were fasted overnight and then the food was replaced for 2 h before the determination of serum glucose level. Serum insulin levels were determined using mouse insulin ELISA kit (catalogue no.: ab277390) obtained from Abcam (Cambridge, MA, USA) after treatment.

Glucose tolerance tests and insulin tolerance tests Glucose tolerance tests (GTTs)
and insulin tolerance tests (ITTs) were performed in mice at 15 and 16 weeks of age as previous described, respectively [1]. Briefly, mice fasted for 6 hr and then were intraperitoneally injected with glucose (2 g glucose/kg body weight; catalogue no.: Y0001745, Sigma-Aldrich, St.Louis, MO, USA) for the GTT assay or with insulin (1 U insulin/kg body weight; Actrapid, Novo Nordisk, Denmark) for ITT assay. And then glucose levels were determined at 0, 15, 30, 60, 90 and 120 min after injection.
During the treatment, the fasting blood glucose were determine with the blood sample withdrawn from mouse tail vein using the OneTouch glucometer and test strips (LifeScan, Milpitas, CA, USA).

Measurement of glycogen and lactate
Mouse skeletal muscle were performed by standard procedure using a glycogen assay kit (catalogue no.: ab65620) and lactated assay kit (catalogue no.: ab169558), respectively, obtained from Abcam (Cambridge, MA, USA).

Hanging test
The mice were placed on a wire (length 100 cm, diameter 2 mm and height 40 cm from the ground). The wire was then rotated to place the mice in hanging state. The time for which the mouse could hold itself was recorded, then the average time for each group was compared with that of other groups. The tests were repeated three times for each mouse and each time with a 2 min rest period.