Kacie Mitchell

 Introduction: Type 2 diabetes (T2D) is a highly prevalent metabolic disease characterized by insulin resistance.¹ In 2015, it was estimated that 1 in 11 adults aged 20-79 were diagnosed with diabetes and over 90% of those were type 2.¹ Underlying causes of T2D include obesity, caloric excess, and sedentary lifestyles.¹ Behaviors such as smoking and alcohol intake are also associated with increased risk of T2D.¹ Recent studies have identified a link between T2D and circadian rhythm disruptions.² The circadian rhythm is the endogenous 24 hour cycle that is governed by a central clock in the hypothalamus.³ The central clock controls peripheral clocks in tissues such as skeletal muscle through proteins like Per2.³ Period circadian regulator 2 (Per2) is expressed in skeletal muscle and controls oscillations in glucose-handling genes in insulin-dependent tissues.⁴ A recent study found that Per2 was significantly decreased in mononuclear cells of T2D patients.⁵ Exercise has been known to ameliorate T2D pathogenesis.¹ Exercise modifies circadian clocks in skeletal muscle through increased expression of Per2 through hypoxic and contractile-mediated mechanisms.^4,5,6  Methods: C2C12 myotubes were isolated from rodents and treated with an HIF1a stabilizing agent to mimic hypoxia during exercise.⁶ Chromatin immunoprecipitation of HIF1a was performed at enhancer boxes of the Per2 promoter.⁶ C2C12 myotubes were isolated and treated with electrical pulsatile stimulation (EPS) to mimic exercise.⁴ Following the EPS treatment, Per2 expression was measured.⁴ Myotubes were also treated with ionomycin (calcium ionophore) and nifedipine (calcium channel blocker).⁴ After this treatment and EPS, Per2 expression was measured.⁴ To determine the mechanism of Per2 upregulation, levels of ubiquitous transcription factor CREB were measured and compared to its active phosphorylated form (pCREB.)⁴ Levels of pCREB were measured following EPS and treatment with ionomycin or nifedipine.⁴ Results: Stabilization of HIF1a led to increased levels and duration of Per2 expression.⁶ Additionally, HIF1a was bound to the enhancer box of the Per2 promoter.⁶ Per2 expression was increased by 1.5 fold 60 minutes following EPS treatment.⁴ Treatment with ionomycin increased Per2 expression, while treatment with nifedipine decreased Per2 expression both before and following EPS.⁴ EPS and ionomycin treatment increased the levels of pCREB significantly while EPS and nifedipine treatment decreased pCREB expression.⁴ Conclusions: These results demonstrate that Per2 expression is increased during exercise through upregulation by HIF1a.⁶ Additionally, these results identify a contractile-mediated mechanism of Per2 upregulation that is calcium-dependent.⁴ Specifically, Per2 expression is increased through calcium-dependent phosphorylation of CREB and subsequent Per2 expression.⁴

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4. Small L, Altıntaş A, Laker RC, et al. Contraction influences Per2 gene expression in skeletal muscle through a calcium-dependent pathway. J Physiol. 2020;598(24):5739-5752. doi:10.1113/JP280428
5. López-Cano C, Gutiérrez-Carrasquilla L, Barbé F, et al. Effect of Type 2 Diabetes Mellitus on the Hypoxia-Inducible Factor 1-Alpha Expression. Is There a Relationship with the Clock Genes?. J Clin Med. 2020;9(8):2632. Published 2020 Aug 13. doi:10.3390/jcm9082632
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