microManaging glucose and lipid metabolism in skeletal muscle: Role of microRNAs

    microManaging Glucose and Lipid Metabolism in Skeletal Muscle: Role of microRNAs Julie Massart, Mutsumi Katayama, Anna Krook PII: DOI: Reference:

S1388-1981(16)30129-9 doi: 10.1016/j.bbalip.2016.05.006 BBAMCB 57975

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BBA - Molecular and Cell Biology of Lipids

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15 March 2016 3 May 2016 10 May 2016

Please cite this article as: Julie Massart, Mutsumi Katayama, Anna Krook, microManaging Glucose and Lipid Metabolism in Skeletal Muscle: Role of microRNAs, BBA - Molecular and Cell Biology of Lipids (2016), doi: 10.1016/j.bbalip.2016.05.006

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microManaging Glucose and Lipid Metabolism in Skeletal Muscle: Role of microRNAs

Department of Molecular Medicine and Surgery, 2Department of Physiology and Pharmacology,

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Julie Massart1, Mutsumi Katayama2, Anna Krook2

Section of Integrative Physiology, Karolinska Institutet, 171 76 Stockholm, Sweden

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Anna Krook Department of Physiology and Pharmacology Section for Integrative Physiology Karolinska Institutet von Eulers väg 4a Stockholm, Sweden E-mail: [email protected]

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Corresponding Author:

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Abstract

MicroRNAs have been described as important regulators of skeletal muscle differentiation

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and development, but the role of microRNAs in glucose and lipid metabolism is less well established. Here we will review the microRNAs involved in insulin resistance and glucose metabolism, as well as microRNAs regulating lipid metabolism and mitochondrial functions in skeletal muscle, with an emphasis on metabolic disorders such as type 2 diabetes and the adaptive response to exercise training. Finally, we will raise some methodological considerations for studying microRNAs, as well as challenges investigators may face when elucidating the direct role of microRNAs in the regulation of glucose and lipid metabolism in skeletal muscle.

Keywords: microRNA; skeletal muscle; type 2 diabetes; metabolism; exercise

Abbreviations: GK, Goto-Kakizaki; HFD, high fat diet; HK2, hexokinase 2; miRNA, microRNA; PGC1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha;

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ACCEPTED MANUSCRIPT RISC, RNA-induced silencing complex; STZ, streptozotocin; T2D, Type 2 Diabetes; TNF,

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tumor necrosis factor alpha; UTR, untranslated region

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ACCEPTED MANUSCRIPT 1. Introduction Skeletal muscle represents about half of the total body mass in healthy people and is therefore quantitatively the largest insulin sensitive organ in the body, constituting a major

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contributor to whole body metabolism [1]. Skeletal muscle is a metabolically flexible organ and

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is able to shift between carbohydrate and lipid utilization. Muscle contraction and physical activity is associated with increased substrate metabolism and energy expenditure, as well as

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enhanced insulin sensitivity.

Type 2 diabetes (T2D) is a major health issue and has now reached epidemic proportions

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worldwide. Diabetes mellitus is a progressive metabolic disease characterized by insulin resistance combined with defect in insulin secretion by the pancreatic -cells, causing chronic

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hyperglycemia. One of the primary events in the development of insulin resistance is metabolic inflexibility, as evidenced by a reduced capacity of skeletal muscle to burn fat in response to

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fasting [1]. In skeletal muscle, insulin resistance is characterized by impaired insulin-stimulated glucose uptake and glucose utilization [1]. The development of insulin resistance in skeletal

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muscle remains not fully understood, increased intramuscular fat deposition as well as fatty acid

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intermediates, such as diacylglycerol and ceramides, have been associated with the development of insulin resistance [2]. Mitochondrial functions have also been reported to be decreased in insulin resistant skeletal muscle [3]. Exercise training has beneficial effects on insulin resistance, increasing insulin sensitivity and mitochondrial functions. However, the precise mechanisms by which exercise training benefits to T2D patients are still not fully understood. Several lines of evidence suggest an important role for microRNAs (miRNAs) in skeletal muscle development and hypertrophy (for review see [4]), however, much less is known regarding miRNA regulation of glucose and lipid metabolism in skeletal muscle. In this respect, miRNAs may fine tune the expression of networks of genes that control glucose and lipid metabolism in health and disease. In skeletal muscle, the role of miRNAs in glucose and lipid metabolism is less well established than in other insulin sensitive tissues and little is known on regulation of miRNAs expression. Here we will review miRNAs involved in glucose homeostasis and lipid metabolism in skeletal muscle, with an emphasis on metabolic disorders such as type 2 diabetes and the adaptive response to exercise training. Additionally we will raise some methodological considerations for studying miRNAs, as well as challenges investigators may 3

ACCEPTED MANUSCRIPT face when elucidating the direct role of miRNAs in the regulation of glucose and lipid metabolism in skeletal muscle.

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2. MicroRNAs biogenesis and regulation MicroRNAs (miRNAs) are short, non-coding RNA molecules regulating gene expression

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post-transcriptionally. miRNAs play crucial roles during various biological processes, from

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embryonic development to glucose homeostasis. miRNAs are evolutionary conserved, with at least 1800 human transcripts identified to date. While the total number of miRNAs in the genome

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is as yet unclear [5], miRNAs may exert posttranscriptional control on up to 30% of all genes [6]. One challenge when considering miRNA-mediated regulation of gene expression is the fact that one miRNA can target hundreds of genes, while at the same time, one transcript may be targeted

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by several miRNAs, highlighting the complexity and also the adaptive capacity of miRNA regulatory networks.

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The biogenesis of miRNA can be divided into two steps, based on the cellular compartment

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in which it occurs. The miRNA gene is first transcribed in the nucleus as primary miRNA (primiRNA) that is next cleaved to a precursor miRNA (pre-miRNA). The second step consists of

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the export of the pre-miRNA to the cytoplasm where it can be cleaved by Dicer, an RNAse III enzyme, as a miRNA duplex. Both miRNA strand of the duplex can act as a functional mature miRNA, and and incorporated into the RNA-induced silencing complex (RISC) where miRNA and target mRNA interact [7]. The miRNA / mRNA interaction can occur in several different ways. The canonical interaction is characterized by binding of the 5’ part of the miRNA, either completely or partially, to complementary sequences located in the 3′ untranslated region (UTR) of target mRNAs. In addition to binding with the 5’ region, pairing of the miRNA to only 3 to 4 nucleotides within the 3’ part of miRNA occurs. This 3’-supplementary site usually has a less profound effect on target recognition. There is also possible mismatch in the seed region, which is compensated by additional pairing in the 3’ part of miRNA, usually referred as 3’ compensatory site [8]. More recently, miRNAs binding within the 5’-UTR of the target mRNA has been associated with mRNA stabilization and enhancement of translation, demonstrating the regulatory potential of miRNAs [9, 10].Moreover, miRNA biogenesis is subjected to fine-tuning regulation at various level, such as pri-miRNA processing by RNA-binding proteins such as Lin28, as well as RNA editing, methylation and adenylation [11]. 4

ACCEPTED MANUSCRIPT 3. miRNAs and glucose homeostasis 3.1 Type 2 diabetes human skeletal muscle miRNA profiling

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miRNAs expression profiles of adipose tissue, pancreas or liver from insulin resistant patients or animal models of type 2 diabetes (T2D) have been derived. Nevertheless, only a few

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studies have focused on skeletal muscle. A global microarray-based miRNA expression analysis

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of skeletal muscle from healthy people with normal glucose tolerance, individuals with impaired glucose tolerance and T2D patients revealed that miRNA levels are altered with disease, despite

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no significant changes in the global mRNA transcriptome [12]. Among the 171 miRNAs found to be expressed in human skeletal muscle, 29 were up-regulated and 33 down-regulated in skeletal muscle from T2D patients. Most of the miRNAs were dysregulated with disease progression (i.e.

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when comparing from impaired glucose tolerance to manifest T2D), with ~15% of the dysregulated miRNAs altered in people with impaired glucose tolerance and only a minority

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additionally altered in type 2 diabetes. Overall, 30% of the expressed miRNAs are dysregulated

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in skeletal muscle from people with T2D, suggesting an important role for miRNAs in the development of skeletal muscle insulin resistance. When comparing skeletal muscle from

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monozygotic twins discordant for T2D, 20 miRNAs were significantly reduced in the twin with T2D, with several of the miRNAs from the miR-15 family being represented [13]. Changes in miRNA could be adaptive or pathologic, however, the specific roles of most of the identified dysregulated miRNAs remain to be assessed. 3.2 Rodent insulin-resistant skeletal muscle miRNA profiling In rodent models of obesity and diabetes, miRNA expression profiles have been determined in skeletal muscle of Goto-Kakizaki (GK) rats, a non-obese spontaneous T2D rat model [14-16], rats rendered diabetic by combination of high fat diet fed and low dose of streptozotocin (HFD/STZ) [17], as well as high fat diet fed mice (HFD) [18, 19]. The differentially expressed miRNAs found to be dysregulated in skeletal muscle of these diabetic animal models are listed in table 1. As the studies used not only different microarray technologies, but also different analysis methods, direct comparison is challenging. More importantly, only few studies specifically report the actual skeletal muscle type investigated, for example, soleus [16] and gastrocnemius [18, 19], with other studies omitting to describe which skeletal muscle was chosen (Table 1) [14, 15, 17]. 5

ACCEPTED MANUSCRIPT The specific skeletal muscle investigated is likely to play a role in miRNA abundance. Previous work has revealed that soleus is the most similar to human skeletal muscle with respect to the global transcriptome, in term of expressed genes [20]. When analyzing the different expression

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patterns of miRNA in skeletal muscle from people with T2D versus rodent models of the disease, 8 upregulated miRNAs found in T2D patients were also found to be upregulated in at least one of

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the rodent arrays. Similarly 16 of the downregulated miRNAs were recapitulated in rodent

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studies (Table 2). Interestingly, two thirds of the commonly regulated miRNAs have been identified in mice rendered insulin resistant in response to a high fat diet. However, some of these

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miRNAs have differential expression depending on the model. For example, miR-99a and miR100, which were found downregulated in T2D patients and HFD mice, were upregulated in

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HFD/STZ rats (Table 2), suggesting that the etiology of the insulin resistance might influence the miRNAs signature. As yet, only few of the listed miRNAs have been studied in vitro and/or in vivo and their targets remain poorly characterized.

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3.3 MyomiRs and Type 2 Diabetes

MyomiR, a term given to a number of miRNAs identified as highly enriched or selectively

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expressed in skeletal and cardiac muscle, play important roles in development and differentiation [21]. In particular miR-1, miR-133a/b and miR-206 are important for skeletal muscle differentiation [22, 23]. In skeletal muscle from T2D patients, miR-133a and miR-206 were downregulated as compared to normal glucose tolerant people and this reduction was correlated with fasting glucose values [12], although the reduction in miR-133a was not observed in a separate cohort [24]. Downregulation of miR-206 and miR-133 has also been reported in skeletal muscle of mice following 12 weeks of high fat diet [18, 19]. In human skeletal muscle, miR-206 was also reduced after 10 days of bed rest [25]. Additionally, in contrast to miR-1 or miR-133, miR-206 expression is highly variable depending on the fiber type composition [26], suggesting that modulation of this miRNA is linked to slow-to-fast fiber type switch. To date, the metabolic consequences of dysregulation of these myomiRs under pathological conditions is unclear and the precise physiological role in mature skeletal muscle remains unknown. 3.4 miRNAs affecting insulin signaling pathway and glucose uptake

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ACCEPTED MANUSCRIPT Among the miRNAs found to be similarly regulated in human and rodents (Table 2), only a few have been functionally validated in skeletal muscle using in vivo or in in vitro systems to determine their effects on glucose or lipid metabolism and insulin sensitivity. A few of these

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miRNAs have been studied in other tissues and found to alter substrate metabolism and insulin action. Direct targeting of the insulin receptor substrate 1 (IRS-1) by miR-144, one of the miRNA

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with increased expression in T2D patient and HFD/STZ rats, has been validated by luciferase

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assay (Fig.1) [17], but the metabolic consequences in skeletal muscle not determined. In ovarian cancer cells, miR-144 directly regulated GLUT1 and overexpression was associated with

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decrease glucose uptake and glycolysis [27]. In C2C12 myotubes, IRS-1 has been proposed to be targeted by miR-128a, influencing the activity of the PI3K/Akt pathway (Fig. 1) [28]. The insulin

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receptor, INSR, has been reported to be targeted by miR-15b and overexpression of miR-15b impaired the insulin signaling cascade, as well as insulin-stimulated glycogen storage in hepatocytes [29]. Together these studies implicate that at least 3 miRNAs upregulated in insulin

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resistant skeletal muscle directly alter insulin signaling and glucose metabolism, supporting a role

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of miRNAs in the onset or the progression of the disease (Fig. 1). However, while miR-15b is upregulated in skeletal muscle from subjects with T2D as compared to healthy (unrelated) control

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subjects [12], mIR-15b was one of the most significantly down-regulated miRNA when comparing monozygotic twins discordant for T2D [13]. Whether this represents a genetic predisposition in the former case, and perhaps an adaptive response to ameliorate insulin resistance in the latter, remains to be determined. Furthermore, miR-15a/b are part of a highly evolutionary-conserved miRNAs paralogues family, whose members include miR-195, reported to be increased in HFD mice (table 1), as well as miR-424, found downregulated in human T2D and GK rats (table 2). Further investigation of the metabolic consequences of these miRNAs specifically in skeletal muscle is needed. Upregulation of miR-223 was reported in skeletal muscle of rats rendered diabetic by combination of high fat diet and low dose of streptozotocin [17], as well as in heart and adipose tissue from insulin resistant humans [30, 31]. While overexpression of miR-223 increased insulin-stimulated glucose uptake and induced GLUT4 expression in cardiomyocytes, the opposite effects have been found in adipocytes, where miR-223 negatively regulates glucose uptake and GLUT4 protein abundance [30, 31]. So far, the precise role of miR-223 in skeletal muscle remains unknown. This is an example of a tissue specific effect of the action of miRNAs, 7

ACCEPTED MANUSCRIPT perhaps due to precise interaction with other miRNAs targeting the same transcripts, which need to be considered when extrapolating the role and targets of a specific miRNA to another tissue. Genetic association studies are suggestive of a role for let-7 in T2D. Let-7 function is in part

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regulated by the miRNA binding protein Lin28. Polymorphisms in the let-7-targeted region of

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Lin28, a miRNA binding protein, is associated with increased T2D risk in Chinese population [32]. Furthermore, Inhibition of let-7 exerts an insulin sensitizing effect in skeletal muscle of

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diet-induced obesity in mouse models [33]. Among the predicted targets, let-7 can bind to INSR and IRS-2, important transducers of insulin action. Loss of function of Lin28 increases let-7

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expression and induces insulin resistance in mouse models [34]. In cultured primary myotubes derived from T2D patients, let-7a is increased when compared to healthy subjects and may play a

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role in the regulation of glucose metabolism by targeting IL-13 [35]. The H19 long non-coding RNA has also been shown to regulate the let-7 family bioavailability [36] and is reduced in human and rodent diabetic skeletal muscle [37]. Let-7, in turn, by targeting H19, reduced insulin-

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stimulated glucose uptake demonstrating a double-negative feedback loop between let-7 and H19

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highlighting the complexity in miRNAs mediated glucose metabolism regulation [37]. The functional role of the majority of the miRNAs found to be dysregulated in metabolic

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disease (Table 2) remain to be functionally characterized in skeletal muscle. However, some of the identified miRNAs have been studied in other tissues and disease states, and appear to play important roles with respect to glucose metabolism. For example, in tumor cells, miR-143 regulates glycolysis by targeting hexokinase 2 (HK2), a rate-limiting enzyme of glycolysis [38]. Experimental validation in skeletal muscle is required to assess whether these miRNAs control glucose metabolism in skeletal muscle, and if so, at what steps in the signaling cascade. 3.5 Factors regulating skeletal muscle miRNAs expression miRNA expression is modulated by different stimuli, and expression levels appear to be important for modulating their target genes under normal and pathological conditions. In skeletal muscle, saturated fatty acids, as well as systemic inflammation, participate in the development of insulin-resistance [39]. Inflammation is known to induce insulin resistance in skeletal muscle in part by inhibiting the insulin signaling pathway. In mouse C2C12 muscle cells, TNF-induced insulin resistance was characterized by an early upregulation of miR-494 and a downregulation of

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ACCEPTED MANUSCRIPT miR-101b [40]. Further characterization demonstrated that overexpression of miR-494 is sufficient to reduce insulin signaling, but inhibition was not sufficient to restore insulin action in cells treated with TNF[40]. Collectively these data suggest that miR-494 contributes to the

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development of insulin resistance, but is unlikely to be the main cause. Further characterization of miR-494 targets could uncover new networks contributing to the inflammation-induced insulin

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resistance. In parallel, let-7 promoter activity was found to be increased upon treatment with

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insulin, glucose and TNF [41] indicating that TNFis likely to coordinately activate expression of several miRNAs.

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The physiological role of acute insulin stimulation on the miRNA signature has been investigated in human skeletal muscle biopsies obtained before and after euglycemic-

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hyperinsulinemic clamp [24]. A total of 39 miRNAs were downregulated following hyperinsulinemia, including a decreased expression of the myomiRs, miR-1, miR-133a and miR206 [24], but these findings were not recapitulated in a similar study [42]. Remarkably, 4

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miRNAs (miR-95; miR-133a; miR-152; miR-27b) were also downregulated in T2D patients [12].

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Interestingly, approximately 800 mRNA transcripts are regulated in skeletal muscle by insulin [43] and more that 300 are regulated by acute hyperglycemia [44]. Therefore, further work is

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required to understand if miRNAs are important mediators of the effects of insulin and glucose on gene expression in skeletal muscle. Identifying the factors responsible of miRNAs expression modulation under normal and pathological conditions will hopefully help, in the future years, the understanding of the physiological role of miRNAs at a dynamic level. 4. miRNAs and physical exercise: the role of mitochondria The beneficial effect of physical exercise on reducing chronic disease such as type 2 diabetes is well documented [45]. Repeated exercise training enhances skeletal muscle mitochondrial function, insulin sensitivity as well as substrate oxidation [46]. Resistance exercise involving progressive overload increases skeletal muscle mass, whereas endurance exercise, consisting in prolonged low to moderate intensity activity, increases mitochondria abundance and aerobic capacity. Both types of exercise promote a number of positive adaptations in skeletal muscle. Numerous studies have explored the effects of different types of exercise on miRNAs levels in human and in mouse skeletal muscle [42, 47-50]. Additionally, a careful mapping of epigenetic

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ACCEPTED MANUSCRIPT changes in skeletal muscle from type 2 diabetic patients in response to both types of exercise has been reported, revealing a reprogramming of the molecular networks [51].

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4.1 PGC1alpha and miRNAs PGC1 is a key regulator of energy metabolism in skeletal muscle, playing an important role

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in promoting mitochondrial biogenesis, as well as glucose and lipid metabolism [52]. Exercise is

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a known inducer of PGC1 expression [53] and reduced expression of PGC1 has been reported in skeletal muscle of T2D patients coincident with decreased fatty acid oxidation concomitant

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with increased accumulation of intramuscular lipids contributing to the insulin resistance state [3]. Endurance training for 16 weeks in T2D patients resulted in increased PGC1 expression

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concomitant with increased mitochondrial enzyme activities [51]. On the contrary, endurance exercise had no effect on glucose and lipid metabolism molecular networks and was associated

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with changes in [51].

Following acute endurance exercise miR-9, -23a, -23b, and -31 were downregulated, whereas

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miR-181 and miR-29b were upregulated in human skeletal muscle [54]. Expression of miR-23a

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is also downregulated by endurance exercise in mice and negatively correlated with PGC1 expression [49]. Several lines of evidence suggest that exercise-mediated regulation of PGC1 may, in part, be dependent on miR-23a, -23b, and -31 (Fig. 2). In vitro reporter assays have demonstrated binding of miR-23a to the 3’-UTR of PGC1 [55]. Whole body transgenic mice overexpressing miR-23a displayed reduced levels of PGC1 in skeletal muscle and this was accompanied by a reduction of mitochondrial enzymes [55]. Conversely, resistance exercise in T2D patients was associated with an increase of miR-23a expression without changes in PGC1 expression [51]. Luciferase assays performed in C2C12 muscle cells confirmed that miR-31 directly targets HDAC4 and NRF1, transcriptional regulators of mitochondrial biogenesis and functions (Fig. 2) [54]. The direct targets of miR-9 in skeletal muscle have not been demonstrated, however, in pancreatic cells, miR-9 regulated SIRT1 [56], a metabolic sensor regulating PGC1 activity by deacetylation (Fig. 2). Molecular characterization of the role of miR-9 on SIRT1 expression, as well as PGC1 activity in skeletal muscle following exercise remains to be established.

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ACCEPTED MANUSCRIPT Four weeks exercise training in mice resulted in upregulation of miR-21 and downregulation of miR-696, -709 and -720 [50]. Modulation of miR-696 in C2C12 cells showed that PGC1 protein abundance is directly regulated by miR-696, concomitant with changes in PDK4 and

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COX2 expression. Of importance, this study demonstrated that reduction of PGC1 abundance by miR-696 led to the metabolic changes, as fatty acid oxidation flux was reduced [50]. In a

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similar study in mice, endurance training resulted in downregulation of miR-696 and miR-761,

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and overexpression of miR-761 in C2C12 cells reduced mitochondrial biogenesis by directly inhibiting translation of PGC1Fig. 2) [57]. To date, these miRNAs have only been studied in

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mouse models and relevance for human physiology remains to be proven.

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4.2 Mitochondrial functions and miRNAs

In healthy young men, seven days of bed rest decreases skeletal muscle mitochondrial DNA content and abundance of oxidative phosphorylation enzymes, as well as miR-1 and miR-133a

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levels [58]. In contrast, one single bout of endurance exercise was sufficient to increase miR-1

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expressions.

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and miR-133a expression [42], linking physical (in)activity with modulation of myomiR

Several miRNAs have been reported to regulate mitochondrial density and function. Interestingly, miR-494, which is upregulated in skeletal muscle from T2D patients, decreased following concurrent endurance and resistance exercise [59]. Similar changes are seen in mouse skeletal muscle following endurance exercise, while inhibition of miR-494 increased mitochondrial biogenesis [60]. In insulin resistant mice, miR-149 was downregulated, whereas its overexpression in mouse C2C12 cells increased mitochondrial biogenesis and oxidative capacities [19]. Expression of miR-106b was increased in T2D patients, as well as HFD-fed mice and its expression was induced by TNF and palmitate in cultured C2C12 cells [61, 62]. Overexpression of miR-106b in cells inhibited insulin-stimulated glucose uptake and this was accompanied with a reduced GLUT4 translocation at the plasma membrane. By direct targeting of mitofusin-2, a GTPase located in the mitochondrial outer membrane necessary for mitochondrial fusion, forced expression of miR-106b led to alteration of mitochondrial morphology and increased oxidative stress [61], mimicking some features characteristic of type 2 diabetes. Interestingly, inhibition of miR-106b increased mitochondrial abundance and alleviated

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ACCEPTED MANUSCRIPT mitochondrial dysfunction induced by TNF treatment [61]. Questions remains as to whether overexpression of miR-149 or inhibition of miR-106b alone in insulin resistant mature skeletal muscle is sufficient to maintain mitochondrial function and if so, to increase oxidative

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metabolism in type 2 diabetes.

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4.3 Fatty acids and miRNAs

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As highlighted above, several miRNAs implicated in the development type 2 diabetes have been shown to play a role in mitochondrial function and lipid metabolism in other tissues and

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disease states (Table 2). Of interest, comparing the miRNAs changed in human T2D skeletal muscle with miRNAs affected by endurance training shows that 4 miRNAs are differentially

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expressed (Fig. 3). This opposite regulation of miR-144, miR-15b, miR-451 and miR-589 in the context of T2D and exercise could implicate these miRNAs in regulation of skeletal muscle insulin sensitivity (Fig. 3). Future work will reveal the role of these miRNAs in skeletal muscle.

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miR-451, which is upregulated in skeletal muscle with T2D, has been shown to affect the

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LKB1/AMPK pathway in glioma cells [63]. AMPK is a key exercise responsive energy sensor in skeletal muscle, and the role of miR-451 on AMPK in exercised skeletal muscle will be of

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interest to understand. Additionally, whole-body deletion of miR-378 led to improved mitochondrial fatty acid metabolism and oxidative capacity [64]. Although initially counterintuitive as miR-378 is downregulated in diabetic muscle, this could reflect an adaptive response to increased fatty acid flux into the cells. 4.4 Exercise, transcription factors and miRNAs The type of exercise performed appears to affect skeletal muscle miRNA. One acute bout of resistance exercise in young healthy men increased pri-miR-206 and reduced pri-miR-1 and primiR-133, however, only miR-1 was repressed at mature level [47]. This reduction was paralleled with a decrease protein abundance of Exportin-5, involved in the transport of pre-miR from the nucleus to the cytoplasm. In contrast, one bout of endurance exercise was characterized by increased miR-1 and miR-133a/b, as well as mRNA levels of Drosha, Dicer and Exportin-5, genes involved in miRNAs biogenesis [54]. Thus exercise type might affect miRNA abundance through modulation of miRNA biogenesis and maturation. Better understanding of the effect of exercise on miRNA biogenesis may give new insight into the mechanisms mediating exercise12

ACCEPTED MANUSCRIPT induced adaptations. In mouse skeletal muscle, two exercise responsive genes, PPARα and PPARβ/δ regulate different miRNAs [65]. Moreover, ERRγ induced the expression of miR-208b and miR-499 which are involved in oxidative capacity whereas PPAR suppressed this process

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[65]. Further research is needed to determine the mediators of exercise-induced miRNAs as well

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as the precise role of miRNAs in the physiological and beneficial effects of exercise.

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5. Considerations for studying miRNAs and Future perspectives 5.1 Approaches to study miRNAs dysregulation

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In this section, we will briefly discuss some methodological considerations as well as the next challenges (for more detailed reviews, see [66, 67]). Application of classical gain/loss of miRNAs function is a popular strategy to address the biological role of a specific miRNA on the

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whole transcriptome or specific targets. Using classical target prediction algorithms, one miRNA is estimated to target hundreds of mRNA sequences. However, considering the low

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complementarity with some targets, in silico prediction is difficult and experimental validation is

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always needed [68]. The interaction between miRNA and mRNA in the RNA-induced silencing complex (RISC) can result either in mRNA degradation or translational repression. As yet, the

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mechanisms determining the degradation versus the repression remains unclear and therefore difficult to predict. In this context, large scale analysis of target mRNAs are less informative and a better approach would be identification of changes in the whole proteome rather than transcriptome. Another approach is to identify miRNAs associated with Argonaute, an important component of RISC where miRNA and target mRNA binds. A biochemical approach to determine immunoprecipitation of Argonaute allows to identify mRNA directly associated with miRNAs in a tissue-specific context [69]. Generation of such data will be of great interest also for improving computational target predictions. Identification of the direct miRNA targets is a challenge that should be overcome in the next years. 5.2 Methodological considerations for studying miRNAs The choice of RNA isolation method from cells is likely to impact results, with the classical guanidinium thiocyanate-phenol-chloroform extraction method or commercially available kits designed specifically or not for small RNA extraction being the most common choices. To date, little is known if extraction method of miRNAs results in different yields and quality of the total 13

ACCEPTED MANUSCRIPT miRNAs in different tissues [70]. Quantitation and detection of miRNA species may be performed using microarray, real-time PCR, or northern blotting. Microarray-based approaches are powerful tool to uncover disease specific dysregulated miRNAs, but compromises sensitivity

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as compared to real-time PCR or Northern blotting, in part due to the short length of miRNAs [67]. Recently, deep sequencing techniques have been employed to quantify miRNAs, and in

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comparison to microarray, allow determination of the relative abundance. The number of

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different arrays available is important and should be taken in consideration while comparing different studies. Northern blotting is still widely used for detecting mature or pre-miRNAs, but

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real-time PCR is usually preferred since it requires less RNA. However, the choice of the appropriate housekeeping genes or normalization method needs to be optimized for each species,

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tissue and disease combination since it must have a stable expression for all samples tested, and normalization alone could affect the data [71]. Altogether, the standardization of the miRNArelated technical procedures might be useful to ensure comparability and reproducibility of the

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available data.

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Bioinformatic algorithms are used to identify potential miRNAs targets and are broadly based on two different models; the rule-based or the data driven models; and it is usually

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recommended to combine results obtained with different target prediction programs [72]. To date a basic assumption is that the miRNA seed pairing is important in the target repression. However, recent studies have revealed alternative modes of miRNAs action, such as 3’ compensatory sites, bulged sites, and 3’-UTR duplex structure as well as miRNA-induced target expression [7, 9, 10]. Better insight and accumulating experimental data on miRNAs biology/functions will help to improve prediction of miRNAs targets. 5.3 Future challenges Besides being composed of different fiber types, skeletal muscle tissue includes a number of different cell types. In addition to muscle fibers, satellite cells, considered as source of myogenic cells, and highly proliferative muscle-derived fibroblasts are quantitatively the most abundant cells in skeletal muscle. Furthermore, skeletal muscle, due to its complex structure contains also connective tissue, nerves, endothelial cells, adipose tissue and others [73]. The relative proportion of each cell type may be affected by the (pathological) condition under investigation and thus affect the miRNA signature. To date, miRNA expression profiles have been characterized only 14

ACCEPTED MANUSCRIPT on whole skeletal muscle preparations and progress in single cell analysis should be considered to be used for more refined analysis. Laser capture microdissection is now a well-established tool for the isolation of a selected cell type from complex tissues. Specific expression profiles could

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be determined by real-time PCR or microarray [74]. The recent development of high-throughput RNA sequencing (RNA-seq) at the single-cell level will provide important insight [75]. Recently,

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expression data with miRNAs profiling in the same fiber.

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single muscle fiber proteomics has been achieved [76] opening the possibility to combine protein

The different modes of miRNAs-mediated mRNA degradation or stabilization are still not

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well characterized. Mature miRNAs are known to be present in the cytoplasm, but the exact localization of action can be indicative of their main mode of regulation. Using in situ

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hybridization, let-7a was mainly localized nearby processing bodies, a subcellular foci identified as sites for mRNA degradation [77]. Detection of miRNA has been possible in formalin-fixed or paraffin-embedded brain tissues using locked nucleic acid -based in situ hybridization (LNA-

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ISH), helping deciphering the cell type localization of miRNAs as well as subcellular localization

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[78]. Dynamic localization associated with its binding activity could also be an alternative and would prove the subcellular action of a miRNA. Single-molecule imaging approaches that allow

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the determination of the spatial localizations of miRNA-binding mRNA target such as errorrobust fluorescence in situ hybridization (MERFISH) [79]. Introducing labelled mature miRNA into the cells will give fluorescence signal that can be recorded in real time and modulation of miRNA activity could also be assessed. However, to date, technical issues still need to be solved and validity of the concept should be determined. 6. Concluding remarks miRNAs are now appreciated as important regulators of metabolic functions in skeletal muscle, and dysregulation of miRNA species can lead to profound impairments of glucose and lipid metabolism. However, in comparison to other metabolically active tissues such as liver, there is a paucity of data on how miRNAs affect metabolic processes in skeletal muscle, with most studies merely reporting on how miRNA abundance is altered with disease state or in response to (patho) physiological challenges. While initial studies focused on the “one miRNA: one target” concept, the integration of large scale analysis allows identification and study of the network affected by one miRNA. Additionally, since a single miRNA can act as a repressor or an 15

ACCEPTED MANUSCRIPT activator of gene expression, and that reciprocally one transcript can be submitted to both kind of regulations, more experimental data are needed to improve in silico prediction. The miRNA research field is now aiming to integrate a full network of miRNAs-targeted genes regulating

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cellular functions. This should herald further insight into the role of miRNAs in skeletal muscle

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physiology and unravel specific miRNAs role in metabolism and energy expenditure.

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Conflict of Interest Authors declare no conflict of interest.

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Acknowledgements

The work in the authors’ laboratory is supported by the Swedish Research Council, Swedish

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Diabetes Association, Diabetes Wellness Sweden, Novo Nordisk Fonden (NNF15CC0018346), Swedish Foundation for Strategic Research, the Strategic Diabetes Research Programme at

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Table 1: Summary of miRNAs significantly dysregulated in rodent diabetic models assessed by miRNAs microarrays Number Upregulated Downregulated Total Muscle Model of miRNAs miRNAs (Up/Down) samples miR-379; miR-127; miR299-5p; miR-434-3p; ? GK rats ? miR-29a/b/c; miR-150 miR-335; miR-130a; miR- 15 (4/11) 19b; miR-451; miR-148a; miR-199a; miR-152 miR-24; miR-126; miR? GK rats 2/2 miR-301; let-7f 424; miR-23a/b; miR-450; 9 (2/7) miR-130a Soleus GK rats 5/5 miR-10b; let-7e 2 (0/2) miR-29a; miR-499; miR-125b-3p; miR29b; miR-223; miR-7a; miR-182; miR-338*; miR-99a; miR-192; miR-135b; miR-146a; HFD/STZ miR-140*; miR-144; miR-487b; miR-181a; ? 6/6 32 (20/12) rats miR-129; miR-24-2*; miR-412; miR-199a-3p; miR-451; miR-320; miR-375; miR-183; miRmiR-146b; miR-10b; 208; miR-363* miR-150; miR-34a; miR-191; miR-100; miR-190; miR-99a; miR133a; miR-133b; miR10a; miR-152; miR-128; miR-125a-3p; miRmiR-206; miR-130a; miR144; miR-301a; miRHFD mouse 374; miR-208a; miRGastroc 3/3 369-3p; miR-551b; 30 (8/22) (12 weeks) 199a-5p; miR-196a; miRmiR-143; miR-106b; 331-3p; miR-126-5p; miR-15b miR-1; miR-10b; miR378; miR-15a; miR-100; miR-24; miR-23b miR-351; miR-207; miR-466; miR-324-3p; miR-484; miR-29a; miRmiR-383; miR-214; 145; miR-27a; miR-149; miR-361; miR-126-3p; miR-328; miR-712*; miR-155; miR-21; HFD mouse miR-127; miR-181a; miRGastroc 9/9 miR-467a; miR-467b; 41 (22/19) (12 weeks) 133a; miR-133b; miRmiR-26a; miR-1; miR689; miR-709; miR-25; 185; miR-195; miRmiR-762; miR-744; miR26b; miR-346; miR99a; miR-690; miR-99b 706; miR-468; miR713; miR-93 HFD: high fat diet; Gastroc: gastrocnemius; STZ: streptozotocin; GK: Goto-Kakizaki 23

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Table 2: Dysregulated miRNAs in human type 2 diabetic muscles (from Gallagher et al., [12]) overlapping with the results from the rodent microarrays. Model Targets/Pathways Comments Glut4 translocation; ↑ miR-106b ↑ HFD mouse [18] Mnf2 (mitochondrial fusion) [61] HK2 – Glycolysis (Cancer ↑ miR-143 ↑ HFD mouse [18] cells) [38] ↓ with endurance training ↑ miR-144 ↑ HFD/STZ rats [17] IRS-1 [17] in human [48] ↓ with endurance training ↑ miR-15b ↑ HFD mouse [18] INSR (Hepatocytes) [29] in human [48] SREBP-2 - Cholesterol ↑ miR-185 ↑ HFD mouse [19] biosynthesis (Hepatocytes) [80] ↑ miR-320 ↑ HFD/STZ rats [17] PFKm [81] ↓ in GK rats [14] LKB1/AMPK pathway ↑ miR-451 ↑ HFD/STZ rats [17] ↓ with endurance training (Glioma cells) [63] in human [48] ↑ miR-93 ↑ HFD mouse [19] ↓ miR-100 ↓ HFD mouse [18] ↑ in HFD/STZ rats [17] ↓ miR-10a ↓ HFD mouse [18] ↓ miR-10b ↓ GK rats [16] ↑ in HFD/STZ rats [17] ↓ miR-126 ↓ GK rats [15] ↓ miR-128a ↓ HFD mouse [18] ↑ after acute exercise in ↓ miR-133a ↓ HFD mouse [18] human [42, 54] ↓ miR-152 ↓ GK rats [14] ↓ miR-15a ↓ HFD mouse [18] ↓ miR-190 ↓ HFD mouse [18] ↓ miR-196a ↓ HFD mouse [18] ↓ miR-199a-3p ↓ HFD/STZ rats [17] ↓ miR-208a ↓ HFD mouse [18] ↓ miR-331-3p ↓ HFD mouse [18] Fatty acid metabolism; ↓ miR-378 ↓ HFD mouse [18] Oxidative capacities [64] ↓ miR-424 ↓ GK rats [15] ↓ miR-99a ↓ HFD mouse [18] ↑ in HFD/STZ rats [17] HFD: high fat diet; STZ: streptozotocin; GK: Goto-Kakizaki

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ACCEPTED MANUSCRIPT Legend to figure 1 Regulation of skeletal muscle metabolism by miRNAs. Several miRNAs have been shown to affect insulin sensitivity through targeting of key insulin signaling mediators. Insulin receptor

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(INSR) is a direct target of miR-15b and let-7; IRS-1 is repressed by miR-128 and let-7; and miR-106b inhibited insulin-stimulated glucose uptake with a reduced GLUT4 translocation. In

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different systems than skeletal muscle, miR-15b reduced insulin-stimulated glycogen synthesis;

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miR-144 reduced glucose uptake and glycolysis; and miR-143 downregulated hexokinase 2 (HK2) which resulted in decreased glycolysis. Collectively, these data suggest an implication of

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these miRNAs in glucose utilization. Furthermore, mitochondrial functions are modulated by miR-378 implicated in mitochondrial fatty acid metabolism and oxidative capacity; and miR-

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106b targets mitofusin-2 (Mfn2) involved in mitochondrial fusion and mitochondrial abundance.

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Regulation of PGC1alpha expression and activity by miRNAs. miR-23a, miR-696,and miR-

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761 have been shown to directly target PGC1. Others miRNAs influenced PGC1 activity by

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affecting regulators expression. miR-451 targets AMPK which phosphorylates PGC1; miR-9 reduces SIRT1 abundance, leading to decreased deacetylation of PGC1 and therefore reduced transcriptional activity. NRF1, a PGC1 co-factor, is targeted by miR-31; highlighting the importance of miRNA in the regulation of PGC1. Alteration of PGC1 abundance or activity led to change in glucose and fatty acid metabolism as well as mitochondrial functions and abundance.

Legend to figure 3 Differential regulation of miRNAs in T2D and endurance training in human skeletal muscle. Four miRNAs, miR-144, miR-15b, miR-451 and miR-589, are upregulated in insulin resistant skeletal muscle from subjects with T2D, and their expression is decreased upon endurance exercise training [12, 48], suggesting an important role in skeletal muscle metabolism. However, the role of these miRNAs on skeletal muscle functions remains to be determined.

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