MiR-206 is expressed in pancreatic islets and regulates glucokinase activity

Glucose homeostasis is a complex indispensable process, and its dysregulation causes hyperglycemia and type 2 diabetes mellitus. Glucokinase (GK) takes a central role in these pathways and is thus rate limiting for glucose-stimulated insulin secretion (GSIS) from pancreatic islets. Several reports have described the transcriptional regulation of Gck mRNA, whereas its posttranscriptional mechanisms of regulation, especially those involving microRNAs (miR), are poorly understood. In this study, we investigated the role of miR-206 as a posttranscriptional regulator of Gck. In addition, we examined the effects of miR-206 on glucose tolerance, GSIS, and gene expression in control and germ line miR-206 knockout (KO) mice fed either with chow or high-fat diet (HFD). MiR-206 was found in Gck-expressing tissues and was differentially altered in response to HFD feeding. Pancreatic islets showed the most profound induction in the expression of miR-206 in response to HFD. Chow- and HFD-fed miR-206KO mice have improved glucose tolerance and GSIS but unaltered insulin sensitivity. In silico analysis of Gck mRNA revealed a conserved 8-mer miR-206 binding site. Hence, the predicted regulation of Gck by miR-206 was confirmed in reporter and GK activity assays. Concomitant with increased GK activity, miR-206KO mice had elevated liver glycogen content and plasma lactate concentrations. Our findings revealed a novel mechanism of posttranscriptional regulation of Gck by miR-206 and underline the crucial role of pancreatic islet miR-206 in the regulation of whole body glucose homeostasis in a murine model that mimics the metabolic syndrome.


Keywords
miR-206; glucose sensing; insulin sensitivity; glucose tolerance; pancreatic islets A surfeit of nutrients combined with a sedentary lifestyle has resulted in an alarming increase in the rate of obesity-associated diseases such as type 2 diabetes (T2DM) and metabolic syndromes (30). T2DM is characterized by hyperglycemia owing to inadequate amounts of glucose-stimulated insulin secretion (GSIS) combined with profound peripheral insulin resistance. T2DM is also a major trigger for cardiovascular diseases (18). Genomewide association and candidate gene studies have identified more than 50 loci associated with T2DMand obesity (19) that underline the complex nature of glucose homeostasis. However, such approaches are largely exclusive to the coding regions of the genome, and regulatory influences of the noncoding genomic regions on disease outcomes are neglected.
Maintenance of glucose homeostasis is mediated by a concerted action involving multiple regulatory mechanisms. More recently, the role of noncoding RNAs, especially micro-RNAs (miRs), in regulating several steps of glucose metabolism has been discovered (24). Various miRs are known to influence multiple aspects of whole body glucose homeostasis and insulin sensitivity. GSIS, one of the most crucial steps in glucose utilization, is also subject to such fine-tuning via β-cell-specific miRs (7). Impaired GSIS is essentially the key to βcell failure and T2DM, with aberrant miR profiles being implicated as an important etiological factor.
The function of pancreatic β-cells is facilitated by the rate-limiting glucose sensor glucokinase (GK) and the low-affinity glucose transporter GLUT2. GK is expressed in liver and various neuroendocrine cells such as pancreatic islets, small intestine, and brain (14,23). The most intriguing evidence supporting the GK-glucose sensor concept arises from the functional consequences of Gck mutations in humans. Most of these mutations manifest as autosomal dominant inherited syndromes, including Gck-linked permanent neonatal diabetes, mature onset diabetes of the young, and others (8). Loss of Gck in rodents leads to severe hyperglycemia and further emphasizes the role of GK in the regulation of glucose homeostasis (17).
The expression levels of GK in the liver and pancreatic islets are stringently controlled by tissue-specific promoters and pathways triggered by insulin and glucagon (3). Binding sites for PPARγ, RXRα, SREBP1c, PDX-1, and HNFs have been identified in the corresponding promoter. More recently, additional transcriptional factors such as LRH-1 and BETA2/ NeuroD1 have been reported to induce the expression of GK in a tissue-specific manner (15,20). GK is also posttranscriptionally regulated by a high-affinity GK regulatory protein (GKRP), which undergoes fructose phosphate and glucose-mediated shuttling between the nucleus and the cytoplasm (9,10). In addition, GK activity is posttranscriptionally controlled by various mechanisms (6,21).
Several miRs have predicted binding sites in the 3′-untranslated region (3′-UTR) of Gck. However, direct evidence of a miR that targets Gck mRNA is lacking. Here, we provide evidence that miR-206 downregulates the expression of Gck by specifically targeting the 3′-UTR of Gck mRNA. miR-206 is expressed in multiple tissues in mice, including pancreatic islets. The transcript levels of miR-206 are upregulated in islets and liver of high-fat diet (HFD)-induced diabetic wild-type mice compared with chow-fed controls. A resistance to impaired glucose metabolism in response to a HFD was observed in miR206-knockout mice. The improvements included improved glucose and insulin tolerance, increased insulin secretion, higher Gck mRNA expression in islets, and increased GK activity in pancreas and liver.

Animals and diet
All animal experiments were carried out in accordance with the guidelines set by the Division of Genetic Engineering and Animal Experiments and were approved by the Austrian Federal Ministry of Science, Research, and Economy (Vienna, Austria). Mice used in this study were all on a 29SvEv-C57BL/6 mixed background. MiR-206-knockout (miR-206KO) mice were originally obtained from Dr. Eric N. Olson's laboratory (Department of Molecular Biology, University of Texas Southwestern Medical Center). All animals were fed ad libitum with normal chow (NC) diet (11.9% calories from fat; Ssniff, Soest, Germany) and maintained in a 12:12-h light-dark cycle in a temperature-controlled environment. To study diet-induced changes, individually housed male miR-206KO mice and control littermates (aged 5 wk) were fed a high-fat/high cholesterol diet (HFD) (D12492 sniff; 60% calories from fat, 21% calories from carbohydrates, and 19% calories from protein; in addition, we added 1 g of cholesterol to 99 g of dry chow). Food intake was measured every 2 wk during the feeding period and was calculated as grams per day per mouse.

Murine islet isolation
Murine islets were isolated from age-and sex-matched control and miR-206KO mice, as described (31). Briefly, mice were euthanized by cervical dislocation. The common bile duct was cannulated using a 27G needle, and 2 ml of ice-cold Liberase II (Roche Diagnostics, Basel, Switzerland) was injected immediately. The perfused pancreas was dislodged from the intestine, spleen, and stomach. To facilitate complete digestion, the pancreas was further incubated in a water bath preheated at 37°C for predetermined time intervals (batch variation, average time 12 min and 30 s). After digestion, islets were purified by filtration and gradient separation. Islets were maintained in RPMI (GIBCO; Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum and 100 U/ml penicillinstreptomycin (GIBCO) for 24 h, and healthy islets were hand-picked for experiments.

Quantitative real-time PCR analysis
Quantitative real-time PCR was carried out as described earlier (28). Data were analyzed using the open access software Relative Expression Software Tool -REST 2010 (http:// www.gene-quantification.com/download.html) (22).

Glucokinase activity assay
GK activity was measured in liver and islets obtained from control and miR-206KO mice. One hundred milligrams of liver tissue was homogenized in RIPA buffer and centrifuged at 13,000 g at 4°C. The supernatant was used for measuring GK activity. Islet GK activity was determined from ~100 hand-picked islets isolated from control and miR-206KO mice. The islets were washed in PBS, lysed in RIPA buffer, sonicated, and centrifuged. The supernatant was used to analyze GK activity as described (4).

In vivo GSIS
To quantify the amount of insulin secreted into the plasma in response to glucose, a solution of D-glucose(2 g/kg body wt) was injected intraperitoneally into control and miR-206KO mice. Blood was collected from the retro-bulbar plexus 0, 15, 30, and 60 min after the injection. Plasma was isolated and analyzed for the amount of secreted insulin.

Microarray meta-analysis
We meta-analyzed gene expression profiles of diabetic vs. nondiabetic human islets using a publicly available data set (accession no. GDS3882) (5). Meta-analyses were carried out using the freely available software package BRB-Array Tools version 4.3.2 that was developed under the direction of R. Simon at the Biometric Research Branch, National Cancer Institute (http://linus. nci.nih.gov/BRB-ArrayTools.html). A list of predicted miR-206 target genes was generated from data obtained from Targetscan (http:// www.targetscan.org/). The microarray data sets were normalized to exclude threshold intensities of <10, with the median array as reference. All genes with >50% missing replicate values on each array were excluded from all analyses. All data obtained were sorted and compared using Microsoft Excel. Scatterplots were generated using BRB Array Tools, and the highlighting gene set function within Array Tools was used to discriminate miR-206 target genes.

Intraperitoneal glucose tolerance test
Intraperitoneal glucose tolerance test (IPGTT) was carried out as described (11). Prior to IPGTT being performed, the animals were starved overnight (12 h), and a solution of Dglucose (2 g/kg body wt) was injected intraperitoneally into control and miR-206KO mice. Blood was drawn from the tail vein for glucose measurements at the designated time points.

Insulin tolerance test
To compare the insulin sensitivity in randomly fed control and miR-206KO mice an insulin tolerance test (ITT) was performed as described earlier (11).

Quantification of pancreatic insulin content
Pancreatic insulin content was measured from 12-wk-old male NC or HFD-fed control and miR-206KO mice after overnight fasting. Posteuthanization, pancreata were isolated, weighed, and homogenized in acid-ethanol solution (1.5% HCl in 70% ethanol) overnight at −20°C, followed by neutralization with 1 M Tris, pH 7.5. Insulin levels were quantified by ELISA (Mercodia, Uppsala, Sweden) according to the manufacturer's protocol. Insulin Vinod  levels were normalized to pancreatic weight. Insulin concentrations in plasma samples after GSIS and randomly fed or fasted control and miR-206KO mice were determined using the same ELISA.

Luciferase reporter assays
The wild-type Gck-3′-UTR with intact miR-206 seed sequence and the Gck-3′-UTR with scrambled miR-206 seed sequence were cloned downstream of a luciferase reporter gene into the pMIR-REPORT-miR expression reporter construct (Ambion, Carlsbad, CA) and βgal expression vector and cotransfected with the miR-206-expressing vector into COS-7 cells using Fugene HD (Roche, Basel, Switzerland) according to the manufacturer's protocol. The relative luciferase activity was measured using the luciferase assay system (Promega, Madison, WI). β-Gal activity was measured using the β-Galactosidase Enzyme Assay System (Promega). The luciferase activity in each sample was normalized to β-gal activity to obtain relative luciferase units (RLU). %RLU in samples transfected with the wild-type Gck-3′-UTR was calculated to the scrambled Gck-3′UTR.

Biochemical assays
Liver glycogen measurements were performed according to Adamo and Graham (1). All protein concentrations were measured using a standard BCA technique. Plasma lactate from HFD-fed control and miR-206KO mice was determined using lactate colorimetric assay kit (BioVision, San Francisco, CA) according to the manufacturer's instructions.

Statistics
For IPGTT, ITT, and GSIS experiments, differences between the control and miR-206KO groups were compared using the two-parameter analysis of variance (2-way ANOVA). Differences in glycogen and lactate content and GK activity were calculated using a twotailed, unpaired Student's t-test. All statistical analyses were performed using GraphPad

Results
miR-206 is expressed in multiple murine tissues and impacts levels of glycolytic transcripts.
Conventionally, miR-206 is considered a muscle-specific miR. We also found the highest expression of miR-206 in muscle, yet the expression was not restricted to it. Relative to muscle, miR-206 expression was 44% in islets, 31% in intestine, and 17% in brain, but only 3.7% in the liver (Fig. 1A). A recent report on the regulation of glycolytic genes by miR-206 prompted us to investigate the relative transcript levels of genes involved in glucose homeostasis in the muscle, islets, intestine, and liver of miR-206KO (25). In miR-206knockout muscles, transcripts of important glycolytic genes such as glucose-6-phosphate dehydrogenase (G6pdh) and glucose transporter 1 (Glut1) were increased by 1.8-and 6.0fold, respectively (Fig. 1B), whereas hexokinase 2 (Hk2) mRNA expression was comparable in miR-206KO and control mice.

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In miR-206KO islets, mRNA expression of G6pdh, Gck, and Glut2 was elevated by 3.4-, 13.6-, and 17.0-fold, respectively (Fig. 1C). In the intestine, the transcript levels of G6pdh, Gck, and Glut1 were increased in miR-206KO mice by 8.7-, 2.0-, and 2.6-fold, respectively, compared with controls ( Fig. 1D). On the chow diet, hepatic transcript levels of G6pdh, Gck, and Glut2 were comparable in miR-206KO and control livers (Fig. 1E), which was probably due to low hepatic miR-206 expression. These data indicate that miR-206 may be involved in regulating glycolytic gene expression in tissues with significant miR-206 abundance.

Genetic deletion of miR-206 improves glucose tolerance and GSIS
To investigate the physiological impact of the changes in glycolytic gene expression, we characterized in vivo glucose handling in miR-206KO and control mice. MiR-206KO mice showed significantly lower plasma glucose excursions 30 and 60 min post-glucose injection and a significant reduction in AUC, indicating increased glucose tolerance (Fig. 2, A and B).
We hypothesized that due to induction in islet Gck mRNA, miR-206KO mice may have an increased insulin secretion. Measurement of GSIS revealed that miR-206KO mice secreted more insulin 15 and 30 min post glucose injection by 22% and 20% compared with controls. This corresponded to an increase of 13% in area under the curve (AUC; Fig. 2D). miR-206KO mice had reduced body weight (Fig. 2E), whereas fasting glucose levels were comparable between control and miR-206KO mice (Fig. 2F). The observed increase in insulin secretion was independent of the total insulin content in miR-206KO pancreas (Fig.  2G). These findings indicate that miR-206 does not affect the regulatory mechanism of insulin production but modulates insulin secretion.

In human type 2 diabetic islets the majority of dysregulated miR-206 predicted targets are repressed
We meta-analyzed publically available microarray data to identify differences in gene expression profiles of predicted miR-206 targets in T2DM vs. nondiabetic islets. Analyses revealed that only a small proportion of the predicted miR-206 targets (17%) were differently regulated in T2DM compared with nondiabetic islets. However, a staggering 90% of these 17% genes were down-regulated in T2DM islets (Tables 1 and 2). This finding indirectly implies that miR-206 may be upregulated in the islets of diabetic individuals (Fig.  3, A and B).

miR-206KO mice are protected from high-fat diet-induced impairment in systemic glucose homeostasis
In view of the results from meta-analysis and the improved GSIS in miR-206KO mice, we next investigated the role of miR-206 in a murine model of metabolic syndrome. Since it is known that high-fat diet (HFD) alters the expression of glycolytic genes and induces a metabolic syndrome in mice, we challenged miR-206KO and control mice with such a diet. In fact, glucose tolerance was significantly greater in HFD-fed miR-206KO compared with control mice 15, 30, and 60 min post-glucose injection, with a 34% lower AUC (Fig. 4, A and B). Independent of the genotype, the maximum reduction in plasma glucose during ITT was 55% in HFD-fed mice (Fig. 4C), whereas chow diet-fed animals achieved 77% (Fig. 2C). This result indicates overall insulin resistance in response to HFD. However, the insulin tolerance was comparable between HFD-fed miR-206KO and control mice.
The 20-wk HFD-feeding paradigm lowered insulin levels in both genotypes. We failed to induce a significant insulin response to ip injected glucose in HFD-fed control mice. Twoway ANOVA did not yield significance in insulin released by the two genotypes. However, relative to baseline, miR-206KO mice showed a significant threefold induction (unpaired ttest, P = 0.048) in insulin secretion 15 min post-ip glucose injection compared with relative secretion in controls at the same time point (Fig. 4D). Notably, the overall AUC for the GSIS in miR-206KO was increased significantly by 2.3-fold (Fig. 4D). Body weight (Fig. 4E) and fasting glucose concentrations (Fig. 4F) were markedly reduced in miR-206KO compared with control mice after HFD feeding. However, compared with chow diet-fed mice, pancreatic insulin content was similar in both genotypes (Fig. 4G). Circulating plasma insulin levels in randomly fed control and miR-206KO mice were not significantly different (Fig. 4H). Thus, increased GSIS in miR-206KO mice is independent of insulin content in the pancreas, indicating that another mechanism is operative in miR-206KO mice.

High-fat diet differentially regulates the expression of mir-206 and glycolytic genes in different tissues
Next, we elucidated the impact of miR-206 deletion on changes in gene expression stimulated by HFD in multiple tissues. Interestingly, miR-206 expression itself was subject to regulation by HFD and was upregulated by 31-, 6.2-, and 1.8-fold in islets, brain, and liver, respectively, compared with the expression in chow diet-fed controls. In contrast, HFD feeding resulted in repression of miR-206 transcripts by 76 and 69% in skeletal muscle and intestine, respectively (Fig. 5A).
Islets showed the most profound induction in the expression of miR-206 in response to HFD (Fig. 5A). Consistent with what we observed in chow diet-fed mice, transcript levels of G6pdh, Gck, and Glut2 were elevated by 3.8-, 12.5-, and 2.3-fold, respectively (Fig. 5B).
Due to the low expression of miR-206 in the liver, we expected only marginal effects of miR-206 in regulating hepatic glycolytic genes. mRNA expression of hepatic Gck and Glut2 were unaltered, whereas G6pdh transcript levels were 2.4-fold higher in miR-206KO compared with control mice (Fig. 5C). Interestingly, unlike in islets, intestinal miR-206 expression was repressed upon HFD. Therefore, we tested whether expression of glycolytic genes varies inversely with respect to miR-206 levels. Transcript levels of intestinal Gck were upregulated 2.1-fold in HFD-fed miR-206KO mice compared with controls (Fig. 5D), whereas G6pdh and Glut1 mRNA expression were unchanged (Fig. 5D). In muscle, HFDfeeding resulted in a 1.8-fold increase in G6pdh mRNA expression, whereas hexokinase 2 (Hk2) and Glut1 levels were comparable between both genotypes (Fig. 5E).

Gck is a direct target of miR-206, and genetic deletion of miR-206 affects GK activity in pancreatic islets
Our results have shown so far that Gck mRNA expression varied most consistently, in accordance with miR-206 transcript levels. Thus, we searched for posttranscriptional mechanisms of miR-206-mediated targeting of Gck by in silico analysis. TargetScan analysis of Gck mRNA revealed a conserved 8-mer target site for miR-206 with a favorable binding energy of (−) 31.0 kcal/mol (Fig. 6A). We next performed luciferase reporter assays in COS-7 cells cotransfected with a miR-206 expression plasmid and the Gck-3′-UTR clone containing the wild-type miR-206 seed sequence or a control Gck-3′-UTR clone containing a scrambled miR-206 seed sequence. The results revealed a 52% decrease in luciferase activity in the Gck-3′UTR with intact miR-206 seed sequence compared with the control clone with scrambled miR-206 seed sequence (Fig. 6B).
To functionally validate this observation, we measured GK enzyme activity in pancreatic islets and liver homogenates. GK activity was increased 2.3-fold in miR-206KO compared with control islets (Fig. 6C). GK activity was also 1.5-fold higher in miR-206KO liver compared with controls (Fig. 6D). An increase in hepatic GK activity directly translates into increased liver glycogen. As predicted, HFD-fed miR-206KO livers showed a threefold increase in glycogen content compared with HFD-fed controls (Fig. 6E). Plasma lactate levels are a covariant with systemic GK activity (26) and were 2.2-fold higher in miR-206KO mice (Fig. 6F).

Discussion
The principle findings of our study are as follows: 1) miR-206 is expressed not only in muscle but also in islets, brain, intestine, and liver, and its expression is tissue specifically altered in response to HFD; 2) miR-206 targets Gck and represses its activity; 3) loss of miR-206 affects important glycolytic genes involved in maintaining systemic glucose homeostasis; and 4) miR-206KO mice have improved glucose tolerance due to an increase in GK activity and insulin secretory capacity in response to glucose.
miRs have emerged as key posttranscriptional regulators of the majority of human genes; they respond to physiological and pathological stress and function mainly to maintain the systemic homeostatic balance (2). Pancreatic β-cells and insulin-sensitive tissues express a distinct set of miRs, and an increasing body of evidence illustrates the role of miRs in glucose homeostasis (7). Several miRs such as miR-7a, miR-29a/b/c, miR-30d, and miR-338p are implicated in pathological processes such as inflammation-mediated β-cell apoptosis, impaired insulin secretion, and insulin production (12,24,29).
Very recently, Singh et. al. (25) reported the reprogramming of glucose metabolism in cancer cells mediated by sustained activation of nuclear factor erythroid-2-related factor 2 (NRF2). NRF2 represses miR-206 expression, resulting in increased expression of its target genes involved in the pentose phosphate pathway, tricarboxylic acid cycle, and fatty acid synthesis. Since cancer cells predominantly express hexoki-nase, the regulation of Gck in this context was not studied. Recent studies have also shown that dietary metabolites influence the expression of β-cell miRs, which in turn orchestrate the changes in gene expression of several targets that influence β -cell function.
Here, we demonstrate that HFD leads to differential regulation of miR-206 in a tissuespecific manner. We describe a previously unknown role of miR-206 in the regulation of glucose homeostasis by modifying expression and activity of the key glycolytic enzyme GK. Glucose sensing in the pancreas is vital for maintaining physiological plasma concentrations of glucose, and β-cell GK has been recognized as a glucose sensor of paramount importance. In hepatocytes, GK serves as a mediator of glucose clearance and catalyzes and regulates important steps in glycogen biosynthesis.
Liver GK activity serves as a cardinal metabolic switch between fed and fasted states of carbohydrate metabolism. During the fed state, increased insulin-mediated uptake and phosphorylation of circulating glucose into liver by GK facilitate the storage of glucose as glycogen. Although the expression of hepatic Gck was only marginally influenced by miR-206, GK activity was increased in the liver of miR-206KO mice, leading to higher hepatic glycogen content. This increase in liver glycogen of HFD-fed miR-206KO mice may also be attributed to increased G6pdh mRNA expression in miR-206KO liver.
Murine Gck mRNA has two transcript variant isoforms, 1 and 2, with different 5′-UTR regions and identical 3′-UTR regions. Gck transcript variant 1 is expressed in pancreatic βcells, whereas transcript variant 2 is expressed in the liver. In silico predictions revealed that miR-206 could putatively bind to both transcript variants and regulate GK repression via its target site in the Gck 3′-UTR. We validated the functionality of the predicted miR-206 target site in the 3′-UTR region of the Gck by miR-206-reporter luciferase assay. The luciferase activity in cells transfected with the Gck-3′-UTR clone containing the wild-type miR-206 seed sequence was halved compared with that transfected with Gck-3′-UTR clone with the selectively scrambled miR-206 seed sequence. This confirmed the sequence specificity for miR-206-mediated regulation of Gck. In addition, our findings of unaltered insulin content in pancreata of miR-206KO mice demonstrate that increased GSIS in miR-206KO mice is independent of insulin synthesis and regulated by GK activity, which controls intracellular ATP production via glucose 6-phosphate availability for glycolysis and K + /Ca 2+ increase. ATP inhibits the K + ATP channel, leading to a depolarization event causing increased intracellular Ca 2+ influx, which then directly activates the synaptotagmin 5 and synaptotagmin-like 4 proteins on the insulin granule, triggering the fusion of the insulin granule with the plasma membrane (27).
Defining the exact mechanism of miR-mediated regulation is daunting given the complex target repertoire and target interaction. For example, although GK activity is important for initiating the glycolytic flux, the most crucial enzyme involved is phosphofructokinase/ fructobisphosphatase (PFK-1). Pfk-1 mRNA is not a predicted target of miR-206. However, the most powerful allosteric activator of PFK-1 is fructose-2,6-bisphosphate (Fru-2,6-P2). The levels of Fru-2,6-P2 are regulated by the enzyme fructose-2,6-biphosphatase 3, the mRNA of which is a predicted target of miR-206 (16). A limitation of our study is the lack of a cell system overexpressing miR-206 to prove the effect of miR-206 overexpression on GK. miR-206 expression in MIN-6 or isolated islets decreases cell viability significantly (unpublished observation). Consistent with our own observations, Liu et al. (13) and others have reported miR-206 as a proapoptotic activator of cell death. Lower levels of baseline (time 0) glucose and increased liver glycogen in HFDfed miR-206KOs are indicative of a role of hepatic miR-206 in regulating systemic glucose levels. In control mice, HFD compared with regular chow leads to a 1.5-fold upregulation in hepatic miR206 expression. The cumulative impact of this upregulation in a metabolically active tissue such as the liver could be significant. As expected, miR-206KOs challenged with HFD showed increased GK activity and higher levels of hepatic glycogen. However, the expected increase in the transcript levels of hepatic Gck upon HFD was not observable.
This leads us to speculate that miR-206 might have other targets that may mediate its effects on glucose metabolism in a tissue-specific manner. Such tissue-specific effects of hepatic vs. islet miR-206 on glucose metabolism are not discernable in a germline knockout model such as the one used in this study. Therefore, future studies aimed at investigating tissue-specific knockouts of miR-206 and the effects on glucose homeostasis are warranted.
Taken together, we have described the novel posttranscriptional regulation of islet Gck. Our data revealed that miR-206 expression is tissue specifically altered in response to HFD, and loss of miR-206 increases GK activity in islets and liver, leading to improved glucose tolerance and GSIS. There is an increasing effort to develop GK activators to treat diabetes. However, in conditions like obesity and diabetes, aberrant expression of posttranscriptional regulators such as miR-206 can repress the levels of GK, making GK activators less effective. miR-206 expression in murine tissues. A: abundance of miR-206 in muscle, islets, intestine, brain, and liver relative to U6 sno-RNA measured by real-time PCR (n = 5/group). B-E: mRNA abundance of key glucose homeostasis genes in muscle (B), pancreatic islets (C), intestine (D), and liver (E), relative to hypoxanthine-guanine phosphoribosyl transferase (Hprt) expression, measured by real-time PCR (n = 5). Data represent means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 by randomization tests with a pairwise reallocation. Hk2, hexokinase; Gck, glucokinase gene; Glut1 and -2, glucose transporter 1 and 2, respectively.     Expression of miR-206 and glycolytic genes in HFD-fed mice. A: relative abundance of miR-206 in islets, brain, liver, muscle, and intestine relative to U6 sno-RNA measured by real-time PCR (n = 5/group). B-E: mRNA abundance of G6pdh, Gck, and Glut2 in islets (B) and liver (C), G6pdh, Gck, and Glut1 in intestine (D), and G6pdh, Hk2, and Glut1 (E) in skeletal muscle of control (open bars) and miR-206KO mice (black bars) fed HFD for 20 wk (n = 5/group) relative to Hprt expression measured by real-time PCR. Data are presented as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 by randomization tests with a pairwise reallocation.  Table 1 List of miR-206 targetgenes (shown in red in Fig. 3 Table 2 List of miR-206 target genes (shown in green in Fig. 3