Articles in PresS. Am J Physiol Endocrinol Metab (December 15, 2015). doi:10.1152/ajpendo.00211.2015 1
Profound hyperglycemia in knockout mutant mice identifies novel function for POU4F2/Brn-3b in regulating
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metabolic processes
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Stavroula Bitsi1, Houda Ali1, Lauren Maskell1, Samir Ounzain1,3, Vidya Mohamed-Ali2 and Vishwanie S. Budhram-
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Mahadeo1*
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Medical Molecular Biology Unit, UCL Institute of Child Health, London UK; Adipokines and Metabolism Research Group, Division of Medicine, University College London, UK.
Experimental Cardiology Unit, University of Lausanne Medical School, Switzerland
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The Rayne Building, University College London, London WC1E 6JF. Tel (44) 2031082160; email: v.budhram-
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[email protected]
To whom correspondence should be addressed: Vishwanie S Budhram-Mahadeo, Medical Molecular Biology Unit,
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Running Title: Loss of Brn-3b linked to metabolic dysfunction
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Keywords: POU4F2/Brn-3b, transcription factor, glucose intolerance, GLUT4
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1 Copyright © 2015 by the American Physiological Society.
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Abstract:
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The POU4F2/Brn-3b transcription factor has been identified as a potentially novel regulator of key metabolic
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processes. Loss of this protein in Brn-3b knockout (KO) mice causes profound hyperglycemia and insulin resistance
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(IR), normally associated with type II diabetes (T2D), whilst Brn-3b is reduced in tissues taken from obese mice fed
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on high-fat diets (HFD), which also develop hyperglycemia and IR. Furthermore, studies in C2C12 myocytes show
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that Brn-3b mRNA and proteins are induced by glucose but inhibited by insulin, suggesting that this protein is itself
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highly regulated in responsive cells. Analysis of differential gene expression in skeletal muscle from Brn-3b KO mice
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showed changes in genes that are implicated in T2D such as increased glycogen synthase kinase-3 beta (GSK3β) and
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reduced GLUT4 glucose transporter. The GLUT4 gene promoter contains multiple Brn-3b binding sites and is directly
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transactivated by this transcription factor in co-transfection assays whereas chromatin immunoprecipitation (ChIP)
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assays confirm that Brn-3b binds to this promoter, in-vivo. In addition, strong correlation between GLUT4 and Brn-3b
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in Brn-3bKO tissues or in C2C12 cells strongly supports a close association between Brn-3b levels and GLUT4
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expression. Since Brn-3b is regulated by metabolites and insulin, this may provide a mechanism for controlling key
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genes that are required for normal metabolic processes in insulin-responsive tissues and its loss may contribute to
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abnormal glucose uptake.
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Introduction:
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Metabolic homoeostasis is achieved by the coordination of complex and highly regulated processes that involve
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crosstalk between different tissues (36). Many of these changes are mediated by circulating metabolites such as
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glucose, which drive genetic changes in different tissues, thereby maintaining blood glucose levels within narrow
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functional ranges. Under normal conditions, elevated blood glucose causes increased insulin secretion by the pancreas,
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which facilitates glucose uptake, in responsive tissues, but inhibits gluconeogenesis and lipolysis in the liver and
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adipose tissue (9). However, deregulation of one or more of these processes can cause elevated blood glucose
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(hyperglycemia), which is characteristic of type II Diabetes Mellitus (T2D). Uncontrolled hyperglycemia can drive
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pathophysiological changes such as inflammation and atherosclerotic plaque formation in the vasculature but also
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contribute to the development of cardiovascular, renal and neuropathic diseases (2; 14; 15; 21; 22). However, the
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molecular mechanisms that drive early pathological changes are not fully elucidated.
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In post-prandial state, skeletal muscles and adipose tissues are responsible for the majority of glucose utilisation and
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this is facilitated by insulin-responsive glucose transporters such as GLUT4, which are translocated from intracellular
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vesicles to the plasma membrane where they facilitate glucose uptake from the bloodstream (27). Hyperglycemia and
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T2D are often associated with insulin resistance, whereby normally insulin-sensitive tissues fail to respond to increase
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circulating insulin (19; 36). Although these complex effects may be attributable to multiple factors, reduced GLUT4
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expression has been implicated in impaired glucose responses and insulin resistance (13; 23; 27). In this regard,
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disruption of one allele in heterozygote (GLUT4+/-) mutants has been sufficient to cause peripheral insulin resistance
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(32). Furthermore, GLUT4 gene transcription is dynamically regulated by feeding in rodent models of streptozocin-
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induced diabetes since fasted animals showed significant reduction of GLUT4 mRNA in adipose tissues, which was
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rapidly reversed upon re-feeding (20; 34). Therefore, transcription factors that regulate GLUT4 expression, in a tissue-
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specific manner, will be important for controlling metabolic processes in insulin-dependent tissues and may contribute
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to initiation or progression of dysfunction/disease (18; 24).
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The POU4F2/Brn-3b transcription factor (Brn-3b), which belongs to subclass IV of POU homeodomain proteins, is
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characterised by the highly conserved DNA binding POU (Pit-Oct-Unc) domain(7) that binds to regulatory regions of
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target genes, thereby controlling the rate of transcription by RNA pol II enzyme. The gene encoding Brn-3b consists
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of two exons separated by an intron, which can give rise to distinct protein isoforms that are completely conserved in
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the DNA binding C’ terminus but differ in N’terminal domain(26; 28). Thus, the shorter Brn-3b(s) protein is encoded 3
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by exon 2 only, whereas Brn-3b(l) protein is encoded by exons 1+2 on therefore contains an additional N’ terminal
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domain, which is not found in Brn-3b(s) (3; 8). However, the functions of distinct isoforms are still to be elucidated.
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Brn-3b mediates diverse effects on cell fate by regulating the expression of multiple target genes but these are highly
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dependent upon the cell types and/or growth conditions. For instance, Brn-3b can transactivate cell cycle proteins
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cyclin D1/CDK4 (3) in epithelial cells and, as such, has been implicated in some cancers, where elevated Brn-3b
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enhances cell proliferation and tumour growth (5; 7; 17). However, Brn-3b also confers drug resistance and migratory
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potential after chemotherapeutic treatment by driving expression of other target genes such as the small heat shock
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protein, HSP27, which protects cells under these conditions (6; 33). On the other hand, Brn-3b is essential for survival
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and specification of retinal ganglion cells (RGC), since KO mutant mice are blind due to loss of RGC after birth (12;
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26). However, if co-expressed with growth inhibitory p53 protein, Brn-3b can promote apoptosis by interacting and
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cooperating with p53 to increase transcription of pro-apoptotic target genes e.g. Bax and Noxa. As such, it is crucial to
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analyse the effects of this transcriptional regulator in the context of cell/tissue specific effects in order to determine its
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mechanism of action on cell growth, behaviour or fate.
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More recently, Brn-3b was identified as the only transcription factor to show significant changes in skeletal muscle
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taken from subjects after 2 months of endurance training, when comparing gene expression in subjects with high or
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low post-training insulin sensitivity(37). The results of these microarray analyses suggested that Brn-3b may be
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important for controlling gene expression and function in metabolically active tissues also and lead us to hypothesize
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that this transcription factor may regulate genes that control important metabolic processes.
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These studies report on the expression of Brn-3b in insulin responsive tissues and investigate how loss of this
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transcription factor affects metabolic function in Brn-3bKO mutant mice. Furthermore, the regulation of Brn-3b
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expression in response to glucose and insulin have been characterised using the skeletal myoblast derived cell line,
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C2C12. Finally, analysis of gene expression changes upon loss of Brn-3b has identified potential target genes such as
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GLUT4, which may be regulated by this transcription factor in skeletal muscle and adipose tissues. The results, herein,
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suggest that Brn-3b represents a novel regulator of target genes that are important for controlling metabolic function
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and glucose homeostasis in insulin-responsive tissues.
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Results:
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Brn-3bKO mutant mice develop increased body weight and glucose intolerance
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Casual observation of differences in the size of Brn-3b KO mice compared with WT littermates, led us to undertake
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longitudinal studies to compare body weight (BW) over time (>12 months). Results showed that mutant mice had
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consistently higher BW when compared with WT controls [Fig1A(i)] although there were no significant changes in
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body length (e.g. Brn-3b KO ~ 9.3+/-0.05 cm VS WT females ~9.1+/-0.1 cm). Closer inspection showed increased
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visceral fat deposits in the abdomen of Brn-3b KO mutants compared with littermate controls [Fig1A(ii)]. To test if
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changes in body weight were caused by differences in calorie consumption, food intake was closely monitored for WT
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and Brn-3b KO mutants fed on a high-fat diet (HFD) (see methods) and used to determine caloric intake. Fig 1A(iii)
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showed that there were no statistically significant differences in consumption by KO mutants and WT controls
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suggesting that increased body weight in Brn-3b KO mutants may result from potential changes in metabolic
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processes. Since Brn-3b was shown to be differentially expressed in skeletal muscle of insulin responsive subjects
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(37), we next tested for changes in glucose handling in Brn-3b KO mutants by undertaking glucose tolerance tests
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(GTT). Therefore, Brn-3bKO and WT control mice were fasted for 12h and blood glucose levels were measured at
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baseline, after which a glucose bolus was administered intraperitoneally. This was followed by half hourly blood
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glucose measurements, up to 120 min. The results (Fig1B) showed no significant differences in blood glucose
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between KO and WT mice at baseline (t=0) but marked differences were evident after administration of the
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intraperitoneal glucose bolus. As expected, WT mice showed increased blood glucose at 30 mins, which decreased by
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60 min and returned to baseline by 120 min. However, Brn-3b KO mice had significantly higher blood glucose levels
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at 30min, which continued to rise at 60 min and remained significantly elevated after 120 min.
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ELISA assays were used to examine insulin levels in serum collected at baseline and after 120 min. Although baseline
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serum insulin levels in the mutant animals were slightly higher than WT, this was not statistically significant.
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However, at the end of experiments (120 min), serum insulin was significantly higher in Brn-3b KO mutants when
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compared with controls (Fig 1C). The observed hyperglycemia, accompanied by increased insulin levels, in mutant
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mice suggested changes in metabolic processes such as insulin production or glucose uptake by insulin-responsive
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tissues e.g. skeletal muscle, adipose tissue and liver. However, since Brn-3b expression was not previously
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investigated in such tissues, western blot analyses were carried out using protein extracts from relevant issues
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including pancreas, skeletal muscle, liver and adipose tissue. Fig 1D shows that Brn-3b was not detectable in pancreas 5
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when significant levels were observed in insulin responsive tissues such as skeletal muscle and liver. The shorter Brn-
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3b(s) isoform was more abundant in skeletal muscle whilst the longer Brn-3b(l) isoform was predominant in the liver
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(Fig 1D) and adipose tissues (fig 2B). Although the potential roles for these distinct isoforms are unclear at present,
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high Brn-3b expression in insulin responsive tissues such as skeletal muscle may suggests that persistent
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hyperglycemia and insulin resistance in Brn-3b KO mutants are likely to be associated with defects in metabolically
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active tissues rather than the pancreas, in which Brn-3b is not readily detected.
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Brn-3b is reduced in insulin responsive tissues of WT mice fed on a diet with high saturated fat content:
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Since loss of Brn-3b was associated with increased body weight and hyperglycemia, we considered if reduction of this
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protein may be linked to diet induced obesity, which can also cause hyperglycemia and insulin resistance in rodent
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models (38). To test this hypothesis, we generated and analysed a diet-induced obese mouse model in which WT mice,
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fed on diet containing 60% saturated fat (HFD) for >12 weeks, were compared with control mice fed on normal diet
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(ND). Most of the mice fed on HFD developed significant increase in BW when compared with age-matched controls
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fed on ND. However, one animal fed on ND developed obesity, whereas one animal fed on HFD did not show
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increased BW. Since these studies aimed to analyse the link between body weight and Brn-3b, these 2 mice were
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excluded. At the end of these studies, GTT was undertaken on fasted mice to analyse blood glucose levels at baseline
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(time 0) and then at 30, 60 and 120 min after administration of a glucose bolus. Although there were no significant
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differences in blood glucose or serum insulin levels, at baseline between these groups (fig 2A), differences were
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detected after 120 min when animals fed on ND showed an expected drop in blood glucose and serum insulin, similar
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to baseline levels. However, animals fed on HFD demonstrated significantly elevated blood glucose and this was
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associated with increased serum insulin levels Fig 2A(ii +iii), similar to the effect seen in Brn-3bKO mutants.
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To examine potential links between changes in Brn-3b expression and development of pathophysiological effects in
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these mice, we analysed for protein expression in insulin responsive tissues taken from mice fed on HFD or ND.
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Analyses were carried out using skeletal muscle, which contributes to postprandial glucose uptake and adipose tissue,
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which was considerably increased in animals fed on HFD. Fig 2B (i) shows protein levels in skeletal muscle and as
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expected, Brn-3b(s) was highly expressed in tissues taken from ND fed mice but was significantly reduced in those
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fed on HFD (ii). Adipose tissues expressed both Brn-3b isoforms but with higher levels of Brn-3b(l) detected in mice
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fed on ND [fig 2B (iii)]. However, both isoforms were significantly reduced in tissues from HFD fed mice. These
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results suggest that Brn-3b proteins are highly regulated in such metabolically active tissues and its reduction may
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contribute to hyperglycemia observed in obese WT animals or in KO mutants.
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Brn-3b expression is regulated by glucose and insulin in C2C12 myoblasts.
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Since loss or reduction of Brn-3b was associated with high serum insulin and hyperglycemia, we next tested if Brn-3b
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expression was affected by glucose and/or insulin in C2C12 myoblasts. Therefore, cells were either grown in medium
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lacking glucose (NG); NG media supplemented with 25mmol glucose in the absence of insulin (NG-GLU); or with
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added insulin (NG-GLU-INS). Control cells were grown in full growth medium (FGM). Fig 3A shows that Brn-3b
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mRNA was reduced in cells grown in glucose-free medium and this was restored upon addition of 25 mmol glucose
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(fig 4A), whereas chronic insulin treatment (10nmol for 24h) down-regulated Brn-3b mRNA, although insulin had no
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effect at early time points (1 or 4h) (data not shown). These results were confirmed by immunoblotting to analyse
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changes in protein levels. Although both Brn-3b isoforms were detectable in these cells, Brn-3b(s) was more abundant
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than Brn-3b(l) (fig 4B), which is similar to previous data showing that Brn-3b(s) was the primary isoform in skeletal
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muscle [fig 2A(ii)]. Cells that were grown in NG medium had reduced levels of both proteins, with Brn-3b(l) being
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undetectable. Brn-3b(s) levels for restored and sustained by adding glucose to the NS medium, whereas addition of
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insulin caused reduction of both Brn-3b isoforms.
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Since Brn-3b expression was stimulated by glucose but reduced by insulin treatment, we next tested if these effects
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were mediated at the level of transcription of the Brn-3b promoter. Therefore, C2C12 myoblasts were transfected with
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Brn-3b reporter construct, then transferred into different growth conditions. Cells were harvested after 24h and dual
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luciferase assays were used to measure promoter activity. Fig 3C shows that Brn-3b promoter activity was
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significantly repressed when cells were grown in medium lacking glucose (NG) compared with FGM, whereas
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addition of 25mM glucose was sufficient to increase promoter activity (>60 fold) compared with NG. However the
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promoter was repressed by insulin. These results suggest that Brn-3b expression is highly regulated by glucose and
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insulin in skeletal muscle.
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Gene expression changes associated with loss of Brn-3b in skeletal muscle:
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To identify gene expression changes in Brn-3bKO mice that might contribute to glucose intolerance, the disease-
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focused RT² Profiler™ PCR 337021 Array was screened with cDNA synthesised using RNA extracted from skeletal 7
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muscle taken from mutant mice or age-matched WT littermates(see methods). Table 1 shows a selection of genes that
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were either over-expressed or under-expressed in Brn-3bKO mutants compared with WT controls. To validate
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changes in selected genes, qRT-PCR was carried out using RNA from skeletal muscle taken from independent Brn-
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3bKO or WT mice. Figure 4 shows that statistically significant changes were reproduced for glycogen synthase kinase
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3 beta (GSK3β) mRNA, which was increased in Brn-3b KO mutants, whereas angiotensin-1 converting enzyme
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(ACE) and the solute carrier family 4 glucose transporter (GLUT4) were both reduced in mutant tissue. However, the
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expected changes in other genes were not significant or reproducible suggesting that GSK3β, ACE and GLUT4 may
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be closely linked to Brn-3b expression.
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Brn-3b binds to and activates the insulin-responsive glucose transporter, GLUT4:
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The profound dysfunction in glucose handling observed in Brn-3b KO mutants was similar to impaired glucose
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response and insulin resistance reported for GLUT4(+/-) or
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might affect glucose uptake by regulating expression of this transporter. Genomatix Transfac analysis identified
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multiple V$BRNF sites in the mouse GLUT4 promoter sequences that could act as potential binding sites for Brn-3b
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transcription factor (fig 5A). These were clustered in two regions that were either proximal to the coding sequence
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(indicated by p1 and p2) and distal sites (p3-p4). Therefore we tested if Brn-3b regulated transcription of this
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promoter, co-transfection studies were carried out, in which the GLUT4 luciferase reporter was co-transfected with
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different concentrations of Brn-3b expression vectors, into C2C12 cells. Results of the luciferase assays showed that at
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1:1 ratio, Brn-3b transactivated the promoter by >4 fold but promoter activity was significantly enhanced (>12 fold)
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when Brn-3b was increased to 2:1 (Fig 5B), confirming that Brn-3b does indeed, regulate GLUT4 promoter activity.
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Since there were multiple potential binding sites spanning relatively large regions of the GLUT4 promoter, it was not
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feasible to undertake site directed mutagenesis to identify specific site(s) that were required for Brn-3b-mediated
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promoter activity. Therefore, to test if Brn-3b binds directly to the GLUT4 promoter in-vivo, in intact cells, we carried
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out chromatin immunoprecipitation (ChIP) assays (26), in which Brn-3b Abs were used to immunoprecipitate the
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protein bound to chromatin in intact C2C12 cells. GAPDH antibody was used as a negative control for binding
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specificity. ChIP DNA isolated using Brn-3b Ab or GAPDH Ab control were used for PCR with primers designed to
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amplify the two regions of GLUT4 promoter that were enriched for Brn-3b binding sites i.e. the proximal sites
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(primers 1+2) or distal sites (primers 3+4) and the PCR products were resolved and 2% agarose gel. As shown in Fig
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5C, amplification with primers 1+2 produced ~500 bp products, which was observed in the positive control (input)
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mutants (13; 23). We therefore considered if Brn-3b
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sample and ChIP DNA immunoprecipitated with Brn-3b Ab but not control Ab. However, although primers 3+4 gave
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rise to ~350 bp products with the input DNA, no significant bands were produced when using the Brn-3b ChIP DNA.
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These results suggest that Brn-3b binds directly to one or more of the sites in the proximal GLUT4 promoter to
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activate gene expression.
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To determine whether Brn-3b was necessary for maximal transcription of this gene, we next analysed for changes in
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GLUT4 mRNA in insulin-responsive tissues such as skeletal muscle and adipose tissue, taken from mutants or age-
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matched WT controls. Therefore, RNA from Brn-3b KO or WT tissues was used for cDNA synthesis and
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quantification of GLUT4 transcripts using qRT-PCR whereas whole-cell protein extracts were used for immunoblots.
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Fig 5D (i) shows that GLUT4 mRNA was significantly lower in skeletal muscle taken from Brn-3b KO mutants when
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compared with levels in WT tissues, confirming that Brn-3b is necessary for transcription of this gene. To test if
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reduced GLUT4 transcripts in Brn-3b KO mutants also affected protein levels, immunoblotting was carried out using
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total cellular protein, extracted from insulin-responsive tissues from Brn-3b KO and WT controls. The representative
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immunoblots shown in Fig 5D (ii) shows that the GLUT4 protein was reduced in Brn-3b KO adipose tissues, with ~
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50% less protein, when compared with controls (iii).
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We next tested if overexpressing Brn-3b affected basal GLUT4 protein levels in myoblasts by transfecting C2C12
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cells with an expression vector containing Brn-3b cDNA (at 1 µg or 2 µg). Untransfected control cells (C) or cells
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transfected with the empty expression vector (V) were used as controls and after 48 hours, protein extracts from
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transfected cells were used for immunoblotting to analyse changes in GLUT4 and Brn-3b expression. Fig 5E shows a
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representative immunoblot from these experiments that demonstrated increased Brn-3b protein in cells transfected
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with the Brn-3b expression vector, which correlated with significant up-regulation of GLUT4 protein, when compared
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with control cells. Furthermore, regression analysis showed statistically significant correlation between Brn-3b
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expression and GLUT4 mRNA expression in C2C12 cells that were grown under different conditions e.g. FGM, NG
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or NG+glucose [Fig 5E(ii)]. These results strongly support Brn-3b as a key regulator of GLUT4 in relevant
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cells/tissues.
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Discussion:
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Hyperglycemia, which is commonly associated with insulin resistance, T2D and obesity, can drive pathological
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processes that contribute to comorbidities such as cardiovascular diseases, renal dysfunction and neurological damage
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(1; 14; 29). These effects result from complex molecular changes that are triggered in different tissues, which lead to 9
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the development and progression of such diseases but the factors to control such changes are not fully understood(36).
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Hyperglycemia, insulin resistance and T2D are set to rise, in line with a predicted global obesity epidemic, which is
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linked to dietary and lifestyle changes (11; 25; 36). Therefore, it is important to understand the molecular mechanisms
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that drive pathological changes since this may help to identify strategies that could be used to block or reverse tissue
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damage, thereby minimising morbidity and mortality.
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In this regard, we have identified Brn-3b as a novel regulator of glucose homeostasis. The first evidence suggesting
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that this transcription factor may be involved in regulating metabolic processes, was inferred from microarray
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analysis, which showed that Brn-3b was significantly altered in skeletal muscle taken from individuals with high or
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low insulin sensitivity, following endurance training (37). Results from our studies have shown that Brn-3b protein is
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expressed in insulin-responsive tissues such as skeletal muscle, adipose tissue as well as in liver, but has not been
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detected in the pancreas. Furthermore, higher body weight in constitutive Brn-3b KO mice are associated with
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increased fat deposition in the abdominal cavity, despite no statistically significant changes in body length or
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differences in calorie consumption, when compared with control mice. Brn-3b KO mutants also developed persistent
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hyperglycemia, in the presence of high serum insulin levels, suggesting that the pancreas continues to produce insulin
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and high blood glucose may be linked to defects in insulin responsiveness and/or glucose uptake in tissues such as
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skeletal muscle and adipose tissues. In line with this, Brn-3b expression is also reduced in skeletal muscle and adipose
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tissues taken from WT mice that develop obesity and hyperglycemia/insulin resistance after being fed on a high
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saturated fat diet. These results suggest that Brn-3b is dynamically regulated in such tissues and that the metabolic
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dysfunction in constitutive KO mutants may result from direct loss of this transcription factor in insulin responsive
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tissues.
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Two distinct Brn-3b isoforms have previously been described (3; 8) and we have shown that Brn-3b(s) is the
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predominant isoform in skeletal muscle, whereas Brn-3b(l) is more abundant in liver and adipose tissues. Moreover
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both isoforms are regulated in response to high-fat diet. Although the functions of these distinct isoforms are still to be
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elucidated, both proteins can be regulated by similar signals but can also regulate each other’s expression (8; 17).
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Therefore, it will be interesting to investigate potential tissue-specific effects for distinct Brn-3b isoforms in insulin
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responsive tissues, in later studies.
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These results have provided evidence to support potential roles for the Brn-3b transcription factor in controlling
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metabolic function and have been reinforced by findings in C2C12 myoblast cell, which show that Brn-3b can, itself, 10
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be regulated by insulin and metabolites such as glucose. In this regard, Brn-3b mRNA and proteins are reduced in
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cells grown in low glucose media or in the presence of insulin but can be restored by addition of physiological levels
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of glucose (25 mM). These effects are likely to be mediated at the level of transcription since low glucose or insulin
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can repress the Brn-3b promoter, whereas addition of glucose strongly stimulated promoter activity. Such tight
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regulation of Brn-3b by insulin and glucose indicate that this transcription factor will have important role(s) for
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regulating genes associated with glucose uptake.
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In line with this, analysis of differential gene expression in skeletal muscle taken from Brn-3bKO and WT mice have
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identified changes in key genes that are associated with metabolic processes. For example, increased GSK3β in Brn-
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3b KO tissues may contribute to insulin resistance by phosphorylating and inhibiting key molecules such as glycogen
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synthase and insulin receptor substrate proteins and importantly, elevated GSK3β has been detected in skeletal muscle
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of patients with T2D (10; 16; 30). On the other hand, reduced GLUT4 in tissues taken from Brn-3b KO may underlie
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the glucose intolerance detected in these mutants since Brn-3b directly regulates transcription of GLUT4 receptor.
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Multiple potential Brn-3b binding sites have been identified by bioinformatic analysis and reporter assays confirm that
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increasing exogenous Brn-3b can strongly transactivate the GLUT4 promoter in C2C12 cells. The presence of
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multiple sites, clustered in 2 regions of the GLUT4 promoter, meant that it has not been possible to use site directed
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mutagenesis to identify the specific sequence(s) required for Brn-3b binding and promoter activation. However, ChIP
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analysis has conclusively demonstrated that Brn-3b binds directly to the proximal sites in the GLUT4 promoter, in-
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vivo, in intact myoblast cells. There is also strong evidence for significant correlation between Brn-3b and GLUT4
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expression from in-vivo and in vitro studies. Thus, GLUT4 mRNA and protein are reduced in insulin responsive
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tissues (skeletal muscle and adipose), taken from Brn-3b KO mutant and altered Brn-3b correlate well with GLUT4
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expression in C2C12 myoblasts, grown under different conditions. In addition, overexpressing Brn-3b in C2C12 cells
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is sufficient to increase basal GLUT4 levels.
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These findings strongly suggest that Brn-3b may be a key regulator of GLUT4 expression in insulin responsive tissues
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and that loss of this receptor may contribute, in part, to hyperglycemia and insulin resistance observed in Brn-3b KO
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mutants or in obese mice with reduced Brn-3b expression. The GLUT4 transporter is important for insulin-mediated
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glucose uptake in adipose tissues and skeletal muscle, which accounts for majority of post-prandial glucose utilisation
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(27). Although GLUT4 translocation from intracellular vesicles to the cell membrane is an important mechanism for
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regulating glucose uptake, changes in expression levels have also been implicated in inefficient glucose utilization. 11
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Thus, reduced GLUT4 expression in heterozygote mice is sufficient to cause defective glucose handling since mutant
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mice develop severe peripheral insulin resistance, which can be reversed by recapitulating expression in skeletal
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muscle (32; 35). Although the relevance of reduced GLUT4 in humans with non-insulin-dependent T2D has been
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brought into question more recently (31), the clear correlation between Brn-3b and GLUT4 expression in insulin
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responsive tissues, in these studies, strongly suggest that this transcription factor will be important for regulating the
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expression of this glucose transporter in different tissues. However, GLUT4 is likely to be one of multiple target genes
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that are regulated by the Brn-3b transcription factor in insulin responsive tissue that give rise to the complex
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phenotypes seen in the constitutive KO mutants.
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Thus, Brn-3b represents a novel and important regulator of metabolic processes in insulin responsive tissues.
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However, since its expression is also controlled by circulating levels of metabolites and insulin, this transcription
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factor may act as part of a feedback loop that maintains glucose homoeostasis, under normal conditions.
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Materials and methods:
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General laboratory reagents- Merck (Nottingham, UK); Sigma (Dorset, UK) unless otherwise stated. Tissue culture
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reagents/plastics: Gibco/Life Technologies (Paisley, UK); Nunc (Paisley, UK); Greiner (Stonehouse, Gloucester, UK)
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or Corning (Scientific Laboratory Supplies, Nottingham, UK). Primary antibodies were sourced as follows: rabbit-
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Brn-3b pAb (Abcam-Cambridge, UK); goat-Brn-3b pAB and goat-actin pAb (Santa Cruz Biotechnology Inc, USA);
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β-tubulin mAb, (Merck Millipore, Darmstad, Germany); Glut4 (1F8) Mouse mAb; Cell Signalling Technology
309
(Danvers, MA, USA) and all secondary Ab (Dako, Cambridgeshire, UK). GLUT4 promoter, cloned in the pGL3
310
luciferase vector, was a generous gift from Prof M. Quon (University of Maryland, Baltimore) and Prof E. Karnieli,
311
(Rambam Medical Center; Haifa).
312
Methods: In vivo models: Studies using rodent models were undertaken in accordance with Home Office guidelines
313
(Animals Scientific Procedures Act 1986) and approved by local Ethics Review Board. C57BL/6J strain was used for
314
studies, with outbred C57BL6 mice obtained from Harlan UK and Brn-3b heterozygote mice used to generate Brn-
315
3bKO mutant mice and aged-matched wild-type (WT) littermates. For longitudinal studies (>1 year), experimental
316
animals were weighed monthly, whereas mice fed on high fat diet (HFD) or normal chow diet (ND) were weighed
317
weekly. Dissected tissues (e.g. skeletal muscle, liver, adipose tissue, pancreas) were either snap frozen (for subsequent
318
RNA/protein extraction) or fixed in 4% paraformaldehyde (PFA) and embedded for subsequent sectioning.
12
319
Glucose Tolerance Tests (GTT) and Serum Insulin measurement: For GTT, experimental animals were fasted for
320
12 hours and baseline blood glucose was evaluated using a glucometer. Glucose bolus (2mg glucose/g BW) was
321
administered by intraperitoneal injection and blood glucose recorded after 15, 30, 60 and 120 min. For serum insulin
322
measurements, whole blood was collected from saphenous vein at the start and end of experiments into serum
323
separator tubes [BD Microtainer® (Becton-Dickinson, Franklin Lakes, NJ, USA)]. Serum insulin levels were
324
measured using Mercodia Mouse Insulin ELISA kit, Upsalla, Sweden).
325
Calorie consumption: Calorie consumption was calculated by monitoring dietary intake and then using the known
326
caloric value of the feed to calculate intake. Briefly mice were housed in separate cages and the chow was weighed at
327
the start of experiments and this was repeated at the end of each week, before topping up or replacing the feed. This
328
was also measured if it was deemed necessary to top up on at other times. The differences in weight (grams) of the
329
feed at the start and end of the week were multiplied by the known caloric value (5.1 kcal/gram) to calculate calorie
330
consumption per week for each mouse. The mean and standard error of each group (n>5) were used to determine
331
differences in calorie consumption over time.
332
Cell culture and treatments: Skeletal muscle satellite cell-derived C2C12 myoblasts were maintained in full growth
333
medium (FGM) [Dulbecco’s Modified Eagle’s Medium (DMEM); 10% foetal bovine serum (FBS); 1%
334
penicillin/streptomycin], grown in 5% CO2 at 37°C. Cells plated onto 6-well (5× 105/well) or 12-well (105/well)
335
culture dishes were transfected or treated as specified. Free fatty acids (FFA) -unsaturated long chain oleic acid and
336
saturated palmitic acid were dissolved in ethanol and used to treat cells. Transfections was carried out using FugeneTM
337
(Promega, Hampshire, UK) as previously described (6; 33) and reporter assays using the Dual Luciferase Reporter
338
Assay System (Promega, Hampshire, UK).
339
RNA extraction, cDNA synthesis, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR):
340
Tissues homogenized in liquid nitrogen were resuspended in TRIZOL® (Invitrogen); C2C12 cells were harvested in
341
Trizol then processed according to the manufacturer’s protocol. DNAse1 treated RNA was used for cDNA synthesis
342
(RNA Superscript™II RT) (Invitrogen,Paisley,UK). QRT-PCR was performed on Opticon 2 DNA engine thermal
343
cycler (BioRad, UK), using SYBR Green master mix (Qiagen, UK) and Brn-3b primers (forward-
344
ATCGCCGAAAAGCTGGAT; reverse-TTCTCTTCTGTTTCTGCCTCTG or QuantiTect Assay primers (Qiagen,
345
Manchester, UK) for selected target genes. Variability between samples or adjusted using GAPDH and fold changes
346
calculated using 2-∆∆CT method (25).
13
347
PCR array analysis: cDNA from Brn-3b KO skeletal muscle and WT controls (see above) were used to screen the
348
Mouse Diabetes RT² (SAB BioSciences, Qiagen, West Sussex, UK), which facilities the screening of 84 genes
349
associated with onset, development and progression of diabetes. Quantitative PCR was undertaken according to the
350
manufacturers’ protocol using the Opticon 2 DNA thermal cycler and analysis done using PCR Array Data Analysis
351
Software https://www.qiagen.com/gb/products/genes and pathways/data-analysis.
352
Protein extraction and immunoblotting: Cells were harvested in Laemmli buffer; mouse tissues were pulverised in
353
liquid nitrogen then resuspended in Laemmli buffer and homogenised. Total protein extraction and polyacrylamide gel
354
electrophoresis (SDS-PAGE) carried out as described(4). Proteins were quantified using densitometry (Quantity One
355
Software-BioRad Laboratories, CA, USA) or Image-J and the invariant β-tubulin protein used to adjust for differences
356
in protein loading.
357
Chromatin Immunoprecipitation (ChIP) Assay was carried out as described by Lee et. al.(22) using anti-goat Brn-
358
3b Ab (Santa Cruz) to immunoprecipitate Brn-3b on chromatin in intact cells. Anti-GAPDH (Abcam) was used as
359
negative. Sonicated ChIP DNA was amplified with PCR primers 1+2 or 3+4, designed to amplify different regions of
360
GLUT4 promoter. Primer 1: CAGGTACACATGTAGTACACA; Primer 2:TGGCTGTTCTGGAACTCACT; Primer
361
3: CTTGAGTATACATGTGGCACAC; Primer 4: TGTCAGCTTCTTGATGGCATC.
362
Transient transfections and reporter assays: To analyse effects of Brn-3b on GLUT4 promoter activity in C2C12
363
cells, 5x104 cells/well (in 6 well plates) were co-transfected with GLUT4 reporter construct and Brn-3b expression
364
vector or control LTR, using FugeneTM reagent (Roche; UK). To analyse effects of glucose +/-insulin on Brn-3b
365
promoter activity, the Brn-3b reporter construct was transfected into C2C12 cells grown in FGM, NG or NG+ glucose
366
with/without insulin. Cells were harvested after 24h and promoter activity measured using Dual-Luciferase reporter kit
367
(Promega, UK) and TD-20/20 luminometer (Turner Designs).
368
Statistics: Statistical analysis was performed using Microsoft Excel or GraphPad Prism 6 (San Diego, CA, USA).
369
Parametric tests (paired t-test) or non-parametric tests (Mann-Whitney; Kruskal-Wallis followed by post-hoc analysis
370
(Dunn’s multiple comparisons test) were used to determine differences between independent samples.
371 372
Acknowledgements: This research was funded by the Dunhill Medical Trust (SA24/0712), British Heart Foundation
373
(PG/08/074/25533) and the Rosetrees Trust. We are grateful to Prof M. Quon, University of Maryland, Baltimore and
374
Prof E. Karnieli, Rambam Medical Center; Haifa for providing GLUT4 promoter constructs; to Gautam Mehta and
375
Katayoon Gardner for technical assistance. 14
376
Author Contributions: S.B., H.A., LM and S.O. undertook experiments to provide data included in the manuscript.
377
V.M-A contributed to discussion and reviewed data, V.B-M obtained funding, supervised the study design and the
378
research and wrote manuscript.
379
Conflict-of-interest: There is no conflict-of-interest associated with this study.
380
Abbreviations: Brn-3b (Brn-3b/POU4F2 transcription factor); POU (Pit-Oct-Unc); T2D (type II diabetes); GSK-3β
381
(glycogen synthase kinase); glucose transporter, GLUT4; PPARα (peroxisome proliferator activated receptor-α); RGC
382
(retinal ganglion cells); FFA (free fatty acids); FGM (full growth medium with glucose); NG (no glucose medium);
383
INS (insulin); GTT (glucose tolerance test); WT (wild type); KO (knockout).
384 385 386 387 388 389 390
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32. Rossetti L, Stenbit AE, Chen W, Hu M, Barzilai N, Katz EB and Charron MJ. Peripheral but not hepatic insulin
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33. Samady L, Dennis J, Budhram-Mahadeo V and Latchman DS. Activation of CDK4 Gene Expression in Human Breast Cancer Cells by the Brn-3b POU Family Transcription Factor. Cancer Biol Ther 3: 317-323, 2004. 34. Sivitz WI, DeSautel SL, Kayano T, Bell GI and Pessin JE. Regulation of glucose transporter messenger RNA in insulin-deficient states. Nature 340: 72-74, 1989. 35. Stenbit AE, Tsao TS, Li J, Burcelin R, Geenen DL, Factor SM, Houseknecht K, Katz EB and Charron MJ. GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes. Nat Med 3: 1096-1101, 1997.
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36. Taubes G. Insulin resistance. Prosperity's plague. Science 325: 256-260, 2009.
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37. Teran-Garcia M, Rankinen T, Koza RA, Rao DC and Bouchard C. Endurance training-induced changes in insulin
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471 472 473
18
474
Figure legends:
475
Fig 1: Metabolic dysfunction in Brn-3b KO mutant mice: A. (i) Change in body weight in Brn-3bKO mutants (red
476
line; n=5) compared with age-matched WT control littermates (blue; n=5). Weights were measured at monthly
477
intervals, for up to 14 mths. (ii) Representative images of abdominal viscera of 3 mth old Brn-3b KO and WT mice. *
478
indicates the increased fatty deposits around internal organs such as the kidney in Brn-3b KO mutants, which is not
479
seen in WT controls (iii) Graph showing calorie consumption by WT mice (dark grey) or Brn-3b KO mutant (light
480
grey). Values represent the mean (+/- standard error) of calorie consumption of 6 individual animals in each group
481
measured at weekly intervals. B. Comparison of blood glucose levels in Brn-3b KO mutant mice (red line) when
482
compared with WT controls (black line). After overnight fasting, baseline blood glucose was measured before
483
administration of peritoneal injection of glucose bolus after-image blood glucose levels were measured half hourly for
484
120 mins. Values represent the mean +/-SD of 6 animals. C. (i) Table showing serum insulin levels in WT or Brn-
485
3bKO mutant mice either at baseline (time 0) and at the end of experiments (120m). (ii) Graph showing mean (+/-SD)
486
of serum insulin levels in WT (dark grey) or mutants (light grey) at two different time points. *indicates statistically
487
significant differences (p3 months. Baseline measurement was
495
undertaken after 12 h fasting and before administration of glucose bolus, after which analyses were done at intervals
496
shown, for up 120 min. (ii) Table showing blood glucose and insulin at baseline (t0) and at the end of experiments
497
(120 m). (iii) graph showing mean (+/-SD) of serum insulin levels in animals fed on ND (dark grey) or HFD (light
498
grey) at two different time points. **indicates statistically significant differences ND control and HFD (n = 6).
499
(B) Representative western blot analysis showing Brn-3b expression in skeletal muscle (i) and adipose tissue (iii)
500
taken from groups of animals fed on normal diet (ND) or high fat diet (HFD). Arrows indicate the position of distinct
501
isoforms, Brn-3b(l) or (s) and β-tubulin blots show differences in protein loading. (ii) and (iv) show quantification of
502
changes in Brn-3b(s) protein in skeletal muscle adipose tissue taken from mice fed on normal diet (ND) or high-fat 19
503
diet (HFD). Values were normalised to β-tubulin in corresponding samples and represent the mean and standard error
504
from four independent animals. * Indicates statistically significant reduction (iii) Brn-3b expression in in Brn-3b
505
protein in tissues taken from animals fed on HFD compared with ND as determined by t-test (n>3).
506
Fig 3: Brn-3b expression is regulated by glucose and insulin in myoblasts derived C2C12 cells: A. Results of
507
qRT-PCR showing relative changes in Brn-3b mRNA in C2C12 grown in full growth medium (FGM); no glucose
508
medium (NG); with NG +25 mmol glucose without insulin (NG-GLU) or with insulin (NG-GLU-INS). Values
509
represent the mean and standard deviation of levels taken from in three independent experiments. Brn-3b levels were
510
adjusted with the house keeping gene, GAPDH.
511
B. Western blot analysis showing changes in Brn-3b protein in C2C12 cells grown in different media including full
512
growth medium of (FGM); no glucose (NG) or with glucose added at different concentrations (as shown). Left panels
513
represent protein levels in the absence of insulin, whereas right panels shows similar glucose levels but in the presence
514
of insulin. The two different isoforms of Brn-3b are indicated (< >) and levels of tubulin protein was used to adjust for
515
variation in protein loading.
516
C. Results of luciferase assays showing changes in activity of the Brn-3b promoter, transfected into C2C12 cells that
517
were treated as indicated. Values were equalized with renilla luciferase activity (internal control) and the data
518
represents mean (+/- SD) from at least 3 independent experiments. * and ** indicates statistically significance (p
WT Brn-3b KO
1
0.5
0
time 0
MW < 46 kD
(ii) 1.5
Pancreas
0
1
Muscle
(B)
*
Serum insulin levels (ug/L)
15
*
Liver
25
Blood glucose (mg/dL)
Body weight (g)
WT
(ii)
(A) (i)
120
Time after glucose bolus (min)
Brn-3b(s) >
< 30 kD
b-tubulin >
< 58 kD
Fig 2:Reduced Brn-3b expression in insulin-responsive tissues in animals fed on a high-fat diet correlates with obesity, hyperglycaemia and insulin resistance
Blood glucose (mg/dL)
25
HFD
20 15 10
ND
5
ND-m1 ND-m2 ND-m3 ND-m4 HFD – m1 HFD – m2 HFD – m3 HFD - m4
0
30
60
Insulin -t0 (ug/L) 0.41 0.35 0.51 0.54 0.39 0.43 0.31 0.53
BG -120 min (mg/dl) 4.9 7.1 4.1 6.5 27.7 23.1 21 15.8
Insulin -120 m (ug/l) 0.49 0.36 0.30 0.53 1.2 2.6 0.94 1.0
Skeletal muscle ND HFD
MW < 46 kD
Brn-3b(l) >
< 30 kD
Brn-3b(s) >
1.5 p = 0.001 1
* 0.5
0 ND
< 58 kD
b-tubulin >
HFD
Diet
Brn-3b(l) > Brn-3b(s) > b-tubulin >
Adipose tissue HFD ND
MW < 46 kD < 30 kD < 58 kD
Brn-3b(l) protein (adjusted with b-tubulin)
(iv)
(iii)
0.45 Brn-3b(l) 0.3
p = 0.01 *
0.15
0 ND
HFD
Diet
p=0.01 1.5
120
Time (min) (i)
BG -t 0 (mg/dl) 4.3 5.3 4.9 4.8 9.2 5.3 5.9 7.3
(ii)
0
(B)
(iii)
(ii)
Serum insulin (ug/L)
30
(i)
Brn-3b(s) protein (SkM) (adjusted with GAPDH)
(A)
** ND HFD
1 0.5 0 time 0
120
Time after glucose bolus (min)
Fig 3: Brn-3b expression is regulated by glucose and insulin in myoblasts derived C2C12 cells (B) (i)
(A)
+ Insulin
no Insulin
NG +Glu NG
FGM
46 kD >
NG +Glu
MW
NG
FGM
**
< Brn-3b (l) > 30 kD >
< Brn-3b(s)>
58 kD >
Brn-3b promoter activity (firefly luciferase activity)
4
*
** 3
2
1
Brn-3b protein (equalised with tubulin)
(ii)
(C)
2 1.5 1 0.5 0
Insulin
0 FGM
NG
NG+ GLU
NG+GLU+INS
FGM +
-
-
NS +
NS + Glu - +
Table 1
Fig 4:Brn-3b KO tissue display significant changes in key metabolic genes
Over-expressed in 3b KO vs WT (RT² Profiler PCR Array) Fold change
Cebpa CEBPa
Thymoma viral proto-oncogene
(+) 321
CCAAT/enhancer binding protein A (+) 35
Ide
Insulin degrading enzyme
(+) 24.9
Ppara
Peroxisome proliferator activated receptor alpha
(+) 17.9
Gsk3b
Glycogen synthase kinase 3 beta
(+) 12
Irs1
Insulin receptor substrate 1
(+) 7
Under-expressed in 3b KO vs WT (RT² Profiler PCR Array) Fold change
Gene Ace
Angiotensin 1 converting enzyme
(-) 12
Slc2a4
Solute carrier family 4 (GLUT4)
(-) 5
** 0.001
mRNA in sk muscles
Akt-2
0.012
2 (equalised with GAPDH)
Gene
WT
**
Brn-3b KO
1.5 0.016
**
1
0.5
0 Ace-1
Akt-2
CEBPa CEBPa
GLUT4
GSK3b GSK3b
PPAR-a PPAR-a
qRT-PCR validation of selected genes in skeletal tissue
Fig 5: Brn-3b can activate GLUT4 expression Mouse GLUT4 gene promoter (A) p3
(E) (i) Transfected C2C12 cells
p1
Brn-3b DNA C 1ug 2ug 2ug
1053 p2
p4
V
Brn-3b>
10
Input
b-tubulin>
3b Ab
GLUT4 >
Input
3b Ab
**
15
(ii)
500bp
*
0 LTR
Input
-ve Ab
3b Ab
5
Glut4 mRNA expression
product size: ~550bp
M
GLUT4 promoter activity (Firefly luciferase)
M
(C)
(B)
-ve Ab
V$BRNF enriched between 1440-1656; 2466- 2993;
Brn-3b (2:1) Brn-3b (4:1) Expression vector
(ratio of Brn-3b to GLUT4 promoter)
300bp product size: ~350bp
2.5 2.0 1.5 1.0
y=0.3057x+0.4777 R2= 0.563 p
1
0.4+/-0.21 0.5
Brn-3b(s) >
1.1+/-0.22
GLUT4>
< 50 kD
0
WT Skeletal muscle
3b KO
b-tubulin >
< 58 kD
GLUT4 protein (adjusted with b-tubulin)
GLUT4 mRNA
(equalised using GAPDH)
(D) (i)
WT
1
3bKO
p= 0.01 *
0.5
0 WT
Brn-3bKO
6