Publications by authors named "Rugivan Sabaratnam"

15 Publications

  • Page 1 of 1

HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution.

Cell Rep 2022 Jul;40(4):111136

Oxford Centre for Diabetes, Endocrinology, and Metabolism, Radcliffe Department of Medicine, University of Oxford, Churchill Hospital, Headington OX3 7LE, UK. Electronic address:

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development.
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http://dx.doi.org/10.1016/j.celrep.2022.111136DOI Listing
July 2022

A Signature of Exaggerated Adipose Tissue Dysfunction in Type 2 Diabetes Is Linked to Low Plasma Adiponectin and Increased Transcriptional Activation of Proteasomal Degradation in Muscle.

Cells 2022 Jun 23;11(13). Epub 2022 Jun 23.

Steno Diabetes Center Odense, Odense University Hospital, DK-5000 Odense C, Denmark.

Insulin resistance in skeletal muscle in type 2 diabetes (T2D) is characterized by more pronounced metabolic and molecular defects than in obesity . There is increasing evidence that adipose tissue dysfunction contributes to obesity-induced insulin resistance in skeletal muscle. Here, we used an unbiased approach to examine if adipose tissue dysfunction is exaggerated in T2D and linked to diabetes-related mechanisms of insulin resistance in skeletal muscle. Transcriptional profiling and biological pathways analysis were performed in subcutaneous adipose tissue (SAT) and skeletal muscle biopsies from 17 patients with T2D and 19 glucose-tolerant, age and weight-matched obese controls. Findings were validated by qRT-PCR and western blotting of selected genes and proteins. Patients with T2D were more insulin resistant and had lower plasma adiponectin than obese controls. Transcriptional profiling showed downregulation of genes involved in mitochondrial oxidative phosphorylation and the tricarboxylic-acid cycle and increased expression of extracellular matrix (ECM) genes in SAT in T2D, whereas genes involved in proteasomal degradation were upregulated in the skeletal muscle in T2D. qRT-PCR confirmed most of these findings and showed lower expression of adiponectin in SAT and higher expression of myostatin in muscle in T2D. Interestingly, muscle expression of proteasomal genes correlated positively with SAT expression of ECM genes but inversely with the expression of in SAT and plasma adiponectin. Protein content of proteasomal subunits and major ubiquitin ligases were unaltered in the skeletal muscle of patients with T2D. A transcriptional signature of exaggerated adipose tissue dysfunction in T2D, compared with obesity alone, is linked to low plasma adiponectin and increased transcriptional activation of proteasomal degradation in skeletal muscle.
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http://dx.doi.org/10.3390/cells11132005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9265693PMC
June 2022

TCF7L2 plays a complex role in human adipose progenitor biology, which might contribute to genetic susceptibility to type 2 diabetes.

Metabolism 2022 08 10;133:155240. Epub 2022 Jun 10.

Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LE, UK; NIHR Oxford Biomedical Research Centre, OUH Foundation Trust, Oxford OX3 7LE, UK. Electronic address:

Introduction: Non-coding genetic variation at TCF7L2 is the strongest genetic determinant of type 2 diabetes (T2D) risk in humans. TCF7L2 encodes a transcription factor mediating the nuclear effects of WNT signaling in adipose tissue (AT). In vivo studies in transgenic mice have highlighted important roles for TCF7L2 in adipose tissue biology and systemic metabolism.

Objective: To map the expression of TCF7L2 in human AT, examine its role in human adipose cell biology in vitro, and investigate the effects of the fine-mapped T2D-risk allele at rs7903146 on AT morphology and TCF7L2 expression.

Methods: Ex vivo gene expression studies of TCF7L2 in whole and fractionated human AT. In vitro TCF7L2 gain- and/or loss-of-function studies in primary and immortalized human adipose progenitor cells (APCs) and mature adipocytes (mADs). AT phenotyping of rs7903146 T2D-risk variant carriers and matched controls.

Results: Adipose progenitors (APs) exhibited the highest TCF7L2 mRNA abundance compared to mature adipocytes and adipose-derived endothelial cells. Obesity was associated with reduced TCF7L2 transcript levels in whole subcutaneous abdominal AT but paradoxically increased expression in APs. In functional studies, TCF7L2 knockdown (KD) in abdominal APs led to dose-dependent activation of WNT/β-catenin signaling, impaired proliferation and dose-dependent effects on adipogenesis. Whilst partial KD enhanced adipocyte differentiation, near-total KD impaired lipid accumulation and adipogenic gene expression. Over-expression of TCF7L2 accelerated adipogenesis. In contrast, TCF7L2-KD in gluteal APs dose-dependently enhanced lipid accumulation. Transcriptome-wide profiling revealed that TCF7L2 might modulate multiple aspects of AP biology including extracellular matrix secretion, immune signaling and apoptosis. The T2D-risk allele at rs7903146 was associated with reduced AP TCF7L2 expression and enhanced AT insulin sensitivity.

Conclusions: TCF7L2 plays a complex role in AP biology and has both dose- and depot-dependent effects on adipogenesis. In addition to regulating pancreatic insulin secretion, genetic variation at TCF7L2 might also influence T2D risk by modulating AP function.
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http://dx.doi.org/10.1016/j.metabol.2022.155240DOI Listing
August 2022

Factors mediating exercise-induced organ crosstalk.

Acta Physiol (Oxf) 2022 02 18;234(2):e13766. Epub 2022 Jan 18.

Steno Diabetes Center Odense, Odense University Hospital, Odense C, Denmark.

Exercise activates a plethora of metabolic and signalling pathways in skeletal muscle and other organs causing numerous systemic beneficial metabolic effects. Thus, regular exercise may ameliorate and prevent the development of several chronic metabolic diseases. Skeletal muscle is recognized as an important endocrine organ regulating systemic adaptations to exercise. Skeletal muscle may mediate crosstalk with other organs through the release of exercise-induced cytokines, peptides and proteins, termed myokines, into the circulation. Importantly, other tissues such as the liver and adipose tissue may also release cytokines and peptides in response to exercise. Hence, exercise-released molecules are collectively called exerkines. Moreover, extracellular vesicles (EVs), in the form of exosomes or microvesicles, may carry some of the signals involved in tissue crosstalk. This review focuses on the role of factors potentially mediating crosstalk between muscle and other tissues in response to exercise.
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http://dx.doi.org/10.1111/apha.13766DOI Listing
February 2022

Adipocyte-Endothelium Crosstalk in Obesity.

Front Endocrinol (Lausanne) 2021 12;12:681290. Epub 2021 Aug 12.

Department of Molecular Medicine, Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark.

Obesity is characterized by pathological adipose tissue (AT) expansion. While healthy AT expansion enhances systemic insulin sensitivity, unhealthy AT expansion through increased adipocyte size is associated with insulin resistance, fibrosis, hypoxia, and reduced adipose-derived adiponectin secretion. The mechanisms causing the unhealthy AT expansion are not fully elucidated; yet, dysregulated crosstalk between cells within the AT is an important contributor. Evidence from animal and human studies suggests a crucial role of the crosstalk between vascular endothelium (the innermost cell type in blood vessels) and adipocytes for metabolic homeostasis. Arterial endothelial cells are directly involved in maintaining normal organ functions through local blood flow regulation. The endothelial-dependent regulation of blood flow in AT is hampered in obesity, which negatively affects the adipocyte. Moreover, endothelial cells secrete extracellular vesicles (EVs) that target adipocytes . The endothelial EVs secretion is hampered in obesity and may be affected by the adipocyte-derived adipokine adiponectin. Adiponectin targets the vascular endothelium, eliciting organ-protective functions through binding to T-cadherin. The reduced obesity-induced adiponectin binding of T-cadherin reduces endothelial EV secretion. This affects endothelial health and cell-cell communication between AT cells and distant organs, influencing systemic energy homeostasis. This review focuses on the current understanding of endothelial and adipocyte crosstalk. We will discuss how obesity changes the AT environment and how these changes contribute to obesity-associated metabolic disease in humans. Particularly, we will describe and discuss the EV-dependent communication and regulation between adipocytes, adiponectin, and the endothelial cells regulating systemic energy homeostasis in health and metabolic disease in humans.
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http://dx.doi.org/10.3389/fendo.2021.681290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387580PMC
December 2021

Effect of long-term testosterone therapy on molecular regulators of skeletal muscle mass and fibre-type distribution in aging men with subnormal testosterone.

Metabolism 2020 11 25;112:154347. Epub 2020 Aug 25.

Steno Diabetes Center Odense, Odense University Hospital, DK-5000 Odense C, Denmark; Department of Clinical Research & Department of Molecular Medicine, University of Southern Denmark, DK-5000 Odense C, Denmark. Electronic address:

Background: Long-term testosterone replacement therapy (TRT) increases muscle mass in elderly men with subnormal testosterone levels. However, the molecular mechanisms underlying this effect of TRT on protein balance in human skeletal muscle in vivo remain to be established.

Methods: Here, we examined skeletal muscle biopsies obtained before and 24-h after the last dose of treatment with either testosterone gel (n = 12) or placebo (n = 13) for 6 months in aging men with subnormal bioavailable testosterone levels. The placebo-controlled, testosterone-induced changes (β-coefficients) in mRNA levels, protein expression and phosphorylation were examined by quantitative real-time PCR and western blotting.

Results: Long-term TRT increased muscle mass by β = 1.6 kg (p = 0.01) but had no significant effect on mRNA levels of genes involved in myostatin/activin/SMAD or IGF1/FOXO3 signalling, muscle-specific E3-ubiquitin ligases, upstream transcription factors (MEF2C, PPARGC1A-4) or myogenic factors. However, TRT caused a sustained decrease in protein expression of SMAD2 (β = -36%, p = 0.004) and SMAD3 (β = -32%, p = 0.001), which was accompanied by reduced protein expression of the muscle-specific E3-ubiquitin ligases, MuRF1 (β = -26%, p = 0.004) and Atrogin-1/MAFbx (β = -20%, p = 0.04), but with no changes in FOXO3 signalling. Importantly, TRT did not affect muscle fibre type distribution between slow-oxidative (type 1), fast-oxidative (type 2a) and fast-glycolytic (type 2×) muscle fibres.

Conclusions: Our results indicate that long-term TRT of elderly men with subnormal testosterone levels increases muscle mass, at least in part, by decreasing protein breakdown through the ubiquitin proteasome pathway mediated by a sustained suppression of SMAD-signalling and muscle-specific E3-ubiquitin ligases.
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http://dx.doi.org/10.1016/j.metabol.2020.154347DOI Listing
November 2020

Urinary extracellular vesicles: Origin, role as intercellular messengers and biomarkers; efficient sorting and potential treatment options.

Acta Physiol (Oxf) 2020 01 1;228(1):e13346. Epub 2019 Aug 1.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

Urinary extracellular vesicles (uEVs) are a heterogenous group of vesicles consisting mainly of microvesicles and exosomes that originate predominantly (99.96%) from kidney, the urinary tract epithelium and the male reproductive tract. Secreted EVs contain molecular cargo from parental cells and provide an attractive source for biomarkers, a potential readout of physiological and pathophysiological mechanisms, and events associated with the urinary system. uEVs are readily enriched and isolated from urine samples and we review 6 standard methods that allow for downstream analysis of the uEV cargo. Although the use of uEVs as a surrogate readout for physiological changes in tissue protein levels is widespread, the protein abundance in uEVs is affected significantly by mechanisms that regulate protein sorting and secretion in uEVs. Data suggest that baseline kidney tissue and uEV levels of apical membrane-associated electrolyte transport proteins are not directly related in human patients. Recent evidence indicates that EVs may contribute to physiological and pathophysiological intercellular signalling and EVs confer protection against renal ischemia-reperfusion injury. The therapeutic use of EVs as information carriers has mainly been explored in vitro and a major hurdle lies in the translation of the in vitro findings into an in vivo setting. Thus, the EV research field is moving from a technical focus to a more physiological focus, allowing for a deeper understanding of human physiology, development of diagnostic tools and potential treatment strategies for precision medicine.
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http://dx.doi.org/10.1111/apha.13346DOI Listing
January 2020

In human nephrectomy specimens, the kidney level of tubular transport proteins does not correlate with their abundance in urinary extracellular vesicles.

Am J Physiol Renal Physiol 2019 09 26;317(3):F560-F571. Epub 2019 Jun 26.

Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

Human urinary extracellular vesicles (uEVs) contain proteins from all nephron segments. An assumption for years has been that uEVs might provide a noninvasive liquid biopsy that reflect physiological regulation of transporter protein expression in humans. We hypothesized that protein abundance in human kidney tissue and uEVs are directly related and tested this in paired collections of nephrectomy tissue and urine sample from 12 patients. Kidney tissue was fractioned into total kidney protein, crude membrane (plasma membrane and large intracellular vesicles)-enriched, and intracellular vesicle-enriched fractions as well as sections for immunolabeling. uEVs were isolated from spot urine samples. Antibodies were used to quantify six segment-specific proteins [proximal tubule-expressed Na-phosphate cotransporters (NaPi-2a), thick ascending limb-expressed Tamm-Horsfall protein and renal outer medullary K channels, distal convoluted tubule-expressed NaCl cotransporters, intercalated cell-expressed V-type H-ATPase subunit G3 (ATP6V1G3), and principal cell-expressed aquaporin 2] and three uEV markers (exosomal CD63, microvesicle marker vesicle-associated membrane protein 3, and β-actin) in each fraction. By Western blot analysis and immunofluorescence labeling, we found significant positive correlations between the abundance of CD63, NaCl cotransporters, aquaporin 2, and ATP6V1G3, respectively, within the different kidney-derived fractions. We detected all nine proteins in uEVs, but their level did not correlate with kidney tissue protein abundance. uEV protein levels showed higher interpatient variability than kidney-derived fractions, indicating that factors, besides kidney protein abundance, contribute to the uEV protein level. Our data suggest that, in a random sample of nephrectomy patients, uEV protein level is not a predictor of kidney protein abundance.
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http://dx.doi.org/10.1152/ajprenal.00242.2019DOI Listing
September 2019

Exercise Induction of Key Transcriptional Regulators of Metabolic Adaptation in Muscle Is Preserved in Type 2 Diabetes.

J Clin Endocrinol Metab 2019 10;104(10):4909-4920

Steno Diabetes Center Odense, Odense University Hospital, Odense C, Denmark.

Context: Type 2 diabetes (T2D) is characterized by insulin resistance in skeletal muscle. Regular exercise improves insulin sensitivity, mitochondrial function, and energy metabolism. Thus, an impaired response to exercise may contribute to insulin resistance.

Objective: We hypothesized that key transcriptional regulators of metabolic adaptation to exercise show an attenuated response in skeletal muscle in T2D.

Design And Patients: Skeletal muscle biopsies were obtained from 13 patients with T2D and 14 age- and weight-matched controls before, immediately after 1 hour acute exercise (70% maximal pulmonary oxygen uptake), and 3 hours into recovery to examine mRNA expression of key transcription factors and downstream targets and activity of key upstream kinases underlying the metabolic adaptation to exercise.

Results: Acute exercise increased gene expression of the nuclear hormone receptor 4A (NR4A) subfamily (∼4- to 36-fold) and other key transcription factors, including ATF3, EGR1, JUNB, SIK1, PPARA, and PPARG (∼1.5- to 12-fold), but with no differences between groups. The expression of NR4A1 (approximately eightfold) and NR4A3 (∼75-fold) was further increased 3 hours into recovery, whereas most muscle transcripts sustained elevated or returned to basal levels, again with no differences between groups. Muscle expression of HKII and SLC2A4 and hexokinase II protein content were reduced in patients with T2D. The phosphorylation of p38 MAPK, Erk1/2, Ca2+/calmodulin-dependent kinase II, and cAMP-responsive element-binding protein was equally increased in response to exercise and/or recovery in both groups.

Conclusion: Acute exercise elicits a pronounced and overall similar increase in expression of key transcription factors and activation of key upstream kinases involved in muscle metabolic adaptation to exercise in patients with T2D and weight-matched controls.
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http://dx.doi.org/10.1210/jc.2018-02679DOI Listing
October 2019

Acute Exercise Increases Plasma Levels of Muscle-Derived Microvesicles Carrying Fatty Acid Transport Proteins.

J Clin Endocrinol Metab 2019 10;104(10):4804-4814

Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark.

Context: Microvesicles (MVs) are a class of membrane particles shed by any cell in the body in physiological and pathological conditions. They are considered to be key players in intercellular communication, and with a molecular content reflecting the composition of the cell of origin, they have recently emerged as a promising source of biomarkers in a number of diseases.

Objective: The effects of acute exercise on the plasma concentration of skeletal muscle-derived MVs (SkMVs) carrying metabolically important membrane proteins were examined.

Participants: Thirteen men with obesity and type 2 diabetes mellitus (T2DM) and 14 healthy male controls with obesity exercised on a cycle ergometer for 60 minutes.

Interventions: Muscle biopsies and blood samples-obtained before exercise, immediately after exercise, and 3 hours into recovery-were collected for the analysis of long-chain fatty acid (LCFA) transport proteins CD36 (a scavenger receptor class B protein) and fatty acid transport protein 4 (FATP4) mRNA content in muscle and for flow cytometric studies on circulating SkMVs carrying either LCFA transport protein.

Results: Besides establishing a flow cytometric approach for the detection of circulating SkMVs and subpopulations carrying either CD36 or FATP4 and thereby adding proof to their existence, we demonstrated an overall exercise-induced change of SkMVs carrying these LCFA transport proteins. A positive correlation between exercise-induced changes in skeletal muscle CD36 mRNA expression and concentrations of SkMVs carrying CD36 was found in T2DM only.

Conclusions: This approach could add important real-time information about the abundance of LCFA transport proteins present on activated muscle cells in subjects with impaired glucose metabolism.
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http://dx.doi.org/10.1210/jc.2018-02547DOI Listing
October 2019

Intact regulation of muscle expression and circulating levels of myokines in response to exercise in patients with type 2 diabetes.

Physiol Rep 2018 06;6(12):e13723

Section of Molecular Diabetes & Metabolism, Institute of Clinical Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.

Regular exercise plays an important role in the prevention and treatment of type 2 diabetes (T2D). The synthesis and secretion of myokines in response to contraction may contribute to the beneficial metabolic effects of exercise. However, some exercise-induced responses may be attenuated in T2D. Here, we investigated whether the effect of acute exercise on selected myokines are impaired in T2D. Skeletal muscle biopsies and blood samples were obtained from 13 men with T2D and 14 weight-matched, glucose-tolerant men before, immediately after and 3-h after acute exercise (60 min cycling) to examine muscle expression and plasma/serum levels of selected myokines. One-hour of exercise increased muscle expression of IL6, FGF21, ANGPTL4, CHI3L1, CTGF and CYR61, of which FGF21, ANGPTL4 and CHI3L1 increased further 3-h into recovery, whereas expression of IL6, CYR61, and CTGF returned to baseline levels. There was no immediate effect of exercise on IL15 expression, but it decreased 3-h into recovery. Plasma IL-6 increased robustly, whereas circulating levels of FGF21, ANGPTL4, IL-15, and CHI3L1 increased only modestly in response to exercise. All returned toward baseline levels 3-h into recovery except for plasma ANGPTL4, which increased further. No significant differences in these responses to exercise were observed between the groups. Our results demonstrate that muscle expression and circulating levels of selected known and putative myokines were equally regulated by acute exercise in patients with T2D and weight-matched controls. This suggests that the potential beneficial metabolic effects of these myokines are not impaired in patients with T2D.
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http://dx.doi.org/10.14814/phy2.13723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009776PMC
June 2018

The prevalent deep intronic c. 639+919 G>A GLA mutation causes pseudoexon activation and Fabry disease by abolishing the binding of hnRNPA1 and hnRNP A2/B1 to a splicing silencer.

Mol Genet Metab 2016 11 27;119(3):258-269. Epub 2016 Aug 27.

Department of Biochemistry and Molecular Biology, the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark. Electronic address:

Fabry disease is an X-linked recessive inborn disorder of the glycosphingolipid metabolism, caused by total or partial deficiency of the lysosomal α-galactosidase A enzyme due to mutations in the GLA gene. The prevalent c.639+919 G>A mutation in GLA leads to pathogenic insertion of a 57bp pseudoexon sequence from intron 4, which is responsible for the cardiac variant phenotype. In this study we investigate the splicing regulatory mechanism leading to GLA pseudoexon activation. Splicing analysis of GLA minigenes revealed that pseudoexon activation is influenced by cell-type. We demonstrate that the wild-type sequence harbors an hnRNP A1 and hnRNP A2/B1-binding exonic splicing silencer (ESS) overlapping the 5'splice site (5'ss) that prevents pseudoexon inclusion. The c.639+919 G>A mutation disrupts this ESS allowing U1 snRNP recognition of the 5'ss. We show that the wild-type GLA 5'ss motif with the ESS is also able to inhibit inclusion of an unrelated pseudoexon in the FGB gene, and that also in the FGB context inactivation of the ESS by the c.639+919 G>A mutation causes pseudoexon activation, underscoring the universal nature of the ESS. Finally, we demonstrate that splice switching oligonucleotide (SSO) mediated blocking of the pseudoexon 3'ss and 5'ss effectively restores normal GLA splicing. This indicates that SSO based splicing correction may be a therapeutic alternative in the treatment of Fabry disease.
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http://dx.doi.org/10.1016/j.ymgme.2016.08.007DOI Listing
November 2016

Intact Regulation of the AMPK Signaling Network in Response to Exercise and Insulin in Skeletal Muscle of Male Patients With Type 2 Diabetes: Illumination of AMPK Activation in Recovery From Exercise.

Diabetes 2016 05 28;65(5):1219-30. Epub 2016 Jan 28.

Section of Molecular Physiology, August Krogh Centre, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Copenhagen, Denmark

Current evidence on exercise-mediated AMPK regulation in skeletal muscle of patients with type 2 diabetes (T2D) is inconclusive. This may relate to inadequate segregation of trimeric complexes in the investigation of AMPK activity. We examined the regulation of AMPK and downstream targets ACC-β, TBC1D1, and TBC1D4 in muscle biopsy specimens obtained from 13 overweight/obese patients with T2D and 14 weight-matched male control subjects before, immediately after, and 3 h after exercise. Exercise increased AMPK α2β2γ3 activity and phosphorylation of ACCβ Ser(221), TBC1D1 Ser(237)/Thr(596), and TBC1D4 Ser(704) Conversely, exercise decreased AMPK α1β2γ1 activity and TBC1D4 Ser(318)/Thr(642) phosphorylation. Interestingly, compared with preexercise, 3 h into exercise recovery, AMPK α2β2γ1 and α1β2γ1 activity were increased concomitant with increased TBC1D4 Ser(318)/Ser(341)/Ser(704) phosphorylation. No differences in these responses were observed between patients with T2D and control subjects. Subjects were also studied by euglycemic-hyperinsulinemic clamps performed at rest and 3 h after exercise. We found no evidence for insulin to regulate AMPK activity. Thus, AMPK signaling is not compromised in muscle of patients with T2D during exercise and insulin stimulation. Our results reveal a hitherto unrecognized activation of specific AMPK complexes in exercise recovery. We hypothesize that the differential regulation of AMPK complexes plays an important role for muscle metabolism and adaptations to exercise.
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http://dx.doi.org/10.2337/db15-1034DOI Listing
May 2016

Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells.

Nucleic Acids Res 2015 May 15;43(9):4627-39. Epub 2015 Apr 15.

Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3'splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.
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http://dx.doi.org/10.1093/nar/gkv275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4482064PMC
May 2015

The ETFDH c.158A>G variation disrupts the balanced interplay of ESE- and ESS-binding proteins thereby causing missplicing and multiple Acyl-CoA dehydrogenation deficiency.

Hum Mutat 2014 Jan 28;35(1):86-95. Epub 2013 Oct 28.

Research Unit for Molecular Medicine, Aarhus University Hospital and Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.

Multiple acyl-CoA dehydrogenation deficiency is a disorder of fatty acid and amino acid oxidation caused by defects of electron transfer flavoprotein (ETF) or its dehydrogenase (ETFDH). A clear relationship between genotype and phenotype makes genotyping of patients important not only diagnostically but also for prognosis and for assessment of treatment. In the present study, we show that a predicted benign ETFDH missense variation (c.158A>G/p.Lys53Arg) in exon 2 causes exon skipping and degradation of ETFDH protein in patient samples. Using splicing reporter minigenes and RNA pull-down of nuclear proteins, we show that the c.158A>G variation increases the strength of a preexisting exonic splicing silencer (ESS) motif UAGGGA. This ESS motif binds splice inhibitory hnRNP A1, hnRNP A2/B1, and hnRNP H proteins. Binding of these inhibitory proteins prevents binding of the positive splicing regulatory SRSF1 and SRSF5 proteins to nearby and overlapping exonic splicing enhancer elements and this causes exon skipping. We further suggest that binding of hnRNP proteins to UAGGGA is increased by triggering synergistic hnRNP H binding to GGG triplets located upstream and downsteam of the UAGGGA motif. A number of disease-causing exonic elements that induce exon skipping in other genes have a similar architecture as the one in ETFDH exon 2.
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http://dx.doi.org/10.1002/humu.22455DOI Listing
January 2014
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