Article. Reference. MicroRNAs 103 and 107 regulate insulin sensitivity. TRAJKOVSKI, Mirko, et al.

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1 Article MicroRNAs and 7 regulate insulin sensitivity TRAJKOVSKI, Mirko, et al. Abstract Defects in insulin signalling are among te most common and earliest defects tat predispose an individual to te development of type diabetes. MicroRNAs ave been identified as a new class of regulatory molecules tat influence many biological functions, including metabolism. However, te direct regulation of insulin sensitivity by micrornas in vivo as not been demonstrated. Here we sow tat te expression of micrornas and 7 (mir-/7) is upregulated in obese mice. Silencing of mir-/7 leads to improved glucose omeostasis and insulin sensitivity. In contrast, gain of mir-/7 function in eiter liver or fat is sufficient to induce impaired glucose omeostasis. We identify caveolin-, a critical regulator of te insulin receptor, as a direct target gene of mir-/7. We demonstrate tat caveolin- is upregulated upon mir-/7 inactivation in adipocytes and tat tis is concomitant wit stabilization of te insulin receptor, enanced insulin signalling, decreased adipocyte size and enanced insulin-stimulated glucose uptake. Tese findings demonstrate te central importance of mir-/7 to [...] Reference TRAJKOVSKI, Mirko, et al. MicroRNAs and 7 regulate insulin sensitivity. Nature,, vol. 7, no. 7, p. 9- DOI :./nature PMID : 7 Available at: ttp://arcive-ouverte.unige.c/unige: Disclaimer: layout of tis document may differ from te publised version.

2 LETTER doi:./nature MicroRNAs and 7 regulate insulin sensitivity Mirko Trajkovski,, Jean Hausser,,Jürgen Soutscek, Bal Bat, Akinc Akin, Miaela Zavolan, Markus H. Heim, & Markus Stoffel, Defects in insulin signalling are among te most common and earliest defects tat predispose an individual to te development of type diabetes. MicroRNAs ave been identified as a new class of regulatory molecules tat influence many biological functions, including metabolism,. However, te direct regulation of insulin sensitivity by micrornas in vivo as not been demonstrated. Here we sow tat te expression of micrornas and 7 (mir-/ 7) is upregulated in obese mice. Silencing of mir-/7 leads to improved glucose omeostasis and insulin sensitivity. In contrast, gain of mir-/7 function in eiter liver or fat is sufficient to induce impaired glucose omeostasis. We identify caveolin-, a critical regulator of te insulin receptor, as a direct target gene of mir-/7. We demonstrate tat caveolin- is upregulated upon mir-/7 inactivation in adipocytes and tat tis is concomitant wit stabilization of te insulin receptor, enanced insulin signalling, decreased adipocyte size and enanced insulin-stimulated glucose uptake. Tese findings demonstrate te central importance of mir-/7 to insulin sensitivity and identify a new target for te treatment of type diabetes and obesity. To identify micrornas (mirnas) tat are deregulated in obesity and insulin resistance, we performed mirna microarray analysis on te livers of two types of obese mice: mice and diet-inducedobese () C7BL/J mice (Supplementary Table a c). Te mir- /mir-7 family was among te five most-upregulated mirnas in te livers of bot obese models, and te expression of tese mirnas was also reportedly increased in diabetic Goto-Kakizaki rats. Te expression levels were validated by nortern blotting, demonstrating a twofold to treefold upregulation in te livers of bot models (Fig. a). Te sequences of mature mir- and mir-7 differ by one nucleotide at position and cannot be discriminated by nortern blotting. By real-time PCR, we could distinguis mir- and mir- 7 and sow tat bot mirnas are upregulated in te livers of and mice (Supplementary Fig. a d). We also measured te expression of tese mirnas in liver biopsies from a coort of uman patients. mir- and mir-7 levels were similar in normal subjects and in subjects wit viral epatitis, but increased in alcoolic liver disease (ALD), non-alcoolic fatty liver disease (NAFLD) and nonalcoolic steatoepatitis (NASH), conditions often associated wit diabetes 7. Furtermore, tere was a positive correlation between te subjects omeostatic model assessment (HOMA) index and mir/ 7 expression levels (Fig. b and Supplementary Fig. a c), indicating an association of tese mirnas wit insulin resistance. To investigate te effect of elevated mir-/7 expression, we generated recombinant adenovirus expressing mir-7 (ad-7/ GFP). Injection of wild-type mice wit ad-7/gfp (Supplementary Fig. a, b) caused a rise in bot random and fasting blood-glucose levels, and also in insulin levels (Fig. c, d). It impaired glucose tolerance after an intra-peritoneal glucose injection and decreased insulin sensitivity relative to tat in control ad-gfp-infected mice (Fig. e, f). Hepatic overexpression of mir-7 resulted in increased glucose production during an intraperitoneal pyruvate-tolerance test (Fig. g). Te increase in epatic glucose production was accompanied by augmented expression of glucose -pospatase, pospoenolpyruvate carboxykinase, pyruvate carboxylase and fructose,- bipospatase, indicating tat increased gluconeogenesis is te primary cause of te elevated glucose levels (Fig. ). Tese data sow tat gainof-function of mir-7 in te liver decreases insulin sensitivity and enances epatic glucose production. To study te effect of mir-/7 silencing, we first tested weter antagomirs would inibit bot mir- and mir-7. Nortern blot analysis of mir- and mir-7 sowed tat antagomir- (ant- ) effectively and specifically silenced bot mirnas in liver and fat (Supplementary Fig. c, d). Markers for liver damage and inflammation were unaffected by te treatment (data not sown). Application of ant- did not affect blood glucose levels in cow-fed wild-type mice but it did lower plasma glucose levels in mice wen compared to mice treated wit or wit te controls ant- (scrambled) or mismatcant- (ant-mm) (Fig. a, b). Similar effects were observed in mice (Fig. c). Glucose-tolerance and insulin-tolerance tests sowed tat tere was improved glucose tolerance and insulin sensitivity in bot and mice tat were injected wit ant- (Fig. d f). A pyruvate-tolerance test revealed tat de novo epatic glucose production was reduced (Fig. g) and tis finding was supported by a reduction in epatic expression of glucose -pospatase, pyruvate carboxylase and fructose,-bipospatase in ant--treated mice (Fig. ). In addition, liver glycogen content was increased and plasma insulin levels were decreased in ant--treated and animals (Fig. i, j). Metabolic and energy-expenditure studies carried out in metabolic cages sowed tat mice lacking mir- expression ad increased O consumption and CO production, as well as moderately elevated body temperature, but tat teir food intake was similar to control mice. Gene expression analysis in adipocytes from ant-- treated mice revealed increased levels of b-oxidation genes (carnitine palmitoyltransferase a (Cpta), peroxisomal acyl-coenzyme A oxidase (Acox) and very long cain acyl-coenzyme A deydrogenase (Acadvl)) but tere were no canges in te lipogenic genes acetylcoenzyme A carboxylase alpa and beta (Acaca and Acacb, also known as Acc and Acc) (Supplementary Fig. a d). Two independent indicators of insulin sensitivity, te glucose infusion rate and clamp glucose turnover, were improved during yperinsulinaemic-euglycaemic clamp studies in ant--treated mice compared to -injected controls. Hepatic glucose production was decreased and glucose uptake in adipose tissue was enanced in ant--treated animals. In contrast, treatment wit ant- did not improve glucose uptake in skeletal muscle (Supplementary Table ). Togeter, tese data demonstrate tat silencing of mir-/7 enances insulin sensitivity in liver and adipose tissue. To test te contribution of te liver to te effect on insulin sensitivity, we delivered ant- and control ant-mm specifically to te liver troug liposomal formulations. Liposomal ant- induced te specific silencing of mir- in te liver, but not in fat and muscle. Silencing of mir-/7 in te livers of mice ad no effect on Institute for Molecular Systems Biology, ETH Zuric, Wolfgang-Pauli Strasse, CH-9 Zuric, Switzerland. Competence Center of Systems Pysiology and Metabolic Disease, ETH Zuric, Scafmattstrasse, HPM F 9. CH-9 Zuric, Switzerland. Biozentrum Basel, University of Basel, Klingelbergstrasse /7, CH- Basel, Switzerland. Regulus Terapeutics Inc., Jon Hopkins Court, San Diego, California 9-, USA. Alnylam Parmaceuticals, Tird Street, Cambridge, Massacusetts, USA. University Hospital Basel, Hebelstrasse, CH- Basel, Switzerland. Macmillan Publisers Limited. All rigts reserved JUNE VOL 7 NATURE 9

3 RESEARCH LETTER a mir-7 mir- Total RNA c Random blood glucose (mm) 9 7 Day WT Day 7 Day Cow Ad-7/GFP Day ( fast) b Relative mir- expression d Insulin (ng ml )..... HOMA Ad-7/GFP fast re-fed re-fed R =. P. Figure Hepatic overexpression of mir-7 induces yperglycaemia. a, Nortern blot of liver RNA from C7BL/J (WT),, cow-fed or mice, as indicated (n ). Te loading control labelled total RNA is stained wit etidium bromide. b, Correlation between relative mir- levels (Rel. mir-) and HOMA index in a group of umans including ealty individuals (n ), cronic epatitis B and epatitis C virus-infected individuals (HBV, n ; HCV, n 7), patients wit alcoolic steatoepatitis (n ), patients wit non-alcoolic fatty liver disease (n ) and patients wit non-alcoolic steatoepatitis (n ). c, Blood glucose levels of C7BL/J mice injected wit e g Ad-7/GFP Ad-7/GFP f Blood glucose (%) Relative expression levels Ad-7/GFP Ad-7/GFP GPc Pepck PC FBPase, ad-gfp or ad-7/gfpbl/ (n ). d, Plasma insulin levels of C7BL/J mice treated as in c, after a fast followed by re-feeding. e g, Glucosetolerance test (e), insulin-tolerance test (f) and pyruvate-tolerance test (g) in mice injected wit ad-gfp or ad-7/gfp., Relative mrna expression of genes encoding glucose -pospatase (GPc), pospoenol-pyruvate carboxykinase (Pepck), pyruvate carboxylase (PC) and fructose,-bipospatase (FBPase) fromlivers of miceas in e g. Expression is normalized to te B gene, encoding te acidic ribosomal pospoprotein P (RPLP) (n ). Means s.d. are sown for all panels., P,.;, P,.;, P,.. a b c Ant-MM d e Blood glucose fast (mm) Day Day Ant- C7BL/ Day Day Ant-MM Ant- Day Day Day Day (random) Day ( fast) Ant- C7BL/, Day Day Day 9 Day Day 7 ( fast) C7BL/ Ant- Ant- Cow f Blood glucose (%) Ant- g C7BL/ Ant GPc Ant-MM Ant- PC FBPase i Liver glycogen content Ant-MM Ant- j Plasma insulin (ng ml ) fast Ant-MM Ant- re-fed 9 7 fast Figure Silencing of mir- and mir-7 alleviates yperglycaemia in diabetic mice. a c, Blood glucose levels of ant--treated or control-treated (, scrambled or ant-mm) (n ), C7BL/J (n ) or (n ) mice. Days after treatment and random or fasting conditions are noted for eac measurement. d, e, Glucose-tolerance tests in control or ant-- injected (d) or (e) mice compared to control C7BL/J or cow-fed mice (n ). f, g, Insulin-tolerance test (f) or pyruvate-tolerance test (g) in control or ant--injected mice (n ). In f, values at te zero time point are normalized to %., Relative mrna expression of te genes for GPc, PC and FBPase in livers of mice d after injection wit ant- MM or ant- (n ). i, Liver glycogen content in mice d after injection wit ant-mm or ant- (n ). j, Insulin levels in mice (left, n ) or mice (rigt, n ) d after injection of ant-mm or ant-. In d, e and g, -injected C7BL/J mice (n ) are sown as controls. Means s.d. are sown for all panels., P,.;, P,.;, P,.. NATURE VOL 7 JUNE Macmillan Publisers Limited. All rigts reserved

4 LETTER RESEARCH blood glucose levels, plasma insulin levels or glucose-tolerance, insulin-tolerance and pyruvate-tolerance tests (Supplementary Fig. a f), indicating tat silencing of mir-/7 in te liver is not sufficient to reverse insulin resistance in obese mice. Because te expression of mir- is about eigtfold iger in adipose tissue tan in liver and muscle, we examined te effect of mir-/7 silencing in adipose tissue. Obese () mice sowed a sligt reduction in body weigt wen mir-/7 were systemically silenced (Supplementary Fig. a). In contrast, specific manipulation of epatic mir-/7 expression using liposomal ant- or Ad-7/GFP did not affect body weigt wen compared to tat of control-treated mice (data not sown). We terefore used computer tomograpy to investigate te fat distribution of and animals after mir- silencing. Bot and mice treated wit ant- sowed reduced levels of total fat, owing to a decrease in bot subcutaneous and visceral adipose tissue (Fig. a, b). Furtermore, organ measurements revealed a decrease in inguinal fat-pad weigts in te ant--treated group but no weigt differences in oter organs (Supplementary Fig. b). To investigate weter tis reduction was due to lower cell numbers or smaller adipocytes, we quantified te mean size of adipocytes from fat tissue sections using automated image-analysis software. Ant-- treated and animals ad smaller adipocytes tan ant- MM-injected controls (Fig. c, d), owing to an increased number of small adipocytes and a decreased number of large ones (Supplementary Fig. c f). A comparison between te decrease in fat-pad size, measured by computer tomograpy, and te average decrease in adipocyte size sowed tat ant--treated mice ad approximately % more adipocytes tan ant-mm-treated controls. Because mir- as been implicated in adipocyte differentiation 9, we explored weter te increase in adipocyte number in mir--depleted mice could be attributed to canges in pre-adipocyte differentiation. We induced adipocyte differentiation of isolated stromal-vascular cells from bot visceral and subcutaneous fat in te presence of eiter ant- or ant-mm. Quantification of mature adipocyte numbers by ig-content imaging after d in culture demonstrated a -fold and.-fold increase in te number of differentiated adipocytes in te ant--treated stromal-vascular cells derived from visceral and subcutaneous fat, respectively. Tis indicates tat te absence of mir- enances adipocyte differentiation in a cell-autonomous fasion. Conversely, overexpression of mir-7 decreased te number of differentiated adipocytes (Fig. e). Te negative effect of mir- on pre-adipocyte differentiation was furter corroborated by gene expression analysis of an early marker of adipocyte differentiation, CCAAT/enancer binding protein (C/ EBP)-b (Cebpb), and of two late markers of adipocyte differentiation, peroxisome proliferator activated receptor-c (Pparg) and adipocyte fatty acid binding protein (Fabp) (Fig. f ). To test weter fat-specific overexpression of mir-7 affects insulin sensitivity, we injected eiter ad-7/gfp or ad-gfp into te inguinal fat pads of wild-type mice. Te relative size of te inguinal fat deposit was.7%.% of total body fat, as determined by computer tomograpy. Expression of mir-7 was increased by about.-fold and was restricted to te fat pad. Levels of blood glucose and insulin in mice injected wit ad-7/gfp were increased and glucosetolerance and insulin-tolerance tests sowed decreased glucose tolerance and insulin sensitivity, respectively, supported by an increase in te HOMA index. Furtermore, adipocyte size was increased in te fat pads injected wit ad-mir-7, compared to te ad-gfp controls (Supplementary Fig. 7a g). Tese data sow tat overexpression of mir- 7 in te fat is sufficient to induce insulin resistance and glucose intolerance. Smaller adipocytes are associated wit increased insulin sensitivity in uman and rodent models. To explore weter insulin-stimulated glucose uptake in adipocytes was affected by mir- silencing, we isolated primary adipocytes from mice injected wit eiter ant- or ant-mm and measured insulin-stimulated D- C-glucose a Visceral fat (g) c f i 7 Ant-MM Ant- Ant-MM Relative [ C]glucose uptake Subcutaneous fat (g) Ant-MM Ant- Ant- Total (g) No insulin nm insulin Ant-MM Ant- Ant-MM g Ant-MM Ant- Cow Adipocyte size Ant- d,,,,, uptake in vitro. Basal and insulin-stimulated glucose uptake was increased in adipocytes from bot subcutaneous and visceral fat of ant--injected animals (Fig. i). Furtermore, adiponectin levels, wic correlate positively wit insulin sensitivity, were increased in ant--injected mice (Fig. j). Togeter, tese data sow tat silencing of mir-/7 increases insulin sensitivity in adipocytes. To address te possible mecanism by wic mir- and mir-7 regulate insulin sensitivity, we performed genome-wide expression analysis using Affymetrix microarrays, comparing livers from C7BL/J mice infected wit ad-7/gfp or ad-gfp. In animals Visceral fat (g) WT-cow j b Ant-MM Adiponectin (μg ml ) Ant-,,,,,, Subcutaneous fat (g) Ant-MM Ant- Adipocyte size SC WT Ant-MM V Ant- Total (g) Ant-MM7 Ant- Ad-7/GFP Ant-MM Ant- Ant-MM Ant-MM Ant-MM Ant- Ant- Ant Time () Time () Time () e Differentiated SV cells Ant-MM Ant- Ad-7/GFP V Figure Silencing of mir- decreases total fat by reducing adipocyte size. a, b, Fat-pad weigts from subcutaneous and visceral adipose tissue of mice injected wit ant-mm or ant-. mice (a, n, respectively) or mice (b, n ) were assessed by computer tomograpy d after injection. c, Haematoxylin staining of paraffin sections from subcutaneous (SC) and visceral (V) fat of mice injected wit ant-mm or ant-. Scale bar: mm. d, Automated quantification of te average adipocyte size from mice treated as in c, wit and wit ant-scrambled controls. e, Automated quantification of differentiated adipocytes after days of differentiation in te presence of ant-, ant-mm, ad-gfp or ad-7/ GFP. Te values sown are normalized to. f, Relative mrna levels (Rel. expression) of Cebp (f), Fabp (g) orpparg () in stromal-vascular cells differentiated in te presence of, ant-mm or ant-, at te indicated time points. Te mrna expression levels at time point were determined before te antagomir/differentiation treatment. i, [ C]Glucose uptake in primary adipocytes isolated from subcutaneous or visceral fat of mice injected wit, ant-mm or ant-. Uptake is normalized to cell numbers. j, Adiponectin levels in ant-mm-injected or ant--injected ob/ ob mice (left, n 7), and in ad-gfp-injected or ad-7/gfp-injected C7BL/J mice (rigt, n 9). Means s.d. are sown for all panels., P,.;, P,.;, P,.. Macmillan Publisers Limited. All rigts reserved JUNE VOL 7 NATURE

5 RESEARCH LETTER infected wit ad-7/gfp, mrnas carrying a seed matc to mir-7 in te 9 untranslated region (9 UTR) were downregulated wen compared to transcripts tat lacked a mir-7 seed. Te data were confirmed by real-time PCR for a subset of mir-7 target genes (Fig. a). Out of over, genes wit a -mer seed matc in te 9 UTR, te predicted top targets of mir-7/ were enriced in membrane-related genes and metabolism genes (Supplementary Fig. a, b). Te gene encoding caveolin- (), a key component of caveolae and a mediator of insulin signalling, was among te mir- /7 seed-containing genes tat were downregulated after overexpression of mir-7 in te liver, and upregulated after its silencing (Fig. a and Supplementary Fig. c). Notably, mir- silencing in te fat resulted in an approximately.-fold upregulation of mrna levels (Fig. b). Murine (m) contains tree mir- sites, wereas uman CAV (CAV) as two seed motifs in te 9 UTR (Supplementary Fig. d, e). Measurements of luciferase activity in HEK 9 cells transfected wit reporter plasmids containing te 9 UTRs of mor CAV sowed reduced expression of tese constructs in te presence of mir- (Fig. c). By mutating te conserved seed, we could fully reverse te mir--induced decrease in luciferase activity in bot m and CAV constructs (Fig. c). Overexpression of mir- also led to an approximately twofold decrease in endogenous levels in HEK 9 cells compared to controls (Fig. d). Conversely, ant-, but not ant-scrambled or controls, increased levels in HEK 9 cells (Fig. e). a b c. Mock. Ad-7/GFP Ant- si-mir-. si-mir Gpnmb Prom LPL Pla (g) Pla (g7) LYPLA LYPLA Pld Pld Relative luciferase activity Taken togeter, tese data demonstrate tat is a direct target of mir- in bot mouse and uman cells. is te principal protein of caveolae, distinct lipid- and colesterol-enriced vascular invaginations at te plasma membrane. activates insulin signalling, probably by stabilizing caveolae and teir associated insulin receptors 7. Peptides corresponding to te scaffolding domain of and Cav potently stimulate insulin-receptorkinase activity. Furtermore, overexpression of Cav augments insulin-stimulated posporylation of insulin receptor substrate (ref. ) and increases epatic insulin-receptor posporylation in response to insulin stimulation, tereby improving te overall glucose metabolism of diabetic mice 9. -null mice are penotypically normal on a cow diet but develop insulin resistance on a ig-fat diet owing to decreased insulin-receptor expression and diminised insulin-receptor signalling in adipose tissue. We investigated weter insulin signalling correlated wit mir-/7-mediated canges in expression. In te fat and liver of mice, silencing of mir- /7 resulted in increased levels, wereas no expression of tis protein could be detected in skeletal muscle (Fig. f ). Te expression of insulin receptor b-subunit (IRb) in adipocytes was increased and insulin-stimulated levels of posporylated Akt and IRb (pakt and pirb) were augmented in te fat and liver of ant--treated mice (Fig. f, g). In contrast, insulin signalling was not enanced in te skeletal muscle of ant--treated mice (Fig. ). In addition, wild-type mice in wic ad-mir-7 was injected into te inguinal fat pad m S L mut S mut L e f g i Group HEK9 Ant- Ad-7/GFP j Insulin ( U kg ) p Lpl () Lpl (C7BL/) () (C7BL/) Plag7 () Plag () Gpnmb () Saa () Ant-MM Ant- Figure Regulation of gene expression and insulin signalling by mir-. a, b, Gene expression analysis (relative expression) in livers from C7BL/J mice d after injection wit ad-gfp or ad-7/gfp (a, n ), or in fat from mice injected wit or ant- (b, n ). c, Relative luciferase activity in HEK 9 cells transfected wit reporter constructs containing te 9 UTR of, co-transfected wit si-mir-, si-mir- (scrambled) or (Mock). S, sort; L, long; mut, mutant. d, Western blotting (WB) and nortern blotting (NB) of HEK 9 cells transfected wit, si-mir- (scrambled) or si-mir-. c-tub, c-tubulin. e, Immunoblotting of protein extracts from HEK 9 cells transfected wit, control ant-scrambled or ant-. f, Immunoblotting of protein extracts from fat (f), liver (g) or muscle () of p k d mir- Insulin ( U kg ) Insulin ( U kg ) Ant-MM Ant- Mock si-mir- si-mir- HEK9 C7BL/ -KO C7BL/ -KO Ant Insulin (.7 U kg ) Ant-MM p p Glut- Ad-7/GFP Ad-7/GFP Ad-7/GFP p WB NB Ant-MM Ant- ant-mm-injected or ant--injected mice stimulated wit U kg insulin for min. i, Western blot analysis of perigonal fat pads in C7BL/J mice surgically injected wit ad-gfp or ad-7/gfp. Eac lane represents a pool of two mice. j, Western blot analysis of liver extracts from C7BL/J or -knockout (-KO) mice injected wit ad-gfp or ad-7/gfp, stimulated wit.7 U kg insulin for min after a fast. k, Immunoblotting of protein extracts from te fat of C7BL/J or -KO mice d after injection wit ant-mm or ant- and stimulated for min wit. U kg insulin after a fast. Animals were kept on a igfat diet for weeks before antagomir injection. Glut-, glucose transporter-. Means s.d. are sown for all panels., P,.;, P,.;, P,.. NATURE VOL 7 JUNE Macmillan Publisers Limited. All rigts reserved

6 LETTER RESEARCH sowed a reduction in expression and decreased IRb and pakt levels (Fig. i). We also studied insulin signalling in te livers of mice wit mir-7 overexpression. and pakt levels were diminised in te livers of wild-type mice infected wit ad-mir-7, wit no canges observed in IRb protein levels (Fig. j). Tis result is in agreement wit our findings sowing tat overexpression of mir-7 can induce epatic insulin resistance, and wit data from -null mice, wic maintain normal IRb levels in te liver but sow reduced IRb and pakt levels in fat 9. Finally, to investigate weter modulation of expression by mir-7 is important for te observed penotypes, we overexpressed or silenced mir-/7 in -null mice. Wereas epatic overexpression of mir-7 in wild-type mice led to impaired glucose tolerance, no significant effects on plasma glucose, glucose-tolerance, insulin-tolerance or pyruvate-tolerance tests were measured wen -null mice were injected wit ad-7/gfp and compared to ad-gfp-treated -null animals (Supplementary Fig. 9a d). Furtermore, no molecular canges in insulin signalling events were detected in te two groups (Fig. j). Administration of ant- to -null mice also did not affect glucose tolerance, insulin sensitivity or posporylation of insulin receptor and Akt upon insulin stimulation (Fig. k and Supplementary Fig. 9e ). However, expression of lipolytic genes in te adipose tissue of -null mice was still responsive to treatment wit ant-, compared to ant-mm (Supplementary Fig. 9i), indicating tat mir-/7 also mediate some -independent metabolic effects. Our findings sow tat mir- and mir-7 are negative regulators of insulin sensitivity. Teir increased epatic expression in rodents and umans wit insulin resistance and epatic steatosis indicates tat tey migt contribute to te aetiology of diabetes. We also sow tat global mir-/7 silencing causes increased insulin signalling in bot liver and adipose tissue, altoug silencing of epatic mir-/7 expression in overt obese and insulin-resistant states is insufficient to reverse te metabolic abnormalities. Tis indicates tat silencing of mir- in adipocytes is te dominant contributor to enanced insulin sensitivity. One mecanism by wic tese mirnas regulate insulin sensitivity is by targeting, tereby diminising te number of insulin receptors in caveolae-enriced plasma membrane microdomains and reducing downstream insulin signaling. It is likely tat also mediates oter effects tat contribute to te penotype because tis protein as many functions in growt-factor signalling, endocytotic patways and lipid regulation. Our finding tat silencing mir-/7 in obese animals improves glucose omeostasis implicates tese mirnas as novel terapeutic targets for te treatment of diabetes. METHODS SUMMARY Animals. All mice were males and were maintained on a C7BL/J background, on a - ligt/dark cycle in a patogen-free animal facility. Antagomirs at doses of mg kg in. ml total volume wit per injection were administered on two consecutive days troug te tail vein of wild-type or mice at between and weeks of age, or to -week-old mice fed on a diet containing % fat (Pvolimi Kliba AG) for weeks. Mice were injected wit adenoviruses troug te tail vein at 9 plaque-forming units in. ml. Injection of ad-7/gfp and antagomirs did not effect food consumption compared to tat of controltreated animals. Mice were killed d after te adenovirus injection. All animal studies were approved by te Kantonale Veterinäramt Züric. Antagomirs. Te single-stranded RNAs and modified RNA analogues used in tis study consisted of nucleotides wit modifications as specified: antagomir-, 9-u s c s auagcccuguacaaugcu s g s c s u s -Col-9; antagomir-7, 9-u s g s auagcccug uacaau gcu s g s c s u s -Col-9; MM-antagomir-, 9-u s g s acagccuuguaccaugcg s g s c s u s -Col-9; antagomir- (scrambled), 9-g s g s cauucaccgcgugcc s u s u s a s -Col-9. Te lower-case letters represent -OMe-modified nucleotides; subscript s represents a posporotioate linkage; Col represents colesterol linked troug a ydroxyprolinol linkage. Lipid nanoparticle formulations. Liver-targeting lipid nanoparticle formulations of antagomirs were prepared using te novel ionizable lipid DLin-KC- DMA (ref. ). Lipid nanoparticles were composed of DLin-KC-DMA, distearoyl pospatidylcoline, colesterol and mpeg-dmg, used at te molar ratio ::.:.. Antagomirs were formulated in lipid nanoparticles at a total lipidto-antagomir weigt ratio of approximately :. Generation of recombinant adenovirus. Ad-7/GFP was generated by inserting te PCR-amplified mirna precursor sequence generated wit primers 9-AATACCCGCATGGAAGCAGGCTAA-9 and 9-AACATGTCTCAAGGA GAGGACGGT-9 into a GFP-expressing suttle vector, AdCMV K-NpA. Ad- GFP (ViraQuest) was used as a control. Statistical analysis. Unless oterwise specified, all bars sow mean s.d. Significance was calculated using student s t-test (, P,.;, P,.;, P,.). Full Metods and any associated references are available in te online version of te paper at Received Marc ; accepted April. Publised online June.. Kan, C. R. Knockout mice callenge our concepts of glucose omeostasis and te patogenesis of diabetes. Exp. Diabesity Res., 9 ().. Taniguci, C. M., Emanuelli, B. & Kan, C. R. Critical nodes in signalling patways: insigts into insulin action. Nature Rev. Mol. Cell Biol. 7, 9 ().. Muoio, D. M. & Newgard, C. B. 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MiRNA expression profile of uman subcutaneous adipose and during adipocyte differentiation. PLoS ONE, e9 ().. Sun, T., Fu, M., Bookout, A. L., Kliewer, S. A. & Mangelsdorf, D. J. MicroRNA let-7 regulates T L adipogenesis. Mol. Endocrinol., 9 9 (9).. Xie, H., Lim, B. & Lodis, H. F. MicroRNAs induced during adipogenesis tat accelerate fat cell development are downregulated in obesity. Diabetes, 7 (9).. Goossens, G. H. Te role of adipose tissue dysfunction in te patogenesis of obesity-related insulin resistance. Pysiol. Beav. 9, ().. Yamauci, T. et al. Te fat-derived ormone adiponectin reverses insulin resistance associated wit bot lipoatropy and obesity. Nature Med. 7, 9 9 ().. Rotberg, K. G. et al. Caveolin, a protein component of caveolae membrane coats. Cell, 7 (99). 7. Nystrom, F. H., Cen, H., Cong, L. N., Li, Y. & Quon, M. J. Caveolin- interacts wit te insulin receptor and can differentially modulate insulin signaling in transfected Cos-7 cells and rat adipose cells. Mol. Endocrinol., (999).. Yamamoto, M. et al. Caveolin is an activator of insulin receptor signaling. J. Biol. Cem. 7, 9 9 (99). 9. Otsu, K. et al. Caveolin gene transfer improves glucose metabolism in diabetic mice. Am. J. Pysiol. Cell Pysiol. 9, C c (9).. Coen, A. W. et al. Caveolin--deficient mice sow insulin resistance and defective insulin receptor protein expression in adipose tissue. Am. J. Pysiol. Cell Pysiol., C C ().. Coen, A. W., Combs, T. P., Scerer, P. E. & Lisanti, M. P. Role of caveolin and caveolae in insulin signaling and diabetes. Am. J. Pysiol. Endocrinol. Metab., E E ().. Parton, R. G. & Simons, K. Te multiple faces of caveolae. Nature Rev. Mol. Cell Biol., 9 (7). Supplementary Information is linked to te online version of te paper at Acknowledgements We would like to tank F. Preitner and B. Torens for te yperinsulinaemic euglycaemic clamp studies. M.T. was supported by a fellowsip from te Juvenile Diabetes Researc Foundation International. Te work was supported in part by te Swiss National Science Foundation (SNF, LiverX), te European Community (SIROCCO, ERC and MetaboloMirs) and te Leducq Foundation. Autor Contributions M.T. and M.S. designed te experiments. M.T. performed te experiments and conducted te data analysis. J.H. and M.Z. performed te bioinformatic analysis. M.H.H. provided liver samples and participated in analysis of clinical data. B.B. syntesized antagomirs. A.A. provided liposomal formulations. M.T. and M.S. wrote te paper wit input from all co-autors. Autor Information Reprints and permissions information is available at Te autors declare no competing financial interests. Readers are welcome to comment on te online version of tis article at Correspondence and requests for materials sould be addressed to M.S. (stoffel@imsb.biol.etz.c). Macmillan Publisers Limited. All rigts reserved JUNE VOL 7 NATURE

7 RESEARCH LETTER METHODS Animals. All mice were males and were maintained on a C7BL/J background, on a - ligt/dark cycle in a patogen-free animal facility. Antagomirs at doses of mg kg in. ml total volume wit per injection were administered on two consecutive days troug te tail vein of wild-type or mice at between and weeks of age, or to -week-old mice fed on a diet containing % fat (Pvolimi Kliba AG) for weeks. Mice were injected wit adenoviruses troug te tail vein at 9 plaque-forming units in. ml. Injection of ad-7/gfp and antagomirs did not effect food consumption compared to tat of controltreated animals. Mice were killed d after te adenovirus injection. All animal studies were approved by te Kantonale Veterinäramt Züric. BL/Adenovirus injection of fat. or ad-7/gfp were injected into te perigonal fat at a concentration of 9 plaque-forming units in ml after surgical exposure. Animals were studied d after injection. Liver biopsies. Liver biopsy specimens from Caucasian patients were obtained during routine diagnostic work-up at te University Hospital, Basel. Blood samples were collected in te fasting state on te day of te liver biopsy for glucose and plasma-insulin measurements. Most study subjects did not take any medications. A specimen was frozen for researc purposes if more tan sufficient material was obtained for istopatological examination and if te patient gave is/er written informed consent in accordance wit te Etics Committee of Basel. Antagomirs. Te single-stranded RNAs and modified RNA analogues used in tis study consisted of nucleotides wit modifications as specified: antagomir-, 9-u s c s auagcccuguacaaugcu s g s c s u s -Col-9; antagomir-7, 9-u s g s auagcccug uacaau gcu s g s c s u s -Col-9; MM-antagomir-, 9-u s g s acagccuuguaccaugcg s g s c s u s -Col-9; antagomir- (scrambled), 9-g s g s cauucaccgcgugcc s u s u s a s -Col-9. Te lower-case letters represent -OMe-modified nucleotides; subscript s represents a posporotioate linkage; Col represents colesterol linked troug a ydroxyprolinol linkage. Lipid nanoparticle formulations. Liver-targeting lipid nanoparticle formulations of antagomirs were prepared using te novel ionizable lipid DLin-KC- DMA (ref. ). Lipid nanoparticles were composed of DLin-KC-DMA, distearoyl pospatidylcoline, colesterol and mpeg-dmg, used at te molar ratio ::.:.. Antagomirs were formulated in lipid nanoparticles at a total lipidto-antagomir weigt ratio of approximately :. Generation of recombinant adenovirus. Ad-7/GFP was generated by inserting te PCR-amplified mirna precursor sequence generated wit primers 9-AATACCCGCATGGAAGCAGGCTAA-9 and 9-AACATGTCTCAAGGA GAGGACGGT-9 into a GFP-expressing suttle vector, AdCMV K-NpA. Ad- GFP (ViraQuest), wic does not contain a transgene, was used as a control. RNA isolation and nortern blotting analysis. mg total RNA, isolated using Trizol reagent (Invitrogen), was separated at W on % polyacrylamide gels containing formamide, as described in ref.. Real-time PCR. Steady-state mrna expressionwas measured by quantitative realtime PCR using te LigtCycler SYBR Green Master I Mix (Roce) wit a MxP Real-Time PCR System (Stratagene). Transcript levels were normalized to glyceraldeyde -pospate deydrogenase (GAPDH) or B, te gene encoding acidic ribosomal pospoprotein P (RPLP). Primer sequences for real-time PCRs are available on request. MiRNA levels were measured using TaqMan microrna Assays (Applied Biosystems) and were normalized to U levels. MicroRNA microarray. We used tree diabetic groups ( or ) and tree control groups of mice for eac diabetic model. Prior to killing, elevated bloodglucose and insulin levels were confirmed in te diabetic mice. Total RNA, isolated and pooled from te livers of ten mice per group, was labelled using te mircury LNA microrna Power Labelling Kit (Exiqon) and ybridized on mirna arrays (mirxplore) tat carry,9 DNA oligonucleotides wit te reverse-complementary sequence of te mature RNAs. Tese arrays cover 7 uman, mouse, rat and viral mirnas, eac spotted on te arrays in quadruplicate. Te Cy-labelled mirnas were normalized to a reference pool of mirnas tat were simultaneously labelled wit Cy. All te data are represented as ratios of logaritmic values between te diabetic and ealty animals s.d. Assay of luciferase activity and cell culture transfection. 9 UTR sequences were PCR-amplified wit specific primers, followed by attb adaptor PCR. Sequences were cloned into te pdonr entry vector using BP Clonase (Invitrogen) and ten cloned beind te stop codon of firefly luciferase in te dual renilla/firefly luciferase pem9 destination vector (gift from E. Miska). HEK 9 cells cultured in -well plates were transfected in quadruplicate using Lipofectamine (Invitrogen) wit ng of te final construct per well, togeter wit or nmol of eiter control or si- double-stranded sirna (Sigma). Cells were collected after transfection and assayed using te Dual-Luciferase Reporter Assay System (Promega). Results were normalized to te renilla luciferase control and expressed relative to te average value of te control, wic was treated wit. HEK 9 cells were transfected wit antagomirs at a concentration of. mgml of medium. Computer tomograpy. Animals were scanned using an animal CT-Scanner (LaTeta) at mm intervals from te ead to te base of te tail. Images were analysed using te LaTeta Software. Isolation of stromal-vascular fraction and primary adipocytes. Primary adipocytes and te stromal-vascular fraction from subcutaneous and visceral fat were prepared as previously described,. Adipocyte differentiation was induced wit insulin, dexametasone, isobutylmetylxantine and rosiglitazone wen stromalvascular cells were % confluent. Cells were treated wit antagomirs at a concentration of. mgml during te induction period on days and. Automated analysis of adipocyte differentiation. Differentiated cells were fixed wit % formaldeyde before staining wit boron-dipyrrometene (BODIPY) for lipid droplets, Hoecst for nuclei and Syto for cytosolic staining (Invitrogen). A total of pictures per well were taken wit an automated microscope imaging system (CellWorx). Pictures were analysed using Cell Profiler Software. Glucose uptake. [ C]-Spiked glucose uptake, wit or witout nm insulin stimulation, was measured as previously described. Hyperinsulinaemic-euglycaemic clamp studies. Clamps were performed on -week-old mice weiging g. An indwelling cateter for infusion of insulin and glucose was placed into te left femoral vein under isoflurane anaestesia. Mice were allowed to recover for d, until tey ad regained 9 % of teir initial body weigt. After a fast, a min yperinsulinaemic-euglycaemic clamp study was conducted in awake, freely moving mice, as previously described,. Adipocyte size. Haematoxylin and eosin staining of mm slices of adipose tissue fixed in % paraformaldeyde was performed according to standard procedures 7 and images were analysed using Cell Profiler Software. At least, adipocytes were measured per animal to determine adipocyte size. Glucose-, insulin- and pyruvate-tolerance tests. Glucose-, insulin- and pyruvatetolerance tests were performed by intraperitoneal injection of glucose ( g kg ), insulin (.7 units kg, unit kg or units kg, as indicated in te figures) or pyruvate ( g kg ) after an overnigt fast for glucose and pyruvate or a fast for insulin. Blood glucose levels were measured before injection (time ) and at,, and min after injection. Antibodies. Te antibodies used were mouse monoclonal anti-c-tubulin (Sigma- Aldric), rabbit polyclonals anti-irb (C-9):sc-7; anti-pirb (Tyr/ ):sc, anti-caveolin- (N):sc-9 (Santa Cruz Biotecnology), anti and anti- (Cell Signaling). Statistical analysis. All bars sow mean s.d. Significance was calculated using student s t-test (, P,.;, P,.;, P,.).. Semple, S. C. et al. Rational design of cationic lipids for sirna delivery. Nature Biotecnol., 7 7 ().. Hansen, L. H., Madsen, B., Teisner, B., Nielsen, J. H. & Billestrup, N. Caracterization of te inibitory effect of growt ormone on primary preadipocyte differentiation. Mol. Endocrinol., 9 (99).. Tozzo, E., Seperd, P. R., Gnudi, L. & Kan, B. B. Transgenic GLUT- overexpression in fat enances glucose metabolism: preferential effect on fatty acid syntesis. Am. J. Pysiol., E9 E9 (99).. Mineira, K. et al. Blocking VLDL secretion causes epatic steatosis but does not affect periperal lipid stores or insulin sensitivity in mice. J. Lipid Res. 9, (). 7. Preitner, F., Mody, N., Graam, T. E., Peroni, O. D. & Kan, B. B. Long-term Fenretinide treatment prevents ig-fat diet-induced obesity, insulin resistance, and epatic steatosis. Am. J. Pysiol. Endocrinol. Metab. 97, E E9 (9). Macmillan Publisers Limited. All rigts reserved