Partial inactivation of Ankrd26 causes diabetes with enhanced insulin responsiveness of adipose tissue in mice

Transcription

1 Diabetologia (11) :911 9 DOI.7/s ARTICLE Partial inativation of Ankrd auses diabetes with enhaned insulin responsiveness of adipose tissue in mie G. A. Raiti & T. K. Bera & O. Gavrilova & I. Pastan Reeived: May 11 /Aepted: July 11 /Published online: 13 August 11 # Springer-Verlag (outside the USA) 11 Abstrat Aims/hypothesis ANKRD is a newly desribed gene loated at p1 in humans, a lous that has been identified with some forms of hereditary obesity. Previous studies have shown that partial inativation of Ankrd in mie auses hyperphagia, obesity and gigantism. Hypothesising that Ankrd mutant () mie ould develop diabetes, we sought to establish whether the observed phenotype ould be (1) solely related to the development of obesity or () aused by a diret ation of ankyrin repeat domain (ANKRD) in peripheral tissues. Methods To test the hypothesis, we did a full metaboli haraterisation of Ankrd mie that had free aess to how or were plaed under two different energy-restrited dietary regimens. Results Highly obese Ankrd mie developed an unusual form of diabetes in whih white adipose tissue is insulin-sensitive, while other tissues are insulin-resistant. When obese mie were plaed on a food-restrited diet, their weight and gluose homeostasis returned to normal. In Eletroni supplementary material The online version of this artile (doi:.7/s ) ontains peer-reviewed but unedited supplementary material, whih is available to authorised users. G. A. Raiti : T. K. Bera : I. Pastan () Laboratory of Moleular Biology, Center for Caner Researh, National Caner Institute, National Institutes of Health, 37 Convent Drive, Room, Bethesda, MD 9-, USA pastani@mail.nih.gov O. Gavrilova Mouse Metabolism Core, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA addition, when young mie were plaed on a pairfeeding diet with normal mie, they maintained normal body weight, but showed better gluose tolerane than normal mie, an inreased responsiveness of white adipose tissue to insulin and enhaned phosphorylation of the insulin reeptor. Conlusions/interpretation These findings show that the ANKRD protein has at least two funtions in mie. One is to ontrol the response of white adipose tissue to insulin; the other is to ontrol appetite, whih when Ankrd is mutated, leads to hyperphagia and diabetes in an obesitydependent manner. Keywords Ankrd. Diabetes. Energy intake restrition. Insulin sensitivity. Obesity. WAT Abbreviations ANKRD Ankyrin repeat domain BAT Brown adipose tissue EGP Endogenous gluose prodution FA Free aess to water and food FR Food restrition GIR Gluose infusion rate GTT Gluose tolerane test ITT Insulin tolerane test Mutant NCI National Caner Institute NIDDK National Institute of Diabetes Digestive and Kidney Diseases NIH National Institutes of Health PF Pair-feeding R d Whole-body gluose disposal rate WAT White adipose tissue

2 91 Diabetologia (11) :911 9 Introdution Type diabetes is the most ommon type of diabetes and aounts for 9% of all forms of the disease. It is a heterogeneous syndrome that is due to the interation of environmental fators with geneti suseptibility to the disease [1] and is haraterised by insulin resistane and/or abnormal insulin seretion, either of whih may predominate []. The prevalene of type diabetes is rapidly inreasing worldwide and is expeted to affet approximately 3 million people by [1]. Obesity is a major risk fator for the development of type diabetes. Indeed, type diabetes is most ommon in people who are older than and overweight; as a onsequene of inreased obesity among young people, it is also beoming more ommon in hildren and young adults [3]. The term diabesity has been oined to denote diabetes ourring in the ontext of obesity [] and desribes one of the main threats to human health in the twenty-first entury []. We reently desribed a new model of obesity resulting from partial inativation of Ankrd gene [], whih is loated at p1 in humans, a geneti lous related to some forms of hereditary obesity [7]. The gene enodes a ~19 kda protein that is highly abundant in the hypothalamus and other regions of the brain, as well as in many tissues and organs, inluding liver, skeletal musle and white adipose tissue (WAT) (T.K. Bera and I. Pastan, unpublished data). The ankyrin repeat domain (ANKRD) protein is assoiated with the inner aspet of the ell membrane, and ontains ankyrin repeats and spetrin helies, motifs known to interat with signalling proteins []. Mie with inativation of Ankrd have marked hyperphagia, whih results in extreme obesity and an inrease in body length []. They also have high leptin levels, suggesting a defet in the feeding entres in the brain. In the present work, we sought to establish whether these Ankrd mutant () mie develop diabetes and whether the observed phenotype is solely related to the development of obesity or is aused by a diret ation of ANKRD in peripheral tissues. Methods Mouse models and energy restrition diets Ankrd mie were bakrossed for eight generations on the C7/ Bl bakground in the animal faility of the National Caner Institute (NCI). Male Ankrd homozygous mie and their normal littermates (wild-type) were used for all studies. All proedures were onduted in aordane with National Institutes of Health (NIH) guidelines, as approved by the Animal Care and Use Committees of the NCI and the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK). Mie were housed one per age on a 1 h light/dark yle (lights on : 1: hours) and had free aess to water and food (FA) (NIH-7 diet, 11% energy from fat; Zeigler Brothers, Gardners, PA, USA) when not under energy restrition dietary regimens. For the food restrition (FR) diet experiment, -month-old wild-type (n=1) and Ankrd (n=1) mie were randomly assigned to two groups: FA and FR. We determined baseline daily food onsumption by weighing the food provided and orreting for food not eaten, inluding spillage. FR was performed for weeks, with eah FR mouse reeiving an alloation of food equal to 33% of the FA (baseline) daily onsumption of mie (3 g of food). For the pairfeeding (PF) diet experiment, -week-old wild-type (n=1) and Ankrd (n=1) mie were randomly assigned to two groups: FA and PF. The PF was performed for a period of months and eah PF mouse reeived an alloation of food equal to the amount of food eaten the day before by the ontrol FA group. For both diets, food was supplied twie a day from Monday to Friday, and one on Saturdays and Sundays. Weight gain and body length of individual mie were measured as desribed []. Biohemial assays were measured as reported in eletroni supplementary material (ESM) Methods. Gluose and insulin tolerane, assessment of insulin and gluagon seretion, hyperinsulinaemi euglyaemi lamps The gluose tolerane test (GTT), insulin tolerane test (ITT) and insulin seretion were performed or measured as desribed [], as was measurement of gluagon seretion [9]. Hyperinsulinaemi euglyaemi lamps were also performed as previously desribed [] and reported in the ESM Methods. In vivo analysis of insulin signalling Mie were fasted overnight, anaesthetised and injeted i.p. with saline or insulin ( U/kg body weight). At min after injetion, musle, adipose tissue and liver were removed and frozen in dry ie. Tissues, homogenates and ell lysates were separated by SDS-PAGE and analysed by western blot as previously desribed [,11,1]. Membranes were probed with antibodies to phospho-igf-i reeptor beta (Tyr113/113)/ insulin reeptor beta (Tyr/11), insulin reeptor beta, phospho- (Ser73) and (Cell Signaling Tehnology, Danvers, MA, USA). Statistial analysis Data are expressed as means ± SEM and statistial signifiane between groups was analysed by two-tailed Student s t test or ANOVA as appropriate. Values

3 Diabetologia (11) : of p<. were onsidered statistially signifiant. The total AUC and the inverse AUC for gluose response during GTT and ITT were alulated using the following equations [13]: AUC ¼ Xx 1 ðt nþ1 T n Þ Gl T n Gl Tnþ1 n¼1 þ min Gl Tn ; Gl Tnþ1 GlT AUC i ¼ Xx 1 ðt nþ1 T n Þ Gl T n Gl Tnþ1 n¼1 þ Gl T max Gl Tn ; Gl Tnþ1 Results Ankrd mie develop diabetes To investigate the role of Ankrd in the regulation of gluose homeostasis in vivo, we did a full metaboli haraterisation of the Ankrd mie. As reported previously [], male Ankrd mie were heavier and longer than agemathed ontrols (wild-type) at, and months (Table 1), and their daily food intake was % higher (wild-type 3.±.1 g/day; Ankrd.±.1 g/day; p<.1). At months of age, Ankrd mie exhibited a slight inrease of fasting and random fed blood gluose levels (Table 1), but with normal GTT (Fig. 1a, b). By ontrast, fasting and random fed blood gluose levels rose in the following months reahing a fasting level of 11.1 mmol/l at months (Table 1). At months of age, gluose loading ( g/kg body weight) made Ankrd mie signifiantly more hyperglyaemi than ontrol mie during the following min (Fig. 1e, f), with gluose tolerane in the former being severely deteriorated at months (Fig. 1i, j). This shows that partial inativation of Ankrd an lead to diabetes. Interestingly, the obese mie had elevated fasting insulin and leptin levels, yet there were no signifiant differenes in fasting NEFA and triaylglyerol onentrations at, and months (Table 1). Ankrd mie show impaired insulin sensitivity and panreati funtion To verify whether Ankrd inativation is aompanied by redued insulin sensitivity, we performed an ITT. Following i.p. injetion of insulin (.7 U/kg body weight) a slight redution of the hypoglyaemi response was observed in Ankrd mie at and months (Fig. 1, d, g, h), and this response was almost abolished in -month-old Ankrd mie (Fig. 1k, l). This shows that a timedependent deterioration of insulin sensitivity ourred in the mie. To assess panreati funtion, we evaluated gluoseindued insulin seretion and insulin-indued gluagon Table 1 Metaboli harateristis of Ankrd mie Variable months months months Body weight (g) 3.±..3±. a 7.±..±1. b.±.9.1±1. Body length (mm) 91.±. 9.±.3 a 9.7±..±. b 9.±..±.9 Metabolites Fasting gluose (mmol/l).±..±. a 7.1±..±.3 b 9.±.9.3±.3 Fed gluose (mmol/l).3±.3 7.±. a.1±. 11.±1.1 b.±. 1.±1.1 Fasting insulin (pmol/l).± ±1. a 91.±..±1.3 b 7.1±.1 3.±.9 Fasting leptin (ng/ml).±. 11.±1. a.±.7 3.7±3. b 7.±.99.±.79 Fasting NEFA (g/l).19±.3.3±..±.3.±..33±.3.±. Fasting triaylglyerol (mmol/l) 1.±.9 3.9±. 9.7±9.7.±.9.3±3. 7.±1.3 Data are means ± SEM of determinations in at least ten mie per group of Ankrd mie () vs wild-type () at age as indiated a p<.1, -month-old vs -month-old ; b p<.1, -month-old vs -month-old ; p<.1, -month-old vs -month-old

4 91 Diabetologia (11) :911 9 Fig. 1 Gluose tolerane, insulin sensitivity, and insulin and gluagon seretion in Ankrd mie. Ankrd (grey squares/lines) and wild-type (blak irles/lines) mie were subjetedtogttat(a, b) months, (e, f) months and (i, j) months. Mie were fasted for 1 h and subjeted to i.p. gluose loading ( g/kg body weight). Blood gluose levels were determined at various times. b, f, j Mean AUC gluose from GTT., d Mie as above underwent ITT at months, (g, h) months and (k, l) months. Random fed mie were injeted i.p. with insulin (.7 U/kg body weight), followed by determination of blood gluose levels at the indiated times. d, h, l Mean AUC insulin from ITT. m Gluose-indued insulin seretion was evaluated in -month-old and wild-type mie. Mie were fasted overnight and then injeted i.p. with gluose (3 g/kg body weight). Serum insulin onentrations were measured at the indiated times by ELISA. For experiments (a m) values are expressed as means ± SEM of determinations in at least ten mie per group. n Gluagon seretion was evaluated in -month-old random fed mie as above (m). Mie were injeted i.p. with insulin (.7 U/kg body weight). Plasma gluagon onentrations were measured by RIA before and min after the insulin injetion. Bars represent the means ± SEM of determinations in five and six ontrol mie. p<., p<.1 and p<.1 vs wild-type a e Gluose (mmol/ll) i m Serum insulin (fold over basal) b AUC f AUC AUC j g k n. 1.. Plasma gluagon (ng/l) Basal Insulin 9 d AUCi h AUCi AUCi l seretion. In ontrol mie, a threefold inrease in insulin seretion was observed 3 min after gluose injetion (3 g/kg body weight), with levels remaining higher than baseline for up to min, indiating a seond-phase response (Fig. 1m). Plasma gluagon levels in ontrol mie were also signifiantly inreased by the hypoglyaemi response indued by insulin (.7 U/kg body weight; Fig. 1n). In ontrast, the aute first-phase insulin seretory response to gluose and the late seond-phase response were ompletely abolished in Ankrd mie (Fig. 1m). In addition, a defetive gluagon response to hypoglyaemia was observed (Fig. 1n), indiating the onomitant presene of impaired insulin and gluagon seretion. Hyperinsulinaemi euglyaemi lamp in Ankrd mie To analyse gluose metabolism and insulin sensitivity of Ankrd mie in more detail, we performed a hyperinsulinaemi euglyaemi lamp. In the basal state, Ankrd mie had signifiantly inreased plasma gluose (Fig. a). During the lamp, the gluose infusion rate (GIR) needed to maintain euglyaemia and also the whole-body gluose disposal rate (R d ) were signifiantly dereased in Ankrd mie, indiating the presene of

5 Diabetologia (11) : Fig. Hyperinsulinaemi euglyaemi lamp studies in 7-month-old Ankrd (grey squares/olumns) and wild-type (; blak squares/ olumns) mie were performed as desribed. a Gluose levels, (b, ) GIR and (d) R d during the lamp proedure. e Basal EGP (monotone bars) and EGP (hathed bars) during the lamp. f Gluose uptake into skeletal musle, (g) BAT and (h) WAT during the lamp. Values are expressed as means ± SEM of determinations in at least eight mie per group. p<.1 and p<.1 vs wild-type. BW, body weight GIR (µmol [kg BW] 1 min 1 ) a Time during lamp (min) d Rd (µmol [kg BW] 1 min 1 ) b GIR (mmol g 1 min ) 1 Time during lamp (min) e EGP (µmol kg 1 min 1 ) Basal Clamp Basal Clamp f Musle gluose uptake (µmol [kg tissue] 1 min 1 ) g BAT gluose uptake (µmol [kg tissue] 1 min 1 ) 1, hwat gluose uptake (µmol [kg tissue] 1 min 1 ) whole-body insulin resistane (Fig. b d). We also evaluated endogenous gluose prodution (EGP) and gluose uptake in the peripheral tissues. Insulin was not able to suppress EGP in Ankrd mie (Fig. e), indiating hepati insulin resistane. In addition, gluose uptake in skeletal musle and brown adipose tissue (BAT) was signifiantly dereased (Fig. f, g), but surprisingly there was no signifiant differene in gluose uptake by WAT (Fig. h). In vivo insulin signalling in Ankrd mie To evaluate at the signalling level whether the progression of wholebody insulin resistane is related to the age of the mie, we injeted i.p. insulin ( U/kg body weight) into - and - month-old fasted Ankrd and ontrol mie, and assessed ativation of the insulin signalling pathway in liver, skeletal musle and WAT by western blot. In -month-old mie the insulin-stimulated phosphorylation of insulin reeptor beta on Tyr /11 and of on Ser73 was omparable to that in wild-type mie in liver and skeletal musle, and inreased vs wild-type in WAT (Fig. 3a f), indiating that insulin signalling in mie was generially intat. On the other hand, in -month-old Ankrd mie, the insulin-indued phosphorylation of insulin reeptor beta and was markedly redued in liver and skeletal musle (Fig. 3g,h,j,k). However, in WAT the insulin response was still slightly inreased ompared with normal mie (Fig. 3i, l), onfirming the observation in lamp studies that WAT remained insulin-responsive. Food restrition diet normalises body weight and gluose tolerane in Ankrd mie To determine whether the insulin resistane and impairment of insulin seretion that led to the onset of diabetes in Ankrd mie were seondary to the development of obesity, we analysed gluose homeostasis of mie under two different energy-restrited feeding regimens (FR diet, PF diet). First, to observe whether the impairment of gluose tolerane ould be reversed by the dietary treatment, FR was performed for weeks in -month-old obese and gluose-intolerant Ankrd and in age-mathed lean wild-type mie, all of whih reeived 3 g food daily. After 1month,Ankrd mie on the FR diet showed a % redution of body weight and a % redution of fasting leptin levels ompared with starting values (ESM Table 1, ESM Fig. 1a). This derease in body weight in Ankrd mie was aompanied by a signifiant derease of fasting and random fed blood gluose levels, as well as of fasting insulin and NEFA levels, with values omparable to those of wild-type mie (ESM Table 1). Gluose levels during GTT performed after months of diet were signifiantly lower in Ankrd mie than before FR; indeed, glyaemia was even lower than in ontrols, indiating higher gluose disposal (ESM Fig. 1b e). In addition, FR Ankrd mie had a near normal response to insulin with values omparable to those of wild-type mie (ESM Fig. 1f i), while their insulin response to gluose injetion beame the same as that of normal mie (ESM Fig. 1j).

6 91 Diabetologia (11) :911 9 Fig. 3 Insulin signalling in Ankrd mie. In vivo insulin signalling in liver (a, d, g, j), tibialis skeletal musle (b, e, h, k) and epididymal WAT (, f, i, l) of -month-old (a f) and -month-old (g l) overnightfasted mie that had been i.p. injeted (+, dark grey) or not (, light grey) with insulin ( U/kg body weight) min before determination along with wild-type () ounterparts; blak, injeted wild-type (); white, non-injeted. The orresponding blots show levels of total and tyrosine (Tyr /11) phosphorylated forms of insulin reeptor (IR), and of total and serine (Ser73) phosphorylated forms of in mie as indiated. Eah blot is representative of three independent experiments. Bars in graphs are expressed as means ± SEM in at least six mie per group. p<. and p<.1 for + insulin vs wild-type + insulin a d g Liver Liver Liver b e h 1 Musle 1 Musle 1 Musle f i WAT WAT 1 WAT Ins + + j Liver k Musle l WAT 1 1 Pair-feeding diet maintains normal body weight and improves gluose tolerane in Ankrd mie To determine whether an impairment of gluose tolerane ould our in lean Ankrd mie, PF was performed for months in -week-old Ankrd and wild-type mie; the former were fed daily the amount of food eaten on the previous day by the ontrol group. At and months, PF Ankrd mie were slightly heavier than PF age-mathed ontrol mie, but no signifiant differenes in body weight and fasting leptin were observed at months (Table, Fig. a). Interestingly, these mie with normal body weight had the same body length as the orresponding wild-type mie, indiating that the hange in length is dependent on some fator that is present only in obese mie (Table, ESM Fig. ). At the age of, and months, PF Ankrd mie had fasting and random fed blood gluose levels, as well as fasting insulin levels within the normal range of ontrol mie (Table ). In ontrast, they had a slight derease of NEFA and triaylglyerol levels ompared with wild-type mie (Table ). Gluose levels during GTT performed in -month-old mie were signifiantly dereased in PF Ankrd mie ompared with those in wild-type mie. This derease was also present in - and -month-old mie, indiating the presene of improved gluose tolerane (Fig. b g). Injetion of insulin evoked a omparable redution of hypoglyaemi responses in PF Ankrd and wild-type mie at, and months (Fig. h m); in addition, the gluose-indued insulin seretion profile was omparable between PF and ontrol mie (Fig. n). All these data from both energy-restrited feeding regimens demonstrate that the severe impairment of gluose tolerane indued by partial inativation of

7 Diabetologia (11) : Table Metaboli harateristis of PF diet-fed Ankrd mie Variable months months months PF PF PF PF PF PF Body weight (g).1±.3.1±. a.9±. 7.±. d 9.1±. 9.9±. Body length (mm) 91.±.7 93.±.3 9.3±.3 9.±. 9.±. 97.±.3 Fat mass (%) NA NA NA NA 19.7±..1±3. Lean mass (%) NA NA NA NA 7.±. 7.±3. Metabolites Fasting gluose (mmol/l).9±.3.±.3.±..±.3.9±.7.±. Fed gluose (mmol/l).7±.7.3±.3.±. 7.±.3 7.9±. 7.±.3 Fasting insulin (pmol/l) 1.±. 33.±. b.±.9 1±7..±19..9±1. Fasting leptin (ng/ml).79±.1.±.3 3.±..±..±1.3.7±1. Fasting NEFA (g/l).1±.1.11±.1 b.39±..±. e.3±.1.3±. f Fasting triaylglyerol (mmol/l) 7.±3..7±19..± ±. 3.±. 3.±. f Data are means ± SEM of determinations in at least five mie per group of Ankrd PF mie vs wild-type ( PF) at age as indiated NA, not available a p<. and b p<.1, -month-old PF vs -month-old PF p<., d p<.1 and e p<.1, -month-old PF vs -month-old PF f p<.1, -month-old PF vs -month-old PF Ankrd is seondary to obesity. Thus, in mie on an energy-restrited diet the inativation of this gene does not impair gluose homeostasis, but, instead, seems to improve it. Improved WAT insulin sensitivity in Ankrd mie on energy-restrited diet We performed hyperinsulinaemi euglyaemi lamps in Ankrd mie under the two different food-intake regimens. During the lamp, FR and PF Ankrd mie had gluose levels, GIR, R d and insulin suppression of EGP omparable to the values of their respetive ontrols (Fig. a e, ESM Fig. 3a e). In addition, insulin-stimulated gluose uptake in skeletal musle and in BAT in mie on the energy-restrited diet was similar to that in ontrols (Fig. f, g, ESM Fig. 3f, g); interestingly, insulin-stimulated gluose uptake was inreased in WAT (Fig. h, ESMFig.3h). Improved WAT insulin signalling in Ankrd mie on energy-restrited diet Consistent with these data was our finding that, in PF Ankrd mie, phosphorylated insulin reeptor beta and levels in response to insulin were omparable to those of wild-type mie in the liver and skeletal musle and higher than those of wild-type mie in WAT (Fig. a f). Similar data were obtained in FR Ankrd mie (ESM Fig. a e). Altogether these data identify WAT as the site responsible for the improvement of gluose tolerane in lean Ankrd mie, indiating a role for this gene in the regulation of insulin sensitivity in adipoytes, as well as in ontrol of appetite in the brain. Disussion In the present work, we report that mie with partial inativation of Ankrd exhibited elevated fasting and random fed blood gluose levels, and developed diabetes when severely obese. mie were also markedly hyperinsulinaemi in the basal state, showed a poor insulin response to a gluose hallenge and a defetive gluagon response to hypoglyaemia, and were also severely insulinresistant upon ITT. These findings show that impaired insulin ation and seretion ontribute to the abnormal gluose tolerane produed by partial inativation of Ankrd. Further, hyperinsulinaemi euglyaemi lamps and studies of insulin signalling in obese Ankrd mie demonstrated the onset of an unusual form of whole-body insulin resistane, in whih WAT remained insulin-sensitive, while the other insulin target tissues beame insulinresistant. These results suggest that Ankrd has a role in the ontrol of insulin sensitivity in WAT. The pathogenesis of type diabetes is known to be omplex, involving not only heightened geneti suseptibility in ertain ethni groups, but also environmental and behavioural fators suh as a sedentary lifestyle, nutrition and obesity [1]. To establish the role of obesity in the glyaemi disorders observed in Ankrd mie, we used two different restrited energy intake feeding strategies. In one approah mature obese mie were plaed on a FR diet, sine hroni moderate redution in energy intake (~ %) results in weight loss and improves whole-body gluose homeostasis in humans, rats and

8 91 Diabetologia (11) :911 9 Fig. Body weight, gluose tolerane, insulin sensitivity and insulin seretion in PF Ankrd mie. a Growth urve of mie subjeted to FA diet (dark grey squares) and PF diet (light grey squares), and of wild-type mie subjeted to FA (blak irles) PF (grey irles). b GTT with () AUC gluose in -month-old, -month-old (d, e) and -month-old (f, g) PF and PF wild-type mie., e, g, grey olumns; wild-type, blak olumns. h ITT with (i) AUC insulin in -month-old, (j, k) -month-old and (l, m) -month-old random fed PF and PF wild-type mie. i, k, m, grey olumns; wild-type, blak olumns. For eah experiment, values are expressed as means ± SEM of determinations in at least six mie per group. n Gluoseindued insulin seretion in -month-old PF (grey) and PF wild-type mie (blak). Data points represent the means ± SEM of determinations in four and five ontrol mie. p<., p<.1 and p<.1 vs PF wild-type b d 9 9 a Body weight (g) AUC e AUC 113 Time (weeks) h j i AUCi k AUCi f l. 9 g AUC m AUCi n Serum insulin (fold over basal) 3 1 mie by inreasing peripheral insulin sensitivity and dereasing glyaemia, insulinaemia and leptinaemia [ 1]. We found that FR ompletely restored normal body weight in mie and improved gluose metabolism by normalising whole-body insulin sensitivity and preserving insulin seretion. FR mie also had inreased insulin sensitivity in WAT. In the other study, young mie were plaed on a PF diet to prevent the onset of obesity and maintain body weight in a normal range [19]. We found that PF was

9 Diabetologia (11) : Fig. Hyperinsulinaemi euglyaemi lamp studies were performed in 7-month-old (grey squares/grey olumns) and wild-type (; grey irles/ blak olumn) mie on PF. a Gluose levels, (b, ) GIR and (d) during the lamp proedure. e Basal EGP (monotone olumns) and EGP during the lamp (hathed olumns). f Gluose uptake into skeletal musle, (g) BAT and (h) WAT during the lamp. Values are expressed as means ± SEM of determinations in at least five mie per group. p<. and p<.1 vs PF wild-type. BW, body weight GIR (µmol [kg BW] 1 min 1 ) f Musle gluose uptake (µmol [kg tissue] 1 min 1 ) a PF PF Time during lamp (min) PF PF d R d (µmol [kg BW] 1 min 1 ) g BAT gluose uptake (µmol [kg tissue] 1 min 1 ) PF PF b GIR (mmol g 1 min 1 ) 1 Time during lamp (min) PF PF e EGP (µmol kg 1 min 1 ) h WAT gluose uptake (µmol [kg tissue] 1 min 1 ) Basal Clamp Basal Clamp PF PF PF PF suffiient to maintain normal body weight in mie; PF mie, moreover, had improved gluose tolerane, and normal insulin seretion and whole-body insulin sensitivity. Like the FR mie, they also showed inreased gluose uptake in WAT. The onset of extreme obesity resulting from the marked hyperphagia of Ankrd mie therefore seems to be essential for the derangement of gluose homeostasis and for the development of diabetes, sine impairment of gluose homeostasis was reversed by dietary regimen and did not our in mie when their food a b PF PF d e f PF PF PF PF PF PF PF PF PF PF Fig. In vivo insulin-signalling in liver (a, d), tibialis skeletal musle (b, e) and epididymal WAT (, f) of -month-old overnight-fasted and wild-type () mie under PF feeding regimens that had been i.p. injeted (+, dark grey and blak olumns) or not (, light grey and white olumns) with insulin ( U/kg body weight) min before determination. The orresponding blots show the levels of total and tyrosine (Tyr /11) phosphorylated forms of the insulin reeptor, and levels of total and serine (Ser73) phosphorylated forms of in mie as indiated. The lanes were run on the same gel but were nonontiguous. Blots are representative of three independent experiments. Bars in graphs are expressed as means ± SEM in at least six mie per group. p<. for + insulin vs wild-type + insulin

10 9 Diabetologia (11) :911 9 intake and body weight were similar to those of ontrol mie. Thus, Ankrd mie should be regarded as a good mouse model of obesity-indued diabetes. However, in marked ontrast to other models of obesity and diabetes, suh as leptin-defiient ob/ob mie, leptin reeptor defiient db/db mie and yellow agouti mie [], Ankrd mie have whole-body insulin resistane in the presene of normal insulin sensitivity in WAT, indiating that peripheral insulin resistane in liver, skeletal musle and BAT, together with a dysfuntion of beta ell funtion, is suffiient to impair gluose homeostasis in these mie when obese. Additionally, Ankrd mie under restrited energy intake regimens show better gluose tolerane than ontrol mie, probably due to their inreased insulin sensitivity in WAT. Indeed, during lamp experiments, insulin-stimulated gluose uptake was slightly higher in WAT of mie on an energy-restrited diet. An enhanement of gluose tolerane assoiated with inreased gluose disposal in WAT has been desribed in several adipose tissue-speifi transgeni mie [1 3]. For example, overprodution of the insulin-responsive gluose transporter, GLUT, in WAT auses enhaned gluose tolerane and inreased gluose uptake in adipose tissue and leads to an inrease in fat ell number [1]. In these transgeni mie and, as with our mie, despite these marked effets at the adipose ell level, obesity indued by high fat feeding leads to a derease in gluose tolerane due to insulin resistane in skeletal musle and liver, where the Glut (also known as Sla) transgene is not expressed []. An additional example is the overabundane of liver gluokinase in WAT, where the inrease of gluose phosphorylation in adipoytes leads to enhaned gluose uptake and metabolism in WAT, thus improving gluose tolerane in vivo [3]. Therefore, an inrease of gluose disposal in WAT ould in itself be suffiient to improve gluose tolerane in mie on an energy-restrited diet. In addition to improved gluose tolerane, Ankrd mie under both energy restrition regimens showed dereased levels of NEFA and triaylglyerol, while these lipid onentrations were in the normal range in obese Ankrd mie. WAT plays a ruial role in buffering the flux of fatty aids in the bloodstream. Insulin modulates this proess, suppressing the release of NEFA into the irulation and inreasing triaylglyerol storage in WAT []. Inreased NEFA release by WAT and subsequent lipid abnormalities in type diabetes are strongly assoiated with insulin resistane [,]. Thus, the observed derease in NEFA and triaylglyerol onentrations in Ankrd mie on an energy-restrited diet ould be explained by their inreased insulin sensitivity in WAT. We also observed an improvement of insulin signalling in WAT from these mie. Indeed, an evaluation of the insulin signalling asade in WAT from FR and PF Ankrd mie showed inreased tyrosine phosphorylation of the beta subunit of the insulin reeptor [] and inreased phosphorylation of, a serine/threonine kinase involved downstream of the insulin signalling [7]. We reently demonstrated that mouse embryoni fibroblast ells from Ankrd mie have a higher rate of spontaneous adipogenesis than wild-type mouse embryoni fibroblasts and that their adipogenesis is greatly inreased when exposed to a mixture of induers []. These findings indiate that Ankrd plays a prominent role in fat ells, regulating their differentiation and insulin sensitivity. Unfortunately, we do not yet know how ANKRD modulates insulin signalling and/or adipogenesis in fat ells. To detet proteins that interat with ANKRD, we are now doing a yeast two hybrid sreen. In addition to obesity, Ankrd mie showed a % inrease in linear growth even though irulating growth hormone and IGF1 are in the normal range []. Differenes in body length were not deteted in -week-old mie (ESM Fig. a), but were deteted at months of age in parallel with an inrease in body weight and serum leptin levels. It is well established that longitudinal growth is regulated by growth hormone and its tissue mediator IGFI [9]. However, reent evidene indiates that signals regulating energy balane also regulate the somatotrophi axis. For example leptin an promote longitudinal growth, sine impairment of leptin signalling auses redued linear growth in ob/ob and db/db mie [], and leptin administration is a potent stimulator of bone growth in ob/ob mie [31]. The hypothesis that leptin an play a ruial role in the inrease of longitudinal growth in Ankrd mie is supported by our finding that PF mie with normal body weight and leptin levels had longitudinal growth omparable to ontrols at, and months of age (ESM Fig. a, b). Defets in Ankrd expression or funtion play a ritial role in ontrolling obesity in Ankrd mie, sine its partial inativation auses hyperphagia and extreme obesity, and does not ause a redution in energy expenditure or ativity []. The important role of hyperphagia is supported by our finding that mie with a post-weaning limitation of food intake had normal body omposition (fat and lean mass; Table ). ANKRD is highly abundant in several regions of the brain, inluding the aruate, paraventriular and ventromedial nulei of the hypothalamus [], a region known to play a key role in regulation of feeding behaviour [3]. Moreover, obese Ankrd mie did not show dereased food intake and body weight in response to leptin (3 mg/kg body weight; data not shown), indiating the presene of a entral leptin resistane. Therefore the obese phenotype of Ankrd mie is very likely to be due to a prominent role of ANKRD in the regulation of food intake in the brain. In support of this onlusion are the

11 Diabetologia (11) : miroarray analysis results obtained by Ko et al. [33], who found that Ankrd mrna levels are inreased in the hypothalamus of C7BL/ mie after administration of three anti-obesity drugs (sibutramine, phendimetrazine, methamphetamine) that are known to suppress appetite by ativating ateholaminergi neurotransmission [3 3]. These findings led the authors to suggest that Ankrd probably aounts for the biologial effets of these drugs. However, the preise mehanism by whih Ankrd ontrols food intake in mie is still not lear and is urrently under investigation in our laboratory. In summary, our results identify Ankrd mie as a new model of obesity-indued diabetes, sine the inativation of this gene in vivo an lead to diabetes by impairing insulin ation and seretion in an obesity-dependent manner. Our results also identify Ankrd as a novel gene involved not only in the regulation of food intake, but also in the regulation of insulin responses in WAT. We propose that ANKRD ould be an attrative moleular target for the generation of new anti-obesity and insulinsensitising drugs. Aknowledgements We thank W. Jou and T. Chanturiya (Mouse Metabolism Core, NIDDK) for invaluable tehnial support with the hyperinsulinaemi euglyaemi lamp proedures. This researh was supported in part by the Intramural Researh Program of the NIH, the NCI, the Center for Caner Researh and in part by the NIDDK. Contribution statement GAR designed the study, researhed data, ontributed to the disussion, wrote the manusript and gave the final approval of the version to be published. TKB researhed data, ontributed to the disussion and gave the final approval of the version to be published. OG researhed data, ontributed to the disussion and gave the final approval of the version to be published. IP designed the study, wrote the manusript and gave the final approval of the version to be published. 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