Siyu Chen,1 Wenxiang Zhang,1 Chunqi Tang,1 Xiaoli Tang,1 Li Liu,2 and Chang Liu1

Vanin-1 Is a Key Activator forHepatic GluconeogenesisDiabetes 2014;63:2073–2085 | DOI: 10.2337/db13-0788

Vanin-1 (VNN1) is a liver-enriched oxidative stresssensor that has been implicated in the regulation ofmultiple metabolic pathways. Clinical investigations in-dicated that the levels of VNN1 were increased in theurine and blood of diabetic patients, but the physiolog-ical significance of this phenomenon remains unknown.In this study, we demonstrated that the hepatic expres-sion of VNN1 was induced in fasted mice or mice withinsulin resistance. Gain- and loss-of-function studiesindicated that VNN1 increased the expression of gluco-neogenic genes and hepatic glucose output, which ledto hyperglycemia. These effects of VNN1 on gluconeo-genesis were mediated by the regulation of the Aktsignaling pathway. Mechanistically, vnn1 transcriptionwas activated by the synergistic interaction of peroxi-some proliferator–activated receptor g coactivator 1a(PGC-1a) and hepatocyte nuclear factor-4a (HNF-4a).A chromatin immunoprecipitation analysis indicatedthat PGC-1a was present near the HNF-4a binding siteon the proximal vnn1 promoter and activated the chro-matin structure. Taken together, our results suggest animportant role for VNN1 in regulating hepatic gluconeo-genesis. Therefore, VNN1 may serve as a potential ther-apeutic target for the treatment of metabolic diseasescaused by overactivated gluconeogenesis.

Hepatic gluconeogenesis plays a crucial role in maintainingblood glucose homeostasis in mammals. Gluconeogenesis isactivated by fasting, which means that the synthesis andsecretion of glucagon and glucocorticoids are induced toenhance hepatic glucose production via the stimulationof pepck and g6pase expression (1,2). In contrast, insulinsuppresses gluconeogenesis via the lipid kinase phosphati-dylinositol 3-kinase pathway and correspondingly activates

glycogen synthesis when sufficient food is available (3).Thus, gluconeogenesis is finely regulated by positive andnegative regulators to achieve a balance. However, gluco-neogenesis is constitutively induced due to insulin resistancein certain pathophysiological conditions, such as type 2diabetes, and leads to excess glucose secretion and aggra-vated hyperglycemia (4,5).

Peroxisome proliferator–activated receptor g coactiva-tor 1a (PGC-1a) is one of the most important metabolicregulators and has been shown to stimulate various phys-iological processes by selectively activating nuclear recep-tors and transcriptional factors (6). Of note, PGC-1arobustly regulates gluconeogenesis. PGC-1a is elevatedin the liver at a fasted or insulin-resistant state whengluconeogenesis is activated (7). In contrast, the liver-specific knockdown of PGC-1a significantly improvesglucose intolerance during hyperglycemia (8). At the mo-lecular level, PGC-1a facilitates the transcriptional activ-ity of the glucocorticoid receptors FOXO1 and hepatocytenuclear factor-4a (HNF-4a) on the promoter of gluconeo-genic genes (9,10). These findings indicate that PGC-1a isa critical factor in the regulation of gluconeogensis andglucose homeostasis.

Vanin-1 (VNN1) is a glycosylphosphatidyl inositol–anchored pantetheinase that is highly expressed in theliver, gut, and kidney (11). Its pantetheinase activityhydrolyzes pantetheine into pantotheic acid (vitaminB5) and cysteamine (12). Pantotheic acid acts as the struc-tural component of CoA (13), which indicates that VNN1may be involved in fatty acid metabolism. Conversely,cysteamine inhibits the g-glutamylcysteine synthetase(g-GCS)–mediated regulation of the endogenous GSHpool and therefore affects the cellular redox status. VNN1-deficient mice exhibit significantly increased resistance to

1Jiangsu Key Laboratory for Molecular and Medical Biotechnology and College ofLife Sciences, Nanjing Normal University, Nanjing, Jiangsu, China2Department of Geriatrics, First Affiliated Hospital of Nanjing Medical University,Nanjing, Jiangsu, China

Corresponding author: Chang Liu, changliu@njnu.edu.cn.

Received 17 May 2013 and accepted 13 February 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0788/-/DC1.

S.C. and W.Z. contributed equally to this work.

© 2014 by the American Diabetes Association. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

Diabetes Volume 63, June 2014 2073

PATHOPHYSIO

LOGY

stress, and their intestinal inflammation is reduced (14–16).Importantly, the link between VNN1 and metabolic dis-eases has been recently revealed. For example, VNN1 pro-tects islet b-cells from streptozotocin-induced injury andregulates the development of type 1 diabetes (17). In theliver, the mRNA expression levels of VNN1, as well as itsactivity, are induced upon fasting (18,19). A clinical studyshowed that the concentrations of VNN1 in pooled humanurine distinguished diabetic patients with macroalbuminu-ria from those with normal albuminuria (20). In addition,the combination of VNN1 and MMP9 has been suggestedas a novel blood biomarker panel to discriminate pancreaticcancer–associated diabetes from type 2 diabetes (21). How-ever, these findings are descriptive, and the detailed mech-anism through which VNN1 regulates energy metabolismremains unknown.

Based on the findings mentioned above, we aimed toexplore whether VNN1 regulates hepatic gluconeogenesisand if this process requires PGC-1a participation. To an-swer this question, we used the gain-of-function and loss-of-function strategy to manipulate the VNN1 expressionlevels both in vivo and in vitro based on adenoviral trans-duction and small interfering RNA (siRNA) delivery. Ourstudies revealed an important role for VNN1 in the regula-tion of gluconeogenesis and suggest that VNN1 inhibitionmay be a therapeutic target for the treatment of metabolicdisorders associated with overactivated gluconeogenesis.

RESEARCH DESIGN AND METHODS

Male C57BL/6 mice and diabetic db/db mice on a C57BKSbackground were purchased from the Model Animal Re-search Center of Nanjing University (Nanjing, Jiangsu,China). All animal procedures in this investigation con-form to the Guide for the Care and Use of LaboratoryAnimals published by the National Institutes of Health(NIH publication 85-23, revised in 1996) and the ap-proved regulations were set by the Laboratory AnimalCare Committee at Nanjing Normal University (permit2090658, issued 20 April 2008). For fasting-induced glu-coneogenesis, the mice were subjected to 2, 5, and 16 hfasting or 16 h fasting followed by 20-h refeeding. Toinduce insulin resistance, 4-week-old mice were feda high-fat diet (HFD, fat content 60%; Research Diets,New Brunswick, NJ) for 2 months (22). To specificallymanipulate the target gene expression in liver, we trans-duced adenoviruses targeting genes or siRNA oligonucleo-tides against the target genes into mice through tail-veininjection. For these experiments, adenoviruses were con-centrated and purified at 1.5 3 109 plaque-forming units.Three days after the injection, all mice were killed and thelivers were collected. Wortmannin (Sigma-Aldrich) was in-traperitoneally injected into mice at a dose of 1.5 mg/kgbody weight 12 h before killing when necessary (23).

Pantetheinase Activity AssayThe substrate, pantothenate-AMC, was chemically syn-thesized using b-alanine 7-amido-4-methylcoumarin

trifluoroacetic acid (TFA) salt (H-b-Ala-AMC.TFA, 36 mg,1 eq) and R-(-)-pantolactone. We used pantothenate-AMC as substrate at 37°C for 30 min to assay the pan-tetheinase activity; the hydrolysis catalyzed by VNN1yields pantothenic acid and detectable free fluorescentAMC (excitation 340 nm; emission 460 nm) (24). Theliver extracts were washed three times with PBS andlysed with potassium phosphate buffer (100 mmol/L,pH 7.5) containing 0.1% Triton X-100 and 0.6% sulfosa-licylic acid. The protein concentrations were determinedwith a bicinchoninic acid protein assay. The enzymaticassay was performed using 10 mg of liver extract in phos-phate buffer (100 mmol/L potassium phosphate buffer,pH 7.5) containing 2 mmol/L pantothenate-AMC, 0.01%BSA, 0.5 mmol/L dithiothreitol, 5% DMSO, and 0.0025%Brij-35 in a total reaction mixture volume of 100 mL. Thereactions were carried out at 37°C in the presence orabsence of liver extract, and the fluorescence (excitation350 nm; emission 460 nm) was recorded every 2 min,with the change in fluorescence measured over a 30-minperiod. A standard curve was generated using purifiedrecombinant VNN1 (Sino Biological, Daxing, China) atthe same buffer conditions described above. The VNN1activity was normalized for total protein content. Thepantetheinase activity was calculated by determining theslope at 30 min from fitting the data to the standardcurve and normalizing it for total protein content.

Statistical AnalysisThe groups of data are presented as the mean 6 SD. Thedata were analyzed using a one-way ANOVA followed byFisher LSD post hoc test. The calculations were performedusing the SPSS for Windows version 12.5S statistical pack-age (SPSS, Chicago, IL). A value of P , 0.05 was consid-ered statistically significant.

RESULTS

Gluconeogenic Signals Induce Hepatic VNN1ExpressionFasting-refeeding cycles are robust nutritional signals toregulate gluconeogensis. As expected, the hepatic mRNAexpression of pgc-1a, an important metabolic regulator,was induced by fasting and peaked at 5 h (Fig. 1A). Sub-sequently, its expression gradually declined. Two gluco-neogenic genes (pepck and g6pase) were induced after2 h of fasting, and this expression persisted during theentire starvation period. Interestingly, the hepatic expres-sion of vnn1 mRNA was also induced in mice subjected tofasting and decreased upon refeeding at 20 h. The proteinexpression levels of VNN1 in the liver showed a similarpattern (Fig. 1B and Supplementary Figure 1A). Con-versely, diabetic db/db mice and HFD feeding–inducedobese mice developed insulin resistance and showed over-activated hepatic gluconeogenesis. We found that theVNN1 mRNA and protein expression levels were increasedin livers of these mice (Fig. 1C–E and Supplementary Fig.1B and C). Furthermore, VNN1 is a pantetheianse, and the

2074 Vanin-1 Activates Gluconeogenesis Diabetes Volume 63, June 2014

Figure 1—Gluconeogenic signals induce hepatic VNN1 expression. RT-qPCR (A) and Western blot (B) analysis of VNN1 and gluconeo-genic gene expression in the liver from mice subjected to fasting (2, 5, and 16 h) or fasting 16 h followed by 20-h refeeding. *P < 0.05 and**P < 0.01 vs. refed group, n = 6. RT-qPCR (C ) and Western blot (D) analysis in the liver from wild-type or db/db mice. **P < 0.01 vs. WT,n = 5. E: mRNA and protein analysis in the liver from mice fed normal diet (ND) or HFD for 2 months. **P < 0.01 vs. ND, n = 6. F:Pantetheinase activity analysis in the liver homogenates from mice described in A. **P < 0.01 vs. refed group, n = 6. G: Pantetheinaseactivity analysis in the liver from wild-type or db/dbmice. **P< 0.01 vs. WT, n = 5. H: Regulation of VNN1 expression by 8-Br cAMP in vitro.AML-12 mouse hepatocytes were treated with 1 mmol/L 8-Br cAMP for an indicated time period, and VNN1 expression was quantified byRT-qPCR (top) and Western blot (bottom) analysis. **P < 0.01 vs. 0 h. I: Reporter gene analysis of transcriptional activity of mouse vnn1promoter. mvnn1-luc was transfected into AML-12 cells. Thirty-six hours later, cells were treated with 8-Br cAMP (1 mmol/L) for another 12 h.**P < 0.01 vs. mvnn1-luc alone. J: Western blot analysis of VNN1 expression in the primary hepatocytes treated with dexamethasone(Dex, 1 mmol/L), 8-Br cAMP (1 mmol/L), or hydrocortisone (HCS, 1 mmol/L) for 6 h. All the data are represented as the mean 6 SD. CTL,control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

diabetes.diabetesjournals.org Chen and Associates 2075

biological functions of VNN1 are thought to depend on itspantetheianse enzymatic activity. In our settings, the pan-tetheianse activity of VNN1 increased in the liver homo-genates of fasted and db/db mice (Fig. 1F and G), which isconsistent with the mRNA accumulation of VNN1.

In vitro studies indicated that 8-Br cAMP, an analog ofthe natural signal molecule cAMP that activates gluconeo-genesis, stimulated vnn1 expression in a time-dependentmanner in AML-12 mouse hepatocytes (Fig. 1H). 8-BrcAMP also increased the transcriptional activity of thevnn1 promoter (Fig. 1I). In addition to cAMP, fasting-induced hormones, such as glucocorticoids, can robustlyactivate gluconeogenesis. However, we found that 8-BrcAMP, but not glucocorticoids (dexamethasone and hy-drocortisone), induced VNN1 protein expression inmouse primary hepatocytes (Fig. 1J). These data sug-gested that cAMP is one of the major players to activateVNN1 expression, whereas glucocorticoids have a modesteffect on VNN1 expression.

VNN3 is another member of the pantetheinase family.In contrast to the robust induction of VNN1 in gluconeo-genic settings, the hepatic expression of vnn3 remainedstable in fasted mice and was only moderately increased(2.1-fold) in db/db mice (Supplementary Fig. 2A and B).Functionally, the knockdown of VNN3 in the livers ofdb/db mice (Supplementary Fig. 2C) did not improve theglucose intolerance (Supplementary Fig. 2D) and insulinresistance (Supplementary Fig. 2E) of these mice. The he-patic expression levels of gluconeogenic genes also remainedunchanged (Supplementary Fig. 2F and G). Taken togeth-er, these results suggested that VNN3 does not regulategluconeogenesis.

VNN1 Activates Hepatic GluconeogenesisAs shown in Fig. 2A and B, the forced expression of ex-ogenous VNN1 in AML-12 cells by adenovirus infectioninduced the mRNA and protein expression levels of glu-coneogenic genes in a dose-dependent manner. In con-trast, VNN1 overexpression did not alter the mRNAexpression levels of baf60a (a subunit of SWI/SNF chro-matin remodeling complex), cyp7a1 (a regulator involvedin bile acid metabolism), and fas (fatty acid synthase).Functionally, the overexpression of VNN1 in mouse pri-mary hepatocytes increased the glucose production rate(Fig. 2C). In vivo studies indicated that the basal bloodglucose levels during starvation were higher in mice thatspecifically overexpressed VNN1 in the liver (Supplemen-tary Table 1). These mice also showed impaired glucosetolerance and insulin sensitivity (Fig. 2D and E). At themolecular level, the hepatic expression levels of gluconeo-genic genes were increased in response to VNN1 over-expression (Fig. 2F and G), whereas baf60a, cyp7a1, andfas remained unchanged. These data suggest that theimpact of VNN1 is specific to the regulation of gluco-neogenesis and is not a general disturbance of the he-patic metabolism. Finally, the pantetheinase activityof VNN1 in the liver homogenates of these mice

significantly increased (Fig. 2H). Taken together, our resultsstrongly suggested that VNN1 activates gluconeogenesis.

Knockdown of VNN1 Ameliorates HyperglycemiaWe next used a loss-of-function strategy to confirm theabove findings. The liver-specific knockdown of VNN1led to improved glucose tolerance in normal fasted mice(Fig. 3A). Consistently, the expression of hepatic gluco-neogenic genes was suppressed upon the depletion ofVNN1 (Fig. 3B and C). In a pathophysiological setting,hepatic VNN1 knockdown reduced the food and waterintake of db/db mice (Fig. 3D). This treatment also re-lieved glucose intolerance and insulin resistance in ani-mals (Fig. 3E and F). Accordingly, the mRNA andprotein expression levels of gluconeogenic genes were re-pressed (Fig. 3G and H). Finally, the pantetheinase activ-ity of VNN1 in the liver homogenates of these micedecreased (Fig. 3I). In agreement with these results,VNN1 knockdown in AML-12 cells repressed the 8-BrcAMP–induced increase of the transcriptional activity ofgluconeogenic gene promoters (Supplementary Fig. 3A).VNN1 knockdown also inhibited gluconeogenic gene ex-pression in AML-12 cells (Supplementary Fig. 3B and C)and decreased the glucose production rate in mouse pri-mary hepatocytes (Supplementary Fig. 3D) upon 8-BrcAMP challenge.

Akt Signal Pathway Mediates VNN1 Regulation ofGluconeogenesisThe insulin-Akt signaling axis plays an important role inthe regulation of gluconeogenesis. Therefore, we investi-gated whether this signaling cascade is involved inmediating VNN1’s effects on glucose control. As shownin Fig. 4A and B, the overexpression of VNN1 reduced theexpression levels of phosphorylated Akt both in vivo andin vitro. Conversely, the liver-specific knockdown ofVNN1 in db/db mice resulted in a marked increase inphospho-Akt levels (Fig. 4C). Similarly, the transfectionof VNN1 siRNA into AML-12 cells increased the basallevel and showed synergistic effects with insulin on Aktphosphorylation (Fig. 4D). To further prove the causalrelationship between the Akt signaling pathway andVNN1 regulation, we used wortmannin (a specific phos-phatidylinositol 3-kinase/Akt inhibitor) to treat db/dbmice with liver-specific VNN1 knockdown. Our data dem-onstrated that wortmannin alone further increased thebasal glucose level and worsened impaired glucose toler-ance in db/db mice. Additionally, the beneficial effects ofVNN1 knockdown on glucose intolerance and insulin re-sistance were attenuated when mice were subsequentlytreated with wortmannin (Fig. 4E and F). These resultsstrongly suggest that the Akt signaling pathway is re-quired to regulate VNN1 in hepatic gluconeogensis. Be-cause VNN1 is a glycosylphosphatidyl inositol–anchoredenzyme, identifying the molecule that mediates the signalcascade from VNN1 to the insulin-Akt signaling pathwayis of interest. Previous studies demonstrated that VNN1

2076 Vanin-1 Activates Gluconeogenesis Diabetes Volume 63, June 2014

prevents peroxisome proliferator–activated receptor g(PPARg) nuclear translocation and antagonizes its tran-scriptional activity in epithelial cells (25). We also ob-served similar phenomena in our system. Confocalimaging and the Western blotting of nuclear extracts in-dicated that VNN1 knockdown led to an increase in

PPARg translocation from the cytoplasm to the nucleiin AML-12 cells (Supplementary Fig. 4A and B). Giventhat PPARg activates the insulin-Akt signaling pathway(26) and inhibits gluconeogenesis (27), PPARg may actas a mediator of signal transduction from VNN1 to theAkt pathway.

Figure 2—VNN1 promotes hepatic gluconeogenesis. A and B: Regulation of gluconeogenic gene expression by VNN1 in vitro. RT-qPCR(A) and Western blot analysis (B) of VNN1 and gluconeogenic gene expression in AML-12 cells transduced with adenoviruses encodingGFP (Ad-GFP, as control) or VNN1 (Ad-VNN1) for 48 h. **P < 0.01 vs. Ad-GFP. C: Glucose output assay in mouse primary hepatocytes.Cells were infected by adenoviruses for 48 h, and glucose production rate was assessed. 8-Br cAMP (1 mmol/L, treatment time 12 h) wasused as a positive control. **P < 0.01 vs. Ad-GFP. D: Glucose tolerance test. Mice were transduced with adenoviruses encoding GFP orVNN1 through tail-vein injection (0.1 absorbance units per mouse). Three days later, mice were fasted for 16 h and the glucose tolerancetest assay was performed. **P< 0.01 vs. Ad-GFP, n = 5. E: Insulin tolerance test. Mice were treated as in D. The fasting time was shortenedto 6 h and the insulin tolerance test assay was performed. *P < 0.05 and **P < 0.01 vs. Ad-GFP group, n = 5. F and G: VNN1 andgluconeogenic gene expression in the liver. RT-qPCR (F ) and Western blot (G) analysis in liver of mice treated as in D. *P < 0.05 and **P <0.01 vs. Ad-GFP group, n = 5. H: Pantetheinase activity analysis in the liver homogenates from mice treated as in D. **P < 0.01 vs. Ad-GFPgroup, n = 5. All the data are represented as the mean 6 SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

diabetes.diabetesjournals.org Chen and Associates 2077

Figure 3—Liver-specific knockdown of VNN1 relieves hyperglycemia. A: Glucose tolerance test analysis. Normal mice were transducedwith scrambled siRNA oligonucleotides (as control) or siRNA oligonucleotides against VNN1 through tail-vein injection (2.5 mg/kg). Threedays later, mice were fasted for 16 h and the glucose tolerance test assay was performed. scra, scrambled siRNA; siVNN1, VNN1 siRNA.B: RT-qPCR analysis of vnn1 and gluconeogenic gene expression in the liver from mice treated as in A. C: Western blot analysis. D:Analysis of food intake (g/mouse/day) and water drinking (mL/mouse/day) in db/db mice transduced with scrambled or VNN1 siRNAoligonucleotides for 3 days. E: Glucose tolerance test analysis. F: Insulin tolerance test analysis. G: RT-qPCR analysis of vnn1 andgluconeogenic gene expression in the liver from db/db mice. H: Western blot analysis. I: Pantetheinase activity analysis. For all the panels,*P < 0.05 and **P < 0.01 vs. scrambled siRNA. Each group included at least six mice. All the data are represented as the mean 6 SD.GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

2078 Vanin-1 Activates Gluconeogenesis Diabetes Volume 63, June 2014

Figure 4—Akt signaling pathway mediates VNN1’s regulation of gluconeogenesis. A: Hepatic Akt phosphorylation levels in mice with liver-specific overexpression of VNN1. Top: Western blot analysis of phospho-Akt levels. Bottom: Quantitative results. **P < 0.01 vs. Ad-GFP,n = 5. B: Akt phosphorylation levels in AML-12 cells. Cells were infected by adenoviruses encoding GFP or VNN1 for 48 h and then treatedwith 100 nmol/L insulin for 0, 5, 10, and 15 min. **P < 0.01 vs. 0 min; #P < 0.05 and ##P < 0.01 vs. Ad-GFP at each time point. C: HepaticAkt phosphorylation levels in db/db mice with liver-specific knockdown of VNN1. *P < 0.05 vs. scrambled siRNA, n = 5. D: Akt phos-phorylation levels in AML-12 cells. Cells were transfected with scrambled or VNN1 siRNA oligonucleotides for 48 h and then treated with100 nmol/L insulin for 0, 5, 10, and 15 min. **P < 0.01 vs. 0 min; #P < 0.05 and ##P < 0.01 vs. scrambled siRNA at each time point.

diabetes.diabetesjournals.org Chen and Associates 2079

PGC-1a and HNF-4a Synergistically Activate VNN1TranscriptionPGC-1a is a key nuclear coactivator of gluconeogensis andmay serve as an upstream regulator for vnn1 transcrip-tion. Indeed, PGC-1a overexpression increased the VNN1expression levels both in vivo (Fig. 5A) and in vitro (Sup-plementary Fig. 5A and B). In contrast, PGC-1a knock-down inhibited VNN1 expression in the liver (Fig. 5B) andin cultured cells (Supplementary Fig. 5C and D). Epigenet-ically, histone hyperacetylation is associated with tran-scriptional activation. Conversely, H3K9me2 is typicallyfound in heterochromatin and silenced genes. Remark-ably, we found that PGC-1a overexpression led to a robustincrease in AcH3 levels with a corresponding decrease inH3K9me2 levels on the vnn1 promoter (Fig. 5C). Theknockdown of PGC-1a caused opposite results (Fig. 5D).These results indicate that PGC-1a stimulates vnn1 tran-scription by altering the local chromatin environmentfrom a repressive to an active state. We next attemptedto identify the transcriptional factors that mediate theactivation of vnn1 transcription by PGC-1a. A bioinfor-matics analysis indicated that the vnn1 promoter (2267to +1) possessed two HNF-4a binding sites (nrmotif.ucr.edu/NRBSScan/H4SBM.htm) (28). Reporter gene assaysdemonstrated that PGC-1a augmented the transcrip-tional activity of HNF-4a on a vnn1 promoter reporter(Fig. 5E). However, when the HNF-4a binding sites weremutated, the synergistic effects of PGC-1a and HNF-4aon the transcription of the vnn1 promoter disappeared(Fig. 5E and Supplementary Fig. 6). To confirm thatHNF-4a is required by PGC-1a to activate VNN1, weknocked down HNF-4a in AML-12 cells and found thatHNF-4a knockdown blocked the PGC-1a–induced epige-netic changes of the vnn1 promoter toward activation(Fig. 5F). Consistently, the activation of vnn1 transcrip-tion and translation by PGC-1a was repressed by HNF-4asiRNA (Fig. 5G). Lastly, we explored whether VNN1 acts asa downstream effector for PGC-1a–controlled gluconeogen-esis. We knocked down VNN1 in PGC-1a–overexpressedAML-12 cells and found that VNN1 knockdown inhibitedthe positive effects of PGC-1a on gluconeogenic gene ex-pression (Fig. 5H). Moreover, the glucose output of mouseprimary hepatocytes was decreased by VNN1 knockdown(Fig. 5I).

DISCUSSION

VNN1 is assumed to primarily function as a pantethei-nase, which hydrolyzes pantetheine into pantothenic acid

(also called vitamin B5 or pantothenate) and cysteamine(12). Recent studies have revealed additional functionsof VNN1. For example, VNN1-deficient mice are resistantto intestinal inflammation, oxidative stress, and experi-mental colitis (15,16). VNN1 also plays a critical role inmalaria susceptibility, psoriasis, carcinogenesis, and car-diovascular disease (29–32). Importantly, the vnn1 gene isone of the major targets of PPARa in the mouse liver,which strongly implies that VNN1 is involved in the reg-ulation of energy metabolism (18). In the current study,we characterized VNN1 as a novel factor that activateshepatic gluconeogenesis. This characterization was ini-tially based on the induction of VNN1 at the transcrip-tional level during fasting. To unmask the impact ofVNN1 during fasting glucose homeostasis in vivo, weadopted VNN1 gain- or loss-of-function manipulationmethods in mouse models. Our data suggested thatVNN1 activates gluconeogenesis and increases glucoseoutput under the control of the PGC-1a/HNF-4a com-plex, which is mediated by the Akt signaling pathway(Fig. 6).

In addition to its role in glucose homeostasis, VNN1also affects lipid metabolism. Hepatic VNN1 expression isinduced by the agonists of PPARa, a key transcriptionalfactor involved in fatty acid oxidation (18,33). The oraladministration of triglycerides or PPARa ligands, such asWY14643 and fenofibrate, leads to a robust increase ofVNN1 expression in the mouse liver (18). In agreementwith previous findings, our study showed that hepaticVNN1 expression was induced in db/db and diet-inducedobese mice, which exhibited severe hepatic steatosis. Moreimportantly, the liver-specific knockdown of VNN1 im-proved the hepatic steatosis in these animal models (S.C.,W.Z., J. Qian, and C.L., unpublished data). These findingssuggested that VNN1 may be a culprit in the pathogenesisof lipid dysregulation and accumulation. Future studies arerequired to fully elucidate the role of VNN1 in lipid me-tabolism, including fatty acid oxidation, lipid synthesis, andthe mobilization and/or conversion of triglycerides, fattyacids, and cholesterol.

The recognition of the physiological functions of VNN1has been significantly extended in recent years. Due to itspantetheinase activity, VNN1 is actively involved in theprogression of inflammatory reactions by generatingcysteamine. Therefore, it plays important roles in the path-ogenesis of diseases related to inflammation. For example,VNN1 protects pancreatic islets from streptozotocin-induced cell death and retards the development of type 1

E: Glucose tolerance test analysis. Scrambled or VNN1 siRNA oligonucleotides were transduced into db/db mice as described above, andthen wortmannin (1.5 mg/kg) was intraperitoneally injected. Twelve hours later, mice were fasted for 16 h and glucose tolerance test analysiswas performed. F: RT-qPCR (top) and Western blot (bottom) analysis in the liver samples from db/db mice treated as above. For E andF, +P < 0.05 and ++P < 0.01, *P < 0.05 and **P < 0.01 vs. db/db plus scrambled siRNA group; #P < 0.05 and ##P < 0.01 vs. db/db plusVNN1 siRNA treatment. n = 5. All the data are represented as the mean 6 SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; INS,insulin; scra, scrambled siRNA.

2080 Vanin-1 Activates Gluconeogenesis Diabetes Volume 63, June 2014

Figure 5—PGC-1a and HNF-4a activate vnn1 transcription. A: Gene expression levels in mice with liver-specific overexpression of PGC-1a. Mice were transduced with adenoviruses encoding GFP or PGC-1a through tail-vein injection (0.1 absorbance units per mouse). Threedays later, the hepatic expression levels of VNN1 and gluconeogenic genes were assessed by RT-qPCR (top) and Western blot (bottom)analysis. *P < 0.05 and **P < 0.01 vs. Ad-GFP, n = 5. B: Gene expression levels in mice with liver-specific knockdown of PGC-1a. Micewere transduced with adenoviruses encoding scrambled siRNA (as control) or siRNA against PGC-1a through tail-vein injection. Threedays later, mice were subjected to 16-h fasting followed by RT-qPCR (top) and Western blot (bottom) analysis. **P < 0.01 vs. scrambledsiRNA, n = 5. C: Chromatin immunoprecipitation assays with indicated antibodies using AML-12 cells infected with adenoviruses encoding

diabetes.diabetesjournals.org Chen and Associates 2081

diabetes (17). In the intestine, VNN1 promotes an in-flammatory reaction and intestinal injury by decreasingthe activity of g-glutamylcysteine synthetase and reduc-ing the stores of reduced glutathione (15,16). Finally, be-cause cysteamine is a transglutaminase inhibitor (34,35),VNN1 is implicated in the development of Huntingtondisease, which is characterized by the accumulation ofhighly cross-linked insoluble proteins. These proteinsmay be associated with an upregulation of transglutami-nase (summarized in Fig. 6). Conversely, recent studiesindicate that glucose homeostasis is modulated by thechronic inflammation associated with metabolic stress(36,37). Genetic models in mice have demonstrated thatthe deletion of key inflammatory mediators improves glu-cose tolerance in obesity-induced insulin resistance (38).The detrimental effects of proinflammatory pathways onglucose homeostasis are partly achieved via the inhibitoryserine phosphorylation of insulin receptor substrate 1 byJun NH2-terminal kinase. In turn, uncontrolled hypergly-cemia could further contribute to chronic inflammation.For example, the activity of nuclear factor-kB is increasedvia the modification of O-GlcNAc under high-glucose con-ditions (39). In our study, we found that the pantethei-nase activity of VNN1 was increased in liver homogenates

from db/db mice, which have chronic inflammation.Therefore, exploring the role of VNN1 pantetheinase ac-tivity in the control of gluconeogenesis is of particularinterest to future studies to integrate inflammation andglucose homeostasis via VNN1.

VNN1 knockout (KO) mice have been generated for.10 years and serve as a useful tool to elucidate thephysiological functions of VNN1. However, Roisin-Bouffayet al. (17) reported that the basal glucose levels and glu-cose tolerance do not change in VNN1 KO mice, whichdiffers from our results. This inconsistency may be due tothe microenvironment difference between whole-body KOand tissue-specific knockdown mice, which has been ob-served in many other studies. For example, Bmal1 isa core component of the mammalian circadian clock ma-chinery. Investigators reported that global bmal1-null miceexhibit impaired glucose tolerance. In contrast, liver-specific bmal1 KO mice show increased glucose tolerance(40). Because we mainly focused on the physiologicalfunction of VNN1 in the liver, we believe that our animalmodels are more straightforward and specific to addressour question.

PGC-1a is intensively involved in the regulation ofgluconeogenesis. In adults, starvation induces PGC-1a ex-pression in the liver via glucagon and glucocorticoidsignaling. Once activated, PGC-1a initiates hepatic gluco-neogenesis by coactivating key transcription factors, suchas HNF-4a, glucocorticoid receptors, and FOXO1 (9,10).In accordance with these observations, PGC-1a KO miceand RNA interference–mediated liver-specific PGC-1aknockdown mice display impaired gluconeogenic gene ex-pression and hepatic glucose production (8,41). Thesemice develop hypoglycemia upon fasting. The strategyto target PGC-1a to regulate glucose homeostasis hasbeen proposed; however, its specificity and safety cannotbe guaranteed because PGC-1a is a versatile molecule. Incontrast, the biological functions of VNN1 are relativelyfocused. In addition, it serves as a downstream effector ofPGC-1a and is required for the action of PGC-1a in glu-coneogenesis. Indeed, PGC-1a expression peaked after 5 hof fasting in our system. The expression of VNN1 in-creased later and peaked after 16 h of fasting. In addition,the fact that the expression levels of PGC-1a and VNN1

Figure 6—A model illustrating the physiological functions of VNN1in various tissues, highlighting the role of VNN1 in the regulation ofhepatic gluconeogenesis in a PGC-1a–dependent manner.

GFP or PGC-1a. The enrichment was quantified by qPCR analysis. *P < 0.05 and **P < 0.01 vs. Ad-GFP. D: Chromatin immunopre-cipitation assays in AML-12 cells. Cells were infected with adenoviruses encoding scrambled siRNA or siRNA against PGC-1a for 36 h andthen treated with 8-Br cAMP for another 12 h. *P < 0.05 and **P < 0.01 vs. 8-Br cAMP group. E: Reporter gene assays in AML-12 cellstransfected with indicated plasmids. **P < 0.01 vs. the basal levels; ##P < 0.01 vs. cotransfection of PGC-1a and HNF-4a. F: Chromatinimmunoprecipitation assays in AML-12 cells. The cells were transfected with scrambled or HNF-4a siRNA oligonucleotides for 12 h andthen infected by adenoviruses encoding GFP or PGC-1a for another 36 h. **P < 0.01 vs. scrambled siRNA plus Ad-PGC-1a. G: RT-qPCR(top) and Western blot (bottom) analysis of PGC-1a, HNF-4a, and VNN1 expression in AML-12 cells treated as in F. **P< 0.01 vs. the basallevels; ##P < 0.01 vs. scrambled siRNA plus Ad-PGC-1a. H: RT-qPCR (top) and Western blot (bottom) analysis of gluconeogenic geneexpression. AML-12 cells were transfected with scrambled or VNN1 siRNA oligonucleotides for 12 h and then infected by adenovirusesencoding GFP or PGC-1a for another 36 h. I: Glucose output assay. For H and I, *P < 0.05 and **P < 0.01 vs. the basal levels; #P < 0.05and ##P < 0.01 vs. scrambled siRNA plus Ad-PGC-1a. All the data are represented as the mean 6 SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; scra, scrambled siRNA.

2082 Vanin-1 Activates Gluconeogenesis Diabetes Volume 63, June 2014

peak at different time points suggests that these twofactors may regulate gluconeogenesis at different stagesof fasting. In the early stage of fasting when PGC-1a isrobustly induced, gluconeogenesis is mainly orchestratedby PGC-1a. When fasting persists and PGC-1a expressionlevels decline, VNN1 will relay and perpetuate gluconeo-genesis. Conversely because it is a membrane protein,VNN1 is more easily manipulated than PGC-1a, whichlocalizes in the nuclei. Collectively, aiming at VNN1, thedownstream target of PGC-1a, without disturbing PGC-1a itself, can lead to a more specific regulation of bodyglucose levels.

Inflammation, as well as oxidative stress, makes impor-tant contributions to the pathogenesis of metabolic dis-eases (37). Obese patients suffer from chronic low-gradeinflammation, as indicated by increased plasma levels ofC-reactive protein, inflammatory cytokines, and multifunc-tional proteins, such as leptin and osteopontin (42). Inflam-mation is also prominent in diabetic elderly patients (43).Inflammatory mediators are known to affect liver function.For instance, tumor necrosis factor-a stimulates hepatic lipo-genesis and promotes hyperlipidemia (44). Conversely, re-active oxygen species are pro-oxidant factors that aregenerated during diabetes development, and lipid peroxide-mediated tissue damage has been observed in both type 1and type 2 diabetes (45). In detail, diabetes-associatedfree radical injury, i.e., the accumulation of lipid peroxi-dation products, depletion of GSH, decreases in the GSH/GSSG ratio, and downregulation of key antioxidant en-zymes, has been detected in various tissues, includingthe liver, with deleterious consequences on hepatic func-tions and cognitive impairments observed in different di-abetes animal models. VNN1 plays a critical role in theregulation of inflammation and oxidative stress. VNN1-deficient mice display downregulated intestinal inflamma-tion and a tissue resistance to oxidative stress (15,16). Thisprotection is a result of the enhanced g-glutamylcysteinesynthetase activity in liver, which is due to the absence ofcysteamine and leads to elevated stores of glutathione,the most potent cellular antioxidant (14). Based on thesefindings, VNN1 could serve as one of the nodes to in-terconnect inflammation, oxidative stress, and metabolicdysregulation. If so, the induction of PGC-1a, an up-stream regulator of VNN1, would provide cell-autonomousprotection to antagonize the deleterious effects of VNN1because PGC-1a is a master player to evoke antioxidantdefense.

Akt is a crucial regulator of glucose transportation,glycolysis, glycogen synthesis, and gluconeogenesis. Im-paired Akt activation is associated with abnormal gluco-neogenesis and glucose intolerance (46). In contrast, therestoration of insulin-induced Akt phosphorylationimproves insulin sensitivity and glucose tolerance inmice that are fed an HFD (47). In the current study, wefound that VNN1 overexpression blocked insulin-inducedAkt phosphorylation in AML-12 cells, whereas VNN1knockdown restored Akt phosphorylation in db/db mice.

More importantly, the Akt inhibitor wortmannin attenu-ated VNN1 knockdown–induced inhibition in gluconeo-genic gene expression. This finding provided correlativeevidence that Akt inactivation may be involved in VNN1signaling and its effects on glucose homeostasis and in-sulin sensitivity. The role of VNN1 in the regulation ofother components involved in the insulin signaling path-way is interesting to study, such as insulin receptorsubstrate 1/2.

Histone undergoes extensive posttranslational modifi-cations in response to external signals, including acetyla-tion, methylation, phosphorylation, and SUMOylation(48,49). These modifications allow the fine tuning of thechromatin structure and gene expression in a context-dependent manner. To date, little is known about theepigenetic regulation of the vnn1 promoter. Our findingsshowed that PGC-1a altered the local chromatin envi-ronment of the HNF-4a binding site on the vnn1 pro-moter from a repressive to an active state. Consistently,PGC-1a positively regulates vnn1 gene expression. Ourfindings extend the current recognition of the epigeneticregulation of gluconeogenesis via VNN1 and highlightthe importance of PGC-1a in this process.

In summary, our results strongly implicate VNN1 as acentral player that orchestrates gluconeogenic programs.The identification of the specific role of VNN1 in hepaticgluconeogenesis would expand our knowledge to un-derstand the important metabolic coordination necessaryfor proper blood glucose control. Conversely, VNN1 mayserve as a pharmaceutical target to treat metabolicdiseases with overactivated gluconeogenesis. In fact, arecent report demonstrated the design, synthesis, andcharacterization of a novel pantetheine analog, RR6,which acts as a selective, reversible, and competitive VNN1inhibitor at nanomolar concentrations. The oral administra-tion of RR6 in rats completely inhibits plasma VNN1 activityand alters the plasma lipid profile (50). More specific inhib-itors of VNN1 will most likely be synthesized to attenuateobesity-related hyperglycemia.

Acknowledgments. The authors thank Dr. Jiandie Lin (Life SciencesInstitute, University of Michigan, Ann Arbor, MI) for providing adenoviruses(Ad-GFP, Ad-PGC-1a, Ad-Scrambled, and Ad-PGC-1a RNAi) and plasmidsencoding PGC-1a, HNF-4a, mpepck-luc, and mg6pase-luc promoters.Funding. This work was supported by grants from the National Basic Re-search Program of China (973 Program) (2012CB947600 and 2013CB911600),the National Natural Science Foundation of China (31171137 and 31271261), theProgram for New Century Excellent Talents in University by the Chinese Ministryof Education (NCET-11-0990), the Program for the Top Young Talents by theOrganization Department of the CPC Central Committee, the Collaborative In-novation Center for Cardiovascular Disease Translational Medicine (Nanjing Med-ical University), and the Priority Academic Program Development of JiangsuHigher Education Institutions.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. S.C. designed and performed research and an-alyzed data. W.Z. performed research and analyzed data. C.T. and X.T. performedresearch. L.L. provided the substrate, pantothenate-AMC, for vanin-1 activity

diabetes.diabetesjournals.org Chen and Associates 2083

analysis. C.L. designed research, analyzed data, and wrote the manuscript. C.L. isthe guarantor of this work and, as such, had full access to all the data in the studyand takes responsibility for the integrity of the data and the accuracy of the dataanalysis.

References1. Pilkis SJ, Granner DK. Molecular physiology of the regulation of hepaticgluconeogenesis and glycolysis. Annu Rev Physiol 1992;54:885–9092. Nordlie RC, Foster JD, Lange AJ. Regulation of glucose production by theliver. Annu Rev Nutr 1999;19:379–4063. Sutherland C, O’Brien RM, Granner DK. Phosphatidylinositol 3-kinase, butnot p70/p85 ribosomal S6 protein kinase, is required for the regulation ofphosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin. Dis-sociation of signaling pathways for insulin and phorbol ester regulation of PEPCKgene expression. J Biol Chem 1995;270:15501–155064. Biddinger SB, Kahn CR. From mice to men: insights into the insulin re-sistance syndromes. Annu Rev Physiol 2006;68:123–1585. Firth RG, Bell PM, Marsh HM, Hansen I, Rizza RA. Postprandial hypergly-cemia in patients with noninsulin-dependent diabetes mellitus. Role of hepaticand extrahepatic tissues. J Clin Invest 1986;77:1525–15326. Liu C, Lin JD. PGC-1 coactivators in the control of energy metabolism. ActaBiochim Biophys Sin (Shanghai) 2011;43:248–2577. Herzig S, Long F, Jhala US, et al. CREB regulates hepatic gluconeogenesisthrough the coactivator PGC-1. Nature 2001;413:179–1838. Koo SH, Satoh H, Herzig S, et al. PGC-1 promotes insulin resistance in liverthrough PPAR-alpha-dependent induction of TRB-3. Nat Med 2004;10:530–5349. Yoon JC, Puigserver P, Chen G, et al. Control of hepatic gluconeogenesisthrough the transcriptional coactivator PGC-1. Nature 2001;413:131–13810. Puigserver P, Spiegelman BM. Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and met-abolic regulator. Endocr Rev 2003;24:78–9011. Pitari G, Malergue F, Martin F, et al. Pantetheinase activity of membrane-bound Vanin-1: lack of free cysteamine in tissues of Vanin-1 deficient mice. FEBSLett 2000;483:149–15412. Maras B, Barra D, Duprè S, Pitari G. Is pantetheinase the actual identity ofmouse and human vanin-1 proteins? FEBS Lett 1999;461:149–15213. Daugherty M, Polanuyer B, Farrell M, et al. Complete reconstitution of thehuman coenzyme A biosynthetic pathway via comparative genomics. J Biol Chem2002;277:21431–2143914. Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymesrepresent a co-ordinately regulated defence against oxidative stress. Free RadicRes 1999;31:273–30015. Berruyer C, Martin FM, Castellano R, et al. Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress. Mol Cell Biol 2004;24:7214–722416. Martin F, Penet MF, Malergue F, et al. Vanin-1(-/-) mice show decreasedNSAID- and Schistosoma-induced intestinal inflammation associated with higherglutathione stores. J Clin Invest 2004;113:591–59717. Roisin-Bouffay C, Castellano R, Valéro R, Chasson L, Galland F, Naquet P.Mouse vanin-1 is cytoprotective for islet beta cells and regulates the developmentof type 1 diabetes. Diabetologia 2008;51:1192–120118. Rakhshandehroo M, Knoch B, Müller M, Kersten S. Peroxisome proliferator-activated receptor alpha target genes. PPAR Res 2010;pii:61208919. Rommelaere S, Millet V, Gensollen T, et al. PPARalpha regulates the pro-duction of serum Vanin-1 by liver. FEBS Lett 2013;587:3742–374820. Fugmann T, Borgia B, Révész C, et al. Proteomic identification of vanin-1 asa marker of kidney damage in a rat model of type 1 diabetic nephropathy. KidneyInt 2011;80:272–28121. Huang H, Dong X, Kang MX, et al. Novel blood biomarkers of pancreaticcancer-associated diabetes mellitus identified by peripheral blood-based geneexpression profiles. Am J Gastroenterol 2010;105:1661–166922. Lee YS, Li P, Huh JY, et al. Inflammation is necessary for long-term but notshort-term high-fat diet-induced insulin resistance. Diabetes 2011;60:2474–2483

23. Ueda J, Saito H, Watanabe H, Evers BM. Novel and quantitative DNA dot-blotting method for assessment of in vivo proliferation. Am J Physiol GastrointestLiver Physiol 2005;288:G842–G84724. Ruan BH, Cole DC, Wu P, et al. A fluorescent assay suitable for inhibitorscreening and vanin tissue quantification. Anal Biochem 2010;399:284–29225. Berruyer C, Pouyet L, Millet V, et al. Vanin-1 licenses inflammatory mediatorproduction by gut epithelial cells and controls colitis by antagonizing peroxisomeproliferator-activated receptor gamma activity. J Exp Med 2006;203:2817–282726. Duan SZ, Usher MG, Foley EL 4th, Milstone DS, Brosius FC 3rd, MortensenRM. Sex dimorphic actions of rosiglitazone in generalised peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-deficient mice. Diabetologia 2010;53:1493–150527. Kim HI, Ahn YH. Role of peroxisome proliferator-activated receptor-gammain the glucose-sensing apparatus of liver and beta-cells. Diabetes 2004;53(Suppl. 1):S60–S6528. Fang B, Mane-Padros D, Bolotin E, Jiang T, Sladek FM. Identification ofa binding motif specific to HNF4 by comparative analysis of multiple nuclearreceptors. Nucleic Acids Res 2012;40:5343–535629. Min-Oo G, Fortin A, Pitari G, Tam M, Stevenson MM, Gros P. Complexgenetic control of susceptibility to malaria: positional cloning of the Char9 locus.J Exp Med 2007;204:511–52430. Jansen PA, Kamsteeg M, Rodijk-Olthuis D, et al. Expression of the vaningene family in normal and inflamed human skin: induction by proinflammatorycytokines. J Invest Dermatol 2009;129:2167–217431. Pouyet L, Roisin-Bouffay C, Clément A, et al. Epithelial vanin-1 controlsinflammation-driven carcinogenesis in the colitis-associated colon cancer model.Inflamm Bowel Dis 2010;16:96–10432. Dammanahalli KJ, Stevens S, Terkeltaub R. Vanin-1 pantetheinase drivessmooth muscle cell activation in post-arterial injury neointimal hyperplasia. PLoSONE 2012;7:e3910633. Schoonjans K, Staels B, Auwerx J. The peroxisome proliferator activatedreceptors (PPARS) and their effects on lipid metabolism and adipocyte differ-entiation. Biochim Biophys Acta 1996;1302:93–10934. Lentile R, Campisi A, Raciti G, et al. Cystamine inhibits transglutaminaseand caspase-3 cleavage in glutamate-exposed astroglial cells. J Neurosci Res2003;74:52–5935. Karpuj MV, Becher MW, Springer JE, et al. Prolonged survival and decreasedabnormal movements in transgenic model of Huntington disease, with adminis-tration of the transglutaminase inhibitor cystamine. Nat Med 2002;8:143–14936. Bhargava P, Lee CH. Role and function of macrophages in the metabolicsyndrome. Biochem J 2012;442:253–26237. Hotamisligil GS. Inflammation and metabolic disorders. Nature 2006;444:860–86738. Osborn O, Olefsky JM. The cellular and signaling networks linking theimmune system and metabolism in disease. Nat Med 2012;18:363–37439. Issad T, Kuo M. O-GlcNAc modification of transcription factors, glucosesensing and glucotoxicity. Trends Endocrinol Metab 2008;19:380–38940. Lamia KA, Storch KF, Weitz CJ. Physiological significance of a peripheraltissue circadian clock. Proc Natl Acad Sci U S A 2008;105:15172–1517741. Lin J, Wu PH, Tarr PT, et al. Defects in adaptive energy metabolism withCNS-linked hyperactivity in PGC-1alpha null mice. Cell 2004;119:121–13542. Maachi M, Piéroni L, Bruckert E, et al. Systemic low-grade inflammation isrelated to both circulating and adipose tissue TNFalpha, leptin and IL-6 levels inobese women. Int J Obes Relat Metab Disord 2004;28:993–99743. Kalofoutis C, Piperi C, Zisaki A, et al. Differences in expression of cardio-vascular risk factors among type 2 diabetes mellitus patients of different age.Ann N Y Acad Sci 2006;1084:166–17744. Endo M, Masaki T, Seike M, Yoshimatsu H. TNF-alpha induces hepaticsteatosis in mice by enhancing gene expression of sterol regulatory elementbinding protein-1c (SREBP-1c). Exp Biol Med (Maywood) 2007;232:614–62145. Feillet-Coudray C, Rock E, Coudray C, et al. Lipid peroxidation and anti-oxidant status in experimental diabetes. Clin Chim Acta 1999;284:31–43

2084 Vanin-1 Activates Gluconeogenesis Diabetes Volume 63, June 2014

46. Whiteman EL, Cho H, Birnbaum MJ. Role of Akt/protein kinase B in me-tabolism. Trends Endocrinol Metab 2002;13:444–45147. Duplain H, Sartori C, Dessen P, et al. Stimulation of peroxynitrite catalysis im-proves insulin sensitivity in high fat diet-fed mice. J Physiol 2008;586:4011–401648. Strahl BD, Allis CD. The language of covalent histone modifications. Nature2000;403:41–45

49. Berger SL. An embarrassment of niches: the many covalent mod-ifications of histones in transcriptional regulation. Oncogene 2001;20:3007–301350. Jansen PA, van Diepen JA, Ritzen B, et al. Discovery of small moleculevanin inhibitors: new tools to study metabolism and disease. ACS Chem Biol2013;8:530–534

diabetes.diabetesjournals.org Chen and Associates 2085