Page 1 of 72 Diabetes

MAP Phosphatase 3 (MKP-3) Deficient Mice

Are Resistant to Diet Induced-Obesity

Bin Feng1, Ping Jiao1,2, Ynes Helou3, Yujie Li1, Qin He1, Matthew S. Walters4, Arthur Salomon5,6, Haiyan Xu1,7*

1 Hallett Center for Diabetes and Endocrinology, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, RI 02903, USA 2 School of Pharmaceutical Sciences, Jilin University, Changchun, Jilin Province, China 3 Department of Molecular Pharmacology and Physiology, Brown University, Providence, RI 02903 4 Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10065 5 Department of Molecular Biology, Biology and Biochemistry, Brown University, Providence, RI 02903 6 Department of Chemistry, Brown University, Providence, RI 02903 7 Pathobiology Program, Brown University, Providence, RI 02903

* To whom correspondence should be addressed.

Haiyan Xu MD PhD Hallett Center for Diabetes and Endocrinology Rhode Island Hospital Department of Medicine Warren Alpert Medical School of Brown University 55 Claverick St., Rm 318 Providence, RI 02903, USA Email: [email protected] Phone: 4014440347 Fax: 4014443784

The authors declare that no conflict of interest exists.

1 For Peer Review Only Diabetes Publish Ahead of Print, published online April 10, 2014 Diabetes Page 2 of 72

Abstract

MAP kinase phosphatase 3 (MKP3) is a negative regulator of ERK signaling. Our laboratory has recently demonstrated that MKP3 plays an important role in obesityrelated hyperglycemia by promoting hepatic output. In this study, we show that MKP3 deficiency attenuates high fat diet (HFD)induced body weight gain and protects mice from developing obesityrelated hepatosteatosis. Triglyceride (TG) contents are dramatically decreased in the liver of MKP3/ mice fed on a HFD compared to wild type controls. Absence of MKP3 also reduces adiposity, possibly through repressing differentiation. In addition, MKP3/ mice display increased energy expenditure, enhanced peripheral glucose disposal, and improved systemic sensitivity. Global phosphoproteomic studies were performed to search for downstream mediators of MKP3 action in liver lipid . Our results reveal that MKP3 deficiency increases of 1 (HDAC1) on 393 by 3.3 fold and HDAC2 on serine 394 by 2.33 fold. Activities of HDAC1 and 2 are increased in the livers of

MKP3/ mice fed on a HFD. Reduction of HDAC1/2 activities is sufficient to restore TG content of MKP3/ primary hepatocytes to the level similar to wild type (WT) cells.

2 For Peer Review Only Page 3 of 72 Diabetes

Introduction

Obesity has become a major public health issue and is mainly caused by sedentary

lifestyle and energydense diet. Massive expansion of white adipose tissue (WAT) is the

hallmark of obesity. Excessive energy is normally stored in WAT as TG. Lipids will be

deposited into nonadipose tissues when the limit of WAT storage capacity is reached,

such as liver and muscle. Lipotoxicity is a major causal factor for the development of

insulin resistance through promoting production of inflammatory mediators in WAT and

liver (1, 2). The effect of insulin on glycemic control is implemented through coordinated

actions in multiple tissues. In the liver, insulin represses glucose output (3); in muscle

and fat, insulin stimulates glucose uptake (4, 5). In the state of insulin resistance, glucose

output from the liver increases and glucose disposal in muscle and fat decreases. As a

result, circulating insulin levels must increase in order to maintain glucose levels

within the normal range. Whether an individual with insulin resistance ultimately develops

diabetes depends on the capacity of pancreatic β cells to sustain increased insulin secretion

(6).

The liver is an important organ for maintaining energy homeostasis. It keeps blood

glucose levels within a normal range by promoting (sequestering glucose by

glycogen synthesis) after a meal, and activating (breaking down of

glycogen) and from noncarbohydrate precursors during starvation (7).

The liver is also central for . It is highly active in oxidizing TGs to

3 For Peer Review Only Diabetes Page 4 of 72

produce energy and exports large quantities of acetoacetate to be metabolized by other tissues through circulation. It is also the main site for converting excessive carbohydrates and into free fatty acids and TGs, which are subsequently transported to adipose tissue for storage. In the setting of overnutrition, liver and adipose tissue have been considered as the primary organs for systemic metabolic disorders whereas skeletal muscle and pancreatic islets are thought as responder systems (8).

MKPs are negative regulators of the MAP kinase pathway in regulation of mitogenesis.

Our study is the first to reveal the involvement of MAP kinase phosphatase in insulinregulated metabolic pathways. We previously reported that MKP3 is highly upregulated in the liver of obese and diabetic mice, and promotes glucose output in both cultured liver cells and in the liver of mice (9, 10). In this study, we characterized the metabolic of MKP3/ mice in response to overnutrition. Global MKP3 deficiency not only decreases blood glucose levels and significantly improves systemic insulin sensitivity but also reduces TG contents in both liver and WAT, indicating that

MKP3 is involved in both glucose and lipid metabolism. Through phosphoproteomic study, we identified that MKP3 regulates in the liver through modulation of

HDAC1/2 phosphorylation.

4 For Peer Review Only Page 5 of 72 Diabetes

RESEARCH DESIGN AND METHODS

Reagents and cells

Hepa16 mouse hepatoma cells were provided by Dr. Gökhan Hotamisligil (Harvard

School of Public Health). 3T3L1 cells were purchased from ATCC. 3T3L1 CAR cells

were provided by Dr. David Orlicky (University of Colorado Health Sciences Center) (11).

Preadipocytes were differentiated as previously described (12). Mouse insulin ELISA kit

was purchased from ALPCO (Salem, NH). MKP3 antibody (Ab), HDAC1 and 2 Abs,

and HA Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Flag Ab

was purchased from Sigma (St Louis, MO). antibody was purchased from Abcam.

Insulin, dexamethasone, isobutyl methyl xanthine, ATP, horse radish peroxidase, glycerol

phosphate oxidase, glycerol kinase, and lipase were purchased from Sigma. Acyl CoA

oxidase was purchased from Wako Diagnostics (Richmond, VA). Amplex red was

purchased from Invitrogen (Carlsbad, CA). Humulin R was purchased from Eli Lilly Inc

(Indianapolis, IN). Glucose test strips and AlphaTRAK meters were purchased from

Abbott Animal Health (Abbott Park, IL). Fluorogenic HDAC Assay Kit was purchased

from BPS Bioscience (San Diego, CA). HDAC1 and 2 constructs were provided by Dr.

Matthew S. Walters (Weill Cornell Medical College). Luciferase kit was purchased from

Promega (Madison, WI). FK228 and SAHA were purchased from Selleck.com (Houston,

TX).

Isolation of primary cells

5 For Peer Review Only Diabetes Page 6 of 72

Adipocytes and stromal vascular cells were isolated from gonadal fat pads of mice as previously described (12). For in vitro , stromal vascular cells were seeded at the density of 4x105 per well on 24well plates and induced by DMEM containing 10% cosmic calf serum, 0.5M isobutyl methyl xanthine, 1M dexamethasone and 1g/ml insulin. Primary hepatocytes were isolated by infusing mouse liver with collagenase buffer and cells were filtered through 150M tissue sieve. Cells were seeded at the density of

1x106 per well in collagencoated 6well plates.

Hyperinsulinemic-euglycemic clamp study

The catheters were implanted into the left jugular vein of mice 510 days before the clamp study. After an overnight fast, hyperinsulinemiceuglycemic clamp study was performed as previously described (10).

RNA extraction and real-time PCR analysis

RNA samples were extracted from tissues or isolated cells using the TRIzol reagent

(Invitrogen ) according to the manufacturer’s instructions. Realtime PCR analysis was performed as previously described (10).

Immunoprecipitation and immunoblot blot analysis

6 For Peer Review Only Page 7 of 72 Diabetes

Tissues and cells were homogenized and lysed with icecold lysis buffer supplemented

with protease inhibitors. MKP3 was immunoprecipitated as previously described (10).

Protein lysates were separated on 412% BisTrisPAGE gel and transferred onto

polyvinylidene difluoride membranes. Following blocking, membranes were incubated

with the corresponding primary antibodies. After thorough washes, membranes were

exposed to corresponding horseradish peroxidaselinked secondary antibodies and signals

were detected with a chemiluminescence western blotting detection solution (PerkinElmer,

Waltham, MA) on the AlphaInotech fluorochem image system.

Mice maintenance and analysis

MKP3 deficient male mice were purchased from the Jackson Laboratory, which were

maintained on a mixed B6/129 background. We backcrossed them with C57BL/6 female

mice purchased from the Jackson Laboratory for nine generations. Mice were kept on a

12hour light/dark cycle and given ad libitum access to food and water. Mice were fed on

either a normal chow diet or a highfat diet (60% kcal from fat, D12492, Research Diets)

at 4 weeks of age. High fat feeding was up to 28 weeks. Body weights were measured

biweekly. At the end of study, mice were sacrificed by CO2 asphyxiation after an

overnight fast or under fed condition. Plasma and tissues were collected for further

analysis. All animal procedures used in our studies were approved by the Institutional

Animal Care and Use Committee of Rhode Island Hospital.

7 For Peer Review Only Diabetes Page 8 of 72

Histological analysis

Livers were fixed in 10% neutrally buffered formalin for one day, then transferred to 70% ethanol and paraffin embedded for sectioning. Hematoxylineosin and oil red O staining were performed at Rhode Island Hospital core laboratory.

Measurement of TG contents

The contents of tissue TG were determined using homogenates of mesenteric WAT and liver samples. Frozen tissues were weighed, pulverized in liquid nitrogen, homogenized in ethanol, vortexed, centrifuged and the supernatant was collected for measurement. For extracting plasma TG, plasma was vortexed in ethanol, centrifuged and supernatant was gathered for measurement. For measuring TG contents, TG standards or samples were mixed with reaction buffer and incubated for 30 minutes at 37ºC.

Indirect calorimetry

Oxygen consumption (VO2), carbon dioxide production (VCO2), and food intake were measured individually for 24 hours using the comprehensive lab animal monitoring system (Columbus Instruments, Columbus, OH) after oneday of acclimation. During the experiment, mice had free access to food and water. Energy expenditure was calculated

0.75 using the following formula: VO2×(3.815+1.232×RQ), and normalized to (body mass) .

8 For Peer Review Only Page 9 of 72 Diabetes

Insulin tolerance test

For insulin tolerance test (ITT), mice were fasted for 6 hours and injected with insulin at

the dose of 1U/kg for HFDfed male mice. Blood glucose levels were measured at 0, 15,

30, 45, 60 and 90 minutes post injection.

Phosphoproteomic study

Hepa16 cells were cultured in SILAC medium for eight doublings and then seeded on

6well plates in SILAC medium. Half of the cells were labeled with light and

and infected with adenovirus expressing shGFP. The other half were labeled with

heavy arginine and lysine and infected with adenovirus expressing shMKP3. For each

sample, 2.5mg of light arginine and lysinelabeled lysates were mixed with 2.5mg

of heavy arginine and lysinelabeled protein lysates. Samples were reduced with 45mM

DTT at 60ºC for 20 minutes, alkylated with 100mM iodoacetimide for 15 min in the dark,

and digested overnight with TPCKtreated trypsin. Peptides were then desalted using

SepPak C18 columns and enriched for phosphopeptides using Titansphere PhosTio kit

before loading to Orbitrap Velos ETD mass spectrometer. Results from five biological

replicates were used for data analysis. Q values for multiple hypothesis tests were also

calculated based on the determined p values using the R package QVALUE as previously

described (13).

9 For Peer Review Only Diabetes Page 10 of 72

HDAC activity assay

HDAC1 or 2 was immunoprecipitated and activity assay was performed using the fluorogenic HDAC assay kit from BPS bioscience according to the manufacturer’s instructions. IP beads were washed with lysis buffer for four times and with HDAC assay buffer twice, then incubated with 50l HDAC substrate at 37°C for 30 min. After centrifugation, 30l supernatant was taken to mix with 30l developer for 15 min incubation at room temperature. Fluorescence was read at 360/450nM.

Statistical analysis.

Results are presented as mean ± standard errors. Statistical significance was determined at

P<0.05. Student’s ttest was used to compare differences between two groups. Twoway

ANOVA and Bonferroni posttests were used to analyze multiple experimental groups.

10 For Peer Review Only Page 11 of 72 Diabetes

Results

MKP-3 deficiency protects mice from developing diet-induced obesity

MKP3/ mice are significantly lighter than wild type (WT) littermate controls at four

weeks of age (Figure 1A). When fed on a chow diet, MKP3/ mice became

indistinguishable from WT mice between six weeks and twenty weeks of age (Figure 1A).

After twentythree weeks of age, male MKP3/ male mice became significantly leaner

than WT mice (supplemental figure 1A). When challenged with a HFD, body weight gain

is attenuated by MKP3 deficiency in both genders (Figure 1B) and mice are protected

from developing dietinduced obesity. Fasting blood glucose levels and fasting plasma

insulin levels are significantly lower in MKP3/ mice fed on a HFD compared to WT

mice (Figure 1CD). There is no difference in fasting blood glucose and fasting plasma

insulin levels between WT and MKP3/ mice fed on a chow diet (supplemental figure

1B). Insulin tolerance test indicates improved systemic insulin sensitivity in HFDfed

MKP3/ mice (Figure 1E).

The weights of liver and total adipose tissue are significantly reduced in HFDfed

MKP3/ mice compared to WT controls (Figure 2A). Weights of adipose tissue are

significantly decreased in all fat depots of HFDfed female MKP3/ mice and in all

except for gonadal fat depot of HFDfed male MKP3/ mice (supplemental figure 2A).

MKP3 deficiency did not change circulating lipid profiles (Figure 2B). On a chow diet,

liver weights are also significantly reduced in MKP3/ mice but adipose tissue weights

11 For Peer Review Only Diabetes Page 12 of 72

are only significantly decreased in male MKP3/ mice without any significant change in female MKP3/ mice (supplemental figure 2BC). TG contents in the livers of HFDfed

MKP3/ mice are drastically reduced to the levels comparable to those in the livers of lean WT mice (Figure 2C). TG contents in the livers of chowfed MKP3/ mice are also significantly reduced (Figure 2C). Histology study reveals that MKP3 deficiency completely protects mice from developing HFDinduced hepatosteatosis (Figure 2D).

Gene expression analysis demonstrates significant reduction of lipogenic expression, including synthase (FAS), stearoyl CoA desaturase 1 (SCD1), acetyl CoA carboxylase 2 (ACC2), and peroxisome proliferatoractivated gamma (PPARγ) in

HFDfed MKP3/ mice compared to HFDfed WT mice (Figure 2E). To determine whether MKP3 is required for of lipogenic , FAS driven luciferase reporter construct was transfected into cultured mouse hepatoma Hepa16 cells expressing interfering RNA for GFP (shGFP) or MKP3 (shMKP3). Transcription of

FAS promoter is significantly repressed when expression of MKP3 is reduced (Figure

2EF).

Absence of MKP-3 increases energy expenditure and improves glucose homeostasis

To understand the effect of MKP3 deficiency on whole body energy homeostasis, metabolic cage study was performed. consumption, carbon dioxide release and energy expenditure are significantly increased in HFDfed MKP3/ mice in both genders during a 24 hour period (Figure 3A). Further analysis indicates that these parameters

12 For Peer Review Only Page 13 of 72 Diabetes

significantly increase during both dark and light cycles (data not shown). Food intake is

not changed in HFDfed male MKP3/ mice but is significantly increased in HFDfed

female MKP3/ mice (Figure 3A). The respiratory exchange ratio (RER) remains

unchanged, indicating that the balance between lipid and carbohydrate oxidation is not

shifted. Oxygen consumption, carbon dioxide release and energy expenditure are also

significantly increased in chowfed MKP3/ male mice during dark period but not

changed in chowfed MKP3/ female mice (data not shown). Food intake does not change

in either gender fed on a chow diet (data not shown).

Hyperinsulinemiceuglycemic clamp study demonstrates significantly increased glucose

infusion rate (GIR) in HFDfed MKP3/ female mice (Figure 3B), supporting our

previous conclusion from insulin tolerance test that systemic insulin sensitivity is

improved in the absence of MKP3. Hepatic glucose production (HGP) can be more

effectively suppressed by insulin in MKP3/ mice (Figure 3B). Glucose disposal rate is

also significantly improved in MKP3/ mice, which is most likely attributable to

increased muscle glucose uptake and (Figure 3B). WAT glucose uptake

increased 54.8% but did not reach statistical significance (Figure 3B). Plasma insulin

levels in HFDfed MKP3/ female mice are significantly lower than those of WT mice

during the clamp period (Figure 3C).

MKP-3 plays a role in adipocyte differentiation

13 For Peer Review Only Diabetes Page 14 of 72

MKP3 mRNA level has been previously reported to increase in WAT of ob/ob and db/db mice (9). In this study, MKP3 mRNA and protein expression levels are shown to significantly increase in WAT of DIO mice (Figure 4AB). Interestingly, MKP3 protein expression level significantly increases at the early stage of adipogenesis (two days post induction, Figure 4C), suggesting that MKP3 may play a role in adipocyte differentiation.

Adipogenesis of 3T3L1 CAR (a subline of 3T3L1 that stably expresses the truncated adenovirus receptor) is impaired when MKP3 expression was reduced in preadipocytes by adenovirusmediated expression of interfering RNA against MKP3

(shMKP3), as shown by decreased TG accumulation (Figure 4D). Analysis of reveals significant reduction of peroxisome proliferatoractivated receptor gamma (PPARγ), FAS and SCD1 (Figure 4E). The stromal vascular cells isolated from gonadal adipose tissue of MKP3/ mice also display impaired adipogenesis reflected by significantly decreased expression of adipocyte genes such as CCAAT/ binding protein α (C/EBPα), FAS and SCD1 (Figure 4F). It has been previously reported that baseline ERK activity mildly increases in the of MKP3/ mice and promotes myocyte proliferation (14). ERK phosphorylation significantly increased in gonadal fat of

HFDfed MKP3/ male mice but unchanged in mesenteric fat (Figure 5A and data not shown). Due to the fact that MKP3 deficiency did not affect the weight of gonadal fat but did decrease mesenteric fat, the reduction of adiposity is unlikely due to ERK activation.

Phosphorylation status of ERK2 but not ERK1 is significantly increased in the livers of

HFDfed MKP3/ mice (Figure 5B).

14 For Peer Review Only Page 15 of 72 Diabetes

HDAC1 and 2 are downstream targets of MKP-3 signaling in hepatic lipid

metabolism

To identify potential downstream mediator(s) of MKP3 action in lipid metabolism, global

phosphoproteomic study was performed using quantitative stable isotope labeling in cell

culture (SILAC) method. Cultured mouse hepatoma Hepa16 cells expressing

shMKP3 via adenovirus mediated gene transfer were labeled with heavy arginine/lysine

and control cells expressing shGFP were labeled with light arginine/lysine. Changes of

serine/ (S/T) phosphorylation caused by reduced MKP3 expression were

expressed as ratios of shMKP3/shGFP. Under the condition of reduced expression of a

phosphatase, we would assume that phosphorylation levels of downstream mediators are

increased if other phosphatases are not sufficient to compensate. Table 1 shows the

average result of five biological replicates, which contains a total of 10 proteins with an

increase of 50% or more on S/T phosphorylation. Among these candidates, HDAC1 stood

out as an interesting target with significantly increased phosphorylation on serine 393

(S393) by 3.3 fold. Interestingly, our labelfree global phosphoproteomic study with livers

from HFDfed WT and MKP3/ mice demonstrates significantly increased

phosphorylation on serine 394 (S394) of HDAC2 by 2.33 fold due to absence of MKP3

(Supplemental table 1).

HDAC1 and 2 proteins are 86% identical and 93% positive. S394 of HDAC2 is the

equivalent of S393 of HDAC1. These two proteins are functionally redundant in many

15 For Peer Review Only Diabetes Page 16 of 72

tissues (1518). To understand the meaning of S393/S394 phosphorylation on HDAC1/2 activity, alanine and mutants were used in activity assay. As shown in figure

6AB, HDAC1 S393A/HDAC2 S394A mutants are significantly less active compared to

WT proteins. Glutamic acid mutants partially mimic phosphorylation and are more active than alanine mutants but less than WT proteins. The endogenous HDAC1/2 activities are also significantly increased in the livers of HFDfed MKP3/ mice compared to WT controls and this activity can be completely blocked by a pan HDAC inhibitor trichostatin

A (Figure 6CD). Furthermore, endogenous HDAC1/2 activities are significantly decreased in the liver of HFDfed WT mice compared to lean controls (Figure 6D), consistent with our previous report that MKP3 expression is markedly increased in the liver of DIO mice (10). These results indicate that MKP3 may repress HDAC1/2 activities by dephosphorylating HDAC1/2 on S393/S394. MKP3 is predominantly localized in the but can shuttle between the nucleus and the (19).

HDAC1/2 are nuclear but can be exported to the cytoplasm under pathological conditions (20, 21). To test whether HDAC1 and 2 interact with MKP3, HDAC1 and 2 were coexpressed with MKP3 in Hepa16 cells. Both HDAC2 and MKP3 could be coimmunoprecipitaed with HDAC1 (Figure 6E). Overexpression of HDAC1 WT and

S393E mutant, but not the S393A mutant, significantly suppresses expression of SCD1 gene in Hepa16 cells (Figure 6F).

16 For Peer Review Only Page 17 of 72 Diabetes

To investigate whether increased HDAC1 and 2 activities in the liver of MKP3/ mice are

responsible for decreased TG accumulation, we used both interfering RNA and a selective

HDAC1/2 inhibitor to repress HDAC1/2 activities in livers cells. HDAC1 knockdown

alone does not significantly increase expression of lipogenic gene expression in Hepa16

cells. Reduction of HDAC2 expression alone significantly induces expression of both

SCD1 and ACC2 genes. Reduction of both HDAC1 and HDAC2 significantly increases

expression of FAS, SCD1 and ACC2 genes (Figure 7A). These results indicate that

HDAC1 and 2 have redundant function in repressing lipogenic gene expression. A potent

and selective HDAC1/2 inhibitor, FK228, was also used to confirm the results obtained

from interfering RNA against HDAC1 and 2. FK228 inhibited class I HDAC activity by

more than 50% at the concentration of 5nM whereas the pan HDAC inhibitor SAHA did

not have any effect at the same concentration (Figure 7B). FK228, not SAHA,

significantly increases expression of FAS, SCD1 and ACC2 genes at 5nM (Figure 7B).

Primary hepatocytes isolated from MKP3/ mice have a significantly reduced TG level by

33% compared to WT hepatocytes (Figure 7C). Treatment with FK228 significantly

increased TG levels by 45% in WT hepatocytes but by 96% in MKP3/ hepatocytes,

which completely abolishes the effect of MKP3 deficiency on reducing TG accumulation

(Figure 7C). These results indicate that HDAC1 and 2 are likely the downstream

mediators of MKP3 action in lipid homeostasis.

17 For Peer Review Only Diabetes Page 18 of 72

Discussion

MKP3 has a wellestablished role in attenuation of MAPK signaling by selectively dephosphorylating ERK1/2 (2224). Expression of hepatic MKP3 drastically increases in response to overnutrition (10). One potential mechanism could be due to overnutritioninduced ERK activation (25). Our previous publications show that MKP3 is an important regulator of hepatic gluconeogenesis by acutely overexpressing and knocking down MKP3 in liver cells and in the liver of lean and DIO mice via adenovirus mediated gene transfer (9, 10, 26). In this study, the role of MKP3 in whole body energy homeostasis was investigated using global MKP3/ mice. As expected, MKP3 deficiency significantly lowered fasting blood glucose levels and improved systemic insulin sensitivity in DIO mice. In addition to the beneficial phenotype on glycemic control,

MKP3 global deficiency protected mice from developing dietinduced obesity, which could attribute to reduced adiposity, decreased hepatic lipid accumulation, and increased energy expenditure. Experiments with cultured adipocytes and isolated preadipocytes demonstrate that MKP3 deficiency impairs in vitro adipocyte differentiation.

The major focus of this study is to investigate the underlying molecular mechanism of the protective effect of MKP3 deficiency on HFDinduced hepatosteatosis. The liver TG contents in HFDfed MKP3/ mice are similar to those in lean WT mice. These results indicate that MKP3 plays a role in lipid homeostasis, which is a previously unrecognized novel function. Considering the fact that RER is not changed in MKP3/ mice, it is more

18 For Peer Review Only Page 19 of 72 Diabetes

likely that MKP3 deficiency decreases lipogenesis. ERK2 activity is significantly

increased in the liver of MKP3/ mice but ERK2 is unlikely responsible for the liver

phenotype because we previously show that overexpression of the constitutively active

MEK actually significantly increases liver weight (25). To search for the downstream

targets of MKP3, we performed two phosphoproteomic studies to identify proteins with

significantly increased phosphorylation in the absence of MKP3: 1) comparing stable

isotopelabeled Hepa16 cells expressing shMKP3 versus cells expressing shGFP; 2)

comparing labelfree liver lysates from HFDfed MKP3/ mice versus liver lysates from

HFDfed WT mice. The first experiment has less variation and represents acute reduction

of MKP3 expression. The second experiment is more physiologically relevant and

reflects the status of chronic MKP3 deficiency. Interestingly, one overlap is significantly

increased phosphorylation of HDAC1 on S393 in experiment 1 and significantly increased

phosphorylation of HDAC2 on S394 in experiment 2. We also demonstrate that activities

of both HDAC1 and 2 are significantly decreased by HFD. The expression of casein

kinase 2α, which was reported to phosphorylate HDAC2 on S394 (27), is also

significantly decreased by HFD (data not shown). This indicates that both MKP3 and

casein kinase 2α maybe responsible for decreased HDAC1/2 activities in response to

overnutrition. Consistent with this hypothesis, HDAC1/2 activities are significantly

increased by MKP3 deficiency in the liver of DIO mice, accompanied by decreased

expression of lipogenic genes. These results indicate that HDAC1 and 2 may repress

19 For Peer Review Only Diabetes Page 20 of 72

lipogenesis by inhibiting expression of lipogenic genes and MKP3 is required to keep

HDAC1/2 in unphosphorylated status on residues S393 and S394, respectively.

HDACs function as transcription through deacetylating lysine residues on histone proteins, which results in chromatin condensation (28). HDACs are divided into three classes based on and function: class I (HDAC13, 8, 10), class II (47, 9,

11) and class III ( 17) (29). Among class I HDACs, HDAC1 and 2 are highly homologous and functionally redundant. Recently, HDAC1 has been shown to deacetylate

AMPK in liver cells, leading to enhanced AMPK/LKB1 interaction, subsequent

AMPK activation and degradation of lipids (30). However, AMPK is not activated in the liver of HFDfed MKP3/ mice by examining S172 phosphorylation (data not shown), indicating that HDAC1/2 most likely regulate lipid metabolism via a different mechanism.

Activities of HDAC1 and 2 can be modified by phosphorylation. It has been reported that phosphorylation on residues S421 and S423 promotes HDAC1 enzymatic activity (31).

Previous publication also showed that casein kinase2α1 promotes hypertrophic response in the heart by phosphorylating HDAC2 on S394. Our study reveals that phosphorylation on residue S393 of HDAC1 and S394 of HDAC2 are essential for their activity. The role of HDAC1 and 2 in liver lipid metabolism has not been investigated previously. Liver specific of HDAC3, also a class I HDAC, has been reported to cause hepatic lipid accumulation (32).

20 For Peer Review Only Page 21 of 72 Diabetes

Our results reveal for the first time that inactivation of HADC1/2 by either interfering

RNA or selective HDAC1/2 inhibitor FK228 is sufficient to promote lipogenic gene

expression in liver cells. Most importantly, inactivation of HDAC1/2 in primary

hepatocytes isolated from MKP3/ mice abolished the protective effect of MKP3

deficiency on lowering TG content. These results support our hypothesis that MKP3 is

involved in lipid metabolism by regulating HDAC1/2 activities through modifying

phosphorylation status on residues S393/S394. In obese state, elevated MKP3 expression

in the liver likely contributes to hepatosteatosis by repressing activities of HDAC1/2.

21 For Peer Review Only Diabetes Page 22 of 72

Author contributions

B.F., P.J., Y.H., Y.L., Q.H., A.S., and H.X research data. H.X. wrote the manuscript. B.F., P.J., Y.H., A.S., and M.W. reviewed/edited manuscript and contributed discussion.

Acknowledgments This work was supported by R01 DK080746 and R01 DK08074602S1 from National Institute of Diabetes & Digestive & Disease. Dr. Haiyan Xu is the guarantor of this work, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. B. Feng and P. Jiao are recipients of George A. Bray Research Scholars Award from Brown University. No potential conflict of interest relevant to this article exists.

22 For Peer Review Only Page 23 of 72 Diabetes

REFERENCES

1. Hotamisligil GS, and Erbay E. Nutrient sensing and in metabolic diseases. Nat Rev Immunol. 2008;8(12):92334. 2. Lomonaco R, Sunny NE, Bril F, and Cusi K. Nonalcoholic fatty liver disease: current issues and novel treatment approaches. Drugs. 2013;73(1):114. 3. Saltiel AR, and Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature. 2001;414(6865):799806. 4. Myers MG, Jr., and White MF. Insulin and the IRS proteins. Annu Rev Pharmacol Toxicol. 1996;36(61558. 5. Holman GD, and Kasuga M. From receptor to transporter: insulin signalling to glucose transport. Diabetologia. 1997;40(9):9911003. 6. Rhodes CJ. a matter of betacell life and death? Science. 2005;307(5708):3804. 7. Pilkis SJ, and Granner DK. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu Rev Physiol. 1992;54(885909. 8. Muoio DM, and Newgard CB. Obesityrelated derangements in metabolic regulation. Annu Rev Biochem. 2006;75(367401. 9. Xu H, Yang Q, Shen M, Huang X, Dembski M, Gimeno R, Tartaglia LA, Kapeller R, and Wu Z. Dual specificity MAP kinase phosphatase 3 activates PEPCK transcription and increases gluconeogenesis in rat hepatoma cells. J Biol Chem. 2005. 10. Wu Z, Jiao P, Huang X, Feng B, Feng Y, Yang S, Hwang P, Du J, Nie Y, Xiao G, et al. MAPK phosphatase3 promotes hepatic gluconeogenesis through dephosphorylation of forkhead box O1 in mice. J Clin Invest. 2010;120(11):390111. 11. Orlicky DJ, DeGregori J, and Schaack J. Construction of stable coxsackievirus and adenovirus receptorexpressing 3T3L1 cells. J Lipid Res. 2001;42(6):9105. 12. Jiao P, Chen Q, Shah S, Du J, Tao B, Tzameli I, Yan W, and Xu H. Obesityrelated upregulation of monocyte chemotactic factors in adipocytes: involvement of nuclear factorkappaB and cJun NH2terminal kinase pathways. Diabetes. 2009;58(1):10415. 13. Storey JD, and Tibshirani R. Statistical significance for genomewide studies. Proc Natl Acad Sci U S A. 2003;100(16):94405. 14. Maillet M, Purcell NH, Sargent MA, York AJ, Bueno OF, and Molkentin JD. DUSP6 (MKP3) null mice show enhanced ERK1/2 phosphorylation at baseline and increased myocyte proliferation in the heart affecting disease susceptibility. J Biol Chem. 2008;283(45):3124655. 15. Montgomery RL, Davis CA, Potthoff MJ, Haberland M, Fielitz J, Qi X, Hill JA, Richardson JA, and Olson EN. Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis, growth, and contractility. Genes Dev. 2007;21(14):1790802. 16. Montgomery RL, Hsieh J, Barbosa AC, Richardson JA, and Olson EN. Histone deacetylases 1 and 2 control the progression of neural precursors to neurons during brain development. Proc Natl Acad Sci U S A. 2009;106(19):787681.

23 For Peer Review Only Diabetes Page 24 of 72

17. Yamaguchi T, Cubizolles F, Zhang Y, Reichert N, Kohler H, Seiser C, and Matthias P. Histone deacetylases 1 and 2 act in concert to promote the G1toS progression. Genes Dev. 2010;24(5):45569. 18. LeBoeuf M, Terrell A, Trivedi S, Sinha S, Epstein JA, Olson EN, Morrisey EE, and Millar SE. Hdac1 and Hdac2 act redundantly to control p63 and functions in epidermal progenitor cells. Dev Cell. 2010;19(6):80718. 19. Karlsson M, Mathers J, Dickinson RJ, Mandl M, and Keyse SM. Both nuclearcytoplasmic shuttling of the dual specificity phosphatase MKP3 and its ability to anchor MAP kinase in the cytoplasm are mediated by a conserved nuclear export signal. J Biol Chem. 2004;279(40):4188291. 20. Kim JY, Shen S, Dietz K, He Y, Howell O, Reynolds R, and Casaccia P. HDAC1 nuclear export induced by pathological conditions is essential for the onset of axonal damage. Nat Neurosci. 2010;13(2):1809. 21. Bakin RE, and Jung MO. HDAC2 cytoplasmic sequestration potentiates terminal differentiation. The Open Cell Dev & Biol J. 2008;1(19. 22. Camps M, Nichols A, and Arkinstall S. Dual specificity phosphatases: a gene family for control of MAP kinase function. Faseb J. 2000;14(1):616. 23. Dickinson RJ, and Keyse SM. Diverse physiological functions for dualspecificity MAP kinase phosphatases. J Cell Sci. 2006;119(Pt 22):460715. 24. Zhao Y, and Zhang ZY. The mechanism of dephosphorylation of extracellular signalregulated kinase 2 by mitogenactivated phosphatase 3. J Biol Chem. 2001;276(34):3238291. 25. Jiao P, Feng B, Li Y, He Q, and Xu H. Hepatic ERK activity plays a role in energy metabolism. Molecular and cellular endocrinology. 2013;375(12):15766. 26. Jiao P, Feng B, and Xu H. Mapping MKP3/FOXO1 interaction and evaluating the effect on gluconeogenesis. PLoS One. 2012;7(7):e41168. 27. Segre CV, and Chiocca S. Regulating the regulators: the posttranslational code of class I HDAC1 and HDAC2. Journal of Biomedicine and Biotechnology. 2011:Article ID 690848. 28. Grunstein M. Histone in chromatin structure and transcription. Nature. 1997;389(6649):34952. 29. Ye J. Improving insulin sensitivity with HDAC inhibitor. Diabetes. 2013;62(3):6857. 30. Lin YY, Kiihl S, Suhail Y, Liu SY, Chou YH, Kuang Z, Lu JY, Khor CN, Lin CL, Bader JS, et al. Functional dissection of lysine deacetylases reveals that HDAC1 and p300 regulate AMPK. Nature. 2012;482(7384):2515. 31. Pflum MK, Tong JK, Lane WS, and Schreiber SL. Histone deacetylase 1 phosphorylation promotes enzymatic activity and complex formation. J Biol Chem. 2001;276(50):4773341. 32. Knutson SK, Chyla BJ, Amann JM, Bhaskara S, Huppert SS, and Hiebert SW. Liverspecific deletion of histone deacetylase 3 disrupts metabolic transcriptional networks. Embo J. 2008;27(7):101728.

24 For Peer Review Only Page 25 of 72 Diabetes

Figure Legends

Figure 1. MKP-3-/- mice are resistant to diet-induced obesity. A. Growth curves of WT

and MKP3/ mice on a chow diet (n=1015 per group). B. Growth curves of WT and

MKP3/ mice on a high fat diet (HFD) (n=11 for male WT, n=10 for male MKP3/,

n=13 for female WT, and n=7 for female MKP3/ mice). C. Fasting blood glucose levels

of HFDfed WT and MKP3/ mice (n=7 per group). D. Fasting plasma insulin levels of

HFDfed WT and MKP3/ mice (n=67 per group). E. Insulin tolerance test of male mice

fed on HFD for 16week (n=7 per group). *P<0.05, WT vs. MKP3/.

Figure 2. MKP-3 deficiency prevents HFD-induced hepatosteatosis. A. Weights of

liver and total adipose tissue of HFDfed wild type (WT) and MKP3/ mice (n=67 per

group). B. Circulating free fatty acid (FFA) and triglyceride (TG) levels in HFDfed WT

and MKP3/ male mice (n=37 per group). C. Liver triglyceride (TG) contents in HFD

and chow fed WT and MKP3/ mice (n=7 for male WT on HFD, n=6 for male MKP3/

HFD, n=5 for male WT chow, n=9 for male MKP3/ chow, n=7 for female WT HFD and

MKP3/ HFD, n=10 for female WT chow and n=8 for female MKP3/ chow). D. Liver

histology of HFDfed male WT and MKP3/ mice. E. Lipogenic gene expression in the

livers of HFDfed male WT and MKP3/ mice (n=78 per group). F. MKP3 is essential

for transcription of (FAS) promoter. Hepa16 cells were transfected

with pCMVRenilla control vector plus pGL3Basic or pGL3FASpromoter using Fugen

HD transfection reagent. The next day, cells were infected with AdshGFP or AdshMKP3.

25 For Peer Review Only Diabetes Page 26 of 72

Luciferase assay was performed using Promega’s duel luciferase assay kit. The ratio of luminescence from FASpromoter (firefly) to luminescence from the control reporter

(pCMVRenilla) was calculated and presented. Result shown here is representative of three independent experiments. Luc, luciferase, FAS, fatty acid synthase; SCD1, stearoyl

CoA desaturase 1; ACC2, acetyl CoA carboxylase 2; PPARγ, peroxisome proliferatoractivated receptor gamma. *P<0.05, WT vs MKP3/, or AdshGFP vs

AdshMKP3; #, P<0.05, DIO vs. lean.

Figure 3. MKP-3 deficiency increases energy expenditure and improves glucose homeostasis. A. Oxygen consumption (VO2), carbon dioxide release (VCO2), energy expenditure, food intake and respiratory exchange ratio (RER) of WT and MKP3/ male mice (n=4 per group) fed on HFD for 18week. B. Hyperinsulinemiceuglycemic clamp study of WT and MKP3/ female mice fed on HFD for 28 week (n=36 per group). C.

Plasma insulin levels during basal and clamp periods (n=36 per group). GIR, glucose infusion rate; HGP, hepatic glucose production; WAT, white adipose tissue. *P<0.05, WT vs. MKP3/.

Figure 4. MKP-3 is involved in adipogenesis. A. MKP3 mRNA expression in WAT of chow and HFDfed male mice (n=4 per group). B. MKP3 protein expression in WAT of chow and HFDfed male mice (n=4 per group). C. MKP3 protein expression in 3T3L1 cells 2 days post induction (D2PI) vs. preadipocytes (pre). D-E. MKP3 knockdown in

26 For Peer Review Only Page 27 of 72 Diabetes

3T3L1 CAR preadipocytes and the effect on adipogenesis. F. Gene expression pattern

during adipogenesis of stromal vascular cells isolated from wild type or MKP3/ mice

(n=34 per group). For cell based experiments, results shown here are representative of

three independent experiments. PPARγ, peroxisome proliferatoractivated receptor gamma;

FAS, fatty acid synthase; SCD1, stearoyl CoA desaturase 1; c/EBPα, CCAAT/enhancer

binding protein alpha. *P<0.05, as indicated.

Figure 5. Effect of MKP-3 deficiency on basal ERK1/2 phosphorylation. A. Basal

ERK1/2 phosphorylation in the mesenteric adipose tissue of male HFDfed WT and

MKP3/ mice (n=67 per group). B. Basal ERK1/2 phosphorylation in the livers of male

HFDfed WT and MKP3/ mice (n=67 per group). *P<0.05, WT vs MKP3/.

Figure 6. MKP-3 deficiency activates HDAC1/2. A. Effects of serine 393 mutants on

HDAC1 activity in Hepa16 cells. B. Effects of serine 394 mutants on HDAC2 activity in

Hepa16 cells. C. Hepatic HDAC1 and 2 activities in wild type (WT) and MKP3/ mice

fed on a high fat diet (n=67 per group). D. Hepatic HDAC1 and 2 activities in WT mice

fed on a chow diet vs WT mice fed on a high fat diet (n=312 per group). E.

HDAC1/HDAC2/MKP3 interaction in Hepa16 cells. F. HDAC1 WT and HDAC1

S393E mutant, but not HDAC1 S393A mutant, reduces the expression level of

endogenous SCD1 gene in Hepa16 cells. For cell based experiments, results shown here

27 For Peer Review Only Diabetes Page 28 of 72

are representative of three independent experiments. βgal, βglactosidase; Vec, vector;

TSA, tricostatin A. *P<0.05 as indicated.

Figure 7. Reduction of HDAC1/2 activity increases lipogenesis in liver cells. A. Effects of HDAC1/2 knockdown on lipogenic gene expression in Hepa16 cells. B. Effects of selective HDAC1/2 inhibitor FK228 on lipogenic gene expression in Hepa16 cells. C.

Effects of selective HDAC1/2 inhibitor FK228 on lipogenesis in primary hepatocytes isolated from wild type or MKP3/ mice. For cell based experiments, results shown here are representative of three independent experiments. *P<0.05 as indicated; ***P<0.001 as indicated.

28 For Peer Review Only Page 29 of 72 Diabetes

Table 1. Fold increase of Ser/Thr phosphorylation in mouse hepatoma cells with reduced MKP3 expression versus control cells

Symbol Name Phosphorylation KD/Control Q Value for sites ratio SILAC

Transcription HDAC1 Histone deacetylase 1 S393 3.3 0.02 THRAP3 Thyroid associated S935 1.5 0.04 protein 3 Translation repression EEF1D Eef1d protein S162 2.7 0.02 Ribosomal biogenesis NOP58 Nucleolar protein 58 S509 1.6 0.03 Protein folding Ero1l Ero1l protein S106 2.6 0.047 PTK9 Twinfilin1 S142 179 0.02 Membrane trafficking SNX2 2 S97T101T104 9.4 0.03 RNA processing PCBP1 Poly(rC)binding protein 1 S173 2.5 0.047 Function unknown RSRC2 Rsrc2 arginine/serinerich coiledcoil S32 2.0 0.02 protein 2 isoform 1 AHNAK nucleoprotein isoform 1 S136 1.5 0.03

29 For Peer Review Only Diabetes Page 30 of 72 Figure 1

A B WT Male chow Female chow Male HFD Female HFD MKP-3 -/-

* * 35 50 * 35 24 45 * 30 * 30 * 22 40 * * 25 20 35 * 25 * 18 30 * 20 16 * 20 * * 25 15 14 20 * 15 * Body weight Body weight (g) Body weight Body weight (g) 12 15 Body weight (g)

10 Body weight (g) 10 10 10 4 6 8 10 12 14 16 18 20 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Age (weeks ) Age (weeks ) Weeks on HFD Weeks on HFD

C D E 250 * 3.5 * 1.2 * 3.0 200 1.0 * 2.5 0.8 150 2.0 * * * * 0.6 * 1.5 100 0.4 1.0 0.2 Fasting Fasting plasma insulin (ng/ml)

Fasting Fasting blood glucose (mg/dL) 50 0.5

Percent change of blood glucose 0.0 0 0.0 0 15 30 45 60 90 WT MKP-3 -/- WT MKP-3 -/- WT MKP-3 -/- WT MKP-3 -/- Time after insulin injection (minutes) Male HFD Female HFD Male HFD Female HFD

For Peer Review Only PageFigure 31 of 272 Diabetes

WT A MKP-3-/- B

2.5 5 12 0.8 Male Male * * * * 0.7 0.4 2.0 10 4 0.6 8 0.5 0.3 1.5 * 3

6 0.4 0.2 1.0 2 0.3 4 0.2 0.1 0.5 1 2 (mM) FFA Plasma 0.1 (mg/ml) TG Plasma Liver weight (g) Liver weight/ Liver weight/ weight body (%) Total fat weight Total fat weight (g) 0.0 0.0 0.0 0 0 WT MKP3-/- WT MKP3-/- Male Female Male Female Male Female HFD HFD HFD HFD HFD HFD HFD HFD

C D Oil red O staining HE staining Male Female 60 * * 30

40 * 20 # * WT 20 # TG (mg/g TG(mg/g liver) # TG(mg/g liver) 10 #

0 0 HFD Chow HFD Chow E MKP-3-/-

1.5 1.2 * 1.5 1.5 1.0 * * * 1.0 0.8 1.0 1.0 / 28S /

γ F 0.6 0.5 0.4 0.5 0.5 FAS/ 28SFAS/ * ACC2/ 28SACC2/ SCD1/ 28S PPAR 0.2 100 0.0 0.0 0.0 0.0 Ad-shGFP HFD HFD HFD HFD Ad-shMKP3 50 For Peer Review Only Firefly/ Firefly/ Renilla

0 pGL-Basic pGL-Fas-Luc Figure 3 Diabetes Page 32 of 72

A WT MKP-3-/-

3000 12 4.0 0.9 4000 * * * * 0.8 3500 2500 10 3.5 * * * 3.0 0.7 3000 /hr) 2000 8 2.5 0.6 2500 0.75 0.5 1500 2.0 2000 (ml/kg/hr) 6 RER RER 2 0.4 (ml/kg/hr) (ml/kg/hr) 2 1500 1000 4 1.5 0.3

VO 1.0 1000 VCO 0.2 500 2 Energy Energy expenditure (kcal/kg 500 Foodintake (g/kg/hr) 0.5 0.1 0 0 0 0.0 0.0 Male Female Male Female Male Female Male Female Male Female HFD HFD HFD HFD HFD HFD HFD HFD HFD HFD

B 40 25 20 4 80 40 * * * * 20 30 * 60 30 15 3 15 20 40 20 10 2 10 10 20 10 5 1

5 Muscle glycolysis (mg/kg/min) GIR (mg/kg/min) GIR (mg/kg/min) Glucose Glucose disposal rate (mg/kg/min) WAT glucose uptake (mg/kg/min) Muscle Muscle glucose uptake (mg/kg/min) 0 % suppression of HGP 0 0 0 0 0 WT MKP-3-/- WT MKP-3-/- WT MKP-3-/- WT MKP-3-/- WT MKP-3-/- WT MKP-3-/- C

6 * 5 WT 4 MKP-3-/- ) ) 3 2 1

Clamp Clamp plasma insulin (ng/mL 0 For Peer Review Only -5 Min 110 Min FigurePage 33 of4 72 Diabetes

A B C 2.5 8 * * MKP-3 10 2.0 * 6 1.5 Tubulin MKP-3 4 5 1.0 Chow HFD Tubulin MKP-3 /28S MKP-3 /Tubulin

0.5 2 Pre D2PI MKP-3 /Tubulin

0.0 0 0 Chow HFD Chow HFD Pre D2PI D E IP:MKP3 WS: MKP-3 1.0 1.0 1.0 1.0

Tubulin 28S / 0.5 * γ 0.5 0.5 * 0.5 shGFP shMKP-3 * 28SFAS/ SCD1/ 28S * MKP-3/ 28S 0.0 PPAR 0.0 0.0 0.0 shGFP shMKP-3 shGFP shMKP-3 shGFP shMKP-3 shGFP shMKP-3

F 1.4 1.0 1.2 1.0 1.0 1.0 1.0 * / 28S/ /28S

 0.8 0.5 0.6 * 0.5 0.5 * 0.5 SCD1/ 28S FAS/ 28SFAS/ MKP-3 /28S PPAR γ C/EBP 0.4 * 0.2 0.0 0.0 0.0 0.0 0.0 WT MKP3 -/- WT MKP3 -/- WT MKP3 -/- WT MKP3 -/- WT MKP3 -/-

For Peer Review Only Diabetes Page 34 of 72 Figure 5

A

4 * * pERK1/2 3 2

tERK1/2 2 1

Tubulin 1 pERK2/ERK2 pERK1/ERK1

-/- WT MKP-3 0 0 WT MKP3-/- WT MKP3-/-

B 1.4 14 * pERK1/2 1.2 12 1.0 10 tERK1/2 0.8 8 0.6 6 0.4 4 Tubulin pERK1/ERK1 pERK2/ERK2 0.2 2 0.0 0 -/- WT MKP3-/- WT MKP3-/- WT MKP3

For Peer Review Only FigurePage 35 of6 72 Diabetes

A B C 3000 1000 * * * * 15000 2500 * * 800 * * * 2000 * 10000 600 1500 400 1000

HDAC2 activity HDAC2 * * 5000

HDAC1 activity HDAC1 500 * 200 activity HDAC1 0 0 0 WT MKP-3-/- WT MKP-3-/- -Gal -Gal Vec Vec

 TSA - - + + HDAC2 HDAC2 HDAC2 HDAC2 S394A HDAC2 S394E HDAC1 HDAC1 HDAC1 HDAC1 S393A HDAC1 S393E

8000 * IP: Flag IP: IgG IP: Flag IP: IgG 6000 HDAC1 HDAC2 4000 Tubulin Tubulin 2000 HDAC2 activity HDAC2 HDAC1 β-gal HDAC1 HDAC1 Vec HDAC2 HDAC2 HDAC2 0 gal S393A S393E - -/- Vec Vec β S394A S394E WT MKP-3 HDAC1 HDAC1 HDAC2 HDAC2

D E F IP: HDAC1 IP: IgG

1.5 HDAC1 * * 9000 20000 WB HDAC2 8000 MKP-3 1.0 7000 15000 * 6000 * * * 5000 HDAC1 10000 4000 28SSCD1/ 0.5 3000 HDAC2 2000 5000 HDAC1 activity HDAC2 activity Input 1000 0 0 MKP-3 0.0 Lean DIO Lean DIO GFP HDAC1 HDAC1 HDAC1 Tubulin S393A S393E

For Peer Review Only Figure 7 Diabetes Page 36 of 72

A 2.0 2.0 2.0 3 3 * * * * * 1.5 * 1.5 1.5 * * 2 2 * * 1.0 1.0 1.0 1 1

HDAC1/ HDAC1/ 28S 0.5 HDAC2/ 28S 0.5 28SFAS/ 0.5 SCD1/ 28S 28SACC2/ 0.0 0.0 0.0 0 0 shGFP + shGFP + shGFP + shGFP + shGFP + shHDAC1 + + shHDAC1 + + shHDAC1 + + shHDAC1 + + shHDAC1 + + shHDAC2 + + shHDAC2 + + shHDAC2 + + shHDAC2 + + shHDAC2 + +

B C

8 12000 *** *** 1.5 3 0.4 * * * * * * 10000 * * 6 0.3 8000 1.0 2 * 6000 4 0.2 FAS/ 28SFAS/ 4000 0.5 SCD1/ 28S 1 ACC2/ 28SACC2/ 2 0.1

2000 TG(mg/mg protein) Class Class I HDAC activity 0 0.0 0 0 0.0 Vehicle + + DMSO + DMSO + DMSO + DMSO + FK228 5nM + + FK228 + FK228 + FK228 + FK228 + WT MKP3-/- 5nM 5nM 5nM 5nM SAHA + SAHA + SAHA + SAHA + 5nM 5nM 5nM 5nM

For Peer Review Only Page 37 of 72 Diabetes Supplemental figure 1

A Male 12 wk Male 23 wk Male 27 wk

30 40 40 ** * 25 30 30 20 15 20 20 10 10 10 5 Body weightBody(g) Body weightBody(g) Bodyweight (g) 0 0 0 WT MKP-3-/- WT MKP-3-/- WT MKP-3-/-

Female 12 wk Female 23 wk Female 27 wk

25 30 30 20 25 25 15 20 20 15 15 10 10 10 5 5 5 Body weightBody(g) Body weightBody(g) 0 weightBody(g) 0 0 WT MKP-3-/- WT MKP-3-/- WT MKP-3-/- B 160 0.7 140 0.6 120 0.5 100 0.4 80 0.3 60 40 0.2 20 0.1 Plasma (ng/ml) Plasma insulin Blood glucose (mg/dL) glucose Blood(mg/dL) 0 0 WT MKP3 -/- WT MKP3 -/- WT MKP3 -/- WT MKP3 -/- Male chow Female chow Male chow Female chow For Peer Review Only Diabetes Page 38 of 72 Supplemental figure 2

A. WT MKP-3-/-

3.0 5 2.5 2.5 * * * * * 2.5 4 2.0 2.0 * 2.0 3 1.5 * 1.5 1.5 2 1.0 1.0 PR fat fat PR(g)

1.0 Mes fat (g) SubQ fat (g) 1 0.5 0.5 Gonadal fat (g) 0.5 0.0 0 0.0 0.0 Male Female Male Female Male Female Male Female HFD HFD HFD HFD HFD HFD HFD HFD

B C WT MKP-3-/- WT MKP-3-/-

1.6 0.9 1.4 * * * 1.2 1.4 0.8 * 0.7 1.0 1.2 1.0 0.6 0.8 0.5 0.8 0.4 0.6 0.6 Liver Liver (g) 0.3 0.4 0.4 SubQ fat (g)

Gonadal fat (g) 0.2 0.2 0.2 0.1 0.0 0.0 0.0 Male Female Male Female Male Female chow chow chow chow chow chow

For Peer Review Only Page 39 of 72 Diabetes

Supplemental table 1. Fold increase of Ser/Thr/Tyr phosphorylation in livers of MKP-3-/- male mice versus wild type male mice fed on a high fat diet (n=5 for each group)

Symbol Name Phosphorylation KO/WT ratio Q Value sites

Apoptosis ACIN1 Acin1 protein S64 11.4 0.02 T66 8.3 0.02 API5 inhibitor 5 S461 2.2 0.03 S462 1.8 0.03 AIFM3 Apoptosis-inducing factor 3 S30 7.4 0.03 TP53BP2 Apoptosis-stimulating of p53 protein 2 S479 3.7 0.02 ACIN1 Apoptotic chromatin condensation S64S70 5.7 0.02 1 S208 7.1 0.02 S210 7.0 0.02 S479S482S491 105.7 0.03 S729 2.8 0.02 PEA15 Astrocytic phosphoprotein PEA-15 S116 10.8 0.02 BAG3 BAG family molecular chaperone regulator S179 3.3 0.02 3 S353S357 2.3 0.03 S360 2.3 0.03 S390 8.4 0.02

BNIP2 BCL2/adenovirus E1B 19 kDa-interacting S114 3.9 0.02 protein 2 alpha BNIP3 BCL2/adenovirus E1B 19 kDa protein- S60 19.8 0.03 interacting protein 3 S85T86 14.5 0.02 S88 6.1 0.02 BCL2L13 Bcl-2-like protein 13 S387 4.0 0.02 T389 3.1 0.02 CAAP1 Caspase activity and apoptosis inhibitor S183 2.3 0.03 CARD6 Card6 caspase recruitment domain family, S809 3.6 0.03 member 6 CASP8 Caspase-8 S188 2.2 0.02 DAP Death-associated protein S51 5.4 0.02 DAPK2 Death-associated protein kinase 2 S299 3.8 0.02 S349 3.5 0.02 FAF1 FAS-associated factor 1 S269 17.1 0.04 GAS2 Growth arrest-specific protein 2 T282 5.3 0.02 S283 7.4 0.02 S287 5.3 0.02 S289 7.4 0.02 GCH1 GTP cyclohydrolase 1 S24 3.9 0.02 HTT Huntingtin S398S409S411 9.7 0.02 KRT18 , type I cytoskeletal 18 T9 2.7 0.02 S31S32S35 2.8 0.02 S43S45 3.1 0.02 PDCD5 MCG128907 S119 10.7 0.02 Y126 4.0 0.02 BNIP3I MCG2480, isoform CRA_b S61S62 12.9 0.03 S63S64 8.1 0.02

1

For Peer Review Only Diabetes Page 40 of 72

RMDN3 regulator of dynamics protein S44 2.8 0.02 3 isoform X1 S46 4.2 0.02 S50 2.8 0.02 SERBP1 Plasminogen activator inhibitor 1 RNA- S197 12.9 0.02 binding protein S243 11.1 0.02 PLEKHB2 Pleckstrin homology-like domain family B S411 4.7 0.03 member 2 S465 3.8 0.03 PDCD4 Programmed cell death protein 4 S76 2.8 0.02 T93 7.8 0.02 S457 4.2 0.02 RRAGC Ras-related GTP-binding protein C S381 6.1 0.02 EBAG9 Receptor-binding antigen expressed S36 11.0 0.02 on SiSo cells RMDN3 Regulator of microtubule dynamics protein S212 1.7 0.03 3 SGPP1 Sphingosine-1-phosphate phosphatase 1 S101 9.9 0.02 UACA Uaca protein S240 13.5 0.02 Autophagy ATG9a Autophagy 9-like 1 protein S735 7.1 0.02 UFL1 E3 UFM1-protein 1 S458 10.2 0.02 S462 213.8 0.02 ZSWIM8 SWIM domain-containing S48 10.3 0.03 protein KIAA0913 ATAD1 ATPase family AAA domain-containing S319 6.5 0.02 protein 1 PGLS 6-Phosphogluconolactonase S46 49.0 0.02 S49 34.0 0.02 CA5A Carbonic anhydrase 5A, mitochondrial S32 4.9 0.02 ENGASE Cytosolic endo-beta-N- S16 5.2 0.04 acetylglucosaminidase ENO1 Enolase alpha S351 2.7 0.02 FBA1 Fructose-bisphosphate aldolase S36 3.5 0.02 T39 2.5 0.02 S161 1.7 0.03 T227T235T241 2.5 0.02 GCGR receptor S433 17.0 0.02 S436 18.0 0.02 GAPDH Glyceraldehyde-3-phosphate S149T151 3.5 0.02 S208 2.4 0.02 GYS2 Glycogen [starch] synthase, liver S8T10S11 5.7 0.02 T626 8.7 0.02 S627 7.2 0.02 GYS1 Glycogen [starch] synthase, muscle S653S657 5.3 0.02 GSK3B kinase 3 beta Y216 4.3 0.02 GSK3A Glycogen synthase kinase-3 alpha T19 1.8 0.03 S21 1.7 0.03 PGAM1 MCG113582 S14 3.0 0.02 PPP1R2 MCG126099, isoform CRA_b S122S123 3.9 0.02 TOMM70A Mitochondrial import receptor subunit S94S99 9.8 0.02 TOM70 S105 3.1 0.02 S108S113 4.8 0.02 PGM3 Phosphoacetylglucosamine mutase S64 3.5 0.02 PGM2 Phosphoglucomutase 2 S135 9.0 0.02

2

For Peer Review Only Page 41 of 72 Diabetes

T171 4.2 0.02 STBD1 Stbd1 protein S161 8.4 0.02 TPI1 Triosephosphate Y127S130 3.6 0.02 UGDH UDP-glucose 6-dehydrogenase T474 2.1 0.03 SLC35A3 UDP-N-acetylglucosamine transporter S115 15.0 0.02 isoform X1 Cell adhesion and extracellular matrix AF6 Afadin S1173 3.2 0.02 S1182 3.9 0.02 S1275 5.4 0.02 FN1 Anastellin S280 6.6 0.02 S283 6.2 0.02 S2475 3.0 0.02 ARVCF Armadillo repeat protein deleted in velo- S269 1.9 0.03 cardio-facial syndrome homolog S345 7.9 0.03 CDHR5 Cadherin-related family member 5 S753 5.3 0.02 CTNNA1 alpha-1 S641 5.9 0.02 S652T654S655 6.2 0.02 T658 6.4 0.02 CD44 CD44 antigen S612 2.4 0.03 CEACAM1 CEA-related 1 T462S469 2.0 0.02 CGN Cingulin S89S90 2.3 0.02 S114 2.2 0.03 S131S134 3.5 0.02 S445 4.7 0.02 CLDN5 Claudin-5 T207 1.9 0.02 EZR Y565 3.4 0.02 GP1BB Glycoprotein Ib, beta polypeptide S194 3.9 0.02 PPFIBP1 Liprin-beta-1 S435 4.1 0.02 PPFIBP2 Liprin-beta-2 S518 1.9 0.02 LMO7 Lmo7 protein S255 5.0 0.02 S453 2.5 0.03 S904S906 3.3 0.02 S993 3.6 0.02 S1069 5.1 0.02 2410018M08RI MCG130675 S45 3.6 0.02 K CLDN3 MCG16699 T203 9.8 0.02 MFAP1 Microfibrillar-associated protein 1 S116S118 15.5 0.02 OCLN Occludin Y367 21.2 0.02 PPHLN1 Periphilin-1 S219 3.8 0.02 PVR Poliovirus receptor S381 5.1 0.02 PCDH1 Protocadherin 1 S962 20.0 0.02 STAB2 Stabilin-2 S2503 6.5 0.02 SYMPK Symplekin S1271 3.1 0.02 S1272 3.3 0.02 TLN1 -1 S2040 3.3 0.02 TROAP Tastin T159T160 3.3 0.02 TJP1 Tight junction protein ZO-1 S125Y132 3.3 0.02 S175S178S179 2.6 0.02 S617 4.2 0.02 S622 5.0 0.02 S912 4.4 0.02 TJP2 Tight junction protein ZO-2 S107 3.4 0.02

3

For Peer Review Only Diabetes Page 42 of 72

S213 1.6 0.03 S404 2.6 0.02 S968 2.3 0.02 S1133 2.2 0.02 TLN2 Tln2 talin-2 T1844 3.4 0.02 TNS1 Tns1 tensin 1 S792 10.6 0.02 S899 8.7 0.02 S1062 4.6 0.02 S1346 2.0 0.02 T1357 4.4 0.02 S1363 5.2 0.02 S1375 1.7 0.02 S1397 5.2 0.02 S1399 2.5 0.02 S1423 3.7 0.02 S1451 3.7 0.02 S1468S1470 4.3 0.02 S1535T1537 5.8 0.02 TFIP11 Tuftelin-interacting protein 11 S96S99 18.5 0.02 VCL S290 5.9 0.02 S346 4.3 0.02

ABI1 Abl-interactor 1 S225 19 0.02 T229S231 6.6 0.02

AHSG Alpha-2-HS-glycoprotein T130 6.6 0.02 S134 6.6 0.02 S138 10.9 0.02 S309S312S314 5.0 0.02 ARMC10 Armadillo repeat-containing protein 10 S43 7.2 0.02 BRAP BRCA1-associated protein S116 58.5 0.02 CALU Calumenin S69 3.8 0.02 CD2BP2 CD2 antigen cytoplasmic tail-binding S196 1.5 0.04 protein 2 CDKN2AIP CDKN2A-interacting protein S168 3.0 0.03 CETN2 Centrin-2 S20 1.8 0.04 CENPA Centromere protein A, isoform CRA_c S15 3.4 0.02 CGNL1 Cingulin-like 1 T250 1.7 0.03 S284 2.3 0.02 CLASP1 CLIP-associating protein 1 S600 15.3 0.02 T650 10.8 0.02 CRIP2 Crip2 protein S114 6.0 0.02 Cutaneous T-cell lymphoma-associated S533 5.2 0.02 antigen 5 homolog S540 7.7 0.02 S628 4.0 0.02 CDK9 -dependent kinase 9 S347 6.1 0.02 CDK11 Cyclin-dependent kinase 11 S47 5.1 0.03 CDK12 Cyclin-dependent kinase 12 Y888T889 6.6 0.02 CDK13 Cyclin-dependent kinase 13 S341S343S349 6.5 0.02 Y862 4.7 0.02 Y870T871 4.4 0.02 T1143S1146 5.5 0.03 CDKN1B Cyclin-dependent kinase inhibitor 1B S7S10 4.2 0.02

4

For Peer Review Only Page 43 of 72 Diabetes

CCNK Cyclin-K S341 19.4 0.02 CCNL1 Cyclin-L1 S358 6.0 0.03 S380 8.9 0.03 CCNL2 Cyclin-L2 S328 13.2 0.02 S345 3.8 0.02 CCNY Cyclin-Y S326 5.7 0.02 SPECC1L Cytospin-A S833 4.6 0.02 MEPCE 7SK snRNA methylphosphate capping S126 2.8 0.02 T188S192 6.8 0.02 DPH1 Diphthamide biosynthesis protein 1 S418 2.0 0.03 DLG1 Disks large homolog 1 S534 10.5 0.02 S537S538S540 9.5 0.02 S542 4.5 0.02 T651 3.9 0.02 S655 9.7 0.02 DNM1L -1-like protein S532 7.0 0.02 DNCLI1 cytoplasmic 1 light intermediate S516 3.5 0.02 chain 1 UHRF2 E3 -protein ligase UHRF2 S668 4.5 0.02 GNL3 Guanine nucleotide-binding protein-like 3 S505 3.8 0.02 LIGL2 Lethal(2) giant larvae protein homolog 2 S1046 8.0 0.02 PCNP MCG130899 T139T141 5.0 0.02 GPS1 MCG2137, isoform CRA_b S509 4.7 0.02 S518S520 7.5 0.02 NPM1 MCG68069 S125 1.8 0.03 MTUS1 Microtubule-associated tumor suppressor 1 S1208 3.3 0.02 homolog NBR1 Nbr1 protein T659 3.3 0.02 NIPBL Nipped-B-like protein S2652 8.6 0.02 HMGN1 Non-histone chromosomal protein HMG-14 T71T80 4.5 0.02 S84 2.8 0.02 S87 4.5 0.02 ZC3HC1 Nuclear-interacting partner of ALK S295 6.1 0.02 S364 7.2 0.02 NUMA1 Numa1 Nuclear mitotic apparatus protein 1 S1739 4.3 0.02 S1835 4.6 0.02 S1974 1.5 0.02 T1982 3.6 0.03 PARD3 Pard3 protein S383 4.0 0.02 PWP1 Periodic tryptophan protein 1 homolog S486S491S493 6.0 0.02 PINX1 PIN2/TERF1-interacting telomerase T228S233 4.3 0.02 inhibitor 1 RRP15 RRP15-like protein S265 3.6 0.02 SASH1 SAM and SH3 domain-containing protein 1 S831 3.4 0.02 SEPT2 -2 S218 5.0 0.02 SEPT7 Septin-7 T425 3.2 0.02 SEPT9 Septin 9 S64 8.6 0.02 SIPA1 Signal-induced proliferation-associated S72 4.7 0.03 protein 1 PDS5B Sister chromatid cohesion protein PDS5 S1160S1165S116 18.5 0.02 homolog B 6 S1356 10.1 0.02 S1360 9.3 0.02 S1381 7.6 0.02

5

For Peer Review Only Diabetes Page 44 of 72

SPHK2 Sphingosine kinase 2 S364 7.2 0.02 WAPAL Wings apart-like protein homolog S77 3.3 0.02 S226 6.7 0.02 ZZEF1 Zinc finger ZZ-type and EF-hand domain- S1538 6.5 0.02 containing protein 1 ZWINT ZW10 interactor S216 6.9 0.02 Cytoskeleton ABLIM1 -binding LIM protein 1 S475 2.2 0.02 S496 2.8 0.02 ABLIM3 Actin-binding LIM protein 3 S373 2.9 0.02 S502S503 3.3 0.02

SVIL Archvillin S857 2.5 0.03 S960 3.3 0.02 RCSD1 CapZ-interacting protein S177S179 3.4 0.02 CDC42EP1 Cdc42 effector protein 1 S205S207 1.7 0.03 S121 8.9 0.02 CDC42EB4 Cdc42 effector protein 4 S64 5.1 0.02 DMTN Dematin S226 2.3 0.02 DSP S2219S2221 4.9 0.02 T2607 3.6 0.02 S2620 11.9 0.02 EPPK1 Epiplakin S1537 2.7 0.02 S2705 5.9 0.02 S2707 4.3 0.02 FNBP1 Formin binding protein 1 S296S299 2.0 0.02 FNBP2 Formin-like protein 2 S171 8.0 0.02 GPHN Gephyrin S327S328 5.9 0.02 S336S338 5.8 0.02 CASK Isoform 3 of Peripheral plasma membrane S569 7.8 0.02 protein CASK S570S571 7.0 0.02 KRT8 Keratin, type II cytoskeletal 8 S24 3.5 0.02 S482S484S489 3.7 0.02 KIF13B Kif13b family member 13B S1380 5.7 0.02 S1390S1395 5.2 0.02 S1409 6.5 0.02 S1414 4.3 0.02 T1653 5.5 0.02 S1654 5.6 0.02 S1705 3.5 0.02 S1794 4.4 0.03 S1795 3.0 0.03 S1812 5.7 0.02 S1814 5.0 0.02 KIF1B Kinesin family member 1B T1406 6.9 0.02 KLC4 Kinesin light chain 4 S590 2.4 0.03 KIF1C Kinesin-like protein KIF1C S674S676 3.5 0.02 KIF21A Kinesin-like protein KIF21A S1241 3.0 0.02 KIF7 Kinesin-like protein KIF7 S1078 8.5 0.02 LIMA1 LIM domain and actin-binding protein 1 T12 3.3 0.02 S488 7.1 0.02 S735 4.1 0.02 MPP5 MAGUK p55 subfamily member 5 S83 1.6 0.02 MAP7D1 MAP7 domain-containing protein 1 S401 10.7 0.03

6

For Peer Review Only Page 45 of 72 Diabetes

MAP1 Microtubule-associated protein S517 3.2 0.02 S761T762 3.1 0.02 T745S748 3.5 0.02 MAP1S Microtubule-associated protein 1S S462 2.9 0.02 PALLD Palladin S1141S1143S114 5.0 0.02 6 S901 4.5 0.03 PALMD Palmdelphin T318T320 5.4 0.02 S370S372 4.3 0.02 S375 8.6 0.02 S384S385 2.8 0.02 PXN Paxillin Y118 3.2 0.02 S137 13.1 0.03 S321 13.3 0.02 PLEC S4392 2.6 0.02 S4393 2.4 0.02 EPB41 Protein 4.1 S552 5.3 0.02 S541S543 4.8 0.02 S678 35.9 0.03 SHROOM1 Protein Shroom1 S188 3.2 0.02 S224 12.4 0.02 ROCK2 Rho-associated protein kinase 2 S1374 5.5 0.02 SLAIN2 SLAIN motif-containing protein 2 S392 1.8 0.03 GW7_15253 beta chain, brain 1 S2102 2.3 0.02 S2159 2.1 0.02 S2163S2164S216 4.8 0.02 8 S2339 2.8 0.02 S2340 2.2 0.02 CTTN Src substrate cortactin S113 3.4 0.02 TJP3 Tight junction protein 3 S103 4.5 0.02 S106S111 4.2 0.02 S343 3.5 0.02 S366 3.7 0.02 TPPP Tubulin polymerization-promoting protein T15S19 4.7 0.02 VASP Vasodilator-stimulated phosphoprotein S235 5.9 0.02 FAM21 WASH complex subunit FAM21 S388 4.5 0.02 S747 3.6 0.02 DNA repair and modification ALKBH3 Alpha-ketoglutarate-dependent dioxygenase T24 1.6 0.03 alkB homolog 3 S28 2.2 0.02 ANKRD17 repeat domain-containing protein 17 S2055 3.7 0.02 APEX1 Apurinic/apyrimidinic endonuclease 1 S18 7.5 0.02 WRNIP1 ATPase WRNIP1 S151 6.2 0.02 S153 5.7 0.02 MSH6 DNA mismatch repair protein Msh6 S62 5.2 0.02 XPC DNA repair protein complementing XP-C S93 6.6 0.02 cells homolog S395S397 8.4 0.02 S382 6.4 0.02 SFR1 DNA repair protein Sfr1 S51T54 3.7 0.02 S99 23.1 0.02 TOP2B DNA topoisomerase 2-beta S1453 9.9 0.02 S1537S1539 11.7 0.02

7

For Peer Review Only Diabetes Page 46 of 72

RNF168 E3 ubiquitin-protein ligase RNF168 T501 4.3 0.02 GEN1 Flap endonuclease GEN homolog 1 T462 5.3 0.02 Junction-mediating and –regulatory protein S58 1.6 0.03 NHEJ1 Non-homologous end-joining factor 1 S243 3.6 0.03 S245 7.7 0.02 DEK Protein DEK S33 2.7 0.02 RAD23A Rad23a protein S123S128 2.4 0.02 RECQL5 RecQ helicase protein-like 5 beta S489S492 13.5 0.03 RNF169 Rnf169 ring finger protein 169 S279 3.6 0.02 ZC3HAV1 Zinc finger CCCH-type antiviral protein 1 S324 3.7 0.02 DNA replication MCM2 DNA replication licensing factor MCM2 S40 2.4 0.03 POLM Polymerase (DNA directed), mu S467 3.3 0.02 RER1 Protein RER1 S95 7.0 0.02 RNASEH1 Ribonuclease H1 S74S80 2.2 0.03 Development EPB41L5 Band 4.1-like protein 5 T387 8.2 0.02 SNTB1 Beta-1-syntrophin T73 5.4 0.02 S86 4.0 0.02 S218 3.3 0.02 BIN1 Bin1 protein S267S273 2.0 0.02 C2CD2 C2cd2 C2 calcium-dependent domain S649 8.4 0.02 containing 2 IBTK Inhibitor of Bruton kinase S1046 4.7 0.02 DLG1 Isoform 2 of Disks large homolog 1 S665 6.6 0.02 T672 3.3 0.02 DLG1 Isoform 3 of Disks large homolog 1 S671 2.0 0.03 IMNA A S17S18 2.6 0.02 S22 2.7 0.02 S390S392T394 2.6 0.03 LUZP1 zipper protein 1 S395 2.2 0.03 S660 2.6 0.02 LSR -stimulated lipoprotein receptor S307 6.3 0.02 S308 6.5 0.02 S436 1.9 0.03 S473Y478 5.1 0.02 S588S591 2.9 0.02 MATR3 Matrin 3 S188 3.1 0.02 S598S604 3.8 0.02 S619 6.0 0.02 S630 36.1 0.03 MOB1A Mps one binder kinase activator-like 1A T35 6.6 0.02 NUCKS1 Nuclear ubiquitous casein and cyclin- S19 4.9 0.02 dependent substrate S50 5.5 0.02 S54S58S61 18.6 0.02 S73S75S79 8.9 0.02 S181 5.6 0.02 S214 5.0 0.02 PDLIM4 PDZ and LIM domain 4 S118S119 4.8 0.02 PDLIM5 PDZ and LIM domain protein 5 S228 1.9 0.03 S308 4.0 0.02 PHACTR4 Phosphatase and actin regulator 4 S117 3.9 0.02 2 S102 3.4 0.02 S131 3.0 0.02

8

For Peer Review Only Page 47 of 72 Diabetes

S132S135 9.5 0.02 S151S154 5.8 0.02 PLEKHA7 Pleckstrin homology domain-containing S481S482 5.1 0.02 family A member 7 COBL Protein cordon-bleu S1103 5.7 0.02 FAM83H Protein FAM83H S522 4.1 0.02 S639 2.0 0.03 S871 4.1 0.02 S904 3.9 0.02 S915 4.6 0.02 S926 5.3 0.02 S948 3.3 0.02 S1041 3.2 0.02 T1042 3.5 0.02 LNP Protein lunapark S411 21.5 0.02 NUMB Protein numb homolog S636 2.9 0.02 TGM1 Protein- gamma- S91S94 7.1 0.02 glutamyltransferase K SHROOM2 SHROOM2 S443 12.1 0.03 S957S959S960S9 2.3 0.02 62T963 SNTB2 Sntb2 protein S88 6.2 0.02 S90 4.7 0.02 SPAG9 Sperm associated antigen 9 (Fragment) S366 3.1 0.02 S368S369 4.6 0.02 UTRN Utrn S933 3.6 0.02 WDTC1 WD and tetratricopeptide repeats 1 S511 3.0 0.03 WDR33 Wdr33 WD repeat domain 33 isoform 1 S1204 4.4 0.02 Heme biosynthesis ALAD Delta-aminolevulinic acid dehydratase S214 2.5 0.03

Immune response CLMN Calmin S317 4.3 0.02 CFH Complement factor H Y1195 2.5 0.03 S1196S1198 3.6 0.03 CRRY Complement regulatory protein Crry Y450 8.8 0.02 S454 6.8 0.02 S483 8.0 0.02 DBNL Drebrin-like protein S277 2.0 0.03 T278 2.9 0.02 EDC3 Enhancer of mRNA-decapping protein 3 S131 1.7 0.02 FYB FYN binding protein S27S30 2.6 0.02 GNL1 Guanine nucleotide-binding protein-like 1 T48S51S55 6.9 0.02 HCK Hemopoietic cell kinase Y388 5.7 0.02 PSMD11 MCG19050, isoform CRA_d S14 3.4 0.02 IFIH1 Melanoma differentiation-associated T286 4.6 0.02 protein 5 S645S648 2.5 0.02 HMHA1 Minor histocompatibility protein HA-1 S577 4.8 0.02 MAVS mitochondrial antiviral-signaling protein S152 13.9 0.02 isoform 1 SERPING1 Plasma protease C1 inhibitor S41 2.0 0.03 Lipid metabolism ACC1 Acetyl-CoA carboxylase 1 S23S29 3.3 0.02 S76 3.1 0.02

9

For Peer Review Only Diabetes Page 48 of 72

S79 2.8 0.02 ACC2 Acetyl-Coenzyme A carboxylase beta S1332 2.3 0.03 ACSS2 Acetyl-Coenzyme A synthetase, S30 3.9 0.02 cytoplasmic S263 3.0 0.02 S267 1.9 0.02 ACOT1 Acyl-coenzyme A thioesterase 1 S327S333 2.7 0.03 ACOT5 Acyl-coenzyme A thioesterase 5 S56 2.1 0.03 ACSM5 Acyl-coenzyme A synthetase 5, S210 12.5 0.03 mitochondrial APOE Apolipoprotein E S319 2.2 0.02 ACLY ATP citrate S455 3.9 0.02 S478S481 11.7 0.02 PCYT1A Choline-phosphate cytidylyltransferase A S319 6.8 0.02 S321 5.2 0.02 S322 6.8 0.02 T342 1.8 0.03 S343 1.8 0.03 ELOVL5 Elongation of very long chain fatty acids S273T281 8.2 0.02 protein 5 S283* 13.8 0.02 PCYT2 Ethanolamine-phosphate S10 5.4 0.02 cytidylyltransferase S163 2.4 0.03 FABP1 Fatty acid-binding protein, liver S11 4.0 0.02 GPAT1 Glycerol-3-phosphate acyltransferase 1, S687S694 83.4 0.02 mitochondrial GPAT3 Glycerol-3-phosphate acyltransferase 3 S66S68 4.0 0.02 HSL Hormone-sensitive lipase S559 2.9 0.02 HACD3 3-hydroxyacyl-CoA dehydratase 3 S114 6.1 0.02 PPAP2A Lipid phosphate phosphohydrolase 1 S273 14.2 0.02 PPAP2B Lipid phosphate phosphohydrolase 3 S19 4.6 0.02 MSR1 Macrophage scavenger receptor types I and S22S23 2.0 0.02 II MCAT Malonyl-CoA-acyl carrier protein S40 4.1 0.02 transacylase, mitochondrial ZDHHC5 Membrane-associated DHHC5 zinc finger S380T385 6.8 0.02 protein PANK2 Pantothenate kinase 2 (Hallervorden-Spatz S42 11.7 0.02 syndrome) PLIN5 Perilipin-5 S264T268 2.2 0.03 CDSA Phosphatidate cytidylyltransferase S20 6.8 0.02 S22 12.5 0.02 T30 10.0 0.02 S32 4.9 0.02 LPIN2 Phosphatidate phosphatase LPIN2 S186S187 3.9 0.02 S245 3.0 0.02 PTGES3 Prostaglandin E synthase 3 S113 3.2 0.02 PDXDC1 Pyridoxal-dependent decarboxylase S30 2.1 0.02 domain-containing protein 1 PPP6R2 SAPS domain family, member 2, isoform S617 12.5 0.03 CRA_a S669 2.3 0.03 SERINC1 Serine incorporator 1 S364 2.7 0.02 PPP4R3A Serine/threonine- 4 S728 72.7 0.02 regulatory subunit 3A HDLBP Vigilin S31 3.9 0.02 Membrane trafficking

10

For Peer Review Only Page 49 of 72 Diabetes

AP1M1 AP-1 complex subunit mu-1 T152 8.7 0.02 T154 8.6 0.02 CLINT1 interactor 1 S227S232 3.5 0.03 DENNBD5B DENN domain-containing protein 5B T1062S1068 25.8 0.03 EPN1 Epsin-1 T415 3.1 0.03 S416 17.2 0.01 S419 16.3 0.01 EPN2 Epsin 2 S192 3.3 0.03 S195Y196 5.2 0.02 GOLGA3 Golgin subfamily A member 3 S60 2.6 0.02 S277 2.1 0.02 SYTL2 Isoform 2 of -like protein 2 S685 3.4 0.03 TMPO Lamina-associated polypeptide 2, isoforms S155 7.0 0.03 alpha/zeta S158T159 6.4 0.02 TMPO Lamina-associated polypeptide 2, isoforms S176Y182 12.5 0.02 beta/delta/epsilon/gamma S183 9.0 0.02 MCG8317 S1076 6.3 0.03 S1566 7.2 0.02 NSFL1C NSFL1 p47 S176 4.9 0.02 OPTN Optineurin S187 3.0 0.02 CASK Peripheral plasma S577 3.4 0.02 CASK S582 6.9 0.02 PICALM Phosphatidylinositol-binding clathrin T301 25.1 0.02 assembly protein LRP1 Prolow-density lipoprotein receptor-related S4521 3.6 0.02 protein 1 S4524T4525 3.7 0.02 TPR Protein Tpr S2141 4.9 0.02 S2223 3.4 0.02 SH3BP19 SH3 domain-containing protein 19 S366 10.1 0.02 SRP72 Srp72 signal recognition particle 72 S620 4.5 0.02 S621S625S630 2.8 0.02 TXLNA Taxilin alpha S515 4.0 0.02 SSR3 Translocon-associated protein subunit S105 104.4 0.04 gamma VAMP4 Vesicle-associated membrane protein 4 S30 8.2 0.02 WDR44 WD repeat domain 44 S50 3.2 0.02 ZFYVE19 Zinc finger FYVE domain-containing S280 3.6 0.02 protein 19 Muscle contraction MPRIP phosphatase Rho interacting S173 2.8 0.02 protein (Fragment) S175 1.6 0.02 MYLK Myosin, light polypeptide kinase S342 5.3 0.02 S362S364 2.2 0.03 S386 4.5 0.02 MYH9 Myosin-9 T1939S1943 8.2 0.02 MYH11 Myosin-11 T1951S1954 2.9 0.02 MYO9B Myosin-IXb S1264 3.6 0.02 MYO6 Myosin-VI T406 4.2 0.02 MYO18A Myosin-XVIIIa S72 6.0 0.02 S1994S1998 9.0 0.02 S2003S2016 2.5 0.02 Y2035S2037S203 5.1 0.02 9 Oxidation and reduction

11

For Peer Review Only Diabetes Page 50 of 72

ASPH Aspartate-beta-hydroxylase S14 6.6 0.03

CATALASE Catalase Y161 2.9 0.02 S166 2.6 0.03 COIL Coilin S146 9.1 0.02 CCS Copper chaperone for superoxide S267 1.8 0.03 dismutase

CYP3A13 CYP3A13 S501 21.5 0.02 HACL1 2-Hydroxyacyl-CoA lyase 1 S4 4.0 0.02 S6 4.0 0.03 S18 4.2 0.03

HAAO 3-Hydroxyanthranilate 3,4-dioxygenase S9 4.4 0.02

HPD 4-Hydroxyphenylpyruvate dioxygenase S250 2.8 0.03 BCKDHA 2-Oxoisovalerate dehydrogenase subunit S334 1.8 0.03 alpha, mitochondrial T335 2.0 0.03 S336 2.1 0.03 S344Y350 3.6 0.02 MTHFD1 C-1-tetrahydrofolate synthase, cytoplasmic S549 6.6 0.02 DNYD Dihydropyrimidine dehydrogenase S905 5.6 0.02 [NADP+] FAM63A Fam63a protein S103 6.2 0.02 S440 2.7 0.02 GLRX5 Glutaredoxin-related protein 5, S151 2.9 0.02 mitochondrial GSTP1 Glutathione S- P 1 S195 21.3 0.02 S196 24.4 0.02 HGD Homogentisate 1,2-dioxygenase S438 10.2 0.02 S440 9.4 0.02 HADH Hydroxyacyl-coenzyme A dehydrogenase, S13 2.6 0.03 mitochondrial PRDX6 Peroxiredoxin-6 T192 3.5 0.03 FAM73B Protein FAM73B T273 5.9 0.02 S276 4.8 0.02 IWS1 Protein IWS1 homolog S295 18.5 0.01 S321 2.3 0.02 S458S460 3.1 0.02 KIAA0284 Protein KIAA0284 S1122 1.6 0.03 S1166 1.8 0.03 PDHA1 E1 alpha 1 T231 2.9 0.02 S293S295S300 3.3 0.02 NDUFV3 RIKEN cDNA 1500032D16, isoform T149S155 7.6 0.02 CRA_a S161S163 7.1 0.02 S165 5.7 0.02 TDO2 Tryptophan 2,3-dioxygenase S19 7.2 0.02 Protein and metabolism APEH Acylamino-acid-releasing enzyme S185 5.7 0.02 PSMD1 26S subunit, non-ATPase T311S315 5.6 0.02 regulatory subunit 1 BHMT Betaine—homocysteine S- S330 9.6 0.02 methyltransferase 1 S333 13.8 0.02

12

For Peer Review Only Page 51 of 72 Diabetes

OASA Cysteine synthase S33 1.7 0.03 DCAF8 DDB1- and CUL4-associated factor 8 S100 2.6 0.02 DIS3L2 DIS3-like exonuclease 2 S864 14.6 0.02 STT3B Dolichyl-diphosphooligosaccharide— S495S496 9.7 0.02 protein glycosyltransferase subunit STT3B DTD1 D-tyrosyl-tRNA(Tyr) deacylase 1 S204S205 3.4 0.02 HECTD1 E3 ubiquitin-protein ligase HECTD1 S632 4.7 0.02 S1383 10.3 0.03 S1575 5.4 0.02 HUWE1 E3 ubiquitin-protein ligase HUWE1 S1395 8.4 0.02 T1579 1.7 0.03 S1903 6.8 0.02 T1905 4.9 0.02 MGRN1 E3 ubiquitin-protein ligase MGRN1 S428 5.1 0.02 NEDD4 E3 ubiquitin-protein ligase NEDD4 T287 3.2 0.02 S309 6.1 0.02 NEDD4L E3 ubiquitin-protein ligase NEDD4-like S477S478 7.7 0.02 UBR3 E3 ubiquitin-protein ligase UBR3 S1055 5.8 0.02 UBR4 E3 ubiquitin-protein ligase UBR4 T2721 8.3 0.02 ZNRF2 E3 ubiquitin-protein ligase ZNRF2 Y17S18S20 2.5 0.02 EML4 Echinoderm microtubule-associated S144S146 3.6 0.02 protein-like 4 NMT1 Glycylpeptide N-tetradecanoyltransferase 1 S15 3.7 0.02 S31 4.3 0.02 S47 3.6 0.02 NMT2 Glycylpeptide N-tetradecanoyltransferase 2 S38 1.8 0.03 HAL Histidine ammonia-lyase S631 7.8 0.02 S635 16.2 0.02 T637 10.8 0.02 S643 7.4 0.02 LARP1 La-related protein 1 S68S81 7.6 0.02 S188 10.4 0.03 T196 17.0 0.02 S198S201 21.3 0.03 T297S302 6.7 0.02 Y488T492 52.2 0.02 S494 34.0 0.02 S498 52.2 0.02 T503 7.6 0.02 S525 33.8 0.02 S743 3.9 0.02 T747S751 4.3 0.02 S1034 1.9 0.02 TGOLN1 MCG129299 S28 4.2 0.02 S31 5.2 0.02 S126 37.8 0.02 T146 4.5 0.02 S210 2.5 0.02 S230 2.7 0.03 S231 2.7 0.02 S266S67 2.9 0.02 NRD1 Nardilysin S85 5.1 0.02 WWP2 NEDD4-like E3 ubiquitin-protein ligase S211 2.3 0.03 WWP2 MME Neprilysin S6 3.9 0.02

13

For Peer Review Only Diabetes Page 52 of 72

OTUD4 OTU domain-containing protein 4 S1016S1017 13.2 0.02 PNPLA7 Patatin-like phospholipase domain- S379 5.3 0.02 containing protein 7 PJA1 Pja1 protein S156 7.0 0.02 PGRMC1 membrane T74 7.4 0.02 component 1 T178 13.7 0.02 Y180S181 26.1 0.02 PSMF1 Proteasome inhibitor PI31 subunit S153 3.8 0.02 PAG1 Proteasome subunit alpha type S250 4.0 0.02 DPY19L1 Protein dpy-19 homolog 1 S23 11.4 0.02 TRIP12 Protein Trip12 T1457 4.2 0.02 T1459 3.7 0.02 HPN Serine protease hepsin S15 12.6 0.02 T16 2.5 0.03 TMEM51 51 S114 1.8 0.02 TAT Tyrosine aminotransferase S448 4.6 0.02 USP39 U4/U6.U5 tri-snRNP-associated protein 2 S81 38.1 0.02 COQ9 Ubiquinone biosynthesis protein COQ9, S81 3.9 0.02 mitochondrial USP Ubiquitin carboxyl-terminal S691 4.0 0.02 S2045 6.2 0.02 S2559 4.4 0.02 USP10 Ubiquitin carboxyl-terminal hydrolase 10 T205 5.2 0.02 S208 6.1 0.02 S359 2.5 0.02 USP36 Ubiquitin carboxyl-terminal hydrolase 36 S435 1.9 0.03 USP47 Ubiquitin carboxyl-terminal hydrolase 47 T933S934 1.6 0.02 UBE4B Ubiquitin conjugation factor E4 B S105 19.1 0.02 UBA1 Ubiquitin-activating enzyme E1, Chr X S13 2.6 0.02 UBAP2L Ubiquitin-associated protein 2-like T114 2.8 0.02 S628 31.3 0.02 UBE2E2 Ubiquitin-conjugating enzyme E2 E2 S18 4.5 0.02 UBE2J1 Ubiquitin-conjugating enzyme E2 J1 S266 11.5 0.02 T267 8.0 0.02 UBL7 Ubiquitin-like protein 7 S230 11.0 0.02 SHP1 UBX domain-containing protein 1 S188 4.2 0.02 Protein folding SNTA1 Alpha-1-syntrophin S194 3.7 0.02 CANX Calnexin S553 8.3 0.02 T561 8.4 0.02 S563 8.3 0.02 S582 7.3 0.02 DNAJC5 DnaJ homolog subfamily C member 5 S10 8.3 0.02 T11 11.3 0.02

DNAJC7 DnaJ homolog subfamily C member 17 S112 2.5 0.03 DNAJC21 Dnajc21 Protein S132 9.4 0.02 FKBP15 FK506-binding protein 15 S1125 4.4 0.02 S1159 6.2 0.02 HSP84B Heat shock protein 84b S226 5.9 0.02 S255S261 3.8 0.02 HSP90A Heat shock protein HSP 90-alpha S231 2.2 0.03 S252 3.5 0.02 S263 3.1 0.02

14

For Peer Review Only Page 53 of 72 Diabetes

PPIG Peptidyl-prolyl cis-trans isomerase G S685 25.9 0.04 S714 15.2 0.03 S742S743T746 1.9 0.03 FAM54B Protein FAM54B S234S235 2.9 0.02 FRA10AC1 Protein FRA10AC1 homolog S250 2.3 0.02 URI1 Unconventional prefoldin RPB5 interactor S440 4.9 0.02 Regulation of GTPase activity ARFGAP2 ADP-ribosylation factor GTPase-activating S236 6.0 0.02 protein 2 ARFGEF2 ADP-ribosylation factor guanine S218T224S227 6.2 0.02 nucleotide-exchange factor 2 (Brefeldin A- inhibited) ALKBH3 Alsin S483 10.1 0.02 AGFG1 Arf-GAP domain and FG repeats- T177S179S181 3.9 0.02 containing protein 1 ASAP2 Arf-GAP with SH3 domain, ANK repeat S704 2.3 0.02 and PH domain-containing protein 2 BCR Breakpoint cluster region protein Y31 150.9 0.02 BCAR3 Breast cancer anti-estrogen resistance S312 3.0 0.02 protein 3 CDC42 GTPase-activating protein S381 3.1 0.02 S1163 4.2 0.02 ARHGAP5 Rho GTPase-activating protein 5 T1172S1174S117 4.9 0.02 7 ARHGEF26 Rho guanine nucleotide exchange factor 26 S390 3.5 0.02

TNKS1BP1 Tankyrase-1-binding protein S1061S1063 3.8 0.02 T533 3.0 0.02 S866 2.3 0.02 Y910S911 4.5 0.02 S1131 4.8 0.02 S1440 7.4 0.02 S1611S1612 2.0 0.02 S1657 4.8 0.02 DENND4A Dennd4a DENN/MADD domain S1586 3.5 0.02 containing 4A DENND4C Dennd4c protein S1096 5.7 0.02 GBF1 Golgi-specific brefeldin A-resistance S1298 5.1 0.02 guanine nucleotide exchange factor 1 T1317S1318 6.7 0.02 GPN1 GPN-loop GTPase 1 S338 4.6 0.02 G3BP2 GTPase activating protein (SH3 domain) T227 3.5 0.02 binding protein 2 GAPVD1 GTPase-activating protein and VPS9 S902 6.6 0.02 domain-containing protein 1 ITSN1 Itsn1 protein S334S335 6.8 0.02 ITSN2 Itsn2 protein S868 2.7 0.02 NOLC1 Nolc1 nucleolar and coiled-body S562 3.7 0.02 phosphoprotein 1 isoform D RABGAP1 GTPase-activating protein 1 S42 4.7 0.02 RABEP1 Rab GTPase-binding effector protein 1 S323 11.5 0.02 T324 5.2 0.02 S326 11.5 0.02 RABGEF1 Rab5 GDP/GTP exchange factor T131 2.2 0.03 S132 7.8 0.02

15

For Peer Review Only Diabetes Page 54 of 72

RANBP10 Ran-binding protein 10 S414 5.7 0.02 S416 14.8 0.03 IQGAP2 Ras GTPase-activating protein-binding T227 2.8 0.02 protein 2 Y14S16 2.0 0.03 RASGRP2 RAS guanyl-releasing protein 2 S554 34.7 0.02 ARHGAP1 Rho GTPase activating protein 1 S91 5.7 0.02 ARHGAP6 Rho GTPase-activating protein 6 S824 5.5 0.02 ARHGAP7 Rho GTPase-activating protein 7 S86S89 11.2 0.02 S818 5.6 0.02 ARHGAP12 Rho GTPase activating protein 12 S163 24.0 0.03 ARHGAP17 Rho GTPase-activating protein 17 S162 3.1 0.02 T164 2.8 0.02 ARHGAP35 Rho GTPase-activating protein 35 S769T771 2.9 0.02 S1179 5.2 0.02 ARHGEF Rho guanine nucleotide exchange factor 7 S132 3.1 0.02 ARHGEF12 Rho guanine nucleotide exchange factor 12 S1327 1.9 0.02 S1389 5.8 0.02 ARHGEF40 Rho guanine nucleotide exchange factor 40 S959 4.0 0.02 RHOJ Rho-related GTP-binding protein RhoJ S9 2.4 0.03 SIPA1L1 Sipa1l1 protein S1564 3.8 0.02 TBC1D10A TBC1 domain family member 10A S18 11.6 0.02 Reproduction ASZ1 Ankyrin repeat and SAM domain S663 2.3 0.02 containing 1 ARG1 -1 T281 5.2 0.02 PGRMC2 Membrane-associated progesterone Y204 7.6 0.02 receptor component 2 T205 8.9 0.02 STARD10 PCTP-like protein S284S289 3.5 0.02

RNA processing ASCC3L1 Activating signal cointegrator 1 complex S225 6.7 0.02 subunit 3-like 1 S339 3.2 0.02 DDX51 ATP-dependent RNA helicase DDX51 S77 5.8 0.02 DDX54 ATP-dependent RNA helicase DDX54 T69 1.9 0.04 S774 5.1 0.02 BUD13 BUD13 homolog T144S148 3.2 0.02 CAST Calpastatin S165 3.0 0.02 S219 3.6 0.02 CMTR1 Cap-specific mRNA (nucleoside-2’-O-)- S27S28 5.9 0.02 methyltransferase 1 PCF11 Cleavage and polyadenylation factor S488S495 6.6 0.02 subunit homolog (S. cerevisiae) CSTF3 Cleavage stimulation factor subunit 3 S691 7.7 0.02 DCUN1D4 Dcun1d4 protein S71 7.8 0.02 GM6793 EG627828 protein S329 4.1 0.02 S331 4.5 0.02 ELAC2 ElaC homolog 2 (E. coli) S814 2.9 0.02 ESYT2 Extended synaptotagmin-2 S661S662S663 4.7 0.02 KHSRP Far upstream element-binding protein 2 S182 7.1 0.02 NOLA4 H/ACA ribonucleoprotein complex subunit S451S453 7.3 0.02 4 S481 4.4 0.02 HNRNPA1 Heterogeneous nuclear ribonucleoprotein S6 4.6 0.02 A1 HNRNPG Heterogeneous nuclear ribonucleoprotein S165 1.6 0.03

16

For Peer Review Only Page 55 of 72 Diabetes

G HNRNPH Heterogeneous nuclear ribonucleoprotein S104 5.4 0.02 H HNRNPLL Heterogeneous nuclear ribonucleoprotein Y36 2.4 0.02 L-like HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins S259 3.2 0.02 A2/B1 HNRNPC Heterogeneous nuclear ribonucleoproteins S231 5.0 0.02 C1/C2 S241 16.7 0.02 S246S249 2.2 0.02 S268 7.9 0.02 S306 4.3 0.02 HNRPK Hnrpk protein Y280 2.9 0.02 S284 5.4 0.02 S379 3.0 0.02 INTS12 Integrator complex subunit 12 S126S127 6.0 0.02 S377 2.4 0.02 DHX9 Isoform 2 of ATP-dependent RNA helicase S137 4.4 0.02 A DBR1 Lariat debranching enzyme T502 2.8 0.02 SRSF7 MCG17902, isoform CRA_a S181S183 4.2 0.02 S233 2.0 0.03 RNMT mRNA cap guanine-N7 methyltransferase S11S15 3.6 0.02 CWC22 pre-mRNA-splicing factor CWC22 S38 1.8 0.03 homolog isoform 2 S829 5.1 0.02 PCBP1 Poly(rC)-binding protein 1 S171S173 3.1 0.02 Y183 34.4 0.02 S189 5.4 0.02 PCBP2 Poly(rC)-binding protein 2 S183S185S266 3.8 0.02 PABP2 Polyadenylate-binding protein 2 S146 20.5 0.02 FIP1L1 Pre-mRNA 3’-end-processing factor FIP1 S479 5.6 0.02 PRPF40A Pre-mRNA-processing factor 40 homolog S879S881S884 3.7 0.02 A SCAF8 Protein SCAF8 T614S617 5.9 0.02 KIAA1429 Protein virilizer homolog S173 9.2 0.02 S1577 6.6 0.02 RAVER1 Ribonucleoprotein PTB-binding 1 S576 2.4 0.02 RBM25 RNA-binding protein 25 S673 5.4 0.02 S675 14.2 0.02 RBM26 RNA-binding protein 26 S127 7.2 0.02 RBM33 RNA-binding protein 33 S243 6.5 0.02 RBM39 RNA-binding protein 39 Y95S97 6.0 0.02 S136 4.2 0.02 RALY RNA-binding protein Raly S135 5.5 0.02 T268S270T274 4.7 0.02 SRRM1 Serine/arginine repetitive matrix 1 S220 4.5 0.02 S260 3.5 0.02 S387S389 3.5 0.02 S400 5.2 0.02 S401 3.8 0.02 T404 5.2 0.02 S412T414 12.0 0.03 S427S429 8.0 0.02 S448S450 6.1 0.02 S461S463 4.2 0.02

17

For Peer Review Only Diabetes Page 56 of 72

S558S560 6.3 0.02 S610S612 4.2 0.02 T619S621 4.4 0.02 S631S633 3.7 0.02 S641S643 3.4 0.03 T698 3.5 0.02 S699 6.1 0.02 S700 3.5 0.02 S717S719 3.1 0.02 S721 3.1 0.02 S742 4.6 0.02 S742S744 15.3 0.02 S747 4.6 0.02 S756S758 3.6 0.02 T876S878 3.4 0.02 SRRM2 Serine/arginine repetitive matrix protein 2 Y335 4.2 0.02 S433S434S435 4.2 0.02 S452S453 2.9 0.02 S848 1.6 0.03 S850 2.3 0.02 S851 1.6 0.03 S1067 13.5 0.02 S1068 5.9 0.02 T1071 13.5 0.02 T1074 5.9 0.02 S1077 9.8 0.02 S1151 5.9 0.02 S1214 2.6 0.02 S1216 2.1 0.03 S1269 4.7 0.02 S1305 2.4 0.02 S1338S1339S134 4.2 0.02 3 S1359S1360 3.2 0.02 S1434S1438S143 2.0 0.03 9 S1630S1631 2.1 0.02 S1647S1649S165 4.4 0.02 0 S1984T1986 4.4 0.02 S2052S2054T205 3.7 0.02 6 S2084 5.3 0.02 S2224 3.6 0.02 T2350S2351 2.8 0.02 S2360T2362 6.2 0.02 S2365 2.2 0.03 S2381 3.1 0.02 S2535 5.3 0.02 S2656S2660 2.3 0.02

SRSF2 Serine/arginine-rich splicing factor 2 S189S191 3.4 0.02 SRSF4 Serine/arginine-rich splicing factor 4 S270 4.8 0.03 S441S443S445 4.0 0.02 S453 2.2 0.03 SRSF9 Serine/arginine-rich splicing factor 9 S205 2.9 0.02

18

For Peer Review Only Page 57 of 72 Diabetes

PPP4R4 Serine/threonine-protein phosphatase 4 S777 11.4 0.02 regulatory subunit 4 SFRS11 Sfrs11 protein Y325 4.3 0.02 S327 4.4 0.02 S342 2.9 0.02 S375 9.2 0.02 SSFA2 Sperm specific antigen 2 S268 1.7 0.02 S736S738 4.5 0.02 T1129 5.8 0.03 S1132 5.9 0.03 SF3B1 Splicing factor 3B subunit 1 T278 6.9 0.03 SF3B2 Splicing factor 3b, subunit 2 S414S418S419 4.8 0.04 SPF45 Splicing factor 45 S155 6.8 0.02 SRSF6 Splicing factor, arginine/serine-rich 6 S303 2.0 0.02 SFSWAP Splicing factor, suppressor of white-apricot S601 1.7 0.03 homolog SREK1 Splicing regulatory glutamine/lysine-rich S486 5.8 0.03 protein 1 SMAP2 Stromal membrane-associated protein 2 S219S224 4.8 0.02 ZCCHC6 Terminal uridylyltransferase 7 S645 4.6 0.02 TRA2A Tra2a transformer-2 protein homolog alpha T200 2.4 0.03 TRA2B Transformer-2 protein homolog beta S95S97S99 2.8 0.02 S264S266 4.4 0.02 NSUN2 tRNA (cytosine(34)-C(5))- S688 1.8 0.02 methyltransferase ZRSR1 U2 small nuclear ribonucleoprotein S417 3.3 0.03 auxiliary factor (U2AF) 1, related sequence S418 18.6 0.02 1 LUC7 LUC7-like S162 2.4 0.02 ZC3H4 Zinc finger CCCH domain-containing S92S94 15.0 0.02 protein 4 ZRANB2 Zinc finger, RAN-binding domain S120 4.3 0.02 containing 2 S153 2.2 0.02 S188 1.9 0.02 Signal transduction PRKAA1 5’-AMP-activated protein kinase catalytic S184S187 4.0 0.02 subunit alpha-1 PRKAA2 5’-AMP-activated protein kinase catalytic S489 7.3 0.02 subunit alpha-2 S491 7.4 0.02

PRKAB1 5’-AMP-activated protein kinase subunit S108 8.2 0.02 beta-1 PRKAG2 5’-AMP-activated protein kinase subunit S87S90 6.5 0.02 gamma-2 S194S195S196 3.7 0.02

AKAP1 A-kinase anchor protein 1, mitochondrial S103 9.9 0.02 T487 9.1 0.02 AKAP2 A-kinase anchor protein 2 S740 4.4 0.02 AKAP8 A-kinase anchor protein 8 S320S325 1.7 0.03 S336 1.9 0.02 APBB1IP Amyloid beta (A4) protein-binding, family S507 9.4 0.02 B, member 1 interacting protein S532 7.0 0.02 AAK1 AP2-associated protein kinase 1 S635 2.9 0.02

19

For Peer Review Only Diabetes Page 58 of 72

T638 2.5 0.02 T672 1.8 0.02 ARRB1 Beta-arrestin-1 T411S413 2.6 0.02 BAIAP2 Brain-specific angiogenesis inhibitor 1- S365 4.0 0.02 associated protein 2 S366 2.1 0.02 BAIAP2L1 Brain-specific angiogenesis inhibitor 1- S421 2.5 0.03 associated protein 2-like protein 1 BCKDK Branched chain ketoacid dehydrogenase T32 6.6 0.02 kinase, isoform CRA_a S33 6.7 0.02 CALCOCO1 Calcium-binding and coiled-coil domain- S460 2.6 0.02 containing protein 1 S466 3.7 0.02 CAMSAP2 Calmodulin-regulated spectrin-associated S456 4.7 0.02 protein 2 PRKACB cAMP-dependent protein kinase catalytic T198 34.4 0.02 subunit beta PRKAR2B cAMP-dependent protein kinase type II- S112 2.1 0.02 beta regulatory subunit CSNK1D , delta S382 4.0 0.02 COMT Catechol O-methyltransferase S261 4.2 0.02 CHN2 Chimerin (Chimaerin) 2 S181 4.0 0.03 CSPG6 Cspg6 S1067 4.7 0.02 PDPK1 3-Phosphoinositide-dependent protein S244 3.7 0.02 kinase 1 DEPTOR Depdc6-001 S244 3.2 0.02 DAB2IP Disabled homolog 2-interacting protein S978 16.8 0.03 DLGAP Dlgap4 protein S699 1.6 0.03 EMR1 EGF-like module-containing mucin-like S926 4.8 0.02 hormone receptor-like 1 EPS8 Epidermal growth factor receptor kinase S658 2.4 0.02 substrate 8 FARP1 Farp1 FERMRhoGEF (Arhgef) and S373 6.2 0.02 pleckstrin domain protein 1 S427 9.2 0.02 T886 4.2 0.02 FRMD4B FERM domain-containing protein 4B Y675S676S678 4.2 0.02 FLT4 FMS-like tyrosine kinase 4 S953 3.1 0.02 GPRC5C -coupled receptor, family C, S434 3.6 0.02 group 5, member C T436 2.1 0.02 GOLPH3 Golph3 protein S35 29.5 0.03 GRB7 Growth factor receptor-bound protein 7 T417 5.5 0.02 S420 13.4 0.02 ITPR1 Inositol 1,4,5-trisphosphate receptor type 1 S1588 11.3 0.02 PPIP5K2 Inositol hexakisphosphate and S44 4.2 0.02 diphosphoinositol-pentakisphosphate kinase 2 INPP5F Inositol polyphosphate-5-phosphatase F S908S910 3.0 0.02 S943 5.7 0.02 IR Insulin receptor S1337 12.5 0.02 PI4K2B Isoform 2 of Phosphatidylinositol 4-kinase S294 10.2 0.02 beta KSR1 Kinase suppressor of ras 1 S297 3.3 0.02 LRRFIP2 Leucine-rich repeat flightless-interacting S111 4.7 0.02 protein 2 T114 3.6 0.02 TMEM57 Macoilin S331 11.4 0.02 STK3 Mammalian sterile twenty 3 kinase T172 3.9 0.03

20

For Peer Review Only Page 59 of 72 Diabetes

MAPKAPK2 MAP kinase-activated protein kinase 2 T320T324 3.5 0.02 MAPK14 Mapk14 protein T180Y182 4.0 0.02 PPP1R12B MCG130490 T645 3.0 0.02 S833T836 1.5 0.04 MCC MCG4620, isoform CRA_b S293 4.3 0.02 MCG5232 T19S20 21.2 0.02 MTMR3 MCG9626, isoform CRA_b S633S636 1.9 0.03 MRAP2 Melanocortin-2 receptor accessory protein S94T97 5.5 0.02 MTSS1 suppressor protein 1 S276 5.6 0.02 S279 4.8 0.02 MINK1 Misshapen-like kinase 1 S729 3.0 0.02 MFF Mitochondrial fission factor S129 13.2 0.02 MAP2K2 Mitogen activated protein kinase kinase 2, S393 5.0 0.02 isoform CRA_d MARCKS Myristoylated alanine-rich C-kinase S46 2.8 0.03 substrate S138 3.3 0.02 S141T143 4.1 0.02 SLC9A3R1 Na(+)/H(+) exchange regulatory cofactor S273S275 4.9 0.02 NHE-RF1 S285S286 3.0 0.02 T288S294S289S2 3.6 0.02 97 PDZK1 Na(+)/H(+) exchange regulatory cofactor S489S492 5.2 0.03 NHE-RF3 NUCB1 Nucleobindin-1 S85 3.8 0.03 S368 3.2 0.02 OSTF1 Osteoclast-stimulating factor 1 Y208 13.0 0.02 GIGYF1 PERQ amino acid-rich with GYF domain- S864 4.4 0.02 containing protein 1 PSD3 PH and SEC7 domain-containing protein 3 S1001 2.1 0.02 S281 49.1 0.03 S1009 2.1 0.02 UHRF1 PHD and RING finger domain-containing S1201 10.6 0.03 protein 1 PI4K2 Phosphatidylinositol 4-kinase beta S428 7.4 0.02 PI4K2A Phosphatidylinositol 4-kinase type 2-alpha S47 3.9 0.02 PAQR9 Progestin and adipoQ receptor family S33 8.5 0.02 member 9 AKT1S1 Proline-rich AKT1 substrate 1 S188 3.1 0.02 PRKD3 Protein kinase D3 S735 11.2 0.02 T739 3.6 0.02 PRKAR1A Protein kinase, cAMP dependent S83 4.4 0.02 regulatory, type I, alpha S97 2.8 0.02 PRKRA Protein kinase, interferon inducible double S18 1.6 0.03 stranded RNA dependent activator LAP2 Protein LAP2 S869 6.1 0.02 NDRG2 Protein NDRG2 S328T330S332 6.2 0.02 T334 4.8 0.02 S335S338 6.1 0.02 S346 3.9 0.02 T348S350 4.2 0.04 S352 4.5 0.02 S355 4.2 0.04 S353 39.5 0.02 T357 3.7 0.02 NDRG3 Protein NDRG3 S331 10.2 0.02

21

For Peer Review Only Diabetes Page 60 of 72

PPP1R12A Protein phosphatase 1 regulatory subunit T443 4.2 0.02 12A S445 3.7 0.02 Y446 4.2 0.02 S507 16.8 0.03 T508 2.7 0.03 S909 1.9 0.03 PPP1R12C Protein phosphatase 1 regulatory subunit S431T432 4.3 0.02 12C PPP1R12A Protein phosphatase 1 regulatory subunit S26 6.9 0.02 14A S28 6.5 0.02 S134S136 3.1 0.02 PPP1R7 Protein phosphatase 1 regulatory subunit 7 S24S27 50.7 0.02 S45S48 2.6 0.02 PPM1K Protein phosphatase 1K, mitochondrial S248 4.2 0.02 SSH1 Protein phosphatase Slingshot homolog 1 S639 3.9 0.02 S927 3.9 0.03 S928 4.3 0.02 S929 4.2 0.02 AKT1 RAC-alpha serine/threonine-protein kinase S124 3.5 0.02 RGL2 Ral guanine nucleotide dissociation S619 2.3 0.02 stimulator-like 2 RGL3 Ral guanine nucleotide dissociation S506S509 3.3 0.02 stimulator-like 3 RALBP1 RalA-binding protein 1 S29 8.7 0.02 REPS2 RALBP1 associated Eps domain S531 2.8 0.02 containing protein 2 RASAL2 Rasal2 Uncharacterized protein S986T987 1.9 0.02 RRAS2 Ras-related protein R-Ras2 S186 6.9 0.02 RIPK1 Receptor-interacting serine/threonine- S313 3.4 0.02 protein kinase 1 RAPTOR Regulatory-associated protein of mTOR S863 3.8 0.02 S877 10.5 0.02 ARAF Serine/threonine--Raf S270 60.9 0.02 S578S580 5.9 0.02 MARK2 Serine/threonine-protein kinase MARK2 T208 7.2 0.02 CDC42BPB Serine/threonine-protein kinase MRCK S1688S1692 5.2 0.02 beta PAK4 Serine/threonine-protein kinase PAK 4 S104 3.2 0.02 PRPF4B Serine/threonine-protein kinase PRP4 S240S242 14.3 0.03 homolog S278 2.5 0.02 S427S437 2.0 0.02 Y849 7.5 0.02 SGK3 Serine/threonine-protein kinase Sgk3 S126S129 10.1 0.02 SIK3 Serine/threonine-protein kinase SIK3 S493 4.6 0.02 TAO3 Serine/threonine-protein kinase TAO3 S324 3.7 0.02 SHE SH2 domain-containing adapter protein E S64 1.7 0.04 SH2B2 SH2B adapter protein 2 S107 1.6 0.03 SH3BP5 SH3 domain-binding protein 5 S422 3.7 0.02 SHC1 SHC-transforming protein 1 S139 6.0 0.02 SNRK SNF-related serine/threonine-protein S541 27.4 0.02 kinase T543 27.2 0.02 S544 1.6 0.04 S569S570 3.6 0.02 TENC1 Tensin-like C1 domain-containing S102 6.7 0.02 phosphatase T103 3.3 0.02

22

For Peer Review Only Page 61 of 72 Diabetes

S813 7.0 0.02 S825 1.6 0.03 S1087 7.5 0.02 TRPM7 Transient receptor potential cation channel T1250 4.8 0.03 subfamily M member 7 S1502 8.7 0.02 FAS Tumor factor receptor superfamily S220 18.5 0.03 member 6 LRRFIP2 leucine-rich repeat flightless-interacting S107 2.2 0.02 protein 2-like WDR26 WD repeat-containing protein 26 S101S103 3.7 0.02 Transcription ATN1 Atrophin-1 S34 6.8 0.03 BCLAF1 Bcl-2-associated 1 S177 9.4 0.02 T256S261 5.1 0.02 S258 5.0 0.02 S263 9.2 0.02 S267 6.3 0.02 Y283 9.4 0.02 S286 9.3 0.02 S289 9.4 0.02 S395 5.6 0.02 S494 4.5 0.02 S510 3.4 0.03 S529 6.1 0.02 S646 33.3 0.02 S656 4.8 0.02 MIIT1 Btk-PH-domain binding protein S463 2.3 0.02 C2CD2L C2 domain-containing protein 2-like S470 7.1 0.02 CARHSP1 Calcium regulated heat stable protein 1 S31 3.1 0.02 CES5B Carboxylesterase 5, isoform CRA_b S552 4.5 0.03 CTNNB1 Catenin beta-1 S191 2.6 0.02 T551 1.6 0.02 S552 8.2 0.05 CTNND1 Catenin delta-1 Y228 4.4 0.02 S230 3.5 0.02 S252 10.4 0.02 Y257 5.7 0.02 S268S269 5.3 0.02 T310 10.1 0.02 S920 3.9 0.02 CBX1 Cbx1 protein S89S91 3.9 0.02 CBX3 Cbx3 protein S93S97 2.3 0.03 S95 48.7 0.02 S99 19.5 0.02 CERS2 Ceramide synthase 2 S341 9.8 0.02 CBX5 Chromobox protein homolog 5 S93 7.0 0.02 CHD4 Chromodomain-helicase-DNA-binding S1528 20.9 0.03 protein 4 CHD5 Chromodomain helicase DNA binding T1077 3.8 0.03 protein 5 CC2D1A Coiled-coil and C2 domain-containing S435 4.3 0.02 protein 1A CRTC2 CREB-regulated transcription 2 S612 4.7 0.02 CRTC3 CREB-regulated transcription coactivator 3 S368S370 5.2 0.02 XRN2 5’-3’ Exoribonuclease 2 S448 5.2 0.02

23

For Peer Review Only Diabetes Page 62 of 72

S470 16.3 0.02 S499S501 6.6 0.02 PSMD9 Proteasome 26S subunit, non-ATPase S126S128 7.2 0.02 regulatory subunit 9 DNA DNA-binding protein A S328 3.4 0.02 BINDING PROTEIN POLR3G DNA-directed RNA polymerase III subunit S158 40.0 0.02 RPC7 EPS8L2 Epidermal growth factor receptor kinase S242 1.8 0.03 substrate 8-like protein 2 S482 4.6 0.02 S483 2.2 0.03 S468 2.1 0.03 ERRFI1 ERBB receptor feedback inhibitor 1 S251S256 5.0 0.02 SSRP2 FACT complex subunit SSRP1 S444 4.3 0.02 FOXK2 Forkhead box protein K2 S329S331 4.6 0.02 FOXO1 S284 3.3 0.02 GCFC2 GC-rich sequence DNA-binding factor 1 S262 19.4 0.02 GTF3C2 General transcription factor 3C polypeptide S163 9.2 0.02 2 HDGF Hepatoma-derived growth factor S132S133 6.2 0.02 S165 6.7 0.02 HDGFRP2 Hepatoma-derived growth factor-related S366S367 14.2 0.02 protein 2 S620 4.1 0.02 S629S635 1.8 0.03 HNRNPD0 Heterogeneous nuclear ribonucleoprotein S71S80S82S83 5.7 0.02 D0 HNRNPUL2 Heterogeneous nuclear ribonucleoprotein S159T163 3.3 0.02 U-like protein 2 S166 1.6 0.03 S183 15.8 0.02 S186 16.0 0.02 S653 3.4 0.02 HMGN5 High mobility group nucleosome-binding S167 2.2 0.02 domain-containing protein 5 HDAC2 S394 2.3 0.02 HIST1H4 Histone H4 S48 5.6 0.02 EHMT1 Histone-lysine N-methyltransferase S81 11.3 0.02 EHMT1 NSD3 Histone-lysine N-methyltransferase NSD3 S560S561 2.8 0.02 HTATSF1 HIV TAT specific factor 1 S388 S408 2.3 0.03 S441 3.7 0.02 S446 4.1 0.03 S613 1.9 0.03 S679 2.4 0.02 ING5 Inhibitor of growth protein 5 S148 12.7 0.03 INO80 INO80 complex subunit C S26 1.9 0.03 IRF2BP1 Interferon regulatory factor 2-binding S384 2.2 0.02 protein 1 S453 4.5 0.03 IRF2BPL Interferon regulatory factor 2-binding S313 5.6 0.02 protein-like S315 3.0 0.03 S526 5.3 0.02 IRF3 Interferon regulatory factor 3 S135 5.9 0.02 HABP4 Intracellular hyaluronan-binding protein 4 S74 3.9 0.03 S108 6.7 0.02 IRF2BP2 Irf2bp2 interferon regulatory factor 2 T73 3.1 0.02

24

For Peer Review Only Page 63 of 72 Diabetes

binding protein 2 S169 5.5 0.02 S229 5.1 0.02 S440 2.1 0.03 KLF12 Krueppel-like factor 12 S202 22.9 0.03 KLF3 Krueppel-like factor 3 S71 4.4 0.02 LDB1 LIM domain-binding protein 1 S305 2.6 0.02 T306 4.6 0.02 PHF2 Lysine-specific demethylase PHF2 S1057 17.0 0.02 S1059 2.8 0.02 MXRA7 Matrix-remodelling associated 7 S79 4.6 0.02 MCG_120563 MCG120563, isoform CRA_a S91S92 19.4 0.02 MCG127409 S366 22.9 0.02 SOX6 MCG141507, isoform CRA_a S98 7.5 0.02 MTFR1 MCG19781, isoform CRA_a S119 6.2 0.02 MED1 Mediator of RNA polymerase II S1157 5.9 0.03 transcription subunit 1 MED19 Mediator of RNA polymerase II S226 4.2 0.02 transcription subunit 19 MAGI1 Membrane-associated guanylate kinase, S733S736 546.6 0.02 WW and PDZ domain-containing protein 1 S737 4.2 0.02 MTA2 Metastasis-associated protein MTA2 S435 17.0 0.02 MBD2 Methyl-CpG-binding protein 2 S80 2.2 0.02 MYCT1 target protein 1 S66 8.8 0.02 NELFE Negative elongation factor E S51 3.0 0.02 S113 4.4 0.02 S115 3.3 0.02 WASL Neural Wiskott-Aldrich syndrome protein S422 3.3 0.03 NKAP NF-kappa-B-activating protein S141 4.8 0.02 NFIB nuclear factor 1 B-type isoform X5 S303 10.6 0.02 S323 6.4 0.02 S324 5.6 0.02 T326 5.1 0.02 S327 5.1 0.02 S331 5.2 0.02 ILF3 Interleukin enhancer-binding factor 3 S382 3.5 0.03 NUFIP2 Nuclear fragile X mental retardation- S213 3.6 0.02 interacting protein 2 S626 3.7 0.02 NCOA2 coactivator 2 T773 23.2 0.02 NCOA5 Nuclear receptor coactivator 5 S29S34 3.5 0.02 NCOR1 Nuclear receptor 1 S2198 3.0 0.02 DDX21 Nucleolar RNA helicase 2 S89 3.5 0.03 T114S118 6.3 0.02 PSIP1 PC4 and SFRS1-interacting protein S105S106 3.6 0.02 T115 4.3 0.02 S118 4.5 0.02 T122S129 4.3 0.02 T134 4.9 0.02 T269S270 3.2 0.02 T271S272S274 19.6 0.02 PNN Pinin S66 5.4 0.02 S100 5.0 0.02 S120 3.8 0.02 RING6 Polycomb group RING finger protein 6 S118 3.9 0.02 PKD2 Polycystin-2 S810 9.2 0.02

25

For Peer Review Only Diabetes Page 64 of 72

PROX1 Prospero protein 1 S177 3.6 0.02 S197 6.5 0.02 S199 4.1 0.02 S511 5.5 0.02 LYRIC Protein LYRIC S417S423 4.6 0.02 T561 7.6 0.02 PML Protein PML S17 5.6 0.02 T535 6.8 0.02 RRP5 Protein RRP5 homolog S1468 3.3 0.02 ZMYND8 Protein Zmynd8 S649 2.9 0.02 S754 1.9 0.02 RPRD1B Regulation of nuclear pre-mRNA domain- S132 4.5 0.02 containing protein 1B S134 3.9 0.02 RPS6KA4 Ribosomal protein S6 kinase alpha-4 S745 2.5 0.02 RBM5 RNA binding motif protein 15 S127 5.6 0.02 S258Y265 4.1 0.02 S291 3.9 0.02 T668S674S678 4.8 0.02 ELL RNA polymerase II elongation factor ELL S299 8.0 0.02 S300 7.7 0.02 RPAP3 RNA polymerase II-associated protein 3 S429 3.1 0.02 CTR9 RNA polymerase-associated protein CTR9 S941S943 8.2 0.02 homolog S970 7.6 0.02 S1039S1041 26.4 0.02 S1079S1083 23.2 0.02 RBM10 RNA-binding protein 10 S718S723 5.5 0.02 RBM14 RNA-binding protein 14 T206 5.0 0.02 RPRD1B Rprd1b Protein S33 3.0 0.02 SLTM SAFB-like transcription modulator S289 5.4 0.02 T535 4.9 0.02 S550 11.6 0.02 S995 7.1 0.02 S996T997S999 3.8 0.02 SRSF10 Serine/arginine-rich splicing factor 10 S131S133 2.5 0.02 STAT5B Signal transducer and activator of Y699 4.6 0.02 transcription 5B SUDS3 Sin3 histone deacetylase corepressor S234S236S237 3.1 0.02 complex component SDS3 SGTA Small glutamine-rich tetratricopeptide T82 2.0 0.02 repeat-containing protein alpha S303 2.1 0.03 T305S307 2.6 0.02 SLIRP SRA stem-loop-interacting RNA-binding T104 5.2 0.02 protein, mitochondrial SKIV2L Superkiller viralicidic activity 2-like (S. S240 6.8 0.02 cerevisiae) SMARCC1 SWI/SNF complex subunit SMARCC1 S327S329 10.2 0.02 SMARCC2 SWI/SNF complex subunit SMARCC2 S283 7.4 0.02 S302S304S306T3 6.0 0.02 08 TBX3 Tbx3 protein S387 9.7 0.02 TCF20 Tcf20 protein S588 5.8 0.02 TCOF1 Tcof1 protein S592S593 4.7 0.02 THRAP3 Thrap3 protein S34S45 8.7 0.02 T47 8.8 0.02 THRAP3 associated S183 4.6 0.02

26

For Peer Review Only Page 65 of 72 Diabetes

protein 3 S240 5.4 0.02 S248S253 5.2 0.02 S669 2.3 0.03 S184 15.7 0.02 S187 10.3 0.02 S190 15.7 0.02 S679 4.7 0.02 S695 16.6 0.03 TNS1 Torsin-1A-interacting protein 1 S140 10.3 0.02 TCEA1 Transcription elongation factor A protein 1 S96 6.1 0.02 S97 4.7 0.02 S100 7.5 0.02 TCEB3 Transcription elongation factor B S195 1.7 0.02 polypeptide 3 ETV6 Transcription factor ETV6 Y17T18 4.0 0.02 PURB Transcriptional activator protein Pur-beta S310S316 3.3 0.03 ATRX Transcriptional regulator ATRX Y89 4.2 0.02 S92T98 5.4 0.02 S912 1.6 0.03 TMCO1 Transmembrane and coiled-coil domains S410 4.3 0.02 protein 1 TRIM28 Tripartite motif-containing 28 S50 2.5 0.02 S51 6.0 0.02 S473 4.8 0.02 T599 9.3 0.02 ZFP36 Tristetraprolin S176 5.7 0.02 IFI204 Interferon-activable protein 204 S190 4.3 0.02 MYG1 UPF0160 protein MYG1, mitochondrial S283 4.7 0.02 GPBP1L1 Vasculin-like protein 1 S76 4.1 0.03 S351 4.2 0.02 YBX1 Y box protein 1 S163 4.8 0.02 S172 4.1 0.02 S174 9.3 0.02 YAP1 Yorkie homolog S94T95 61.2 0.02 S112 45.4 0.03 ZC3H3 Zc3h13 Zinc finger CCCH type containing S921T926Y927 3.8 0.03 13 S953 3.1 0.03 ZFP869 Zfp869 zinc finger protein 869 S155 7.3 0.02 ZBTB7A Zinc finger and BTB domain-containing S537 3.5 0.02 protein 7A ZC3H14 Zinc finger CCCH domain-containing S515 4.4 0.02 protein 14 ZC3H18 Zinc finger CCCH domain-containing S45 1.7 0.03 protein 18 T62S64S71 4.0 0.02 S92 1.7 0.03 S507 1.5 0.04 S552 4.0 0.02 S849 5.9 0.02 S890 2.3 0.02 ZNF326 Zinc finger protein 326 S270 9.5 0.02 T274 6.3 0.03 ZNF687 Zinc finger protein 687 S226 5.1 0.02 DPF2 Zinc finger protein ubi-d4 S142 3.4 0.02 Translation CTIF CBP80/20-dependent translation initiation S18 2.7 0.02

27

For Peer Review Only Diabetes Page 66 of 72

factor CLASP2 Clasp2 protein S376 10.5 0.02 EEF1D Eef1d protein T124 4.4 0.02 S133 4.8 0.02 T142 3.4 0.02 S157 3.0 0.02 EFHD2 EF hand domain containing 2 S74 3.9 0.02 EFB1 Elongation factor 1-beta S82 4.6 0.02 S106 3.1 0.02 S112 6.8 0.02 EFT2 Elongation factor 2 T54 9.4 0.02 T57T59 11.0 0.02 EEF2K Eukaryotic elongation factor 2 kinase S437 11.2 0.02 Y442 6.0 0.02 S473 6.2 0.02 EIF3B Eukaryotic translation initiation factor 3 S90 3.0 0.02 subunit B T74S75S79 4.2 0.02 S120S123 5.2 0.02 EIF3C Eukaryotic translation initiation factor 3 S39 5.8 0.02 subunit C EIF3G Eukaryotic translation initiation factor 3 T38 7.5 0.02 subunit G T41 7.8 0.02 S42 5.3 0.02 EIF4G1 Eukaryotic translation initiation factor 4 S1096 11.8 0.02 gamma 1 S1187 16.9 0.02 S1189 6.5 0.02 S1211 9.6 0.02 EIF4G2 Eukaryotic translation initiation factor 4 T507 5.0 0.02 gamma 2 EIF4G3 Eukaryotic translation initiation factor 4 S267 6.2 0.02 gamma 3 S1150 7.1 0.02 S1151 3.4 0.02 EIF4B Eukaryotic translation initiation factor 4B S418T420S422 10.6 0.03 S424 5.5 0.03 S497 4.1 0.02 S498 5.0 0.02 T500 4.6 0.02 S504 2.9 0.03 EIF5B Eukaryotic translation initiation factor 5B T107S108S114* 1.9 0.02 S114 3.2 0.02 S137S139 4.4 0.02 S165 81.1 0.04 S215 4.3 0.02 INADL InaD-like protein S455S459 3.2 0.02 S457 3.0 0.03 LRRC47 Leucine rich repeat containing 47 S293 1.9 0.03 MCG10050 S102S105 16.2 0.02 RPLP1 MCG10168 S101S104 7.9 0.02 MCG1128, isoform CRA_c S237 5.1 0.02 RPS27A MCG13441 S115 4.7 0.02 MCG142467 S44 4.0 0.02 S45 3.8 0.02 MES1 Methionyl-tRNA synthetase, cytoplasmic S827 10.9 0.02 NOP56 Nucleolar protein 56 S513 75.0 0.03 S528S529 6.6 0.02

28

For Peer Review Only Page 67 of 72 Diabetes

S536S543 33.9 0.02 NOP58 Nucleolar protein 58 S509 8.0 0.02 T515 13.3 0.02 S516 5.3 0.02 S521 8.0 0.02 NIBAN Protein Niban S595 4.3 0.02 PUM2 Pumilio homolog 2 S136 5.2 0.02 S181 4.5 0.02 RPL13 Ribosomal protein L13 S140 3.5 0.02 RPS6 Ribosomal protein S3 T220 5.3 0.02 RPS6 Ribosomal protein S6 S235S236S240 12.3 0.02 T241 1.8 0.02 P0 Ribosomal protein, large, P0 S304S307 7.8 0.02 TNRC6B Trinucleotide repeat-containing gene 6B S600 2.7 0.02 protein

Transport SLC33A1 Acetyl-coenzyme A transporter 1 S42 16.9 0.02 ACBP5 Acyl-CoA-binding domain-containing S252S253 17.9 0.02 protein 5 HBA-A1 Alpha globin 1 S4S9 4.0 0.02 ADD1 Alpha-adducin S586 14.4 0.02 ENSA Alpha-endosulfine S108 44.1 0.02 AP3B1 AP-3 complex subunit beta-1 Y274 2.6 0.02 S276 3.6 0.02 AP3D1 AP-3 complex subunit delta-1 S755T758S760 4.6 0.02 S784 7.4 0.02 AQP7 -7 S295 5.6 0.02 ABCA1 ATP-binding cassette sub-family F S107 5.5 0.02 member 1 S138 4.2 0.02 S193 6.4 0.03 S225 9.1 0.02 BETA- Beta-globin S45 5.5 0.02 GLOBIN S51 5.5 0.02 S53 5.0 0.02 T70 3.2 0.02 S81 3.9 0.02 ABCB11 Bile salt export pump S688Y689 4.5 0.02 S696 6.9 0.02 ABCC1 Canalicular multispecific organic anion S279 5.1 0.02 transporter 1 IGF2R Cation-independent mannose-6-phosphate S2401 5.2 0.02 receptor COPB2 subunit beta’ S845 4.2 0.02 UQCRC1 b-c1 complex subunit 1, S212T214 9.2 0.02 mitochondrial PITPNC1 Cytoplasmic phosphatidylinositol transfer S274 14.7 0.02 protein 1 RANBP E3 SUMO-protein ligase RanBP2 S1603 19.6 0.02 S2088 4.4 0.02 S2640S2641T264 4.8 0.02 6 SLC4A4 Electrogenic sodium bicarbonate S239 4.4 0.02 cotransporter 1 S245 6.7 0.02

29

For Peer Review Only Diabetes Page 68 of 72

T254S255S256S2 6.6 0.02 57 S1069 5.2 0.02 ERC1 ELKS/Rab6-interacting/CAST family S14 7.4 0.02 member 1 S15 7.2 0.02 S17S21 7.7 0.02 S37 3.9 0.02 ADD3 Gamma-adducin S679 2.2 0.02 GJB1 protein beta1 S233 101.3 0.02 S258 21.6 0.02 S266 378.6 0.02 USO1 General vesicular transport factor p115 S940 4.8 0.02 GOLIM4 Golgi integral membrane protein 4 S540 3.4 0.02 Isoform 2 of Sorbin and SH3 domain- S409S410S411S4 13.7 0.04 containing protein 1 13 T414 12.3 0.03 KLHDC7A Kelch domain-containing protein 7A S361 11.5 0.02 LBR Lamin-B receptor S101 1.8 0.03 LARP4B La-related protein 4B T735S739 3.1 0.03 BAG6 Large proline-rich protein BAG6 S995 6.4 0.02 SLC7A2 Low affinity cationic amino acid S645 7.7 0.02 transporter 2 MCG11503 T230S231 3.3 0.02 STIM2 MCG1293 S661 6.4 0.02 ATP2B1 MCG13663, isoform CRA_a S1140 7.8 0.02 SLCO1A1 MCG6998, isoform CRA_b S649 34.3 0.02 MIA3 Melanoma inhibitory activity protein 3 S615S617 5.3 0.02 S766 33.7 0.02 SLC16A1 Monocarboxylate transporter 1 S210S213 2.2 0.03 S461T462 2.8 0.02 S491 6.9 0.02 SLC16A10 Monocarboxylate transporter 10 S498 48.2 0.02 S501 48.6 0.02 BLT Multidrug resistance protein 2 S665T666 4.2 0.03 NACA Nascent polypeptide-associated complex S151 5.2 0.02 subunit alpha T157 118.6 0.02 T159 1.6 0.04 T161 57.6 0.02 S166 7.6 0.02 SCP2 Non-specific lipid-transfer protein S8 4.8 0.02 S420 1.8 0.03 T424S425 5.2 0.03 NUP98 Nucleoporin 98 S888 1.8 0.02 OSBPL9 Oxysterol-binding protein 9 S319 3.9 0.02 T327S329S330 4.8 0.02 OSBPL11 oxysterol-binding protein-related protein S21 8.3 0.02 11 S188 4.0 0.02 S189S192 4.2 0.02 PEX1 Peroxisome biogenesis factor 1 S1182 2.2 0.03 S1212 2.6 0.03 S1217 5.6 0.02 FXYD1 Phospholemman S82S83 23.5 0.02 KCNK5 TASK2 S266 1.6 0.04 KCNQ2 Potassium voltage-gated channel, S406 3.5 0.02 subfamily Q, member 2

30

For Peer Review Only Page 69 of 72 Diabetes

PPFIA1 Ppfia1 protein S239S242 6.1 0.02 PSEN2 Presenilin-2 S52 3.8 0.02 CASC3 Protein CASC3 S145 10.0 0.02 SET Protein SET S30 4.0 0.02 SEC16B Protein transport protein Sec16B S137 2.5 0.02 S939 14.0 0.02 S944 11.0 0.03 T950 14.0 0.02 SEC24A Protein transport protein Sec24A T227 4.6 0.02 SEC31A Protein transport protein Sec31A S526 7.6 0.02 RANBP3 Ran-binding protein 3 T56 7.3 0.02 S57 5.0 0.02 S58 5.3 0.02 T60 5.0 0.02 RRBP1 Ribosome binding protein 1 S134 6.0 0.02 S959 2.6 0.02 ATP2A2 Sarcoplasmic/endoplasmic reticulum S661S663 6.1 0.02 calcium ATPase 2 SEC16A SEC16 homolog A (S. cerevisiae) S2101 5.6 0.02 SEC23IP SEC23-interacting protein S735 8.8 0.03 Slc16a7 Slc16a7 protein S214 3.2 0.02 S218T219 3.3 0.02 SLC6A13 Sodium- and chloride-dependent GABA T587 15.3 0.02 transporter 2 T590 16.3 0.02 SLC10A1 Sodium/bile acid cotransporter T330 6.9 0.02 SLC38A3 Sodium-coupled neutral amino acid S53 11.1 0.02 transporter 3 SLC38A4 Sodium-coupled neutral amino acid S49 2.5 0.02 transporter 4 SLC20A2 Sodium-dependent phosphate transporter 2 S316 9.2 0.02 SLC2A2 Solute carrier family 2, facilitated glucose S520S522 5.3 0.02 transporter member 2 SLC4A1AP Solute carrier family 4 (Anion exchanger), S50 2.3 0.02 member 1, adaptor protein SLCO1A4 Solute carrier organic anion transporter S657 3.4 0.02 family member 1A4 T659 3.3 0.02 SLCO1B2 Solute carrier organic anion transporter S290 29.0 0.02 family member 1B2 S288 8.6 0.02 T662 1.5 0.03 SLCO2B1 Solute carrier organic anion transporter S21S27 6.8 0.02 family member 2B1 S312T313S315 11.7 0.03 SORBS1 Sorbin and SH3 domain-containing protein S62 4.2 0.02 1 T1207S1209 2.8 0.02 SORBS2 Sorbs2 protein S27 5.5 0.02 S274S276 1.8 0.03 S338 1.8 0.03 S357 10.2 0.02 S377S378S380 3.9 0.02 S567S570 3.6 0.02 SNX17 Sorting nexin-17 S433S434S437S4 4.8 0.02 40 STIM1 Stromal interaction molecule 1 S512 6.0 0.02 T517 5.5 0.02 S519S521S523 28.0 0.02 SNAP Synaptosomal-associated protein S110S112 4.4 0.02

31

For Peer Review Only Diabetes Page 70 of 72

SNAP29 Synaptosomal-associated protein 29 S163 3.8 0.02 STX4 -4 S15 5.8 0.02 S117 5.9 0.02 STX12 Syntaxin 12 S142 2.2 0.02 TMX1 Thioredoxin-related transmembrane protein S245 31.0 0.02 1 TRAM1 Tram1 protein S365 12.4 0.02 SEC62 Translocation protein SEC62 T158 3.3 0.02 S356 9.3 0.02 T375 11.4 0.02 TCOF1 Treacle protein S1252 17.6 0.03 VPS4B 4b (Yeast) S92 3.2 0.02 S102 3.8 0.02 VPS4A Vacuolar protein sorting-associated protein S90 8.9 0.02 4A S95 5.7 0.03 S97 8.9 0.02 S99 7.9 0.03 VPS26B Vacuolar protein sorting-associated protein S304 4.2 0.02 26B Tumorigenesis FLCN Folliculin S62 10.4 0.02 PRCC Papillary renal cell carcinoma S157S159 4.6 0.02 (Translocation-associated) TPD52 Tumor protein D52 S175 3.0 0.02 TPD54 Tumor protein D54 S180 5.5 0.02 TP53I11 Tumor protein p53-inducible protein 11 S14 5.8 0.02 Function unknown GPR182 Adrenomedullin receptor S364 4.6 0.02 CB1_00008902 Ahnak AHNAK nucleoprotein isoform 1 S136 3.5 0.03 9 S217 2.6 0.02 S5504 2.8 0.02 ANKRD57 Ankyrin repeat domain-containing protein S82 2.9 0.02 57 RSRC2 Arginine/serine-rich coiled-coil protein 2 S45 4.6 0.02 ASL1/Dennd1b fusion protein S163S164 8.4 0.02 Cobl-like 1 T266 4.6 0.02 S268T272 3.0 0.02 T287 2.8 0.02 S334 7.7 0.02 S476T480 3.5 0.02 S483 3.4 0.02 T594S596 3.4 0.02 S724 5.6 0.02 S826 6.8 0.02 S827 4.8 0.02 S1022 8.4 0.02 S1144 6.7 0.02 S1145T1147 3.5 0.02 S1196 2.0 0.03 FAM104a DNA segment, Chr 11, Wayne State S15 1.6 0.04 University 99, expressed FAM208A Isoform 2 of Protein FAM208A S653 7.3 0.02 Isoform 2 of Uncharacterized protein S66S69 4.6 0.02 C18orf25 homolog

32

For Peer Review Only Page 71 of 72 Diabetes

KIDINS220 Kidins220 Kidins220 protein S1583 13.7 0.02 LARP1B Larp2 protein S60 6.9 0.02 Likely ortholog of H. sapiens T311 4.1 0.03 9 open reading frame 138 (C9orf138) RAPH1 MCG116540, isoform CRA_b S192 3.4 0.02 WDR20 MCG14935, isoform CRA_a S434 5.3 0.02 S491S492 3.9 0.02 PDAP1 MCG17262 S48 3.3 0.03 S60S63 7.1 0.02 MCG19001 S15S17 4.1 0.02 S261 4.6 0.02 9430020K01RI MCG55337 S542 3.6 0.02 K MCG7378 S7 8.3 0.02 S13S17S19 12.0 0.02 NAV2 Nav2 neuron navigator 2 isoform 1 S1262 5.1 0.02 NCK1 Non-catalytic region of tyrosine kinase S91 2.9 0.02 adaptor protein 1 Novel protein S25 2.2 0.02 PCDH1 Pcdh1 Protocadherin 1 isoform 2 S1010S1012 9.7 0.02 PLEKHA6 Pleckstrin homology domain-containing S901 4.2 0.03 family A member 6 S967 5.3 0.02 ALKBH5 Probable alpha-ketoglutarate-dependent S384S385 4.3 0.02 dioxygenase ABH5 FAM122A Protein FAM122A S34 2.0 0.02 S73 6.6 0.02 S140S144 4.2 0.02 FAM40A Protein FAM40A S335 3.5 0.02 GM7694 Protein Gm7694 S47 2.2 0.02 S219 3.4 0.02 KRI1 Protein KRI1 homolog S148 5.2 0.02 KTI12 Protein KTI12 homolog S157 2.8 0.02 MB21D2 Protein MB21D2 T371 3.1 0.02 PRRC2C Protein PRRC2C T353 2.6 0.02 S897 16.7 0.03 S899 24.7 0.02 Putative monooxygenase p33MONOX S158 3.0 0.03 isoform 3 S159T160 3.6 0.02 S164 3.2 0.02 RSRC2 Rsrc2 arginine/serine-rich coiled-coil S27 2.7 0.02 protein 2 isoform 1 S30 2.8 0.02 D623_1003269 Run domain Beclin-1 interacting and S412 2.7 0.02 3 33ysteine-rich containing protein Testis development-related S178 10.1 0.02 X3 T183 9.3 0.02 T185 9.2 0.02 TEX-2 Testis expressed gene 2 S743 5.6 0.02 S749 2.2 0.03 TEX264 Tex264 testis expressed gene 264 T237 3.8 0.02 S302T303 6.0 0.02 THUMPD1 THUMP domain-containing protein 1 S86S88 6.1 0.02 UBN2 Ubinuclein-2 S570 119.3 0.02 1700021F05Rik Uncharacterized protein S106 2.5 0.03 C6orf203 homolog

33

For Peer Review Only Diabetes Page 72 of 72

UHRF1BP1L UHRF1-binding protein 1-like S418T420 4.1 0.02 Uncharacterized protein C10orf47 T222S223 2.9 0.02 homolog KIAA1671 Uncharacterized protein KIAA1671 S205 5.4 0.02 CX026 UPF0368 protein Cxorf26 homolog S184 3.3 0.02 CX052 UPF0690 protein C1orf52 homolog T153 3.8 0.02 WDR81 WD repeat-containing protein 81 S85 2.6 0.02

34

For Peer Review Only