MG149 | Glut Signal

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce
Molecular and Cellular Endocrinology 537 (2021) 111425

Mof acetyltransferase inhibition ameliorates glucose intolerance and islet Image dysfunction of type 2 diabetes via targeting pancreatic α-cells
Xinghong Guo a, Chen Cui a, Jia Song a, Qin He a, Nan Zang a, Huiqing Hu a, Xiaojie Wang c, Danyang Li g, Chuan Wang a, Xinguo Hou a, Xiangzhi Li b, Kai Liang a, d, e, f, Fei Yan a, d, e, f,**,
Li Chen a, d, e, f,*
a Department of Endocrinology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
b Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, Life Science School of Shandong University, Qingdao, 266237, Shandong, China
c Department of Pharmacology, Basic Medicine School of Shandong University, Jinan, 250012, Shandong, China
d Institute of Endocrine and Metabolic Diseases of Shandong University, Jinan, 250012, Shandong, China
e Key Laboratory of Endocrine and Metabolic Diseases, Shandong Province Medicine & Health, Jinan, 250012, Shandong, China
f Jinan Clinical Research Center for Endocrine and Metabolic Disease, Jinan, 250012, Shandong, China
g Department of Rehabilitation, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China

A R T I C L E I N F O

Keywords: Mof H4K16ac
α-cell
mg149 Acetylation T2DM
A B S T R A C T

Background: Previously, we reported that Mof was highly expressed in α-cells, and its knockdown led to ameliorated fasting blood glucose (FBG) and glucose tolerance in non-diabetic mice, attributed by reduced total α-cell but enhanced prohormone convertase (PC)1/3-positive α-cell mass. However, how Mof and histone 4 lysine 16 acetylation (H4K16ac) control α-cell and whether Mof inhibition improves glucose handling in type 2 diabetes (T2DM) mice remain unknown.

Methods: Mof overexpression and chromatin immunoprecipitation sequence (ChIP-seq) based on H4K16ac were applied to determine the effect of Mof on α-cell transcriptional factors and underlying mechanism. Then we administrated mg149 to α-TC1-6 cell line, wild type, db/db and diet-induced obesity (DIO) mice to observe the impact of Mof inhibition in vitro and in vivo. In vitro, western blotting and TUNEL staining were used to examine α-cell apoptosis and function. In vivo, glucose tolerance, hormone levels, islet population, α-cell ratio and the co- staining of glucagon and PC1/3 or PC2 were examined.

Results: Mof activated α-cell-specific transcriptional network. ChIP-seq results indicated that H4K16ac targeted essential genes regulating α-cell differentiation and function. Mof activity inhibition in vitro caused impaired α-cell function and enhanced apoptosis. In vivo, it contributed to ameliorated glucose intolerance and islet dysfunction, characterized by decreased fasting glucagon and elevated post-challenge insulin levels in T2DM mice.

Conclusion: Mof regulates α-cell differentiation and function via acetylating H4K16ac and H4K16ac binding to Pax6 and Foxa2 promoters. Mof inhibition may be a potential interventional target for T2DM, which led to decreased α-cell ratio but increased PC1/3-positive α-cells.

1. Introduction
T2DM has become a global social burden because of its soaring morbidity and mortality. Regarding the pathological mechanism, T2DM is thought as a bi-hormonal disease, which is characterized by hyper- glucagonemia and hypoinsulinaemia, emphasizing the importance of pancreatic α-cells (Gromada et al., 2018). Recent studies have proved
that α-cell participates in glucose regulation via glucagon and glucagon-like peptide-1 (GLP-1), which are cleaved from the same precursor by different prohormone convertase (PC) and exert contrary functions. Glucagon is cleaved by PC2, raising blood glucose via binding to glucagon receptor (GcgR) in liver and promoting hepatic glucose output (Sandoval and D’Alessio, 2015). In obese and T2DM patients, increased α-cell mass is observed, resulting in hyperglucagonemia (Ellenbroek et al., 2017). GLP-1 is originally found in intestinal L-cells,

* Corresponding author. Qilu Hospital of Shandong University, Shandong, China Department of Endocrinology, Institute of Endocrinology and Metabolism.
** Corresponding author. Qilu Hospital of Shandong University, Shandong, China, Department of Endocrinology, Institute of Endocrinology and Metabolism.
E-mail addresses: [email protected] (F. Yan), [email protected] (L. Chen).

https://doi.org/10.1016/j.mce.2021.111425

Received 6 April 2021; Received in revised form 10 August 2021; Accepted 12 August 2021
Available online 13 August 2021
0303-7207/© 2021 Elsevier B.V. All rights reserved.

Abbreviations
H4K16ac histone 4 lysine 16 acetylation T2DM type 2 diabetes
ChIP-seq chromatin immunoprecipitation sequence DIO diet-induced obesity
WT wild type
ELISA enzyme-linked immunosorbent assay
FBG fasting blood glucose
DMSO HE
IF IPGTT
IPITT
dimethyl sulfoxide hematoxylin-eosin immunofluorescence
intraperitoneal glucose tolerance test
Intraperitoneal insulin tolerance test
PC
GLP-1 glucagon-like peptide-1 GcgR glucagon receptor
prohormone convertase

ROS reactive oxygen species
NAFLD non-alcoholic fatty liver disease
q-RT PCR quantitative reverse transcriptional-polymerase chain reaction
IRS insulin receptor substrate GCK glucokinase
MSL male-specific lethal NSL non-specific lethal
H3K27ac histone 3 lysine 27 acetylation HDAC histone deacetylase

while latest research demonstrate that a small number of α-cells express PC1/3 and secret GLP-1. The physiological and therapeutic roles of α-cells expressing PC1/3 are being gradually uncovered. Physiologi- cally, lack of α-cell-derived GLP-1 leads to impaired glucose tolerance and β-cell function in aging mice (Traub et al., 2017). Therapeutically, transplanting PC1/3-expressing, but not PC2-expressing α-cell can effectively reverse hyperglycemia in db/db (Wideman et al., 2009) and streptozotocin-induced diabetes mice (Wideman et al., 2007). There- fore, reducing PC2-positive α-cell mass and increasing PC1/3-positive α-cell mass are potent strategies for glucose handling in diabetes.
Mof (Kat8, Myst1) is a highly-conserved histone acetyltransferase, responsible for global H4K16ac. Plenty of researches proved pivotal roles of Mof in oncogenesis, DNA damage response, proliferation, and stem cell development (Singh et al., 2020). Recently, the regulatory mechanism of Mof in endocrine and metabolism is indicated, from the aspects of endocrine cell differentiation (Ma et al., 2020), reactive ox- ygen species (ROS) production (Liu et al., 2019), inflammation (den- Dekker et al., 2020), cell mass maintenance (Guo et al., 2020) and fatty acid β-oxidation (Khoa et al., 2020; Sheikh et al., 2020). In non-alcoholic fatty liver disease (NAFLD), Mof acetyltransferase inhibition by mg149 could suppress NADPH oxidase transcription, thus lowering ROS accu- mulation in hepatic cells (Liu et al., 2019). In diabetic wound, Mof knockdown in macrophages effectively accelerated wound healing via decreasing TNF-α (denDekker et al., 2020). In addition, Mof signifi- cantly correlated with waist circumference in north Han males in China (Du et al., 2019).

We reported Mof was highly expressed in α-cells. Mof knockdown
mice exhibited reduced α-cell ratio, lower FBG and ameliorated glucose tolerance. Further investigation demonstrated Mof knockdown led to a vast drop in PC2-positive α-cell mass and raised PC1/3-positive α-cell mass, contributing to lower glucagon but elevated intra-islet GLP-1 levels. Since GLP-1 exerts insulinotropic effect and promotes β-cell proliferation, we observed enhanced β-cell proliferation, β-cell mass, and post-glucose challenge insulin level in α-cell-specific Mof deficiency mice (Guo et al., 2020). Theoretically, these results implied reduced Mof activity may be a potent method in glucose control in T2DM. Besides, we also reported Mof knockdown in α-TC1-6 cells resulted in increased apoptosis, impaired glucagon secretion signal pathway, and altered differentiation makers’ expressions. However, how Mof and H4K16ac regulate α-cell behaviors and whether Mof could be a target for T2DM therapy remain unexplored. Here, we uncover how Mof directly regu- lates α-cell gene expression and demonstrate that Mof inhibition is beneficial to glucose control in T2DM mice.

2. Materials and methods
2.1. Animal experiments

Animal experiments were approved by the Animal Care and Use Committee of Shandong University. All animals had free access to diet and water, and no mice became severely ill or died until the end of experiments.
WT and db/db mice were purchased from SPF Biotechnology Co., Ltd (Beijing) and given chow diet. WT and db/db mice were randomly divided into 2 groups, respectively. From 8 weeks, one group was given 1 mg/kg mg149 (Selleck) intra-peritoneally every other day (Liu et al., 2018a), while the other was given dimethyl sulfoxide (DMSO) as con- trol. To establish DIO model, WT mice about 4 weeks old were given 60% fat diet for 10 weeks. Then DIO mice were randomly divided into mg149 or control group as described. WT and DIO mice were C57BL/6J background and db/db mice were C57BL/BKS background.
After mice being sacrificed under anesthesia, serums were immedi- ately used or stored in —80 ◦C. Pancreas tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm thick
—slides. Then these slides were used for hematoxylin-eosin (HE) and immunofluorescence (IF) staining. Livers were stored in 80 ◦C and used in western blotting to examine Mof inhibition efficacy.

2.2. Cell culture and treatment
Mice α-cell line α-TC1-6 and β-cell line Min6 were cultured as pre- viously described (Guo et al., 2020). To inhibit Mof activity, 35 nM mg149 (Li et al., 2020) was added to the medium for 2 d. To overexpress Mof, mus-Mof was cloned into pcDNA3.1 plasmid (Supplementary Table 1), and transfected as described via Lipofectamine 2000 Trans- fection Reagent (Invitrogen, USA).

2.3. Intraperitoneal glucose and insulin tolerance test

Intraperitoneal glucose tolerance test (IPGTT) was carried out in WT, db/db and DIO mice as previously described (Wang et al., 2017), with the glucose dosage of 1.5 g/kg body weight. Fasting and 30 min post glucose challenge blood samples were obtained from angular vein for insulin level measurement. Intraperitoneal insulin tolerance test (IPITT) was executed in WT and DIO mice as described with the insulin of 0.75 U/kg body weight. Tail vein blood glucose was measured by Accu– Chek® Performa.

2.4. HE, IF and TUNEL staining

HE staining was conducted according to the standard protocol. Then pictures were taken and islet areas were measured by CellSens Standard
A. Western blotting showed that Mof was overexpressed in α-TC1-6. B. q-PCR indicates that when Mof was overexpressed (red column), expres- sions of Nkx2.2, Nkx6.1, Pax6, MafB, NeuroD1, Arx,
and Gcg were increased, but not ins1, ins2, pdx1 (n =
3). C. Western blotting showed that Mof was over- expressed in Min6. D. q-PCR indicates that when Mof was overexpressed (red column), expressions of Nkx2.2, Pax6, MafB, NeuroD1, Arx, and Gcg were
increased, but not ins1, ins2, pax4 (n = 3). (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
according to the manufacturer’s instructions. Islets were divided into different groups based on the size as described (Guo et al., 2020; Sato et al., 2020 For IF staining, after antigen retrieval in citrate buffer (pH 6.0) and blocking unspecific sites, primary antibodies were incubated overnight at 4 ◦C. In the following day, fluorophore-conjugated secondary anti-
bodies (ZSGB-Bio, China) were applied at 37 ◦C for 1 h. After 4′,6-Dia-
midino-2-phenylindole (DAPI) staining, pictures were taken under OLYMPUS fluorescence microscopy. Primary antibodies were utilized as following: insulin (1:1000, 15848-1-AP, proteintech, China), insulin (1:1000, 66198-1-Ig, proteintech), glucagon (1:200, 15954-1-AP, pro- teintech), glucagon (1:200, 67286-1-Ig, proteintech), PC1/3 (1:200, ab233397, Abcam, USA) and PC2 (1:200, 14013, CST, USA).
For islet morphological analyses, α cell ratio in islet was measured by
glucagon-positive area/(glucagon-positive + insulin-positive) area. PC1/3-positive α cell ratio was measured by PC1/3-positive α cell number/total α cell number.TUNEL staining of α-TC1-6 were conducted as standard protocol provided by KeyGEN BioTECH (KGA7061, China) to show cell apoptosis, as previously described (Guo et al., 2020).

2.5. Islet isolation and culturing

Pancreas from WT mice was isolated after anesthesia. After cutting pancreas into small pieces, we digested it by collagenase P (Roche). Then, islets were hand-picked, and about 70 islets of the similar size were seeded in one well of 48-well plate. After overnight culturing in RPMI 1640 medium, to inhibit Mof activity, 35 nM mg149 was added to the medium for 2 d. To measure the glucagon secretion ex vivo, islets were first balanced in Krebs-Ringer bicarbonate HEPES (KRBH) buffer (120 mM NaCl, 0.75 mM CaCl2⋅2H2O, 4 mM KH2PO4, 10 mM NaHCO3,
1 mM MgSO4⋅7H2O, 30 mM HEPES, 1% BSA) containing no glucose for
0.5 h in 37 ◦C, then incubated in KRBH buffer containing 2.5 mM glucose for 3 h. Supernatant were collected for glucagon ELISA test.

2.6. RNA extraction and quantitative reverse transcriptional-polymerase chain reaction(q-RT PCR)
RNA extraction, cDNA synthesis and q-PCR were implemented as we described before (Guo et al., 2020). Primers were synthesized by Gene Pharma (Shanghai, China). Primer sequences used in this manuscript were listed in Supplementary Table 1.

2.7. Western blotting

Briefly, total protein was extracted via lysis buffer and separated by SDS-PAGE. Next, we transferred protein onto PVDF membranes. After being blocked by 5% skim milk, membranes were incubated with
primary antibodies with rotation in 4 ◦C overnight. Next day, secondary antibodies (ZSGB-Bio) conjugated with horseradish peroxidase were incubated. The immune complexes were detected with enhanced chemiluminescence kit (Millipore, USA). Primary antibodies used are as following: GAPDH (1:2000, 60004-1-Ig, proteintech), Mof (1:1000, ab200660, abcam), H4K16ac (1:1000, YM3317, Immunoway, USA), H4K8ac (1:1000, YK0012, Immunoway), caspase-3 (1:1000, 9662,CST), cleaved-capsase3 (1:1000, 9664, CST), PARP (1:1000, 9542, CST),cleaved-PARP (1:1000, 9548, CST), Nkx2.2 (1: 500, YN0790, Immuno-
way), Pax6 (1:1000, 12323-1-AP, Proteintech), mTOR (1:1000, 2972,CST), p-mTOR (1:1000, 5536, CST), GcgR (1:1000, 26784-1-AP, Pro-teintech), Foxa2 (1:1000, 22474-1-AP, Proteintech), Glucokinase (GCK, 1:1000, ab37796, abcam), glucagon (1:1000, EP3070, Abcam).

2.8. ELISA

For circulatory insulin, glucagon and total GLP-1 levels, blood sam- ples were collected into no anti-coagulant tubes for serum. 0.1% Diprotin A was added when GLP-1 levels were measured (Biovision, USA) (Guo et al., 2020; Liu et al., 2018b). ELISA tests were carried out with insulin (ALPCO, USA), glucagon (Cloud-Clone Corp, USA) or total GLP-1 ELISA kit (Millipore) in accordance with the protocols provided. For in vitro islet glucagon secretion, ELISA tests were performed with glucagon (Blue Gene, China) kit.

2.9. ChIP-seq and raw data analysis
α-TC1-6 cells were cross-linked using 1% formaldehyde. Chromatin samples were sonicated for 10 min of 5 s on/10 s off sonication cycle.
ChIP was achieved with protein A + G magnetic beads (Magna
ChIP™16-663) and DNA was immunoprecipitated with anti-H4K16ac antibody (Active Motif, 39168). Then DNA library was established via PCR and sequenced on the Illumina Novaseq (Illumina, USA). For the obtained raw data, we filtered out sequencing adaptors and low-quality data, performed peak-calling and motif test. Finally, we made annota- tions to the peaks based on the GO and KEGG database. Enrichment calculations were conducted using Fisher’s exact test.

2.10. Data analysis

Data were shown as mean standard error of mean (SEM). Statis- tical analyses were achieved by Graph-Pad Prism 6.0. Statistical com- parisons of means between 2 groups were performed by unpaired t-test. p value＜ 0.05 was taken to indicate a statistically significant difference.
*p stands for ＜0.05, **p for ＜0.01, and ***p for ＜0.001. A. Distribution of H4K16ac peaks in α-TC1-6. B. Some key genes playing roles in α cell or β cell differentiation and maturation are regulated by H4K16ac. C. The KEGG results of H4K16ac peaks. D. The GO results of H4K16ac peaks.

3. Result
3.1. Mof regulated α-cell specific transcriptional network
Previously, we reported α-cell-specific Mof knockdown mice exhibited greatly declined total α-cell and enhanced PC1/3-positive α-cell mass. In vitro, Mof knockdown did not alter PC1/3 or PC2 expression, but was involved in expression of α-cell fate-related factors. Because Mof is a transcriptional activator via acetylating H4K16, we overexpressed Mof in α-TC1-6 to observe how Mof activated cell-specific transcriptional network (Fig. 1A).
When we overexpressed Mof in α-TC1-6, mRNA levels of transcrip- tional factors commonly used in endocrine differentiation, Nkx2.2, Nkx6.1, NeuroD1 and α-cell markers Pax6, MafB, Arx were greatly increased, but not β-cell marker Pdx1. Interestingly, the glucagon level was increased, but amounts of insulin1, insulin2 were not altered (Fig. 1B), suggesting Mof activated α-cell-specific transcriptional network in α-TC1-6.
To verify whether Mof activated α-cell-specific transcriptional
network in β-cells, we also overexpressed Mof in Min6 (Fig. 1C). Simi- larly, in Mof-overexpressed Min6, the relative quantities of Pax4, insu- lin1, insulin2 were not changed, while those of Pax6, Nkx2.2, mafB, NeuroD1, Arx, and most importantly, glucagon were elevated (Fig. 1D). Hence, Mof activated α-cell specific transcriptional network in both α and β-cells.

3.2. ChIP-seq of H4K16ac in α-cells
Because Mof is H4K16ac-specific histone acetyltransferase (Taipale et al., 2005), and further we intended to investigate the effect of Mof inhibition on α-cells, we undertook ChIP-seq of H4K16ac in α-TC1-6 to find the promoters regulated by Mof activity. Peaks distribution was shown in Fig. 2A. Peak annotations were listed in Supplementary
Table 2. H4K16ac directly bind to Akt1, Akt2, Akt3, insulin receptor substrate (Irs) 1, Irs2, mTOR, Nkx2.2 and Nkx6.1, consistent with our previous report that Mof knockdown elicited down-regulation of above genes (Guo et al., 2020).
We found that Mof regulated α-cell-specific transcriptional network,
thus we focused on peaks annotated as key genes regulating endocrine cell differentiation (Fig. 2B). Interestingly, many genes essential for α-cell differentiation and maturation, such as Pax6, Paupar, Nkx2.2, and Foxa2 were targets of H4K16ac. In addition, some key genes in β-cell maturation were also found in ChIP-seq result, such as Pdx1, Pax4 and Ins2. However, when Mof was overexpressed, Pdx1, Pax4 and Ins2 expression remained unchanged, suggesting Mof-mediated H4K16ac might not be the pivotal regulator of their capacity.
Furthermore, we performed KEGG annotations (Fig. 2C, Supple- mentary Table 3), and found H4K16ac peaks were enriched in tran- scription and translation, metabolic pathways, such as “oxidative phosphorylation” “citrate cycle” “fatty acid metabolism” “insulin/ glucagon signal pathway” “non-alcoholic fatty liver disease” “biosyn- thesis of cofactors” and some other already investigated pathways, including “autophagy” “signal pathways regulating pluripotency of stem cell”. These results advised that Mof might be critical in metabolic process, but how Mof regulates metabolic disease requires further investigation. GO analyses (Fig. 2D, Supplementary Table 4) implied that H4K16ac also had great impact on catabolic process regulation and complexes responsible for histone modification.

3.3. Mof inhibition led to increased α-cell apoptosis and impaired α-cell function in vitro
We aimed to investigate whether Mof inhibition might be a thera- peutic target for T2DM, so the effect of Mof inhibition in vitro was studied first. We used a commercially available Mof inhibitor, mg149. Because mg149 was indicated to inhibit Mof and Tip60 with the IC50 at A. Western blotting showed that 35 nM mg149 is the optimal concentration for Mof inhibition. B. When mg149 is administrated to α-TC1-6, enhanced cleaved-caspase3/caspase3 and cleaved PARP/PARP indicated increased apoptosis. C. TUNEL staining demonstrated that mg149 administration led to increased apoptosis. Apoptotic cells were indicated as red staining. D. When mg149 is administrated to α-TC1-6, expressions of key regulators of α cell maturation and function, including p-mTOR/mTOR, FoxA2, Pax6, Nkx2.2 and Gck were decreased. E. When mg149 is administrated to α-TC1-6, glucagon expression was decreased. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)47 and 74 nm, respectively, we performed experiments and confirmed that 35 nM was the optimal concentration to inhibit Mof (decreased H4K16ac) but not Tip60 (unchanged H4K8ac) in vitro, consistent with previous report (Li et al., 2020) (Fig. 3A).
We evaluated the effect of Mof inhibition on α-cells apoptosis.

Administration of mg149 did not change Mof expression, but promoted apoptosis similar to Mof knockdown, featured by elevated cleaved- caspase3/caspase3 and cleaved-PARP/PARP (Fig. 3B). TUNEL staining also confirmed enhanced α-cell apoptosis (Fig. 3C). mTOR signal is important in α-cells proliferation via targeting Foxa2. Intriguingly, Mof
inhibition effectively reduced p-mTOR/mTOR (Fig. 3D), which might also contribute to α-cell loss. In addition, mg149 administration did not result in cell loss in Min6 as in α-TC1-6 (Supplementary Figure 1).
Then α-cell differentiation and function were evaluated. Pancreatic α-cell differentiation and maturation are controlled by three indepen- dent pathways, namely Arx, Pax6 and Foxa2 (Gosmain et al., 2010). Our ChIP-seq data suggested H4K16ac targeted both Pax6 and Foxa2. When Mof was inhibited, the quantities of H4K16ac targeted genes, Pax6, Foxa2 and Nkx2.2 were down-regulated (Fig. 3D). Alterations of such genes implicated impaired terminal differentiation status. Accordingly, A. WT mice were administrated with mg149. Western blotting of liver tissue showed that mg149 inhibited H4K16ac, but not H4K8ac. In addition, Mof inhibition led to decreased GcgR expression. B. IPGTT of WT + DMSO (black, n = 5) and WT + mg149 (red, n = 5) mice before administration. C. IPGTT of WT + DMSO (black, n = 5) and WT + mg149 (red, n = 5) mice (7 d post administration). D. IPGTT of WT + DMSO (black, n = 5) and WT + mg149 (red, n = 5) mice (14 d post administration). E. IPITT of WT + DMSO (black, n = 5) and WT + mg149 (red, n = 5) mice. F: No statistically significance in fasting glucagon level was observed between WT + DMSO (white, n = 5) and WT + mg149 (red, n = 5) mice. G: No statistically significance in fasting insulin level was observed between WT +
DMSO (white, n = 5) and WT + mg149 (red, n = 5) mice. H: No statistically significance in fasting circulatory GLP-1 level was observed between WT + DMSO (white, n = 5) and WT + mg149 (red, n = 5) mice. I: No statistically significance in post-challenge insulin level was observed between WT + DMSO (white, n = 4) and WT + mg149 (red, n = 4) mice. J: No statistically significance in glucagon level was observed between primary islets isolated from WT + DMSO (white, n = 3) and WT + mg149 (red, n = 3) mice. K: The islet size distribution of WT + DMSO (white, n = 4) and WT + mg149 (red, n = 4) mice. L: In WT + mg149 mice, the α-cell ratio (red) was decreased. M: The co-staining of PC1/3 (green) and glucagon (red). Upper is WT + DMSO mice, lower is WT + mg149 mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
the glucagon levels in Mof inhibited cells were also decreased, indicating impaired α-cell function (Fig. 3E). In conclusion, Mof inhibition contributed to increased α-cell apoptosis and impaired α-cell function in vitro.

3.4. Mof inhibition decreased α-cell ratio and increased PC1/3-positive
α-cell in WT mice
We then investigated whether Mof inhibition could reduce blood glucose and improve islet function in T2DM mice. First, we studied the effect of Mof inhibition in healthy mice. Livers were used to examine the inhibition efficacy (Fig. 4A). H4K16ac levels were decreased but not H4K8ac. In the whole process of mg149 administration, body weight showed no difference between control and Mof inhibited WT mice (Supplementary Figure 2A). IPGTT were performed before mg149 de- livery (Fig. 4B), and 7 d (Figure 4C), 14 d (Fig. 4D) post initial dosage. No difference was observed before or 7 d post mg149 injection. How- ever, 14 d post mg149 injection, IPGTT showed better glucose level in 120 min. FBG levels exhibited no difference. We also performed IPITT, and found that in 90 min, the glucose levels in Mof inhibited mice were obviously lower, implying impaired glucose elevating (Fig. 4E). Because no difference was observed in fasting glucagon levels, we compared the GcgR levels and observed less GcgR in Mof inhibited mice (Fig. 4A).
=Furthermore, we detected the circulatory hormone levels. Interest- ingly, fasting glucagon of Mof inhibited WT mice showed a tendency to increase (Fig. 4F). Fasting insulin (Fig. 4G) and GLP-1 levels (Fig. 4H) were similar between two groups. In IPGTT, insulin levels at 30 min post glucose challenge (Fig. 4I) in mg149 group were slightly higher than control, without statistically significance(p 0.0799). To further verify whether Mof inhibition leads to a tendency of increased glucagon secretion, which was not consistent with our previous report that Mof knockdown contributed to decreased glucagon level, we isolated pri- mary islets of WT mice. After mg149 incubation for 2 d, the glucagon secretion of primary islets also showed a tendency to increase (Fig. 4J).

Previously, we reported that Mof knockdown resulted in declined α-cell but exaggerated β-cell mass. Here, we conducted HE staining, and then divided islets into different groups according to their sizes (Fig. 4K). Although the percentage of islet area ﹤1000 μm2 were lower
after mg149 utilization, ratios of other groups exhibited no difference. Similar to previous report, IF staining (Supplementary Figure 2B) showed evident decline of α-cell ratio in mg149 group (Figure 4L). Although statistically significant increase in β-cell ratio was shown (Data not shown), it is comparatively minor and mainly due to α-cell ratio change. We then carried out co-staining of glucagon and PC1/3 or PC2. Although almost all α-cell were PC2-positive (Supplementary Figure 2C), mg149 group exhibited greatly enhanced PC1/3-positive α-cells (Figure 4M). Therefore, Mof acetyltransferase inhibition aroused decreased total α-cell ratio and increased PC1/3-positive α-cells.

3.5. Mof inhibition ameliorated glucose tolerance and islet function in db/ db mice
We administrated mg149 in T2DM model, db/db mice. During this process, no difference in body weight between db/db + mg149 and
control mice were seen (Supplementary Figure 3A). Mof inhibition was verified via liver tissues (Fig. 5A). IPGTT were performed as in WT mice. In IPGTT, blood glucose levels post-glucose challenge was so high that Accu-Chek® Performa showed “HIGH” rather than a specific number. All the “HIGH” were recorded as 33.3 mmol/L in raw data by default.

Before mg149 delivery, no difference was exhibited between two groups (Fig. 5B). However, 7 d post first dosage, FBG levels of db/db mg149 decreased by about 6 mol/L. In 210 min post glucose adminis- tration, approximately 5 mmol/L reduction was observed (Fig. 5C). 14 d post initial mg149 injection, about 3 mmol/L reduction in FBG and 1–2 mmol/L reduction in 240- and 270-min post glucose challenge were exhibited in db/db mg149 (Fig. 5D). The therapeutic effect became less dominant, which may be attributed to rapid T2DM progression of db/db mice. Considering the greatly impaired insulin sensitivity, IPITT was not executed in db/db mice.
We evaluated the influence of mg149 on islet function of db/db mice. Intriguingly, fasting glucagon levels were reduced (Fig. 5E), but fasting insulin (Fig. 5F) and GLP-1 (Fig. 5G) levels were not changed. In IPGTT, before glucose was given, insulin levels were similar (Fig. 5H), but higher insulin levels were seen in mg149 group at 30 min post-glucose challenge (Fig. 5I). Consequently, mg149 injection improved islet dysfunction in T2DM db/db mice.

Subsequently, we observed the islet size and different cell ratio al- terations. HE staining suggested declined ratio of islets whose size were between 1000 and 5000 μm2 and elevated proportion of islet whose size
were ﹥ 20000 μm2 (Fig. 5J). IF staining (Supplementary Figure 3B)
demonstrated decreased α-cell ratio in mg149 group (Fig. 5K). Similarly, β-cell ratio elevation was not that apparent, though statistically mean- ingful, which was contributed by α-cell loss. PC1/3-positive α-cells were more in mg149 group (Figure 5L), but still, most α-cells were PC2- positive (Supplementary Figure 3C).

3.6. Mof inhibition ameliorated glucose tolerance and islet function in DIO mice
Mof inhibition was confirmed via western blotting of liver tissues (Fig. 6A). DIO mice were established as diet-induced T2DM model, characterized by increased body weight (Supplementary Figure 4A) and impaired glucose tolerance (Fig. 6B). During mg149 administration process, no difference in body weight between DIO mg149 and control mice were seen (Supplementary Figure 4B).
7 d post initial mg149 dosage, glucose tolerance was significantly improved (Fig. 6C), but not including FBG. 14 d later, both FBG and glucose tolerance were improved (Fig. 6D). IPITT showed no dif- ference between mg149 and control (Fig. 6E). Therefore, the ameliorated FBG and glucose tolerance were due to ameliorated islet dysfunction.Similar to db/db mice, lower fasting glucagon (Fig. 6F), unaltered fasting insulin (Fig. 6G) and fasting GLP-1 (Fig. 6H) but increased post- challenge insulin levels (Fig. 6I) were shown, suggesting amelioratedislet dysfunction. HE staining indicated lower proportion of islets with the size ＜1000 μm2 and enhanced ratio of those between 10000 and

A. db/db mice were administrated with mg149. Western blotting of liver tissue showed that mg149 inhibited H4K16ac. B. IPGTT of db/db + DMSO (black, n
= 4) and db/db + mg149 (red, n = 4) mice before administration. C. IPGTT of db/db + DMSO (black, n = 4) and db/db + mg149 (red, n = 4) mice (7 d post administration). D. IPGTT of db/db + DMSO (black, n = 4) and db/db + mg149 (red, n = 4) mice (14 d post administration). E: db/db + mg149 mice (red) showed decreased fasting glucagon level (n = 4). F: No statistically significance in fasting insulin level was observed between db/db + DMSO (white, n = 4) and db/db + mg149 (red, n = 4) mice. G: No statistically significance in fasting circulatory GLP-1 level was observed between db/db + DMSO (white, n = 4) and db/db + mg149 (red, n = 4) mice. H: No statistically significance in insulin level was observed between db/db + DMSO (white, n = 4) and db/db + mg149 (red, n = 4) mice at 0 min in IPGTT. I. db/db + mg149 mice (red) showed increased post-challenge insulin level in IPGTT (n = 4). J: The islet size distribution of db/db + DMSO (white, n = 4) and db/db + mg149 (red, n = 4) mice. K: In db/db + mg149 mice, the α-cell ratio (red) was decreased. L: The co-staining of PC1/3 (green) and glucagon (red). Upper is db/db + DMSO mice, lower is db/db + mg149 mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)2000 μm2 (Fig. 6J). Similar with db/db mice, reduced α-cell ratio (Fig. 6K, Supplementary Figure 4C) and enhanced PC1/3-positive α-cells (Figure 6L) were exhibited when Mof was inhibited.
To sum up, Mof inhibition ameliorated glucose intolerance and islet dysfunction, characterized by lower fasting glucagon but increased post- challenge insulin levels. Such improvement was mainly attributed to decreased overall α-cell ratio and increased PC1/3-positive α-cells.

4. Discussion
Metabolism is closely related with acetylation, especially histone acetylation (Menzies et al., 2016), thus acetylation may be a promising target for the treatment of metabolic disorders, such as T2DM and NAFLD. Histone acetylation mediated by acetyltransferases and deace- tylases (HDAC), is usually site-specific, and exert tissue-specific out- comes by regulating clusters of gene expression (Shahbazian and Grunstein, 2007). For example, increased histone 3 lysine 27 acetylation (H3K27ac) in β-cell is a consequence of fatty-acid signal and regulates obesity-induced β-cell function alterations (Nammo et al., 2018). In NAFLD, Mof-induced H4K16ac activates the expressions of NADPH ox- idases and promote ROS production. Mg149 treatment to liver cells could decrease H4K16ac and lower oxidative stress injury (Liu et al., 2018a). In diabetic foot, more H4K16ac depositions in NF-кB binding sites of the promoters of Il1b and Tnf accelerate inflammation in mac- rophages (denDekker et al., 2020). Besides, the usage of HDAC inhibitor, although not site-specific, promotes β-cell development, proliferation, function and positively affects diabetic microvascular complications via elevating overall acetylation levels (Christensen et al., 2011). In addi- tion to observed effectiveness above, histone acetylation modification is dynamic and reversible, making it a safe interventional target.
Previously, we reported Mof regulate glucose level via altering different α-cell subset mass and intra-islet GLP-1 and glucagon secretion (Guo et al., 2020). Considering intestinal L cells-derived GLP-1 is rapidly degraded in circulation, α-cells are regarded as the main source of GLP-1 which exert effect on pancreatic islet (Whalley et al., 2011). Emerging evidence also supported that α-cell derived GLP-1 was essential in β-cell proliferation (Guo et al., 2020; Pereira de Arruda et al., 2020) and function (Traub et al., 2017; de Souza et al., 2020; Liu et al., 2011). α-cell derived GLP-1 is secreted from PC1/3-positive α-cells, and the transplantation of PC1/3-posotive α-cells could improve the glucose tolerance of diabetic mice (Wideman et al., 2007, 2009). In this study, we showed that mg149 administration contributed to enhanced PC1/3-positive α-cells and decreased overall α-cells, which was the basis of ameliorated glucose intolerance and islet dysfunction in this study.
In tamoxifen-induced global Mof knockdown mice, insulin resistance was observed. But when we administrated mg149 to inhibit Mof ace- tyltransferase activity, insulin resistance was not impaired. This differ- ence suggested that Mof inhibition is better than Mof knockdown, because Mof is the core component of many complex (Ravens et al., 2014), participating not only in H4K16ac, but also in other histone modifications, such as H3K4 di-methylation (Zhao et al., 2013), H3K79 methylation (Valencia-Sa´nchez et al., 2021)and H2B ubiquitylation (Wu et al., 2014). But our previous western blotting results and ChIP-seq results both indicated that H4K16ac regulated Irs1 and Irs2 amounts. Therefore, further investigation regarding Mof and insulin sensitivity isnecessary.

ChIP-seq of Mof or H4K16ac has been studied in many cell types, such as embryonic stem cell (Khoa et al., 2020; Li et al., 2012), thyroid (Ma et al., 2020; Li et al., 2020), erythroid (Pessoa Rodrigues et al., 2020), neurons (Sheikh et al., 2020) and Hela (Chatterjee et al., 2016). Our ChIP-seq results showed that H4K16ac regulated key genes regu- lating α-cell mass, maturation, and function, including Pax6 (Gosmain et al., 2010), Paupar (Singer et al., 2019), Nkx2.2 (Doyle et al., 2007), and Foxa2 (Lee et al., 2005). In addition, H4K16ac also affected mTOR signaling pathway, which is also vital for α-cell mass and function (Bozadjieva et al., 2017). We also observed that Mof-mediated H4K16ac regulated genes mastering β cell function. This was consistent with previous report that H4K16ac binds to many genes’ promoters, but the activation of certain gene was cell-type specific (Horikoshi et al., 2013). Both our data and other published data indicate that Mof and H4K16ac regulate certain biological process and signaling pathways in different cell types. KEGG indicated Mof and H4K16ac had profound impact on cellular metabolism, including fatty acid metabolism, oxidate phos- phorylation, citrate cycle, coenzyme metabolism and glycer- ophospholipid metabolism. Although Mof and H4K16ac directly control
metabolism, the study on Mof-mediated metabolic process and meta- bolic disease is still a blank page. GO indicated Mof and H4K16ac are important in post-translational modification, protein catabolic process, DNA repair, etc. Considering endocrine cell differentiation, our data showed that H4K16ac regulated Nkx2.2, Pax6 and Foxa2 mastering α-cell differentiation. ChIP-seq data in thyroid cell proved Mof controlled the differentiation from hES cells to thyroid cell via Nkx2.1 and Pax8 (Ma et al., 2020). It seems H4K16ac regulates endocrine cell differentiation following similar patterns, which is an intriguing but less-investigated field.

Inhibiting glucagon (Lef`ebvre et al., 2015) or elevating
PC1/3-positive α-cell ratio (Davis and Sandoval, 2020) is effective in glucose handling. Overall, our research showed inhibition of Mof-mediated H4K16ac may provide a new method for T2DM therapy, although the long-term effect, safety and side effect should be further deeply investigated. Interestingly, although post mg149 administration, both db/db and DIO mice showed lower circulatory fasting glucagon level, WT mice seemed to show a tendency for increased glucagon level, although not statistically significant. To further confirmed this pheno- type, we administrated mg149 to primary islets isolated from WT mice. The glucagon secretion from Mof-inhibited WT mice also showed a tendency to increase. Such phenomena indicated that the effect of Mof inhibition may vary in non-diabetic and diabetic model. In our study, we found that in WT mice, mg149 administration led to decreased hepatic glucagon receptor expression. We proposed that Mof-inhibition medi- ated GcgR knockdown might also contribute to the phenotypes we observed. GcgR antagonism (Lang et al., 2020; Bagger et al., 2011) or knockout (Conarello et al., 2007) is reported to decrease blood glucose level, increase GLP-1 secretion from α cell and L cells, increase β cell mass but increase circulatory glucagon levels. As for α cell mass, the effect of GcgR remains debatable (Mu et al., 2011; Lam et al., 2019). We have previously reported the effect of α cell-specific Mof knockdown led to decreased α cell mass and glucagon level, resulting in lower glucose level. However, when global Mof was inhibited, glucagon receptor expression level was also decreased. The effects of Mof inhibition and

A. DIO mice were administrated with mg149. Western blotting of liver tissue showed that mg149 inhibited H4K16ac. B. IPGTT of DIO + DMSO (black, n = 5) and DIO + mg149 (red, n = 10) mice before administration. C. IPGTT of DIO + DMSO (black, n = 5) and DIO + mg149 (red, n = 10) mice (7 d post administration).
D. IPGTT of DIO + DMSO (black, n = 5) and DIO + mg149 (red, n = 10) mice (14 d post administration). E. IPITT of DIO + DMSO (black, n = 5) and DIO + mg149 (red, n = 10) mice. F: DIO + mg149 mice (red, n = 10) showed decreased fasting glucagon level. G: No statistically significance in fasting insulin level was observed between DIO + DMSO (white, n = 5) and DIO + mg149 (red, n = 10) mice. H: No statistically significance in fasting circulatory GLP-1 level was observed between DIO + DMSO (white, n = 5) and DIO + mg149 (red, n = 10) mice. I. DIO + mg149 mice (red, n = 5) showed increased post-challenge insulin level in IPGTT. J. The islet size distribution of DIO + DMSO (white, n = 5) and DIO + mg149 (red, n = 6) mice. K. In DIO + mg149 mice, the α-cell ratio (red) was decreased. L. The co- staining of PC1/3 (green) and glucagon (red). Upper is DIO + DMSO mice, lower is DIO + mg149 mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

GcgR knockdown may partly explain why WT mice administrated with mg149 showed a tendency for increased glucagon secretion. Except for glucagon secretion, other phenotypes we observed in Mof inhibited diabetic mice, including decreased α cell ratio, increased post-challenge but not fasting insulin levels, and increased PC1/3-positive α cells are similar with our previous report in Mof knockdown mice, further indi- cating that these phenotypes should be attributed to Mof inhibition.
CRediT authorship contribution statement
Xinghong Guo: Data curation, Investigation, Methodology, Project administration, Writing – original draft. Chen Cui: Investigation, Methodology. Jia Song: Investigation, Methodology. Qin He: Formal analysis. Huiqing Hu: Formal analysis. Xiaojie Wang: Methodology. Danyang Li: Methodology. Chuan Wang: Writing – original draft. Xinguo Hou: Writing – original draft. Xiangzhi Li: Writing – review & editing. Kai Liang: Project administration, Funding acquisition. Fei Yan: Funding acquisition, Writing – review & editing, Project admin- istration. Li Chen: Conceptualization, Funding acquisition, Writing – review & editing.

Declaration of competing interest
None.

Acknowledgements
This work was supported by National Nature Science foundation (grant number 81873632, 81900756, 82070800, 81770818), Taishan Scholars Project (grant number ts201712089), the Natural Science Foundation of Shandong Province (grant number ZR2019PH078) and National Key R&D program of China (2016YFC0901204, 2018YFC1311801).

Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/j.mce.2021.111425.

References
Bagger, J.I., Knop, F.K., Holst, J.J., et al., 2011. Glucagon antagonism as a potential therapeutic target in type 2 diabetes. Diabetes Obes. Metabol. 13, 965–971. https:// doi.org/10.1111/j.1463-1326.2011.01427.x.
Bozadjieva, N., Blandino-Rosano, M., Chase, J., Dai, X.Q., Cummings, K., Gimeno, J., et al., 2017. Loss of mTORC1 signaling alters pancreatic α cell mass and impairs glucagon secretion. J. Clin. Invest. 127, 4379–4393. https://doi.org/10.1172/ JCI90004.
Chatterjee, A., Seyfferth, J., Lucci, J., et al., 2016. MOF acetyl transferase regulates transcription and respiration in mitochondria. Cell 167, 722–738. https://doi.org/ 10.1016/j.cell.2016.09.052 e23.
Christensen, D.P., Dahllo¨f, M., Lundh, M., et al., 2011. Histone deacetylase (HDAC) inhibition as a MG149 novel treatment for diabetes mellitus. Mol. Med. 17, 378–390. https://doi.org/10.2119/molmed.2011.00021.
Conarello, S.L., Jiang, G., Mu, J., et al., 2007. Glucagon receptor knockout mice are resistant to diet-induced obesity and streptozotocin-mediated beta cell loss and hyperglycaemia. Diabetologia 50, 142–150. https://doi.org/10.1007/s00125-006-
0481-3.
Davis, E.M., Sandoval, D.A., 2020. Glucagon-like peptide-1: actions and influence on pancreatic hormone function. Comp. Physiol. 10, 577–595. https://doi.org/ 10.1002/cphy.c190025.
de Souza, A.H., Tang, J., Yadev, A.K., et al., 2020. Intra-islet GLP-1, but not CCK, is necessary for β-cell function in mouse and human islets. Sci. Rep. 10, 2823. https:// doi.org/10.1038/s41598-020-59799-2.
denDekker, A.D., Davis, F.M., Joshi, A.D., et al., 2020. TNF-α regulates diabetic
macrophage function through the histone acetyltransferase MOF. JCI Insight 5, e132306. https://doi.org/10.1172/jci.insight.132306.
Doyle, M.J., Loomis, Z.L., Sussel, L., 2007. Nkx2.2-repressor activity is sufficient to specify alpha-cells and a small number of beta-cells in the pancreatic islet.
Development 134, 515–523. https://doi.org/10.1242/dev.02763.
Du, Z., Ma, L., Qu, H., et al., 2019. Whole genome analyses of Chinese population and de novo assembly of A northern han genome. Dev. Reprod. Biol. 17, 229–247. https:// doi.org/10.1016/j.gpb.2019.07.002.
Ellenbroek, J.H., To¨ns, H.A.M., Hanegraaf, M.A.J., et al., 2017. Pancreatic α-cell mass in obesity. Diabetes Obes. Metabol. 19, 1810–1813. https://doi.org/10.1111/ dom.12997.
Gosmain, Y., Marthinet, E., Cheyssac, C., et al., 2010. Pax6 controls the expression of critical genes involved in pancreatic {alpha} cell differentiation and function. J. Biol. Chem. 285, 33381–33393. https://doi.org/10.1074/jbc.M110.147215.
Gromada, J., Chabosseau, P., Rutter, G.A., 2018. The α-cell in diabetes mellitus. Nat. Rev.
Endocrinol. 14, 694–704. https://doi.org/10.1038/s41574-018-0097-y.
Guo, X., Li, D., Song, J., et al., 2020. Mof regulates glucose level via altering different
α-cell subset mass and intra-islet glucagon-like peptide-1, glucagon secretion. Metabolism 109, 154290. https://doi.org/10.1016/j.metabol.2020.154290.
Horikoshi, N., Kumar, P., Sharma, G.G., et al., 2013. Genome-wide distribution of histone H4 Lysine 16 acetylation sites and their relationship to gene expression. Genome Integr. 4, 3. https://doi.org/10.1186/2041-9414-4-3.
Khoa, L.T.P., Tsan, Y.C., Mao, F., et al., 2020. Histone acetyltransferase MOF blocks acquisition of quiescence in ground-state ESCs through activating fatty acid oxidation. Cell Stem Cell 27, 441–458. https://doi.org/10.1016/j.stem.2020.06.005 e10.
Lam, C.J., Rankin, M.M., King, K.B., et al., 2019. Glucagon receptor antagonist- stimulated α-cell proliferation is severely restricted with advanced age. Diabetes 68, 963–974. https://doi.org/10.2337/db18-1293.
Lang, S., Wei, R., Wei, T., et al., 2020. Glucagon receptor antagonism promotes the production of gut proglucagon-derived peptides in diabetic mice. Peptides 131, 170349. https://doi.org/10.1016/j.peptides.2020.170349.
Lee, C.S., Sund, N.J., Behr, R., Herrera, P.L., Kaestner, K.H., 2005. Foxa2 is required for the differentiation of pancreatic alpha-cells. Dev. Biol. 278, 484–495. https://doi. org/10.1016/j.ydbio.2004.10.012.
Lef`ebvre, P.J., Paquot, N., Scheen, A.J., 2015. Inhibiting or antagonizing glucagon: making progress in diabetes care. Diabetes Obes. Metabol. 17, 720–725. https://doi. org/10.1111/dom.12480.
Li, X., Li, L., Pandey, R., et al., 2012. The histone acetyltransferase MOF is a key regulator of the embryonic stem cell core transcriptional network. Cell Stem Cell 11, 163–178. https://doi.org/10.1016/j.stem.2012.04.023.
Li, D., Yang, Y., Chen, B., et al., 2020. MOF regulates TNK2 transcription expression to promote cell proliferation in thyroid cancer. Front. Pharmacol. 11, 607605. https:// doi.org/10.3389/fphar.2020.607605.
Liu, Z., Stanojevic, V., Avadhani, S., et al., 2011. Stromal cell-derived factor-1 (SDF-1)/ chemokine (C-X-C motif) receptor 4 (CXCR4) axis activation induces intra-islet glucagon-like peptide-1 (GLP-1) production and enhances beta cell survival.
Diabetologia 54, 2067–2076. https://doi.org/10.1007/s00125-011-2181-x.
Liu, L., Wu, X., Xu, H., et al., 2018a. Myocardin-related transcription factor A (MRTF-A) contributes to acute kidney injury by regulating macrophage ROS production.
Biochim. Biophys. Acta (BBA) – Mol. Basis Dis. 1864, 3109–3121. https://doi.org/ 10.1016/j.bbadis.2018.05.026.
Liu, P., Song, J., Liu, H., et al., 2018b. Insulin regulates glucagon-like peptide-1 secretion by pancreatic alpha cells. Endocrine 62, 394–403. https://doi.org/10.1007/s12020- 018-1684-3.
Liu, L., Hong, W., Li, M., et al., 2019. A cross talk between BRG1 and males absent on the first contributes to reactive oxygen species production in a mouse model of nonalcoholic steatohepatitis. Antioxidants Redox Signal. 30, 1539–1552. https:// doi.org/10.1089/ars.2016.6822.
Ma, R., Morshed, S., Latif, R., et al., 2020. Epigenetic changes during human thyroid cell differentiation. Thyroid 30, 1666–1675. https://doi.org/10.1089/thy.2019.0772.
Menzies, K.J., Zhang, H., Katsyuba, E., et al., 2016. Protein acetylation in metabolism – metabolites and cofactors. Nat. Rev. Endocrinol. 12, 43–60. https://doi.org/ 10.1038/nrendo.2015.181.
Mu, J., Jiang, G., Brady, E., et al., 2011. Chronic treatment with a glucagon receptor antagonist lowers glucose and moderately raises circulating glucagon and glucagon- like peptide 1 without severe alpha cell hypertrophy in diet-induced obese mice. Diabetologia 54, 2381–2391. https://doi.org/10.1007/s00125-011-2217-2.
Nammo, T., Udagawa, H., Funahashi, N., et al., 2018. Genome-wide profiling of histone H3K27 acetylation featured fatty acid signalling in pancreatic beta cells in diet- induced obesity in mice. Diabetologia 61, 2608–2620. https://doi.org/10.1007/ s00125-018-4735-7.
Pereira de Arruda, E.H., Vieira da Silva, G.L., da Rosa-Santos, C.A., et al., 2020. Protein restriction during pregnancy impairs intra-islet GLP-1 and the expansion of β-cell mass. Mol. Cell. Endocrinol. 518, 110977. https://doi.org/10.1016/j. mce.2020.110977.
Pessoa Rodrigues, C., Herman, J.S., Herquel, B., et al., 2020. Temporal expression of MOF acetyltransferase primes transcription factor networks for erythroid fate. Sci Adv 6. https://doi.org/10.1126/sciadv.aaz4815 eaaz4815.
Ravens, S., Fournier, M., Ye, T., et al., 2014. Mof-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation. Elife 3, e02104. https://doi.org/10.7554/eLife.02104.
Sandoval, D.A., D’Alessio, D.A., 2015. Physiology of proglucagon peptides: role of glucagon and GLP-1 in health and disease. Physiol. Rev. 95, 513–548. https://doi. org/10.1152/physrev.00013.2014.
Sato, Y., Rahman, M.M., Haneda, M., et al., 2020. HNF1α controls glucagon secretion in
pancreatic α-cells through modulation of SGLT1. Biochim. Biophys. Acta (BBA) – Mol. Basis Dis. 1866, 165898. https://doi.org/10.1016/j.bbadis.2020.165898.
Shahbazian, M.D., Grunstein, M., 2007. Functions of site-specific histone acetylation and deacetylation. Annu. Rev. Biochem. 76, 75–100. https://doi.org/10.1146/annurev. biochem.76.052705.162114.
Sheikh, B.N., Guhathakurta, S., Tsang, T.H., et al., 2020. Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of

vasculature. Nat. Cell Biol. 22, 828–841. https://doi.org/10.1038/s41556-020- 0526-8.
Singer, R.A., Arnes, L., Cui, Y., et al., 2019. The long noncoding RNA paupar modulates PAX6 regulatory activities to promote alpha cell development and function. Cell Metabol. 30, 1091–1106. https://doi.org/10.1016/j.cmet.2019.09.013 e8.
Singh, M., Bacolla, A., Chaudhary, S., et al., 2020. Histone acetyltransferase MOF orchestrates outcomes at the crossroad of oncogenesis, DNA damage response, proliferation, and stem cell development. Mol. Cell Biol. 40 https://doi.org/ 10.1128/MCB.00232-20 e00232-20.
Taipale, M., Rea, S., Richter, K., et al., 2005. hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells. Mol. Cell Biol. 25, 6798–6810. https://doi.org/10.1128/MCB.25.15.6798-6810.2005.
Traub, S., Meier, D.T., Schulze, F., et al., 2017. Pancreatic α cell-derived glucagon-related
peptides are required for β cell adaptation and glucose homeostasis. Cell Rep. 18, 3192–3203. https://doi.org/10.1016/j.celrep.2017.03.005.
Valencia-Sa´nchez, M.I., De Ioannes, P., Wang, M., et al., 2021. Regulation of the Dot1 histone H3K79 methyltransferase by histone H4K16 acetylation. Science 371, eabc6663. https://doi.org/10.1126/science.abc6663.
Wang, L., Qing, L., Liu, H., et al., 2017. Mesenchymal stromal cells ameliorate oxidative stress-induced islet endothelium apoptosis and functional impairment via Wnt4-
β-catenin signaling. Stem Cell Res. Ther. 8, 188. https://doi.org/10.1186/s13287- 017-0640-0.
Whalley, N.M., Pritchard, L.E., Smith, D.M., et al., 2011. Processing of proglucagon to GLP-1 in pancreatic α-cells: is this a paracrine mechanism enabling GLP-1 to act on β-cells? J. Endocrinol. 211, 99–106. https://doi.org/10.1530/JOE-11-0094.
Wideman, R.D., Covey, S.D., Webb, G.C., et al., 2007. A switch from prohormone convertase (PC)-2 to PC1/3 expression in transplanted alpha-cells is accompanied by differential processing of proglucagon and improved glucose homeostasis in mice. Diabetes 56, 2744–2752. https://doi.org/10.2337/db07-0563.
Wideman, R.D., Gray, S.L., Covey, S.D., et al., 2009. Transplantation of PC1/3-Expressing alpha-cells improves glucose handling and cold tolerance in leptin-resistant mice. Mol. Ther. 17, 191–198. https://doi.org/10.1038/mt.2008.219.
Wu, L., Li, L., Zhou, B., et al., 2014. H2B ubiquitylation promotes RNA Pol II processivity via PAF1 and pTEFb. Mol. Cell. 54, 920–931. https://doi.org/10.1016/j. molcel.2014.04.013.
Zhao, X., Su, J., Wang, F., et al., 2013. Crosstalk between NSL histone acetyltransferase and MLL/SET complexes: NSL complex functions in promoting histone H3K4 di- methylation activity by MLL/SET complexes. PLoS Genet. 9, e1003940 https://doi. org/10.1371/journal.pgen.1003940.