Am J Physiol Endocrinol Metab 310: E91–E102, 2016.
First published November 10, 2015; doi:10.1152/ajpendo.00285.2015.
The MAFB transcription factor impacts islet ␣-cell function in rodents
and represents a unique signature of primate islet ␤-cells
Elizabeth Conrad,1 Chunhua Dai,2 Jason Spaeth,1 Min Guo,1 Holly A. Cyphert,1 David Scoville,1
Julie Carroll,3 Wei-Ming Yu,4 Lisa V. Goodrich,4 David M. Harlan,5 Kevin L. Grove,3
Charles T. Roberts, Jr.,3 Alvin C. Powers,1,2,6 Guoqiang Gu,7 and Roland Stein1
1
Submitted 19 June 2015; accepted in final form 21 October 2015
Conrad E, Dai C, Spaeth J, Guo M, Cyphert HA, Scoville D,
Carroll J, Yu W, Goodrich LV, Harlan DM, Grove KL, Roberts
CT Jr, Powers AC, Gu G, Stein R. The MAFB transcription factor
impacts islet ␣-cell function in rodents and represents a unique
signature of primate islet ␤-cells. Am J Physiol Endocrinol Metab
310: E91–E102, 2016. First published November 10, 2015;
doi:10.1152/ajpendo.00285.2015.—Analysis of MafB⫺/⫺ mice has
suggested that the MAFB transcription factor was essential to islet ␣and ␤-cell formation during development, although the postnatal
physiological impact could not be studied here because these mutants
died due to problems in neural development. Pancreas-wide mutant
mice were generated to compare the postnatal significance of MafB
(MafB⌬panc) and MafA/B (MafAB⌬panc) with deficiencies associated
with the related ␤-cell-enriched MafA mutant (MafA⌬panc). Insulin⫹
cell production and ␤-cell activity were merely delayed in MafB⌬panc
islets until MafA was comprehensively expressed in this cell population. We propose that MafA compensates for the absence of MafB in
MafB⌬panc mice, which is supported by the death of MafAB⌬panc mice
soon after birth from hyperglycemia. However, glucose-induced glucagon secretion was compromised in adult MafB⌬panc islet ␣-cells.
Based upon these results, we conclude that MafB is only essential to
islet ␣-cell activity and not ␤-cell. Interestingly, a notable difference
between mice and humans is that MAFB is coexpressed with MAFA
in adult human islet ␤-cells. Here, we show that nonhuman primate
(NHP) islet ␣- and ␤-cells also produce MAFB, implying that MAFB
represents a unique signature and likely important regulator of the
primate islet ␤-cell.
diabetes; transcription factor; nonhuman primate; islet; ␣-cell
␣- AND ␤-CELLS are major hormone-producing cell
types of the islets of Langerhans. The hormones secreted by
these cells act in a counterregulatory manner to control the
glucose concentration in the bloodstream, with ␣-cell-produced glucagon raising and ␤-cell-produced insulin lowering
levels. Loss or dysfunction of islet ␤-cells leads to insulin
insufficiency and hyperglycemia in type 1 (T1DM) and type
2 diabetes mellitus (T2DM) (44). The healthcare costs
associated with diabetes care are enormous and rising, and
consequently, efforts are focused on developing therapies to
minimize these complications, including developing ␤-like
PANCREATIC
Address for reprint requests and other correspondence: R. Stein, Dept. of
Molecular Physiology and Biophysics, Vanderbilt University Medical School,
723 Light Hall, Nashville, TN 37232 (e-mail: [email protected]).
http://www.ajpendo.org
cells from human embryonic stem cells or induced-pluripotent stem cells (26, 33, 35).
Identifying the key factors that mediate islet ␣- and ␤-cell
formation is essential to understanding T1DM and T2DM
disease processes and for improving cell-based interventions.
Notably, many transcription factors serve as critical regulators
of islet cell development and function (32), with alterations in
their activity contributing to ␣- and ␤-cell dysfunction in
T2DM (19). For example, pancreatic duodenal homeobox-1
(Pdx1) is expressed in all pancreatic progenitors before being
restricted to islet ␤-cells. Mice (2, 25, 30) and humans (42)
completely lacking this transcription factor fail to develop a
pancreas, whereas cell identity is severely compromised upon
conditional deletion from adult mouse islet ␤-cells (2). Ngn3,
which is produced later during embryogenesis, is essential for
the differentiation of endocrine progenitor cells into each of the
hormone⫹ cell subtypes [i.e., ␤- and ␣-cells, ␦-cells (which
secrete somatostatin), pancreatic polypeptide cells (which secrete pancreatic polypeptide), and ε-cells (which secrete ghrelin)] (16, 17). MafA and MafB are unusual in not being
expressed until the onset of hormone⫹ cell formation (21), with
MafA exclusively in insulin⫹ cells (27, 29) and MafB also in
glucagon⫹ cells (6).
MAFA, MAFB, and PDX1 levels were specifically decreased in human T2DM islet ␣- and ␤-cells, and these factors
have been proposed to be major contributors to islet cell
dysfunction (19). Interestingly, the combination of Pdx1,
Ngn3, and MafA was sufficient to convert nonendocrine cells
to ␤-like cells in mice (12, 47), whereas adenovirus-driven
expression of MafA was able to induce glucose-stimulated
insulin secretion (GSIS) in neonatal rat islets (1). Collectively,
these data not only illustrate the stage-specific activities of key
islet transcription factors but also provide support for MafA
and MafB contributing to events involved in ␤-cell formation.
MafB⫺/⫺ pancreata exhibited an ⬃50% loss of insulin⫹ and
glucagon⫹ cells by embryonic day (E)15.5, whereas expression of key ␤-cell-enriched transcription factors was reduced
by E18.5 (e.g., Pdx1, Nkx6.1). Islet ␤-cell-specific MafA
production correlated with insulin⫹ cell formation in the
MafB⫺/⫺ animals, and there was no reduction in total endocrine cell number (3). Although these results strongly suggested that MafB was a key islet cell regulator in vivo, this
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Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee; 2Division
of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University School of Medicine, Nashville,
Tennessee; 3Division of Diabetes, Obesity, and Metabolism, Oregon National Primate Research Center, Oregon Health and
Science University, Beaverton, Oregon; 4Department of Neurobiology, Harvard Medical School, Boston, Massachusetts;
5
Department of Medicine, University of Massachusetts, Worcester, Massachusetts; 6Veterans Affairs Tennessee Valley
Healthcare System, Nashville, Tennessee; and 7Department of Cell and Developmental Biology, Vanderbilt University
Medical Center, Nashville, Tennessee
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CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
MATERIALS AND METHODS
Generation of the pancreas-specific and endocrine-specific knockout mice. Pancreas-wide deletion mutants of MafA and MafB were
generated by crossing MafAfl/fl (5) and MafBfl/fl (45) mice with
Pdx15.5-Cre mice (17), which produces Cre in pancreatic progenitor
cells prior to MafA and MafB expression. MafAfl/fl;MafBfl/fl;Pdx15.5Cre and MafBfl/fl;Pdx15.5-Cre were referred to as MafAB⌬panc and
MafB⌬panc mice, respectively. MafABfl/fl and MafBfl/fl mice were used
as controls. Pancreatic endocrine cell-specific MafA and MafB deletion mutant mice were generated with neurogenin 3 (Ngn3)-Cre mice
(39), referred to as MafAB⌬endo. Chi2 analysis of expected vs. actual
genotypes at time of genotyping was performed on MafAB⌬panc and
MafAB⌬endo litters. For embryonic samples, day 0.5 was counted as
the day the vaginal plug was observed. All studies with mice were in
compliance with protocols approved by the Vanderbilt Institutional
Animal Care and Use Committee.
Islet isolation conditions. Two-week-old mouse pancreata were
partially digested with 1 mg/ml collagenase, followed by handpicking
of islets; adult islets were collected as described previously (22). NHP
(rhesus macaque) pancreata were obtained from five females and five
males (average age 8.8 ⫾ 1.3 yr, range 0.32–13 yr) as excess material
under unrelated protocols approved by the Oregon National Primate
Research Center Institutional Animal Care and Use Committee. Islet
isolation was initiated within 10 –15 min of exsanguination by cannulation and perfusion through the pancreatic duct with collagenase/
neutral protease solution in a Ricordi apparatus. When islet release
was evident by dithizone staining, islets in digestion solution were
concentrated and washed by centrifugation and purified using a COBE
2991 cell processor. Human islets were provided by the Integrated
Islet Distribution Network [http://iidp.coh.org/; 30 total preparations,
11 female and 19 male donors, age 38.4 ⫾ 2.4 yr (range: 17– 60), BMI
25.99 ⫾ 0.55 kg/m2 (range: 18.8 –29.7)]. Cause of death was head
trauma (n ⫽ 11), neurological events (stroke, subarachnoid hemorrhage, etc.; n ⫽ 10), anoxia (n ⫽ 2), or unknown (n ⫽ 7). The cold
ischemia time before pancreas isolation was 9.9 ⫾ 1.1 h (range:
1.5–24.9 h). Human and NHP islets were handpicked on the day of
arrival as described (14). All studies with human and NHP islets were
in compliance with the Vanderbilt Institutional Animal Care and
Review Board Committee.
RNA analysis. Quantitative real-time PCR was performed on RNA
isolated from NHP, human, and mouse (e.g., C57BL/6J) islets (14) as
well as MafB⌬panc and MafBfl/fl islets (22), using previously described
conditions. Preloaded arrays (Applied Biosystems) of 16 genes were
used to determine expression levels in NHP, human, and mouse islets
in Fig. 7, with the 18S/18s, ACTB/ActB, TFRC/Tfrc, and TBP/Tbp
genes used for normalization. Primer sequences are available upon
request. Primer efficiency was similar between species as guaranteed
by the manufacturer and verified empirically. For assessing the data,
the Minimum Information for Publication of Quantitative Real-Time
PCR Experiments was followed (10).
Tissue collection and immunohistochemistry. The paraformaldehyde-embedding conditions for NHP, human, and mouse islets (14) as
well as mouse pancreata (22) have been described. Primary antibodies
utilized for mouse sections were as follows: insulin (guinea pig;
Dako), glucagon (mouse; Sigma), glucagon (rabbit; Linco), MafB
(rabbit; Bethyl), MafA (rabbit; Novus), Pax6 (rabbit; Covance), Pdx1
(goat; provided by Chris Wright, Vanderbilt), Nkx6.1 (rabbit; ␤-Cell
Biology Consortium), Slc30A8 (rabbit; Pierce), Glut2 (goat; Santa
Cruz Biotechnology), ghrelin (goat; Santa Cruz Biotechnology), Ki67, and (mouse; BD Pharmingen). Primary antibodies used for NHP
and human sections were as follows: insulin (guinea pig; Dako or
Linco), glucagon (mouse; Sigma), glucagon (rabbit; Cell Signaling
Technology), MafB (rabbit; Novus or Bethyl), MafA (rabbit; Novus),
and somatostatin (goat; Santa Cruz Biotechnology). Briefly, the primary antibody-antigen complex was visualized on 6-␮m sections by
immunofluorescence using secondary antibodies conjugated with
Cy2, Cy3, or Cy5 fluorophores (1:500; Jackson ImmunoResearch,
West Grove, PA). Nuclear costaining was conducted with DAPI
Fluoromount G (Southern Biotech). Immunofluorescent images were
acquired with a Zeiss LSM510 confocal microscope or a Zeiss
Axioimager M2 microscope.
Islet cell population analysis. Mouse pancreatic sections at 133-,
288-, and 354-␮m spacing were prepared from E15.5, postnatal
day 1 (P1), and 2-wk-old samples, respectively. Insulin⫹, glucagon⫹, Pax6⫹, Pdx1⫹, and Nkx6.1⫹ cell images were counted
manually and divided by the total number of pancreatic DAPI⫹
nuclei. At least 10,000, 20,000, and 100,000 pancreatic nuclei were
counted for E15.5, P1, and 2-wk-old samples, respectively. The
percentage of MafA⫹insulin⫹ and Ki-67⫹insulin⫹ cells was de-
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could not be assessed postnatally since MafB⫺/⫺ mice die at
birth due to renal failure and central apnea (7, 37). In contrast
to MafB⫺/⫺ mice, there was no impact on endocrine cell
formation in null [MafA ⫺/⫺ (46)], pancreas-specific
[MafA⌬panc (22)], or ␤-cell-specific [MafA⌬␤ (41)] MafAknockout mice. However, these MafA mutants postnatally
develop glucose intolerance, changes in islet cell architecture,
reduced ␤-cell mass, and compromised ␤-cell gene expression
(5, 22, 41, 46). Because MafB is not produced in rodent ␤-cells
after birth (6), these results indicated that MafB was necessary
for ␤-cell development, and MafA was essential to adult islet
␤-cell function.
In contrast to mouse ␤-cells, MAFB is coexpressed with
MAFA in adult human islet ␤-cells (14). However, the expression and distribution of other islet-enriched transcription factors (i.e., PDX1, NKX6.1) are similar between rodents and
humans (14). Because islet-enriched transcription factors profoundly influence mouse islet cell function and identity, the
distinctive human MAFB expression pattern could be contributing to dissimilarities in islet cell characteristics between
humans and mice (14). This hypothesis derives from the
biochemical and functional differences reported for the MafA
and MafB dimeric activators (20). For example, misexpression
of MafA in a mouse islet ␣-cell line activated insulin gene
expression (4), whereas MafB induced glucagon in a mouse
␤-cell line (6). Similarly, only MafA stimulated insulin production in chick in ovo electroporation assays, although exchanging the MafB COOH-terminal DNA-binding dimerization (basic leucine zipper) spanning region with that of MafA
enabled insulin induction (4).
Here, we produced pancreas-wide deletion mutants of MafB
(MafB⌬panc) and MafA/B (MafAB⌬panc) to compare the postnatal contributions of these transcription factors to islet cell
formation and function. Elevated blood glucose levels were
observed just after birth in MafB⌬panc mice, which soon resolved upon comprehensive production of MafA within the
insulin⫹ cell population. In addition, there was a profound
reduction in glucagon secretion levels from adult islet ␣-cells.
In contrast, MafAB⌬panc mice died from hyperglycemia shortly
after birth due to loss of insulin⫹ cells. These results demonstrated that MafB primarily affects mouse islet ␣-cell function
and not ␤-cell activity. Notably, MAFB was coexpressed with
MAFA in nonhuman primate (NHP) islet ␤-cells, suggesting
that this factor imparts distinct control properties in primates.
The significance of MAFB to primate ␤-cells is supported by
the recent observation showing that knockdown of this transcription factor suppressed GSIS in the human EndoC-␤H1
␤-cell line (41).
CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
cell content hormone levels were determined by ELISA (insulin,
ALPCO; glucagon, RayBiotech). Hormone secretion was expressed as the percentage of total hormone content. The NHP,
human, and mouse perifusion studies were conducted in the Vanderbilt Islet Isolation and Analysis Core under standard conditions
(14), using as baseline 5.6 mM glucose and 16.7 mM glucose alone
or with 500 ␮M 3-isobutyl-1-methylxanthine (IBMX) for stimulating insulin secretion.
Statistics. Statistical significance was determined by one-way
ANOVA between NHP, human, and mouse samples. The data with
MafAB⌬panc and MafB⌬panc mice were presented as means ⫾ SE,
and statistical significance assessed by comparing data points
between mutants and controls using the Student two-tailed unpaired t-test.
RESULTS
Loss of MafB alone results in delayed ␤-cell maturation.
The role of MafB in postnatal ␣- and ␤-cells remains
unclear, since MafB⫺/⫺ mice die at birth (37). As a result,
floxed MafB mice were crossed with early pancreas-driven
Pdx1-Cre (MafB⌬panc) transgenic mice to determine the
specific influence on postnatal islet cells. MafB was effectively removed from MafB⌬panc ␣- and ␤-cells by E15.5
Fig. 1. Postnatal ␤-cell maturation is delayed in MafB⌬panc mice. A: postnatal day 1 (P1) and 2-wk-old random blood glucose measurements in MafB⌬panc and
control MafBfl/fl littermates (n ⫽ 12–14). B: reduction in embryonic day (E)15.5 MafB⌬panc insulin⫹ and glucagon⫹ cell numbers is ameliorated within 2 wk after
birth. Approximately 10,000 and 100,000 pancreatic nuclei were counted at E15.5 and 2 wk, respectively (n ⫽ 3). C: MafB⌬panc animals exhibit normal glucose
tolerance by 3 wk. Glucose tolerance tests were performed at 3, 6, and 8 wk (n ⫽ 11–21). Statistical significance between MafBfl/fl and MafB⌬panc was analyzed
by Student’s t-test. *P ⬍ 0.05 and **P ⬍ 0.01 compared with MafBfl/fl.
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termined by dividing the number of copositive cells by the total
count of insulin⫹ cells for MafA at P1 and 2 wk and for Ki-67 at
P1. The percentage of Pdx1⫹insulin⫺ cells was determined by
dividing the number of Pdx1⫹insulin⫺ cells by total Pdx1⫹ cells at
P1 and 2 wk. Cell counting was performed blinded to prevent bias.
Intraperitoneal glucose tolerance test. Three-, six-, and eight-wkold mice (n ⱖ 7) were fasted for 6 h, and blood glucose level from tail
blood was determined using a FreeStyle glucometer (Abbott Diabetes
Care). The mice were then weighed, and 2 mg dextrose/g body wt
(Fisher Biotech) in sterile PBS was injected intraperitoneally. Blood
glucose levels were measured at 0, 15, 30, 60, 90, and 120 min
postinjection. Fed blood glucose levels were determined prior to
fasting.
Stimulated hormone secretion. Handpicked islets from 2- and
8-wk-old MafB⌬panc and control MafBfl/fl mice were cultured overnight in RPMI 1640, 10% FBS, and penicillin-streptomycin supplemented with 5.6 (for insulin secretion) or 11 mM D-glucose (for
glucagon secretion). The next day, islets were subjected to hormone secretion conditions in KRBH buffer (1.25 mM CaCl2, 0.6
mM MgS04, 0.6 mM KH2PO4, 2.4 mM KCl, 64.0 mM NaCl,
20 mM HEPES, pH 7.9, and 5 mM NaHCO3) with either 2.8 or
16.7 mM glucose for insulin secretion and 1 mM glucose or 1 mM
glucose plus 10 mM arginine for glucagon secretion. Following 45
min of stimulation at 37°C, secretion media were collected and
islets lysed in 1.5% HCl and 70% ethanol. Secreted (media) and
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CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
(i.e., 3.65 ⫾ 2.15% remains MafB⫹insulin⫹ and 6.62 ⫾
1.63% remains MafB⫹glucagon⫹), and there was no effect
on mortality. However, MafB⌬panc mice were mildly hyperglycemic at P1, a situation that resolved by 2 wk (Fig. 1A).
Hormone⫹ and pan-endocrine Pax6⫹ cell numbers were
quantified at E15.5 and 2 wk to determine whether insulin⫹
and/or glucagon⫹ cell production was impacted in
MafB⌬panc mice. Although there was no change in the Pax6
population that marks all islet cells, there was a significant
decrease in the insulin⫹ (⬃75%) and glucagon⫹ (⬃50%)
cell numbers at E15.5 (Fig. 1B). These results were expected
from the analysis of the MafB⫺/⫺ mutant (3).
Notably, MafB⌬panc insulin⫹ and glucagon⫹ cell numbers
were indistinguishable from wild-type littermate controls by
2 wk (Fig. 1B). However, the islet ␤-cell-enriched Pdx1
transcription factor was aberrantly detected in hormonenegative cells in earlier P1 MafB⌬panc samples, which was
resolved by 2 wk (Fig. 2, A and B). Since MafB is normally
produced in embryonic insulin⫹ cells prior to MafA (6, 27),
we examined whether MafA or another related large Maf
transcription factor, c-Maf (28, 29), could be acting in a
compensatory manner in MafB⌬panc mice. MafA was detected only in MafB⌬panc insulin⫹ cells (Fig. 2, A and C),
whereas c-Maf was undetectable in both mutant and control
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Fig. 2. Pancreatic duodenal homeobox-1
(Pdx1) is not produced in all newly produced
insulin⫹ cells, and Glut2 and Slc30a8 expression is also delayed in these cells. A:
white arrows denote the Pdx1⫹insulin⫺ nuclei and yellow arrows MafA⫺insulin⫹ nuclei in MafBfl/fl and MafB⌬panc islets. B–D:
percentage of Pdx1 ⫹ insulin ⫺ (B),
MafA⫹insulin⫺ (C), and Ki-67⫹insulin⫺
cells (D). There was no difference in the
proliferation rate of P1 MafB fl/fl and
MafB⌬panc ␤-cells. E: islet-enriched Slc30a8
and Glut2 levels are reduced on P1 and
normal by 2 wk in MafB⌬panc islets; Glut2
(white) and Slc30a8 (red). Dashed yellow
lines mark the endocrine⫹ cells. Scale bars,
10 ␮m. The same confocal settings were
used to produce these images.
CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
samples (data not shown). Moreover, the inability to detect
a change in the Ki-67⫹ cell numbers between MafB⌬panc and
MafBfl/fl insulin⫹ cells at P1 suggests that enhanced proliferation did not lead to recovery of the ␤-cell population
(Fig. 2D). Considering the importance of MafA to mouse
␤-cell function after birth (22, 46), we conclude that MafA
compensates for MafB in MafB⌬panc mice, with the later
expression of MafA influencing the delayed production of
insulin⫹ cell numbers and ␤-cell function. In support of this
conclusion, the expression levels of two other known MafAand MafB-activated gene targets, Slc30a8 and Glut2 (5, 22),
were also only transiently compromised in neonatal
MafB⌬panc islets (Fig. 2E).
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Our results indicate that embryonic loss of MafB delays
islet ␤-cell maturation. Consequently, we analyzed whether
GSIS was compromised in 2-wk-old compared with 8-wkold MafB⌬panc islets (Fig. 3A), when essentially no MafB⫹
␤- or ␣-cells were present (data not shown). Insulin secretion was slightly elevated under nonstimulating 2.8 mM
glucose conditions in 2-wk-old MafB⌬panc islets, a basal
secretion pattern of immature ␤-cells (8, 24, 36).
Conversely, the insulin secretion profile was normal in
8-wk-old MafB⌬panc islets. MafB⌬panc and control mice also
had indistinguishable blood glucose tolerance profiles at 3,
6, and 8 wk (Fig. 1C). In addition, the expression levels of
a variety of MafA- and MafB-regulated genes were similar
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Fig. 3. Glucagon secretion is compromised
in adult MafB⌬panc islets but not insulin secretion. Static culture analysis of insulin (A)
and glucagon secretion (B) in MafB⌬panc
(black bars) and MafBfl/fl (open bars) islets.
In B, the ratio of 1 mM glucose to 1 mM
glucose ⫹ 10 mM Arg is 3.5 in MafBfl/fl and
1.6 in MafB⌬panc. *P ⬍ 0.05, ***P ⬍ 0.001,
and ****P ⬍ 0.0001, basal vs. stimulating
conditions (n ⫽ 4 – 6). C: glucagon and
ghrelin mRNA levels are selectively reduced
in 8-wk-old MafB⌬panc islet ␣-cells (n ⱖ 4).
In contrast, many other ␣-cell, ␤-cell, and
islet control factors are unchanged. *P ⬍
0.05 compared with MafBfl/fl controls.
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CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
was altered. Both glucagon content (Fig. 3B) and ␣-cell
numbers (Fig. 1B) were unchanged from controls, although
glucagon secretion was reduced in MafB⌬panc islets upon
exposure to a stimulating low glucose concentration and the
potent secretagogue, arginine (Fig. 3B). There were no
changes in islet ␣-cell-enriched transcription factors [i.e.,
Arx (13) and Brn4 (22a)] or several other ␣-cell-enriched
gene products in MafB⌬panc islets [i.e., HigD1a, Ndst4,
Pdk4, and Ptprd (15); Fig. 3C]. These analyses demonstrate
that MafB controls postnatal islet ␣-cell function, which is
quite interesting considering that MAFB levels are reduced
in T2DM islet ␣-cells and glucagon secretion is dysregulated in this state (19, 43).
Loss of MafA and MafB in islet cells results in death soon
after birth due to hyperglycemia. Floxed MafA and MafB
mice were crossed with early pancreas-driven Pdx1-Cre
(MafAB⌬panc) transgenic mice to compare their phenotype to
the individual mutants. In contrast to MafA⌬panc (5) or
MafB⌬panc mutants, no viable MafAB⌬panc mice were found
within 2.5 wk of birth (Table 1), although these mice were
born at the predicted frequencies (data not shown). This
same property was found in MafAB⌬endo animals, where
expression of Ngn3-driven Cre removes each transcription
factor gene prior to expression in embryonic islet cell
Fig. 4. MafB is expressed in mouse P1 islet ghrelin⫹
ε-cells, although there is no apparent change in the P1
MafB⌬panc ghrelin⫹ cell population. A: representative
images of P1 MafBfl/fl islets stained for ghrelin (white),
MafB (red), and insulin (green). White arrows denote
MafB⫹ghrelin⫹ cells. B: P1 MafBfl/fl and MafB⌬panc
islets stained for insulin (green), glucagon (blue), and
ghrelin (white). Scale bars, 20 ␮m.
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to controls in 8-wk-old MafB⌬panc islets, including insulin,
Nkx6.1, Glut2, Pdx1, and Slc30a8 (Fig. 3C).
MafB is important to ␧-cell ghrelin expression. Interestingly, glucagon and ghrelin mRNA levels were decreased
significantly in 8-wk-old MafB⌬panc islets, whereas somatostatin, pancreatic polypeptide, and insulin levels were
similar to controls (Fig. 3C). The impact on glucagon
expression was not surprising, since MafB binds to and
activates at the G1 element found between ⫺71 and ⫺55
base pairs relative to the transcription start site (6). In
contrast, it is unclear how MafB regulates ghrelin expression. Unfortunately, we could not determine whether there
was a change in ghrelin staining levels due to the very few
ε-cells in adult MafBfl/fl or MafB⌬panc islets. Immunostaining
confirmed that MafB is present in wild-type ghrelin⫹ cells at
P1 (Fig. 4), when ε-cells make up a small but larger
proportion of the islet cell population than in the adult (34).
There was no obvious change in the P1 islet ghrelin
cell population (Fig. 4). Interestingly, MafB expression was
observed in the gut but not in ghrelin⫹ cells (Fig. 5), the
principal location of this hormone-expressing population.
MafB is important to islet ␣-cell function. Glucose-stimulated glucagon secretion was measured in 8-wk-old
MafB⌬panc islets to determine whether secretion capacity
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CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
Fig. 5. MafB is produced in the developing
gut, albeit not in gut ghrelin⫹ cells. A representative image of P1 wild-type mouse
duodenum; MafB (red), ghrelin (white), and
DAPI (blue). White arrow denotes a
ghrelin⫹MafB⫺ cell. Scale bar, 20 ␮m.
Table 1. MafAB⌬panc and MafAB⌬endo mice die soon after birth
Potential Genotypes
Mendelian Ratio
fl/fl
MafAB⌬panc
MafA⌬panc/⫹ B⌬panc or MafA⌬panc B⌬panc/⫹
MafA⌬panc/⫹ MafB⌬panc/⫹
Pdx1-Cre⫺
Total
⌬endo
MafAB
MafA⌬endo/⫹ MafB⌬endo
MafAfl/⫹ Bfl/fl
MafAfl/fl Bfl/fl
Total
No. of Pups Expected (Out of 54)
fl/fl
MafA MafB
0.125
0.25
0.1
0.50
⫻ MafA
⌬panc/⫹
Actual No. of Pups
Actual Ratio
⌬panc/⫹
MafB
6.75
13.50
6.75
27.00
MafAfl/flMafBfl/fl ⫻ MafA⌬endo/⫹MafB⌬endo
0.25
14.75
0.25
14.75
0.25
14.75
0.25
14.75
0*
16
12
26
54
0
0.30
0.22
0.48
0***
21
18
20
59
0
0.36
0.31
0.34
The expected and actual no. of animals obtained from the MafAfl/flMafBfl/fl ⫻ MafA⌬panc/⫹MafB⌬panc/⫹ mating or MafAfl/flMafBfl/fl ⫻ MafA⌬endo/⫹MafB⌬endo
mating at 2.5 wk. *P ⫽ 0.0438 for MafAB⌬panc; ***P ⫽ 0.000171 MafAB⌬endo.
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␤-cell formation but that MafB alone (unlike MafA) has little
(if any) impact on mouse ␤-cells.
MAFB is expressed in NHP islet ␤-cells, with islet architecture and cell function analogous to human. Human islets are
dissimilar to mice in several ways, including the presence of
MAFB in human ␤-cells, GSIS properties, and islet cell composition and distribution (9, 11, 14). We next determined
whether these islet properties were shared with NHPs. Like
humans, MAFB was present in ␤-cells as well as ␣-cells (Fig.
7, A and B). MAFB mRNA expression was relatively high in
NHP and human islets compared with mice, whereas NHP
MAFA was present at a level similar to mice (Fig. 7A). PDX1
and pan-endocrine NEUROD1 transcription factor levels were
comparable across species.
The relative mRNA levels of NHP INSULIN, GLUCAGON, and SOMATOSTATIN were similar to human islet
levels (Fig. 7D). The differences in INSULIN, GLUCAGON,
and ␣-cell-enriched ARX (Fig. 7A) transcription factor levels with mice closely correlated with the increased proportion of ␣-cells in human and NHP islets, with fewer ␤-cells
and more ␣-cells in primates (Fig. 7E) (9). Moreover, the
distribution of human (9) and NHP ␣-, ␤-, and ␦-cells was
progenitors (Table 1). The blood glucose levels in
MafAB⌬panc P1 mice were extremely high (Fig. 6A),
strongly indicating that deficiencies in ␤-cell mass and/or
function led to their early death. In addition, blood glucose
levels were elevated in MafA⌬pancMafB⌬panc/⫹ and
MafA⌬panc/⫹MafB⌬panc mice.
To investigate how insulin⫹ and glucagon⫹ cell formation
was affected in MafAB⌬panc mice, the number of hormone⫹
and Pax6⫹ cells was quantified at E15.5 and P1. The
MafAB⌬panc insulin⫹ and glucagon⫹ cell population was significantly reduced (Fig. 6B), whereas total Pax6⫹ endocrine
cell numbers were unchanged. Note that very few Pax6⫹ cells
produce either insulin or glucagon in MafAB⌬panc islets (i.e.,
⬃75% change in hormone⫹ cell numbers; Fig. 6, B and C).
However, ␤-cell-enriched Nkx6.1⫹ and Pdx1⫹ cell numbers
were decreased by only ⬃25% (Fig. 6D). These results suggest
that MafA and MafB are the principal drivers of insulin gene
expression and not Nkx6.1 (38) and Pdx1 (31). The overall
developmental impact on hormone and transcription factor
levels was comparable between MafAB⌬panc and MafB⫺/⫺ (3)
mice. Collectively, these data demonstrate not only that the
combined actions of MafA and MafB are required for postnatal
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CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
scattered throughout the islet, which was different from
mouse islets where the ␣- and ␦-cells surround the ␤-cellrich core (Fig. 7E). Insulin secretion from NHP and human
islets was indistinguishable at 5.6, 16.7, and 16.7 mmol/l
glucose plus IBMX and significantly different from mouse
islets (Fig. 8). The higher basal and lower insulin secretion
response to high glucose is a defining feature of human and
NHP islets. These results suggest that MAFB expression in
islet ␤-cells represents a unique feature of the primate islet
and is consequential to cell function.
DISCUSSION
Mouse MafA and/or MafB were removed specifically in
early pancreatic progenitor cells to obtain insight into the
contribution of MafB individually and MafA and MafB in
combination with postnatal islet ␤- and ␣-cells. Only transient
deficiencies in MafB⌬panc ␤-cell activity were found, although
adult islet ␣-cell glucagon secretion levels as well as ghrelin
mRNA were compromised. Compensation by MafA likely
explains the limited effect on MafB⌬panc ␤-cells, which is
supported by the tight expression linkage with mutant insulin⫹
cells (Fig. 2). This proposal is also consistent with the substantially reduced islet insulin⫹ cell population in MafAB⌬panc
islets, which caused overt hyperglycemia and death. We conclude that MafB is essential only to postnatal mouse ␣-cell
activity. This seems paradoxical since MAFB in human islet
␤-cells represents a unique transcription factor signature. Here
we show that NHP islet ␤-cells also produce MAFB, and islet
cell composition and ␤-cell function are essentially equivalent
to that of humans.
MafB was broadly expressed in the P1 duodenum (Fig. 5),
wherein a variety of metabolically interesting hormones are
produced [e.g., cholecystokinin (CCK), gastric inhibitory
peptide (GIP), glucagon-like peptide-1 (GLP-1), and ghrelin] (18). Notably, MafB was not expressed in ghrelin⫹ cells
in the duodenum, presumably because these cells are not
derived from Ngn3⫹ progenitors (23), as in the pancreas.
With this consideration, it will be of interest to determine
whether MafB is necessary for regulating Ngn3-derived
CCK, GIP, and/or GLP-1 expression, with positive results
supporting a wider role for MafB in defining cell identity
decisions.
Notably, whereas MafA and MafB influence transcription of
genes associated with glucose sensing and hormone secretion
in mouse islet ␣- and ␤-cells (5, 22), both have distinct effects
on cell activity (1, 5) and hormone gene expression (4). For
example, induction of glucose-responsive insulin secretion is
obtained upon misexpression of MafA in neonatal rat islets,
with high MafB expression associated with immature rodent
GSIS properties (1). Amino acid differences within the NH2-
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Fig. 6. MafAB⌬panc mice have profoundly reduced insulin⫹ and glucagon⫹ cell numbers. A: neonatal MafAB⌬panc animals are severely hyperglycemic at P1.
Statistical significance between MafAfl/flMafBfl/fl and MafA⌬pancMafB⌬panc/⫹, MafA⌬panc/⫹MafB⌬panc, or MafAB⌬panc is shown. *P ⬍ 0.05, **P ⬍ 0.005, and ***P ⬍
0.0001. B: insulin⫹ and glucagon⫹ cell numbers are decreased in MafAB⌬panc animals (n ⫽ 3). *P ⬍ 0.05 and **P ⬍ 0.01 compared with MafABfl/fl controls. C: most
MafAB⌬panc Pax6⫹ cells do not produce insulin at P1; insulin (Ins; green), glucagon (Gluc; white), and Pax6 (red). Scale bars, 10 ␮m. D: MafAB⌬panc animals have fewer
␤-cell-enriched Pdx1⫹ and Nkx6.1⫹ cells (n ⫽ 3). Approximately 10,000 and 20,000 pancreatic nuclei were counted at E15.5 and P1, respectively. **P ⬍ 0.01 compared
with MafABfl/fl control.
CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
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Fig. 7. MAFB is expressed with MAFA in adult nonhuman primate (NHP) islet ␤-cells. A: relative gene expression of various transcription factors in NHP
(n ⫽ 5), human (n ⫽ 10), and mouse (C57BL/6J) (n ⫽ 6) islets. *P ⬍ 0.05 and ***P ⬍ 0.001, NHP and mouse vs. human; ⫹⫹P ⬍ 0.01 and ⫹⫹⫹P ⬍ 0.001,
mouse vs. NHP. The islet-enriched gene mRNA signal was normalized to the 18S, ACTB, TFRC, and TBP signal. B: representative images of a NHP islets; MAFB
(red), MAFA (red), Ins (green), and Gluc (blue). White arrows denote MAFB⫹INS⫹, blue arrows MAFB⫹GCG⫹, and yellow arrows MAFA⫹INS⫹ cells. Scale
bar, 10 ␮m. C: illustration of functional domains in human MAFA and MAFB. Protein alignment and %identity of human MAFA and MAFB was determined
using the EMBOSS Matcher tool. Amino acids of the transactivation domain are aligned below with residues that are common between the 2 proteins highlighted
in gray. Amino acids 76 –108 of human MAFA are not present in human MAFB. D: the endocrine hormone mRNA expression pattern is analogous between
human and NHP islets, whereas mouse is different. NHP (n ⫽ 5), human (n ⫽ 10), and mouse (C57BL/6J) (n ⫽ 6) islets. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001,
mouse vs. human; ⫹P ⬍ 0.05 and ⫹⫹P ⬍ 0.01, mouse vs. NHP. No significant difference seen between NHP and human (P ⬎ 0.05). E: representative image of a
NHP and mouse (C57BL/6J) islet; Ins (green), Gluc (red), and somatostatin (SOM, blue). Scale bars, 100 ␮m.
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CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
terminal transactivation domain and COOH-terminal spanning
basic leucine zipper DNA-binding/dimerization region likely
contribute to the distinct functions of each of these proteins
(see Fig. 7C). For example, exchanging the COOH-terminal
region of MafA with MafB enabled this chimera to activate
insulin levels in chick in ovo electroporation assays (4). Consequently, the increased level of MAFB in primate ␤-cells
could contribute to differences in insulin secretion levels in
rodents islets (Fig. 8) (14). Importantly, recent evidence suggests that both MAFA and MAFB mediate GSIS in human
␤-cells (41). Thus, insulin secretion was compromised upon
reducing the levels of either MAFA or MAFB in human
EndoC-␤H1 cells. Furthermore, candidate gene analysis demonstrated that several genes important to human ␤-cell activity
were regulated by MAFA and MAFB, but only MAFB activated SLC2A1 (GLUT1) levels, the primary glucose transporter
of human ␤-cells. Future efforts will be focused on obtaining a
better understanding of how MAFA and MAFB control human
islet ␤- and ␣-cell functions.
Our data suggest that reduced MAFB levels in islet ␣-cells
would contribute to the dysregulated glucagon secretion associated with T2DM patients (44). However, we predict that
T2DM ␣-cell dysfunction does not derive solely from MAFB
loss but rather from a compilation of changes that occur in the
islet populations, including counterregulatory insulin signaling
to the ␣-cell. It will be of value to explore the mechanism(s) by
which MAFB controls glucagon secretion and whether these
are dysregulated in diabetic islet ␣-cells.
GRANTS
This work was supported by grants from the National Institutes of Health
(RO1-DK-090570 to R. Stein; DK-66636, DK-68854, DK-72473, DK-89572,
and DK-089538 to A. C. Powers; T32-DK-007563 to J. Spaeth; R24-DK093437 to D. M. Harlan, K. L. Grove, C. T. Roberts, Jr., A. C. Powers, and R.
Stein; and P51-OD-0110921 to K. L. Grove and C. T. Roberts, Jr.) and the
Vanderbilt Diabetes Research and Training Center (DK-20593). This work
was also supported by a Merit Review Award from the Veterans Affairs
Research Service (BX000666 to A. C. Powers) and the March of Dimes
(1-FY08-381 to L. V. Goodrich) as well as grants from the Juvenile Diabetes
Research Foundation (JDRF; 26-2008-863 to A. C. Powers). Imaging was
performed with National Institutes of Health support from the Vanderbilt
University Medical Center Cell Imaging Shared Resource (CA-68485, DK20593, DK-58404, HD-15052, DK-59637, and EY-08126) and the Vanderbilt
University Medical Center Islet Procurement and Analysis Core (DK-20593).
Human islets were obtained from the Integrated Islet Distribution Program,
which is supported by the National Institute of Diabetes and Digestive and
Kidney Diseases and the JDRF.
DISCLOSURES
No potential conflicts of interest relevant to this article, financial or
otherwise, are reported.
AUTHOR CONTRIBUTIONS
E.C., C.D., J.S., H.A.C., D.S., J.C., W.-M.Y., and R.S. conception and design
of research; E.C., C.D., J.S., M.G., H.A.C., D.S., J.C., and W.-M.Y. performed
experiments; E.C., C.D., J.S., H.A.C., D.S., W.-M.Y., L.V.G., and D.M.H. analyzed data; E.C., J.S., H.A.C., D.S., W.-M.Y., L.V.G., K.L.G., C.T.R.J., A.C.P.,
G.G., and R.S. interpreted results of experiments; E.C. and J.S. prepared figures;
E.C., J.S., and R.S. drafted manuscript; E.C., J.S., H.A.C., D.M.H., K.L.G.,
C.T.R.J., A.C.P., G.G., and R.S. edited and revised manuscript; E.C., C.D., J.S.,
M.G., H.A.C., D.S., J.C., W.-M.Y., L.V.G., D.M.H., K.L.G., C.T.R.J., A.C.P.,
G.G., and R.S. approved final version of manuscript.
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Fig. 8. Insulin secretion properties of NHP and human islets are indistinguishable. A–C: glucose-stimulated insulin secretion characteristics of NHP (n ⫽ 9; A),
human (n ⫽ 30; B), and C57 mouse (n ⫽ 12; C) islets in perifusion assays. D: basal insulin secretion levels. E and F: area under the curve (AUC) analysis of
the 16.7 mM glucose (E) and 16.7 mM glucose ⫹ IBMX (F) data. ***P ⬍ 0.001, mouse vs. human; ⫹P ⬍ 0.05 and ⫹⫹⫹ P ⬍ 0.001, mouse vs. NHP. No
significant differences were found between NHP and human. G, glucose. IEQ, islet equivalent.
CHARACTERIZATION OF MAFB IN MOUSE AND PRIMATE ISLET CELLS
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