Fructose-1,6-bisphosphatase overexpression in pancreatic beta-cells results in reduced insulin secretion: a new mechanism for fat-induced impairment of beta-cell function.

Abstract

Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme that is upregulated in islets or pancreatic beta-cell lines exposed to high fat. However, whether specific beta-cell upregulation of FBPase can impair insulin secretory function is not known. The objective of this study therefore is to determine whether a specific increase in islet beta-cell FBPase can result in reduced glucose-mediated insulin secretion.
To test this hypothesis, we have generated three transgenic mouse lines overexpressing the human FBPase (huFBPase) gene specifically in pancreatic islet beta-cells. In addition, to investigate the biochemical mechanism by which elevated FBPase affects insulin secretion, we made two pancreatic beta-cell lines (MIN6) stably overexpressing huFBPase.
FBPase transgenic mice showed reduced insulin secretion in response to an intravenous glucose bolus. Compared with the untransfected parental MIN6, FBPase-overexpressing cells showed a decreased cell proliferation rate and significantly depressed glucose-induced insulin secretion. These defects were associated with a decrease in the rate of glucose utilization, resulting in reduced cellular ATP levels.
Taken together, these results suggest that upregulation of FBPase in pancreatic islet beta-cells, as occurs in states of lipid oversupply and type 2 diabetes, contributes to insulin secretory dysfunction.

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Fructose-1,6-Bisphosphatase Overexpression in
Pancreatic ?-Cells Results in Reduced Insulin Secretion
A New Mechanism for Fat-Induced Impairment of ?-Cell
Function
Melkam Kebede,1Jenny Favaloro,1Jenny E. Gunton,2,3D. Ross Laybutt,2Margaret Shaw,1
Nicole Wong,1Barbara C. Fam,1Kathryn Aston-Mourney,1Christian Rantzau,1Anthony Zulli,1
Joseph Proietto,1and Sofianos Andrikopoulos1
OBJECTIVE—Fructose-1,6-bisphosphatase (FBPase) is a glu-
coneogenic enzyme that is upregulated in islets or pancreatic
?-cell lines exposed to high fat. However, whether specific ?-cell
upregulation of FBPase can impair insulin secretory function is
not known. The objective of this study therefore is to determine
whether a specific increase in islet ?-cell FBPase can result in
reduced glucose-mediated insulin secretion.
RESEARCH DESIGN AND METHODS—To test this hypothe-
sis, we have generated three transgenic mouse lines overexpress-
ing the human FBPase (huFBPase) gene specifically in
pancreatic islet ?-cells. In addition, to investigate the biochemi-
cal mechanism by which elevated FBPase affects insulin secre-
tion, we made two pancreatic ?-cell lines (MIN6) stably
overexpressing huFBPase.
RESULTS—FBPase transgenic mice showed reduced insulin
secretion in response to an intravenous glucose bolus. Compared
with the untransfected parental MIN6, FBPase-overexpressing
cells showed a decreased cell proliferation rate and significantly
depressed glucose-induced insulin secretion. These defects were
associated with a decrease in the rate of glucose utilization,
resulting in reduced cellular ATP levels.
CONCLUSIONS—Taken together, these results suggest that
upregulation of FBPase in pancreatic islet ?-cells, as occurs in
states of lipid oversupply and type 2 diabetes, contributes to
insulin secretory dysfunction. Diabetes 57:1887–1895, 2008
T
cardiovascular disease and stroke. Although insulin resis-
tance may be the initiating defect, hyperglycemia in type 2
diabetes results from a relative deficiency of circulating
ype 2 diabetes is characterized by a chronic
elevation of plasma glucose concentration, caus-
ing complications such as retinopathy, neuropa-
thy, and nephropathy and increasing the risk of
insulin (1). Progressive deterioration in ?-cell function is
likely to result from exposure to the diabetic milieu (i.e.,
hyperglycemia and hyperlipidemia), thus setting up a
positive feedback loop in which hyperglycemia and/or
hyperlipidemia impairs ?-cell function, leading to further
hyperglycemia (2–7).
Chronic high fatty acid exposure results in increased
basal and blunted glucose-mediated insulin secretion and
reduced ?-cell mass (2,5,8,9). This is associated with the
pancreatic ?-cells undergoing adaptive changes such that
genes that are highly expressed under normal conditions,
for example, insulin, PDX-1, and GLUT2, are underex-
pressed and genes that are poorly expressed in the pan-
creatic
?-cells,suchas
phosphatase, c-Myc, and acetate dehydrogenase, are
shown to be upregulated (10,11).
One of the genes that is upregulated in ?-cell lines under
conditions of high fatty acid exposure is fructose-1,6-
bisphosphatase (FBPase) (10–12), a regulated enzyme in
the gluconeogenic pathway that catalyzes the dephosphor-
ylation of fructose-1,6-bisphosphate to fructose-6-phos-
phate. FBPase is abundant in the liver and the kidneys but
is poorly expressed in the pancreatic ?-cells under normal
conditions. In addition, FBPase was upregulated fivefold
in islets from the diabetes-susceptible obese BTBR mouse
compared with the diabetes-resistant C57BL/6 mouse (13).
We have previously shown that FBPase is upregulated in
the liver of mice or rats fed a high-fat diet (14,15) and in
the New Zealand Obese (NZO) mouse, an obese model of
type 2 diabetes (14,16). We have recently demonstrated
that transgenic mice with specific overexpression of
FBPase in the liver displayed increased glycerol glucone-
ogenesis (17).
From the abovementioned studies, it is evident that
upregulation of FBPase is induced by fatty acids. How-
ever, whether an increase in FBPase alone can be detri-
mental to cellular function and in particular to ?-cell
insulin secretory rates has not been investigated.
To determine whether an increase in FBPase impairs
insulin secretion, we generated both transgenic mice and
stably transfected pancreatic ?-cell lines (MIN6) overex-
pressing the human FBPase (huFBPase) gene. We demon-
strated that overexpression of FBPase in ?-cells results in
impaired glucose-stimulated insulin secretion, which is
associated with decreased glucose metabolism, resulting
in reduced cellular ATP levels and cell proliferation.
hexokinaseI,glucose-6-
From the1Department of Medicine, Heidelberg Repatriation Hospital, Univer-
sity of Melbourne, Heidelberg Heights, Victoria, Australia; the
Institute of Medical Research, Darlinghurst, New South Wales, Australia;
and
South Wales, Australia.
Corresponding author: Sofianos Andrikopoulos, sof@unimelb.edu.au.
Received 17 September 2007 and accepted 23 March 2008.
Published ahead of print at http://diabetes.diabetesjournals.org on 28 March
2008. DOI: 10.2337/db07-1326.
© 2008 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
2Garvan
3Diabetes and Endocrinology, Westmead Hospital, Westmead, New
ORIGINAL ARTICLE
DIABETES, VOL. 57, JULY 20081887

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RESEARCH DESIGN AND METHODS
MIN6 cells were obtained from Prof. Jun-ichi Miyazaki (Osaka University,
Osaka, Japan). The RIP7 expression vector was provided by Prof. Thomas Kay
(St. Vincent’s Institute of Medical Research, Victoria, Australia). The FBPase
primary antibody was a gift from Dr. Hideo Mizunuma (Akita University,
Akita, Japan). Oligonucleotide primers were synthesized by Gene Works
(Hindmarsh, South Australia, Australia). Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum, glutamine, and penicillin/streptomycin were
from Invitrogen Australia (Mount Waverley, Victoria, Australia).
Human islet preparation. Human islets were prepared as previously de-
scribed (18). Briefly, pancreata were removed from heart-beating deceased
donors and disaggregated by infusing the ducts with cold Liberase enzyme
(Liberase Human Islet; Roche Applied Science, Indianapolis, IN). Dissociated
islets and acinar tissue was separated on a continuous Biocoll (Biochrom,
Berlin) density gradient (polysucrose 400 and amidotrizolic acid).
Generation of transgenic construct. Standard molecular biology tech-
niques were used to generate the transgenic construct (19) (Fig. 1A).
Pancreatic ?-cell specificity of transgene expression was directed by a
segment of the rat insulin II gene promoter (RIP), generated by PCR
amplification of the RIP7 expression vector (20). The amplified fragment
included a 659-bp promoter/enhancer region, the first exon and intron of the
rat insulin II gene, a ClaI cloning site in the second exon, and the class II E?
poly A terminator sequence (20). This fragment was then ligated into a
pUC-based expression vector, designated the insulated transgenic vector, in
which the promoter fragment was flanked on each side by two tandem copies
of the chicken Hyper-Sensitive-4 (HS4) insulator sequence (21). The
huFBPase fragment was generated by PCR amplification of the human liver
cDNA as previously described (17) and ligated into the ClaI cloning site. The
complete DNA sequence of the insulated RIP-huFBPase plasmid was deter-
mined, and expression of huFBPase was confirmed by transient transfection
in a ?TC3 cell line (22) and Western blot analysis. The insulated RIP-huFBPase
expression fragment (Fig. 1A) was separated from the pUC-19 backbone by
AvrII and SpeI restriction enzyme digestion and gel purification.
Transgenic mice. Transgenic mice were generated at the Central Microin-
jection Service of the Walter and Eliza Hall Institute of Medical Research
(Parkville, Victoria, Australia) by injecting the insulated RIP-huFBPase DNA
fragment (Fig. 1A) into the pronuclei of C57BL/6 fertilized oocytes, following
established procedures (23). Offspring were genotyped for the presence of the
transgene by PCR amplification of tail DNA using a forward primer from the
RIP promoter (5?-CCTAAGTGACCAGCTACAGTC-3?) and a reverse primer
from the coding region of huFBPase (5?-GCTGGGTCAACTCGCCCGTG-3?).
Primers amplifying a glyceraldehyde-3-phosphate dehydrogenase fragment of
650 bp were included as an internal control for the PCR analysis. PCR reagents
were obtained from New England Biolabs (Beverly, MA). The amplified DNA
products were resolved on a 2% agarose gel.
Physiological studies were carried out on third- and fourth-generation
heterozygous mice. Control mice for all experiments were the transgene-
negative littermates. Mice were fed ad libitum with a standard rodent
laboratory diet and kept under a 12-h light/12-h dark cycle (light on at 0700 h).
RNA and cDNA preparation. Total RNA was extracted from tissues and
cultured cells using the Chomczynski and Sacchi procedure (24). RNA was
treated with DNaseI (RNase-free; Ambion) before cDNA synthesis using 2 ?g
RNA and random primers with the Promega Reverse Transcription kit.
Real-time PCR analysis. Primers were designed for SYBR-green real-time
PCR using the Primer-Express software (Applied Biosystems, Foster City,
CA). The primers for ?-actin (GenBank accession no. NM_007393) were
forward, 5?-CGTGAAAAGATGACCCAGATCA-3?, and reverse, 5?-CACAGCCT
GGATGGCTACGT-3?. The primers for total FBPase were homologous to both
mouse (GenBank accession no. NM_019395) and human (GenBank accession
no. NM_000507) liver FBPase sequences. The sequences were forward,
5?-AGC CTT CTG AGA AGG ATG CTC-3?, and reverse, 5?-GTC CAG CAT GAA
GCA GTT GAC-3?. The transgene-specific primers for RIP-huFBPase mRNA
but not the endogenous mouse FBPase sequence were forward, 5?-GTG TTG
ACG TCC GTG TCG AA-3?, and reverse, 5?-ACC AGC TAC AGT CGG AAA
CCA-3?. All primer sets were designed to amplify a product spanning an
exon-intron boundary of the genomic DNA.
Each sample was run in triplicate for each set of primers (i.e., FBPase and
?-actin). Gene expression was determined using ABI PRISM 7900 HT system
(Applied Biosystems). The relative quantification comparative Ct, also known
as the ??Ctmethod using the ABI sequence detection software (SDS), was
used.
Western blot analysis. FBPase protein levels were determined by immuno-
blotting as previously described (25). Protein lysates were resolved by
SDS-PAGE under reducing conditions, transferred to polyvinylidene difluoride
membranes (PolyScreen PVDF membrane; Perkins Elmer), and probed with
the appropriate secondary antibody by standard protocols. Immunoreactive
proteins were visualized with ECL Western blotting detection reagents
(Amersham Bioscience).
Intravenous glucose tolerance test. The intravenous glucose tolerance test
(IVGTT) was performed as previously described (26). A bolus of glucose (1
g/kg) was injected via a carotid artery catheter and blood was sampled at 0, 2,
5, 10, 15, and 30 min after the injection. Plasma was stored at ?20°C for future
glucose and insulin analysis.
Intraperitoneal glucose tolerance test. The intraperitoneal glucose toler-
ance test was performed as previously described (26). A bolus of glucose (2
g/kg) was injected intraperitoneally and blood was sampled at 0, 15, 30, 60,
and 120 min after the injection. Plasma was stored at ?20°C for future glucose
and insulin analysis.
FBPaseRIPII2 x cHS42 x cHS4
1
23 Mouse Liver
37 kDa
B
A
FIG. 1. Generation and testing of the transgenic construct. A: The complete 7,645-bp linear-insulated RIP-huFBPase construct, which consists of
695 bp of RIPII, the first intron of the insulin gene, a cDNA encoding huFBPase, and a downstream polyadenylation site (Poly A) flanked by two
sets of the cHS4 insulator sequences. B: Transient transfection of FBPase in ?TC-3 cells with the construct depicted in A, showing its capacity
to produce the expected 37-kDa protein immunoreactive with the FBPase antibody in tissue culture. Lane 1, untransfected ?TC-3 cells; lanes 2
and 3, transiently transfected ?TC-3 cells, compared with a mouse liver sample.
0
2
4
6
ControlType 2 Diabetes
Islet FBPase mRNA
(Arbitrary Units)
*
FIG. 2. Islet FBPase expression in patients with type 2 diabetes.
FBPase mRNA levels in islets isolated from patients with type 2
diabetes (n ? 3) and in age- and weight-matched control subjects (n ?
6) was determined using real-time PCR. Results are presented as
means ? SE. *P < 0.05.
ELEVATED ISLET FBPase IMPAIRS INSULIN SECRETION
1888DIABETES, VOL. 57, JULY 2008

Page 3

Islet isolation and culture. Islets were isolated from the pancreas by
collagenase digestion as previously described (27,28). After islets were freed
from exocrine tissue, they were hand-picked under a stereomicroscope
(Olympus, Tokyo, Japan) and transferred for overnight culture in RPMI 1640
with 10% (vol/vol) heat-inactivated fetal calf serum (FCS), in a 37°C humidified
atmosphere of 95% air:5% CO2.
After overnight culture, batches of islets were preincubated for 90 min in
Krebs-Ringer bicarbonate buffer (KRBB) with 2.8 mmol/l glucose. Triplicate
batches of five islets were then transferred to tubes containing 1 ml KRBB
supplemented with 2.8 mmol/l in the absence or presence of 10 mmol/l
L-arginine or 20 mmol/l glucose. After a 60-min incubation of the islets at 37°C,
tubes were centrifuged at 2,000 rpm for 5 min, and 0.5-ml supernatant was
removed for insulin analysis. The remaining 0.5 ml containing the islets was
treated with 0.18 mol/l HCl/95% ethanol followed by sonication to determine
insulin content.
0.0
2.0
4.0
6.0
8.0
10.0
12.0
Line 1
(High)
Line 32
(Medium)
Line 44
(Low)
Negative
Littermate
Human Islet FBPase mRNA
(relative to Low)
Line 1
(High)
Line 32
(Medium)
Line 44
(Low)
Negative
Littermate
37 kDa
A
B
Negative Low Medium High
Littermate (line 44) (line 32) (line 1)
Insulin
FBPase
C
FIG. 3. Expression of the FBPase gene in transgenic mice. A: huFBPase mRNA levels in three transgenic mouse lines (1, 32, and 44), showing high,
medium, and low expression levels of the transgene, relative to the lowest expressing transgenic line 44. As expected, the transgene was not
detected (ND) in negative littermate mice. B: Immunoblotting of pancreatic samples from transgenic lines 1, 32, and 44, showing high, medium,
and low levels of the 37-kDa FBPase protein, in accordance with the mRNA data shown in A above. C: Immunohistochemistry of pancreata from
the three FBPase transgenic mouse lines (1, 32, and 44) and a negative littermate, showing insulin immunostaining in all sections, whereas
FBPase immunostaining was only seen in the transgenic pancreata, localized to the insulin staining.
TABLE 1
FBPase upregulation in mouse models of obesity and diabetes
Model
n
FBPase levels
(fold increase of
corresponding control)
NZO
High-fat–fed C57BL/6
C57BL/KsJ-db/db
3
3
5
1.9 ? 0.2*
2.1 ? 0.3*
3.5 ? 1.0†
Data are means ? SE for the number of mice indicated. Pancreatic
FBPase was measured by immunoblotting in NZO and high-fat–fed
(60% ?wt/wt? for 16 weeks) C57BL/6 mice, and islet FBPase mRNA
levels were determined in C57BL/KsJ-db/db mice using real-time
PCR. *P ? 0.05, †P ? 0.01.
M. KEBEDE AND ASSOCIATES
DIABETES, VOL. 57, JULY 20081889

Page 4

Insulin levels were determined with a double antibody radioimmunoassay
using a rat-specific insulin antibody and rat insulin as a standard (Linco
Research, St. Charles, MO).
Cell culture. Pancreatic cell lines were routinely cultured in DMEM supple-
mented with 25 mmol/l glucose, 15% FCS, 100 units/ml penicillin, 100 ?g/ml
streptomycin, and 50 ?mol/l ?-mercaptoethanol at 37°C with 5% CO2.
Transient transfection. Cells were seeded into six-well plates at 2 ? 106
cells/well 2 days before transfection. Lipofectamine 2000 (Invitrogen) was
used to cotransfect cells with 1–2 ?g supercoiled insulated RIP-huFBPase
plasmid (Fig. 1A) and 0.1–0.2 ?g pEGFP-C1 (Clontech Laboratories) reporter
plasmid per well according to the manufacturer’s protocol. Cells were
harvested 48 h posttransfection, and cell pellets were stored at ?80°C for
subsequent analysis.
Stable transfection. Cells were transfected with the linear-insulated RIP-
huFBPase fragment (Fig. 1A) using Lipofectamine 2000 as above, but the
cotransfecting plasmid was pPGK-puro to enable selection with the antibiotic
puromycin. Forty-eight hours after transfection, the cell medium was supple-
mented with 6 ?g/ml puromycin, and resistant cells started to form colonies
after ?7 days. Colonies were picked, amplified, and screened for FBPase
expression by RNA and protein analysis.
Cell proliferation assay. Cell proliferation was assessed over a 7-day period
using trypan blue dye exclusion. At day 0, cells were seeded at 1 ? 105
cells/well in 0.5 ml DMEM in 24-well plates. Cells were then counted manually
at days 1, 3, 5, and 7.
FBPase enzyme activity assay. Proteins were extracted by homogenizing
1 ? 106cells in 0.5 ml homogenization buffer (50 mmol/l triethanolamine, pH
7.2, 1 mmol/l dithiothreitol, and 10 mmol/l EDTA) followed by centrifugation
at 40,000g for 40 min at 4°C. FBPase enzyme activity was determined using a
spectrophotometric-coupled enzyme assay as previously described (16,25).
Insulin secretion and content in MIN6 cells. Cells were seeded at 3 ? 105
cells/well in 0.5 ml DMEM in 24-well plates. After 24 h, cultured cells were
washed in modified KRBB containing 2.8 mmol/l glucose and preincubated for
30 min in 0.5 ml of the same medium at 37°C. This medium was then replaced
with 0.5 ml prewarmed KRBB containing 2.8 mmol/l glucose, 2.8 mmol/l
glucose plus 10 mmol/l methyl-pyruvate, 16.8 mmol/l glucose, or 16.8 mmol/l
glucose plus a nonglucose secretagogue cocktail. The secretagogue cocktail
contained 0.1 mmol/l 3-isobutyl-1-methylxanthine (IBMX), 10 mmol/l arginine,
and 5 ?mol/l carbamylcholine chloride (carbachol) (29,30). The cells were
incubated for 60 min at 37°C, and an aliquot of medium was removed for
analysis of insulin. To measure the total insulin content, the cell monolayers
were washed twice in PBS and harvested in 0.5 ml 0.18 mol/l HCl/95% ethanol
per well followed by sonification for 20 min in a Branson 2200 bath sonicator
(Branson Ultrasonics, Danbury, CT). The cell lysates were centrifuged at
13,000 rpm for 10 min, and supernatants were retained and stored at ?20°C
until analysis. Insulin levels were determined with a double-antibody radio-
immunoassay using rat-specific insulin antibody and rat insulin as a standard
(Linco Research).
Measurement of glucose utilization and glucose oxidation. Glucose
utilization of cells was measured using the production of tritiated water from
the metabolism of D-[5-3H]glucose as previously described (31,32). Tritiated
H2O was then measured by liquid scintillation counting. Glucose oxidation
was estimated by assessing the formation of14CO2from the metabolism of
[U-14C]glucose (33). The amount of14CO2was determined by liquid scintilla-
tion counting.
Measurement of glycolytic intermediates. Fructose 1,6-bisphosphate and
fructose 6-phosphate assays were performed on the supernatant as described
before (34,35).
Measurement of cellular ATP content. The amount of ATP was measured
fluorimetrically using the ATP bioluminescent assay kit from Sigma (St. Louis,
MO) as previously described (30).
Immunohistochemistry. For immunohistochemical detection of FBPase and
insulin, pancreata from control and transgenic mice were fixed in freshly
made 4% paraformaldehyde, processed for paraffin, embedded, and sectioned
at 5 ?m. Slides were incubated with either a guinea-pig anti-porcine insulin
antibody (DAKO, Carpentaria, CA) diluted 1:100 or rabbit-anti-rat FBPase
antibody diluted 1:100. The sections were then washed with Tris hydrochlo-
ride (10 mmol/l, pH 7.4) for 10 min and then incubated with the DAB
(3,3?-diaminobenzidine) chromagen for 1 min. Sections were counterstained
with hematoxylin and mounted with distyrene plasticizer and xylene mount-
ing media and observed under a microscope.
Ethics approval. All of the animal studies were approved by the Austin
Health Animal Ethics committee. Consent for collection of the human tissue
samples was obtained by the Red Cross Transplantation Service, and St.
Vincent’s Sydney Hospital Human Research Ethics Committee gave approval
for the human studies.
Statistical analysis. Area under the curve was calculated using the trape-
zoidal rule. Results are presented as means ? SE. Statistical significance was
assessed using the Student’s t test. Differences were considered statistically
significant at P ? 0.05.
RESULTS
To confirm the literature reports that FBPase is upregu-
lated by lipid oversupply, we first assessed the pancreatic
FBPase levels in a naturally occurring obese animal
model, the NZO mouse; in a diet-induced obesity animal
model, the C57BL/6 mouse fed a 60% fat diet for 16 weeks;
and in the diabetic C57BL/KsJ/-db/db mouse. We found
that both the NZO and high-fat–fed C57BL/6 mice had a
twofold increase in pancreatic FBPase protein levels,
whereas there was a 3.5-fold increase in FBPase mRNA
levels in db/db mice compared with their respective con-
trols (Table 1).
To determine whether patients with type 2 diabetes
have elevated FBPase, pancreatic islets were isolated from
three diabetic and six age-matched (50.6 ? 3.4 vs. 51 ? 4.4
years) and BMI-matched (26.5 ? 0.5 vs. 27.8 ? 4.0 kg/m2)
control subjects as previously described (18). FBPase
mRNA levels were measured by real-time PCR and were
corrected for expression using the housekeeping gene
TATA-box binding protein. FBPase levels were increased
by twofold in the human diabetic islets (P ? 0.05; Fig. 2).
Taken together, these data support previous experimental
evidence that FBPase is upregulated by lipid oversupply/
diabetes in animal models and suggests for the first time
that islet FBPase may also be increased in patients with
type 2 diabetes.
To determine whether a specific upregulation of FBPase
in islet ?-cells can result in defective insulin secretion,
transgenic mice were generated. A construct was gener-
ated with the human liver FBPase cDNA, driven by the
minimal (654 bp) RIP and flanked by the chicken HS4
(cHS4) insulator sequences (Fig. 1A). To test whether this
construct expressed protein, it was transiently transfected
into the immortalized pancreatic ?-TC3 cell line. Immuno-
blotting of transiently transfected cells showed a band
immunoreactive with the anti-FBPase antibody at the
expected size of 37 kDa (Fig. 1B), confirming that the
construct was capable of producing FBPase. The RIPII-
TABLE 2
Body weight, fasting plasma glucose, and plasma insulin concentrations; pancreatic insulin content levels; and glucose tolerance in
10-week-old ?-cell FBPase transgenic mice
Line 1 (high)Line 32 (medium) Line 44 (low)Negative littermate
Male body weight (g)
Female body weight (g)
Plasma glucose (mmol/l)
Plasma insulin (ng/ml)
Pancreatic insulin content (ng/?g protein)
Glucose area under the curve (mmol/l ?120 min)
21.4 ? 1.0
17.2 ? 0.51
7.0 ? 0.4
0.3 ? 0.02*
883 ? 197
2,500 ? 101*
22.9 ? 0.4
16.7 ? 0.35
7.5 ? 0.3
0.5 ? 0.10*
739 ? 95
1,773 ? 103
24.4 ? 0.5
17.6 ? 0.10
10.3 ? 0.5
0.3 ? 0.10*
694 ? 61
1,460 ? 125
22.5 ? 0.8
17.7 ? 0.30
8.0 ? 0.6
0.8 ? 0.16
661 ? 72
1,863 ? 113
Data are means ? SE (n ? 10). *P ? 0.05 compared with the negative littermate.
ELEVATED ISLET FBPase IMPAIRS INSULIN SECRETION
1890 DIABETES, VOL. 57, JULY 2008

Page 5

huFBPase chimeric gene was microinjected into mouse
embryos, and four founder mice (on the C57BL/6 genetic
background) were obtained. One of the lines did not
express the transgene and was therefore excluded from
further analysis. The studies presented here were per-
formed using three transgenic mouse lines denoted 1, 32,
and 44. The three lines expressed the transgene at different
levels. The highest expresser (line 1) exhibited an ?9
times higher transgene mRNA level than the lowest ex-
presser (line 44), as estimated by real-time PCR (Fig. 3A).
No expression of the transgene was detected in the liver,
muscle, and brain of the transgenic mice (data not shown).
Immunoblotting confirmed line 1 as the highest expresser
of FBPase, followed by lines 32 and 44, whereas very low
levels of FBPase protein were detected in the pancreata of
negative littermate mice (Fig. 3B). Although it is not
quantitative, immunohistochemistry of FBPase transgenic
and negative littermate pancreata showed islet FBPase
staining in the same region as insulin immunostaining,
suggesting ?-cell–specific expression of the transgene in
the transgenic mice, whereas the negative littermate mice
showed no islet FBPase staining (Fig. 3C).
Body weight of the three FBPase transgenic lines was
not different to the negative littermates (Table 2). Fasting
plasma glucose levels were not different, except for a
slight increase in line 44 compared with the negative
littermate mice (Table 2). However, fasting plasma insulin
concentrations were significantly lower in all three
FBPase transgenic lines compared with negative litter-
mates (Table 2). Pancreatic insulin content was compara-
ble between transgenic mice and that of the negative
littermate mice (Table 2).
When subjected to an IVGTT, all three lines showed a
significant impairment in insulin secretion (Fig. 4A). Con-
sequently, the area under the insulin curve was reduced in
all three transgenic lines compared with the negative
littermates (Fig. 4B). Glucose tolerance was significantly
impaired only in line 1 (Table 2), which had the highest
expression of FBPase and most significant defect in insu-
lin secretion compared with the negative littermates.
Insulin secretion was also determined in vitro in isolated
islets from line 32 and the data presented in Fig. 4C. In
0.0
1.0
2.0
3.0
4.0
5.0
0 5 10 15 20 25 30
Time (min)
Plasma Insulin Levels
(ng/ml)
Negative Littermate
Low (Line 44)
Medium (Line 32)
High (Line 1)
0.0
20.0
40.0
60.0
80.0
Negative
Littermate
Low (Line
44)
Medium
(Line 32)
High (Line
1)
Total Insulin Secretion
(ng/ml x 30 min)
*
*
*
A
B
0.0
100.0
200.0
300.0
400.0
500.0
ControlTransgenic
Islet Insulin Secretion
(% fold 2.8 mM glucose)
2.8 mM glucose
20 mM glucose
2.8 mM glucose + arginine
*
*
*
*,#
C
FIG. 4. Insulin secretory responses in FBPase transgenic and negative
littermate mice. A: Plasma insulin levels in FBPase transgenic and
negative littermate mice during the IVGTT. B: Total insulin secretion,
calculated as the area under the insulin curve over the 30 min of the
IVGTT in the three transgenic FBPase and negative littermate mice.
Results are presented as means ? SE (n ? 6–14). *P < 0.05; **P < 0.01.
C: Insulin secretion measured from islets isolated from line 32 trans-
genic and negative littermate control mice. Results are presented as
means ? SE (n ? 3). *P < 0.05 compared with 2.8 mmol/l glucose; #P <
0.05 compared with control islets.
12345
37 kDa
0
2
4
6
8
10
Parental pPGK-
puro
LowHigh
FBPase enzyme activity
(µ µmol/min/µ µg protein)
NDND
*
A
B
FIG. 5. Generation of MIN6 cells overexpressing huFBPase. A: MIN6
cells were cotransfected with 1–2 ?g transgene construct shown in Fig.
1A and 0.1–0.2 ?g pPGK-puro selection plasmid. Eighteen puromycin-
resistant colonies were isolated, of which three colonies were selected
to be extensively studied with no (pPGK-puro), low, and high levels of
the FBPase protein, respectively, as assessed by immunoblotting. Lane
1, parental MIN6 cells; lane 2, low FBPase; lane 3, high FBPase; lane 4,
pGK-puro (no FBPase); lane 5, mouse liver control. B: FBPase enzyme
activity in the cell lines depicted in A, showing increased activity in the
cells with the highest FBPase protein levels, whereas the parental
MIN6 and pPGK-puro cells had no detectable (ND) enzyme activity.
Results are presented as means ? SE (n ? 3). *P < 0.05 compared with
low-expressing FBPase transgenic cells.
M. KEBEDE AND ASSOCIATES
DIABETES, VOL. 57, JULY 20081891

Page 6

response to high glucose, line 32 transgenic islets secreted
significantly less insulin compared with negative littermate
islets. Furthermore, in response to arginine, which is
proposed to act distal to the ATP-sensitive K?channel by
directly depolarizing the plasma membrane, there was no
difference in insulin release between transgenic and neg-
ative littermate islets.
To investigate the biochemical mechanism by which
FBPase upregulation resulted in significantly reduced glu-
cose-mediated insulin secretion, transgenic cell lines
(MIN6) overexpressing the huFBPase gene were gener-
ated and characterized as follows. The FBPase and pPGK-
puro constructs were stably transfected into MIN6 cells.
Eighteen colonies, which grew in puromycin, were se-
lected and checked for FBPase expression and protein
levels. From this screen, three colonies were chosen to be
more extensively studied: a colony that had no FBPase
expression but grew in puromycin as a result of the
pPGK-puro transgene, a colony with relatively low expres-
sion, and a colony with relatively high expression of
FBPase (Fig. 5A). FBPase enzyme activity was also as-
sayed and shown to be elevated in the high-expressing
cells compared with the low-expressing cells (Fig. 5B).
Furthermore, the parental and pPGK-puro cells had unde-
tectable activity, concurring with the immunoblotting data
(Fig. 5A).
To determine whether the overexpression of FBPase
affected insulin secretory function, the parental MIN6,
pPGK-puro, and the two overexpressing cell lines were
incubated with basal glucose (2.8 mmol/l), high glucose
(16.8 mmol/l), or high glucose plus a cocktail of secreta-
gogues (arginine, IBMX, and carbachol), and insulin re-
lease was determined. All cell lines released similar levels
of insulin under basal conditions, although there was a
trend for insulin secretion to be lower in the FBPase-
transfected cells (Fig. 6A), reflecting the lower basal
plasma insulin levels in the transgenic mice (Table 2).
Both the parental MIN6 and pPGK-puro cells showed
increases in insulin secretion in response to high glucose
and high glucose plus the secretagogue cocktail, respec-
tively (Fig. 6A). In contrast, FBPase-overexpressing cells
did not increase insulin release in response to 16.8 mmol/l
glucose and showed a much-blunted response to glucose
plus the cocktail secretagogue compared with the parental
cell line (Fig. 6A). Insulin content was determined and
found to be not significantly different in the transfected
cells compared with the parental MIN6 (Fig. 6B). There-
fore, overexpression of FBPase significantly reduced
glucose-mediated insulin secretion, reproducing and con-
firming what was found in vivo in the transgenic mice
overexpressing FBPase in ?-cells.
A mechanism by which increased FBPase may reduce
insulin secretion is by decreasing glucose utilization and
energy production. Because islets from patients with type
2 diabetes have markedly decreased expression of phos-
phofructokinase-1 (PFK-1) (18), increased FBPase would
be expected to further impair glycolysis. Therefore, glu-
cose utilization and glycolytic intermediates were mea-
sured. All cell lines had comparable glucose utilization
when cultured at basal (2.8 mmol/l) glucose concentra-
tions (Fig. 7A, left). However, in response to high (16.8
mmol/l) glucose, glucose utilization (assessed using 5-3H-
glucose) was significantly lower in cells overexpressing
FBPase compared with the parental and pPGK-puro con-
trols (Fig. 7A, right). Furthermore glucose oxidation (mea-
sured using [U-14C]glucose) was reduced in the high-
overexpressing FBPase compared with the parental MIN6
cells when incubated with both 2.8 and 20 mmol/l glucose
(Fig. 7B). FBPase catalyzes the conversion of fructose-1,6-
bisphosphate to fructose-6-phosphate and is opposed by
PFK-1 in the glycolytic pathway. Overactivity of FBPase
would be expected to result in overabundance of the
product and a reduction of the substrate. Figure 7C shows
that the substrate (fructose-1,6-bisphosphate) was lower
and the product (fructose-6-phosphate) was higher in the
FBPase-overexpressing cell lines compared with the pa-
rental MIN6 cells.
To determine whether the decrease in glucose utiliza-
tion caused a reduction in energy levels, cellular ATP
levels were measured and found to be comparable among
the four cell lines at basal (2.8 mmol/l) glucose (Fig. 7D).
However, with high (16.8 mmol/l) glucose culture, the
FBPase-overexpressing cell lines showed decreased ATP
levels compared with the parental and control cell lines
(Fig. 7D). Together, these data suggest that decreased
glucose utilization and energy production may be respon-
sible for the reduced insulin secretion, in ?-cells overex-
pressing the human liver FBPase gene.
A reduction in energy availability may affect cell growth.
To study this, the proliferation rate of the four cell lines
was determined over 7 days. There was an increase in cell
number in all cell lines over the 7-day test period (Fig. 8A).
However, the cell lines overexpressing FBPase showed a
slower rate of cell proliferation compared with the paren-
0
15
30
45
60
75
2.8mM glucose 16.8mM glucose 16.8mM glucose
+Cocktail
Insulin secretion
(% of Insulin content)
Parental
pPGK-puro
Low
High
* *
* *
0.0
50.0
100.0
150.0
200.0
250.0
Parental pPGK-
puro
Low High
Insulin Content
(ng/100,000 cells)
A
B
FIG. 6. Glucose- and non–glucose-induced insulin secretion in FBPase
transgenic MIN6 cells. A: The cells were incubated in KRBB containing
2.8 mmol/l glucose, 16.8 mmol/l glucose, or 16.8 mmol/l glucose plus a
cocktail of secretagogues, including 10 mmol/l arginine, 0.1 mmol/l
IBMX, and 5 ?mol/l carbachol for 1 h. Medium was taken to determine
levels of insulin secretion. B: Total cellular insulin content was deter-
mined by lysing the cells with 0.18 mol/l HCl/95% ethanol, followed by
sonification. Insulin secretion was then expressed as relative to total
cellular insulin content of cells. Results are presented as means ? SE
of three independent experiments. *P < 0.05 compared with parental
MIN6 cells.
ELEVATED ISLET FBPase IMPAIRS INSULIN SECRETION
1892 DIABETES, VOL. 57, JULY 2008

Page 7

tal and the pPGK-puro control cell lines, respectively (Fig.
8A). This difference in cell proliferation was particularly
pronounced at days 5 and 7.
To confirm that the reduction in growth rate was due to
a deficiency in energy availability secondary to reduced
rate of glycolysis, cells were supplemented with sodium
pyruvate to a final concentration of 2.5 mmol/l. As can be
seen in Fig. 8B, pyruvate supplementation normalized
growth rates at 5 and 7 days in the low-expressing and at
5 days in the high-expressing FBPase-MIN6 cells. These
data further support the assertion that increased expres-
sion of FBPase results in impaired cellular function by
reducing energy availability.
To investigate whether pyruvate stimulation improves
the insulin secretory function of FBPase-overexpressing
MIN6 cells, the parental and low-expressing cells were
pretreated with pyruvate (to a final concentration of 2.5
mmol/l), and insulin secretory function was determined at
basal glucose (2.8 mmol/l) concentrations. We chose the
low-expressing cell line because we have shown in Fig. 8B
that in this cell line, 2.5 mmol/l pyruvate supplementation
rescued the cell proliferation defects in FBPase-overex-
pressing cells. In concordance with these data, pyruvate
stimulation also resulted in significantly higher insulin
secretory rates in the FBPase-transfected cells, compared
with control cells transfected with the puromycin gene only
(insulin content 68.3 ? 2.6 vs. 16.4 ? 1.5%, n ? 4, P ? 0.05).
DISCUSSION
Islet ?-cell dysfunction is a key characteristic of patients
with type 2 diabetes that results in hyperglycemia. Hyper-
glycemia, in conjunction with the dyslipidemia, which is
often present in patients with type 2 diabetes, can further
exacerbate the defects in insulin secretion. A number of
studies have shown that FBPase is upregulated in re-
sponse to high glucose or fatty acid conditions (10–12,36).
Importantly, direct evidence for the stimulatory effect of
fat on FBPase expression has been provided in vitro using
pancreatic ?-cell lines incubated with palmitate (10,11).
Although the mechanism for this induction is not known,
we speculate that fatty acids increase the transcription of
the FBPase gene. We have extended these results and have
shown here that in obese (NZO) or diabetic (C57BL/KsJ-
db-db) mice and in a high-fat–fed mouse model, there is a
two- to threefold increase in pancreatic/islet FBPase lev-
els. Furthermore, we show for the first time that increased
FBPase expression has direct relevance to human disease
because its expression was increased twofold in islets
from patients with type 2 diabetes. These studies, how-
ever, do not provide any information on whether this
increase in FBPase has functional consequences in islet
?-cells. To determine whether overexpression of FBPase
impairs insulin secretion, we generated three lines of
transgenic mice with different levels of islet FBPase over-
0
5
10
15
20
25
30
2.8mM Glucose16.8mM Glucose
Glucose Utilization
(3H2O/60 min/µ µg protein)
Parental
pGK-puro
Low
High
* *
0
50
100
150
200
250
2.8 mM Glucose16.8 mM Glucose
Glucose Oxidation
(14CO2/60 min/µ µg protein)
Parental
High
*
*
A
B
0
50
100
150
200
Frucose-1,6 bisphosphate Fructose-6-phosphate
Percentage of parental
Parental
pGK-puro
Low
High
*
*
0.0
200.0
400.0
600.0
800.0
1000.0
2.8 mM glucose 16.8 mM glucose
ATP Levels
(nmol/mg protein)
Parental
Low
High
*
*
C
D
FIG. 7. Glucose utilization, glucose oxidation, glycolytic intermediate, and cellular ATP levels in MIN6 cells overexpressing FBPase. A: Glucose
utilization was measured by the conversion of [5-3H]glucose into3H2O as described in RESEARCH DESIGN AND METHODS. Results are expressed as
means ? SE of three independent experiments. *P < 0.05 compared with parental MIN6 cells. B: Glucose oxidation was measured by the
conversion of [U-14C]glucose into14CO2as described in RESEARCH DESIGN AND METHODS. Results are expressed as means ? SE of five independent
experiments. *P < 0.05 compared with parental MIN6 cells. C: Fructose-1,6-bisphosphate and fructose-6-phosphate levels in parental, pGK-puro
(no FBPase), low-FBPase, and high-FBPase MIN6 cells. Results are expressed as means ? SE of three independent experiments. *P < 0.05
compared with control MIN6 cells. D: Cellular ATP content was measured in parental, low-FBPase, and high-FBPase MIN6 cells. Results are
expressed as means ? SE of three independent experiments. *P < 0.05 compared with parental cells.
M. KEBEDE AND ASSOCIATES
DIABETES, VOL. 57, JULY 20081893

Page 8

expression. We demonstrated that insulin secretion in
response to glucose was reduced in a dose-dependent
manner with greater overexpression, causing more severe
impairment of secretion. Interestingly, despite a reduction
in insulin secretion in all three transgenic lines tested,
glucose tolerance was impaired only in line 1 with the
highest FBPase overexpression and the greatest reduction
in secretion. This probably reflects the robust nature of the
mouse and that a severe reduction in insulin secretion is
required before it affects glucose tolerance. All of this sug-
gests that upregulation of islet FBPase by high glucose/lipid
levels can result in reduced glucose-mediated insulin secre-
tion in vivo. Using MIN6 cells, an in vitro ?-cell model, we
went on to show that the most likely mechanism by which
FBPase overexpression impairs insulin secretion is a reduc-
tion in glycolytic flux resulting in reduced energy production.
Arginine-induced secretion was not impaired in the trans-
genic islets. Because arginine acts by directly depolarizing
the plasma membrane, this result suggests that the secretory
machinery is functioning appropriately and supports the
notion of decreased glycolytic flux as the cause of reduced
insulin secretion in the transgenic mice.
Many mechanisms for ?-cell dysfunction and death have
been proposed, including high glucose–induced oxidative
stress (8), fatty acid–induced defects in secretion and
activation of apoptotic pathways (30,37), islet amyloid–
mediated ?-cell death (38,39), and others (40). Some of
these may be inherent defects that are exposed when the
islet is stressed with obesity and excess nutrients. It is
clear from our own data and that of other researchers that
the upregulation of FBPase is a consequence of the
diabetic milieu. The question then arises: What role does
this particular abnormality play in the pathogenesis of type
2 diabetes? We show in this study that FBPase is not
normally expressed at high levels in the islet. Furthermore,
we and others have shown that this enzyme is upregulated
by fat. Noting this, together with the data in this report that
a specific upregulation of FBPase causes impaired insulin
secretion, we suggest that the obese/diabetic milieu of
increasing lipidemia stimulates the expression of islet FB-
Pase, which contributes to the deteriorating function of the
?-cell, resulting in further diminution of insulin release.
We show in this report that FBPase expression is
increased in patients with type 2 diabetes and that a
specific increase in this gene can lead to impaired insulin
secretory function. However, this is not the only protein
whose expression is altered in diabetes. It has been shown
that there are ?200 genes upregulated in islets from
patients with type 2 diabetes compared with control
subjects (18), and Busch et al. (10) have shown that ?100
genes are overexpressed ?1.9-fold when MIN6 cells are
cultured in high-fat conditions (including FBPase). One
approach to determine the functional consequences would
be to systematically inhibit each of the overexpressed
genes in diabetes and to determine whether cellular func-
tion is improved. Although for most genes, this would have
to be done in vitro using, for example, siRNA technology,
for FBPase, this can now be done in vivo because specific
inhibitors of this enzyme have been developed as thera-
peutic targets to treat type 2 diabetes (41,42). MB06322
(CS-917) is one such compound that efficiently inhibits the
enzyme and reduces gluconeogenesis in hepatocytes and
in vivo in ZDF rats, resulting in a substantial lowering of
plasma glucose levels in these diabetic animals (41,42). It
would be of interest to determine whether this or other
compounds that inhibit islet FBPase can improve insulin
secretory function and the diabetic phenotype of animal
models, such as the ZDF rat or C57BL/KsJ-db/db mouse, or
even in patients with type 2 diabetes.
It is of interest that reduction in glycolytic rate and the
ensuing reduction in ATP levels resulted in a slowing of
?-cell growth in vitro. It is important to note that the
insulin secretory experiments were performed after 24 h
of seeding the same amount of cells, a time that there was
no difference in cell number regardless of whether FBPase
was expressed (Fig. 8). We therefore do not believe that
differences in cell number can explain the blunting in
glucose-mediated insulin release with FBPase overexpres-
sion. As stated above, we provide evidence that the
decreased insulin release is due to reduced glycolytic flux.
Taken together, we have demonstrated that obese and
hyperglycemic mice and patients with type 2 diabetes
display an increase in the expression of pancreatic islet
FBPase. We consequently generated transgenic mice and
cell lines with a specific increase in islet ?-cell FBPase,
and these were shown to have reduced insulin secretion
associated with impaired glucose metabolism and ATP
generation. This study identifies islet FBPase as a possible
therapeutic target for the improvement of insulin secre-
tory function in type 2 diabetes.
0
2
4
6
8
10
12
1357
Time (Days)
Cells Number (x105per well)
Parental
pPGK-puro
Low
High
*
*
*
*
0
5
10
15
20
25
30
35
1357
Time (Days)
Cells Number (x105per well)
Parental
pPGK-puro
Low
High
*
A
B
FIG. 8. Cell proliferation rates in MIN6 cells overexpressing FBPase. A:
Cells were counted on days 1, 3, 5, and 7 after seeding. Results are
expressed as means ? SE of three independent experiments. *P < 0.05
compared with parental cells. B: The cell culture medium was supple-
mented so that the final concentration of pyruvate was 2.5 mmol/l, and
cells were counted on days 1, 3, 5, and 7 after seeding. Results are
expressed as means ? SE of three independent experiments. *P < 0.05
compared with parental cells.
ELEVATED ISLET FBPase IMPAIRS INSULIN SECRETION
1894DIABETES, VOL. 57, JULY 2008

Page 9

ACKNOWLEDGMENTS
J.E.G. has received a National Health and Medical Research
Council of Australia C.J. Martin Fellowship. S.A. has received
a National Health and Medical Research Council of Australia
R.D. Wright Biomedical Career Development Award. This
study was supported by projects grants from the National
Health and Medial Research Council of Australia and Diabe-
tes Australia Research Trust.
We thank Amy Blair for excellent technical assistance
and Dr. July Blassioli for help with the ATP assay.
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