Published in Volume
119, Issue 1
(January 5, 2009)J Clin Invest.
Copyright © 2009, American Society for Clinical
Deletion of the von Hippel–Lindau gene in pancreatic
β cells impairs glucose homeostasis in mice
1Centre for Diabetes and Endocrinology and
Cell Signalling and Molecular Genetics, Faculty of Medicine, Rayne Institute, and
3Department of Physiology and Mitochondrial Biology Group, University
College London, London, United Kingdom.
4Beta Cell Development and
Function Group, Division of Reproductive Health, Endocrinology and Development,
King’s College London, London, United Kingdom.
Genetic Medicine and Development, Faculty of Medicine, University of Geneva, Geneva,
Address correspondence to: Dominic J. Withers, Centre for Diabetes and
Endocrinology, University College London, Rayne Institute, 5 University Street,
London WC1E 6JJ, United Kingdom. Phone: 00442076796586; Fax: 00442076796583; E-mail:
firstname.lastname@example.org. Or to: Patrick H. Maxwell, Centre for Cell
Signalling and Molecular Genetics, University College London, Rayne Institute, 5
University Street, London WC1E 6JJ, United Kingdom. Phone: 00442076796351; Fax:
00442076796211; E-mail: email@example.com.
Authorship note: James Cantley, Colin Selman, and Deepa Shukla
contributed equally to this work.
First published December 8, 2008
Received for publication September 21,
2005, and accepted in revised form October 29,
Defective insulin secretion in response to glucose is an important component of the
β cell dysfunction seen in type 2 diabetes. As mitochondrial oxidative
phosphorylation plays a key role in glucose-stimulated insulin secretion (GSIS),
oxygen-sensing pathways may modulate insulin release. The von
Hippel–Lindau (VHL) protein controls the degradation of hypoxia-inducible
factor (HIF) to coordinate cellular and organismal responses to altered oxygenation.
To determine the role of this pathway in controlling glucose-stimulated insulin
release from pancreatic β cells, we generated mice lacking
Vhl in pancreatic β cells
(βVhlKO mice) and mice lacking Vhl in
the pancreas (PVhlKO mice). Both mouse strains developed glucose
intolerance with impaired insulin secretion. Furthermore, deletion of
Vhl in β cells or the pancreas altered expression of
genes involved in β cell function, including those involved in glucose
transport and glycolysis, and isolated βVhlKO and
PVhlKO islets displayed impaired glucose uptake and defective
glucose metabolism. The abnormal glucose homeostasis was dependent on upregulation of
Hif-1α expression, and deletion of Hif1a in
Vhl-deficient β cells restored GSIS. Consistent with this, expression of
activated Hif-1α in a mouse β cell line impaired GSIS. These
data suggest that VHL/HIF oxygen-sensing mechanisms play a critical role in glucose
homeostasis and that activation of this pathway in response to decreased islet
oxygenation may contribute to β cell dysfunction.
Blood glucose levels are normally tightly controlled by the regulation of insulin
release from the pancreatic β cells. Glucose-stimulated insulin secretion
(GSIS) is a complex metabolic process involving the uptake and phosphorylation of
glucose via GLUT2 transporters and glucokinase (Gck), respectively, metabolism of
glucose-6-phosphate via the glycolytic pathway, and subsequent activation of
mitochondrial metabolism to produce coupling factors such as ATP (1). A rise in the cytoplasmic ATP/ADP ratio leads to closure of
KATP channels, depolarization of the plasma membrane, opening of
voltage-sensitive Ca2+ channels, and activation of Ca2+-dependent
exocytotic mechanisms, resulting in insulin secretion (1). This metabolic sensing mechanism requires molecular oxygen for the
quantitative generation of ATP from glucose. Understanding the complex physiology of
this mechanism may give insights into both the pathogenesis and treatment of the
β cell dysfunction seen in type 2 diabetes.
Hypoxia-inducible factor (HIF) is a transcription control complex containing a
constitutive β subunit and regulatory α subunit, which acts as a
master regulator of the responses to altered cellular and tissue oxygen concentration
(2). In the presence of oxygen,
HIF-α subunits are hydroxylated, enabling capture by the von
Hippel–Lindau (VHL) tumor suppressor gene product, which is the substrate
recognition component of an ubiquitin E3 ligase complex (3, 4). At low oxygen concentrations,
HIF-α is stabilized and active. In the absence of VHL, HIF is constitutively
active. Key processes regulated by HIF include erythropoiesis, angiogenesis, and
cellular energy metabolism, thereby adapting the organism, tissue, and cell to hypoxia
(4). HIF is responsive within the range of
oxygen tensions encountered in normal tissues and is increasingly recognized as an
important physiological regulator rather than a simple stress response mechanism,
playing roles, for example, in innate immunity (5), neutrophil survival (6), muscle
performance (7), and skin oxygen sensing (8).
HIF upregulates expression of the high-affinity glucose transporter GLUT1 and glycolytic
enzymes and decreases mitochondrial oxygen consumption in a range of cell types (4). Since glucose uptake, glycolysis, and
mitochondrial respiration are key steps in β cell glucose sensing,
activation of the HIF pathway has the potential to provide a major input modulating
GSIS. This could potentially be important in a wide range of disease states in which
oxygen delivery is altered, including obstructive sleep apnea and acute and chronic
respiratory disease, or when islet oxygenation is directly compromised, such as in islet
transplantation. VHL disease is associated with pancreatic tumors believed to be of
endocrine origin, also indicating a potential role for this pathway in islet endocrine
cell growth and function. Furthermore, small-molecule HIF activators are currently under
evaluation for the treatment of anemia, and understanding the potential effects of
pharmacological manipulation of this pathway on pancreatic islet function is also of
Therefore, to determine the effect of activating HIF, we investigated the effect of
deleting the Vhl gene, specifically in β cells or the
pancreas in mice. After we initiated these studies, it was reported that islets of
patients with type 2 diabetes show reduced expression of the HIF-α
dimerization component aryl hydrocarbon receptor nuclear
translocator/HIF1b (ARNT/HIF1B) and that knockdown of
Arnt/Hif1b in β cells or mice impaired GSIS (9). In light of these studies, and assuming a
monotonic relationship between GSIS and HIF activation, it was reasonable to predict
that HIF activation would have the opposite effect of deletion of
Arnt/Hif1b and therefore enhance GSIS. Our genetic experiments showed
that Hif activation in mice results in profound disruption of β cell
function and, in contrast, that β cell deletion of Hif1a
does not impair glucose homeostasis.
Deletion of Vhl in β cells or in the pancreas impairs glucose
homeostasis in mice. Initially, we crossed mice expressing Cre under control of the rat insulin II
promoter (RIPCre mice; ref. 10) and mice with a floxed allele of Vhl (11) to generate mice lacking Vhl
in pancreatic β cells (βVhlKO mice) and in a
small population of hypothalamic neurons (12).
βVhlKO mice were viable and born with expected
Mendelian ratios (data not shown). The Vhl allele was deleted in
islets and hypothalami from βVhlKO mice (Figure 1A), and Hif-1α stabilization and
expression was induced in more than 95% of β cells of
βVhlKO mice, consistent with Vhl
deletion (Figure 1B and Supplemental Figure 1A;
supplemental material available online with this article; doi:
10.1172/JCI26934DS1). However, βVhlKO mice
were proportionate dwarfs with reduced pituitary growth hormone (GH) levels
(Supplemental Figure 1, B and C), while relative food intake and fat mass were normal
in these animals (Supplemental Figure 1, D and E). These findings suggest that Vhl
acts in RIPCre hypothalamic neurons to regulate Gh secretion. This
endocrine phenotype may have had an impact on subsequent studies of glucose
homeostasis in βVhlKO mice. Therefore, to test the
possibility that potential β cell phenotypes in
βVhlKO mice resulted wholly or partly from
hypothalamic deletion of Vhl, we used mice expressing Cre under
control of the mouse pancreatic and duodenal homeobox 1 promoter
(PdxCre mice) to generate PVhlKO mice, which lacked
Vhl only in the pancreas (13, 14). PVhlKO mice
were born with the expected Mendelian frequency and, in contrast to
βVhlKO mice, displayed normal body weight
(Supplemental Figure 1F). Recombination of the Vhl allele was
observed in PVhlKO islets but not in hypothalami (Figure 1A). Stabilization and expression of
Hif-1α was induced in the pancreas of PVhlKO mice
(Figure 1C) with greater than 70% of
β cells expressing Hif-1α and a 70% reduction in
Vhl mRNA (Supplemental Figure 1, G and H). We did not detect
Hif-2α in the pancreas of βVhlKO or
PVhlKO mice, and pancreatic weight was normal in
PVhlKO mice with no clinical evidence of exocrine dysfunction in
these animals (data not shown).
Deletion of Vhl in β cells or the pancreas
impairs glucose homeostasis in mice. (A) Recombination of the Vhl allele was assayed by
PCR in islets, cerebral cortex (cortex), and hypothalami (hyp) from WT and KO mice
for βVhlKO and PVhlKO mice. Positive
and negative PCR controls for deletion were performed using DNA from
Vhl-null cells, Vhl control (cont) cells, WT
cells, and without addition of DNA. Arrows indicate the deleted allele.
(B and C) Hif-1α staining using chromogenic
detection (left and middle panels) in islets from control,
βVhlKO, and PVhlKO mice and
combined chromogenic/immunofluorescence staining (right panels) co-localizing
Hif-1α and insulin in islets from βVhlKO
and PVhlKO mice. (D and E) Fed blood
glucose levels in 12-week-old female control, βVhlKO,
and PVhlKO mice. n = 8. (F and
G) Blood glucose after an intraperitoneal injection (2 g/kg body
weight) of glucose in 12-week-old female control,
βVhlKO, and PVhlKO mice.
n = 8 for null alleles; n = 24 for control
animals. (H and I) Fed plasma insulin levels in
12-week-old female control, βVhlKO, and
PVhlKO mice. n = 8. *P
< 0.05, **P < 0.01, ***P <
0.001 compared with control.
Generation of βVhlKO and PVhlKO mice
permitted examination of the role of Vhl in glucose homeostasis in vivo.
Twelve-week-old female βVhlKO and
PVhlKO mice displayed significantly elevated fed glucose levels and
impaired glucose tolerance (Figure 1,
D–G). Glucose intolerance was also seen in 12-week-old male
βVhlKO and PVhlKO mice (Supplemental
Figure 2, A and B). Fed insulin levels were reduced in both
βVhlKO and PVhlKO mice (Figure 1, H and I), fasting insulin levels in
PVhlKO mice were similar to those seen in control animals, and
βVhlKO mice displayed a mild fasting hypoinsulinemia
compared with control mice (Supplemental Figure 2, C and D). Insulin tolerance tests
in PVhlKO mice demonstrated no impairment of insulin sensitivity
(data not shown). The similarity of the glucose homeostasis phenotypes in both
βVhlKO and PVhlKO mice indicated
that this was not due to the hypothalamic deletion of Vhl seen in
the βVhlKO animals.
Deletion of Vhl in β cells or in the pancreas does not alter
β cell mass, proliferation, or survival or cause pancreatic tumors. The VHL/HIF pathway has been implicated in both growth factor–mediated
cell proliferation and cell survival. Therefore, we undertook islet morphometric
analysis in βVhlKO and PVhlKO mice to
exclude reduction of β cell mass as the cause for the impaired glucose
homeostasis. Absolute β cell mass was reduced in
βVhlKO animals, but when expressed as a percentage of
body mass was equivalent to that in control mice (Supplemental Figure 3, A and B).
β cell area in βVhlKO mice was equivalent to
that seen in control animals (Supplemental Figure 3C). Likewise, we detected no
significant difference in β cell mass (either expressed as an absolute
value or as a function of body weight) or β cell area in
PVhlKO mice compared with control animals (Supplemental Figure 3,
D–F). Consistent with these findings, there were no alterations in
β cell apoptosis or proliferation rates in both strains (Supplemental
Figure 3, G and H). In both mouse lines, organization of α and
β cells was preserved, but in βVhlKO mice
occasional α cells were scattered among the β cells
(Supplemental Figure 4, A and B). Taken together, these findings imply that the in
vivo defects in glucose handling were not the result of ablation of β
cells. We did detect alteration in the vascularization of the pancreas in
βVhlKO and PVhlKO mice by staining
for Cd31, although expression of this vascular marker as determined by RT-PCR was not
increased in βVhlKO and PVhlKO islets
compared with those of controls (Supplemental Figure 4, C–E).
Interestingly, however, and consistent with the normal cell proliferation and
survival parameters, we did not detect tumors or cyst formation in the pancreases of
βVhlKO or PVhlKO mice up to 12
months old (data not shown), suggesting that these pathologies arise from cell types
other than those in which we deleted Vhl (i.e., β cells or
Pdx1-expressing cells) or involve additional events such as expression of
Deletion of Vhl impairs GSIS in vivo and in vitro. The absence of changes in β cell mass in the islets of
βVhlKO or PVhlKO mice suggested that
functional defects in β cells underlie the alterations in glucose
homeostasis. Consistent with this idea, in vivo GSIS was severely blunted in
PVhlKO mice (Figure 2A). To
define the nature of the defects in GSIS seen with the deletion of
Vhl, we undertook further in vitro analysis of β cell
function using isolated islets and β cells. In perifusion studies,
insulin release was equivalent in isolated control and
βVhlKO islets under basal conditions (2 mmol/l glucose;
Figure 2B). As the perifusate glucose
concentration was increased to 20 mmol/l, peak and sustained GSIS were markedly
impaired in βVhlKO islets (Figure 2B). When the same islets were challenged with 20 mmol/l glucose
and 500 nmol/l phorbol ester to induce potentiated insulin release,
βVhlKO islets had a response that reached 60% of
control islets, suggesting significant preservation of the response to a non-glucose
potentiator of GSIS (Figure 2C). In static
incubation experiments, GSIS was also markedly impaired in PVhlKO
islets (Figure 2D). To further probe the site of
the defective GSIS we used the non-glucose secretagogues α-ketoisocaproic
acid (which is exclusively metabolized in mitochondria) and potassium chloride (which
depolarizes β cell membranes independently of glucose metabolism).
Stimulated insulin secretion in βVhlKO islets was normal
with both of these agents, while GSIS was again impaired, suggesting that the defect
is proximal to mitochondrial metabolism (Figure 2E).
Defective GSIS in mice lacking Vhl in β cells or
the pancreas. (A) Plasma insulin levels before and after an intraperitoneal
injection of glucose (3 g/kg body weight) in 12-week-old female control (open
squares) and PVhlKO (filled squares) mice. n =
7–9 per genotype. (B and C) Insulin
secretion from isolated control (open squares) and
βVhlKO islets (closed squares) in response to
perifusion with 2 and 20 mmol/l glucose and 500 nmol/l phorbol ester (PMA).
n = 3. (D) Insulin secretion from isolated
control and PVhlKO islets in static cultures in response to 2 and
20 mmol/l glucose. n = 5. (E) Insulin secretion from
isolated control and βVhlKO islets in static cultures
in response to 2 and 20 mmol/l glucose, 10 mmol/l α-ketoisocaproic
acid (αKIC), and 25 mmol/l potassium chloride (KCl).
n = 8. **P < 0.01.
Increased expression of constitutively activated HIF-1α in
β cells impairs GSIS. We next examined whether the defects in GSIS could result from increased activation
of HIF-1α using a mouse β cell line. Elevated expression of
HIF-1α was achieved in Min6 cells transduced with a retrovirus expressing
a constitutively active HIF-1α (Figure 3A). Examination of these cells revealed impaired insulin release in response
to glucose, indicating that activation of HIF-1α perturbs GSIS (Figure
Dependence of abnormal glucose homeostasis on upregulation of
Hif-1α and phenotype of βHif1aKO mice. (A) Western blot for Hif-1α and tubulin loading control
in Min6 cells transfected with activated Hif-1α mutant.
α-tubulin was resolved on a 10% gel, and Hif-1α was
analyzed on a separate 6% gel after equal amounts of the same cell lysate were
loaded. Blots are typical of 2 independent experiments. (B) Insulin
secretion from Min6 cells transfected with activated Hif-1α mutant or
empty vector in response to 2 and 30 mmol/l glucose. n = 5.
(C) Blood glucose after an intraperitoneal injection (2 g/kg body
weight) of glucose in 4- to 6-month-old male control (open squares) and
βVhlHif1aKO (filled squares) mice.
n = 8. (D) Blood glucose after an intraperitoneal
injection (2 g/kg body weight) of glucose in 4- to 6-month-old male control (open
squares) and βHif1aKO (filled squares) mice.
n = 8. (E) Insulin secretion from isolated
control, βHif1aKO, and
βVhlHif1aKO islets from 4- to 6-month-old male
mice in static cultures in response to 2 and 20 mmol/l glucose. n
= 8. *P < 0.05, **P < 0.01
compared with control.
Rescue of defective glucose homeostasis in βVhlKO mice by concomitant
deletion of Hif1a and lack of glucose homeostasis phenotype in βHif1aKO
mice. To test genetically in vivo the role of Hif-1α in mediating the
perturbation in glucose homeostasis following Vhl deletion, we
generated mice lacking both Vhl and Hif1a in
pancreatic β cells (βVhlHif1aKO mice) using
a mouse with a floxed allele of Hif1a. These mice had normal fasted
and fed blood glucose levels (Supplemental Figure 4, F and G) and normal glucose
tolerance at 4–6 months of age (Figure 3C). Furthermore the dwarf phenotype seen in
βVhlKO mice was reversed in these animals (Supplemental
The generation of βHif1aKO mice, which lack
Hif1α in RIPCre-expressing cells as part of the genetic
crosses used to breed βVhlHif1aKO mice, also permitted
us to analyze the impact of reduced Hif-1α activity upon glucose
homeostasis. These animals, which had normal body weight (Supplemental Figure 4I),
displayed normal glucose handling on glucose tolerance testing at both 12 weeks and
4–6 months of age (Figure 3D and
Supplemental Figure 4J). Studies in isolated islets from these mice revealed a slight
but significant increase in insulin release under basal (2 mmol/l) glucose
conditions, but no difference in GSIS compared with control mice (Figure 3E). Furthermore, when we examined GSIS in islets
from βVhlHif1aKO mice, this was slightly enhanced
compared with that seen in control animals (Figure 3E). Together, these findings suggest that Hif-1α may have a mild
restraining effect upon β cell function and that the abnormal GSIS seen
in βVhlKO mice is dependent upon upregulation of
These results indicate that appropriate expression of Hif-1α is required
for normal β cell function, and for GSIS in particular. The rescue of the
phenotype of βVhlKO mice by concomitant deletion of
Hif1a, the defective GSIS in cells expressing constitutively
active HIF-1α, and the concordant phenotype seen in
PVhlKO mice suggest that this abnormality is not the result of other
features that may be present in the βVhlKO mice due to
the hypothalamic deletion.
Alteration in expression of key components of the glucose-sensing apparatus and
glycolytic pathway in islets lacking Vhl. The profound defects in GSIS in the absence of abnormalities in β cell
mass and the preserved insulin release in response to α-ketoisocaproic
acid suggested that β cells lacking Vhl had
abnormalities in glucose-sensing or in the proximal elements of glucose metabolism.
We therefore examined expression of components of the glucose-sensing and glycolytic
apparatus, which are known targets of the VHL/HIF pathway. In both
βVhlKO and PVhlKO islets, there was
a significant reduction of Glut2 mRNA expression and a concomitant
induction in Glut1 mRNA expression with parallel changes in Glut2
and Glut1 production in pancreatic sections and in isolated islets (Figure 4, A–F). In particular, there was a
striking loss of cell membrane Glut2 immunofluorescence and protein production in
βVhlKO and PVhlKO islets (Figure
4, D–F). Expression of
Gck mRNA was also reduced in islets of both genotypes (Figure
4, A and B). Glut1
expression was increased and Glut2 expression was reduced in Min6
cells expressing constitutively active HIF-1α or exposed either to
hypoxia or the HIF activator dimethyloxalylglycine (16) (Figure 4G and Supplemental
Figure 5, A and B), supporting the conclusion that the observed expression changes in
βVhlKO and PVhlKO islets are
mediated by increased Hif-1α activity.
Abnormal expression of glucose-sensing apparatus and glycolytic genes in
βVhlKO and PVhlKO mice. (A and B) Expression of Glut1,
Glut2, and Gck mRNA in
βVhlKO and PVhlKO islets relative
to control islets. n = 5. (C) Glut1 immunostaining
in control, βVhlKO, and PVhlKO
islets. Representative images are shown. Scale bars: 150 μm.
(D and E) Glut2 (green) and insulin (red) staining in
control, βVhlKO, and PVhlKO islets.
Representative images are shown. Scale bars: 100 μm. (F)
Western blotting for Hif-1α, Glut1, Glut2, and α-tubulin
(loading control) in islets isolated from PVhlKO mice. Glut1,
Glut2, and α-tubulin were resolved on a 10% gel, and blots were probed
and stripped for each of the 3 antibodies. Hif-1α was analyzed on a
separate 6% gel with an equal amount of the same cell lysate loaded.
Representative blots are shown and are typical of 2 independent experiments.
(G) Expression of Glut1 and
Glut2 mRNA in Min6 cells transfected with activated
HIF1α mutant relative to empty
vector–transfected control cells. n = 3.
(H and I) Expression of Gapdh,
Aldola, Pfk, and Pdk1 mRNA
in isolated βVhlKO and PVhlKO islets
relative to control islets. n = 5. *P <
0.05, **P < 0.01, ***P < 0.001
compared with control.
Analysis of the expression of a panel of glycolytic genes revealed that expression of
pyruvate dehydrogenase kinase 1 (Pdk1), phosphofructokinase
(Pfk), and Gapdh was increased in islets
isolated from βVhlKO and PVhlKO mice,
while aldolase expression was reduced (Figure 4,
H and I). Increased Pdk1 expression inhibits pyruvate dehydrogenase
activity and restricts the entry of pyruvate to mitochondrial oxidative pathways,
while reduced aldolase expression further contributes to reducing flux through
We next examined the expression of key β cell transcription factors. In
both βVhlKO and PVhlKO islets, there
was a significant reduction in the expression of Nkx6.1, a
homeodomain transcription factor that regulates GSIS in β cells (17) (mRNA expression as a percentage of control:
βVhlKO, 51.5% ± 11.5% vs. control, 100.0%
± 10.1%, P < 0.01, n = 6;
PVhlKO, 56.6% ± 3.4% vs. control, 100.0%
± 13.8%, P < 0.05, n = 5). In
contrast, the expression of hepatocyte nuclear factor 1a (Hnf1a),
Hnf3b, Hnf4a, and NeuroD was
unaltered (data not shown). The glycoprotein E-cadherin (Ecad) has
been implicated in β cell function (18), and we have recently demonstrated that the VHL/HIF pathway suppresses
its expression in renal cancer cells (19). In
both βVhlKO and PVhlKO islets,
expression of Ecad was reduced (mRNA expression as a percentage of
control: βVhlKO, 46.6% ± 5.3% vs. control,
100% ± 10%, P < 0.01, n = 6;
PVhlKO, 39.7% ± 2.0% vs. control, 100% ±
10.7%, P < 0.01, n = 5).
Islets lacking Vhl have impaired glucose uptake and metabolism. The net effect of the expression changes in glucose-sensing and glycolytic genes
might be expected to reduce coupling of glucose uptake and glycolysis to
mitochondrial ATP production and insulin secretion. Therefore, we undertook further
functional analysis in islets isolated from βVhlKO and
PVhlKO mice, initially studying glucose uptake by isolated
β cells. Control β cells incubated in 2.5 mmol/l glucose
rapidly took up
(2-NBDG), a fluorescent D-glucose derivative (20) (Figure 5A). In
contrast, both total steady-state fluorescence and the rate of 2-NBDG uptake were
significantly reduced in βVhlKO and
PVhlKO islets (Figure 5,
A–C, and data not shown).
Glucose uptake and metabolism is impaired in β cells lacking
Vhl, but mitochondrial function is preserved. (A) Continuous monitoring of 2-NBDG uptake (18 nmol/l added at the
time points indicated by vertical arrows) into control and
βVhlKO β cells incubated in 2.5 mmol/l
D-glucose. (B and C) Rate of uptake of 2-NBDG into
β cells from control, βVhlKO, and
PVhlKO mice. n = 8 groups of β
cells from 3 animals of each genotype. *P < 0.05,
**P < 0.01 compared with control.
(D–G) NADH and FAD++
autofluorescence was monitored by confocal microscopy for control (D
and F), βVhlKO (E), and
PVhlKO (G) β cells at the indicated
glucose concentrations. Results are expressed as a percentage of basal values
± SEM for 8 groups of β cells from 3 animals of each
genotype. (H and I) Ca2+ signals in response
to indicated glucose concentrations and tolbutamide (200 μmol/l) in
control, βVhlKO, and PVhlKO
β cells. Recordings were made from 50 cells of each genotype from 2
animals of each genotype. (J and K) Ca2+
signals in response to methyl succinate (10 mmol/l) in control,
βVhlKO, and PVhlKO β
cells. Recordings were made from 50 cells of each genotype from 2 animals of each
Next we monitored the increase in metabolic flux as glucose is metabolized by
β cells, by measuring cellular NADH and flavin adenine dinucleotide
(FAD++) levels using autofluorescence (21). In response to increasing glucose concentrations, control
β cells exhibited a robust increase in NADH and a concomitant fall in
FAD++ fluorescence (Figure 5, D
and F). In contrast, these responses were essentially absent in
βVhlKO and PVhlKO β
cells, indicating altered mitochondrial metabolism in response to glucose (Figure
5, E and G). To probe the site of this
defect, we monitored Ca2+ fluxes in Fura-2AM–loaded
β cells from βVhlKO and
PVhlKO mice in response to glucose, methyl-succinate (which enters
the mitochondrial respiratory chain at complex II) and the KATP channel
blocker tolbutamide. Glucose-stimulated Ca2+ mobilization was markedly
attenuated in βVhlKO and PVhlKO
β cells compared with controls (Figure 5, H and I). In contrast, responses to methyl-succinate and tolbutamide were
preserved in βVhlKO and PVhlKO
β cells (Figure 5, J and K), which,
in combination with the normal secretory response to α-ketoisocaproic
acid (Figure 2E), places the defect in GSIS
between the glucose uptake machinery and mitochondrial metabolism. Taken together
with the abnormalities in the proximal components of the glucose-sensing apparatus,
these findings demonstrate that the VHL/HIF pathway is critical in regulating
mammalian pancreatic β cell function.
Pancreatic β cells have high rates of aerobic metabolism compared with other
cell types, and islet oxygenation is markedly higher than in the exocrine pancreas or
other tissues (22). In the current studies, we
investigated whether inactivation of Vhl in β cells and
concomitant Hif activation alters glucose sensing. Although available transgenic lines
are effective at β cell deletion, they are not completely selective (12, 13). We
therefore used 2 inactivation strategies, both of which inactivated Vhl
in β cells. The concordant defects in GSIS that we observed in
βVhlKO and PVhlKO mice, combined with
our analyses of isolated islets, imply that loss of Vhl in the
β cell results in a profound defect in GSIS.
The most extensively studied function of VHL is its role in the destruction of
HIF-1α and HIF-2α in the presence of oxygen (3). VHL also interacts with a number of other
proteins and influences diverse intracellular pathways (reviewed in ref. 23). Proteins that have been reported to interact
with VHL in other cell types, and which could alter glucose uptake and GSIS in
β cells, include PKC-λ/ι and -δ (24, 25) and
Sp1 (26). To determine whether the effects of
Vhl loss of function were mediated by Hif-1α, we
therefore generated βVhlHif1aKO mice. Our finding of normal
glucose homeostasis and body weight in βVhlHif1aKO mice
suggests that the alteration in both these parameters caused by deletion of
Vhl is mediated via activation of Hif-1α. This experiment
does not formally exclude the possibility that hypothalamic deletion of
Hif1a in βVhlHif1aKO mice contributes to
the correction of glucose homeostasis. Nevertheless, our data on β cells
expressing a constitutively activated HIF-1α show that HIF-1α
activation is sufficient to have major effects on GSIS and gene expression. Together
with the data from PVhlKO mice, we conclude that the β cell
phenotypes most likely arise from increased β cell Hif-1α
activation and consequent engagement of the Hif-1α–dependent
gene expression program.
The VHL/HIF pathway has many downstream targets, and therefore abnormal β
cell function in βVhlKO and PVhlKO mice is
likely to be due to a combination of effects. We demonstrate marked abnormalities in
GSIS in Vhl-deficient islets but no alterations in either β
cell proliferation or survival. The molecular mechanisms underlying this defect include
reduced Glut2 expression, which has been shown to be sufficient to
perturb glucose homeostasis in mice and humans (27, 28), and attenuated
Gck expression, which alone (10,
29) or in combination with additional
β cell defects (30) causes
β cell dysfunction. Our findings also suggest that increased
Glut1 expression compromises appropriate glucose handling by
β cells and does not compensate for the marked reduction in Glut2
expression. The Km for D-glucose of rodent Glut2 is
17 mmol/l, allowing a wide range of physiological blood glucose levels to be sensed by
the β cell. GLUT1, however, has been shown to have a
Km for D-glucose of 2.3–2.6 mmol/l
in humans and to confer a human β cell Vmax of 3
mmol/l/min, compared with a rodent β cell Vmax
of 32 mmol/l/min for glucose uptake via Glut2. This suggests that
βVhlKO and PVhlKO islets relying on
Glut1 to supply glucose into the β cell sense little difference between 2
and 20 mmol/l glucose, since Glut1 facilitates glucose transport at near maximum
capacity at these concentrations. The absolute levels of glucose uptake depend on the
absolute Glut1 expression levels, but our data show the increased Glut1 expression does
not normalize glucose uptake in an appropriate manner to restore GSIS in
We also demonstrate alterations in the expression of glycolytic genes that have an
additional negative impact upon GSIS. Increased Pdk1 expression
inhibits pyruvate dehydrogenase activity and would restrict the entry of pyruvate to
mitochondrial oxidative pathways, while reduced aldolase expression would be anticipated
to reduce flux through glycolysis as well. Our studies using non-glucose secretagogues
together with metabolic and imaging studies place the defects in GSIS proximal to the
mitochondria. The combination of markedly reduced glucose uptake and impaired glycolytic
flux would prevent the burst of mitochondrial oxidative phosphorylation required for
quantitative coupling of glucose uptake to insulin release when glucose levels rise.
Taken together, this combination provides a sufficient explanation for the abnormalities
in βVhlKO and PVhlKO mice.
Recently it was reported that mice with β cell–specific deletion
of the Hif-1α dimerization component Arnt/Hif1b show
impaired glucose homeostasis (9). In contrast, our
genetic manipulation of deleting Vhl in insulin-producing cells led to
increased β cell Hif activity. While it might have been predicted that these
2 distinct perturbations of the Hif pathways would have opposite effects upon glucose
homeostasis, a number of explanations could underlie the similar phenotypes resulting
from deletion of Vhl or Arnt in mouse β
cells. Firstly, in addition to HIFα subunits, ARNT dimerizes with the aryl
hydrocarbon receptor, and loss of the AHR/ARNT response may influence β cell
function. Indeed, siRNA for Ahr was reported to decrease GSIS in the
Min6 β cell line (9). Secondly,
Arnt will have been deleted not only in β cells, but
also in the hypothalamus, which could contribute to the reported abnormalities in
glucose homeostasis seen in mice generated using the RIPCre animal
(9). Third, glucose intolerance has been
demonstrated in the pure RIPCre strain on certain genetic backgrounds,
and our own use of PdxCre animals (14) provides evidence that the effect we observed was not due to the
RIPCre transgene. We also demonstrate normal glucose tolerance in
the RIPCre strain in our hands. Our studies of mice lacking
Hif1a in insulin-producing cells give further insights into the role
of this pathway in β cells and suggest that Hif-1α may have a
mild restraining role on β cell function under basal conditions. There are
additional considerations in evaluating the potential pathological relevance of our
findings compared with those of Gunton et al. (9),
which suggested that decreased HIF could contribute to human type 2 diabetes. First,
type 2 diabetes is clearly multifactorial, and it is plausible that perturbations of HIF
in either direction could contribute to its pathophysiology, although our studies with
Hif1a deletion in β cells suggest that this may not be
the case. Second, HIF activation by tissue hypoxia is a well-understood
pathophysiological construct, whereas the circumstances under which HIF would be
suppressed — other than by genetic manipulation — are unclear.
In this context it is also important to appreciate that HIF is activated, at least in
the kidney, in response to very minor alterations in oxygen delivery that are within the
physiological range. Finally, our results indicate that although engagement of the
HIF-1α–dependent transcriptional program would be anticipated to
protect β cells from hypoxia, the consequence is perturbation of the
intimate relationship between oxidative phosphorylation and GSIS, highlighting the
complexity of the role of the VHL/HIF pathway in β cell function.
Our findings are likely to have clinical relevance independent of the putative role of
the VHL/HIF pathway in human type 2 diabetes. Tissue oxygen delivery is reduced in many
settings, including sepsis syndrome, acute and chronic pulmonary conditions, and
obstructive sleep apnea. Indeed, sepsis and obstructive sleep apnea are associated with
impaired glucose metabolism, and acute hypoxia causes glucose intolerance in humans
(31). Our findings suggest that HIF activation
in the β cell in these settings would impair GSIS. However, understanding
the overall effects of the VHL/HIF pathway and hypoxia on metabolic networks in these
human illnesses represents a complex challenge in systems biology, as it is unclear
precisely which tissues may contribute to abnormal glucose handling in the setting of
hypoxia in humans. However, a particular clinical scenario in which islet hypoxia may be
relevant is islet transplantation. This potentially attractive therapy for diabetes is
hampered by hypoxic graft failure, and it has been suggested that impaired graft
function may occur in part through activation of the HIF pathway (32). Our studies show that this is likely and provide mechanisms by
which this would occur. Our genetic experiments also suggest that pharmacological
manipulation of the HIF system, which is under investigation as a therapeutic strategy
in cancer, anemia, cardiac ischemia and failure (33), and inflammatory conditions such as glomerulonephritis (34), may have profound effects on β cell
function unless this treatment is targeted to specific organs. Vhl is a
tumor suppressor gene, and pancreatic cysts and tumors are a feature of human VHL
disease (35). However, the
βVhlKO and PVhlKO mice in our studies
did not develop these types of tumors, although they did show some increase in
vascularization. One explanation would be that the human tumors and cysts do not arise
from β cells or other cells expressing Pdx1, which would
reinforce the cell and tissue specificity of the tumor suppressor action of
Vhl. An alternative possibility is that further events are required for
the development of cysts and tumors in the pancreas, which typically present in the
third and fourth decades in humans. An important question has been the extent of
redundancy in the VHL/HIF system, and whether differential redundancy of HIF components
between cell types might account for this striking tissue specificity of tumors in VHL
disease (36). Our results may help to clarify
this by showing that in β cells Vhl loss is sufficient for
Hif-1α stabilization and activation, does not result in detectable
Hif-2α activation, and does not produce an increase in β cell
mass. The lack of Hif-2α activation is likely to reflect cell-type
specificity in Hif2a mRNA expression and is consistent with a previous
study in which acute hypoxia did not activate Hif-2α in rat islets (37). Interestingly in kidney cells, the site of
malignant tumors in VHL patients, acquisition of Hif2a expression
appears to be crucial in progression and tumor growth (15). Finally, it is becoming increasingly clear that the VHL/HIF pathway also
has important physiological roles that may not necessarily be manifest as disease
processes, such as involvement in innate immunity (5), in muscle performance (7), in
neutrophil survival decisions (6), in stem cell
differentiation and homing (38–40), and in the role of skin oxygen sensing (8).
In summary, our studies show that VHL/HIF oxygen-sensing mechanisms play a critical role
in glucose homeostasis, and activation of this pathway causes β cell
Mice and animal care. Floxed Vhl allele (Vhl+fl; The Jackson
Laboratory) mice were crossed with RIPCre mice (The Jackson
Laboratory) and PdxCre mice (14) to generate compound heterozygote mice for both Cre strains. Double
heterozygote mice were crossed with Vhl+fl mice to obtain
WT, Vhlfl/fl, Cre, and Cre
Vhlfl/fl mice for each Cre strain. Mice lacking
Vhl in RIPCre-expressing cells were designated
βVhlKO mice, and those lacking Vhl
in PdxCre-expressing cells were designated PVhlKO
mice. Hif1a+fl mice (41) were intercrossed with RIPCre mice and compound
heterozygote mice mated with Hif1a+fl mice to obtain mice
lacking Hif1a in insulin-producing cells, designated
βHif1aKO mice. βHif1aKO
mice were intercrossed with RIPCreVhlfl/fl, and compound heterozygote mice for all alleles
were subsequently crossed to generate mice lacking both Vhl and
Hif1a in insulin-producing cells, designated
βVhlHif1aKO mice. Mice were maintained on a 12-hour
light/12-hour dark cycle with free access to water and standard mouse chow (4% fat,
RM1; Special Diet Services) and housed in specific pathogen–free barrier
facilities. Mice handling and all in vivo studies were performed in accordance with
the United Kingdom Home Office Animal Procedures Act of 1986 and with approval of the
University College London Animal Ethics Committee. All mice were studied on a mixed
129S/C57BL/6 background with appropriate littermate controls. WT,
Cre transgenic, and Vhlfl/fl mice were
phenotypically indistinguishable with no differences in glucose tolerance between WT,
Cre transgenic, and Vhlfl/fl mice for
both βVhlKO and PVhlKO strains
(Supplemental Figure 5, C and D). Balanced numbers of mice of these genotypes were
therefore used as controls. All phenotypes described for the mice in Results were
present in both male and female animals. Genotyping of the mice was performed by PCR
amplification of tail DNA as previously described (11, 12, 14). Cre-mediated excision of Vhl was detected
by PCR on genomic DNA isolated from pancreatic islets and hypothalami. DNA (150 ng)
was assayed on the Opticon 2 system (MJ Research) using the primers mVHLPCR5
(5′-CAAACTGCATGCCTGGTACCCAC-3′) and mVHLPCR8
Physiological measurements. Body and tissue weights were determined using a Sartorius BP610 balance. Blood
samples were collected from mice via tail vein bleeds or from cardiac puncture on
terminally anesthetized mice. Blood glucose was measured using an Ascensia Elite
Glucometer (Bayer Corp.). Plasma insulin levels were determined using an
ultrasensitive rat insulin ELISA (Crystalchem Inc.) using a mouse standard or with a
mouse ELISA (Linco Research). Glucose tolerance tests and GSIS tests were performed
on mice after a 16-hour fast as previously described (14). Animals were injected intraperitoneally with 2 g/kg or 3 g/kg
D-glucose, and blood glucose or insulin levels, respectively, were determined.
Immunohistochemistry and morphometric analysis. Immunohistochemistry for insulin and glucagon and morphometric analysis were
performed using methods described previously (12, 14). Antibodies used were as
follows: mouse anti-insulin antibody (clone K36aC10; Sigma-Aldrich), rabbit
anti-glucagon antibody (Abcam), chicken anti-mouse IgG–Alexa Fluor 488
conjugate, and anti-rabbit IgG–Alexa Fluor 594 (Invitrogen). For
immunostaining of Hif-1α (rabbit polyclonal; Novus Inc.), Glut1 (rabbit
polyclonal; Alpha Diagnostics Ltd.), Cd31 (rabbit polyclonal; Santa Cruz
Biotechnology Inc.), and Glut2 (rabbit polyclonal, gift from B. Thorens, University
of Lausanne, Lausanne, Switzerland) in paraffin-embedded sections, antigen retrieval
with DAKO target retrieval was performed, and for visualization either a DAKO CSA kit
or DAKO Envision kit or anti-rabbit Alexa Fluor 594–conjugated secondary
antibodies were used according to the manufacturers’ instructions. For
Hif-1α and insulin double-staining, paraffin-embedded sections were first
immunostained and visualized for Hif-1α (rabbit polyclonal, Alpha
Diagnostics) using the DAKO CSA kit. After visualization, sections were immunostained
with mouse anti-insulin antibody (clone K36aC10; Sigma-Aldrich) and Alexa Fluor
488–conjugated chicken anti-mouse IgG. Mounting solution containing DAPI
(VECTASHIELD DAPI; Vector Laboratories) was used to identify nuclei. For detection of
apoptosis, rabbit anti–active caspase-3 antibody (BD Biosciences
— Pharmingen) was used, and to detect proliferation, rabbit anti-Ki67
antibody (Abcam) was used, both as previously described (14). Sections were imaged via light, fluorescence, or confocal
microscopy, and images were captured as previously described (12, 14).
Western blotting. Urea-SDS lysis buffer was used to prepare protein lysates, and protein concentrations
were measured using BCA (Pierce Biotechnology). Lysates were run on SDS/PAGE gels and
then transferred to Immobilon-P PVDF membranes (Millipore). Membranes were blocked in
5% milk/1% BSA for 45 minutes and primary antibodies applied for 2 hours at room
temperature. Membranes were then washed and incubated with an HRP-linked secondary
antibody (DAKO) for 1 hour at room temperature. Following further washes, the
membrane was developed with ECL plus (Amersham Biosciences). Primary antibodies used
were Hif-1α, Glut1, and Glut2 as described above, and
Islet and Min6 cell experiments. Mice were sacrificed by cervical dislocation, and the common bile duct was cannulated
and its duodenal end occluded by clamping. Liberase solution (2 ml at 0.25 mg/ml in
HBSS) was injected into the duct to distend the pancreas. The pancreas was excised,
incubated at 37°C for 15 minutes, and mechanically disrupted in 10 ml of
HBSS (supplemented with 1% BSA). Cellular components were collected by centrifugation
(201 g for 1 minute), washed, and resuspended in 10 ml of HBSS.
Islets were hand-picked under a microscope and washed once in HBSS. Prior to DNA or
protein extraction, islets were collected by centrifugation at 5,724
g for 2 minutes and stored at –80°C. For
insulin secretion studies, isolated islets were cultured overnight in DMEM with 11
mmol/l glucose. Medium was replaced with KRBH (Krebs-Ringer buffer containing 2
mmol/l D-glucose and 10 mmol/l HEPES) 1 hour prior to study. Dynamic insulin release
was then assessed using a multichamber perifusion system at 37°C in a
temperature-controlled environment, as previously described (42). Insulin was measured in perifusate samples by
radioimmunoassay, as previously described (42). Static insulin release was assessed using batch cultures of 5 islets in
150 μl KRBH plus additional D-glucose, 25 mmol/l KCl, or 10 mmol/l
α-ketoisocaproic acid (Sigma-Aldrich) for 1 hour at 37°C.
Insulin release was measured by ELISA. Min6 cells were cultured as previously
described (42). To activate
Hif-1α, cells were incubated in 1% oxygen or 0.5 mmol/l
dimethyloxalylglycine for 16 hours. For examination of gene expression in Min6 cells,
retroviral transduction with active HIF-1α (P402A and P564A) was
performed as previously described (19). GSIS
from infected Min6 cells was measured by static culture in a 96-well plate (3
× 104 cells per well). Following overnight culture, media was
replaced with KRBH for 1 hour before static secretion assays were performed (as
Gene expression studies. Isolated islets or Min6 cells were harvested and RNA extracted immediately with
RNABee reagent (Biogenesis) or RNeasy kits (Qiagen) according to the
manufacturers’ instructions. Total RNA (0.5–2 μg)
was reverse transcribed using a first-strand cDNA synthesis kit (Roche). Quantitative
PCR was carried out with first-strand cDNA and commercial TaqMan Assays (Applied
Biosystems) on the Opticon 2 System (MJ Research Inc.). Expression levels of
Glut1, Glut2, Gck,
Ecad, Nkx6.1, NeuroD1,
Hnf3b, Hnf1a, Hnf4a,
Pfk, Aldolase, Gapdh,
Pdk1, Vhl, and Cd31 were
normalized to hypoxanthine-guanine phosphoribosyl transferase
(Hprt), and data were analyzed using the
2–ΔΔCT method (12). Details of the primers used are presented in
Imaging studies in isolated islets. Islets isolated as described above were dissociated into single cells by incubation
in trypsin at 21°C for 2.5 min with gentle agitation. Cells were washed
once before being allowed to attach to poly-l-lysine–treated
glass coverslips. Cells were incubated for 48 hours in DMEM culture medium with
l-glutamine supplemented with 10% heat-inactivated FBS and 5.5 mmol/l
D-glucose. The medium was changed to HBSS supplemented with 20 mmol/l HEPES and 2
mmol/l D-glucose 1 hour prior to imaging. Cells were imaged on a 37°C
heated stage on a Zeiss LSM 510 confocal microscope. β cells were stained
following analysis, using dithizone for identification (43). Uptake of the fluorescent glucose analogue 2-NBDG
(Invitrogen) was measured by addition of 18 nmol/l 2-NBDG in the presence of 2.5
mmol/l D-glucose during continuous imaging with an excitation wavelength of 458 nm
and collection of light emitted at greater than 505 nm (20). Reduced NADH and oxidized flavoprotein (FAD++)
autofluorescence in response to the addition of D-glucose in 9 mmol/l increments were
captured as previously described (21, 44). The mitochondrial uncoupler carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP) was used to artificially evoke maximally
oxidized states of the NADH/flavoproteins to establish the dynamic range of the
signals. Ca2+ imaging using Fura-2AM was performed as previously described
(21, 44). Measurements were obtained by averaging the signal collected in a region
of interest drawn across the maximum area of the β cell. Mono-methyl
succinate (Fluka) and tolbutamide (Sigma-Aldrich) were used to stimulate
Ca2+ influx via mitochondrial metabolism and pharmacological
KATP channel closure respectively.
Statistics. Data are presented as mean ± SEM, unless otherwise indicated. All
statistics were performed using Minitab (version 13) and GraphPad (Prism 4) software.
Paired and unpaired t tests and 1- and 2-way ANOVA were performed,
using the post-hoc Newman-Keuls test where appropriate. P <
0.05 was regarded as statistically significant.
View Supplemental data
We thank Randall Johnson (UCSD, La Jolla, California, USA) for the mice with a floxed
allele of Hif1a and Keval Chandarana and Gemma Pearson for technical
assistance. This work was supported by grants from the Wellcome Trust (to D.J. Withers
and M.R. Duchen), the British Heart Foundation (to P.H. Maxwell), Diabetes UK (to D.J.
Withers), the Medical Research Council (to D.J. Withers), Cancer Research UK (to P.H.
Maxwell), and the Biotechnology and Biological Sciences Research Council (to D.J.
Withers). Work was performed in part in the Functional Genomics of Ageing Consortium,
which is supported by the Wellcome Trust Functional Genomics programme (to D.J. Withers
and others), and in the BetaCellTherapy consortium, which is supported as an integrated
project by the sixth European Union framework program (to D.J. Withers, P.L. Herrera,
and others). Min6 cells were a kind gift from J. Miyazaki (Osaka University Medical
School, Osaka, Japan).
Conflict of interest: P.H. Maxwell is a director and stockholder of ReOx
Nonstandard abbreviations used: ARNT, aryl hydrocarbon receptor nuclear
βHIF1αKO mice, mice lacking
HIF1α in pancreatic β
mice, mice lacking Vhl and
HIF1α in pancreatic β
cells; βVhlKO mice, mice lacking
Vhl in pancreatic β cells; Ecad,
E-cadherin; FAD++, flavin adenine dinucleotide; Gck, glucokinase; GSIS,
glucose-stimulated insulin secretion; 2-NBDG,
PdxCre mice, mice expressing Cre under control of the mouse
pancreatic and duodenal homeobox 1 promoter; Pfk, phosphofructokinase;
PVhlKO mice, mice lacking Vhl in the pancreas;
Pdk1, pyruvate dehydrogenase kinase 1; RIPCre
mice, mice expressing Cre under control of the rat insulin II promoter; VHL, von
Citation for this article:J. Clin. Invest.119:125–135 (2009). doi:10.1172/JCI26934
James Cantley’s present address is: Diabetes and Obesity Research
Program, Garvan Institute of Medical Research, Sydney, New South Wales,
Colin Selman’s present address is: Institute of Biological and
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