Article

Free Fatty Acids Block Glucose-Induced -Cell Proliferation in Mice by Inducing Cell Cycle Inhibitors p16 and p18

Division of Endocrinology and Metabolism, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Diabetes (Impact Factor: 8.1). 03/2012; 61(3):632-41. DOI: 10.2337/db11-0991
Source: PubMed

ABSTRACT

Pancreatic β-cell proliferation is infrequent in adult humans and is not increased in type 2 diabetes despite obesity and insulin resistance, suggesting the existence of inhibitory factors. Free fatty acids (FFAs) may influence proliferation. In order to test whether FFAs restrict β-cell proliferation in vivo, mice were intravenously infused with saline, Liposyn II, glucose, or both, continuously for 4 days. Lipid infusion did not alter basal β-cell proliferation, but blocked glucose-stimulated proliferation, without inducing excess β-cell death. In vitro exposure to FFAs inhibited proliferation in both primary mouse β-cells and in rat insulinoma (INS-1) cells, indicating a direct effect on β-cells. Two of the fatty acids present in Liposyn II, linoleic acid and palmitic acid, both reduced proliferation. FFAs did not interfere with cyclin D2 induction or nuclear localization by glucose, but increased expression of inhibitor of cyclin dependent kinase 4 (INK4) family cell cycle inhibitors p16 and p18. Knockdown of either p16 or p18 rescued the antiproliferative effect of FFAs. These data provide evidence for a novel antiproliferative form of β-cell glucolipotoxicity: FFAs restrain glucose-stimulated β-cell proliferation in vivo and in vitro through cell cycle inhibitors p16 and p18. If FFAs reduce proliferation induced by obesity and insulin resistance, targeting this pathway may lead to new treatment approaches to prevent diabetes.

Full-text

Available from: Rachel Stamateris
Free Fatty Acids Block Glucose-Induced b-Cell
Proliferation in Mice by Inducing Cell Cycle Inhibitors
p16 and p18
Jordan Pascoe,
1
Douglas Hollern,
1
Rachel Stamateris,
1
Munira Abbasi,
1
Lia C. Romano,
2
Baobo Zou,
2
Christopher P. ODonnell,
2
Adolfo Garcia-Ocana,
1
and Laura C. Alonso
1
Pancreatic b-cell proliferation is infrequent in adult humans and
is not increased in type 2 diabetes despite obesity and insulin
resistance, suggesting the existence of inhibitory factors. Free
fatty acids (FFAs) may inuence proliferation. In order to test
whether FFAs restrict b-cell proliferation in vivo, mice were in-
travenously infused with saline, Liposyn II, glucose, or both, con-
tinuously for 4 days. Lipid infusion did not alter basal b-cell
proliferation, but blocked glucose-stimulated proliferation, with-
out inducing excess b-cell death. In vitro exposure to FFAs in-
hibited proliferation in both primary mouse b-cells and in rat
insulinoma (INS-1) cells, indicating a direct effect on b-cells. Two
of the fatty acids present in Liposyn II, linoleic acid and palmitic
acid, both reduced proliferation. FFAs did not interfere with cyclin
D2 induction or nuclear localization by glucose, but increased ex-
pression of inhibitor of cyclin dependent kinase 4 (INK4) family cell
cycle inhibitors p16 and p18. Knockdown of either p16 or p18 res-
cued the antiproliferative effect of FFAs. These data provide evi-
dence for a novel antiproliferative form of b-cell glucolipotoxicity:
FFAs restrain glucose-stimulated b-cell proliferation in vivo and in
vitro through cell cycle inhibitors p16 and p18. If FFAs reduce
proliferation induced by obesity and insulin resistance, targeting
this pathway may lead to new t reatment approaches to prevent
diabetes. Diabetes 61:632641, 2012
b
- Cell mass and insulin secretory function are both
reduced in type 2 diabetes (13). Despite robust
adaptive b-cell proliferation in some rodent strains,
this phenomenon is variable, suggesting the exis-
tence of restraining inuences (1). The signals driving
adaptive b-cell proliferation remain poorly understood. Al-
though existing modelsobesity, insulin resistance, partial
pancreatectomy, pregnancy, and hyperglycemiashare in-
creased metabolic load on the b-cell, a common mechanism
has not been identied (4). One potential link may be in-
tracellular glucose metabolism, which is increased in hy-
perglycemic models but also drives b-cell proliferation in
certain normoglycemic conditions (510).
Factors limiting adaptive b-cell proliferation are even
less well unde rstood. Free fatty acids (FFAs) exert toxic
effects on b-cell survival a nd function and are predictive
of progression to type 2 diabetes independently of insulin-
mediated glucose uptake (1116). Although it has been
postulated that FFAs might stimulate b-cell proliferation in
the context of obesity (16), other proliferation drivers, such
as insulin resistance and hyperinsulinemia, are also present.
In fact, FFAs may inhibit b-cell proliferation (17,18). Data
remain discordant. In b-cell culture models, for example,
FFAs are neutral or stimulate proliferation during nutrient-
starvation, such as low glucose and serum starvation (19,20),
whereas FFAs block proliferat ion and cause apoptosis in
nutrient-stimulatory conditions (18,21). Studies addressing
this question in vivo have mostly concluded that FFAs do
not limit b-cell proliferation ( 2225). However, no in vivo
study has yet systematically evaluated the effect of high
FFAs on b-cell proliferation in both control and stimulated
conditions.
On the basis of work by others in rats (24,26,27), we
previously developed a 4-day glucose infusion model in
mice and showed that hyperglycemia stimulates both mouse
and human b-cell proliferation in vivo (2830). We have now
used our infusion hyperglycemia model to test whether FFAs
alter mouse b-cell proliferation in vivo in both basal and
glucose-stimulatory conditions. Our ndings illustrate a
novel form of in vivo glucolipoto xicity: FFAs block glu-
cose-mediated adaptive b-cell proliferation via induction of
cell cycle inhibitors p16 and p18.
RESEARCH DESIGN AND METHODS
Surgical catheterization. Mouse studies were approved by the University of
Pittsburgh Institutional Animal Care and Use Committee. Mice were housed in
controlled temperature, humidity, and 12-h light-dark cycle with free access to
chow and water. Detailed protocols for surgical catheterization and blood
sampling can be found in the online supplement to Alonso et al. (28). Ten- to
twelve-w eek-old male C57BL/6 J mice were anesthetized with inhaled 2%
isourane, and microrenathane catheters (MRE-025; Braintree Scientic) were
inserted into the left femoral artery and vein, tunneled subcutaneously to exit
the skin a t the upper back, tape d to a wire attached to posterior cervical
muscles (792500; A-M Systems), and conne cted to a 360° dual c hanne l swivel
(375/D/22QM; Instech). Catheter patency was maintained by continuous 7 mL/h
infusion of sterile saline containing 20 units/mL unfractionated heparin (APP
Pharmaceuticals) using a syringe pump (R99-EM; Razel ScienticInstruments).
Intravenous infusions. Intravenous infusions were begun 3 days after cath-
eterization (Fig. 1A). All blood samples were taken from the arterial catheter in
unhandled, awake mice; infusions were via the venous catheter. Chow was
provided throughout the experiment. Nonfasting morning arterial blood sam-
ples were taken at time 0, and at 8, 24, 48, 72, and 96 h. Glucose was measured
on whole blood, plasma was frozen for future measurement of insulin, and
RBCs were resuspended in 20 mL saline containing 100 units/mL heparin and
reinfused. After the time 0 blood sample, the infusio ns were started: SAL (0.9%
saline, 200 mL/h), LIP (Liposyn II 20%, 100 mL/h plus 0.9% saline, 100 mL/h),
GLU (50% dextrose, 100 mL/h plus 0.9% saline, 100 m L/h), or L+G (Liposyn II,
100 mL/h plus 50% dextrose, 100 mL/h). All infusates contained heparin
(2 units/h) and bromodeoxyuridine (BrdU, 100 mg/h). Immediately following
infusion, mice were killed and organs dissected for histological analyses or
islets isolated for molecular analyses.
From the
1
Division of Endocrinology and Metabolism, University of Pittsburgh,
Pittsburgh, Pennsylvania; and the
2
Division of Pulmonary, Allergy and Criti-
cal Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
Corresponding author: Laura C. Alonso, lca4@pitt.edu.
Received 18 July 2011 and accepted 18 October 2011.
DOI: 10.2337/db11-0991
J.P., D.H., and R.S. contributed equally to this work.
Ó 2012 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 prot,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
See accompanying commentary, p. 560.
632 DIABETES, VOL. 61, MARCH 2012 diabetes.diabetesjournals.org
ORIGINAL ARTICLE
Page 1
FIG. 1. Intravenous lipid infusion elevated FFA levels in both basal and hyperglycemic conditions. A : Timeline: mice with femoral artery and vein
catheters received continuous intravenous 4-day infusions of 0.9% saline, Liposyn II, 50% glucose, or Liposyn II plus 50% glucose. Arterial blood
was sampled daily. B and C: Glucose infusion elevated blood glucose (BG) levels consistently to moderate levels; coinfusion of lipid did not alter
the degree of hyperglycemia (n =2634). D and E: Glucose infusion transiently elevated plasma insulin levels; coinfusion of lipid did not alter the
degree of hyperinsulinemia (n =1315). F: Lipid infusion increased plasma FFA levels to a similar degree with and without coinfusion of glucose
(n =713). G: Oil Red O stain of liver showed end-organ lipid deposition in mice receiving intravenous Liposyn II. Data are mean 6 SEM; P values
by ANOVA. ns, nonsignicant. (A high-quality digital representation of this gure is available in the online issue.)
J. PASCOE AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 61, MARCH 2012 633
Page 2
Biochemical assays. Blood glucose was measured using an Ascencia XL
glucometer. Plasma insulin was measured by radioimmunoassay (Linco sen-
sitive rat insulin RIA kit; Millipore). FFAs were measured by colorimetric assay
(Roche) on terminal blood samples obtained by cardiac puncture into pre-
chilled tubes on ice.
Histological analyses. Pancreata were xed in Bouins xative for 4 h and
parafn embedded. TUNEL, BrdU, and cyclin D2 staining were performed as
described (28). For Oil Red O, livers were frozen in optimal cutting temper-
ature compound; 10 mm cryosections were xed in formalin, rinsed in 60%
isopropanol, stained 15 min with 0.3% Oil Red O in 60% isopropanol, and he-
matoxylin counterstained. For proliferating cell nuclear antigen (PCNA) and
Ki67 staining, parafn sections were blocked in 1% BSA/5% goat serum/0.1%
triton X-100, incubated overnight at 4°C with anti-PCNA (1:500; Santa Cruz
Biotechnology) or anti-Ki67 (1:200; Neomarkers) and anti-insulin (1:500,
Dako), incubated 30 min with secondary antibodies, and mounted with
Hoechst. The number of b-cells quantied was as follows: 2,147 6 116 (BrdU),
1,814 6 144 (TUNEL), 1,675 6 147 (PCNA), and 1,970 6 204 (Ki67). Micros-
copy was performed using an Olympus Fluoview confocal microscope or an
Olympus Provis microscope.
Islet experiments. Islets were isolated by ductal collagenase injection and
Ficoll separation (28). For immunoblot, islets were washed in PBS containing
100 nmol/L sodium orthovanadate immediately after isolation and frozen for
future analysis. For islet cell culture experiments, trypsinized islet cells were
plated on glass coverslips in RPMI containing 10% FBS and penicillin/strep-
tomycin (islet medium) containing 5.5 mmol/L glucose. Dispersed islet cells
were cultured for 72 h in islet medium containing 2 mmol/L or 15 mmol/L
glucose, with either 0.5% BSA or 0.4 mmol/L FFAs conjugated to 0.5% BSA (19);
BrdU was added for the nal 24 h.
Cultured insulinoma cell experiments. Rat insulinoma (INS-1) cells (kindly
provided by Doris Stoffers, University of Pennsylvania) were cultured in INS-1
medium: RPMI with 10% FBS, 11 mmol/L glucose, 10 mmol/L HEPES, 2 mmol/L
L-glutamine, 1 mmol/L sodium pyruvate, penicillin/streptomycin, and 50 mmol/
L b-mercaptoethanol. For proliferation analyses, INS-1 cells were plated on
coverslips, then cultured for 24 h in INS-1 medium containing 2 or 11 mmol/L
glucose and 0.5% BSA or 0.4 mmol/L linoleic, oleic, or palmitic acid, or a mixture
containing 7:2:1 linoleic:oleic:palmitic acid, all conjugated to 0.5% BSA. BrdU
was added for the nal hour of culture; cells xed in 4% paraformaldehyde were
immunostained for BrdU and Hoechst as above except antigen retrieval was by
DNAse. For immunoblots, INS-1 cells were cultured in INS-1 medium with 0.5%
BSA or BSA-conjugated FFAs for 24 h. For small interfering RNA (siRNA)
experiments, siRNA pools (Dharmacon) targeting rat p16 or p18 were applied at
100 nmol/L for 24 h prior to proliferation assay or immunoblot.
Immunoblots. Whole islets or INS-1 cells were sonicated in lysis buffer con-
taining 125 mmol/L Tris pH 6.8, 2% SDS, 1 mmol/L dithiothreitol, 20 mg/mL 4-
Amidinophenylmethanesulfonyl uoride hydrochloride (APMSF), and protease
inhibitors; separated by SDS-PAGE; transferred to nitrocellulose; and blocked in
5% milk/PBS Tween. Antibodies include cyclin D2 (Neomarkers), p16, p18, p21
(Santa Cruz Biotechnology), p27 (BD Pharmingen), tubulin (Calbiochem), anti-
mouse and anti-rabbit (Jackson ImmunoResearch). Data were collected on lm
by ECL or ECL+ (Amersham Pharmacia Biotech).
Statistical analyses. Data are expressed as mean 6 SE. All statistical analyses
were perf or med u si ng Prism (Grap hPa d So ft war e). P values were calculated
by two-tailed Student t te st when two groups were com pa red , by one-way
ANOVA with Newman-Keuls post hoc analysis when more than two groups
were compared, using log-transformed data when Bartlett test showed P , 0.01
for the vari anc e amo ng gr oups t o be un equ al , or by lin e ar re gre ssio n wh en
two continuous variables were tested for interaction. P , 0.05 was con-
sidered signicant .
RESULTS
Lipid infusion increases circulating FFAs in basal and
glucose-stimulated conditions. To test whether FFAs
alter glucose-stimulated b-cell proliferation, mice were in-
travenously infused continuously for 4 days (Fig. 1A)with
saline (SAL), Liposyn II (LIP), glucose (GLU) or Liposyn II
and glucose (L+G). Glucose infusion resulted in a moderate,
sustained elevation in blood glucose (Fig. 1B), to a degree
previously shown to stimulate b-cell proliferation (28). Al-
though infusion of lipid alone (LIP) reduced blood glucose,
coinfusion of lipid with glucose (L+G) did not alter blood
glucose relative to glucose alone (GLU). Comparison of av-
erage blood glucose over the 4-day infusion (Fig. 1C) showed
that GLU and L+G mice had similar average circulating
glucose levels, signicantly higher than SAL and LIP mice.
Plasma insulin levels were transiently elevated in GLU
and L+G mice relative to SAL and LIP mice (Fig. 1D).
Average insulin levels over the 4-day infusion were sig-
nicantly higher in GLU and L+G mice than SAL controls
(Fig. 1E). Average plasma insulin levels did not differ be-
tween GLU and L+G mice.
Lipid infusion increased plasma FFA levels equivalently
in LIP and L+G mice to levels higher than those measured
in SAL and GLU mice (Fig. 1F). Hepatic steatosis was
readily apparent in LIP and L+G mice, conrming lipid de-
livery to tissues (Fig. 1G). Therefore, continuous intrave-
nous infusion of lipids successfully elevated FFA levels under
control and glucose-stimulated conditions.
Elevated FFAs do not increase b-cell death in vivo in
the context of mild hyperglycemia. Decades of work
from multiple investigators has produced incontrovertible
evidence that FFAs cause b-cell death in vitro when glu-
cose levels are also elevated (11). To determ ine whether
4-day infusion of lipids and glucose increased b-cell death
in vivo, sections from pancreata obtained after infusion
were analyzed for cell death by TUNEL staining (Fig. 2A).
Although cell death was readily detectable in positive con-
trol sections, cell death was rare in L+G sections and was
not increased over the other groups (Fig. 2B).
FFAs block glucose-stimulated b-cell proliferation in
vivo in mice. b-Cell proliferation was measured using
histochemistry on pancreas sections after infusion (Fig. 3A
and B). To learn whether elevating FFAs altered b-cell
proliferation under basal conditions, LIP and SAL mice
were compare d. Cells costained for insulin and BrdU were
present at similar frequency in sections from LIP and SAL
mice, suggesting that, under basal conditions, elevation of
FFAs neither increased nor decreased cumulative b-cell
proliferation (Fig. 3C).
As expected (28), moderate continuous 4-day hypergly-
cemia increased b-cell proliferation in GLU mice (Fig. 3C).
To determine whether FFAs alter proliferation under
glucose-stimulatory conditions, L+G mice were compared
with GLU mice. As measured by BrdU incorporation, b
-cell
proliferation was lower in L+G mice than GLU mice (Fig.
3C), equivalent to SAL and LIP controls, despite having
similar blood glucose and plasma insulin levels to GLU mice
(Fig. 1BE). These data suggest that circulating FFAs have
antiproliferative effects on b-cells under glucose-stimulatory
conditions in vivo.
To conrm the antiproliferative e ffect of lipid infusion in
vivo, b-cell replication was measured by immunostaining
for cell-cycl e indicators PCNA and Ki67. These markers
measure proliferation at the time of death rather than cu-
mulatively over the 4-day infusion. For both PCNA and
Ki67, the percent b-cells immunoreactive for both pro-
liferation and insulin was not different between SAL and
LIP mice, but was signicantly higher in GLU mice than
L+G mice, conrming that lipid infusion did not alter b-cell
proliferation under basal conditions but blocked glucose-
induced proliferation (Fig. 3D and E). Over the 4-day in-
fusion, b-cell mass was not signicantly altered in any of
the groups (Fig. 3FH); intriguingly, the cross-sectional
area of individual b-cells was smaller in LIP mice and
larger in L+G mice (Fig. 3I).
FFAs block glucose-stimulated b-cell proliferation in
vitro. The antiproliferative effects of lipid infusion on
b-cells could result indirectly from effects on other tissues
and could result from mediators other than FFAs. To nd
out whether FFAs have the capacity to block glucose-
stimulated b-cell proliferation directly, primary mouse islet
FREE FATTY ACIDS BLOCK b-CELL PROLIFERATION
634 DIABETES, VOL. 61, MARCH 2012 diabetes.diabetesjournals.org
Page 3
cells were cultured in low (2 mmol/L) or high (15 mmol/L)
glucose with BSA control or high glucose in the presence of
BSA-conjugated FFAs. The FFA mixture, engineered to
mimic the fatty acid content of Liposyn II, contained a 7:2:1
ratio of linoleic, oleic, and palmitic acids. b -Cell proliferation
was readily detectable and was more frequent in high glu-
cose (Fig. 4A). Quantication conrmed that glucose in-
creased primary b-cell proliferation (Fig. 4B). Importantly,
treatment with the FFA mixture blocked glucose-stimulated
b-cell proliferation in vitro, conrming that FFAs exert an-
tiproliferative effects directly on primary islet cell cultures.
FFAs might act on other cell types within primary islet
cell cultures, such as other endocrine cells or broblasts,
which could send secondary antiproliferative signals to the
b-cell. To determine whether FFAs act directly on b-cells,
a b-cellderived transformed cell line (INS-1) was cultured
in low or high glucose with BSA control or FFA mixture,
and proliferation was measured (Fig. 4C). Basal proliferation
was frequent in this cell line. How ever, glucose signi-
cantly increased proliferation above baseline, and again,
the FFA mixture blocked the glucose- dependent increase
in proliferation (Fig. 4D ).
To learn which component of the FFA mixture exerted
antiproliferative effects on b-cells, INS-1 cells were cul-
tured in high glucose with each of the individual compo-
nent FFAs. Quantication revealed that both linoleic and
palmitic acids blocked proliferation in high glucose; the
reduction by oleic acid treatment was nonsignicant (Fig.
4D). Therefore, FFAs, specically linoleic and palmitic
acids, act directly on the b-cell to block glucose-induced
proliferation.
FFAs do not prevent glucose-mediated induction of
islet cyclin D2 protein. Cyclin D2, an impor tant regulator
of rodent b-cell proliferation (3135), is upregulated and
relocates to the nucleus in response to hyperglycemia (28).
We hypothesized that FFAs might interfere with glucose-
mediated cyclin D2 protein induction or nuclear localiza-
tion. To determine whether FFAs blocked the increase in
cyclin D2 expression in islets in response to hyperglycemia
in vivo, islets were isolated after 4-day infusion and sub-
jected to immunoblot. Unexpectedly, although islet cyclin
D2 levels were lower in LIP mice than SAL controls, cyclin
D2 protein upregulation in GLU mice was unaffected by
coinfusion of lipid (Fig. 5A and B). When islet cyclin D2
protein expression by immunoblot was analyzed in the
context of prevailing average blood glucose levels during
infusion in mice without lipid exposure (SAL or GLU),
a signicant positive correlation was observed (Fig. 5C).
Supporting the concept that FFAs did not alter glucose
induction of islet cyclin D2 protein, a similar positive
correlation between blood glucose and cyclin D2 expres-
sion was observed in mice with lipid exposure (LIP or L+G;
Fig. 5C). As observed previously (28), the increase in cyclin
D2 by hyperglycemia appeared to be mediated post-
translationally, since cyclin D2 mRNA was not signi
cantly
increased in GLU mice relative to SAL mice (Fig. 5D). Al-
though lipid infusion reduced cyclin D2 mRNA levels un-
der low-gl ucose conditions, lipids d id not signi cantly
lower cyclin D2 mRNA expression under glucose-stimulatory
conditions. In sum, elevating FFA levels in vivo did not
prevent glucose-mediated induction of cyclin D2 protein.
To nd out whether FFA exposure might block pro-
liferation by preventing cyclin D2 from relocalizing to the
nucleus in response to hyperglycemia, pancreas sections
from infused mice were immunostained for cyclin D2 (Fig.
5E). In fact, cyclin D2 protein was readily detectible in islet
nuclei from L+G mice, suggesting that FFAs did not block
nuclear local ization, but might, instead, act downstream of
cyclin D2 to reduce proliferation.
FFAs increas e b-cell expression of cell cycle inhibi-
tors p16 and p18, both in vivo and in vitro. D-cyclins
bind to and activate cyclin-dependent kinase (Cdk) -4 and -6,
which regulate entry into the G1-S transition of the cell
cycle (36,37). Cell cycle inhibitors act downstream of
D-cyclins to block cell cycle progression; both cyclin in-
hibitory protein/kinase inhibitory protein (Cip/Kip) inhibitors
p21/p27 and INK4 family members p16/p18 are known to
regulate proliferation in b-cells (31,3843). We hypothesized
that FFAs might block proliferation by inducing expression
FIG. 2. Four days of elevated FFAs did not increase b-cell death in vivo.
Pancreas sections obtained after 4-day infusion were stained for
TUNEL (green), insulin (red), and Hoechst (blue). A: Positive control
sections obtained by injecting a mouse with streptozotocin 50 mg/kg i.p.
and killing them 16 h later showed readily detectable TUNEL staining.
B: Elevated FFAs did not increase b-cell TUNEL staining in either basal
(LIP) or mildly hyperglycemic (L+G) conditions (n =711). Data are
mean 6 SEM; P values by ANOVA. (A high-quality digital representa-
tion of this gure is available in the online issue.)
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Page 4
of cell cycle inhibitors. Immunoblot of islets isolated after
infusion showed that neither p21 nor p27 were signi-
cantly induced in L+G islets (data not shown). However,
both p16 (Fig. 6A and B) and p18 (Fig. 6C and D) were
signicantly induced at the protein level in islets isolated
from L+G mice, relative to all other groups. To conrm
that FFAs increase cell cycle inhibitor expression in b-cells
directly, INS-1 cells were treated with fatty acids and sub-
jected to immunoblot. Although the linoleic:oleic:palmitic
mixture did not increase expression of either p16 or p18,
possibly because of a protective effect of oleate in INS-1
cells, treatment with palmitic acid alone induced both p16
FIG. 3. FFAs blocked glucose-stimulated b-cell proliferation in vivo in mice. A and B: Pancreas sections obtained after infusion were immunos-
tained for insulin (green) and BrdU (red) (A) or for insulin (green), PCNA (red), and Hoechst (blue) (B). CE: Quantication of b-cell BrdU (C ),
PCNA (D), and Ki67 (E ) staining conrmed that glucose increased proliferation (GLU vs. SAL). Elevated FFAs did not alter basal proliferation
(LIP vs. SAL) but blocked glucose-stimulated (L+G vs. GLU) prolife ration. FH: b-Cell mass, the product of pancreas weight (F) and % islet area
(G) was not statistically different in any of the groups (H). I: The cross-sectional area of individual b-cells was reduced in LIP mice and increased
in L+G mice. Data are mean 6 SEM; P values by ANOVA. ns, nonsignicant. *P < 0.05; **P < 0.01; ***P < 0.001. (A high-quality digital repre-
sentation of this gure is available in the online issue.)
FREE FATTY ACIDS BLOCK b-CELL PROLIFERATION
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Page 5
and p18 protein expression (Fig. 6EG). Therefore, the
antiproliferative effect of FFAs in vivo and in vitro may
be mediated by induction of cell cycle inhibitors p16
and p18, which act to block the cell cycle downstream of
D-cyclins.
Both p16 and p18 are required for the antiprolifer-
ative effect of FFAs. To nd out whether either p16 or
p18 was required for FFAs to block proliferation, a knock-
down approach was used. INS-1 cells treated with siRNA
directed against either p16 (Fig. 7A and B) or p18 (Fig. 7C
and D) showed a modest but signicant knockdown of
each inhibitor relative to scrambled control siRNA. When
INS-1 cell proliferation was measured in the presence of
scrambled siRNA, glucose-induced proliferation was blocked
by palmitic acid (Fig. 7E) as seen in prior experiments
(Fig. 4D). Intriguingly, knockdown of either p16 or p18
completely eliminated the antiproliferative effect of palmitic
acid in INS-1 cells, suggesting that both p16 and p18 may be
required for FFAs to block b-cell proliferation.
DISCUSSION
These studies describe a new form of in vivo lipotoxicity:
inhibition of glucose-stimulated b-cell proliferation. If FFAs
restrict b-cell proliferation in response to other stimuli, such
as obesity and insulin resistance, this process could in-
uence b-cell mass accrual and type 2 diabetes risk. This
nding could also be relevant to type 1 diabetes; when
immune-protective tools become sufciently developed to
pursue b-cell regenerative treatments, the elevated FFAs
that occur with acute insulin insufciency could impair the
b-cell proliferative response to treatment.
Strengths of this study include the use of a carefully
controlled intrav enous infusion system to directly assess
FIG. 4. Linoleic acid and palmitic acid inhibit b-cell proliferation in vitro. A: Primary mouse islet cells were cultured for 72 h in low (2 mmol/L) or
high (15 mmol/L) glucose in the presence of either BSA control or a FFA mixture designed to mimic the components of Liposyn II (7:2:1 linoleic:
oleic:palmitic acids) conjugated to BSA; proliferation was measured by immunostaining for BrdU (red), insulin (green), and Hoechst (blue).
B: Quantication showed that glucose increased b-cell proliferation; glucose-induced proliferation was block ed by addition o f FFAs (n =710).
C and D: INS-1 cells cultured for 24 h with BSA or FFAs also showed that the FFA mixture blocked glucose-induced proliferation. Culturing INS-1
cells with the individual components of the FFA mixture indicated that both linoleic and palmitic acids blocked proliferation in high glucose; the
reduction by oleic acid was nonsignicant (n =45). Mean 6 SEM; P values by ANOVA. (A high-quality digital representation of this gure is
available in the online issue.)
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Page 6
the impac t of elevating FFAs under basal and proliferation-
stimulatory conditions in vivo, in vitro studies to verify that
FFAs act directly on the b-cell, and the novel identication
of two cell cycle inhibitors required for the antiproliferative
effect. Our infusion system allows manipulation of a single
variable in an in vivo setting; inter ventions such as genetic
alteration or diet-induced obesity introduce multiple vari-
ables. Although it is not yet known whether the mechanism
of adaptive b-cell proliferation in response to hyperglyce-
mia is similar to obesity or insulin resistance, various par-
allels, such as increased insulin secretory load on the b-cell,
downstream signals such as insulin receptor substrate 2 and
FIG. 5. FFAs did not prevent glucose-mediated induction of islet cyclin D2 protein in vivo. A and B: Immunoblot of islets isolated after 4-day
infusion showed that cyclin D2 protein was increased by hyperglycemia with or without elevated FFAs; data are quantied (B); n =56. One point
was excluded from the LIP group on the basis of abnormally high blood glucose (BG) in this mouse. C: A positive correlation was observed between
islet cyclin D2 expression by immunoblot and average bloo d gl ucose during the preceding 4-day infusion, bo th in the absence (SAL and GLU;
open symbols) and presence (LIP and L+G; shaded symbols) of FFAs. D: Islet cyclin D2 mRNA was not induced by glucose infusion, and there was
no effect of coinfusion of lipids, although cyclin D2 mRNA levels were signicantly lower in islets isolated from mice infused with lipid alone (n =34).
E: Immunostaining of pancreas sections after infusion showed that coinfusion of lipid did not prevent nuclear accumulation of cyclin D2 induced by
glucose. Data are mean 6 SEM; P values by ANOVA. ns, nonsignicant. (A high-quality digital representation of this gure is available in the online
issue.)
FREE FATTY ACIDS BLOCK b-CELL PROLIFERATION
638 DIABETES, VOL. 61, MARCH 2012 diabetes.diabetesjournals.org
Page 7
cyclin D2, and a role for intracellular glucose metabolism
in obesity-related proliferation, suggest areas of potential
overlap (4,7,10,28,35).
Although an antiproliferative effect of FFAs on b-cells has
been hypothesized for a decade (17) and was observed in
vitro (18,21), in vivo evidence to date has not supported this
hypothesis (2225). An interesting distinction raised by our
data set is the difference between basal- and proliferation-
stimulated settings. Our ndings suggest that FFAs do not
alter basal proliferation, but prevent glucose-stimulated
proliferation. Several experiments have observed a propro-
liferative effect of FFAs on b-cells under basal nutrient
conditions, both in vitro (19,20) and in vivo (24). In our
experiments, glucose and insulin levels were lower in LIP
mice than SAL mice, possibly due to reduced chow intake
and lower overall caloric and carbohydrate load in this group,
raising the possibility that a proproliferative effect of lipids
in the basal state was counteracted by an antiproliferative
effect of hypoglycemia, hypoinsulinemia, or undernutrition.
Several studies have examined the effect of elevated
lipids on b-cell proliferation in rats; however, none allows
direct comparison of stimulated proliferation without and
with elevated FFAs. One important study found robust
b-cell mass recovery after pancreatectomy in Zucker fatty
rats, which have elevated plasma lipids (22). However,
b-cell proliferation was not increased by pancreatectomy,
and islet neogenesis was thought to be responsible for the
b-cell mass expansion, limiting conclusions regard ing the
effect of FFAs on b-cell proliferation. Three studies have
measured b-cell proliferation in rats after intravenous in-
fusion of lipids. In one, lipid effects on proliferation were
FIG. 6. Cell cycle inhibitors p16 and p18 are induced in b-cells by FFAs
in combination with high glucose . Islets isolated after infusion showed
increased expression of p16 (A)(n =56; data are quantied [B]) and
p18 (C)(n=56; data are quantied [D]), only in mice infused with
both lipids and glucose. EG: INS-1 cells cultured with glucose and BSA
control or BSA-conjugated palmitic acid had increased expression
of both p16 and p18 by immunoblot (E ); data are quantied (FG), n =
1213. Data are mean 6 SEM; P values by ANOVA (B and D) and by t
test (FG).
FIG. 7. Both p16 and p18 are required for the antiproliferative effects
of FFAs. By immunoblot, INS-1 cells treated with siRNA directed
against p16 (si-p16; A and B) or p18 (si-p18; C and D) had reduced
expression of the targeted proteins relative to siRNA targeting non-
specic sequence (si-scr), n =34. E: INS-1 cells treated with scramble
siRNA again showed that palmitic acid blocked proliferation in high
glucose, but knockdown of either p16 or p18 rescued the anti-
proliferative effects of palmitic acid. Mean 6 SEM; P values by t test
(C and D) and by ANOVA (E). * P < 0.05; ** P < 0.01; §P < 0.01 vs. si-Scr
11 mmol/L-PA. tub, tubulin.
J. PASCOE AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 61, MARCH 2012 639
Page 8
examined only in the unstimulated state (24). In the sec-
ond, cyclical glucose exposure did not induce pro-
liferation, preventing conclusions regarding the effect of
cyclical FFA exposure on stimulated proliferation (25). In
the third study, lipids were continuously coinfused with
glucose into 2- or 6-mo nth-old rats for 72 h (23). In-
triguingly, b-cell proliferation in L+G rats was equivalent
to saline controls at 2 months of age, similar to our present
mouse data, but was increased relative to saline controls at
6 months of age. Since a glucose-alone control was not
presented, effects of coinfusion of lipid on glucose-stimulated
proliferation cannot be inferred. Therefore, our study is
the rst to isolate lipids as a variable and compare in vivo
stimulated b-cell proliferation in low and high FFA states.
Acute elevation of FFAs causes insulin resistance (rev.
in 15); insulin resistance is a poten t stimulus for b-cell
proliferation (35). Although we have not measured insulin
sensitivity in this study, the antiproliferative effect of FFAs
cannot be related to changes in peripheral insulin re-
sistance, because the effect occurs when b-cells are ex-
posed to FFAs in culture and because insulin resistance
would be expected to increase rather than decrease b-cell
proliferation. The intriguing hypo thesis that FFAs might
induce insulin resistance at the level of the b-cell, and
thereby block a proliferative effect of insulin, remains to
be tested. Since the proliferative effect of glucose requires
glucose metabolism (8), the observed negative impact of
lipids on glucose-induced proliferation may be related to the
metabolic impact of FFAs, which includes inhibition of
glucose oxidation (16,44).
At rst glance, an antiproliferative effect of FFAs seems
inconsistent with the marked proproliferative effect of high-
fat diet exposure. However, overnutrition is associated with
other potent proliferation drivers (4). Elevated circulating
FFA levels are not a consistent featur e of early, compen-
sated high-fat diet exposure, since insulin potently sup-
presses lipolysis (45). Teleogically, one might predict no
evolutionary pressure to expand b-cell mass in the setting
of high FFAs, which would occur during nutrient depri-
vation on a hunter-gatherer diet.
b-Cell death was not increased by combined hypergly-
cemia and FFAs in vivo in our model. This may be due to
the degree of hyperglycemia, which is lower than glucose
concentrations used to elicit glucolipotoxicity in vitro (11),
or to the timing. The deleterious effects of FFAs on b-cells
vary with exposure duration (11); 4 days represents an
intermediate duration which may produce different effects
than either acute or chronic exposure.
How FFAs increase expression of INK4 family cell cycle
inhibitors remains unknown; however, the observation that
genomic polymorphisms near the p16 locus predict risk of
type 2 diabetes adds importance to our nding (46). Per-
oxisome proliferatoractivated receptors, nuclear receptors
activated by lipids, increase p16 expression in mesenchymal
cells (47). Intriguingly, p16 is a primary mediator of se-
nescence, and senescence was observed in b -cells after
prolonged high-fat diet exposure (48). On the other hand,
after 8 weeks of high-fat diet, at a time when obesity- or
insulin resistancemediated b-cell proliferation was mark-
edly increased, islet p16 levels were reduced (49). Future
studies are needed to dissect the mechanisms of the anti-
proliferative effect of FFAs.
Surprisingly, knockdown of either p16 or p18 was suf-
cient to relieve proliferation repression by FFAs, sug-
gesting that both are required for the antiproliferative
effect. We speculate that signaling downstream of p16 and
p18 interacts in such a way that reduction of one disal lows
action of the other, resulting in the apparent requirement
for both. Intriguingly, glucose-stimulated proliferation lost
statistical signicance in INS-1 cells after p16 knockdown,
suggesting a possible basal suppression of proliferation in
low glucose by p16. Based on the modest increase in INK4
expression by palmitate and the modest reduction by
siRNA, it seems that relatively small changes in cellular
INK4 content are able to signicantly inue nce th e rate of
proliferation. In mice, loss of either p16 or p18 is compen-
sated by the presence of the other, as measured by islet cell
proliferation under basal conditions (50). Future experi-
ments will determine whether both p16 and p18 are required
for the antiproliferative effect of FFAs in
b-cells in vivo.
In conclusion, modest elevations of FFAs and glucose
cause in vivo glucolipotoxicity with respect to adaptive
b-cell proliferation. If FFAs also restrict proliferation in
response to obesity and insulin resistance, this may be an
important mechanism driving failure of b-cell mass expan-
sion in prediabetes. A logical extension of our ndings is to
speculate that the antiproliferative effect of FFAs might
represent the elusive connection between p1 6 and genetic
risk of type 2 diabetes. It is our hope that this work leads to
therapeutic interventions that expand b-cell mass to pre-
vent diabetes.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health
grants HL063767 (C.P.O.), DK067351 and DK077096 (A.G.-O.),
DK076562 and DK046204 (L.C.A.), and by American Diabetes
Association grant 7-11-BS-04 (L.C.A.).
No potential conicts of interest relevant to this article
were reported.
J.P., D.H., R.S., and M.A. performed the experiments.
L.C.R. efciently organized the mouse infusion room. B.Z.
catheterized the mice. C.P.O. and A.G.-O. contributed to dis-
cussion and revised the manuscript. L.C.A. conceived and
directed the experiments, contributed to data collection,
andwroteandrevisedthemanuscript.
The authors thank Andrew Stewart, Rupangi Vasavada,
Don Scott, and Robert ODoherty, from the University of
Pittsburgh, for their thoughtful input.
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  • Source
    • "Various cell-cycle regulators have been suggested to be involved in beta-cell replication. p27, p57, and p16 have been implicated in cell-cycle arrest induction as well as cell senescence [19,20,26,27]. In the present study, p16 and p57 were strongly upregulated in " quiescent " adult beta-cells, whereas no difference in p27 expression was observed between the groups. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction: The low frequency of beta-cell replication in the adult human pancreas limits beta-cell regeneration. A better understanding of the regulation of human beta-cell proliferation is crucial to develop therapeutic strategies aiming to enhance beta-cell mass. Methods: To identify factors that control beta-cell proliferation, cell-cycle regulation was examined in human insulinomas as a model of increased beta-cell proliferation (n=11) and healthy pancreatic tissue from patients with benign pancreatic tumors (n=9). Tissue sections were co-stained for insulin and cell-cycle proteins. Transcript levels of selected cell-cycle factors in beta-cells were determined by qRT-PCR after performing laser-capture microdissection. Results: The frequency of beta-cell replication was 3.74±0.92% in the insulinomas and 0.11±0.04% in controls (p=0.0016). p21 expression was higher in insulinomas (p=0.0058), and Rb expression was higher by trend (p=0.085), whereas p16 (p<0.0001), Cyclin C (p<0.0001), and p57 (p=0.018) expression levels were lower. The abundance of Cyclin D3 (p=0.62) and p27 (p=0.68) was not different between the groups. The reduced expression of p16 (p<0.0001) and p57 (p=0.012) in insulinomas and the unchanged expression of Cyclin D3 (p=0.77) and p27 (p=0.55) were confirmed using qRT-PCR. Conclusions: The expression of certain cell-cycle factors in beta-cells derived from insulinomas and healthy adults differs markedly. Targeting such differentially regulated cell-cycle proteins may evolve as a future strategy to enhance beta-cell regeneration.
    Full-text · Article · Feb 2016 · Metabolism: clinical and experimental
  • Source
    • "We speculate that the loading of Mcm proteins onto the origins of replication is an important determinant of the responsiveness of quiescent b cells to mitogenic stimuli. Consistent with this idea, it was shown before that p16/Ink4A, a negative regulator of b cell replication (and a proposed mediator of the negative effect of lipids on glucose-stimulated b cell replication) (Pascoe et al., 2012), negatively regulates the loading of Mcm proteins on origins of replication (Braden et al., 2006). Furthermore, Mcm2 was identified as a marker of nondividing neuronal stem cells resting between two divisions (Maslov et al., 2004). "
    [Show abstract] [Hide abstract] ABSTRACT: Because tissue regeneration deteriorates with age, it is generally assumed that the younger the animal, the better it compensates for tissue damage. We have examined the effect of young age on compensatory proliferation of pancreatic β cells in vivo. Surprisingly, β cells in suckling mice fail to enter the cell division cycle in response to a diabetogenic injury or increased glycolysis. The potential of β cells for compensatory proliferation is acquired following premature weaning to normal chow, but not to a diet mimicking maternal milk. In addition, weaning coincides with enhanced glucose-stimulated oxidative phosphorylation and insulin secretion from islets. Transcriptome analysis reveals that weaning increases the expression of genes involved in replication licensing, suggesting a mechanism for increased responsiveness to the mitogenic activity of high glucose. We propose that weaning triggers a discrete maturation step of β cells, elevating both the mitogenic and secretory response to glucose. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Feb 2015 · Developmental Cell
  • Source
    • "In this context, it could be very intriguing to mention that genome-wide association studies revealed an association between SNPs near Cdk2a (the locus encoding p16INK4a) and increased risk of type 2 diabetes (113, 127, 128). It has also been shown that free fatty acids, whose levels were typically increased in type 2 diabetes and that could be responsible for beta-cell damage (129), can induce p16INK4a expression in islets (130). Thus, p16INK4a could represent a potential link between aging, metabolic derangements, and beta-cell failure in type 2 diabets (131). "
    [Show abstract] [Hide abstract] ABSTRACT: The incidence of type 2 diabetes significantly increases with age. The relevance of this association is dramatically magnified by the concomitant global aging of the population, but the underlying mechanisms remain to be fully elucidated. Here, some recent advances in this field are reviewed at the level of both the pathophysiology of glucose homeostasis and the cellular senescence of pancreatic islets. Overall, recent results highlight the crucial role of beta-cell dysfunction in the age-related impairment of pancreatic endocrine function and delineate the possibility of new original therapeutic interventions.
    Full-text · Article · Sep 2014 · Frontiers in Endocrinology
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