diabetes of the liver
TRANSCRIPT
7/27/2019 Diabetes of the Liver
http://slidepdf.com/reader/full/diabetes-of-the-liver 1/7
Diabetes of the Liver: The Link Between Nonalcoholic
Fatty Liver Disease and HFCS-55
Kate S. Collison1, Soad M. Saleh1, Razan H. Bakheet1, Rana K. Al-Rabiah1, Angela
L. Inglis1
, Nadine J. Makhoul1
, Zakia M. Maqbool1
, Marya Zia Zaidi1
, Mohammed A. Al-Johi1 and Futwan A. Al-Mohanna1
1Cell Biology and Diabetes Research Unit, Department of Biological and Medical Research,
King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
Correspondence: Kate S. Collison ([email protected])
Received 13 April 2008; Accepted 16 February 2009; Published online 12 March 2009.
Top of page
Abstract
Nonalcoholic fatty liver disease (NAFLD) is associated with obesity and insulin resistance. It
is also a predisposing factor for type 2 diabetes. Dietary factors are believed to contribute to
all three diseases. NAFLD is characterized by increased intrahepatic fat and mitochondrial
dysfunction, and its etiology may be attributed to excessive fructose intake. Consumption of
high fructose corn syrup-55 (HFCS-55) stands at up to 15% of the average total daily energy
intake in the United States, and is linked to weight gain and obesity. The aim of this study
was to establish whether HFCS-55 could contribute to the pathogenesis of NAFLD, by
examining the effects of HFCS-55 on hepatocyte lipogenesis, insulin signaling, and cellular function, in vitro and in vivo. Exposure of hepatocytes to HFCS-55 caused a significant
increase in hepatocellular triglyceride (TG) and lipogenic proteins. Basal production of
reactive oxygen metabolite (ROM) was increased, together with a decreased capacity to
respond to an oxidative challenge. HFCS-55 induced a downregulation of the insulin
signaling pathway, as indicated by attenuated ser473 phosphorylation of AKT1. The c-Jun
amino-terminal kinase (JNK), which is intimately linked to insulin resistance, was also
activated; and this was accompanied by an increase in endoplasmic reticulum (ER) stress and
intracellular free calcium perturbation. Hepatocytes exposed to HFCS-55 exhibited
mitochondrial dysfunction and released cytochrome C (CytC) into the cytosol. Hepatic
steatosis and mitochondrial disruption was induced in vivo by a diet enriched with 20%
HFCS 55; accompanied by hypoadiponectinemia and elevated fasting serum insulin andretinol-binding protein-4 (RBP4) levels. Taken together our findings indicate a potential
mechanism by which HFCS-55 may contribute to the pathogenesis of NAFLD.
Top of page
Introduction
Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorder of
industrialized countries, affecting ~15 – 25% of the general population (1). Previously
unrecognized until the early 1980s, NAFLD has an etiology related to recent changes in diet
and lifestyle. Nonalcoholic steatohepatitis (NASH), the more severe form of NAFLD, isassociated with obesity, insulin resistance (2), and mitochondrial dysfunction (3). Estimations
7/27/2019 Diabetes of the Liver
http://slidepdf.com/reader/full/diabetes-of-the-liver 2/7
of the incidence of NASH in the general population vary from 2 to 3% (4), with indications
that this condition is becoming increasingly prevalent (5). The limited data, existing on the
incidence of pediatric NASH in the United States and Asia, suggest an overall prevalence of
at least 3% (6). Overconsumption of simple carbohydrates is associated with the incidence of
NASH (7), and the bulk of these carbohydrates are in the form of various sugars (8). Dietary
components have been demonstrated to play an important role in the development of themetabolic syndrome (9), obesity (10), and type 2 diabetes (11). Insulin resistance is a
common occurrence in all three diseases and occurs primarily in the liver and in skeletal
muscle (12). Insulin resistance can be induced by diets overrepresented with fats (13) or
simple sugars (14). Intake of dietary fructose, either as a free monosaccharide or bound to
glucose in the form of sucrose, has increased 1,000% during the past 40 years (15), and the
majority of this is consumed in the form of high fructose corn syrup (HFCS), a refined
product of corn that was introduced into the human diet from 1970 onwards. HFCS
consumption during the past decade accounts for a per capita mean of 53.8 g per day (~200
calories or 10% of caloric food intake), according to data from the US Department of
Agriculture (USDA) (16). This figure was recently confirmed in the third National Health
and Nutrition Examination Survey, which estimated the mean consumption of fructose to be54.7 g/day (17). HFCS can be a mixture of various concentrations of free fructose and free
glucose, and according to the USDA (16), around 60% of HFCS is in the form of 55%
fructose (termed HFCS-55), with the remainder being typically 42% fructose (HFCS-42).
Dietary fructose is primarily metabolized in the liver, where it has been demonstrated to
induce elevated plasma triglyceride (TG) (13) and increased adiposity (18). Epidemiological
studies have indicated that the development of NAFLD may be associated with excessive
dietary fructose consumption (4,19,20). Whereas several animal models of hepatic
lipogenesis and insulin resistance have used high amounts of fructose not typically
encountered in the daily diet (21,22) or combination diets involving a mixture of sugars and
fats (23,24), HFCS-55 on its own has not previously been directly linked to liver dysfunction.
In humans, short-term dietary consumption of 30% total of daily kilocalories as fructose
results in higher TG and ghrelin levels and lower plasma insulin and leptin levels, when
compared to glucose (25). In recent epidemiological studies, HFCS consumption has been
linked to a rise in obesity (15,26), however other studies have suggested that HFCS does not
contribute to obesity any differently than other types of energy sources (27). In view of the
global increase in fructose consumption (16,17), ~50% of which is in the form of sugar-
sweetened beverages and fruit juices (17), and due to the paucity of data addressing the effect
of HFCS-55 on hepatic metabolism, we set out to investigate the effect of HFCS-55 on TG
production and hepatic function in vitro together with hepatic steatosis and markers of insulin
resistance in vivo. The amount of HFCS-55 used in our animal model (20% wt/wt,
corresponding to roughly 10% fructose and 10% glucose) is comparable with the estimatedaverage daily per capita intake of fructose-containing carbohydrates (16,17). The aim is to
establish whether or not HFCS-55 could contribute to the pathogenesis of NAFLD and
NASH — conditions which are associated with obesity, metabolic syndrome, and type 2
diabetes.
Top of page
Methods and Procedures
HFCS-55 was a kind gift from Hanseland, Groningen, Holland. Fetal bovine serum was from
HyClone (Logan, UT). Electrophoresis reagents were purchased from Invitrogen (Carlsbad,CA). MitoTracker Green FM (M7514) was from Molecular Probes (Eugene, OR). The water-
7/27/2019 Diabetes of the Liver
http://slidepdf.com/reader/full/diabetes-of-the-liver 3/7
soluble antioxidant epigallocatechin gallate (EGCG, no. 50299) was from Fluka (Sigma-
Aldrich, St Louis, MO); and the inhibitor of c-Jun amino-terminal kinase (JNK)
phosphorylation (SP600125) was purchased from BioSource International (Camarillo, CA).
Cytochrome C (CytC) release assay kit (QIA87) was purchased from Calbiochem
(Gibbstown, NJ). Antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Cell
culture reagents and human recombinant insulin, protease inhibitors, and other analyticalgrade reagents were purchased from Sigma-Aldrich (Ontario, CA).
Animals and diets
C57BL/6J mice were from the Jackson Laboratory and were housed/caged and fed a standard
chow diet until 6 weeks of age, when they were separated, and half were given ad libitum a
diet containing 20% HFCS-55 (5C4K; TestDiet Purina, Richmond, IN) and the remainder
continued on the standard chow diet (control). After a 3-week period of adjustment, 20 male
and 20 female F1 animals per diet group were bred, weaned, and maintained on these diets
for a period of 32 weeks. The breeding and care of the animals are in accordance with the
protocols approved by the Animal Care and Use Committee of the King Faisal SpecialistHospital and Research Centre.
Measurement of murine serum lipids, glucose, insulin, adiponectin, leptin, and RBP4
levels
Serum TG, total cholesterol, HDL, and LDL concentrations were measured in overnight
fasted animals using the Reflovert Plus instrument, according to the manufacturer's
instructions (Roche, F. Hoffmann-La Roche, Basel, Switzerland). Fasting blood glucose
levels were measured in 32-week mice using the Ascensia Contour (Bayer HealthCare,
Mishawaka, IN). Fasting serum insulin was measured using the ultrasensitive mouse insulin
ELISA kit from Mercodia (Uppsala, Sweden: assay range 0.188 –
6.9 g/l; sensitivity > 0.188
g/l), according to the manufacturer's instructions. Fasting serum adiponectin/Acrp30 and
leptin were measured by ELISA using commercial assay kits (MRP300 mouse
adiponectin/Acrp30: (assay range 1 – 10 ng/ml, sensitivity > 0.003 ng/ml), MOB00 mouse
leptin, (assay range 62.5 – 4,000 pg/ml, sensitivity >22 pg/ml): both from R&D Systems,
Minneapolis, MN). Fasting serum retinol-binding protein-4 (RBP4) was measured in diluted
serum using the Dual mouse/rat RBP4 ELISA kit (RB0642EK; AdipoGen, Seoul, Korea:
assay range 0.19 – 12 ng/ml, sensitivity >60 pg/ml). Data from the manufacturers of these kits
indicated that they were 100% specific for murine insulin, leptin, adiponectin, and RBP4,
respectively.
Histology and Oils-Red-O staining of murine liver tissue
Formalin-fixed, paraffin-embedded liver tissue from eight 32-week-old mice were processed
and 4 m thick serial sections were cut and stained with hematoxylin and eosin or Oils-Red-
O for lipid analysis, according to standard pathology laboratory procedures. After mounting
with glycerol gelatin, images were captured using AxioVision Rel 4.5 software (Carl Zeiss
Micro Imaging, Jena, Germany).
Hepatocellular culture
HepG2 cells were grown to a concentration of 50% confluency in Hepes Modified RPMIsupplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic solution containing
7/27/2019 Diabetes of the Liver
http://slidepdf.com/reader/full/diabetes-of-the-liver 4/7
penicillin G sodium, streptomycin sulfate and amphotericin B in a humidified air and 5%
CO2 incubator at 37 °C. The following day, the medium was changed to RPMI with 10%
heat-inactivated fetal bovine serum and HFCS-55 (0.625 – 2.5%) for the times stated. Where
indicated, sucrose or glucose (0.625 – 2.5%) replaced HFCS-55. Media was replaced every 48
h. Following overnight culture in serum-free medium, in some experiments, cells were
exposed to 100 nmol/l insulin (Sigma-Aldrich, St Louis, MO) for 5 min prior to lysis.
Intracellular and hepatic TG quantitation
Levels of HepG2 or mouse liver TG were quantified using the Triglyceride Determination
Kit TRO100, which is specific and linear between the range 1 – 10 mg/ml (Sigma-Aldrich, St
Louis, MO). Lipids were first extracted using a 1:1 mixture of chloroform: ether, and the
dried pellets resuspended in the Reaction Buffer provided in the Kit. Protein was determined
from serial dilutions of cell lysates using the protein-specific Quant-iT kit, according to the
manufacturer's instructions (range 0.25 – 5 g; Invitrogen, Eugene, OR). Results were
expressed as TG ( g/mg cellular protein) s.e.m.
Lipid staining of cultured HepG2 cells
Monolayer cultures of HepG2 cells were rinsed twice with phosphate buffered solution (PBS)
and fixed in 3.7% formaldehyde in PBS for 10 min. Following a 1-min incubation in 50%
ethanol, cells were rinsed in distilled water and stained with a saturated solution of SudanBlack in 75% ethanol for 15 min at 37 °C. Cells were then washed twice with 50% ethanol
for 2 min each time and rinsed in distilled water prior to viewing using the AxioVision
Digital Imaging System (Carl Zeiss Micro Imaging, Jena, Germany).
Live cell imaging of ROMs and Ca2+
Cells cultured at low density on glass cover slips were incubated for 15 min at room
temperature with 2', 7'-dichlorofluorescein diacetate for detection of reactive oxygen
metabolite (ROM), or 30 min at room temperature with FLUO-3 AM for detection of Ca2+
(both Molecular Probes). ROM production was induced in control and HFCS-treated cells
using platelet-derived growth factor (PDGF, 10 nmol/l). Stained cells were rinsed and
examined with a LSM5 META laser scanning microscope (Carl Zeiss Micro Imaging, Jena,
Germany), as previously described (28).
Indirect immunofluorescence
Monolayer cultures of HepG2 cells were fixed in 3.7% formaldehyde in PBS for 14 min at
room temperature. Cells were permeabilized in a solution of 0.05% Triton X-100 in PBS,
blocked in 1% bovine serum albumin for 30 min, before incubation with the following
primary antibody diluted in PBS for 40 min: Thr183/Tyr185 p-JNK (no. 36-9300; Zymed
Laboratories, Invitrogen, Carlsbad, CA), protein disulfide isomerase – endoplasmic reticulum
(ER) marker (ab2792; Abcam, Cambridge, MA). Cells were washed three times in PBS and
incubated with FITC-conjugated antimouse (715-095-151) or TRITC-conjugated antirabbit
(711-025-152) antibodies (Jackson ImmunoResearch, West Grove, PA) diluted in PBS for 40
min before washing, mounting, and visualization using the Ultra View Imaging System
(Perkin Elmer, Cambridge, UK).
Cell lysis and western blotting
7/27/2019 Diabetes of the Liver
http://slidepdf.com/reader/full/diabetes-of-the-liver 5/7
Monolayer cultures of HepG2 cells were serum starved for 18 h unless otherwise stated and
washed with ice-cold PBS before lysis in ice-cold RIPA buffer (1% sodium deoxycholate,
1% Triton X-100 and 0.1% sodium dodecyl sulfate, 50 mmol/l Tris-HCl, pH 7.4 and 150
mmol/l sodium chloride) containing protease inhibitors (10 mmol/l sodium orthovanadate, 1
mmol/l phenylmethylsulfonyl fluoride, 100 mol/l Pepstain A, 0.5 mmol/l leupeptin, 1
mmol/l chymotrypsin, and 10 mol/l aprotinin). Phosphatase inhibitor (5 mmol/l sodiumfluoride) was also added where appropriate. Similarly, RIPA lysates were made from snap-
frozen blocks of murine liver. For SDS-PAGE, lysates containing ~10 g of protein were
separated on 4 – 12% gradient NuPAGE gels (Invitrogen, Carlsbad, CA) and transferred onto
Hybond ECL nitrocellulose (GE Healthcare, Glyfada, Greece), using the Trans-Blot semi-dry
transfer system (BioRad, Hercules, CA). Blots were immunoprobed with the appropriate
primary antibody diluted in 3% nonfat milk in TBS-Tween buffer overnight at 4 °C; followed
by washing in TBS-Tween buffer and incubating with an appropriate horseradish peroxidase
secondary antibody (Promega, Madison, WI) diluted in 3% nonfat milk in TBS-Tween buffer
for 3 h at room temperature. Following ECL and autoradiography, images were scanned, then
gray scaled and cropped using Adobe Photoshop.
Quantitative analysis of western blot data
Measurement of signal intensity on ECL films after western blotting with various antibodies
was performed using a scanner and image processing and analysis software (Alpha Innotech,
San Leandro, CA). For statistical analysis, all data were expressed as integrated density
values. For acetyl-CoA carboxylase alpha (ACC- ), apoB, glutathione reductase, PDI, and
CytC, integrated density values were calculated as a ratio of the density values of the specific
protein bands/ -actin density values, and expressed as % controls s.e.m. For phospho-ser473
AKT and phospho-Thr183, Tyr185-JNK, integrated density values were calculated as the ratio of
the density values of the specific protein bands/total AKT and total JNK density values,
respectively. All figures showing quantitative analysis include data from at least three
independent experiments.
RNA isolation and RT-PCR
Total RNA was prepared from HepG2 cells using the phenol/guanidine/isothiocyanate
commercial reagent (TRI-Regent; Sigma Chemical, St Louis, MO) according to the
manufacturer's instructions, and stored at -80 °C until use. The RNA concentration was
measured by microspectrophotometry (NanoDrop Technologies, Wilmington, DE). ACC- ,
PDI, and GAPDH receptor mRNA were quantified by RT-PCR, using the following primers
to amplify the relevant products: ACC- F: CTG GAG CCC TCA ACA AAG TC; ACC- R:CCA GGG CTG CAT AAT CTC T. PDIF CGC CCT GTG GTA TCC C; PDIR: ACT CTG
CGC GTT CCT TCG TC; GAPDHF: GGT GGA GGT CGG AGT CAA C; GAPDHR: ATG
GGT GGA ATC ATA TTG GA. RT-PCR was performed using PCT-200 thermal cycler (MJ
Research, Waltham, MA) and the following conditions: denaturing: 94 °C 2 min; 30 cycles of
94 °C 30 s, 60 °C 1 min, 72 °C 1 min; followed by 72 °C 10 min.
Imaging of mitochondria
The mitochondria-specific fluorescent dye MitoTracker Green FM (M7514; Molecular
Probes, Invitrogen, Carlsbad, CA) was used to assess mitochondrial integrity in cultured
HepG2 cells. Formaldehyde-fixed monolayer cultures of HepG2 cells were incubated with100 nmol/l MitoTracker Green FM for 15 min before washing and mounting for viewing with
7/27/2019 Diabetes of the Liver
http://slidepdf.com/reader/full/diabetes-of-the-liver 6/7
the Ultra View System (PerkinElmer). For imaging of mitochondria in paraffin-embedded
mouse liver samples, sections were first deparafinized in three washes of xylene followed by
rehydration of sections to PBS pH 7.4. Liver sections from 32-week mice in the control and
HFCS-55 diet groups were incubated with 100 nmol/l MitoTracker Red CMXRos (M7512)
for 15 min before washing and mounting for viewing with the Ultra View System.
CytC release assay
A commercial kit was used to isolate mitochondrial and cytosolic fractions from cultured
form of HepG2 cells by differential centrifugation, according to the manufacturer's protocol
(Calbiochem, EMD Biosciences, San Diego, CA). Fractions were subjected to SDS-PAGE
and western blotting analysis using the anti-CytC antibody supplied in the kit.
Statistics
Values are expressed as mean s.e.m. The significance of the differences in mean values
among different treatment groups were analyzed by one-way ANOVA or unpaired t -test. P values <0.05 were considered significant. All statistics were performed with GraphPad InStat
3 (GraphPad software; GraphPad, La Jolla, CA).
Top of page
Results
Effect of HFCS-55 on lipogenesis and the insulin signaling pathway
HFCS-55 was analyzed by HPLC-UV analysis and was shown to contain fructose andglucose monomers at a ratio of 55:42, with no significant contaminants. Exposure of HepG2
cells to HFCS-55 (0.625 – 2.5%) caused a dose increase in hepatocellular TG levels to a
maximum of 2080 77 g/mg cellular protein from a resting level of 346 3 g/mg ( P <
0.01, n = 3) after 96 h of treatment (Figure 1a). Increased lipid deposition was clearly
apparent in cells stained with the lipid-specific dye Sudan Black (Figure 1b). Incubation of
HepG2 cells with sucrose or glucose solution (0.625 – 2.5%) under identical conditions failed
to result in significantly increased lipid production, suggesting that the effect was not due to
osmotic shift (Figure 1a). Cellular viability was not affected by the presence of HFCS-55
(0.625 – 2.5%) throughout the time-course of the experiment (as tested by Trypan blue
exclusion, data not shown). Concomitant addition of the antioxidant EGCG (5 – 50 mol/l) to
HFCS-treated cells did not significantly reduce the levels of hepatocyte TG (data not shown).Levels of ACC- , a key enzyme in the biogenesis of fatty acids and a regulator of fatty acid
-oxidation (29), were increased by up to 130.9 2.1% after 72 h of treatment with HFCS-55
(Figure 1c,d; P < 0.001, n = 3). Additionally, apolipoprotein B (apoB), the structural
component of TG-rich lipoproteins such as very low-density lipoproteins, increased by 180.3
11.7% in the same time frame ( P < 0.001, n = 3). The increase in ACC- protein
concentration appeared to be regulated in part at the transcriptional level, as HFCS-55
induced an increase in ACC- mRNA levels relative to GAPDH housekeeping gene of
128.01 2.39% (Figure 1e,g, mean s.e.m., P < 0.01, n = 3). Increased apoB mRNA was not
detected. Insulin signaling was impaired by HFCS-55, with an inhibition of insulin-
stimulated ser473-phosphorylation of AKT1 and 2 of up to ~70% occurring in cells treated with
2.5% HFCS-55 for 72 h (Figure 1c,f , P < 0.05, n = 3).
7/27/2019 Diabetes of the Liver
http://slidepdf.com/reader/full/diabetes-of-the-liver 7/7