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Background: Dietary intake of saturated fat is a likely contributor to nonalcoholic fatty liver disease (NAFLD) and insulin resistance, but the mechanisms that initiate these abnormalities in humans remain unclear. We examined the effects of a single oral saturated fat load on insulin sensitivity, hepatic glucose metabolism, and lipid metabolism in humans. Similarly, initiating mechanisms were examined after an equivalent challenge in mice. Methods: Fourteen lean, healthy individuals randomly received either palm oil (PO) or vehicle (VCL). Hepatic metabolism was analyzed using in vivo 13C/31P/1H and ex vivo 2H magnetic resonance spectroscopy before and during hyperinsulinemic-euglycemic clamps with isotope dilution. Mice underwent identical clamp procedures and hepatic transcriptome analyses. Results: PO administration decreased whole-body, hepatic, and adipose tissue insulin sensitivity by 25%, 15%, and 34%, respectively. Hepatic triglyceride and ATP content rose by 35% and 16%, respectively. Hepatic gluconeogenesis increased by 70%, and net glycogenolysis declined by 20%. Mouse transcriptomics revealed that PO differentially regulates predicted upstream regulators and pathways, including LPS, members of the TLR and PPAR families, NF-κB, and TNF-related weak inducer of apoptosis (TWEAK). Conclusion: Saturated fat ingestion rapidly increases hepatic lipid storage, energy metabolism, and insulin resistance. This is accompanied by regulation of hepatic gene expression and signaling that may contribute to development of NAFLD.REGISTRATION. NCT01736202. Funding: Germany: Ministry of Innovation, Science, and Research North Rhine-Westfalia, German Federal Ministry of Health, Federal Ministry of Education and Research, German Center for Diabetes Research, German Research Foundation, and German Diabetes Association. Portugal: Portuguese Foundation for Science and Technology, FEDER - European Regional Development Fund, Portuguese Foundation for Science and Technology, and Rede Nacional de Ressonância Magnética Nuclear.
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The Journal of Clinical Investigation CLINICAL MEDICINE
695 Volume 127 Number 2 February 2017
The pandemic of obesity, type  diabetes mellitus TDM and non-
alcoholic fatty liver disease NAFLD has frequently been associated
with dietary intake of saturated fats  and speciically with dietary
palm oil PO . According to current paradigms, chronic insulin
resistance is the common feature of these diseases ,  and relates
to intracellular concentrations of triglycerides TG and lipotoxins
. There is evidence that a chronic high-fat diet in mice and humans
leads to insulin resistance via similar mechanisms , . Chronic
insulin resistance comprises not only impaired muscle insulin action
but also increased rates of endogenous glucose production EGP
and gluconeogenesis GNG in obese and TDM patients .
Still, only a few studies have addressed the initial effects of
high-fat loading using either intravenous or enteral administration
of lipids. These studies mainly focused on the role of skeletal mus-
cle by assessing intramyocellular TG content , substrate oxi-
dation, glycogen synthesis , or glucose disposal . Stud-
ies using parenteral administration of unsaturated lipids  or
high-calorie mixed meals yielded conflicting results with regard to
hepatic energy metabolism. One mixed-meal study found greater
de novo lipogenesis without affecting hepatic glycogen metabo-
lism , while an intravenous lipid infusion study failed to detect
any effect on hepatic insulin sensitivity . Another study com-
BACKGROUND. Dietary intake of saturated fat is a likely contributor to nonalcoholic fatty liver disease (NAFLD) and insulin
resistance, but the mechanisms that initiate these abnormalities in humans remain unclear. We examined the effects of a
single oral saturated fat load on insulin sensitivity, hepatic glucose metabolism, and lipid metabolism in humans. Similarly,
initiating mechanisms were examined after an equivalent challenge in mice.
METHODS. Fourteen lean, healthy individuals randomly received either palm oil (PO) or vehicle (VCL). Hepatic metabolism
was analyzed using in vivo 13C/31P/1H and ex vivo 2H magnetic resonance spectroscopy before and during hyperinsulinemic-
euglycemic clamps with isotope dilution. Mice underwent identical clamp procedures and hepatic transcriptome analyses.
RESULTS. PO administration decreased whole-body, hepatic, and adipose tissue insulin sensitivity by 25%, 15%, and 34%,
respectively. Hepatic triglyceride and ATP content rose by 35% and 16%, respectively. Hepatic gluconeogenesis increased
by 70%, and net glycogenolysis declined by 20%. Mouse transcriptomics revealed that PO differentially regulates predicted
upstream regulators and pathways, including LPS, members of the TLR and PPAR families, NF-κB, and TNF-related weak
inducer of apoptosis (TWEAK).
CONCLUSION. Saturated fat ingestion rapidly increases hepatic lipid storage, energy metabolism, and insulin resistance. This is
accompanied by regulation of hepatic gene expression and signaling that may contribute to development of NAFLD.
FUNDING. Germany: Ministry of Innovation, Science, and Research North Rhine–Westfalia, German Federal Ministry of Health,
Federal Ministry of Education and Research, German Center for Diabetes Research, German Research Foundation, and German
Diabetes Association. Portugal: Portuguese Foundation for Science and Technology, FEDER – European Regional Development
Fund, Portuguese Foundation for Science and Technology, and Rede Nacional de Ressonância Magnética Nuclear.
Acute dietary fat intake initiates alterations in
energy metabolism and insulin resistance
Elisa Álvarez Hernández,1,2 Sabine Kahl,1,2,3 Anett Seelig,4 Paul Begovatz,1,2 Martin Irmler,5 Yuliya Kupriyanova,1,2
Bettina Nowotny,1,2,3 Peter Nowotny,1,2 Christian Herder,1,2 Cristina Barosa,6 Filipa Carvalho,6 Jan Rozman,4,5 Susanne Neschen,2,4,5
John G. Jones,6,7 Johannes Beckers,2,5,8 Martin Hrabě de Angelis,2,5,8 and Michael Roden1,2,3
1Institute for Clinical Diabetology, German Diabetes Center, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany. 2German Center for Diabetes Research, Munich-Neuherberg,
Germany. 3Department of Endocrinology and Diabetology, Medical Faculty, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany. 4Helmholtz Zentrum Munich, German Research Center for Environmental
Health (GmbH), Neuherberg, Germany. 5Institute of Experimental Genetics, Helmholtz Zentrum Munich, German Research Center for Environmental Health (GmbH), Neuherberg, Germany. 6Center for
Neurosciences and Cell Biology, UC Biotech, Cantanhede, Portugal. 7Portuguese Diabetes Association (APDP), Lisbon, Portugal. 8Technische Universität Munich, Chair of Experimental Genetics, Freising, Germany.
Related Commentary: p. 454
Authorship note: E. Álvarez Hernández, S. Kahl, A. Seelig, and P. Begovatz contributed
equally to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Submitted: July 27, 2016; Accepted: November 10, 2016.
Reference information: J Clin Invest. 2017;127(2):695–708.
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PO results in increased circulating TG, glucagon, and incretins.
After PO administration, TG in plasma rose by  area under
the time curve AUC, P  . and by  in chylomicrons
AUC, P  . Figure A. The AUC for plasma free fatty acids
FFA Figure B and insulin concentrations Figure C was
unchanged, while the AUC for plasma C-peptide was  high-
er after PO ingestion versus VCL P  ., Figure D. Of note,
FFA were increased at , , and  minutes. Blood glucose
levels were not different between PO- and VCL-treated groups
Figure E. Plasma glucagon rose by  AUC, P  .
only after PO ingestion Figure F. Also, glucagon-like peptide 
GLP and gastric inhibitory polypeptide GIP levels were mark-
edly increased and remained elevated after PO ingestion both
P  . Supplemental Figure ; supplemental materi-
al available online with this article;./
JCIDS. Circulating levels of TNF-α, IL, fetuin-A, chem-
erin, omentin, and cortisol were not different between PO and
VCL groups P  . for all Supplemental Table .
PO induces insulin resistance at whole-body, liver, and adipose
tissue levels. Insulin sensitivity was measured using hyperinsu-
linemic-euglycemic clamp tests in healthy humans. Steady state
was reached Supplemental Figure , and pertinent parameters
were analyzed during this time. PO ingestion reduced WBIS by
 compared with VCL treatment P  ., Figure A. Fur-
thermore, after PO, volunteers also showed a decrease of 
P  . in the rate of glucose disappearance Rd, mostly due
to a  P  . reduction in glucose oxidation GOX, while
the rate of nonoxidative glucose disposal remained unchanged
paring subacute oral ingestion of fatty acids with different com-
positions found an increase in the glucose infusion rate only after
polyunsaturated fatty acid ingestion .
To overcome possible limitations of the previous studies, such
as the use of nonphysiological routes of lipid administration or
mixed meals, which introduce protein and carbohydrates as con-
founders, we designed a translational study concept comprising a
clinical trial involving healthy humans and a corresponding study
involving nonobese nondiabetic mice. The randomized crossover
clinical trial examined the effects of a single oral challenge with
PO, which is mainly composed of saturated fatty acids , versus
vehicle VCL ingestion on whole-body insulin sensitivity WBIS
and hepatic insulin sensitivity. Moreover, the contributions of
hepatic glucose fluxes, i.e., GNG, net glycogenolysis GLYnet,
and the futile exchange between glycogenogenic and glycogeno-
lytic pathways glycogen cycling to EGP as well as the effects of
these fluxes on hepatocellular lipids HCL and phosphorous-
containing metabolites were analyzed using combined in vivo
multinuclear C/P/H and stable isotope tracers to assess plasma
glucose appearance rates and sources of EGP. In the mouse study,
we examined the effects of a similar oral saturated fat challenge on
insulin sensitivity and hepatic transcriptome changes.
Studies in humans. A total of  young, lean, insulin-sensitive male
volunteers Figure  and Table  received either an oral dose of PO
~. g/kg BW or an identical volume of VCL on  occasions in
random order, spaced by an -week interval.
Figure 1. CONSORT flow diagram. Forty-four
patients underwent screening, which included a
medical history, BMI analyses, and bioimpedance
and an oral glucose tolerance tests. Of the 44
participants, 19 did not meet the inclusion crite-
ria, 2 declined to participate, and 2 were excluded
for other reasons. Twenty-one participants were
allocated to receive the intervention, two of
whom did not receive the allocated intervention.
An additional 5 volunteers were excluded due
to changes in inclusion and exclusion criteria
(n = 2), as well as changes in the experimental
procedure, which yielded data that could not be
compared with subsequently acquired data
(n = 3). Ultimately, data from 14 individuals were
analyzed, except for hepatic lipid and energy
analyses (n = 12) and some hepatic glucose flux
measurements (n = 9).
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by  AUC, P  ., while TG levels were not signiicantly
different AUC, P  . after PO Figure , A and B. The post-
intervention AUC for blood glucose and insulin was comparable
between groups Figure , C and D.
PO preferably induces hepatic insulin res istance. Insulin sensitiv-
ity was measured using hyperinsulinemic-euglycemic clamp tests
in unrestrained mice. WBIS in mice trended toward a reduction
P  . Figure A. Whole-body glucose uptake, given by Rd,
was not different between interventions Figure B. Residual EGP
during the clamp tended to be higher P  . Figure C, and
insulin-mediated suppression of EGP was  lower P  .
after PO Figure D, reflecting marked hepatic insulin resistance.
PO ingestion did not affect insulin-induced FFA suppression
Figure E but reduced insulin-induced TG suppression by 
P  . Figure F. Of note, glucose uptake in the gastrocne-
mius muscle Supplemental Figure A and white adipose tissue
Supplemental Figure B as well as rates of glycolysis did not dif-
fer between the PO- and VCL-treated groups Supplemental Fig-
ure C. Interestingly, PO administration did not change hepatic
TG content in mice Supplemental Figure D.
PO causes changes in the expression of hepatic transcription
factors. Livers from both mouse cohorts were used for analysis
on Affymetrix Gene . ST microarrays. A total of  and 
differentially regulated probe sets were obtained from each
cohort. Array data were deposited in the NCBI’s Gene Expres-
sion Omnibus GEO database GEO GSE. Canonical
pathways predicted to be differentially regulated by Ingenuity
software after PO included TNF-like weak inducer of apop-
tosis TWEAK and aryl hydrocarbon receptor AHR under
insulin-stimulated and noninsulin-stimulated conditions; phos-
pholipase and D-myoinositol- phosphate signaling under insu-
lin-stimulated conditions; and p MAPK, NF-κB, PPARα, and
OX under noninsulin-stimulated conditions Figure , A and
B, all P  .. Several upstream regulators involved in hepat-
ic fatty acid metabolism and inflammatory processes were pre-
dicted by Ingenuity software to be regulated by PO ingestion.
These upstream regulators included LPS, which was activated
with the most certainty, with a Z score of . and . under insu-
lin- and noninsulin-stimulated conditions, respectively, TLR
family members TLR and TLR under insulin-stimulated
conditions as well as TLR under noninsulin-stimulated condi-
tions, PPARα, and FOXO all P  . Figure , C and D.
Figure B. EGP increased by  P  . at  minutes and
by  P  . at  minutes after PO compared with VCL
treatment Figure C. After PO, insulin-stimulated suppression of
EGP was  lower than that detected after VCL P  ., Fig-
ure D. Finally, insulin-stimulated suppression of FFA Figure E
and TG Figure F was  P  . and  P  .
lower after PO than after VCL treatment.
PO augments the contribution of GNG to EGP. In order to fur-
ther analyze the PO-induced increase in EGP, we measured the
contributions of gluconeogenic and glycogenolytic fluxes. We
found that GNG increased by approximately  P  ., while
GLYnet decreased by approximately  P  . after PO. Gly-
cogen phosphorylase GP flux tended to be lower in the PO arm
P  .. The contribution of glycogen cycling to total GP flux
was negligible in both study arms Figure .
PO increases lipid oxidation rates. The respiratory quotient
R, deined as the rate of CO production/O consumption,
was comparable in the period after PO or VCL ingestion but was
reduced during the clamp only after PO Figure A. Resting ener-
gy expenditure REE and lipid oxidation LOX rates increased
markedly at  minutes after PO ingestion and remained elevat-
ed into the clamp at  min after PO Figure , B and C. GOX
decreased during the clamp following PO ingestion compared
with GOX after VCL ingestion Figure D.
PO raises hepatic ATP and lipid content. HCL and hepatic γATP
increased by  P  . and  P  . from – min-
utes to  minutes after PO, respectively. At  minutes, γATP
tended to be higher in the VCL arm P  ., while HCL lev-
els did not differ between PO and VCL arms. Hepatic inorganic
phosphate Pi and γATP/Pi did not change after PO or VCL Table
. When comparing the mean difference between – and 
minutes after PO or VCL between interventions, only the change
in HCL levels tended to be increased Δ  HCL PO vs. VCL,
P  .; Δ γATP, P  .; Δ Pi, P .; Δ γATP/Pi, P  ..
Studies in mice. Two mouse cohorts received PO or VCL via
gavage. One cohort then underwent hyperinsulinemic-euglycemic
clamp tests under unrestrained conditions, whereas tissue and
blood sample analysis was done for the other cohort.
PO increases circulating lipid levels. Plasma FFA levels increased
Table 1. Anthropometric and blood parameters of study participants
Parameter Mean ± SEM
n (men/women) 14 (14/0)
Age (yr) 25.8 ± 1.4
BMI (kg/m2)22.5 ± 0.3
Waist circumference (cm) 79.5 ± 1.4
Lean BW (kg) 59.2 ± 2.1
TG (mg/dl) 78.4 ± 9.8
FFA (μmol/l) 386.9 ± 32.9
ALT (U/l) 24.4 ± 3.2
AST (U/l) 26.0 ± 2.5
Fasting blood glucose (mg/dl) 76.1 ± 2.0
2-hour postprandial blood glucose (mg/dl) 76.4 ± 4.7
HCL (% H2O) 0.60 ± 0.09
ALT, alanine aminotransferase; AST, aspartate.
Table 2. Hepatocellular lipids and hepatic phosphorus-
containing metabolites
Time (min) –120 240
HCL (% H2O) 0.97 ± 0.20 0.93 ± 0.23 1.04 ± 0.21 1.26 ± 0.32A,D
γATP (mmol/l) 2.86 ± 0.17 2.79 ± 0.14 3.17 ± 0.16C3.25 ± 0.16B
Pi (mmol/l) 2.35 ± 0.04 2.61 ± 0.07 2.61 ± 0.15 2.65 ± 0.20
γATP/Pi1.21 ± 0.07 1.07 ± 0.04 1.26 ± 0.10 1.35 ± 0.17
Data represent the mean ± SEM. n = 12. AP < 0.01, BP < 0.05, and CP = 0.066,
for –120 minutes compared with 240 minutes; DP = 0.085, for the mean
difference from –120 to 240 minutes between groups.
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698 Volume 127 Number 2 February 2017
expression and signaling, which could contribute to the
promotion of NAFLD.
PO induced a marked increase in plasma FFA con-
centrations in both humans and mice, but no alterations
in circulating inflammatory markers or adipokines, such
as TNF-α, IL, fetuin A, chemerin, and omentin. This
inding indicates that the acute effects of PO are medi-
ated by metabolic rather than endocrine changes and is
partly in line with indings obtained after intravenous
infusion of soybean oil, which showed no changes in
circulating cytokines , or after ingestion of cream,
which resulted in increased expression of TNF-α but not
IL . Interspecies differences could also be due to
the administration of emulsiied versus pure PO.
Notably, a single dose of PO markedly altered
hepatic glucose fluxes and resulted in increased rates
of GNG, reduced GLYnet, and a corresponding trend
toward reduced GP flux. This inding extends our pre-
vious results on the effects of parenteral administration
of polyunsaturated lipids . The present study also
provides a comprehensive analysis of in vivo hepatic
glucose and glycogen fluxes in humans that includes an
assessment of glycogen cycling, which ensured correct
estimation of GLY and GNG contributions to EGP .
In chronic insulin-resistant states, such as occurs in
TDM or type  diabetes mellitus TDM, elevation of
GNG and EGP coexists with enhanced glycogen cycling
. Surprisingly, oral lipid loading stimulated GNG
and hepatic insulin resistance without affecting glyco-
gen cycling, which remained negligible, as was report-
ed for healthy humans in the fasted state , . This
indicates that healthy humans can rapidly downreg-
ulate GLY under conditions of elevated GNG to avoid
futile cycling. The absence of any effect on glycogen
cycling might be due to the prevailing euglycemia and
basal peripheral insulinemia compared with the hyper-
glycemic hypoinsulinemic conditions in the aforementioned
study. Consequently, the augmented glycogen cycling observed
in insulin-resistant states reflects chronically abnormal hepat-
ic energy metabolism rather than an immediate physiologi-
cal response to changes in dietary lipid supply. Of note, these
changes occur in the presence of higher glucagon concentra-
tions, which are likely due to FFA-induced glucagon stimula-
tion . Speciically, the increase in GNG and the decrease in
GLYnet upon PO administration could, at least in part, result
from the increase in circulating glucagon. Even small changes in
plasma glucagon can modify GLYnet independently of insulin in
healthy humans .
Interestingly, ingestion of PO increased hepatic γATP and
HCL concentrations, along with the induction of LOX and insulin
resistance. Recently, we reported that a test involving ingestion of a
mixed-meal with  fat content increases hepatic γATP exclusive-
ly in insulin-resistant obese humans but not in lean, nondiabetic or
TDM individuals . This suggests that the stimulatory effect of
saturated fat on hepatic energy metabolism is dose dependent and
may be linked to the onset of insulin resistance, but not the insulin
sensitivity state per se. In line with this, recent studies report the
Except for LPS, the analysis did not reveal whether the pathways
and upstream regulators were activated or inhibited.
Under insulin- and noninsulin-stimulated conditions, PO
resulted in upregulation of the following transcripts: miR,
LPS-regulated genes Iit, Cleca, Slca, and Car ,
Gs, and Ar, while Tweak gene expression was downregulated.
Opposing regulatory transcripts, i.e., those that were upregulated
under insulin-stimulated conditions and downregulated under
noninsulin-stimulated conditions, were also found and includ-
ed, for example, transcripts involved in cell growth Mapk and
Slcaf ,  and FFA metabolism and development of NAFLD
fatty acid–binding protein  or Fabp , as well as the predict-
ed pseudogenes Gm and HK Figure E.
This study demonstrates that a single oral dose of saturated fat
increases hepatic TG accumulation, insulin resistance, GNG, and
ATP concentrations in the human liver. Ingestion of saturated fat
also induces peripheral insulin resistance in skeletal muscle and
adipose tissue. In mice, a single saturated fat load preferentially
induces hepatic insulin resistance and also affects hepatic gene
Figure 2. Time courses of circulating metabolites and hormones in humans. VCL
(gray triangles) or PO (black circles) was administered at 0 minutes to lean, healthy
men, and the hyperinsulinemic-euglycemic clamp was started at 360 minutes. TG cir-
culating in plasma (solid line) and in chylomicrons (dashed line). The AUC was 59% and
156% higher after PO ingestion, respectively (A). Circulating FFA (B). Time courses for
insulin (C), C-peptide (AUC 28% higher after PO) (D), blood glucose (E), and glucagon
(AUC 41% higher after PO) (F). Values represent the mean ± SEM. n = 14; chylomicron
TG n = 6. ***P < 0.001, **P < 0.005, and *P < 0.05, by paired, 2-tailed t test. Large
asterisks refer to AUC differences; small asterisks refer to differences per time point.
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insulin resistance. This may have resulted from spe-
cies differences in insulin-sensitive tissues and lip-
id flux–buffering capacities of adipose tissue. Under
physiological conditions, adipose tissue can buffer
fatty acid flux into the bloodstream, thereby avoiding
excessive exposure to lipotoxic stimuli. This is accom-
plished by suppression of FFA and TG release as well
as increased clearance of circulating TG . Under
the present experimental conditions, the high-dose PO
likely impaired all  mechanisms in humans and to some
extent in mice. The higher plasma TG concentrations
observed in our study could have been due to higher rates
of intestinal lipid absorption, altered hepatic lipid han-
dling, or lower insulin sensitivity of the adipose tissue,
as reflected by reduced insulin-induced suppression of
FFA. Humans and mice both featured impaired insulin-
induced TG suppression after PO ingestion. This is part-
ly in line with indings in humans after a fat-free test
meal, in which TDM patients were also unable to sup-
press plasma TG in contrast to lean, healthy men .
The inability of our cohort to suppress TG secretion may
have therefore at least partly resulted from an altered
apolipoprotein metabolism associated with insulin-
resistant states and with decreased insulin-mediated
fatty acid trapping by adipose tissue . In order to
maintain low levels of HCL, the liver can only make use
of either LOX or of lipid export of apolipoproteins .
Here, we show that a single dose of PO induces both
mechanisms, but nonetheless leads to increased HCL
content. Of note, these changes occurred in the face of
increased C-peptide, but unchanged insulin concen-
trations. The mismatch between insulin and C-peptide
concentrations is likely explained by increased insulin
clearance and turnover. While lower concentrations
of palmitate decrease receptor-mediated insulin deg-
radation in rat hepatocytes, higher concentrations of
palmitate concentrations enhance this degradation
. This can also explain the lack of increased insulin
concentrations in the face of increased GLP and GIP.
In humans, most studies on insulin extraction used infusions of
mainly polyunsaturated lipids, which revealed either unchanged or
lower splanchnic insulin extraction , . Likewise, oral intake of
soybean oil containing  polyunsaturated fatty acids resulted in
increased concentrations of both insulin and C-peptide .
The marked alterations in hepatic glucose fluxes in humans
and the predominant hepatic insulin resistance in mice raise inter-
est in the effects of saturated fatty acids on hepatic gene expres-
sion, which has been previously examined mostly upon exposure
to polyunsaturated fatty acids . The present study showed that
a single dose of PO differentially regulated the canonical path-
ways TWEAK and AHR. The AHR pathway promotes NAFLD via
upregulation of fatty acid transport , which is in line with the
observed upregulation of Fabp and increase in HCL content in
humans. The TNF-related TWEAK is known to promote cell turn-
over homeostasis through the NF-κB and p MAPK pathways and
could serve as a biomarker of obesity and TDM . TWEAK was
also found to be associated with reduced TG accumulation in pal-
upregulation of hepatic mitochondrial capacity in obese, insulin-
resistant, but not nondiabetic, humans in the absence of progressive
NAFLD  and that TDM patients have decreased ATP turnover
associated with increased HCL . Collectively, these data suggest
that an acute rise in lipid availability and oxidation can upregulate
γATP production and HCL deposition in young, healthy individu-
als with conserved mitochondrial plasticity, even after the develop-
ment of acute insulin resistance. The minor increase in HCL has to
be considered in the context of lean, insulin-sensitive individuals.
In this case, it is reasonable to assume that an increase in HCL after
just  dose of PO probably contributes to altered hepatic metabo-
lism. Furthermore, PO, like meal ingestion, probably induced indi-
vidual time courses of increases, so that maximum γATP increases
may have been missed in the absence of continuous magnetic res-
onance spectroscopy MRS monitoring, which would have been
impossible, given the current experimental design.
Upon PO ingestion, human volunteers developed generalized
insulin resistance, while mice responded primarily with hepatic
Figure 3. Parameters of insulin resistance in human volunteers after VCL or PO during
clamp experiments. VCL, white bars; PO, black bars. (A) WBIS, reflected by the M value.
(B) Rd and its components GOX and nonoxidative glucose disposal (NOXGD). (C) EGP
denoting hepatic insulin sensitivity at baseline (–180 min), after PO or VCL ingestion
(240 min), and under insulin-stimulated conditions during the clamp (480 min). (D)
Insulin-induced EGP suppression as an indicator of hepatic insulin sensitivity. (E)
Insulin-induced FFA suppression reflecting adipose tissue insulin sensitivity and (F) the
percentage of insulin-induced TG suppression. Data shown represent the mean ± SEM.
n = 14. ****P = 0.0005, **P < 0.01, and *P < 0.05; #P < 0.05, for GOX PO versus VCL.
P values were determined by 2-tailed t test and ANOVA.
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The Journal of Clinical Investigation
700 Volume 127 Number 2 February 2017
mitic acid–treated hepatocytes and may be involved in hepatic tis-
sue repair . The present study cannot prove the activation sta-
tus of TWEAK, thus making it dificult to interpret the observed
downregulation of the Tweak gene and its receptor Tnfrsfa.
However, recent indings of decreased circulating TWEAK con-
centrations correlating with obesity and concomitant NAFLD
 could suggest that inhibition of the TWEAK pathway might
increase susceptibility to hepatic injury.
The expression analyses further predicted PO-induced acti-
vation of LPS with or without insulin stimulation. This study also
found upregulation of Iit, Cleca, Slca, and Car, genes
regulated by LPS-stimulated macrophages. These data are in line
with the reported increase in LPS concentrations upon high-fat
ingestion ,  resulting from lipid-induced disruption of the
intestinal barrier . Interestingly, LPS is also known to decrease
TWEAK signaling .
Downstream from LPS, NF-κB was found to be differentially
regulated by PO. Generally known for its proinflammatory proper-
ties, NF-κB is also an important antiapoptotic factor , . LPS
activation and TNF collectively lead to increased TLR expres-
sion, proinflammatory cytokine production, and inflammation,
on the one hand, and NF-κB activation and cytoprotection on the
other . NF-κB activation leads to modest and short-lived JNK
activation, in turn inducing antiapoptotic genes, such as c-FLIP
a caspase  inhibitor and X-linked inhibitor of apoptosis .
As a result, the active NF-κB pathway is critical for LPS-induced
resistance to hepatotoxicity . Additionally, a high-fat diet and
obesity are associated with prolonged JNK activation and TNF-
induced cell death , . This alludes to an adaptation, which is
lost upon repeated and/or sustained exposure to hepatotoxic stim-
uli leading to NAFLD and steatohepatitis. Furthermore, the consti-
tutive activation of NF-κB has been associated with severe hepatic
and moderate peripheral insulin resistance . The present data
suggest that a single PO challenge promotes pathways of LPS-
and TLR-mediated inflammation and cytotoxicity, which are
buffered by the activation of NF-κB, which in turn contributes to
insulin resistance. This study also found altered regulation of other
putative cytoprotective mechanisms including the phospholipase
C pathway, which is important for hepatic regeneration , and
PPARα, which serves as both a canonical pathway and an upstream
regulator protecting against NAFLD progression .
Our analyses of the differential regulation of single genes by
saturated fat revealed several genes of interest. The observed
upregulation of Gs may contribute to decreased TG clearance,
thereby promoting NAFLD . The observed greater expression of
Arl in the present study may also serve to protect against NAFLD,
Figure 5. Time course of parame-
ters of energy metabolism in lean,
healthy volunteers after VCL and
PO. VCL, white bars; PO, black bars.
Parameters were obtained by perform-
ing an indirect calorimetry. The time
points indicated are basal (–5 min),
300 minutes after intervention, and
420 minutes under insulin-stimulated
conditions. Effects on RQ (A), REE (B),
LOX (C), and GOX (D). Data represent
the mean ± SEM. n = 14. ***P < 0.001,
**P < 0.005, and *P < 0.05, by ANOVA.
Figure 4. Hepatic glucose fluxes in humans. The rates of GNG, GP
flux, glycogen cycling (cycling), and GLYnet were analyzed using in vivo
13C/31P/1H and ex vivo 2H MRS combined with 2H2O ingestion, after either
VCL (white bars) or PO (black bars) treatment, in lean, insulin-sensitive,
male volunteers. Data represent the mean ± SEM. n = 14; GP and cycling
n = 9. *P < 0.05 and #P = 0.085, by 2-tailed t test.
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The Journal of Clinical Investigation CLINICAL MEDICINE
701 Volume 127 Number 2 February 2017
because loss of function of this gene has been implicated in obesity,
NAFLD, and diabetes in Bardet-Biedl syndrome . Of note, the
descriptive nature of the transcriptome analyses and expression
proiling carried out in our study does not allow inal conclusions
to be drawn as to causality. That is to say, given the results of this
study, it is impossible to single out  gene, transcription factor, or
pathway that is activated by acute exposure to PO and that can be
causally linked to either insulin resistance or steatosis.
This study offers the advantages of the translational approach
in mice and humans, the use of near-physiologic administration
of saturated fat, and the comprehensive phenotyping of in vivo
hepatic glucose and energy metabolism. This study’s limitations
include the relatively small number of humans and mice, which
might have prevented the detection of discrete effects of an acute
lipid challenge. Nevertheless, the results demonstrate marked
metabolic and transcriptional changes associated with PO treat-
ment, even in these small groups. Another caveat of this study is
that the hepatic expression data obtained in mice are not neces-
sarily transferable to humans, as we could not obtain liver speci-
mens from our human participants for ethical reasons. The lack of
proteomic analysis of the targets identiied by Ingenuity software
constitutes another limitation of this study. Each pathway and reg-
ulator necessitates a thorough study of their role in the pathogen-
esis of insulin resistance and NAFLD, a task that must be tackled
in subsequent studies.
The practical implication of this work is that the PO challenge
used in this study most likely resembles the effects of ingestion of
a meal rich in saturated fat, e.g., an -slice pepperoni pizza, con-
taining approximately . g of saturated fat/ g  or a meal
consisting of a -g cheeseburger and a large portion of French
fries, containing  g and  g per , kcal of saturated fat,
respectively . One such meal would probably be suficient to
induce transient insulin resistance and impair hepatic metabo-
lism, which necessitates the activation of hepatic defense mecha-
nisms. Other simultaneously ingested biomacromolecules would
exacerbate this metabolic challenge . The amount and types
of fatty acids and carbohydrates in one such meal are in contrast to
the diet recommendations of the American Diabetes Association
ADA. In this diet, daily intake of saturated fatty acids should not
exceed  of total caloric intake. Furthermore, intake of mono-
unsaturated fatty acids and carbohydrates from vegetables, fruits,
whole grains, and legumes is recommended . We presume
that lean, healthy individuals are able to compensate adequately
for excessive intake of saturated fatty acids, however, sustained
and repeated exposure to such nutrients will ultimately lead to
chronic insulin resistance and NAFLD. Recent studies have shown
hepatic energy metabolism alterations and induction of insulin
resistance in obese and lean patients after ingestion of a simple
hypercaloric mixed meal containing  g of fat or a drink con-
taining  g/kg BW of a mixture of palmitate and soybean oil ,
, . These results suggest that even lower doses of fatty acids
are capable of inducing alterations similar to those observed with
ingestion of pure PO.
In conclusion, the initial effects of ingestion of saturated fat
include a augmented hepatic energy metabolism and lipid stor-
age; b impaired hepatic insulin sensitivity, along with increased
GNG flux; and c altered hepatic expression of genes regulating
inflammatory and protective pathways, which predispose to and
protect against the development of NAFLD.
Studies in humans
Volunteers. Fourteen lean, young male volunteers were enrolled in this
randomized, controlled crossover trial Figure  and Figure . Partic-
ipants were recruited from March  through December . The
sample size calculation was based on a -sided, paired t test, assum-
ing a mean difference of EGP of . and a standard deviation of .,
resulting in a sample size of  to reach a power of . The random
allocation sequence was generated using SAS software SAS Institute
by our in-house statistician. The possible order of treatments was
randomly permuted in  blocks, with  extra block being generated
to account for dropouts. Allocation was not concealed. Participants
were enrolled and assigned to their treatment order by the study phy-
Figure 6. Circulating metabolites and hormones in mice. VCL
(white bars) or PO (black bars) was administered via gavage
to identical mouse cohorts at minute 0, after a 6-hour fast.
Hyperinsulinemic-euglycemic clamp experiments were per-
formed from 120 to 240 minutes. (A) The TG AUC tended to be
higher after PO than after VCL. (B) The FFA AUC was increased
after PO administration. Blood glucose (C) and insulin (D)
levels were not different between groups. Data represent the
mean ± SEM. n = 6–10. **P < 0.005 and #P = 0.08, by 2-tailed
t test.
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The Journal of Clinical Investigation
702 Volume 127 Number 2 February 2017
sician. Inclusion criteria were age between  and  years and a BMI
between  and  kg/m. Exclusion criteria included dysglycemia,
a family history of TDM, acute, or chronic diseases, and the use
of pharmacological agents known to affect insulin sensitivity, lipid
metabolism, or immunological function. All participants underwent
screening that included recording of clinical history, physical exam-
ination, bioimpedance assessment of lean body mass, routine labo-
ratory tests, and a -g oral glucose tolerance test. Upon inclusion,
participants were instructed to maintain their usual physical activity
during the study period and to ingest a carbohydrate-rich diet for 
days before each study day. They were then randomly assigned to 
intervention and  weeks later to the other intervention. Basal hepatic
ATP and HCL values from the study by Gancheva et al. were used as
part of the control group data .
Experimental design. Participants arrived at the Clinical Research
Center at  pm and received a standardized dinner containing approxi-
mately  kcal at : pm. At : pm, : pm, and : am, vol-
unteers drank . g/kg body water of HO .; Sigma-Aldrich, up
to a total dose of  g/kg body water in order to assess GNG  Figure
. Body water was assumed to be  in all of our male study cohort
participants . Starting at : am deined as time point
– minutes of the next day, participants drank  ml
of water containing . HO every  minutes through-
out the experiment to maintain an isotopic equilibrium of
body water. At –, , and  minutes, participants
received an oral dose of acetaminophen  mg. For EGP
calculation, participants received a -minute priming bolus
. mg/kg BW/min  fasting plasma glucose mg/dl of
D-,-Hglucose  enriched in H glucose; Cam-
bridge Isotope Laboratories at – minutes, followed by
a continuous infusion . mg/kg BW/min . At zero
time, participants drank either VCL or PO within  min-
utes. Patients with more than  kg BW drank  g, and
those with less than  kg BW drank  g of PO ~.g/kg
BW; Biopalm; Landkrone . To facilitate ingestion, PO
was heated to °C, mixed with . g or . g emulsiier
Glice, Texturas; Albert y Ferran Adria,  or  g sugar-free
vanilla syrup Torani, and . or . ml bottled still water,
for a PO mix of  g and  g, respectively. Oil test drinks
were stirred constantly and served hot. For VCL administra-
tion, PO was substituted with . ml or . ml bottled
still water, respectively. At  minutes, a hyperinsulinemic-
euglycemic clamp began -min insulin bolus:  U/hour;
continuous insulin infusion:  mU/m/min; Insuman Rap-
id; Sanoi. Blood glucose concentration was adjusted to 
mg/dl by adapting the glucose infusion rate GIR using 
glucose B. Braun AG enriched with  D-,-Hglucose,
as described previously . Urine was sampled from –
to  minutes, from  to  minutes, and from  to 
minutes for the quantiication of GNG and GLY. Blood was
sampled at – and  minutes for assessment of GNG.
Indirect calorimetry. Indirect calorimetry IC was
performed in the canopy mode using Vmax Encore n
CareFusion, as described previously , during base-
line at – min, intervention at  min, and steady-
state clamp conditions at  min for  minutes after a
-minute adaptation period. R, REE, and substrate oxi-
dation rates were calculated as reported previously . Nonoxidative
glucose disposal was calculated from the difference between rates of
glucose disappearance and carbohydrate oxidation.
Metabolites and hormones. Blood samples were immediately
chilled, centrifuged, and the supernatants stored at either –°C or
–°C until analysis. Venous blood glucose concentration was mea-
sured immediately using the glucose oxidase method EKF Biosen
CLine glucose analyzer; EKF Diagnostics . TG concentration
was analyzed enzymatically on a Roche Cobas c  Analyzer Roche
Diagnostics. Serum chylomicron content was determined from the
TG concentration in the irst fraction of density-gradient ultracentrif-
ugation . FFA were assayed enzymatically Wako using orlistat to
prevent in vitro lipolysis . Serum C-peptide, insulin, and plasma
glucagon levels were measured by radioimmunoassay EMD Milli-
pore. Cortisol levels in serum samples were measured by immunoas-
say using a Siemens Immulite XPi Analyzer . GLP and GIP
were measured by ELISA TECOmedical; EMD Millipore . ELISA
was used to measure plasma concentrations of IL, TNF-α, fetuin-A
all using Quantikine HS ELISA kits from R&D Systems, omentin,
and chemerin  both using ELISA kits from BioVendor. Intra-
Figure 7. Parameters of insulin resistance in mice. VCL (white bars) or PO (black bars)
was administered via gavage to identical mouse cohorts at minute 0, after a 6-hour
fast. Hyperinsulinemic-euglycemic clamp experiments were performed from 120 to
240 minutes. The M value trended toward a reduction after PO (A), while the Rd was
unchanged (B). EGP at basal (0 min) and at clamp steady state (210–240 min) (C). EGP
suppression was impaired after PO (D). Insulin-induced FFA suppression (E) and TG
suppression (F) are also shown. Data represent the mean ± SEM. n = 9–10. **P < 0.005,
*P < 0.05, and #P = 0.07, by 2-tailed t test.
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703 Volume 127 Number 2 February 2017
Gas chromatography–mass spectrometry. Determination of atom
percentage enrichment APE of H in blood glucose was done after
deproteinization and derivatization to the aldonitrile-pentaacetate.
The analyses were performed on a Hewlett-Packard  gas chro-
matograph equipped with a  m/. mm/. μm CPSilCB capil-
lary column Chrompack; Varian and interfaced with a Hewlett Pack-
ard  mass selective detector. Selected ion monitoring was used to
determine enrichments of fragments C to C Supplemental Figure
, A and B. Average mass units were  for endogenous glucose and
 for D-,-Hglucose as described previously .
MRS. All measurements were conducted with the volunteers lying
in a supine position within a whole-body . T Achieva MRI machine
Philips Healthcare. Twelve volunteers were studied, including all par-
ticipants for whom flux measurements were obtained. The effects of PO
or VCL on HCL and hepatic ATP concentrations were assessed at base-
line and  minutes after intervention. For hepatic H MRS, a Q-body
coil was used for shimming and HCL acquisition. Clinical T-weighted
turbo spin-echo TSE images were obtained in the transverse and cor-
onal planes for localization and repositioning of the voxels used for
HCL and ATP measurements. Respiratory-triggered H spectra were
acquired with a single-voxel      mm stimulated echo acqui-
sition mode STEAM sequence. The variables were as follow: repetition
time TR , ms, echo time  ms, and signal averages . To accu-
assay and interassay coeficients of variation CV for all cytokines
were  to . and  to ., respectively.
Glucose and glucuronide H enrichment measurements by ex vivo H
MRS. The positional enrichment of urinary acetaminophen glucuron-
ide and plasma glucose, resulting from ingestion of HO and acetamin-
ophen at the level of glucose -phosphate GP, was assessed as previ-
ously described  to estimate the contributions of GNG and GLY to
EGP. Plasma glucose was derivatized to monoacetone glucose MAG,
while urinary acetaminophen glucuronide was converted into -O-
acetyl monoacetone glucuronic lactone MAGLA . When plasma
glucose enrichment was inadequate, urinary glucuronide enrichment
was analyzed instead, since both methods yield identical estimates of
EGP contributions , . In total,  participants yielded suficient
data for NMR analysis,  of them for glucuronide. H spectra were
obtained with a Bruker Avance III HD  spectrometer equipped
with a H-selective -mm probe incorporating a F lock channel. For
MAGLA samples, ,, free-induction decays FIDs were
collected. For MAG samples, ,, FIDs were collected.
Positional H enrichments of MAG and MAGLA derived from plasma
glucose and urinary anion gap AG were determined using the methyl
signals as an intramolecular standard . All spectra were analyzed
using the curve-itting routine supplied with the NUTS PC-based NMR
Spectral Analysis Program Acorn NMR.
Figure 8. Transcriptome analysis after PO in murine
hepatic tissue. Tissue was harvested from clamped and
nonclamped murine cohorts. (A) Relevant canonical
pathways predicted by Ingenuity software in hepatic
samples from mice after PO under insulin- (black bars) and
noninsulin-stimulated (gray bars) conditions. (B) Predicted
canonical pathways with a P value of less than 0.05 during
insulin-stimulated (turquoise) and noninsulin-stimulated
conditions (blue). (C) Noteworthy predicted upstream
regulators and (D) predicted upstream regulators with a
P value of less than 0.01 (asterisk denotes an activation
Z score above 1.9). (E) Genes that were upregulated in both
cohorts (green), downregulated in both cohorts (yellow),
and downregulated under insulin-stimulated, but upregu-
lated under noninsulin-stimulated, conditions (green and
yellow, respectively) after PO treatment, with a P value
of less than 0.05, a fold change greater than 1.3, and an
average expression in at least 1 group of greater than 4
(n = 9–10, 273, and 327 probe sets, respectively).
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704 Volume 127 Number 2 February 2017
Calculations. Total AUC were calculated using the trapezoidal
method. FFA and TG suppression was calculated from  – clamp
FFA or TG concentrations  /basal FFA or TG concentrations,
respectively. Glycogen cycling, i.e., simultaneous fluxes through gly-
cogen synthase GS and GP, was assessed. In order to measure glyco-
gen cycling, isotopic tracer measurements of EGP, GNG, and GP fluxes
must be supplemented by a measurement of GLYnet in this case, C
MRS . GLYnet was calculated from the linear regression of hepatic
glycogen content at –, , , and  hours of PO or VCL ingestion using
the least mean squares method. Rates of GLYnet were normalized to
liver volume and BW and are expressed in μmol/kg/min .
Whole-body glucose disposal M value was calculated from glu-
cose infusion rates during the clamp steady state. The rates of EGP
in μmol/kg/min were calculated by dividing the tracer infusion
rate of D-,-Hglucose times its enrichment to the hydrogens
bound to carbon , by the mean percentage of enrichment of plas-
ma D-,-Hglucose and then subtracting the tracer infusion rate
. To account for the incorporation of H from HO by GNG during
the overnight fast, background D-,-Hglucose was determined
before administration of HO as well as at – minutes on the day
of the study. Consequently, for determination of basal EGP and EGP
at the end of the intervention, the background D-,-Hglucose
enrichment from the –-minute time point was used for calcula-
tions, whereas, during clamp conditions, with GNG being close to
zero, the D-,-H value before administration of HO was used.
GNG in μmol/kg/min was calculated from the difference between
EGP and GLYnet. The fractional GP flux contribution to EGP, i.e.,
the fraction of EGP originating from GLY, was calculated as  – H/
H; where H/H is the ratio of glucuronide position  to position
 enrichment from HO, keeping in mind that glucose derived from
GLY is enriched with H in position , while glucose derived from
GNG is enriched in positions  and . Absolute GP flux in μmol/kg/
min was calculated from the equation: GP  EGP ×  – H/H. Gly-
cogen cycling was then calculated as: GP – GLYnet.
rately assess hepatic lipid volume, sets of non–water-suppressed and
water-suppressed H spectra were acquired, using a STEAM sequence
TR , ms, echo time  ms, signal averages  ms and variable
power and optimization relaxation VAPOR STEAM sequences TR,
echo time, and signal averages ,, , and  ms, respectively.
Water and lipid peaks were itted and quantiied using the NUTS soft-
ware package Acorn NMR, and lipid was expressed as the summa-
tion of the methyl and methylene fat peaks relative to water content
using the equations described in ref. . For hepatic P MRS, a -cm
P circular surface coil Philips Healthcare was placed over the liver
for the acquisition of hepatic P spectra. Afterwards, PMRS proton-
decoupled liver ATP measurements were conducted with a D image-
selected in vivo spectroscopy DISIS localized sequence voxel:
     mm, TR: , ms, averages: , decoupling: WALTZ 
wideband alternating-phase low-power technique for zero-residual
splitting –, time:  min. Absolute concentrations of γATP CV  
and Pi CV   were quantiied using the jMRUI v. software pack-
age EC Human Capital and Mobility Networks, France, as described
previously . Liver volume measurements were made from the cor-
onal plane T-weighted TSE images. For liver glycogen measurements,
C spectra were obtained with a -cm dual-tuned C/H coil PulseTeq
Ltd. , via a proton-decoupled pulse acquisition sequence TR: 
ms; bandwidth:  KHz, averages:   ,; data points: ; decou-
pling: continuous wave; time:  min. Liver glycogen spectra were
acquired with a block pulse  μs that produced an Ernst angle at a
distance of  mm. Coil loading was corrected via integration of the
right-most peak of a C-enriched sample of formic acid placed within
the coil housing. Glycogen concentrations were determined from the
integration of the C-glycogen resonance zero illing: k, effective line
broadening:  Hz after the addition of  scans and baseline correc-
tion within NUTS software Acorn NMR Inc.. The glycogen signal was
corrected for distance and quantiied via aqueous glycogen phantom
measurements of  and  mmol/l measured at a distances of  to
 mm. The CV from repeated hepatic glycogen measurements was .
Figure 9. Human study design. Lean, healthy male adults randomly received either PO or VCL on 2 occasions. Hepatic metabolism was measured using
in vivo 13C, 31P, 1H and ex vivo 2H MRS combined with 2H2O and acetaminophen ingestion before and during hyperinsulinemic-euglycemic clamps with
D-[6,6-2H2]glucose–labeled 20% glucose infusion.
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deoxyglucose--phosphate was separated from -Cdeoxyglucose
via ion-exchange columns Poly-Prep AGX; Bio-Rad as previ-
ously described . Glucose uptake was calculated by multiplying
the mean plasma glucose levels between  and  minutes of the
clamp mmol/ml by - Cdeoxyglucose tissue content dpm/ g
tissue, divided by the -Cdeoxyglucose plasma AUC in the same
time frame. Radioisotopes were purchased from PerkinElmer and
samples measured in an Ultima-Gold Scintillation Cocktail Tri-
CarbTR; PerkinElmer Figure . Whole-body glucose dispos-
al M value was calculated from the tracer infusion rate, the speciic
activity of -Hglucose, and BW.
Biochemical analyses. Blood glucose concentrations were assessed
using a Contour hand-held glucometer Bayer Vital. Plasma TG lev-
els were determined by a colorimetric assay Cayman Chemical, and
plasma FFA levels were assessed with the FFAHR-Test Wako.
Hepatic TG levels were measured in whole-liver homogenates bio-
chemically with the BioVision Assay.
Experiments under noninsulin-stimulated conditions. CBL/N Tac
mice n   with characteristics identical to those described above
were fasted for  hours and given  g/kg BM PO Landkrone or
water VCL per gavage. Lateral tail vein blood samples were obtained
prior to treatment and  hours afterward. Six hours after treatment,
mice were euthanized with isoflurane, and a vena cava blood sample
was collected and centrifuged at °C, and plasma aliquots were imme-
diately frozen in liquid nitrogen. Liver was dissected and immediately
snap-frozen in liquid nitrogen Figure .
RNA isolation
Snap-frozen liver samples from both cohorts were processed after
administration of PO. Total RNA was isolated using the mRNeasy
Mini Kit QIAGEN. The Agilent  Bioanalyzer was used to assess
Figure 10. Mouse study design. Lean, adult male C57BL/6NTac mice were matched for BM and littermates and then divided into 2 cohorts. One cohort
underwent hyperinsulinemic-euglycemic clamps after receiving either PO or vehicle via gavage (A), whereas another identical cohort underwent analysis of
tissue and blood samples (B).
Studies in mice
Animals. Studies were conducted in lean, male,  week-old
CBL/N Tac mice Taconic. Animals had ad libitum access to water
and a standard chow diet. Mice were kept on a low-fat LF diet  of
calories derived from fat,  kJ/g, Standard Diet ; Altromin, and
were matched for body mass BM and littermates. BM and compo-
sition MiniSpec LF; Bruker Optics were measured  day prior to
the start of the experiment. Animals were bred and housed in a tem-
perature- and humidity-controlled environment including a -hour
light/-hour dark cycle, in compliance with the Federation of Euro-
pean Laboratory Animal Science Associations protocols.
Experiments under insulin-stimulated conditions. A permanent jug-
ular vein catheter was placed under ketamine/xylazine anesthesia into
a cohort of  mice with the aforementioned characteristics. Six to
seven days later, the mice were fasted for  hours and then received
 g/kg BM PO or VCL via gavage. Six hours later, unrestrained, con-
scious mice underwent hyperinsulinemic-euglycemic clamps.
After  minutes of primed-continuous -Hglucose infusion
. kBq/min, a blood sample was collected to determine plasma
insulin, glucose, and -Hglucose concentrations for the calcula-
tion of basal EGP. A -Hglucose infusion . kBq/min containing
insulin  pmol/kg/min; HumulinR; Lilly was started. Blood glucose
concentrations were measured every  minutes and target glycemia
established by adjusting the GIR. At minute , -Cdeoxyglucose
 kBq was injected intravenously to assess tissue-speciic Rg rates.
At the end of the experimental procedure, mice were euthanized by
means of an intravenous ketamine/xylazine injection. Livers were
collected, immediately snap-frozen in liquid nitrogen, and stored at
–°C. Blood was collected at culling, and plasma H and C radioac-
tivity was determined in deproteinized plasma before and after HO
evaporation to estimate glycolysis rates. In hepatic lysates, -C
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The Journal of Clinical Investigation
706 Volume 127 Number 2 February 2017
study, obtained MRS data, and reviewed and edited the manuscript.
EÁH, SK, AS, and PB contributed equally to this project. YK inter-
preted MRS data. BN researched the clinical data and reviewed and
edited the manuscript. CB and FC performed derivatization exper-
iments and enrichment analysis for HMRS. JGJ performed the
HMRS analyses and reviewed and edited the manuscript. PN con-
ducted analyses and reviewed and edited the manuscript. CH con-
ducted laboratory analyses and reviewed and edited the manuscript.
SN and JR supervised the mouse studies and edited and reviewed
the manuscript. MI analyzed transcriptomics data and edited and
reviewed the manuscript. JB and MHdA supervised transcriptome
analyses and edited and reviewed the manuscript. All authors gave
inal approval of the version to be published.
We thank Ulrike Partke and Anika Morcinietz at the German Diabe-
tes Center Düsseldorf, Germany as well as Anke Bettenbrock, Jür-
gen Schultheiß, Moya Wu, and Anne Junker at the Helmholtz Cen-
ter Munich, Germany for their excellent technical support. We
also thank Alessandra Bierwagen at the German Diabetes Center
for her guidance with MRS data analysis. This study was supported
in part by the Ministry of Innovation, Science, and Research North
Rhine–Westphalia MIWF NRW; the German Federal Ministry of
Health BMG; as well as by a grant from the Federal Ministry of
Education and Research BMBF to the German Center for Diabe-
tes Research DZD e.V.; DZD grant , and by grants from the
Helmholtz portfolio theme: Metabolic Dysfunction and Common
Diseases, and the Helmholtz Alliance to Universities: Imaging and
Curing Environmental Metabolic Diseases ICEMED; the Ger-
man Research Foundation DFG; SFB ; the German Diabetes
Association DDG; and the Schmutzler-Stiftung. Financial sup-
port was also provided by the Portuguese Foundation for Science
and Technology research grant EXCL/DTP//. Struc-
tural funding for the Center for Neurosciences and the UCNMR
facility was provided in part by the FEDER – European Regional
Development Fund through the COMPETE Program; by grants
from the Portuguese Foundation for Science and Technology
PEst-C/SAU/LA/, REE//QUI/, RECI/QEQ-
QFI//, and CENTROCTFEDER; and
by the Rede Nacional de Ressonância Magnética Nuclear.
Address correspondence to: Michael Roden, Auf’m Henneka-
mp , , Düsseldorf, Germany. Phone: .....;
BN’s present address is: Clinical Research Services GmbH,
Mönchengladbach, Germany.
SN’S present address is: Sanoi-Aventis Deutschland GmbH,
R&D Diabetes Research & Translational Medicine, Frankfurt am
Main, Germany.
RNA quality, and only high-quality RNA RNA integrity number RIN
 was used for microarray analysis.
Expression profiling
Total RNA ~ ng was ampliied using the Ovation PicoSL WTA
System V in combination with the Encore Biotin Module both from
NuGEN. Ampliied cDNA was hybridized on Affymetrix Mouse Gene
. ST array plates containing approximately , probe sets. Stain-
ing and scanning GeneChip Scanner  G; Affymetrix were per-
formed according to the Affymetrix Gene Titan expression protocol
and modiied according to NuGEN’s Encore Biotin protocol.
Statistical transcriptome analysis
Expression Console software version ...; Affymetrix was used
for quality control and to obtain annotated normalized robust multiar-
ray average RMA gene-level data standard settings included median
polish and sketch-quantile normalization. Statistical analyses were
performed using the statistical programming environment R imple-
mented in CARMAweb .
Genewise testing for differential expression was done using the limma t
test, and a P value of less than . was set as the threshold to deine sets
of regulated genes. Filters for a fold change greater than . times and a
linear average expression greater than  were applied. Pathway analy-
ses were generated using the Ingenuity Pathway Analysis QIAGEN;, where the overlapping P value identiies
transcriptional regulators that can explain observed gene expression
changes. The activation Z score helps infer activation states of predicted
transcriptional regulators, with values of  or more indicating activation
and values of – or less indicating inhibition.
General statistical analyses
Results are presented as the mean  SEM and were compared using a
-tailed Student t test or ANOVA adjusted for repeated measures, with
Bonferroni’s testing as appropriate. Calculations were performed using
GraphPad Prism, version . GraphPad Software. A P value of less than
. was considered statistically signiicant, unless otherwise indicated.
Study approval
All participants provided written informed consent before inclusion in
the study, which was performed according to the Declaration of Hel-
sinki of  and approved by the ethics board of the Heinrich Heine
University Düsseldorf. All animal experiments were approved by the
Upper Bavarian district government AZ ....
Author contributions
MR initiated the investigation, led the clinical experiments and
wrote, reviewed, and edited the manuscript. EÁH obtained and
analyzed the data and wrote, edited, and reviewed the manuscript.
SK obtained and analyzed the data, aided in designing the clinical
study, and edited and reviewed the manuscript. AS obtained data
and edited and reviewed the manuscript. PB designed the MRS
. van Dam RM, Willett WC, Rimm EB, Stampfer
MJ, Hu FB. Dietary fat and meat intake in relation
to risk of type  diabetes in men. Diabetes Care.
. Mancini A, et al. Biological and Nutritional Prop -
erties of Palm Oil and Palmitic Acid: Effects on
Health. Molecules. ;:.
. Szendroedi J, et al. Abnormal hepatic energy
homeostasis in type  diabetes. Hepatolog.
. Koliaki C, Roden M. Hepatic energy metabolism
in human diabetes mellitus, obesity and non-
alcoholic fatty liver disease. Mol Cell Endocrinol.
. Erion DM, Shulman GI. Diacylglycerol-mediated
Downloaded from on February 2, 2017.
The Journal of Clinical Investigation CLINICAL MEDICINE
707 Volume 127 Number 2 February 2017
insulin resistance. Nat Med. ;: .
. Magkos F, et al. Effects of Moderate and Sub-
sequent Progressive Weight Loss on Metabolic
Function and Adipose Tissue Biology in Humans
with Obesity. Cell Metab. ;:.
. Winzell MS, Ahrén B. The high-fat diet-fed
mouse: a model for studying mechanisms and
treatment of impaired glucose tolerance and type
 diabetes. Diabetes. ; Suppl :S S.
. Gastaldelli A, et al. Influence of obesity and
type  diabetes on gluconeogenesis and glucose
output in humans: a quantitative study. Diabetes.
. Groop LC, et al. Glucose and free fatty acid
metabolism in non-insulin-dependent diabetes
mellitus. Evidence for multiple sites of insulin
resistance. J Clin Invest. ;:.
. Lewis GF, Carpentier A , Vranic M, Giacca A.
Resistance to insulin’s acute direct hepatic effect
in suppressing steady-state glucose production
in individuals with type  diabetes. Diabetes.
. Bachmann OP, et al. Effects of intravenous and
dietary lipid challenge on intramyocellular lipid
content and the relation with insulin sensitivity
in humans. Diabetes. ;: .
. Roden M, et al. Mechanism of free fatty acid-
induced insulin resistance in humans. J Clin
Invest. ;: .
. Szendroedi J, et al. Lipid-induced insulin resis-
tance is not mediated by impaired transcapillary
transport of insulin and glucose in humans. Dia-
betes. ;:.
. Itani SI, Ruderman NB, Schmieder F, Boden G.
Lipid-induced insulin resistance in human mus-
cle is associated with changes in diacylglycerol,
protein kinase C, and IkappaB-alpha. Diabetes.
. Kashyap SR, et al. Discordant effects of a chronic
physiological increase in plasma FFA on insulin
signaling in healthy subjects with or without a
family history of type  diabetes. Am J Physiol
Endocrinol Metab. ;:EE.
. Boden G, et al. Effects of fat on insulin-
stimulated carbohydrate metabolism in normal
men. J Clin Invest. ;:.
. Shah P, et al. Effects of free fatty acids and glycer-
ol on splanchnic glucose metabolism and insulin
extraction in nondiabetic humans. Diabetes.
. Clore JN, Stillman JS, Li J, O’Keefe SJ, L evy JR.
Differential effect of saturated and polyunsatu-
rated fatty acids on hepatic glucose metabolism
in humans. Am J Physiol Endocrinol Metab.
. Nowotny B, et al. Mechanisms underlying
the onset of oral lipid-induced skeletal mus-
cle insulin resistance in humans. Diabetes.
. Xiao C, Giacca A, Carpentier A, Lewis GF.
Differential effects of monounsaturated, poly-
unsaturated and saturated fat ingestion on glu-
cose-stimulated insulin secretion, sensitivity and
clearance in overweight and obese, non-diabetic
humans. Diabetologia. ;: .
. Lee CG, Demarquoy J, Jackson MJ, O’Brien
WE. Molecular cloning and characterization
of a murine LPS-inducible cDNA. J Immunol.
 . Ban JY, Kim BS, Kim SC, Kim DH, Chung JH.
Microarray Analysis of Gene Expression Proiles
in Response to Treatment with Melatonin in
Lipopolysaccharide Activated RAW . Cells.
Korean J Physiol Pharmacol. ;:.
. Li L, et al. A solute carrier family  member 
variant rs GA associated with coro-
nary heart disease inhibits lipopolysaccharide-in-
duced inflammatory response. J Biol Chem.
. Sivapalaratnam S, et al. Identiication of can-
didate genes linking systemic inflammation to
atherosclerosis; results of a human in vivo LPS
infusion study. BMC Med Genomics. ;:.
. Masaki M, Ikeda A, Shiraki E, Oka S, Kawasaki
T. Mixed lineage kinase LZK and antioxidant
protein- activate NF-kappaB synergistically. Eur
J Biochem. ;:.
. Noh H, Paik HY, Kim J, Chung J. The alteration
of zinc transporter gene expression is associated
with inflammatory markers in obese women. Biol
Trace Elem Res. ;:.
 . Westerbacka J, et al. Genes involved in fatty acid
partitioning and binding, lipolysis, monocyte/
macrophage recruitment, and inflammation are
overexpressed in the human fatty liver of insulin-
resistant subjects. Diabetes. ;: .
 . Deopurkar R, et al. Differential effects of cream,
glucose, and orange juice on inflammation,
endotoxin, and the expression of Toll-like recep-
tor- and suppressor of cytokine signaling-.
Diabetes Care. ;:.
. Roden M, et al. Effects of free fatty acid elevation
on postabsorptive endogenous glucose produc-
tion and gluconeogenesis in humans. Diabetes.
. Kacerovsky M, et al. Postprandial and fasting
hepatic glucose fluxes in long-standing type 
diabetes. Diabetes. ;: .
. Hundal RS, et al. Mechanism by which metformin
reduces glucose production in type  diabetes.
Diabetes. ;:.
. Wang L, et al. Acute stimulation of glucagon
secretion by linoleic acid results from GPR
activation and Cai increase in pancreatic islet
alpha-cells. J Endocrinol. ;: .
. Roden M, et al. The roles of insulin and glu-
cagon in the regulation of hepatic glycogen
synthesis and turnover in humans. J Clin Invest.
 . Fritsch M, et al. Time course of postprandial
hepatic phosphorus metabolites in lean, obese,
and type  diabetes patients. Am J Clin Nutr.
. Koliaki C, et al. Adaptation of hepatic mitochon-
drial function in humans with non-alcoholic
fatty liver is lost in steatohepatitis. Cell Metab.
. Schmid AI, Szendroedi J, Chmelik M, Krssák M,
Moser E, Roden M. Liver ATP synthesis is lower
and relates to insulin sensitivity in patients with
type  diabetes. Diabetes Care. ;: .
 . Frayn KN. Adipose tissue as a buffer for daily
lipid flux. Diabetologia. ; :.
. Søndergaard E, Sørensen LP, Rahbek I, Gormsen
LC, Christiansen JS, Nielsen S. Postprandial
VLDL-triacylglycerol secretion is not suppressed
in obese type  diabetic men. Diabetologia.
. Perry RJ, Samu el VT, Petersen KF, Shulman
GI. The role of hepatic lipids in hepatic insu-
lin resistance and type  diabetes. Nature.
. Hennes MM, Shrago E, Kissebah AH. Receptor
and postreceptor effects of free fatty acids FFA
on hepatocyte insulin dynamics. Int J Obes.
. Wiesenthal SR, et al. Free fatty acids impair
hepatic insulin extraction in vivo. Diabetes.
 . Vallim T, Salter AM. Regulation of hepatic gene
expression by saturated fatty acids. Prostaglandins
Leukot Essent Fatty Acids. ;:.
. Maecker H, et al. TWEAK attenuates the tran-
sition from innate to adaptive immunity. Cell.
. Burkly LC. TWEAK/Fn axis: the current
paradigm of tissue injury-inducible function
in the midst of complexities. Semin Immunol.
 . Lozano-Bartolomé J, et al. Reduced circulating
levels of sTWEAK are associated with NAFLD
and may affect hepatocyte triglyceride accumu-
lation. Int J Obes Lond. ;:.
. Cani PD, et al. Metabolic endotoxemia initi-
ates obesity and insulin resistance. Diabetes.
. Chicheportiche Y, Fossati-Jimack L, Moll S,
Ibnou-Zekri N, Izui S. Down-regulated expres-
sion of TWEAK mRNA in acute and chronic
inflammatory pathologies. Biochem Biophys Res
Commun. ;:.
. Wullaert A, van Loo G, Heyninck K, Beyaert
R. Hepatic tumor necrosis factor signaling and
nuclear factor-kappaB: effects on liver homeosta-
sis and beyond. Endocr Rev. ;:.
. Chang G, et al. Hepatic TLR signaling is
activated by LPS from digestive tract during
SARA, and epigenetic mechanisms contribute
to enforced TLR expression. Oncotarget.
. Tang F, Tang G, Xiang J, Dai , Rosner MR, Lin A.
The absence of NF-kappaB-mediated inhibition
of c-Jun N-terminal kinase activation contributes
to tumor necrosis factor alpha-induced apopto-
sis. Mol Cell Biol. ;: .
. Cai D, et al. Local and systemic insulin resistance
resulting from hepatic activation of IKK-beta and
NF-kappaB. Nat Med. ;:.
. Jelenik T, et al. Tissue-speciic differences
in the development of insulin resistance in
a mouse model for type  diabetes. Diabetes.
. Santos SH, et al. Oral Angiotensin- prevented
obesity and hepatic inflammation by inhibition
of resistin/TLR/MAPK/NF-κB in rats fed with
high-fat diet. Peptides. ;:.
. Marion V, et al. BBS-induced ciliary defect
enhances adipogenesis, causing paradoxical
higher-insulin sensitivity, glucose usage, and
decreased inflammatory response. Cell Metab.
. Zhang X, Xie X, Heckmann BL, Saarinen AM,
Czyzyk TA, Liu J. Targeted disruption of G/
G switch gene  enhances adipose lipolysis,
Downloaded from on February 2, 2017.
The Journal of Clinical Investigation
708 Volume 127 Number 2 February 2017
alters hepatic energy balance, and alleviates
high-fat diet-induced liver steatosis. Diabetes.
. Fan Y, et al. Mutations in a member of the Ras
superfamily of small GTP-binding proteins
causes Bardet-Biedl syndrome. Nat Genet.
. Haytowitz DB, Pehrsson PR, Holden JM. The
National Food and Nutrient Analysis Program:
A decade of progress. J Food Compost Anal.
. Urban LE, Roberts SB, Fierstein JL, Gary CE,
Lichtenstein AH. Sodium, saturated fat,
and trans fat content per , kilocalories:
temporal trends in fast-food restaurants,
United States, . Prev Chronic Dis.
. Teff KL , et al. Endocrine and metabolic effects
of consuming fructose- and glucose-sweetened
beverages with meals in obese men and women:
influence of insulin resistance on plasma tri-
glyceride responses. J Clin Endocrinol Metab.
. American Diabetes Association. . Foundations
of Care and Comprehensive Medical Evaluation.
Diabetes Care. ; Suppl :S S.
. Krssak M, et al. Alterations in postprandial hepat-
ic glycogen metabolism in type  diabetes. Diabe-
tes. ;: .
. Beau doin MS, Robinson LE, Graham TE. An oral
lipid challenge and acute intake of caffeinated
coffee additively decrease glucose tolerance in
healthy men. J Nut r. ;:.
. Gancheva S, et al. Variants in Genes Con-
trolling Oxidative Metabolism Contribute
to Lower Hepatic ATP Independent of Liver
Fat Content in Type  Diabetes. Diabetes.
. Jones JG, et al. NMR derivatives for quantiica-
tion of H and Cenrichment of human glucuro-
nide from metabolic tracers. J Carbohydr Chem.
 . Nowotny B, Nowotny PJ, Strassburger K, Roden
M. Precision and accuracy of blood glucose
measurements using three different instruments.
Diabet Med. ; :.
. Herder C, et al. Adiponectin may mediate the
association between omentin, circulating lipids
and insulin sensitivity: results from the KORA F
study. Eur J Endocrinol. ;:.
. Brehm A, Krssak M, Schmid AI, Nowotny
P, Waldhäusl W, Roden M. Increased lipid
availability impairs insulin-stimulated ATP
synthesis in human skeletal muscle. Diabetes.
. Jones J, Kahl S, Carvalho F, Barosa C, Roden M.
Simpliied analysis of acetaminophen glucu-
ronide for quantifying gluconeogenesis and
glycogenolysis using deuterated water. Anal
Biochem. ;:.
. Barosa C, Jones JG, Rizza R, Basu A, Basu R.
Acetaminophen glucuronide and plasma glucose
report identical estimates of gluconeogenesis
and glycogenolysis for healthy and prediabetic
subjects using the deuterated water method.
Magn Reson Med. ;:.
. Bischof MG, et al. Hepatic glycogen metabolism
in type  diabetes after long-term near normogly-
cemia. Diabetes. ;:.
. Kraegen EW, James DE, Jenkins AB, Chisholm
DJ. Dose-response curves for in vivo insulin sen-
sitivity in individual tissues in rats. Am J Physiol.
; Pt :E E.
 . Neschen S, et al. Prevention of hepatic ste-
atosis and hepatic insulin resistance in mito-
chondrial acyl-CoA:glycerol-sn--phosphate
acyltransferase  knockout mice. Cell Metab.
Downloaded from on February 2, 2017.
... We recently demonstrated that a single oral SAFA-rich lipid load initiates hepatic insulin resistance (HEP-IR) and fat accumulation in healthy lean men (5), likely resulting from lipid-mediated inhibition of insulin signaling (6). This lipid load also raised hepatic gluconeogenesis (GNG), which is possibly due to lipid-induced allosteric stimulation of hepatic mitochondrial activity, as reported in rodent models (6). ...
... This study demonstrates that a single oral dose of oleic acid-rich OIL induces insulin resistance in skeletal muscle and liver during hyperinsulinemia but also already under preclamp insulinemia in whole body and liver. Additionally, OIL increased the rate of hepatic GNG and its contribution to EGP, but -in contrast to a former study on saturated fat intake -affected neither hepatic energy metabolism nor lipid deposition (5). ...
... observed upon SAFA-rich lipid loading (5). During clamp, reduced insulin action is presumed by the lower insulin-stimulated EGP suppression also reported for SAFA but not PUFA (5,9). ...
BACKGROUND While saturated fat intake leads to insulin resistance and nonalcoholic fatty liver, Mediterranean-like diets enriched in monounsaturated fatty acids (MUFA) may have beneficial effects. This study examined effects of MUFA on tissue-specific insulin sensitivity and energy metabolism.METHODSA randomized placebo-controlled cross-over study enrolled 16 glucose-tolerant volunteers to receive either oil (OIL, ~1.18 g/kg), rich in MUFA, or vehicle (VCL, water) on 2 occasions. Insulin sensitivity was assessed during preclamp and hyperinsulinemic-euglycemic clamp conditions. Ingestion of 2H2O/acetaminophen was combined with [6,6-2H2]glucose infusion and in vivo 13C/31P/1H/ex vivo 2H-magnet resonance spectroscopy to quantify hepatic glucose and energy fluxes.RESULTSOIL increased plasma triglycerides and oleic acid concentrations by 44% and 66% compared with VCL. Upon OIL intervention, preclamp hepatic and whole-body insulin sensitivity markedly decreased by 28% and 27%, respectively, along with 61% higher rates of hepatic gluconeogenesis and 32% lower rates of net glycogenolysis, while hepatic triglyceride and ATP concentrations did not differ from VCL. During insulin stimulation hepatic and whole-body insulin sensitivity were reduced by 21% and 25%, respectively, after OIL ingestion compared with that in controls.CONCLUSIONA single MUFA-load suffices to induce insulin resistance but affects neither hepatic triglycerides nor energy-rich phosphates. These data indicate that amount of ingested fat, rather than its composition, primarily determines the development of acute insulin resistance.TRIAL NCT01736202.FUNDINGGerman Diabetes Center, German Federal Ministry of Health, Ministry of Culture and Science of the state of North Rhine-Westphalia, German Federal Ministry of Education and Research, German Diabetes Association, German Center for Diabetes Research, Portugal Foundation for Science and Technology, European Regional Development Fund, and Rede Nacional de Ressonancia Magnética Nuclear.
... cumulation in the liver. [31,32] Interestingly though, there are also studies that do not support a role for a high fat diet per se in the development of liver steatosis. For example, a diet that was in energy balance but very high in fat and saturated fat had no effect on hepatic triglyceride levels in overweight individuals, [33] whereas diets that were in energy balance but high in MUFA even decreased levels of liver fat in patients with pre-T2D, [34] T2D, [35] and NAFLD. ...
... In line with this, it is interesting to note that also in a human study, infusion with palm oil resulted in increased hepatic ATP levels in healthy lean individuals. [32] These findings are consistent with the finding that respiratory chain activity is initially increased in individuals with hepatic steatosis, as mentioned previously in this review. [50,51] The in vitro studies also found that ROS accumulation occurred downstream of altered mitochondrial oxidative metabolism and this preceded the initiation of apoptosis, as shown by an increased caspase 3 activity, and reduced cell viability. ...
Full-text available
Non‐Alcoholic fatty liver disease (NAFLD) is the most common form of liver disease and is characterized by fat accumulation in the liver. Hypercaloric diets generally increase hepatic fat accumulation, whereas hypocaloric diets decrease liver fat content. In addition, there is evidence to suggest that moderate amounts of unsaturated fatty acids seems to be protective for the development of a fatty liver, while consumption of saturated fatty acids (SFA) appears to predispose towards hepatic steatosis. Recent studies highlight a key role for mitochondrial dysfunction in the development and progression of NAFLD. We propose that changes in mitochondrial structure and function are key mechanisms by which SFA lead to the development and progression of NAFLD. In this review, we describe how SFA intake is associated with liver steatosis and decreases the efficiency of the respiratory transport chain. This results in the production of reactive oxygen species and damage to nearby structures, eventually leading to inflammation, apoptosis and scarring of the liver. Furthermore, we present studies demonstrating that SFA intake affects the composition of mitochondrial membranes, and this process accelerates the progression of NAFLD. It is likely that events are intertwined and reinforce each other, leading to a constant deterioration in health. This article is protected by copyright. All rights reserved
... resistance, non-alcohol fatty liver disease (NAFLD), and so on (2)(3)(4)(5). Fat-rich diets not only lead to NAFLD but can also impair carbohydrates metabolism, manifesting in impaired glucose tolerance curves (6,7), alterations in gut microbiota (8), and disrupt molecular systems responsible for signaling to the liver (9). ...
Full-text available
Background: Dietary oils differ in their fatty acid composition and the presence of additional microcomponents (antioxidants, etc.). These differences are thought to invoke different biochemical pathways, thus affecting fats and carbohydrates metabolism differently. Olive oil (OO) and soybean oil (SO) are common vegetable oils in the local cuisine. Peanuts oils of local varieties are viewed as potential sources of dietary vegetable oils, especially in the food industry. Objective: We examined the effect of four different dietary vegetable oils on carbohydrate and lipid metabolism in mice. The selected oils were OO, high in oleic acid, extracted from cultivated high oleic acid peanut (C-PO), regular peanut oil (PO), and SO. Design: In this study, 32 male C57BL/6J mice were randomly divided into four groups (n = 8 in each group) and were fed with four different diets enriched with 4% (w/w) dietary vegetable oils (OO, C-PO, PO, or SO). After 10 weeks, the mice were sacrificed. Western blot was used to examine proteins such as phospho-AMP-activated protein kinase (p-AMPK), ace-tyl-CoA carboxylase (ACC), cluster of differentiation 36 (CD36), and Sirtuin 1 (SIRT1), whereas real-time polymerase chain reaction (PCR) was used to examine the expression of sterol regulatory element-binding protein-1c (SREBP-1C), fatty acid synthase (FAS), glucose-6-phosphatase (G6Pase), and CD36 transcripts. Results: In mice-fed SO, lipid accumulation was predominately in adipose tissue, accompanied a tendency decrease in insulin sensitivity. Mice-fed OO had lower plasma triglycerides (TG) and increased hepatic CD36 gene expression. The C-PO group presented lower messenger RNA (mRNA) levels in the liver for all examined genes: SREBP-1c, FAS, G6Pase, and CD36. There were no significant differences in weight gain, plasma cholesterol and high-density lipoprotein (HDL) cholesterol levels, hepatic ACC, SIRT1, AMPK, and CD36 protein levels or in liver function among the diets. Discussion: It seems that as long as fat is consumed in moderation, oil types may play a lesser role in the metabolism of healthy individuals. Conclusion: This finding has the potential to increase flexibility in choosing oil types for consumption.
... In the current study, unhealthy diet was also found to be a risk factor for NAFLD. Overconsumption of fried foods is associated with an increase in the intake of calories, saturated fatty acids, and cholesterol, and this in turn, may lead to an increase in hepatic lipid storage and insulin resistance (28,29). On the other hand, tubers contain high amounts of starch and antioxidants, which are known to have beneficial effects on health. ...
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Nonalcoholic fatty liver disease (NAFLD) is a common liver disease globally, but there are no optimal methods for its prediction or diagnosis. The present cross-sectional study proposes a non-invasive tool for NAFLD screening. The study included 2,446 individuals, of whom 574 were NAFLD patients. Multivariable logistic regression analysis was used to identify risk factors for NAFLD and incorporate them in a risk prediction nomogram model; the variables included both clinical and lifestyle-related variables. Following stepwise regression, BMI, waist circumference, serum triglyceride, high-density lipoprotein cholesterol, alanine aminotransferase, presence of diabetes and hyperuricemia, tuber and fried food consumption were identified as significant risk factors and used in the model. The final nomogram was found to have good discrimination ability (area under the receiver operating characteristic curve = 0.843 [95% CI: 0.819-0.867]), and reasonable accuracy for the prediction of NAFLD risk. A cut-off score of <180 for the nomogram was found to have high sensitivity and predictivity for the exclusion of individuals from screening. The model can be used as a non-invasive tool for mass screening.
... The earliest events initiating NAFLD may reside in hypercaloric energy-dense dietary habits [23]. A single oral intake of saturated fat has been shown to rapidly induce skeletal muscle, adipose tissue and hepatic insulin resistance along with 70% increased gluconeogenesis and upregulated inflammatory pathways [24]. Chronically positive energy balance will enlarge adipose tissue compartments and lead to dysfunctional adipose tissue with excessive lipolysis, overflow of free fatty acids (FFA) elevated gluconeogenesis and again insulin resistance [25]. ...
Non-alcoholic fatty liver disease (NAFLD) comprises fatty liver (steatosis), non-alcoholic steatohepatitis (NASH) and fibrosis/cirrhosis and may lead to end-stage liver failure or hepatocellular carcinoma. NAFLD is tightly associated with the most frequent metabolic disorders, such as obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM). Both multisystem diseases share several common mechanisms. Alterations of tissue communications include excessive lipid and later cytokine release by dysfunctional adipose tissue, intestinal dysbiosis and ectopic fat deposition in skeletal muscle. On the hepatocellular level, this leads to insulin resistance due to abnormal lipid handling and mitochondrial function. Over time, cellular oxidative stress and activation of inflammatory pathways, again supported by multiorgan crosstalk, determine NAFLD progression. Recent studies show that particularly the severe insulin resistant diabetes (SIRD) subgroup (cluster) associates with NAFLD and its accelerated progression and increases the risk of diabetes-related cardiovascular and kidney diseases, underpinning the critical role of insulin resistance. Consequently, lifestyle modification and certain drug classes used to treat T2DM have demonstrated effectiveness for treating NAFLD, but also some novel therapeutic concepts may be beneficial for both NAFLD and T2DM. This review addresses the bidirectional relationship between mechanisms underlying T2DM and NAFLD, the relevance of novel biomarkers for improving the diagnostic modalities and the identification of subgroups at specific risk of disease progression. Also, the role of metabolism-related drugs in NAFLD is discussed in light of the recent clinical trials. Finally, this review highlights some challenges to be addressed by future studies on NAFLD in the context of T2DM.
... In the present study, ATP was found to inhibit MECR protein in the LA synthetic pathway in three models: DIO mice, cellular model and BBR model. The increase in intracellular ATP is associated with insulin resistance in several tissues of DIO mice [2,18,19] and in liver of healthy individuals by overfeeding [22]. The role of ATP in the pathogenesis of insulin resistance is supported by the activities of metformin [3] and chemical uncouplers [23,24], both of which improve insulin sensitivity in vivo. ...
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MECR (mitochondrial 2-enoyl-acyl-carrier protein reductase) is an enzyme in the mitochondrial fatty acid synthase (mtFAS) pathway. MECR activity remains unknown in the mechanism of insulin resistance in the pathogenesis of type 2 diabetes. In this study, MECR activity was investigated in diet-induced obese (DIO) mice. Mecr mRNA was induced by insulin in cell culture, and was elevated in the liver of DIO mice in the presence hyperinsulinemia. However, MECR protein was decreased in the liver of DIO mice, and the reduction was blocked by treatment of the DIO mice with berberine (BBR). The mechanism of MECR protein regulation was investigated with a focus on ATP. The protein was decreased in the cell lysate and DIO liver by an increase in ATP levels. The ATP protein reduction was blocked in the liver of BBR-treated mice by suppression of ATP elevation. The MERC protein reduction was associated with insulin resistance and the protein restoration was associated with improvement of insulin sensitivity by BBR in the DIO mice. The data suggests that MECR protein is regulated in hepatocytes by ATP in association with insulin resistance. The study provides evidence for a relationship of MECR protein and insulin resistance.
... Copyright ©2019, P-ISSN : 2337-6236; E-ISSN : 2622-884X jenuh secara cepat meningkatkan penyimpanan lemak hepar, metabolisme energi, serta resistensi insulin. 24 Tingginya kadar insulin dalam darah menurunkan ekskresi asam urat melalui ginjal, sehingga kadar asam urat serum akan mengalami peningkatan. 25 Selain itu, jaringan lemak juga dapat mensekresikan asam urat. ...
Latar Belakang : Obesitas adalah suatu kelainan metabolisme, dimana terjadi ketidakseimbangan antara energi yang masuk dan energi yang keluar. Obesitas pada lansia merupakan salah satu masalah kesehatan paling serius didunia. Pada penderita obesitas, resiko untuk mengalami penyakit gout lebih tinggi. Penyakit gout lebih sering menyerang pada orang yang mengalami kelebihan berat badan lebih dari 30%. Penelitian ini bertujuan untuk mengetahui perbedaan kadar asam urat pada wanita lansia obesitas dan non obesitas.Metode :Jenis penelitian ini adalah observasional dengan rancangan cross-sectional. Jumlah subjek penelitian adalah 56 orang wanita dengan usia 60-74 tahun. Kadar asam urat diperoleh menggunakan metode enzimatik, sedangkan status obesitas diperoleh menggunakan alat BIA (Bioelectrical Impedance Analyser).Hasil :Kadar asam urat sebagian besar kadar asam urat subjek (78,6%) berada dalam rentang normal, yaitu antara 2,6 – 6 mg/dl, sementara 12 orang subjek (21,4%) mengalami hiperurisemia. Pada subjek obesitas ditemukan 7 orang subjek hiperurisemia (> 6mg/dl), sedangkan pada subjek non obesitas ditemukan 5 orang subjek hiperurisemia ( 6mg/dl). Hasil statistik menunjukkan bahwa tidak terdapat perbedaan kadar asam urat antara subjek obesitas dan non obesitas (p>0,05).
... The pooled results of our meta-analysis are in accordance with other systematic review and meta-analysis, indicating that consumption of red meat and sugar-and artificially sweetened soda is positively associated with NAFLD (53,54) . Firstly, red meat rich in saturated fat increases hepatic lipid accumulation and insulin resistance via reducing lipid oxidation and increasing lipid synthesis (55,56) . Additionally, heme-iron intake reduces insulin sensitivity through cellular oxidation stress (57) . ...
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Dietary habits have been implicated in the development and severity of nonalcoholic fatty liver disease (NAFLD). Several epidemiologic studies attempted to assess the relationship between food groups and the likelihood of NAFLD, but these results were conflicting. The present meta-analysis was conducted to assess the association between food groups and the likelihood of NAFLD. Published literature were retrieved and screened from MEDLINE, EMBASE and Web of Science. Out of 7892 retrieved articles, 24 observational studies (15 cross-sectional studies and 9 case-control studies) met our eligibility criteria and were finally included in this systematic review and meta-analysis. Consumption of both red meat and soft drinks contributed to a positive association with NAFLD. Inversely, nut consumption was negatively associated with NAFLD. There were no significant influences on the likelihood of NAFLD about consuming whole grains, refined grains, fish, fruits, vegetables, eggs, dairy, and legumes. This meta-analysis suggests that individuals who consumed more red meat and soft drinks may have a significantly increased likelihood of NAFLD, whereas higher nut intake may be negatively associated with NAFLD. Further prospective studies are required to assess the association between food patterns and NAFLD.
... Diet has a relevant role in the development and progression of NAFLD, since a high energy intake and consumption of specific nutrients have a direct impact on the abnormal accumulation of TG in the liver, a hallmark of NAFLD [4]. High intake of nutrients that include saturated fatty acids (FA) such as palmitic acid (C16:0) [17][18][19] and trans FA of industrial origin [20] decrease FA oxidation (FAO), stimulate the synthesis and secretion of TGs, and trigger lipotoxic effects in the liver [4,17,18]. Moreover, high intake of n-6 PUFA, especially linoleic acid (C18:2n-6), and low consumption of n-3 LCPUFA (EPA and DHA) also appear to favor the development of hepatic steatosis [21]. ...
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Nonalcoholic fatty liver disease (NAFLD) is present in approximately 25% of the population worldwide. It is characterized by the accumulation of triacylglycerol in the liver, which can progress to steatohepatitis with different degrees of fibrosis, stages that lack approved pharmacological therapies and represent an indication for liver transplantation with consistently increasing frequency. In view that hepatic steatosis is a reversible condition, effective strategies preventing disease progression were addressed using combinations of natural products in the preclinical high-fat diet (HFD) protocol (60% of fat for 12 weeks). Among them, eicosapentaenoic acid (C20:5n-3, EPA) and docosahexaenoic acid (C22:5n-3, DHA), DHA and extra virgin olive oil (EVOO), or EPA plus hydroxytyrosol (HT) attained 66% to 83% diminution in HFD-induced steatosis, with the concomitant inhibition of the proinflammatory state associated with steatosis. These supplementations trigger different molecular mechanisms that modify antioxidant, antisteatotic, and anti-inflammatory responses, and in the case of DHA and HT co-administration, prevent NAFLD. It is concluded that future studies in NAFLD patients using combined supplementations such as DHA plus HT are warranted to prevent liver steatosis, thus avoiding its progression into more unmanageable stages of the disease.
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Peroxisome proliferator activated receptor α (PPARα) acts as a fatty acid sensor to orchestrate the transcription of genes coding for rate-limiting enzymes required for lipid oxidation in hepatocytes. Mice only lacking Pparα in hepatocytes spontaneously develop steatosis without obesity in aging. Steatosis can develop into non alcoholic steatohepatitis (NASH), which may progress to irreversible damage, such as fibrosis and hepatocarcinoma. While NASH appears as a major public health concern worldwide, it remains an unmet medical need. In the current study, we investigated the role of hepatocyte PPARα in a preclinical model of steatosis. For this, we used High Fat Diet (HFD) feeding as a model of obesity in C57BL/6 J male Wild-Type mice (WT), in whole-body Pparα-deficient mice (Pparα −/−) and in mice lacking Pparα only in hepatocytes (Pparα hep−/−). We provide evidence that Pparα deletion in hepatocytes promotes NAFLD and liver inflammation in mice fed a HFD. This enhanced NAFLD susceptibility occurs without development of glucose intolerance. Moreover, our data reveal that non-hepatocytic PPARα activity predominantly contributes to the metabolic response to HFD. Taken together, our data support hepatocyte PPARα as being essential to the prevention of NAFLD and that extra-hepatocyte PPARα activity contributes to whole-body lipid homeostasis.
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Context: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome and is strongly associated with obesity, dyslipidaemia and altered glucose regulation. Previous data demonstrated that low circulating levels of tumour necrosis factor weak inducer of apoptosis (sTWEAK) were associated with obesity, diabetes and insulin resistance, all traits associated with an increased risk of NALFD. Circulating sTWEAK levels are expected to be reduced in the presence of NAFLD. Objective: We aimed to explore the relationship between NAFLD and circulating sTWEAK levels in obese patients, and to evaluate the effect of sTWEAK on hepatocyte triglyceride accumulation.Design setting and patients:This is an observational case-control study performed in n=112 severely obese patients evaluated for NAFLD by abdominal ultrasound and n=32 non-obese patients without steatosis. Serum sTWEAK concentrations were measured by ELISA. Multivariable analyses were performed to determine the independent predictors of NAFLD. We analysed TWEAK and Fn14 protein expression in liver biopsies by western blotting and immunohistochemistry. An immortalized primary human hepatocyte cell line (HHL) was used to evaluate the effect of sTWEAK on triglyceride accumulation. Results: We observed a reduction in serum circulating sTWEAK concentrations with the presence of liver steatosis. On multivariable analysis, lower sTWEAK concentrations were independently associated with the presence of NAFLD (odds ratio (OR)=0.023; 95% confidence interval: 0.001-0.579; P<0.022). In human hepatocytes, sTWEAK administration reduced fat accumulation as demonstrated by the reduction in palmitic acid-induced accumulation of triglyceride and the decreased expression of cluster of differentiation 36 (CD36) and perilipin 1 and 2 (PLIN1 and PLIN2) genes. Conclusions: Decreased sTWEAK concentrations are independently associated with the presence of NAFLD. This is concordant with the observation that TWEAK reduces lipid accumulation in human liver cells.
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Although 5%–10% weight loss is routinely recommended for people with obesity, the precise effects of 5% and further weight loss on metabolic health are unclear. We conducted a randomized controlled trial that evaluated the effects of 5.1% ± 0.9% (n = 19), 10.8% ± 1.3% (n = 9), and 16.4% ± 2.1% (n = 9) weight loss and weight maintenance (n = 14) on metabolic outcomes. 5% weight loss improved adipose tissue, liver and muscle insulin sensitivity, and β cell function, without a concomitant change in systemic or subcutaneous adipose tissue markers of inflammation. Additional weight loss further improved β cell function and insulin sensitivity in muscle and caused stepwise changes in adipose tissue mass, intrahepatic triglyceride content, and adipose tissue expression of genes involved in cholesterol flux, lipid synthesis, extracellular matrix remodeling, and oxidative stress. These results demonstrate that moderate 5% weight loss improves metabolic function in multiple organs simultaneously, and progressive weight loss causes dose-dependent alterations in key adipose tissue biological pathways.
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Subacute ruminal acidosis (SARA) is known to trigger a systemic inflammatory response that is possibly caused by the translocation of lipopolysaccharides (LPS) from the gastrointestinal tract into the bloodstream. The aim of this study is to investigate this causal relationship between the increases of circulating LPS and liver inflammation. Here we found that SARA goats exhibited significantly increased LPS concentrations in both the rumen and portal vein. The livers of these goats exhibited increased mRNA concentrations of pro-inflammatory genes that indicated inflammation. Meanwhile, the occurrence of liver inflammation was further validated by the enhanced protein expression of those cytokines in the livers of SARA goats. These increased expressions of detected pro-inflammatory genes were likely mediated by enforced TLR4 signaling because SARA increased the concentrations of TLR4 mRNA and protein in the liver and the abundance of both the NF-kB-p65 factor and its active phosphorylated variant. We also verified that the enhanced TLR4 expression was accompanied by chromatin decompaction and demethylation of the proximal TLR4 promoter. Hence, epigenetic mechanisms are involved in the enforced expression of immune genes during SARA, and these findings open innovative routes for interventions via the modulation of these epigenetic mechanisms.
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Background: Impaired energy metabolism is a possible mechanism that contributes to insulin resistance and ectopic fat storage. Objective: We examined whether meal ingestion differently affects hepatic phosphorus metabolites in insulin sensitive and insulin resistant humans. Design: Young, lean, insulin-sensitive humans (CONs) [mean ± SD body mass index (BMI; in kg/m(2)): 23.2 ± 1.5], insulin-resistant, glucose-tolerant, obese humans (OBEs) (BMI: 34.3 ± 1.7), and type 2 diabetes patients (T2Ds) (BMI: 32.0 ± 2.4) were studied (n = 10/group). T2Ds (61 ± 7 y old) were older (P < 0.001) than were OBEs (31 ± 7 y old) and CONs (28 ± 3 y old). We quantified hepatic γATP, inorganic phosphate (Pi), and the fat content [hepatocellular lipids (HCLs)] with the use of (31)P/(1)H magnetic resonance spectroscopy before and at 160 and 240 min after a high-caloric mixed meal. In a subset of volunteers, we measured the skeletal muscle oxidative capacity with the use of high-resolution respirometry. Whole-body insulin sensitivity (M value) was assessed with the use of hyperinsulinemic-euglycemic clamps. Results: OBEs and T2Ds were similarly insulin resistant (M value: 3.5 ± 1.4 vs. 1.9 ± 2.5 mg · kg(-1) · min(-1), respectively; P = 0.9) and had 12-fold (P = 0.01) and 17-fold (P = 0.002) higher HCLs than those of lean persons. Despite comparable fasting hepatic γATP concentrations, the maximum postprandial increase of γATP was 6-fold higher in OBEs (0.7 ± 0.2 mmol/L; P = 0.03) but only tended to be higher in T2Ds (0.6 ± 0.2 mmol/L; P = 0.09) than in CONs (0.1 ± 0.1 mmol/L). However, in the fasted state, muscle complex I activity was 53% lower (P = 0.01) in T2Ds but not in OBEs (P = 0.15) than in CONs. Conclusions: Young, obese, nondiabetic humans exhibit augmented postprandial hepatic energy metabolism, whereas elderly T2Ds have impaired fasting muscle energy metabolism. These findings support the concept of a differential and tissue-specific regulation of energy metabolism, which can occur independent of insulin resistance. This trial was registered at as NCT01229059.
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A growing body of evidence highlights the close association between nutrition and human health. Fat is an essential macronutrient, and vegetable oils, such as palm oil, are widely used in the food industry and highly represented in the human diet. Palmitic acid, a saturated fatty acid, is the principal constituent of refined palm oil. In the last few decades, controversial studies have reported potential unhealthy effects of palm oil due to the high palmitic acid content. In this review we provide a concise and comprehensive update on the functional role of palm oil and palmitic acid in the development of obesity, type 2 diabetes mellitus, cardiovascular diseases and cancer. The atherogenic potential of palmitic acid and its stereospecific position in triacylglycerols are also discussed.
Type 1 diabetes has been recently linked to non-alcoholic fatty liver disease (NAFLD), which is known to associate with insulin resistance, obesity and type 2 diabetes. However, the role of insulin resistance and hyperglycemia for hepatic energy metabolism is yet unclear. To analyze early abnormalities in hepatic energy metabolism, we examined 55 patients with recently diagnosed type 1 diabetes. They underwent hyperinsulinemic-normoglycemic clamps with [6,6-(2)H2]glucose to assess whole-body and hepatic insulin sensitivity. Hepatic γ-adenosine triphosphate (γATP), inorganic phosphate (Pi) and triglyceride concentrations (HCL) were measured with multinuclei magnetic resonance spectroscopy ((31)P/(1)H-MRS). Glucose-tolerant humans served as control (CON; n=57). Whole-body insulin sensitivity was 44% lower in patients than in age- and BMI-matched CON. Hepatic γATP was 15% reduced (2.3±0.6 vs 2.7±0.6 mmol/l, P<0.001), while hepatic Pi and HCL were similar in patients when compared with CON. Across all participants, hepatic γATP correlated negatively with glycemia and oxidized low-density lipoproteins. Carriers of the PPARG G allele (rs1801282) and non-carriers of PPARGC1A A allele (rs8192678) had 21% and 13% lower hepatic ATP concentrations. Variations in genes controlling oxidative metabolism contribute to reduction in hepatic ATP in the absence of NAFLD suggesting that alterations in hepatic mitochondrial function may precede diabetes-related liver diseases.
Diabetes and obesity are two metabolic diseases characterized by insulin resistance and a low-grade inflammation. Seeking an inflammatory factor causative of the onset of insulin resistance, obesity, and diabetes, we have identified bacterial lipopolysaccharide (LPS) as a triggering factor. We found that normal endotoxemia increased or decreased during the fed or fasted state, respectively, on a nutritional basis and that a 4-week high-fat diet chronically increased plasma LPS concentration two to three times, a threshold that we have defined as metabolic endotoxemia. Importantly , a high-fat diet increased the proportion of an LPS-containing microbiota in the gut. When metabolic endotoxemia was induced for 4 weeks in mice through continuous subcutaneous infusion of LPS, fasted glycemia and insulinemia and whole-body, liver, and adipose tissue weight gain were increased to a similar extent as in high-fat–fed mice. In addition, adipose tissue F4/80-positive cells and markers of inflammation, and liver triglyceride content, were increased. Furthermore, liver, but not whole-body, insulin resistance was detected in LPS-infused mice. CD14 mutant mice resisted most of the LPS and high-fat diet–induced features of metabolic diseases. This new finding demonstrates that metabolic endotoxemia dysregulates the inflammatory tone and triggers body weight gain and diabetes. We conclude that the LPS/CD14 system sets the tone of insulin sensitivity and the onset of diabetes and obesity. Lowering plasma LPS concentration could be a potent strategy for the control of metabolic diseases.
The association of hepatic mitochondrial function with insulin resistance and non-alcoholic fatty liver (NAFL) or steatohepatitis (NASH) remains unclear. This study applied high-resolution respirometry to directly quantify mitochondrial respiration in liver biopsies of obese insulin-resistant humans without (n = 18) or with (n = 16) histologically proven NAFL or with NASH (n = 7) compared to lean individuals (n = 12). Despite similar mitochondrial content, obese humans with or without NAFL had 4.3- to 5.0-fold higher maximal respiration rates in isolated mitochondria than lean persons. NASH patients featured higher mitochondrial mass, but 31%-40% lower maximal respiration, which associated with greater hepatic insulin resistance, mitochondrial uncoupling, and leaking activity. In NASH, augmented hepatic oxidative stress (H2O2, lipid peroxides) and oxidative DNA damage (8-OH-deoxyguanosine) was paralleled by reduced anti-oxidant defense capacity and increased inflammatory response. These data suggest adaptation of the liver ("hepatic mitochondrial flexibility") at early stages of obesity-related insulin resistance, which is subsequently lost in NASH. Copyright © 2015 Elsevier Inc. All rights reserved.
Measurement of Acetaminophen glucuronide (AG) (2)H-enrichment from deuterated water ((2)H2O) by (2)H NMR analysis of its monoacetone glucose (MAG) derivative provides estimation of gluconeogenic and glycogenolytic contributions to endogenous glucose production (EGP). However AG derivatization to MAG is laborious and unsuitable for high-throughput studies. An alternative derivative, 5-O-acetyl monoacetone glucuronolactone (MAGLA), was tested. Eleven healthy subjects ingested (2)H2O to 0.5% body water enrichment and 500 mg Acetaminophen. Plasma glucose and urinary glucuronide positional (2)H-enrichments were measured by (2)H NMR spectroscopy of MAG and MAGLA, respectively. A Bland-Altman analysis indicated agreement at the 95% confidence level between glucose and glucuronide estimates. Copyright © 2015. Published by Elsevier Inc.