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Effect of a ketogenic diet on hepatic steatosis and hepatic mitochondrial metabolism in nonalcoholic fatty liver disease


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Weight loss by ketogenic diet (KD) has gained popularity in management of nonalcoholic fatty liver disease (NAFLD). KD rapidly reverses NAFLD and insulin resistance despite increasing circulating nonesterified fatty acids (NEFA), the main substrate for synthesis of intrahepatic triglycerides (IHTG). To explore the underlying mechanism, we quantified hepatic mitochondrial fluxes and their regulators in humans by using positional isotopomer NMR tracer analysis. Ten overweight/obese subjects received stable isotope infusions of: [D 7 ]glucose, [ ¹³ C 4 ]β-hydroxybutyrate and [3- ¹³ C]lactate before and after a 6-d KD. IHTG was determined by proton magnetic resonance spectroscopy ( ¹ H-MRS). The KD diet decreased IHTG by 31% in the face of a 3% decrease in body weight and decreased hepatic insulin resistance (−58%) despite an increase in NEFA concentrations (+35%). These changes were attributed to increased net hydrolysis of IHTG and partitioning of the resulting fatty acids toward ketogenesis (+232%) due to reductions in serum insulin concentrations (−53%) and hepatic citrate synthase flux (−38%), respectively. The former was attributed to decreased hepatic insulin resistance and the latter to increased hepatic mitochondrial redox state (+167%) and decreased plasma leptin (−45%) and triiodothyronine (−21%) concentrations. These data demonstrate heretofore undescribed adaptations underlying the reversal of NAFLD by KD: That is, markedly altered hepatic mitochondrial fluxes and redox state to promote ketogenesis rather than synthesis of IHTG.
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Effect of a ketogenic diet on hepatic steatosis and
hepatic mitochondrial metabolism in nonalcoholic
fatty liver disease
Panu K. Luukkonen
, Sylvie Dufour
, Kun Lyu
, Xian-Man Zhang
, Antti Hakkarainen
, Tiina E. Lehtimäki
Gary W. Cline
, Kitt Falk Petersen
, Gerald I. Shulman
, and Hannele Yki-Järvinen
Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520;
Minerva Foundation Institute for Medical Research, Helsinki 00290,
Department of Medicine, University of Helsinki and Helsinki University Hospital, Helsinki 00290, Finland;
Yale Diabetes Research Center, Yale
School of Medicine, New Haven, CT 06520;
Department of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06520;
Department of
Radiology, HUS Medical Imaging Center, University of Helsinki and Helsinki University Hospital, Helsinki 00290, Finland; and
Department of Neuroscience
and Biomedical Engineering, Aalto University School of Science, 00076 Espoo, Finland
Contributed by Gerald I. Shulman, January 31, 2020 (sent for review December 26, 2019; reviewed by Fredrik Karpe and Roy Taylor)
Weight loss by ketogenic diet (KD) has gained popularity in
management of nonalcoholic fatty liver disease (NAFLD). KD rapidly
reverses NAFLD and insulin resistance despite increasing circulating
nonesterified fatty acids (NEFA), the main substrate for synthesis of
intrahepatic triglycerides (IHTG). To explore the underlying mecha-
nism, we quantified hepatic mitochondrial fluxes and their regula-
tors in humans by using positional isotopomer NMR tracer analysis.
Ten overweight/obese subjects received stable isotope infusions of:
]glucose, [
]β-hydroxybutyrate and [3-
C]lactate before and
after a 6-d KD. IHTG was determined by proton magnetic resonance
spectroscopy (
H-MRS). The KD diet decreased IHTG by 31% in the
face of a 3% decrease in body weight and decreased hepatic insulin
resistance (58%) despite an increase in NEFA concentrations
(+35%). These changes were attributed to increased net hydrolysis
of IHTG and partitioning of the resulting fatty acids toward keto-
genesi s (+232%) due to reductions in serum insulin concentra-
tions (53%) and hepatic citrate synthase flux (38%), respectively.
The former was attributed to decreased hepatic insulin resistance
and the latter to increased hepatic mitochondrial redox state
(+167%) and decreased plasma leptin (45%) and triiodothyronine
(21%) concentrations. These data demonstrate heretofore unde-
scribed adaptations underlying the reversal of NAFLD by KD: That is,
markedly altered hepatic mitochondrial fluxes and redox state to
promote ketogenesis rather than synthesis of IHTG.
carbohydrate restriction
citrate synthase
insulin resistance
pyruvate carboxylase
Nonalcoholic fatty liver disease (NAFLD) is the most common
chronic liver disease and can progress from steatosis to ad-
vanced liver disease, including liver cirrhosis and hepatocellular
carcinoma (13). It is strongly associated with insulin resistance,
which is characterized by excessive hepatic glucose production and
compensatory hyperinsulinemia (410). In adipose tissue of sub-
jects with NAFLD, insulin fails to suppress lipolysis, which leads to
increased hepatic delivery of nonesterified fatty acids (NEFA), the
main substrate for synthesis of intrahepatic triglycerides (IHTG)
(411). Excess substrate and hyperinsulinemia may stimulate re-
esterification and de novo lipogenesis (DNL) of fatty acids, which
can further increase IHTG content and overproduction of very low-
density lipoprotein (VLDL)-TG into circulation (1216). Together,
these features of NAFLD increase the risk of type 2 diabetes and
cardiovascular disease (1, 2).
Since obesity is an important cause of NAFLD, its management
is underpinned by weight loss (1722). Recently, low-carbohydrate
ketogenic diets (KD) have gained popularity in the treatment of
obesity, type 2 diabetes, and NAFLD (2325). While long-term data
comparing different weight loss regimens in NAFLD are virtually
nonexistent, a low-carbohydrate diet has been reported to induce a
threefold greater IHTG loss than a low-fat, high-carbohydrate diet
after 48 h of caloric restriction (26). We previously showed that
a hypocaloric, KD induces an 30% reduction in IHTG content in
6 d despite increasing circulating NEFA (27).
While the antisteatotic effect of KD is well-established, the
underlying mechanisms by which it does so remain unclear. KD
increases plasma NEFA concentrations, the main substrate of IHTG
(11). In the liver, NEFA can either be re-esterified into complex
lipids, such as TGs, or be transported to the mitochondria to be
metabolized by β-oxidation into acetyl-CoA, which in turn can
either be irreversibly condensed with oxaloacetate by citrate syn-
thase to form citrate and enter the TCA cycle for terminal oxi-
dation to CO
(28, 29) or it can enter the ketogenic pathway,
where it is converted into acetoacetate (AcAc) and β-hydroxybutyrate
(β-OHB) (28). These mitochondrial fluxes are tightly regulated
by substrate availability and product inhibition (29), mitochon-
drial redox state (30), and hormones, such as leptin (31) and
triiodothyronine (T3) (32).
Ketogenic diet is an effective treatment for nonalcoholic fatty
liver disease (NAFLD). Here, we present evidence that hepatic
mitochondrial fluxes and redox state are markedly altered
during ketogenic diet-induced reversal of NAFLD in humans.
Ketogenic diet for 6 d markedly decreased liver fat content and
hepatic insulin resistance. These changes were associated with
increased net hydrolysis of liver triglycerides and decreased
endogenous glucose production and serum insulin concentra-
tions. Partitioning of fatty acids toward ketogenesis increased,
which was associated with increased hepatic mitochondrial
redox state and decreased hepatic citrate synthase flux. These
data demonstrate heretofore undescribed adaptations un-
derlying the reversal of NAFLD by ketogenic diet and highlight
hepatic mitochondrial fluxes and redox state as potential
treatment targets in NAFLD.
Author contributions: P.K.L., G.I.S., and H.Y.-J. designed research; P.K.L. recruited partic-
ipants, performed clinical studies and drafted the manuscript; P.K.L., S.D., K.L., and X.-M.Z.
analyzed plasma samples; A.H. and T.E.L. obtained magnetic resonance imaging data;
P.K.L., S.D., K.L., X.-M.Z., A.H., T.E.L., G.W.C., K.F.P., G.I.S., and H.Y.-J. analyzed data; and
P.K.L., K.F.P., G.I.S., and H.Y.-J. wrote the paper.
Reviewers: F.K., Oxford Centre for Diabetes; and R.T., Newcastle University.
The authors declare no competing interest.
This open access article is distributed under Creative Commons Attribution-NonCommercial-
NoDeriv atives Licen se 4.0 (CC BY-NC-N D).
G.I.S. and H.Y.-J. contributed equally to this work.
To whom correspondence may be addressed. Email: or
This article contains supporting information online at
doi:10.1073/pnas.1922344117/-/DCSupplemental. PNAS Latest Articles
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In this study we examined the effects of a short-term KD on
hepatic steatosis by assessing IHTG content and liver stiffness by
magnetic resonance spectroscopy/elastography (
in 10 overweight/obese participants before and after a 6-d KD. In
order to examine the effect that a short-term KD diet might have
on rates of hepatic mitochondrial fat oxidation and gluconeo-
genesis, we applied a positional isotopomer NMR tracer analysis
(PINTA) method (3335) to assess rates of hepatic mitochon-
drial flux through pyruvate carboxylase (V
) relative to citrate
synthase flux (V
), as well as rates of endogenous glucose,
β-OHB, and lactate production by stable isotope infusions of
]glucose, [
]β-OHB, and [3-
C]lactate, respectively. Fi-
nally, in order to gain insights into how these hepatic mito-
chondrial fluxes might be regulated during a KD, we also
assessed some key potential regulators of these mitochondrial
fluxes (i.e., hepatic mitochondrial redox state as reflected by
plasma [β-OHB]/[AcAc], plasma leptin, and T3 concentrations) in
these same subjects (Fig. 1 and SI Appendix,Fig.S1).
The Study Diet Was Ketogenic and Participants Were Compliant.
Characteristics of the participants are shown in Table 1. Their
dietary intakes were assessed by 3-d food records at baseline and
at the end of the 6-d KD (Fig. 1A). Compliance was verified by
measuring plasma ketone bodies (β-OHB and AcAc). Compared
to the habitual diets of the participants, the study diet was very
low in carbohydrates (183 ±20 vs. 23 ±1 g/d, before vs. after,
P<0.000001) (Fig. 2A), while intake of fat and protein remained
unchanged (Fig. 2A). This resulted in a decrease in energy intake
(2,019 ±177 vs. 1,444 kcal/d, before vs. after, P<0.01). Plasma
concentrations of β-OHB increased 10-fold from 0.1 ±0.1 to
1.0 ±0.2 mmol/L (P<0.001) (Fig. 2B) and AcAc 6-fold from
0.1 ±0.1 to 0.6 ±0.1 mmol/L (P<0.001) (Fig. 2C). Body weight
decreased on the average by 3.0 ±0.3% from 93.5 ±5.3 to 90.7 ±
5.2 kg (P<0.00001) (Fig. 2Dand Table 1).
KD Decreased IHTG Content. IHTG content decreased by 31%
from 10.3 ±2.3 to 7.1 ±2.0% (P<0.001) (Fig. 3A)asdetermined
H-MRS. Liver stiffness as determined by MRE remained un-
changed (2.6 ±0.1 vs. 2.5 ±0.1 kilopascals [kPa], before vs. after,
P=0.18) (Fig. 3B). Activities of plasma γ-glutamyltransferase
(GGΤ) decreased from 48 ±10 to 38 ±7 U/L (P<0.05) and
alkaline phosphatase (ALP) from 82 ±8to73±7U/L(P<0.05)
(Fig. 3), while plasma alanine aminotransferase (ALT) and as-
partate aminotransferase (AST) remained unchanged during the
diet (Table 1). The AST/ALT ratio increased significantly by
34% from 0.84 ±0.09 to 1.13 ±0.15 (P<0.05) during the diet
(Table 1).
KD Improved Plasma Glucose, TGs, and Insulin Sensitivity. Fasting
plasma glucose concentrations decreased by 13% from 112 ±3
to 98 ±3mg/dL(P<0.01) (Fig. 4A), while fasting NEFA
concentrations increased by 35% from 0.55 ±0.02 to 0.74 ±0.02
mmol/L (P<0.001) (Fig. 4B). Plasma TG concentration, which
in the fasting state reflects predominantly liver-derived VLDL-
TGs, decreased by 25% from 1.26 ±0.14 to 0.94 ±0.10 mmol/L
(P<0.01) (Fig. 4C), while plasma total, LDL, or high-density
lipoprotein (HDL) cholesterol concentrations remained unchanged
(Table 1). The 6-d KD induced a marked improvement in insulin
sensitivity, as determined from decreases in fasting serum insulin
concentrations (53%, 10.9 ±1.8 vs. 5.1 ±0.8 mU/L, before vs.
after, P<0.01) (Fig. 4D), C-peptide concentrations (36%, 0.75 ±
0.07 vs. 0.48 ±0.06 nmol/L, P<0.001) (Fig. 4E), and homeostasis
assessment of insulin resistance (HOMA-IR) (57%, 3.0 ±0.5 vs.
1.3 ±0.2 AU, P<0.01) (Fig. 4F).
Fig. 1. Study design. (A) Before and after the 6-d KD, participants visited an imaging center for measurement of IHTG content and liver stiffness (days 1and
6) and underwent metabolic studies at the Clinical Research Unit (days 0 and 7). Participants wore portable accelerometers between days 0 and 7 for de-
termination of physical activity and recorded 3-d food intake starting at days 3 and 4 for determination of dietary composition and compliance. (B) During
metabolic study visits, 180-min tracer infusions of lactate, β-OHB, and glucose were given for determination of rates of substrate fluxes. Indirect calorimetry
was performed to measure energy expenditure and rates of substrate oxidation. An Xdenotes blood sample.
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KD Altered Hepatic Mitochondrial Fluxes. The rate of endogenous
glucose production decreased by 22% from 948 ±60 to 743 ±
45 μmol/min (P<0.001) (Fig. 5A) and the rate of endogenous
lactate production decreased by 18% from 1,045 ±83 to 860 ±
60 μmol/min (P<0.001) (Fig. 5B) during the diet. In contrast, the
rate of β-OHB production (i.e., ketogenesis) increased threefold
from 174 ±30 to 579 ±58 μmol/min (P<0.001) (Fig. 5C)during
the diet. The ratio of the rates of hepatic V
and mitochondrial
oxidation (V
) increased by 52% from 2.4 ±0.3 to 3.6 ±0.2 (P<
0.001) (Fig. 5D). This increase in the V
ratio could entirely
be attributed to a marked reduction in rates of V
, which de-
creased by 38% from 188 ±20 to 116 ±8μmol/min (P<0.001)
(Fig. 5E), since rates of hepatic V
remained unchanged (410 ±
50 vs. 408 ±23 μmol/min, P=0.97) (Fig. 5F).
Potential Mechanisms Underlying the Reduction in V
.The rate of
is highly regulated and can be inhibited by an increase in the
mitochondrial redox state (29) and stimulated by hormones, such
as leptin (31) and T3 (32). Hepatic mitochondrial redox state, as
illustrated from an increase in the ratio of plasma β-OHB and
AcAc ([β-OHB]/[AcAc]) (30, 36), increased markedly by 2.7-fold
from 0.6 ±0.1 to 1.6 ±0.1 (P<0.001) (Fig. 6A) during the diet.
The decrease in V
was also associated with reduction in plasma
concentrations of leptin by 45% from 46.5 ±16.7 to 25.6 ±
9.5 ng/mL (P<0.05) (Fig. 6B) and total T3 by 21% from 0.85 ±
0.08 to 0.67 ±0.03 ng/mL (P<0.05) (Fig. 6C).
KD Increased Protein Catabolism. Energy expenditure and non-
protein respiratory quotient remained unchanged, but rates of whole-
body protein oxidation as assessed by urinary urea nitrogen ex-
cretion (37) increased by 13% during the diet, corresponding to
an average of 9 g more protein being oxidized per day (Table 1).
In the present study, we investigated the antisteatotic effects of a
short-term KD by measuring IHTG content and hepatic mito-
chondrial fluxes by
H-MRS and PINTA. IHTG content de-
creased by 31% in 6 days (Fig. 3), whereas body weight
decreased by 3%, and hepatic insulin resistance decreased
markedly despite increases in circulating NEFA concentrations
(Fig. 4), consistent with previous studies (26, 27). The decrease
in IHTG content could be attributed to increased net hydrolysis
of IHTG and partitioning of the resulting FA toward ketogenesis
Fig. 2. The study diet was ketogenic and participants were compliant. (A) Macronutrient intakes, (B) plasma β-OHB and (C) plasma AcAc concentrations, and
(D) body weight before (orange bars) and after (yellow bars) the 6-d KD (n=10). Data are shown as mean ±SEM. Pvalues were determined using paired
Table 1. Clinical characteristics of the participants before and
after the 6-d KD
Before After Pvalue
Age (y) 58.2 ±2.8
Gender (n, women/men) 5/5
Body mass index (kg/m
) 31.6 ±2.0 30.6 ±2.0 <0.000001
Body weight (kg) 93.5 ±5.3 90.7 ±5.2 <0.00001
Fat mass (kg) 33.5 ±4.7 32.0 ±4.7 <0.001
Fat free mass (kg) 59.5 ±3.8 58.5 ±3.8 <0.01
Total body water (kg) 43.7 ±2.8 42.7 ±2.8 <0.001
Waist circumference (cm) 105.5 ±4.4 102.9 ±4.7 <0.01
Hip circumference (cm) 112.9 ±4.3 110.3 ±4.2 <0.001
P-ALT (IU/L) 42 ±838±7 0.36
P-AST (IU/L) 31 ±337±5 0.15
P-AST/ALT ratio 0.84 ±0.09 1.13 ±0.15 <0.05
P-Total cholesterol (mmol/L) 5.3 ±0.5 5.1 ±0.6 0.46
P-HDL cholesterol (mmol/L) 1.27 ±0.10 1.22 ±0.11 0.25
P-LDL cholesterol (mmol/L) 3.7 ±0.4 3.6 ±0.6 0.71
P-GDF15 (pg/mL) 285.9 ±25.7 280.2 ±27.3 0.37
P-Alanine (mmol/L) 0.32 ±0.02 0.26 ±0.01 0.055
Energy expenditure (kcal/24 h) 1722 ±62 1626 ±67 0.076
Nonprotein respiratory
0.69 ±0.02 0.65 ±0.01 0.094
Protein oxidation (g/24 h) 72.8 ±6.7 82.0 ±6.1 <0.05
Data are in nor means ±SEM. Significances were determined by paired
Studentsttests. GDF15, growth/differentiating factor 15; P, plasma; S, serum.
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due to the accompanying reductions in serum insulin concen-
trations and hepatic V
, respectively (Fig. 5). Reductions in
serum insulin could be attributed to decreased hepatic insulin
resistance, and reductions in V
to increased hepatic mito-
chondrial redox state and decreased plasma leptin and T3 con-
centrations (Fig. 6).
The antisteatotic effect of KD is well-established, but the
underlying intrahepatic adaptations are poorly understood (21,
2527). Since better understanding of these could help to develop
new approaches to treat NAFLD, we applied a PINTA method,
which allows comprehensive assessment of intrahepatic mito-
normality in NAFLD and initially is characterized by compensatory
hyperinsulinemia and later by increased hepatic glucose pro-
duction (510). In the present study, the 6-d KD markedly de-
creased fasting serum glucose and insulin concentrations, as well
as rates of endogenous glucose production (Figs. 4 and 5), im-
plying enhanced hepatic insulin sensitivity, consistent with pre-
vious studies (26). Rates of endogenous lactate turnover also
decreased by 18% during the KD, likely reflecting decreased Cori
cycling due to substrate deprivation (Fig. 5B) (38). Theoretically,
the decrease in rates of glucose production could be due to gly-
cogen depletion or decreased gluconeogenesis. In support of the
former, KD has been shown to decrease hepatic glycogen content
as measured using repeated liver biopsies in humans (39). We
have previously shown that liver volume decreases by 22% during
an identical 6-d KD, and that 70% of this decrease was attributed
to loss of glycogen (40), similar to previous observations in 72-h
fasted human individuals (41).
Oxaloacetate, a key gluconeogenic intermediate, is produced
in hepatic mitochondria from glucose, lactate, and amino acids
by pyruvate carboxylase (42). The rate of V
is controlled by
substrate availability and allosteric activation by acetyl-CoA (42).
In the present study, V
as determined by PINTA remained
unchanged (Fig. 5F), despite decreased substrate availability (Fig.
5Aand Band Table 1), possibly due to increased allosteric ac-
tivation by acetyl-CoA, as assessed by β-OHB turnover (Fig. 5C)
(43). In addition to gluconeogenesis, oxaloacetate can be utilized
in the TCA cycle, which decreased during the KD as determined
by reduced V
(Fig. 5E) (44). Since V
remained unchanged
(Fig. 5F), the decreased utilization of oxaloacetate in the TCA
cycle implies that oxaloacetate was preferentially partitioned to-
ward gluconeogenesis than the TCA cycle (Fig. 5D). Moreover,
the unchanged rates of hepatic V
(Fig. 5F) suggest that the
decreased rates of endogenous glucose production (Fig. 5A)was
due to hepatic glycogen depletion.
Insulin resistance of white adipose tissue lipolysis is another
hallmark of NAFLD (49). Circulating concentrations and turn-
over of NEFA are increased in NAFLD and the antilipolytic action
of insulin is impaired (410). In the present study, plasma NEFA
concentrations were increased (Fig. 4B) while IHTG content was
decreased (Fig. 3A) during the KD, in keeping with previous
studies (27). Since the antilipolytic action of insulin, as determined
using a glycerol tracer combined with hyperinsulinemic-euglycemic
clamp technique, increased during an identical KD diet (27), the
increase in NEFA during the KD appears to be caused by reduced
serum insulin concentrations rather than increased white adipose
tissue insulin resistance.
One possible fate of FA in the liver is re-esterification, which
is the major pathway contributing IHTG in NAFLD (11). In the
present study, both IHTG content (Fig. 3A) and plasma TG con-
centrations (Fig. 4C) decreased markedly during the KD, consistent
with previous data (27). The marked fall in IHTG is also likely to
underrepresent a considerable fall in intrahepatocellular diac-
ylglycerol content, a key mediator of hepatic insulin resistance (1,
45), under conditions of reduced net re-esterification. Insulin is a
key regulator of TG metabolism in the liver and in adipose tissue, as
it inhibits the hydrolysis of existing TGs and stimulates the synthesis
of new TGs (14). The decrease in serum insulin, IHTG content, and
fat mass, and the increase in plasma NEFA concentrations (Figs. 3
and 4 and Table 1) imply that net TG hydrolysis is accelerated by
the KD diet, resulting in increased hepatic availability of FA.
An alternative hepatic fate of FA is mitochondrial β-oxidation
into acetyl-CoA (28, 29). Hepatic concentrations of acetyl-CoA,
as determined by β-OHB turnover (43), were increased during
Fig. 4. KD improved plasma glucose, TGs, and insulin sensitivity. (A) Plasma
glucose, (B) plasma NEFA, (C)plasmaTG,(D) serum insulin, (E) serum C-peptide
concentrations, and (F) HOMA-IR before (orange bars) and after (yellow bars)
the 6-d KD (n=10). Data are shown as mean ±SEM. Pvalues were determined
using paired Studentsttests.
Fig. 3. KD decreased IHTG content. (A) IHTG content (n=8), (B) liver stiffness
(n=10), (C)plasmaGGT(n=10), and (D)plasmaALP(n=9) before (orange
bars) and after (yellow bars) the 6-d KD. Data are shown as mean ±SEM. P
values were determined using paired Studentsttests.
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the diet (Fig. 5C), suggesting that transport of FA to mitochondria
and β-oxidation to acetyl-CoA was increased. Mitochondrial
acetyl-CoA has two alternative metabolic fates: The oxidative
pathway (i.e., the TCA cycle) and ketogenesis (28, 29). A key
reaction that determines this fate is citrate synthase (46). The rate
of V
flux was markedly decreased during the KD (Fig. 5E),
which could explain why the acetyl-CoA derived by β-oxidation of
FA was preferentially channeled toward ketogenesis (Fig. 5B)
rather than the TCA cycle.
We next wished to examine the potential mechanisms underly-
ing the reduction in V
is inhibited by increased mitochon-
drial redox state (29). In the present study, hepatic mitochondrial
redox state, as reflected by the observed increase in the [β-OHB]/
[AcAc] ratio (30, 36), increased by 2.7-fold (Fig. 6A) during the
KD. This suggests that the decrease in V
during the KD could be
attributed to increased mitochondrial redox state in the liver. In
addition, V
can be stimulated by hormones such as leptin (31)
and T3 (32). Indeed, the decrease in V
was associated with re-
duced plasma concentrations of these hormones (Fig. 6 Band C),
which may also have contributed to the observed reduction in V
by the KD.
Another key characteristic of NAFLD and hyperinsulinemia is
increased DNL (15). We have previously shown that overfeeding
of carbohydrates increases DNL and IHTG (47, 48). KD has an
opposite effect (48, 49). The primary substrate in the DNL
pathway is citrate, which is produced by mitochondrial citrate
synthase (12). Thus, an additional contributing mechanism by
which KD decreases IHTG could be a decrease in DNL due to
decreases in serum insulin concentrations and V
Although the KD improved all metabolic abnormalities of
NAFLD in just 6 d, there were also some adverse effects. The
AST/ALT ratio increased by 34% during the diet (Table 1),
suggesting that such a rapid weight loss could induce a transient
hepatocellular injury, consistent with a previous study (50). In ad-
dition, the metabolic changes induced by the 6-d KD closely re-
sembledthoseseeninstarvation(35). Whole-body protein oxidation
increased by 13% (Table 1), which in the face of unchanged dietary
protein intake (Fig. 2A) implies that protein catabolism was in-
creased during the KD. These data are consistent with a previous
Fig. 6. Potential mechanisms underlying the reduction in V
.(A) Ratio of plasma β-OHB and AcAc concentrations, which reflect mitochondrial redox state,
(B) plasma leptin concentrations, and (C) plasma total T3 concentrations before (orange bars) and after (yellow bars) the 6-d KD (n=10). Data are shown as
mean ±SEM. Pvalues were determined using paired Studentsttests.
Fig. 5. KD altered hepatic mitochondrial fluxes. Rates of endogenous (A) glucose, (B) lactate, and (C)β-OHB production; (D) ratio of hepatic V
and V
fluxes, (E) hepatic V
flux, and (F) hepatic V
flux before (orange bars) and after (yellow bars) the 6-d KD (n=10). Data are shown as mean ±SEM. Pvalues
were determined using paired Studentsttests.
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study showing increased urinary nitrogen excretion during KD
(51), and with decreased serum concentrations of insulin (Fig.
4D), which stimulates protein synthesis and inhibits proteolysis in
skeletal muscle (52). While the present study was designed to
investigate the antisteatotic effect of short-term weight loss due to
KD, it would be of interest to determine whether a more mod-
erate, long-term and weight-stable KD has similarly beneficial
effects on NAFLD and insulin sensitivity with less adverse effects
as in this short-term study.
In summary, we show that a short-term (6-d) KD decreased
IHTG and hepatic insulin resistance despite an increase in plasma
NEFA concentrations. These changes could be attributed to in-
creased net hydrolysis of IHTG and partitioning of the resulting
FA toward ketogenesis due to reductions in serum insulin con-
centrations and hepatic citrate synthase flux, respectively. Taken
together, these data demonstrate heretofore undescribed hepatic
mitochondrial adaptations underlying the reversal of NAFLD by a
short-term KD to promote ketogenesis rather than synthesis of
IHTG (Fig. 7).
Materials and Methods
Participants. A total of 10 participants were recruited among individuals who
had previously participated in our metabolic studies at the Clinical Research
Unit (47, 53). Inclusion criteria included: 1) Age 18 to 70 y and 2) alcohol
consumption <20 g/d for women and <30 g/d for men. Exclusion criteria in-
cluded: 1) Known acute or chronic disease other than hepatic steatosis based
on medical history, physical examination, and laboratory tests; 2) history or
current use of agents associated with hepatic steatosis; 3) pregnancy or lac-
tation; (4) intolerance to foods in the study diet; and 5) contraindications for
magnetic resonance imaging (e.g., claustrophobia, metal implants). Details of
therecruitmentareshowninSI Appendix,Fig.S1and clinical characteristics
of the study subjects in Table 1. The Ethics Committee of the Hospital District
of Helsinki and Uusimaa (Helsinki, Finland) approved the study protocol. The
studies were performed in accordance with the Declaration of Helsinki. Each
participant received an explanation of the nature and potential risks of the
study prior to obtaining their written informed consent. The study was reg-
istered at (NCT03737071).
Diet. The study was designed to investigate the antisteatotic effect of a 6-d
KD, which we have previously shown to result in 3kgweightlossand
30% reduction in IHTG content (27). Thus, the KD diet in the present study
was similar as in the previous study and provided 1,440 kcal energy per day,
6% as carbohydrate (25 g/day), 64% as fat, and 28% as protein. All meals
were prepared in the catering kitchen of the Helsinki University Hospital and
provided to the participants as four frozen microwave-safe meals. The partici-
pants were contacted by phone during the intervention to ensure compliance
and to address any issues. In addition, compliance was verified by 3-d food
Fig. 7. Model of the antisteatotic mechanisms of KD. Glucose production decreased during the 6-d KD, which could be attributed to hepatic glycogen
depletion. The decrease in glucose production was accompanied by reduced serum insulin concentrations, which promoted net hydrolysis of TGs in the liver
and adipose tissue. This increased hepatic availability of fatty acids underwent β-oxidation to produce acetyl-CoA. Consequently, the hepatic mitochondrial
redox state increased, which inhibited V
and diverted mitochondrial acetyl-CoA toward ketogenesis: That is, production of AcAc and β-OHB rather than into
oxidation to carbon dioxide (CO
). The reduction in V
was also associated with decreased plasma leptin and T3 concentrations. Mitochondrial V
unchanged during the KD, despite decreases in availability of substrates (glucose, lactate, and alanine), likely due to allosteric activation by acetyl-CoA. The
increase in V
diverted mitochondrial oxaloacetate (OAA) toward gluconeogenesis rather than oxidation. In addition, by limiting the availability of the
primary substrate (citrate) and decreasing insulin concentrations, KD resulted in reduced hepatic DNL. Taking these data together, we find that KD improves
steatosis by markedly altering hepatic mitochondrial fluxes and redox state to promote partitioning of FA to ketogenesis rather than re-esterification and
| Luukkonen et al.
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records and an increase in fasting blood ketone concentrations upon admission
as determined by FreeStyle Precision Neo (Abbott).
Study Design. The study consisted of 1) a screening visit, 2) visits to the im-
aging center for quantification of IHTG content using
H-MRS and liver
stiffness using MRE, and 3) metabolic study visits (Fig. 1A).
Screening visit. The screening visit was performed after an overnight fast. A
history and physical examination were performed to review the inclusion and
exclusion criteria. Blood samples were obtained for measurement of blood
count, blood hemoglobin A
, plasma glucose, creatinine, thyroid-stimulating
hormone, albumin, thromboplastin time, ferritin, C-reactive protein (CRP),
sodium, potassium, bilirubin, AST, ALT, ALP, GGT, serum hepatitis A and C
virus, antinuclear, mitochondrial and smooth muscle antibodies, and hepatitis
B surface antigen concentrations. After the screening visit, the participants
were asked to collect a 3-d dietary record to determine their baseline dietary
composition. The dietary records were analyzed using the AivoDiet software
(v2.0.2.3; Aivo Finland, Turku, Finland).
Imaging visit. Before the metabolic study visits, the participants underwent
imaging visits, which were performed after a 4-h minimum fast. IHTG content
was determined by
H-MRS using Signa HDXt 1.5T scanner (GE Medical
System). MR spectra were acquired using a point resolved spectroscopy se-
quence (TE of 30 ms, TR of 3,000 ms) and analyzed using the jMRUI v5.2
software with AMARES algorithm. Resonances of methylene groups in the
FA chains and water were determined using line-shape fitting with prior
knowledge. Signal intensities were corrected for T2 relaxation using the
equation I
exp(TE/T2). T2 values of 46 ms and 58 ms were used for
water and fat, respectively. IHTG content was expressed as a ratio of signal
from methylene group to total signal of methylene and water, and con-
verted from signal ratio to a weight fraction, applying method validated by
Longo et al. (54) and Szczepaniak et al. (55). The following experimentally
determined factors were used: 1) The ratio of the number of lipid protons in
the fitted (CH2)
signal to the total number of lipid protons is 0.6332 (56);
2) proton densities of fat and water are 110 and 111 mol/l, respectively; 3)
1 g liver tissue contains 711 mg water; 4) densities of the liver tissue, fat in the
liver, and water are 1.051 g/mL, 0.900 g/mL, and 1.000 g/mL; respectively. Liver
stiffness was assessed using MRE (M7000MT, Resoundant Inc.). Participants
were imaged lying in a supine position with an acoustic driver device on the
anterior body wall overlying the liver. The acoustic driver device generated
60-Hz amplitude mechanical waves and produced shear wave motion in the
abdomen. Four noncontiguous axial slices (10-mm thick, 10-mm interslice gap)
were acquired through the widest transverse section of the liver. MR elasto-
grams were generated from the wave images at the slice locations displaying
stiffness in units of kilopascal. Stiffness values were calculated as a median of
four consecutive measurements.
Metabolic study visit. For 3 d prior to the metabolic study day, the participants
were asked to avoid foods naturally enriched in
C (such as sea food, corn,
and sugar), alcohol, and strenuous physical exercise. The participants came
to the clinical research center after an overnight fast. A timed urine collec-
tion was started for 260 min for determination of urinary urea excretion.
Body weight was measured to the nearest 0.1 kg using a calibrated digital
scale (Soehnle) with the participant wearing light indoor clothing without
shoes. Height was measured to the nearest 0.5 cm using a nonstretching
tape. Waist circumference was measured from the midway between the
lower rib margin and the superior iliac spine, and hip circumference at the
greater trochanter level. Body fat mass, fat free mass, and total body water
were determined using the bioelectric impedance method (InBody 720,
An intravenous line was inserted into an antecubital vein for infusion of
the stable isotope tracers and another intravenous line was placed in a dorsal
hand vein of the heated contralateral hand for sampling of arterialized
venous blood (Fig. 1B). At baseline, blood samples were taken for mea-
surement of plasma AST, ALT, ALP, GGT, CRP, LDL, and HDL cholesterol and
TG, as well as serum C-peptide concentrations. Plasma β-OHB, glucose, NEFA,
and serum insulin concentrations were determined at baseline and 90 and
180 min after the start of the infusions.
After the baseline blood sampling, 180-min tracer infusions of [D
(administered as a priming dose of 105 mg/m
over 5 min followed by a
constant infusion of 2.1 mg/m
/min), [
]β-OHB (0.01 mg/kg/min), and [3-
lactate (8.7 micromol/kg/min) were started (Fig. 1B). Arterialized plasma
samplesweretakenat5, 0, 140, 150, 160, 170, and 180 min for measurement
of plasma enrichments of lactate, β-OHB, and glucose using GC-MS (Agilent)
for determination of rates of turnover. In addition, 30 mL of plasma was
collected at 180 min for analyses of positional
C isotopomer enrichments in
glucose using
C MRS (Bruker Avance III HD, 500 UltraShield, TopSpin 3.2,
Bruker) in combination with GC-MS and LC-MS/MS analyses, as previously
described (34), for determination of hepatic rates of V
and V
Sixty minutes after the start of the triple tracer infusion, 40 min of indirect
calorimetry was performed using a computerized flow-through canopy
system (Deltatrac, Datex) to measure respiratory gas exchange and resting
energy expenditure. The hood was placed over the participantshead 10 min
before starting the measurements. Protein oxidation was calculated from
urea concentration in urine collected for 260 min assuming that 1 mol of
urea contains 28 g of urea nitrogen, and that oxidation of 6.25 g of protein
produces 1 g of urea nitrogen (37). Nonprotein respiratory quotient (NPRQ)
was calculated assuming that 1 g of protein requires 966 mL O
and pro-
duces 782 mL CO
. Hence, NPRQ =(VCO
[782 ×P
[966 ×P
where VCO
is the production rate of carbon dioxide, VO
is the consump-
tion rate of oxygen, and P
is protein oxidation rate in grams per minute
(37). Rates of energy expenditure were calculated using the following ad-
ditional assumptions: Oxidation of 1 g of carbohydrate requires 746 mL O
and produces 746 mL CO
, oxidation of 1 g of lipid requires 2,029 mL of O
and produces 1,430 mL of CO
, and that oxidation of 1 g carbohydrate
produces 3.74 kcal, 1 g lipid 9.50 kcal, and 1 g protein 4.10 kcal (37).
At the end of the first metabolic study visit, the participants were provided
with all meals of the study diet to be consumed during the 6-d dietary in-
tervention and they were asked to collect another 3-d dietary record to
determine their dietary intake during the study. The participants were also
given a portable accelerometer (GT3X, Actigraph) to be worn for 6 d to
measure physical activity during the 6-d KD.
The imaging and metabolic study visits were repeated with identical
protocols after the 6-d KD (Fig. 1).
Laboratory Analyses. Concentrations of blood Hb
, plasma glucose, creat-
inine, albumin, ferritin, CRP, sodium, potassium, bilirubin, ALT, AST, ALP, GGT,
β-OHB, TGs, total cholesterol, HDL cholesterol, and serum insulin, C-peptide,
hepatitis A antibody, hepatitis B surface antigen, and hepatitis C antibody
were determined using Architect C16000 autoanalyzer (Abbott) (57).
Blood counts were assessed by impedance, flow cytometric, and photometric
assay (XN10, Sysmex). Plasma thyroid-stimulating hormone concentration was
assessed using Architect i2000SR autoanalyzer (Abbott). Plasma thromboplastin
time was determined using the Owren method by Nycotest PT (Axis Shield).
Serum antinuclear, antimitochondrial, and antismooth muscle antibodies were
assessed using indirect immunofluorescence assays. Plasma growth/differentiat-
ing factor 15 (GDF15) concentration was determined using GDF15 DuoSet ELISA
kit (R&D Systems). Plasma AcAc concentration was determined by a colori-
metric assay (Biovision). Plasma leptin and T3 concentrations were assessed
using double antibody radio-immunoassays (Linco). Plasma NEFA concentrations
were measured using enzymatic, colorimetric assay (Wako Diagnostics). Plasma
alanine concentrations were measured by GC-MS after spiking the samples with
H-alanine internal standard and comparing the ratio of labeled to unlabeled
substrate to a standard curve. Urinary urea was determined by photometric,
enzymatic assay using Architect C16000 (Abbott). Concentrations of glucose and
lactate infusates were assessed by YSI 2700 analyzer (YSI Inc), and those of β-OHB
were determined by COBAS MIRA Plus (Ro che) . HOMA -IR w as cal culated
using the formula: HOMA-IR =fS-insulin (mU/l) ×fP-glucose (mg/dL)/405.
Calculations. Rates of [D
]glucose, [
]β-OHB and [3-
C]lactate turnover
were calculated during isotopic steady state as the tracer infusion rate ×[(infusate
enrichment/plasma enrichm ent) 1]. V
and V
were calculated
from the
C glucose enrichments: m+1, m+2, [4-
C]glucose, [5-
as previously derived (34, 35).
Statistics. Continuous variables were tested for normality using the Shapiro
Wilk test. Nonnormally distributed data were log-transformed for analysis
and back-transformed for presentation. The paired Studentsttest was used
to compare data at the end of the study to the baseline. Data were reported
in means ±SE of means. Statistical analyses were using GraphPad Prism 8.1.2
for Mac OS X (GraphPad Software). A Pvalue of less than 0.05 indicated
statistical significance.
Material and Data Availability. Sources for materials used in this study are
described in Materials and Methods. The raw data obtained for this study
are presented in Dataset S1.
ACKNOWLEDGMENTS. The authors thank Aila Rissanen for advice in diet
design; Aila Karioja-Kallio, Päivi Ihamuotila, and Kimmo Porthan for their
excellent clinical assistance; Gina Butrico, Irina Smolgovsky, John Stack,
Maria Batsu, Codruta Todeasa, and the Yale Hospital Research Unit for excellent
technical assistance; Anni Honkala, Niina Laihanen, Maria Riihelä, Maria
Luukkonen et al. PNAS Latest Articles
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Rautamo, and Kaisa Jousimies for assistance with infusates; Titta Kaukinen
and Jussi Perkiö for assistance with imaging; Heini Oksala and Karri Mikkonen
for assistance with diets; Siiri Luukkonen for graphical assistance; and the
volunteers for their help. This study was supported by Academy of
Finland Grant 309263 (to H.Y.-J.); EU H2020 project Elucidating Pathways of
SteatohepatitisEPoS Grant 634413 (to H.Y.-J.); and H2020-JTI-IMI2 EU project
777377-2 Liver Investigation: Testing Marker Utility inSteatohepatitis (LITMUS)
(to H.Y.-J.), Erityisvaltionosuus (H.Y.-J.); Sigrid Jusélius Foundation (H.Y.-J.,
P.K.L.); Finnish Diabetes Research Foundation (P.K.L.); Instrumentarium Foun-
dation (P.K.L.); Novo Nordisk (P.K.L.) Foundation; and the United States Public
Health Service Grants R01 DK113984 (to G.I.S.), P30 DK45735 (to G.I.S.), and
UL1 RR024139 (to Yale Hospital Research Unit).
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| Luukkonen et al.
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... While several weight-loss diets can help with fatty liver, this review will discuss studies showing that LCHF diets may be more helpful than Low-Fat High-Carbohydrate calorierestricted diets. While weight loss is effective for reducing liver fat content, there is evidence that the benefits of LCHF diets on this front may be-at least, to some extent-independent of it (Gepner et [7,9,11,17,38] . Moreover, significant improvements may be achieved through carbohydrate restriction rather than calorie restriction alone (Browning et al., 2011;Holmer et al., 2021) [4,11] . ...
Full-text available
This study aims to equip health professionals with essential directions for their practice and research in fatty liver disease. In fatty liver disease, excess fat is accumulated in the liver cells, and it mainly has two types: Alcoholic Fatty Liver Disease (AFLD) and Non-Alcoholic Fatty Liver Disease (NAFLD). AFLD is widely accepted to be associated with excessive alcohol consumption. However, in NAFLD's case, although the name acknowledges that it is not associated with excessive alcohol consumption, the exact cause of NAFLD remains ambiguous. Furthermore, for almost four decades, without specialised treatments and prevention strategies, NAFLD has constantly increased, affecting more than a quarter of the world's population. Meanwhile, although the current dietary recommendations for NAFLD patients orbit around the conventional High-Carbohydrate Low-Fat diets, mounting evidence advocates the broader benefits of Low-Carbohydrate High-Fat (LCHF) diets in this regard. Therefore, focusing on the databases such as PubMed, Cochrane Library, and Google Scholar, the authors have carried out an advanced literature search reporting on the efficacy of LCHF diets on NAFLD. After a comprehensive search—using appropriate "keywords and Boolean operators" and "inclusion & exclusion criteria"—the authors selected a potentially relevant set of existing peer-reviewed articles for this narrative review. After critically investigating the LCHF-NAFLD theme, the authors found that LCHF diets may significantly decrease liver fat and may even reverse the disease by targeting the key causes of hepatic fat storage, i.e., high insulin levels, excessive calorie consumption (mainly from carbohydrates), and excessive consumption of refined carbohydrates. Additionally, LCHF diets may provide a comprehensive health benefit beyond liver health. The authors conclude that the LCHF- NAFLD theme represents a rich vein of research opportunities. The authors encourage and call researchers, doctors, nutritionists, dieticians, and related-health professionals to engage more with the LCHF-NAFLD theme.
... Indeed, in hyperleptinemic rodents, increased NEFA uptake by hepatocytes is associated with hepatic steatosis and depletion of apoB-100 [66,68]. However, hepatic steatosis is not commonly reported in humans on CRD and has been shown to be beneficial for non-alcoholic fatty liver disease (NAFLD) [69]. It is therefore feasible that humans, who evolved to transition between carbohydrate and fat-based metabolic states, are evolutionarily equipped to export NEFAs taken up by hepatocytes in the form of VLDLs. ...
Full-text available
When lean people adopt carbohydrate-restricted diets (CRDs), they may develop a lipid profile consisting of elevated LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) with low triglycerides (TGs). The magnitude of this lipid profile correlates with BMI such that those with lower BMI exhibit larger increases in both LDL-C and HDL-C. The inverse association between BMI and LDL-C and HDL-C change on CRD contributed to the discovery of a subset of individuals—termed Lean Mass Hyper-Responders (LMHR)—who, despite normal pre-diet LDL-C, as compared to non-LMHR (mean levels of 148 and 145 mg/dL, respectively), exhibited a pronounced hyperlipidemic response to a CRD, with mean LDL-C and HDL-C levels increasing to 320 and 99 mg/dL, respectively, in the context of mean TG of 47 mg/dL. In some LMHR, LDL-C levels may be in excess of 500 mg/dL, again, with relatively normal pre-diet LDL-C and absent of genetic findings indicative of familial hypercholesterolemia in those who have been tested. The Lipid Energy Model (LEM) attempts to explain this metabolic phenomenon by positing that, with carbohydrate restriction in lean persons, the increased dependence on fat as a metabolic substrate drives increased hepatic secretion and peripheral uptake of TG contained within very low-density lipoproteins (VLDL) by lipoprotein lipase, resulting in marked elevations of LDL-C and HDL-C, and low TG. Herein, we review the core features of the LEM. We review several existing lines of evidence supporting the model and suggest ways to test the model’s predictions.
... These NEFA undergo either re-esterification into complex lipids as TG or transport into the mitochondria to be oxidized by β-oxidation into acetyl-CoA and link the TCA cycle for complete oxidation to CO 2 . Another important fate of acetyl CoA is the ketogenic pathway, as it is converted into acetoacetate (AAA) and β-hydroxybutyrate (β-OHB) [60]. Another important mechanism that explains the antisteatotic effect of the LCHF diet is the decreased glucose influx into the liver cells, with decreased insulin to the basal level. ...
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Metabolic syndrome (MetS) risks cardiovascular diseases due to its associated Dyslipidemia. It is proposed that a low-carbohydrate, high-fat (LCHF) diet positively ameliorates the MetS and reverses insulin resistance. Therefore, we aimed to investigate the protecting effect of the LCHF diet on MetS-associated Dyslipidemia in an experimental animal model. Forty male Sprague-Dawley rats were divided into four groups (10/group): the control group, dexamethasone-induced MetS (DEX) (250 µg/kg/day), LCHF-fed MetS group (DEX + LCHF), and High-Carbohydrate-Low-Fat-fed MetS group (DEX + HCLF). At the end of the four-week experiment, fasting glucose, insulin, lipid profile (LDL-C, HDL-C, Triglyceride), oxidized-LDL, and small dense-LDL using the ELISA technique were estimated. HOMA-IR, Apo B/Apo A1 ratio, and TG/HDL were calculated. Moreover, histological examination of the liver by H & E and Sudan III stain was carried out. In the DEX group, rats showed a significant (p < 0.05) increase in the HOMA-IR, atherogenic parameters, such as s-LDL, OX-LDL, Apo B/Apo A1 ratio, and TG/HDL. The LCHF diet significantly improved the parameters of Dyslipidemia (p < 0.05) by decreasing the Apo B/Apo A1 and TG/HDL-C ratios. Decreased steatosis in LCHF-fed rats compared to HCLF was also revealed. In conclusion, the LCHF diet ameliorates MetS-associated Dyslipidemia, as noted from biochemical results and histological examination.
... A decrease in lipid accumulation in HMGCS2overexpressed HepG2 cells further confirmed a hepatocyte-specific ketogenic role in lipid metabolism. Moreover, our findings suggest a possible mechanism underlying the protective effects of dietary interventions against NAFLD, as a ketogenic diet [23] and intermittent fasting [39,79,80] are known to activate ketogenesis. Although no liver-targeting pharmacotherapeutics against NAFLD are presently in use, current drugs, such as metformin [81], GLP1R agonists [3] and SGLT2 inhibitors [4], being clinically used for the treatment of other metabolic diseases (i.e., diabetes), have shown benefits in alleviating different aspects of NAFLD pathology [4,82]. ...
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Objective Aberrant ketogenesis is correlated with the degree of steatosis in NAFLD patients, and an inborn error of ketogenesis (mitochondrial HMG-CoA synthase deficiency) is commonly associated with the development of the fatty liver. Here we aimed to determine the impact of Hmgcs2-mediated ketogenesis and its modulations on the development and treatment of fatty liver disease. Methods Loss- and gain-of-ketogenic function through in vivo and in vitro models, achieved by Hmgcs2 knockout and overexpression, respectively, were examined to investigate the role of ketogenesis in the hepatic lipid accumulation during neonatal development and the diet-induced NAFLD mouse model. Results Ketogenic function was decreased in NAFLD mice with a reduction in Hmgcs2 expression. Mice lacking Hmgcs2 developed spontaneous fatty liver phenotype during postnatal development, which was rescued by a shift to a low-fat dietary composition via early weaning. Hmgcs2 heterozygous mice, which exhibited reduced ketogenic activity, were more susceptible to diet-induced NAFLD development, whereas HMGCS2 overexpression in NAFLD mice improved hepatosteatosis and glucose homeostasis. Conclusions Our study adds new knowledge to the field of ketone body metabolism and shows that Hmgcs2-mediated ketogenesis modulates hepatic lipid regulation under a fat-enriched nutritional environment. The regulation of hepatic ketogenesis may be a viable therapeutic strategy in the prevention and treatment of hepatosteatosis.
... For instance, infusion of [6,6-2 H 2 ]glucose into mice with a fat-and sugarrich diet showed that NAFLD progresses to nonalcoholic steatohepatitis (NASH) through upregulation of hepatic tricarboxylic acid (TCA) flux and anaplerosis [38]. A ketogenic diet, in comparison, decreased hepatic mitochondrial flux and alleviated NAFLD in obese human subjects [39]. In a double-blind randomized human trial, long-term consumption of beverages sweetened with fructose and sucrose, but not glucose, altered lipid metabolism to increase liver fat, a hallmark of developing NAFLD [40]. ...
Complete understanding of a biological system requires quantitation of metabolic fluxes that reflect its dynamic state. Various analytical chemistry tools, enzyme-based probes, and microscopy enable flux measurement. However, any method alone falls short of comprehensive flux quantitation. Here we show that integrating these techniques results in a systems-level quantitative map of absolute metabolic fluxes that constitute an indispensable dimension of characterizing phenotypes. Stable isotopes, mass spectrometry, and NMR spectroscopy reveal relative pathway fluxes. Biochemical probes reveal the physical rate of environmental changes. FRET-based and SRS-based microscopy reveal targeted metabolite and chemical bond formation. These techniques are complementary and can be computationally integrated to reveal actionable information on metabolism. Integrative metabolic flux analysis using various quantitative techniques advances biotechnology and medicine.
... The ketogenic diet is a high fat, low carbohydrate with moderate protein diet [18]. It has been used in multiple diseases, such as epilepsy, obesity, type2 diabetes, and so on [19][20][21][22][23][24][25][26]. Recent studies showed that the benefits of ketogenic diet were mostly due to the ability to regulate inflammation by the ketone body β-hydroxybutyrate (BHB) [18,[27][28][29][30]. ...
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Background Aseptic Loosening (AL) following periprosthetic osteolysis is the main long-term complication after total joint arthroplasty (TJA). However, there is rare effective treatment except for revision surgery, which is costly and painful to the patients. In recent years, the ketone body β-hydroxybutyrate (BHB) has attracted much attention and has been proved to be beneficial in many chronic diseases. With respect to the studies on the ketone body β-hydroxybutyrate (BHB), its anti-inflammatory ability has been widely investigated. Although the ketone body β-hydroxybutyrate has been applied in many inflammatory diseases and has achieved considerable therapeutic efficacy, its effect on wear particles induced osteolysis is still unknown. Results In this work, we confirmed that the anti-inflammatory action of β-hydroxybutyrate (BHB) could be reappeared in CoCrMo alloy particles induced osteolysis. Mechanistically, the ketone body β-hydroxybutyrate (BHB) deactivated the activation of NLRP3 inflammasome triggered by CoCrMo alloy particles. Of note, this inhibitory action was independent of Gpr109a receptor as well as histone deacetylase (HDAC) suppression. Furthermore, given that butyrate, one kind of short chain fatty acid (SCFA) structurally related to β-hydroxybutyrate (BHB), has been reported to be an inhibitor of osteoclast, thus we also investigate the effect of β-hydroxybutyrate (BHB) on osteoclast, which was contributed to bone resorption. It was found that β-hydroxybutyrate (BHB) did not only affect osteoclast differentiation, but also inhibit its function. Unlike the inflammasome, the effect of β-hydroxybutyrate (BHB) on osteoclast may mainly rely on histone deacetylase (HDAC) suppression. Conclusions In general, our study showed that the alleviation of osteolysis may owe to the effect of β-hydroxybutyrate (BHB) on inflammasome deactivation and osteoclast. Graphical Abstract
Non-alcoholic fatty liver disease (NAFLD) has a high global prevalence with a heterogeneous and complex pathophysiology that presents barriers to traditional targeted therapeutic approaches. We describe an integrated quantitative systems pharmacology (QSP) platform that comprehensively and unbiasedly defines disease states, in contrast to just individual genes or pathways, that promote NAFLD progression. The QSP platform can be used to predict drugs that normalize these disease states and experimentally test predictions in a human liver acinus microphysiology system (LAMPS) that recapitulates key aspects of NAFLD. Analysis of a 182 patient-derived hepatic RNA-sequencing dataset generated 12 gene signatures mirroring these states. Screening against the LINCS L1000 database led to the identification of drugs predicted to revert these signatures and corresponding disease states. A proof-of-concept study in LAMPS demonstrated mitigation of steatosis, inflammation, and fibrosis, especially with drug combinations. Mechanistically, several structurally diverse drugs were predicted to interact with a subnetwork of nuclear receptors, including pregnane X receptor (PXR; NR1I2), that has evolved to respond to both xenobiotic and endogenous ligands and is intrinsic to NAFLD-associated transcription dysregulation. In conjunction with iPSC-derived cells, this platform has the potential for developing personalized NAFLD therapeutic strategies, informing disease mechanisms, and defining optimal cohorts of patients for clinical trials.
Nonalcoholic fatty liver disease (NAFLD) is associated with high carbohydrate (HC) intake. We investigated whether the relationship between carbohydrate intake and NAFLD is mediated by interactions between gut microbial modulation, impaired insulin response, and hepatic de novo lipogenesis (DNL). Stool samples were collected from 204 Korean subjects with biopsy-proven NAFLD (n = 129) and without NAFLD (n = 75). The gut microbiome profiles were analyzed using 16S rRNA amplicon sequencing. Study subjects were grouped by the NAFLD activity score (NAS) and percentage energy intake from dietary carbohydrate. Hepatic DNL-related transcripts were also analyzed (n = 90). Data from the Korean healthy twin cohort (n = 682), a large sample of individuals without NAFLD, were used for comparison and validation. A HC diet rather than a low carbohydrate diet was associated with the altered gut microbiome diversity according to the NAS. Unlike individuals from the twin cohort without NAFLD, the abundances of Enterobacteriaceae and Ruminococcaceae were significantly different among the NAS subgroups in NAFLD subjects who consumed an HC diet. The addition of these two microbial families, along with Veillonellaceae, significantly improved the diagnostic performance of the predictive model, which was based on the body mass index, age, and sex to predict nonalcoholic steatohepatitis in the HC group. In the HC group, two crucial regulators of DNL (SIRT1 and SREBF2) were differentially expressed among the NAS subgroups. In particular, kernel causality analysis revealed a causal effect of the abundance of Enterobacteriaceae on SREBF2 upregulation and of the surrogate markers of insulin resistance on NAFLD activity in the HC group. Consuming an HC diet is associated with alteration in the gut microbiome, impaired glucose homeostasis, and upregulation of hepatic DNL genes, altogether contributing to NAFLD pathogenesis.
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The ketogenic diet (KD) is a low-carbohydrate, high-fat diet regarded as a potential intervention for cancers owing to its effects on tumor metabolism and behavior. Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer, and its management is worth investigating because of the high fatality rate. Additionally, as the liver is the glucose and lipid metabolism center where ketone bodies are produced, the application of KD to combat HCC is promising. Prior studies have reported that KD could reduce the energy supply and affect the proliferation and differentiation of cancer cells by lowering the blood glucose and insulin levels. Furthermore, KD can increase the expression of hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in hepatocytes and regulate lipid metabolism to inhibit the progression of HCC. In addition, β-hydroxybutyrate can induce histone hyperacetylation and reduce the expression of inflammatory factors to alleviate damage to hepatocytes. However, there are few relevant studies at present, and the specific effects and safety of KD on HCC warrant further research. Optimizing the composition of KD and combining it with other therapies to enhance its anti-cancer effects warrant further exploration.
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Energy restriction reduces liver fat, improves hepatic insulin resistance and lipid metabolism. However, temporal data in which these metabolic improvements occur and their interplay is incomplete. By performing repeated MRI scans and blood analysis at day 0, 3, 7, 14 and 28 the temporal changes in liver fat and related metabolic factors were assessed at five times during a low-calorie diet (LCD, 800–1100 kcal/day) in ten obese non-diabetic women (BMI 41.7 ± 2.6 kg/m²) whereof 6 had NAFLD. Mean weight loss was 7.4 ± 1.2 kg (0.7 kg/day) and liver fat decreased by 51 ± 16%, resulting in only three subjects having NAFLD at day 28. Marked alteration of insulin, NEFA, ALT and 3-hydroxybuturate was evident 3 days after commencing LCD, whereas liver fat showed a moderate but a linear reduction across the 28 days. Other circulating-liver fat markers (e.g. triglycerides, adiponectin, stearoyl-CoA desaturase-1 index, fibroblast growth factor 21) demonstrated modest and variable changes. Marked elevations of NEFA, 3-hydroxybuturate and ALT concentrations occurred until day 14, likely reflecting increased tissue lipolysis, fat oxidation and upregulated hepatic fatty acid oxidation. In summary, these results suggest linear reduction in liver fat, time-specific changes in metabolic markers and insulin resistance in response to energy restriction.
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The common patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant I148M predisposes to nonalcoholic liver disease but not its metabolic sequelae. We compared the handling of labeled polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFA) in vivo in humans and in cells harboring different PNPLA3 genotypes. In 148M homozygous individuals, triglycerides (TGs) in very low-density lipoproteins (VLDL) were depleted of PUFAs both under fasting and postprandial conditions compared with 148I homozygotes, and the PUFA/SFA ratio in VLDL-TGs was lower relative to the chylomicron precursor pool. In human PNPLA3-148M and PNPLA3-KO cells, PUFA but not SFA incorporation into TGs was increased at the expense of phosphatidylcholines, and under lipolytic conditions, PUFA-containing diacylglycerols (DAGs) accumulated compared with PNPLA3-148I cells. Polyunsaturated TGs were increased, while phosphatidylcholines (PCs) were decreased in the human liver in 148M homozygous individuals as compared with 148I homozygotes. We conclude that human PNPLA3-I148M is a loss-of-function allele that remodels liver TGs in a polyunsaturated direction by impairing hydrolysis/transacylation of PUFAs from DAGs to feed phosphatidylcholine synthesis.
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Liver fat can be non-invasively measured by proton magnetic resonance spectroscopy (¹H-MRS) and fibrosis estimated as stiffness using transient elastography (FibroScan). There are no longitudinal data on changes in liver fat in Europids or on predictors of liver stiffness using these methods. We determined liver fat (¹H-MRS) and clinical characteristics including features of insulin resistance at baseline and after a median follow-up period of 11.3 (range 7.3–13.4) years in 97 Finnish subjects. Liver stiffness was measured at 11.3 years. Liver fat content decreased by 5% (p < 0.05) over time. Values at baseline and 11.3 years were closely interrelated (r = 0.81, p < 0.001). Baseline liver fat (OR 1.32; 95%CI: 1.15–1.50) and change in BMI (OR 1.67; 95%CI: 1.24–2.25) were independent predictors of liver fat at 11.3 years (AUROC 0.90; 95%CI: 0.83–0.96). Baseline liver fat (AUROC 0.84; 95%CI: 0.76–0.92) predicted liver fat at 11.3 years more accurately than routinely available parameters (AUROC 0.76; 95%CI: 0.65–0.86, p = 0.02). At 11.3 years, 29% of the subjects had increased liver stiffness. Baseline liver fat (OR 2.17; 95%CI: 1.05–4.46) was an independent predictor of increased liver stiffness. These data show that liver fat is more important than the associated metabolic abnormalities as the predictor of future liver fat and fibrosis.
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Hepatic mitochondria play a central role in the regulation of intermediary metabolism and maintenance of normoglycemia, and there is great interest in assessing rates of hepatic mitochondrial citrate synthase flux (VCS) and pyruvate carboxylase flux (VPC) in vivo. Here, we show that a positional isotopomer NMR tracer analysis (PINTA) method can be used to non-invasively assess rates of VCS and VPC fluxes using a combined NMR/gas chromatography-mass spectrometry analysis of plasma following infusion of [3-¹³C]lactate and glucose tracer. PINTA measures VCS and VPC fluxes over a wide range of physiological conditions with minimal pyruvate cycling and detects increased hepatic VCS following treatment with a liver-targeted mitochondrial uncoupler. Finally, validation studies in humans demonstrate that the VPC/VCS ratio measured by PINTA is similar to that determined by in vivo NMR spectroscopy. This method will provide investigators with a relatively simple tool to non-invasively examine the role of altered hepatic mitochondrial metabolism.
Nonalcoholic fatty liver disease (NAFLD) is estimated to affect up to one-third of the general population, and new therapies are urgently required. Our laboratory previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides. Although we previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, hepatic inflammation, and fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess its safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of longer-term CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in high-fat, fructose-fed cynomolgus macaques ( n = 6) and spontaneously obese dysmetabolic rhesus macaques ( n = 12). Using positional isotopomer nuclear magnetic resonance tracer analysis (PINTA), we demonstrated that acute CRMP treatment (single dose, 5 mg/kg) increased rates of hepatic mitochondrial fat oxidation by 40%. Six weeks of CRMP treatment reduced hepatic triglycerides in both nonhuman primate models independently of changes in body weight, food intake, body temperature, or adverse reactions. CRMP treatment was also associated with a 20 to 30% reduction in fasting plasma triglycerides and low-density lipoprotein (LDL)–cholesterol in dysmetabolic nonhuman primates. Oral administration of CRMP reduced endogenous glucose production by 18%, attributable to a 20% reduction in hepatic acetyl–coenzyme A (CoA) content [as assessed by whole-body β-hydroxybutyrate (β-OHB) turnover] and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of liver-targeted mitochondrial uncouplers for the treatment of metabolic syndrome in humans.
In order to determine whether the glucose-alanine cycle regulates rates of hepatic mitochondrial oxidation in humans, we applied positional isotopomer NMR tracer analysis (PINTA) to assess rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in healthy volunteers following both an overnight (12 hours) and a 60-hour fast. Following the 60-hour fast, rates of endogenous glucose production and mitochondrial oxidation decreased, whereas rates of hepatic pyruvate carboxylase flux remained unchanged. These reductions were associated with reduced rates of alanine turnover, assessed by [3-13C]alanine, in a subgroup of participants under similar fasting conditions. In order to determine whether this reduction in alanine turnover was responsible for the reduced rates of hepatic mitochondrial oxidation, we infused unlabeled alanine into another subgroup of 60-hour fasted subjects to increase rates of alanine turnover, similar to what was measured after a 12-hour fast, and found that this perturbation increased rates of hepatic mitochondrial oxidation. Taken together, these studies demonstrate that 60 hours of starvation induce marked reductions in rates of hepatic mitochondrial oxidation, which in turn can be attributed to reduced rates of glucose-alanine cycling, and reveal a heretofore undescribed role for glucose-alanine in the regulation of hepatic mitochondrial oxidation in humans.
Objective: Nonalcoholic fatty liver disease (i.e., increased intrahepatic triglyceride [IHTG] content), predisposes to type 2 diabetes and cardiovascular disease. Adipose tissue lipolysis and hepatic de novo lipogenesis (DNL) are the main pathways contributing to IHTG. We hypothesized that dietary macronutrient composition influences the pathways, mediators, and magnitude of weight gain-induced changes in IHTG. Research design and methods: We overfed 38 overweight subjects (age 48 ± 2, BMI 31 ± 1 kg/m2, liver fat 4.7 ± 0.9%) 1,000 extra kcal/day of saturated (SAT) or unsaturated (UNSAT) fat or simple sugars (CARB) for 3 weeks. We measured IHTG (1H-MRS), pathways contributing to IHTG (lipolysis ([2H5]glycerol) and DNL (2H2O) basally and during euglycemic hyperinsulinemia, insulin resistance, endotoxemia, plasma ceramides, and adipose tissue gene expression at 0 and 3 weeks. Results: Overfeeding SAT increased IHTG more (+55%) than UNSAT (+15%, P < 0.05). CARB increased IHTG (+33%) by stimulating DNL (+98%). SAT significantly increased while UNSAT decreased lipolysis. SAT induced insulin resistance and endotoxemia and significantly increased multiple plasma ceramides. The diets had distinct effects on adipose tissue gene expression. Conclusions: Macronutrient composition of excess energy influences pathways of IHTG: CARB increases DNL, while SAT increases and UNSAT decreases lipolysis. SAT induced greatest increase in IHTG, insulin resistance, and harmful ceramides. Decreased intakes of SAT could be beneficial in reducing IHTG and the associated risk of diabetes.
A carbohydrate-restricted diet is a widely recommended intervention for non-alcoholic fatty liver disease (NAFLD), but a systematic perspective on the multiple benefits of this diet is lacking. Here, we performed a short-term intervention with an isocaloric low-carbohydrate diet with increased protein content in obese subjects with NAFLD and characterized the resulting alterations in metabolism and the gut microbiota using a multi-omics approach. We observed rapid and dramatic reductions of liver fat and other cardiometabolic risk factors paralleled by (1) marked decreases in hepatic de novo lipogenesis; (2) large increases in serum β-hydroxybutyrate concentrations, reflecting increased mitochondrial β-oxidation; and (3) rapid increases in folate-producing Streptococcus and serum folate concentrations. Liver transcriptomic analysis on biopsy samples from a second cohort revealed downregulation of the fatty acid synthesis pathway and upregulation of folate-mediated one-carbon metabolism and fatty acid oxidation pathways. Our results highlight the potential of exploring diet-microbiota interactions for treating NAFLD.
This summer, 25 overweight and obese adults participating in a tightly controlled feeding study will take up full-time residence for 3 months at a wooded lakefront center in Ashland, Massachusetts. However, before checking in at Framingham State University’s Warren Conference Center and Inn, they will have to lose 15% of their body weight on a calorie-restricted diet with home-delivered meals.
NAFLD is closely linked with hepatic insulin resistance. Accumulation of hepatic diacylglycerol activates PKC-ε, impairing insulin receptor activation and insulin-stimulated glycogen synthesis. Peripheral insulin resistance indirectly influences hepatic glucose and lipid metabolism by increasing flux of substrates that promote lipogenesis (glucose and fatty acids) and gluconeogenesis (glycerol and fatty acid-derived acetyl-CoA, an allosteric activator of pyruvate carboxylase). Weight loss with diet or bariatric surgery effectively treats NAFLD, but drugs specifically approved for NAFLD are not available. Some new pharmacological strategies act broadly to alter energy balance or influence pathways that contribute to NAFLD (e.g., agonists for PPAR γ, PPAR α/δ, FXR and analogs for FGF-21, and GLP-1). Others specifically inhibit key enzymes involved in lipid synthesis (e.g., mitochondrial pyruvate carrier, acetyl-CoA carboxylase, stearoyl-CoA desaturase, and monoacyl- and diacyl-glycerol transferases). Finally, a novel class of liver-targeted mitochondrial uncoupling agents increases hepatocellular energy expenditure, reversing the metabolic and hepatic complications of NAFLD.