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Effect of a ketogenic diet on hepatic steatosis and
hepatic mitochondrial metabolism in nonalcoholic
fatty liver disease
Panu K. Luukkonen
a,b,c
, Sylvie Dufour
a,d
, Kun Lyu
e
, Xian-Man Zhang
a,d
, Antti Hakkarainen
f,g
, Tiina E. Lehtimäki
f
,
Gary W. Cline
a,d
, Kitt Falk Petersen
a,d
, Gerald I. Shulman
a,d,e,1,2
, and Hannele Yki-Järvinen
b,c,1,2
a
Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520;
b
Minerva Foundation Institute for Medical Research, Helsinki 00290,
Finland;
c
Department of Medicine, University of Helsinki and Helsinki University Hospital, Helsinki 00290, Finland;
d
Yale Diabetes Research Center, Yale
School of Medicine, New Haven, CT 06520;
e
Department of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06520;
f
Department of
Radiology, HUS Medical Imaging Center, University of Helsinki and Helsinki University Hospital, Helsinki 00290, Finland; and
g
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:
[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 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
|
redox
|
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 (1–3). It is strongly associated with insulin resistance,
which is characterized by excessive hepatic glucose production and
compensatory hyperinsulinemia (4–10). 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)
(4–11). 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 (12–16). 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 (17–22). Recently, low-carbohydrate
ketogenic diets (KD) have gained popularity in the treatment of
obesity, type 2 diabetes, and NAFLD (23–25). 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
2
(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).
Significance
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).
1
G.I.S. and H.Y.-J. contributed equally to this work.
2
To whom correspondence may be addressed. Email: gerald.shulman@yale.edu or
Hannele.Yki-Jarvinen@helsinki.fi.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1922344117/-/DCSupplemental.
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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 (
1
H-MRS/MRE)
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 (33–35) to assess rates of hepatic mitochon-
drial flux through pyruvate carboxylase (V
PC
) relative to citrate
synthase flux (V
CS
), as well as rates of endogenous glucose,
β-OHB, and lactate production by stable isotope infusions of
[D
7
]glucose, [
13
C
4
]β-OHB, and [3-
13
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).
Results
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
by
1
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 “X”denotes 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
PC
and mitochondrial
oxidation (V
CS
) increased by 52% from 2.4 ±0.3 to 3.6 ±0.2 (P<
0.001) (Fig. 5D). This increase in the V
PC
/V
CS
ratio could entirely
be attributed to a marked reduction in rates of V
CS
, which de-
creased by 38% from 188 ±20 to 116 ±8μmol/min (P<0.001)
(Fig. 5E), since rates of hepatic V
PC
remained unchanged (410 ±
50 vs. 408 ±23 μmol/min, P=0.97) (Fig. 5F).
Potential Mechanisms Underlying the Reduction in V
CS
.The rate of
V
CS
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
CS
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).
Discussion
In the present study, we investigated the antisteatotic effects of a
short-term KD by measuring IHTG content and hepatic mito-
chondrial fluxes by
1
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
Student’sttests.
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
2
) 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
quotient
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
Student’sttests. 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
CS
, respectively (Fig. 5). Reductions in
serum insulin could be attributed to decreased hepatic insulin
resistance, and reductions in V
CS
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,
25–27). 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-
chondrialmetabolisminvivo(34).Insulinresistanceisakeyab-
normality in NAFLD and initially is characterized by compensatory
hyperinsulinemia and later by increased hepatic glucose pro-
duction (5–10). 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
PC
is controlled by
substrate availability and allosteric activation by acetyl-CoA (42).
In the present study, V
PC
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
CS
(Fig. 5E) (44). Since V
PC
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
PC
(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 (4–9). Circulating concentrations and turn-
over of NEFA are increased in NAFLD and the antilipolytic action
of insulin is impaired (4–10). 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 Student’sttests.
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 Student’sttests.
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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
CS
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
CS
.V
CS
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
CS
during the KD could be
attributed to increased mitochondrial redox state in the liver. In
addition, V
CS
can be stimulated by hormones such as leptin (31)
and T3 (32). Indeed, the decrease in V
CS
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
CS
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
CS
.
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
CS
.(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 Student’sttests.
Fig. 5. KD altered hepatic mitochondrial fluxes. Rates of endogenous (A) glucose, (B) lactate, and (C)β-OHB production; (D) ratio of hepatic V
PC
and V
CS
fluxes, (E) hepatic V
CS
flux, and (F) hepatic V
PC
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 Student’sttests.
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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 ClinicalTrials.gov (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
CS
and diverted mitochondrial acetyl-CoA toward ketogenesis: That is, production of AcAc and β-OHB rather than into
oxidation to carbon dioxide (CO
2
). The reduction in V
CS
was also associated with decreased plasma leptin and T3 concentrations. Mitochondrial V
PC
remained
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
PC
/V
CS
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
lipogenesis.
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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
1
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
1c
, 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
1
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
m
=I
0
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)
n-2
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
13
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,
Biospace).
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
7
]glucose
(administered as a priming dose of 105 mg/m
2
over 5 min followed by a
constant infusion of 2.1 mg/m
2
/min), [
13
C
4
]β-OHB (0.01 mg/kg/min), and [3-
13
C]
lactate (8.7 micromol/kg/min) were started (Fig. 1B). Arterialized plasma
samplesweretakenat−5, 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
13
C isotopomer enrichments in
glucose using
13
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
PC
and V
CS
fluxes.
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 participants’head 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
2
and pro-
duces 782 mL CO
2
. Hence, NPRQ =(VCO
2
–[782 ×P
ox
])/(VO
2
–[966 ×P
ox
]),
where VCO
2
is the production rate of carbon dioxide, VO
2
is the consump-
tion rate of oxygen, and P
ox
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
2
and produces 746 mL CO
2
, oxidation of 1 g of lipid requires 2,029 mL of O
2
and produces 1,430 mL of CO
2
, 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
A1c
, 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
a
2
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
7
]glucose, [
13
C
4
]β-OHB and [3-
13
C]lactate turnover
were calculated during isotopic steady state as the tracer infusion rate ×[(infusate
enrichment/plasma enrichm ent) –1]. V
PC
/V
EGP
and V
PC
/V
CS
were calculated
from the
13
C glucose enrichments: m+1, m+2, [4-
13
C]glucose, [5-
13
C]glucose,
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 Student’sttest 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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MEDICAL SCIENCES
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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
Steatohepatitis’EPoS 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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