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Hepatic Notch Signaling Correlates With Insulin
Resistance and Nonalcoholic Fatty Liver Disease
Luca Valenti,
1
Rosa M. Mendoza,
2
Raffaela Rametta,
1
Marco Maggioni,
3
Chris Kitajewski,
4
Carrie J. Shawber,
4
and Utpal B. Pajvani
2
Hepatic Notch signaling is inappropriately activated in obese/
insulin-resistant mouse models. Genetic or pharmacologic in-
hibition of hepatic Notch signaling in obese mice simultaneously
improves glucose tolerance and reduces hepatic triglyceride
content. As such, we predicted that Notch signaling in human
liver would be positively associated with insulin resistance and
hepatic steatosis. Here, we systematically survey Notch signaling
in liver biopsy specimens, and show active Notch signaling in
lean and obese adults, with expression of multiple Notch receptors
and ligands. In morbidly obese patients undergoing bariatric
surgery, we show that Notch activation positively correlates with
glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxy-
kinase (PCK1) expression, key regulators of hepatic glucose out-
put. We used immunofluorescence to identify active Notch
signaling in hepatocytes and show highest activity in hyperglyce-
mia, which we confirmed is a direct effect of hyperglycemia and
insulin resistance. In a validation cohort of leaner individuals
undergoing percutaneous liver biopsy for suspected nonalcoholic
fatty liver disease (NAFLD), Notch activity showed independent
positive association with insulin resistance and hepatic steatosis.
Notably, Notch activity showed stronger correlation with the
NAFLD activity score and alanine aminotransferase levels than
with steatosis alone, suggesting that Notch activity is associated
with nonalcoholic steatohepatitis. In summary, this study estab-
lishes that Notch signaling is activated in and may represent
a therapeutic target for patients with obesity-related liver disease.
Diabetes 62:4052–4062, 2013
O
besity manifests as multiple pathologic states in
the liver. Insulin resistance in adipocytes
results in unrestrained lipolysis, with conse-
quent excess free fatty acid flux to the liver (1).
In a parallel pathogenic process, ex cess adiposity leads to
insulin resistance, which begets the fasting hyperglycemia
of type 2 diabetes (T2D) (2). Compensatory hyper-
insulinemia drives de novo lipogenesis (3), and coupled
with an impaired ability to catabolize and export fatty
acids (4), results in excess hepatocyte triglyceride accu-
mulation, or nonalcoholic fatty liver disease (NAFLD), in
the presence of a predisposing genetic background (5).
Steatosis may be associated with hepatocellular damage
and necroinflammatory changes, defining nonalcoholic
steatohepatitis (NASH), which predisposes to cirrhosis
and hepatocellular cancer (1). Interestingly, NASH further
exacerbates hepatic insulin resistance through activation
of forkhead box class O (FoxO) 1, the key transcriptional
activator of glucose-6-phosphatase (G6PC) and phospho-
enolpyruvate carboxykinase (PCK1), rate-limiting enzymes
of gluconeogenesis and glycogenolysis, which combined
regulate hepatic glucose output. This vicious cycle results in
coincident NAFLD and T2D, which show independent asso-
ciations with cardiovascular disease (6). No approved phar-
macologic therapy is approved for NALFD, and although
multiple T2D therapies are available, few show durability and
long-term efficacy (7). As such, novel therapeutic directions
are necessary to reduce overall obesity-related morbidity .
We previously showed that inhibition of hepatic Notch
signaling protects from both obesity-induced glucose in-
tolerance, by suppressing hepatic glucose output (8), and
fatty liver, by reducing de novo lipogenesis (9). Notch
signaling is highly conserved from lower organisms to
primates and is critical for cell fate decision making, in-
cluding regulation of cell specification and lineage restric-
tion, depending on the cellular context (10). In mammals,
cell surface Notch ligands of the Jagged (-1 and -2) and
Delta-like (-1, -3, -4) families bind one of four Notch re-
ceptors (Notch1–4) on a neighboring cell, resulting in a se-
ries of cleavage events that culminate in the transcription of
canonical Notch targets, the hairy enhancer of split (HES)
and Hes-related (HEY) family of genes (11). Homozygous
null alleles in this pathway result in embryonic lethality in
mice (12–14) and loss-of-function mutations in severe de-
velopmental defects in affected individuals (15,16), proving
the critical role of Notch signaling to regulate cell fate
decisions in normal development.
Less is known about Notch signaling in matur e tissue.
Increased Notch expression and fun ction has been shown
in cancer and tumor angiogenesis (17–19), but there are
few reports of expression analysis of Notch pathway pro-
teins in developed, non-neoplastic tissue. In the liver,
Notch proteins are constitutively expressed in multiple cell
types (20,21), with increased expression in hepatocytes
after partial hepatectomy (22). In rat models, normal liver
regeneration was prevented by genetic inhibition of he-
patocyte Notch signaling (23). As such, we pred icted that
the Notch-signaling apparatus is present in mature liver
and may be induced by obesity-induced hepatocyte dam-
age. Our initial characterization of Notch signaling in mu-
rine liver demonstrated that Notch target gene expression
is, in fact, increased in mouse models of obesity and in-
sulin resistance (8). Interestingly, genetic (8) or pharma-
cologic (11,24,25) blockade of hepatocyte Notch signaling
resulted in parallel inhibition of hepatic glucose pro-
duction (8) as well as triglyceride accumulation (9), low-
ering the overall atherosclerotic burden in obese mice (26).
From the
1
Department of Pathophysiology and Transplantation, Università
degli Studi di Milano, Internal Medicine, Fondazione Istituto di Ricovero e
Cura a Carattere Scientifico Ca’ Granda, Milano, Italy; the
2
Department of
Medicine, Columbia University, New York, New York; the
3
Department of
Pathology, Università degli Studi di Milano, Internal Medicine, Fondazione
Istituto di Ricovero e Cura a Carattere Scientifico Ca’ Granda, Milano, Italy;
and the
4
Department of Obstetrics and Gynecology, Columbia University,
New York, New York.
Corresponding author: Utpal B. Pajvani, up2104@columbia.edu.
Received 13 May 2013 and accepted 22 August 2013.
DOI: 10.2337/db13-0769
Ó 2013 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
4052 DIABETES, VOL. 62, DECEMBER 2013 diabetes.diabetesjournals.org
ORIGINAL ARTICLE
Conversely, constitutive activation of hepatocyte Notch
signaling caused glucose intolerance and fatty liver (8,9).
These studies suggested that the Notch pathway is active
through adulthood in rodent liver, is inappropriately
stimulated by obesity, and may be manipulated to reduce
the obesity-related metabolic disease burden.
On the basis of these rodent studies, we hypothesized
that Notch signaling is similarly functional and may cor-
relate with disease severity in patients with hepatic insulin
resistance an d NAFLD. In this study, we show that Notch
proteins and ligands are expressed in lean and obese
subjects and that increased activation of this pathway, as
assessed by expression of Notch target genes of the HES/
HEY family, positively correlates with gluconeogenic gene
expression and hyperglycemia in a cohort of morbidly
obese patients undergoing bariatric surgery. In a validation
cohort across a range of BMIs, we confirmed the positive
association between HES/HEY family genes and insulin
resistance as well as demonstrated an independent pos i-
tive association with hepatic fat content. Finally, we show
that hepatic Notch signal activation correl ates better with
measures of liver inflammation than with simple steatosis
(SS), suggestin g that it may represent a marker of the
transition from SS to NASH. This work establishes that
Notch signaling correlates and potentially represents
a novel therapeutic target for both arms of obesity-related
liver disease, T2D and NAFLD.
RESEARCH DESIGN AND METHODS
Subjects. The study conforms to the ethical guidelines of the 1975 Declaration
of Helsinki and was approved by the institutional review board and ethical
committee of the Fondazione Istituto di Ricovero e Cura a Carattere Scientifico
(IRCCS) Ca’ Granda. Each subject gave written informed consent. Demographic
and anthropo met ric feat ures, ar teria l blood pr essu re, med ical hi story, an d
medication s were r ecorde d for a ll patients and are summarized in Table 1.
A needle liver biopsy was performed in all patients. The specimen was
formalin-preserved for histological and immunofluorescence analysis. Part of
the sample was included in RNAlater (Ambion, Carlsbad, CA), immediately
frozen in liquid nitrogen, and stored at 280°C for RNA analysis.
Bariatric surgery clinic. We recruited 44 of 48 conse cutive patients who
underwent ba riatric surgery at the IRCCS Ca’ Granda Ospedale Policlinico d i
Milano, between 2006 and 2008. Indications for bariatric surgery included
BMI .40 kg/m
2
or BMI .35 kg/m
2
in the prese nce of metabolic complica -
tions (T2D, uncontro lled hypertensi on, severe dyslipidemia, obstructive
sleep apnea). We e xclude d subjects with alcohol consumption .30 g/day for
men and 20 g/day for wome n (n = 1) and chronic viral h epatitis (n =3).
Fasting glucose, HDL and total chole sterol, triglycerides, alanine amino-
transferase (ALT), and as partate aminotransferase (AST) were assessed the
day of surgery, and ne edle liver biopsy (16 -gaug e) was p erformed during
the bar iatric surgery. Insulin-resistance status was c lassi fie d according to
fasting glucose levels and oral glucose tolerance test results. These patients
are part of a previously reported c ohort in which we characterized insulin-
dependent signaling and the re gulation of lipid metabolism according to liver
histology (27).
Hepatology clinic patients. We recruited 38 unrelated patients followed up
at the Metabolic Liver Diseases outpatient service, Fondazione IRCCS Ca’
Granda, who underwent percutaneous liver biopsy because of suspected
NAFLD due to persistently abnormal liver enzymes/serum ferritin test results
or a history of steatosis associated with severe metabolic abnormalities, be-
tween January 2011 and January 2012. Other causes of liver disease were
excluded, including increased alcohol intake (.30 g/day for men or 20 g/day
for women), viral and autoimmune hepatitis, hereditary hemochromatosis,
and a1-antitrypsin deficiency. Fasting glucose and insulin levels, HDL and total
cholesterol, triglycerides, and ALT and AST levels were assessed the day of
the biopsy. Patients were classified as insulin-sensitive based on homeostasis
model assessment–insulin resis tanc e (HOMA-I R) ,2.5 ([fasting insulin
(mU/mL) 3 fasting glucose (mmol/L)]/22.5) (28) without a history of impaired
glucose tolerance or impaired fasting glucose, insulin resistance (HOMA-IR
.2.5, or impaired fasting glucose or impaired glucose tolerance, but no di-
agnosis of diabetes) or T2D.
Histological analysis. A single expert pathologist, unaware of gene expres -
sion and immunofluorescence data, evaluated all biopsy specimens according
to Kleiner et al. (29), based on the determination of the NAFLD activity score
(NAS) as the result of the sum of steatosis severity (0–3), intralobular necro-
inflammation (0–3), and hepatocellular ballooning (0–2). Steatosis percentage
was determined in at least 10 hepatic lobules per patient. Subjects were clas-
sified in three groups according to liver histology: histologically normal liver, SS,
and NASH.
Immunofluorescence. Paraffinized sections were deparaffinized, rehydrated,
and stained as previously described (30). To determine Notch signal activation,
tissues were stained with Hey1 (#5714, 1:100 dilution) or HeyL (#10094, 1:150
dilution) antibodies from Millipore and detected with donkey anti-rabbit Alexa
Fluor 488 (1:1,000 dilution) or donkey anti-mouse Alexa Fluor 594 (1:1,000)
from Invitrogen. Slides were mounted with Vectashield with DAPI (Vector
TABLE 1
Demographic and clinical features of subjects included in the study subdivided according to the case series (bariatric surgery and
hepatology clinic) and liver histology
Bariatric surgery Hepatology clinic
Normal SS NASH P value Normal SS NASH P value
n (%) 5 (12) 14 (33) 23 (55) 7 (18) 11 (29) 20 (53)
Sex (n) 0.035 0.23
Female 5 13 14 0 1 0
Male 0 1 9 7 10 20
Age (years) 44 6 4406 9426 10 0.73 45 6 12 54 6 9516 12 0.33
BMI (kg/m
2
) 40.8 6 10 40.5 6 10 43.5 6 8 0.54 25.1 6 2.4 27.5 6 3.4 28.7 6 4.2 0.11
Abdominal circumference (cm) —— ——94 6 6 102 6 6 112 6 21 0.05
Glucose (mg/dL) 83 6 8886 5 117 6 56 0.09 92 6 7 116 6 35 111 6 30 0.24
Insulin (IU/mL) —— ——8.4 6 2.2 14.1 6 1.9 16.0 6 1.5 0.034
HOMA-IR —— ——1.7 6 0.4 3.6 6 1.7 3.8 6 1.7 0.018
Glucose tolerance — 0.001
IS [n (%)] —— — 7 (100) 2 (18) 4 (20)
IR [n (%)] —— — 0 5 (46) 10 (50)
T2D [n (%)] 0 0 9 (39) 0 4 (36) 6 (30)
Cholesterol (mg/dL) 215 6 31 211 6 36 214 6 41 0.96 191 6 47 201 6 37 188 6 40 0.66
Triglyceride (mg/dL) 99 6 54 155 6 95 157 6 54 0.30 94 6 27 98 6 48 115 6 54 0.51
HDL (mg/dL) 73 6 11 52 6 9466
12 ,0.0001 55 6 15 57 6 28 44 6 14 0.17
Steatosis (%) 2 6 1176 13 61 6 21 ,0.0001 3 6 2196 14 52 6 23 ,0.0001
ALT (IU/mL) 18 6 5266 9396 25 0.03 22 6 8406 32 66 6 46 0.02
Data represent average 6 SD unless indicated otherwise. IR, insulin-resistant; IS, insulin-sensitive.
L. VALENTI AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 62, DECEMBER 2013 4053
Laboratories). Images were captured with a Nikon ECLIPSE E800 microscope
and a Nikon DXM 1200 digital camera and processed with Image ProPlus
software. Staining was quantitated in a blinded fashion from 1 (low expres-
sion) to 4 (high expression), independently by three investigators, and scores
were averaged.
Quantitative RT-PCR. We isolated RNA with Trizol (Invitrogen), synthesized
cDNA with Superscript III RT (Invitrogen), and performed quantitative PCR
with a DNA Engine Opticon 2 System (Bio-Rad) and GoTaq SYBR Green
(Promega). Absolute mRNA levels were determine for each gene using species-
and primer-specific standard curves, then normalized to 18S, and are presented
as relative transcript levels (fg/ng 18S). Primer sequences are available upon
request.
Luciferase assays. Hepa1c1c7 cells were transfected with the Rbp-Jk reporter
luciferase construct, as previously described (8), and then incubated in serum-
free medium with variable glucose content in the presence or absence of
10 nmol/L insulin or 10 nmol/L glucagon.
Hepatocyte studies. We isolated and cultured primary mouse hepatocytes as
described (8) and obtained human primary hepatocytes from Invitrogen. For
gene and protein expression studies, we treated hepatocytes with variable
concentrations of glucose, and/or 10 nmol/L insulin (Sigma-Aldrich) for 3 h,
with all experiments completed by 24 h after isolation.
Statistical analysis. Results are shown as mean 6 SEM. ANOVA was used for
comparison of means among groups. Gene expression levels were correlated
by the Pearson correlation. The association between HES1 expression (above
the median) and insulin resistance of T2D was evaluated by multivariate lo-
gistic regression analysis adjusted for age, steatosis, and BMI. Independent
predictors of HES1 expression were evaluated at multivariate regression
analysis (generalized linear model), including NAS and insulin levels, the
variables most significantly associated at univariate analysis. Differences were
considered significant at P , 0.05 (two-tailed).
RESULTS
Human liver has a functional, evolutionarily conserved
Notch-signaling apparatus. To clarify the role of
Notch signaling in the postdevelopment human liver,
we characterized liver Notchandligandexpressionin
insulin-sensitive subjects with a histologically normal
liver. We found that in these subjects, all four Notch
receptors were expressed, with relative higher lev els of
NOTCH1 and NOTCH2 (Fig. 1A, left panel). In addition, all
five Notch ligands were detectable by quantitative PCR,
with JAG1 and JAG2 showing greater expression than
Delta-like ligands. Of note, this expression pattern is
broadly similar to the distribution observed in murine liver
(Fig. 1A, right panel) as well as in mouse hepatocytes (8).
Generally, expression of NOTCH1 and other Notch genes
covaried (Fig. 1B)aswellaswithHES/HEY family target
genes (Fig. 1C), but showed no correlation with house-
keeping genes (ACTB, GAPDH)andanonsignificant trend
toward a negative correlation with Notch ligand expres-
sion (data not shown). These data show that Notch sig-
naling components are present in mature liver and that
relative expression is evolutionarily conserved.
Hepatic Notch signaling correlates with G6PC/PCK1
expression and is increased in pat ients with T2D.
Because Notch proteins and ligands are expressed in hu-
man liver, we hypothesized that Notch signaling may be
FIG. 1. Notch pathway in human liver. A: Notch receptor and ligand expression is similar in human (left) and mouse liver (right). Hepatic NOTCH1
expression positively correlates with the expression of other Notch receptors (P < 0.001 by ANOVA for all comparisons) (B) and canonical Notch
target HES1 (P < 0.001 by ANOVA for all comparisons) (C). Data show means 6 SEM. AU, arbitrary units.
NOTCH ACTIVATION IN DIABETES AND FATTY LIVER
4054 DIABETES, VOL. 62, DECEMBER 2013 diabetes.diabetesjournals.org
increased in obesity and insuli n resistance as in mouse
models. We performed a pilot study in 42 morbidly obese
patients undergoing liver biopsy at time of gastric banding.
Full demographic and clinical information is summarized
in Table 1, but most of the subjects were female (76%),
with SS or NASH (88%), and nondiabetic (79%). Expres-
sion of canonical Notch target genes of the HES/HEY
family positively correlated with hepatic expression of
G6PC and PC K1 (Table 2 a nd Fig. 2A). Of note, we
saw no correlation in severel y obese patients between
Notch signaling and age, sex, or BMI (data not shown),
which suggests that the correlations between HES/HEY
and G6PC/ PCK1 were in dependen t of overall adiposity,
but correlations persisted or even strengthened when
FIG. 1. Continued.
L. VALENTI AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 62, DECEMBER 2013 4055
diabetic patients were excluded from the analysis
(Table 2).
Liver consists primarily of hepatocytes, but also non-
hepatocyte residents, including endothelial, phagocytic
Kupffer, and stellate cells (31). The relative contribution of
hepatocytes to overall hepatic Notch signaling was unclear
from gene expression studies from the whole liver, so we
performed immunofluorescence for Notch targets on
a representative subset of patients across a range of gly-
cemic control. We observed predominantly hepatocyte
staining of Notch targets HEY1 and HEYL (Fig. 2B). HEY1
hepatocyte staining and HEY1 gene expression was very
well correlated (Fig. 2C), as was HEYL staining and HE YL
expression (not shown), suggesting he patic gene expres-
sion is a good surrogate for hepatocyte protein levels.
Further, HEY1 gene expression strongly correlated with
HES1, HEY1, and other Notch targets (F ig. 2D and data
not shown), allowing HEY1 staining as a surrog ate for
global hepatocyte Notch activation. Having validated
the technique, we next examined sections from age-
and BMI-matched normoglycemic and diabetic patients
and noted a marked increase in HEY1 and HEYL
staining in hyperglycemic patients (Fig. 2E and F and
data not shown ). These data establish that Notch sig-
naling is present in adult hepatocytes and increased in
T2D.
FIG. 2. Notch activity in liver increases with hyperglycemia. A: Correlation of Notch targets of the HES/HEY family with the expression of genes
controlling hepatic glucose production, G6PC and PCK1. B: Representative liver sections, stained with antibodies to HEY1 (green, left) or HEYL
(red, middle) and counterstained with DAPI (blue), with merged image (right). Hepatic HEY1 expression correlates with Hey1 staining (P < 0.001
by ANOVA) (C) and HES1 expression (P < 0.001 by ANOVA) (D). Immunofluorescence (E) and quantitation (F) of staining for HEY1 in formalin-
fixed liver sections from non-T2D and T2D patients undergoing liver biopsy during gastric bypass surgery. The very bright spots are auto-
fluorescent erythrocytes and are not included in the quantitation. Data show means 6 SEM. *P < 0.05 vs. non-T2D patients. AU, arbitrary units.
TABLE 2
Correlation of Notch gene targets with G6PC and PCK1 expression in 42 patients undergoing bariatric surgery
Overall series Patients without diabetes
G6PC PCK1 G6PC PCK1
Gene r Pearson r Pearson r Pearson r Pearson
HES1 +0.39 0.011 +0.47 0.0016 +0.44 0.006 +0.35 0.03
HES6 +0.24 NS +0.33 0.018 +0.35 0.03 +0.46 0.003
HES7 +0.27 NS +0.35 0.033 +0.51 0.007 +0.45 0.008
HEY1 +0.27 NS +0.42 0.002 +0.44 0.006 +0.41 0.01
NOTCH ACTIVATION IN DIABETES AND FATTY LIVER
4056 DIABETES, VOL. 62, DECEMBER 2013 diabetes.diabetesjournals.org
Hepatic Notch signaling is increased in insulin
resistance. The positive correlation of HES/HEY family
genes with G6PC/PCK1 in obese patients (Table 2), cou-
pled with increased HEY1 staining in hyperglycemia,
suggested that Notch s ignaling is i ncreased in insulin-
resistant li ver. To d etermine w hether hepat ic Notch
signaling was similarly induced in leaner patients and
m ight precede development of frank diabetes in insulin
FIG. 2. Continued.
TABLE 3
HES1 expression correlates independently with HOMA-IR as well as NAS scores
Correlation coefficient SE Unadjusted P value* Adjusted P value**
Age (per 10 years) 0.03 0.05 NS NA
BMI (kg/m
2
) 0.01 0.02 NS NA
Glucose (per 10 mg/dL increase) 0.00 0.02 NS NA
Insulin 0.02 0.01 0.002 0.03
ALT (per 10 IU/L increase) 0.07 0.02 0.003 NA
HOMA-IR 0.11 0.03 0.002 NA
% Steatosis (per 10% increase) 0.05 0.02 0.011 NA
NAS 0.11 0.03 ,0.0001 0.03
NA, not addressed. *At univariate analysis (generalized linear model). **At multivariate analysis (generalized linear model) including insulin
levels and NAS score, the strongest variables related to insulin resistance and histological damage, respectively, at univariate analysis.
L. VALENTI AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 62, DECEMBER 2013 4057
resistance, we analyzed specimens from 38 consecutive
outpatients undergoing percutaneous liver biopsy. Full
demographics can be found in Table 1, but this group was
97% male, with an average BMI in the overweight range
(25–30 kg/m
2
). In these patients, we again noted that HES/
HEY family genes correlated with G6PC and PCK1, as well
as with each other (data not shown), suggesting that Notch
and gluconeogenic gene expression coregulation is not
specific to obese patients. As in the bariatric surgery co-
hort, Notch signaling did not vary by age, BMI, or abdominal
circumference, but as predicted, we found a significant
positive correlation with Notch targets (HES1) and plasma
FIG. 3. Notch activity in hepatocytes increases with hyperglycemia. Notch-luciferase reporter (Csl-luc) expression in Hepa1c1c7 hepatoma cells
(A), and Notch target gene expression in primary hepatocytes from mice (B) or human donors (C) is increased with transient exposure to hy-
perglycemic (25 mmol/L glucose) conditions. Insulin reduces Notch activation in Hepa1c1c7 hepatoma cells when cultured in normoglycemic (D),
but not hyperglycemic conditions (E), even as glucagon has a synergistic effect. F: Insulin fails to repress Hes1 expression in hepatocytes derived
from mice lacking hepatic Notch signaling. G: Hyperglycemic-induced Notch reporter expression in Hepa1c1c7 hepatoma cells is abrogated by
transduction with N1-decoy, which blocks ligand-dependent Notch signaling. Data show means 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs.
5 mmol/L glucose or control (Cre- or GFP-transduced) cells. AU, arbitrary units.
NOTCH ACTIVATION IN DIABETES AND FATTY LIVER
4058 DIABETES, VOL. 62, DECEMBER 2013 diabetes.diabetesjournals.org
insulin levels (not shown) and HOMA-IR, independent of
confounding factors (Table 3), which was not mitigated by
exclusion of diabetic patients (data not shown). Multivari-
ate logistic regression analysis showed increased HES1
expression was positively associated with the presence of
insulin resistance of diabetes (odds ratio [OR] 2.11 [95% CI
1.5–38]), together with age (OR 1.17 [95% CI 1.05–1.40]) and
steatosis (OR 5.5 [95% CI 1.3–53]), independently of BMI.
These data establish that Notch activation is found in the
insulin-resistant liver, preceding frank hyperglycemia and
the development of diabetes.
Insulin and hyperglycemia directly and reciprocally
affect Notch signaling. To test the hypot hesis that
the apparent regulation of hepatic Notch signaling by
insulin resistance and hyperglycemia is direct and cell-
autonomous, we transfected hepatoma cells with a Notch-
reporter luciferase construct and found increased Notch
activation in hyperglycemic as opposed to basal conditions
(Fig. 3A). Similarly, endogenous Notch target expression
was higher in primary mouse or human hepatocytes tran-
siently exposed to hyperglycemia (Fig. 3B and C). Insulin
treatment had the opposite effect on Notch signaling, with
insulin-treated cells showing decreased reporter activation
(Fig. 3D). Interestingly, the inhibitory effect of insulin, but
not a synergistic effect of glucagon, was lost in cells
chronically cultured in hyperglycemic conditions (Fig. 3E),
Further, insulin was no longer able to repress Hes1 ex-
pression in primary hepatocytes derived from mice lacking
Rbp-Jk (8), the common transcriptional effector of Notch1–4
signaling (Fig. 3F). Similarly, pharmacologic application
of a novel Notch antagonist, an ectodomain “decoy” that
quenches ligand-dependent Notch signaling (24), abrogated
hyperglycemia-induced Notch-reporter activity (Fig. 3G). In
sum, these data suggest a cell-autonomous, dynamic regu-
lation of hepatic Notch signaling by metabolic stimuli.
Hepatic Notch signaling is positively correlated with
hepatic steatosis and inflammation. Beyond the posi-
tive correlation between HES/HEY gene expression and
measures of insulin resistance and hyperglycemia, we
predicted that Notc h signaling would correlate with he-
patic lipid content and markers of necroinflammation. In-
deed, when patients are subdivided by liver histology, we
find that HES1 and other Notch target genes are strongly
upregulated across the spectrum from normal liver to SS
to NASH, and further still in insulin resistance (Fig. 4A)or
in patients with T2D (Fig. 4B). Notch target staining was
similarly increased in hepatocytes of NASH patients com-
pared with SS pa tients (Fig. 4C).
In fact, HES1 expression more closely correlated to
measures of hepatic inflammation than to steatosis, with
stronger coefficients of correlation with NAS and ALT
levels than with the percentage of steatosis (Fig. 5A–C and
Table 3). As such, patients with a NAS score of 0–2, cor-
relating with a low risk of NASH (29,32), had lower Notch,
ligand, and HES/HEY family gene expression than those
with scores of 3 or higher (Fig. 5D–F). Furthermore, the NAS
score was associated with HES1 expression independently of
insulin resistance (Table 3). In sum, these data suggest that
hepatic Notch signaling is elevated in insulin resistance as
well as in NASH .
FIG. 4. Notch-dependent gene expression is progressively increased in insulin resistance and NAFLD severity. Quantitative PCR for HES1 and
other Notch target genes from liver biopsy samples from patients with pathologically confirmed SS, NASH, or normal liver, further subdivided as insulin-
sensitive (HOMA-IR <2.5) vs. insulin-resistant (HOMA-IR >2.5) (A), or T2D patients (B), are shown. C: Hepatocyte Notch target expression is in-
creased in patients with NASH. Scale bars are 50 mm long. Data show means 6 SEM. *P < 0.05 vs. T2D/SS; ***P < 0.001 by ANOVA. AU, arbitrary units.
L. VALENTI AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 62, DECEMBER 2013 4059
DISCUSSION
Notch signaling has been extensively studied in the con-
text of differentiation or cancer (10), but its metabolic
functions are novel. Rodent studies have demonstrated
a postdevelopment role for Notch in the regulation of
obesity-induced insulin resistance/diabetes (8,26) and in
liver fat accumulation (9), but whether this would translate
to human disease was unclear. This work answers two
important questions: hepatic Notch signaling is 1) present
in human liver/hepatocytes, and 2) its activation is tied
FIG. 5. Notch activity correlates with hepatocyte necroinflammation. Liver HES1 expression was plotted against NAS (A), serum ALT level (B),
and percentage of hepatic steatosis in patients undergoing percutaneous liver biopsy (C). Notch target (D), protein (E), and ligand gene expression
(F) are increased in liver from patients with higher NAS (NAS 3+) compared with patients at low risk for steatohepatitis (NAS 0–2). Data show
means 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NAS 0–2. AU, arbitrary units.
NOTCH ACTIVATION IN DIABETES AND FATTY LIVER
4060 DIABETES, VOL. 62, DECEMBER 2013 diabetes.diabetesjournals.org
to the metabolic state of the organism, with an inde-
pendent positive correlation with measures of insulin
resistance and hepatic steatosis/inflammation. Notch sig-
naling appears to be inappropriately reactivated in the
insulin-resistant liver, reprising its developmental role and
reassociating with its molecular partners from differentia-
tion that drives the metabolic effects of Notch signaling. For
instance, the Notch transcriptional effector Rbp-Jk binds to
and activates FoxO1 (33), a key transcriptional activator of
hormone-stimulated hepatic glucose production (2,34), in-
creasing functional hepatic insulin resistance (8). Similarly,
Notch signaling activates the nutrient-sensitive mTorc1
pathway in the liver, increasing de novo lipogenesis and
hepatic triglyceride (9). Mouse models with reduced hepatic
glucose production often have compensatory fatty liver due
to reoriented carbon flux; for instance, FoxO-knockout
mice (35,36) or mice treated with glucokinase activators
(37). Reduced Notch action allows the dissociation of
insulin-signaling pathways in the liver, redressing insulin
resistance without causing undue nutrient sensitivity, al-
lowing for this rare dual therapeutic benefit independent of
effects on body weight or adiposity.
It is intriguing that Notch activation more strongly cor-
relates with markers of steatohepatitis, including the NAS
and ALT levels, than with steatosis itself. With better im-
aging techniques (38), clinicians can increasingly di-
agnose excess hepatic fat (5). NAFLD is extraordinarily
common, with a prevalence approaching 30% in many
populations (39), leaving a therapeutic dilemma because
only a subset of patients progress to NASH (40). As such,
increased Notch signaling may represent a biomarker or
even a causative factor for progression from SS to NASH. It
is noteworthy that Notch pathway activation has similarly
been found in human hepatocellular carcinoma (41) and
causes hepatic fibrosis and tumor formation in mice (41–43),
which potentially explains the predisposition of patients
with NASH to develop cirrhosis and hepatocellular carci -
noma (44,45).
One of the major limitations of this type of observational
study is that we are able to detect Notch signaling, as well
as markers of insuli n sensitivity and hepatic fat, at one
moment in time. Longitudinal studies (46) are required to
determine whether altered Notch signaling predates or
predicts the development of worsening steatosis or pro-
gression to NASH and hepatocellular carcinoma. Similarly,
whether increased Notch signaling precedes developm ent
of insulin resistance or heralds the transition to frank di-
abetes is unknown. In addition, it would be of interest to
know whethe r interventions to reduce insulin resistance,
or steatosis and associated inflammation (47,48), also re-
duce hepatic Notch signaling. Finally, whether higher
Notch signaling seen in NASH patients reflects both he-
patocyte and nonhepatocyte contribution requires clarifi-
cation, because this may shed light on how Notch signals
are transduced. These future studies will inform the
question whether Notch may be a therapeutic target for
insulin-resistance/diabetes or NASH.
It is premature, given these lingering questions and po-
tential safety concerns, to propose a clinical trial with the
use of Notch inhibitors, which are already in advanced
clinical development for cancer (49,50), for treatmen t of
T2D or NASH. This temptation exists, however, in the
current era of pandemic obesity. Our data suggest the
possibility of alternative uses for these existing therapeu-
tics to combat the various faces of obesity-related meta-
bolic disease in the 21st century.
ACKNOWLEDGMENTS
The work was partly funded by National Institutes of
Health grants DK-093604 (U.B.P.) and 5R01-CA136673
(C.J.S.), a Columbia Diabetes and Endocrinology Research
Center Pilot and Feasibility Grant (U.B.P.), the Edward
Mallinckrodt, Jr., Foundation Grant (U.B.P.), and Ricerca
Corrente Fondazione IRCCS Ca’ Granda, Milano (L.V.).
No potential conflicts of interest relevant to this article
were reported.
L.V. and C.J.S. analyzed data and wrote the manuscript.
R.M.M. and C.K. designed and performed experiments and
analyzed data. R.R. collected and managed biological
samples for RNA analysis. M.M. selected and collected
histological samples and reviewed histological samples.
U.B.P. designed and performed experiments, analyzed data,
and wrot e the manuscript. U.B.P. is the g uarantor of this
work and, as such, had full access to all of the data in the
study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
The authors thank Domenico Accili and members of the
Accili Laboratory at Columbia University, as well as mem-
bers of the Metabolic Liver Diseases Laboratory at the
University of Milan , for insightful discussion of the data,
and Robin Goland at Columbia University for assistance in
study design and implementation. The authors acknowl-
edge excellent technical support from Jessie Lee and Taylor
Lu at Columbia University, and Rosa Lombardi, Anna
Ludovica Fracanzani, Enrico Mozzi, and Silvia Fargion at
the University of Milan for colle ction of clinical data and
biological samples.
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