Increased Plasma Proneurotensin Levels Identify NAFLD in Adults With and Without Type 2 Diabetes
J Clin Endocrinol Metab, June
Increased Plasma Proneurotensin Levels Identify NAFLD in Adults With and Without Type 2 Diabetes
Ilaria Barchetta 0
Flavia Agata Cimini 0
Frida Leonetti 0
Danila Capoccia 0
Claudio Di Cristofano 1
Gianfranco Silecchia 1
Marju Orho-Melander 2
Olle Melander 2
Maria Gisella Cavallo 0
0 Department of Experimental Medicine, Sapienza University of Rome , 00161 Rome , Italy
1 Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome , 00161 Rome , Italy
2 Department of Clinical Sciences, Lund University , 20502 Malm o ? , Sweden
Context: Neurotensin (NT), an intestinal peptide released by fat ingestion, promotes lipid absorption; higher circulating NT levels are associated with type 2 diabetes (T2D), obesity, and cardiovascular disease. Whether NT is related to nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) has not been fully investigated. Objective: To study the relationship between plasma proneurotensin 1 to 117 (pro-NT), a stable fragment of the NT precursor hormone, and the presence/severity of NAFLD/NASH and to unravel correlates of increased pro-NT levels. Design/Setting/Participants: For this cross-sectional study, 60 obese individuals undergoing bariatric surgery for clinical purposes were recruited. The association between pro-NT and NAFLD was further investigated in 260 consecutive subjects referred to our outpatient clinics for metabolic evaluations, including liver ultrasonography. The study population underwent complete metabolic characterization; in the obese cohort, liver biopsies were performed during surgery. Main Outcome Measures: Plasma pro-NT levels in relation to NAFLD/NASH. Results: Obese subjects with biopsy-proven NAFLD (53%) had significantly higher plasma pro-NT than those without NAFLD (183.6 6 81.4 vs 86.7 6 56.8 pmol/L, P , 0.001). Greater pro-NT correlated with NAFLD presence (P , 0.001) and severity (P , 0.001), age, female sex, insulin resistance, and T2D. Higher pro-NT predicted NAFLD with an area under receiver operating characteristic curve of 0.836 [95% confidence interval (CI), 0.73 to 0.94; P , 0.001]. Belonging to the highest pro-NT quartile correlated with increased NAFLD risk (odds ratio, 2.62; 95% CI, 1.08 to 6.40) after adjustment for confounders. The association between higher pro-NT and NAFLD was confirmed in the second cohort independently from confounders. Conclusions: Increased plasma pro-NT levels identify the presence/severity of NAFLD; in dysmetabolic individuals, NT may specifically promote hepatic fat accumulation through mechanisms likely related to increased insulin resistance. (J Clin Endocrinol Metab 103: 2253-2260, 2018)
Nlogical condition characterized by the macrovesicular
onalcoholic fatty liver disease (NAFLD) is a
pathoaccumulation of triglycerides within hepatocytes (hepatic
steatosis); in a number of cases, necroinflammatory activity
and fibrosis coexist [nonalcoholic steatohepatitis (NASH)];
furthermore, cirrhosis and liver failure may occur in 20%
to 25% of affected individuals (
). Nowadays, NAFLD
represents the most common cause of chronic liver disease
in developed countries (3), being detectable in 20% to 30%
of the general population (
) in almost 75% of patients
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body
mass index; MS, metabolic syndrome; NAFLD, nonalcoholic fatty liver disease; NAS,
nonalcoholic fatty liver disease activity score; NASH, nonalcoholic steatohepatitis; NT,
neurotensin; pro-NT, proneurotensin 1 to 117; T2D, type 2 diabetes; US, ultrasonography.
with type 2 diabetes (T2D) (
) and in up to 90% of obese
individuals with T2D (
). In dysmetabolic conditions,
NAFLD worsens inflammatory and metabolic outcomes
) and is associated with a greater prevalence and
severity of microvascular and macrovascular
complications in patients with T2D (
). Indeed, NAFLD is
universally recognized as an independent risk factor for
cardiovascular mortality (16). Nowadays, despite the
impressively high number of pharmacological interventions
proposed, the identification of an effective therapy of
NAFLD beyond standard lifestyle measures is still an open
issue and represents a major challenge (
Neurotensin (NT), a 13?amino acid peptide mainly
secreted by neuroendocrine cells in the small intestine
), displays an important role in regulating food
ingestion and fat absorption (
). By doing so, NT
influences energy balance and body weight (
). NT mainly
acts as a neurotransmitter in the central nervous system
and as a hormone in the periphery, exerting its
physiological action by binding the specific NT receptors,
NTSR1, NTSR12, and NTSR13 (
evidence has shown that the NT/NTSR1 system is
involved in adaptive energy balance (
). Loss of the
leptin action mediated by NT neurons coexpressing the
long form of the leptin receptor determines overweight
and impairs the ability to appropriately respond to energy
deprivation in experimental mice (24), pointing out a
crucial role of NT in mediating, among others, leptin
) and ghrelin (
) pathways. Indeed, the
leptinmediated systems regulating appetite are controlled by
NT-expressing neurons (
). In the periphery, NT
influences body weight by controlling macronutrient
absorption. Physiology studies have described an acute
increase of intestinal NT release immediately after food
ingestion, directly associated with meal fat content (
In addition, several data have been produced on the
role of NT in facilitating lipid digestion and fat
absorption in the small intestine (
). The refined,
complex control of energy balance exerted by NT at
different levels provides a possible pathophysiological
explanation about a correlation between its circulating
levels and increased prevalence and incidence of
obesityrelated diseases (
). In particular, within the large
cohort of the Malm o? Diet and Cancer Study (30), the
fasting concentration of pro-NT, the circulating
peptide secreted at equimolar levels to NT, was associated
with the incidence of T2D, cardiovascular disease, breast
cancer, and total and cardiovascular mortality (
association between pro-NT and incident major
cardiovascular events has been confirmed in the
Framingham Heart Study Offspring cohort, independently of the
presence of traditional cardiovascular risk factors (
Very recently, an extensive investigation on a putative
causal role of NT in determining aberrant fat
accumulation and metabolic diseases has been carried out (
showing reduced intestinal fat absorption, along with
protection from obesity and NAFLD, in NT-deficient
mice fed with a high-fat diet. Furthermore, the same
study demonstrated that in humans, higher plasma
proNT levels were associated with features of insulin
resistance and doubled the risk of developing obesity later
in life in nonobese individuals.
Despite the strong rationale behind and encouraging
evidence from animal models, little is known on
circulating pro-NT levels and NAFLD/NASH in humans.
Therefore, aims of this study were to investigate the
relationship between plasma NT concentration and the
presence and severity of NAFLD/NASH in adult obese
individuals with or without T2D and to determine clinical
correlates of impaired NT levels in this population.
Materials and Methods
For these purposes, we recruited 60 consecutive obese
candidates for bariatric surgery referring to the endocrinology
and diabetes outpatient clinics of Sapienza University of Rome
for preoperative evaluations. The presence of an association
between circulating pro-NT levels and NAFLD was further
explored in a cohort of individuals (n = 260) referring to the
same outpatient clinics for metabolic evaluations, including
upper abdomen ultrasonography (US) for assessing the presence
of fatty liver. To be eligible for the study, all study participants
had to fulfill the following criteria: male and female aged
between 20 and 65 years; no history of current or past excessive
alcohol drinking, as defined by an average daily consumption of
alcohol .30 g/d in men and .20 g/d in women; negative tests
for the presence of hepatitis B surface antigen and antibody to
hepatitis C virus; absence of history and findings consistent with
cirrhosis and other causes of liver diseases (autoimmune
hepatitis, hemochromatosis, Wilson disease); and no treatment with
drugs known to cause liver steatosis (e.g., corticosteroids,
estrogens, methotrexate, tetracycline, calcium channel blockers,
or amiodarone). Furthermore, patients belonging to the
morbidly obese cohort had a clinical indication for bariatric surgery.
All participants underwent a complete workup, including
medical history collection, clinical examination,
anthropometric measurements, and laboratory tests.
Clinical and laboratory assessment
Weight and height were measured with patients wearing
light clothing and no shoes. Body mass index (BMI, kg/m2) was
calculated as weight in kilograms divided by height in meters
squared. Waist circumference was measured midway between
the 12th rib and the iliac crest. Systemic systolic blood pressure
and diastolic blood pressure were measured after 5 minutes of
rest; three measurements were taken, and the average of the
second and third measurements was recorded and used in the
analyses. Individuals without a previously formulated diagnosis
of diabetes mellitus underwent a standard oral glucose
tolerance test measuring blood glucose and insulin at baseline and
30, 60, 90, and 120 minutes after glucose ingestion. A 12-hour
overnight fasting blood sample was obtained before surgery for
metabolic profiling. Fasting blood glucose (mg/dL),
glycosylated hemoglobin (%, mmol/mol), total cholesterol (mg/dL),
high-density lipoprotein cholesterol (mg/dL), triglycerides
(mg/dL), aspartate aminotransferase (AST, IU/L), alanine
aminotransferase (ALT, IU/L), and creatinine (mg/dL) were
measured by centralized standard methods. Fasting blood insulin
(IU/mL) was measured by radioimmunoassay (ADVIA Insulin
Ready Pack 100; Bayer Diagnostics, Milan, Italy), with intra-assay
and interassay coefficients of variation ,5%. Low-density
lipoprotein cholesterol value was obtained the Friedewald formula.
For our purposes, we measured the circulating concentration
of pro-NT in plasma frozen immediately after separation and
stored at 280?C. Pro-NT was measured using a
chemiluminometric sandwich immunoassay to detect pro-NT amino acids 1 to
117 as described previously (
). The analytical assay sensitivity
(mean relative light units of 10 determinations of sheep serum
plus two standard deviations) was 4.8 pmol proNT/L. The
interassay (10 assay runs) coefficient of variability was 6.2% at
48 pmol proNT/L and 4.1% at 191 pmol/L. Recovery and
dilution was .85% in a measurement range of 25 to 850 pmol/L.
The homeostasis model assessment of insulin resistance and
homeostasis model assessment of insulin secretion were
calculated as previously described (
). Diabetes mellitus was
defined according to the American Diabetes Association 2009
) and metabolic syndrome (MS) by the modified
National Cholesterol Education Program Adult Treatment
Panel III criteria (
Liver biopsy and histology
Study patients underwent intraoperative liver biopsy during
surgery for sleeve gastrectomy. All procedures were conducted
in accordance with recommendations set by the American
Association for the Study of Liver Diseases (
). Liver fragments
were fixed in buffered formalin for 2 to 4 hours and embedded
in paraffin, and sections were cut and stained with hematoxylin
and eosin and Masson trichrome stains. A single pathologist
(C.D.C.) blinded to patients? medical history and biochemistry
performed the overall histological evaluations. A minimum
biopsy length of 15 mm or the presence of 10 complete portal
tracts was required (
). Liver biopsy samples were classified
based on the presence of NASH by Brunt et al. (
) and graded
according to the NAFLD activity score (NAS) (
was quantified on the basis of the NASH Clinical Research
Network Scoring System Definition (
NAFLD assessment in individuals not candidates for surgery
In individuals who were not candidates for surgery, NAFLD
was evaluated through liver US. This was performed using an
Esaote (Genoa, Italy) instrument with a convex 3.5-MHz probe
by the same operator blinded to laboratory values. Liver
steatosis was defined according to Saverymuttu et al. (
) on the
basis of abnormally intense, high-level echoes arising from the
hepatic parenchyma, liver-kidney difference in echo amplitude,
echo penetration into the deep portion of the liver, and clarity of
liver blood vessel structure.
SPSS version 23 was used to perform statistical analyses.
Continuous variables are reported as the mean 6 standard
deviation, and categorical variables are reported as percentages.
The Student t test for continuous variables and x2 test for
categorical variables were used to compare mean values
between two independent groups; skewed variables underwent
natural logarithmic transformations before performing the
analyses. Correlations between parameters were explored by
Pearson (continuous variables) or Spearman (categorical
variables) coefficients or by age-, sex-, and BMI-adjusted partial
correlations. Histological parameters are expressed by ordinal
scales for NAS and Steatosis Activity Fibrosis score (
was used as a continuous scale for activity assessment, and
comparisons between more than two were obtained by a
Bonferroni-adjusted analysis of variance test. The predictive
value of plasma pro-NT for NAFLD identification was
estimated by the area under the receiver operating characteristic
curve, with a 95% confidence interval (CI). Multivariate logistic
regression models were built to identify determinants of NAFLD
(yes/no, dependent variable) in our study population, entering
all the variables significantly associated with the bivariate
analyses as covariates. Data are shown as mean 6 standard
deviation. For all the above, a two-tailed P value ,0.05 was
To our knowledge, no study has investigated circulating
pro-NT levels in relation to NAFLD. Therefore, to confirm
the statistical power of this study, we performed a post hoc
sample size calculation considering the mean pro-NT
concentration in individuals with and without NAFLD, and we
calculated that 15 patients per subgroup would be enough to
reach the statistical significance with power = 90% and
a error = 0.05. For all the above, a P value ,0.05 was
The study protocol was reviewed and approved by the
Ethics Committee of Policlinico Umberto I, Sapienza
University of Rome and conducted in conformance with the
Helsinki Declaration. Written consent was obtained from all
patients before the study.
Pro-NT and biopsy-proven NAFLD/NASH
Within our study population, 32 of 60 patients (53%)
had histological features of NAFLD; clinical and
biochemical characteristics of the study population in
relation to the presence of NAFLD are shown in Table 1,
along with results from age-, sex-, and BMI-adjusted
partial correlation analyses.
Plasma pro-NT levels were significantly higher in
patients with NAFLD than in those without NAFLD
(183.6 6 81.4 vs 86.7 6 56.8 pmol/L, P , 0.001;
Fig. 1A) and directly correlated with the diagnosis of
NASH, severity of steatosis, intrahepatocyte ballooning,
and, subsequently, higher NAS and Steatosis Activity
Fibrosis score, as shown in Table 2. In particular,
proNT levels were significantly higher throughout
increasing NAS severity subgroups (P , 0.001; Fig. 1B),
and this association was strongly significant after
correcting for sex and age in the partial correlation analysis
(r = 0.62, P , 0.001). Among clinical parameters,
Values are presented as mean 6 standard deviation unless otherwise indicated. DBP, diastolic blood pressure; FBG, fasting blood glucose; FBI, fasting
blood insulin; HbA1c, glycosylated hemoglobin; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, insulin resistance; HOMA-b, homeostasis model
assessment of insulin secretion; LDL-C, low-density lipoprotein cholesterol; NS, not significant; SBP, systolic blood pressure.
aStudent t test. P values refer to the age-, sex-, and BMI-adjusted partial correlation analyses unless differently specified.
greater pro-NT levels correlated with age, female sex,
T2D, and parameters associated with insulin resistance
and impaired glucose metabolism, as detailed in Table 2.
In contrast, no association was found for BMI and waist
The presence of biopsy-proven NAFLD was
associated with female sex (r = 0.31, P = 0.02), higher pro-NT
(r = 0.56, P , 0.001), higher ALT (r = 0.30, P = 0.03),
and lower AST/ALT (r = -0.37, P = 0.009). In the
multivariate logistic regression analysis, higher pro-NT
levels were associated with biopsy-proven NAFLD
independently from possible confounders (Table 3).
Higher pro-NT concentration predicts the presence of
NAFLD, with an area under the receiver operating
characteristic curve of 0.836 (95% CI, 0.73 to 0.94, P ,
0.001; Fig. 2).
Pro-NT and US-detected NAFLD
Of the 260 consecutive individuals undergoing
metabolic characterization and liver US, 60% (n = 157) had a
diagnosis of NAFLD; patients with NAFLD had significantly
higher plasma pro-NT levels than non-NAFLD individuals
(190.78 6 116.6 vs 154.3 6 88.9 pmol/L, P = 0.003). Clinical
and metabolic characteristics of this study cohort, according
to the presence of NAFLD, are shown in Supplemental Data
1. In the bivariate analyses, greater pro-NT levels
correlated with the presence of NAFLD (r = 0.19, P = 0.002), T2D
(r = 0.25, P = 0.001), and female sex (r = 0.15, P = 0.05),
whereas a trend toward a positive association that did
not reach statistical significance was observed between
higher pro-NT levels and the number of MS components
(r = 0.11, P = 0.08). No association was found between
pro-NT, age, and indexes of body adiposity, such as
BMI and waist circumference (Supplemental Data 2).
The multivariate logistic regression analysis confirmed
that higher pro-NT correlated with the presence of
NAFLD independently from age, sex, presence of T2D,
and number of MS components (Supplemental Data 3).
This study demonstrates the existence of an association
between circulating pro-NT levels and the presence and
severity of biopsy-proven NAFLD and NASH in obese
adults. The relationship between higher pro-NT and
NAFLD was confirmed in a larger population of adults
with a diagnosis of fatty liver made with US
examination but without signs of severe liver damage, thus
reinforcing the evidence obtained in patients evaluated
with liver histology.
Recently Li et al. (
), in an extensive investigation on
mechanisms behind the association between higher
proNT and the development of obesity and cardiometabolic
), found significantly reduced intestinal
fat absorption in NT-deficient mice and protection
toward high-fat diet-induced obesity, hepatic steatosis, and
insulin resistance in comparison with wild-type mice
(29). As NT-deficient mice (
), NTR3-deficient mice are
protected from high-fat diet-induced obesity and fatty
), indicating that NT-induced hepatic fat
accumulation is mediated by both NTR1 and NTR3. In our
study, higher pro-NT correlated with T2D and signatures
of impaired glucose metabolism and insulin resistance,
but not with adiposity per se, in line with previous reports
). NAFLD represents an established
cardiovascular risk factor (16) and may determine and worsen
insulin resistance, systemic inflammation (
metabolic complications of obesity (
). Indeed, we
observed a linear association between pro-NT, hepatic
damage in NASH, and parameters related to glucose
A possible weakness of this novel observation can be
represented by the limited sample size of the cohort
undergoing liver biopsy. However, obtaining samples for
Multivariate Logistic Regression Analysis
Pro-NT Bivariate Correlation Analyses
liver histology implies the use of invasive procedures,
reasonably representing per se a limiting factor for study
On the other hand, all the study participants
underwent accurate metabolic characterization; the study
was monocentric, and all the procedures were performed
by the same operator, strengthening the study design and
the reliability of our results. Finally, the findings obtained
in the main study population have been confirmed in an
additional cohort undergoing hepatic US and metabolic
phenotyping, reinforcing our results and making them
applicable also in individuals with different degrees of
body adiposity and nonclinically relevant hepatic
damage. Indeed, the association between plasma pro-NT and
different measurements of NAFLD broadens the clinical
utility of our findings.
Although the cross-sectional design of our study
does not allow us to establish a causal nexus between
these findings, it is plausible to hypothesize that
increased pro-NT levels facilitate the absorption of fatty
acids from the small intestine, promoting fat
accumulation in specific sites, such as the liver. Thus, NT may
lead to NAFLD/NASH in a dose-dependent manner and
may act both directly and indirectly?through
hepatic fat accumulation?in worsening insulin resistance
and the metabolic profile. Gut hormone regulation is
currently considered an appealing target for
antiobesity treatment (
); in this context, our findings
are intriguing and may provide the basis for
further investigation on novel therapeutic approaches to
NAFLD. Moreover, pro-NT may represent a novel
biomarker of NAFLD in individuals with and
without obesity, with relevant implications in clinical
practice. In conclusion, our study demonstrates for the
first time, to our knowledge, that pro-NT levels predict
the presence of biopsy-proven NAFLD in obese
individuals and are associated with insulin resistance
and a detrimental metabolic profile. Studies on larger
cohorts and longitudinal designs are warranted to
investigate the possible role of NT in the development,
progression, and prognosis of NAFLD and NASH.
Financial Support: This study was founded by grants from
Sapienza University (to M.G.C.).
Author Contributions: I.B., O.M., M.O.-M., and M.G.C.
conceived the study. I.B., M.G.C., F.L., D.C., and F.A.C.
coordinated the study, oversaw patient recruitment, and
finalized the data set. F.A.C., I.B., and D.C. oversaw collection
and analysis of biological samples. M.O.-M. and O.M.
performed the experiments. I.B., M.C.G., and O.M. performed the
statistical analyses. G.S. performed all the liver biopsies. C.D.C.
read all the biopsies and finalized the data set. and I.B. and
M.G.C. drafted the paper, which was reviewed by all authors.
All authors read and approved the final manuscript.
Correspondence and Reprint Requests: Maria Gisella
Cavallo, MD, PhD, Policlinico Umberto I, Sapienza University,
Viale Regina Elena 324, 00161 Rome, Italy. E-mail: gisella.
Disclosure Summary: The authors have nothing to
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