SCIENCE ADVANCES | RESEARCH ARTICLE

IMMUNOLOGY Copyright © 2021

The Authors, some
Adipose saturation reduces lipotoxic systemic rights reserved;
exclusive licensee
inflammation and explains the obesity paradox American Association
for the Advancement
of Science. No claim to
Biswajit Khatua1, Bara El-Kurdi1, Krutika Patel1, Christopher Rood2, Pawan Noel1, original U.S. Government
Michael Crowell1, Jordan R. Yaron1, Sergiy Kostenko1, Andre Guerra1, Douglas O. Faigel1, Works. Distributed
Mark Lowe3, Vijay P. Singh1* under a Creative
Commons Attribution
Obesity sometimes seems protective in disease. This obesity paradox is predominantly described in reports from NonCommercial
the Western Hemisphere during acute illnesses. Since adipose triglyceride composition corresponds to long-term License 4.0 (CC BY-NC).
dietary patterns, we performed a meta-analysis modeling the effect of obesity on severity of acute pancreatitis, in
the context of dietary patterns of the countries from which the studies originated. Increased severity was noted in
leaner populations with a higher proportion of unsaturated fat intake. In mice, greater hydrolysis of unsaturated
visceral triglyceride caused worse organ failure during pancreatitis, even when the mice were leaner than those
having saturated triglyceride. Saturation interfered with triglyceride’s interaction and lipolysis by pancreatic
triglyceride lipase, which mediates organ failure. Unsaturation increased fatty acid monomers in vivo and aqueous

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

media, resulting in greater lipotoxic cellular responses and organ failure. Therefore, visceral triglyceride saturation
reduces the ensuing lipotoxicity despite higher adiposity, thus explaining the obesity paradox.

INTRODUCTION We therefore approached the obesity paradox by examining vis-

While quantitative parameters such as body mass index (BMI) (1), waist
circumference, and amount of visceral adipose (2, 3) are well-studied
risk factors for acute disease severity, the impact of adipose compo-
sition on severity is unclear. Organ failure, the hallmark of acute
pancreatitis (AP) severity, has been associated with long-chain non-
esterified fatty acid (NEFA) lipotoxicity in humans (4–7). Clues to
the role of adipose composition in severity come from studies from
Western populations showing a BMI of >30 mostly, but not always
(2, 3) associated with severity, while in Eastern populations, much
lower BMIs, sometimes ≥23 (8, 9), are associated with severity. This
“obesity paradox” (10) also occurs in other acute scenarios such as burns
(11), acute heart failure (12), after trauma (13), cardiovascular surgery
(14), and during critical illnesses (15) in which elevated pancreatic
enzymes (16–18) and long-chain NEFA (19, 20) have been associated
with severity. More recent studies associate such an enzyme leak (21)
and dietary fat composition (22), along with elevated NEFA levels,
to the severity (23) and mortality from coronavirus disease 2019
(COVID-19) (22, 24). However, the mechanisms determining NEFA
generation and how NEFA mediate outcomes such as the cytokine
storm (24) and organ failure (19, 20, 22) are unclear.
Most individual studies supporting the obesity paradox come
from the Western Hemisphere (12–14), where dietary and adipose
fat are more saturated (25, 26). We therefore aimed at understanding
this problem in different populations, by modeling AP, wherein the
pancreatic lipases leak into the surrounding fat (6, 7, 27) and hydrolyze
the neutral lipids (7). Principal among these lipases is pancreatic
triglyceride lipase (PNLIP), which enters visceral adipocytes by various
mechanisms (7) and mediates lipotoxic systemic injury along with
organ failure (7, 28) via the generated long-chain NEFA (7, 28) inhib-
iting mitochondrial complexes I and V (6). However, the impact of
long-chain NEFA saturation on systemic inflammation is unknown.
1
Department of Medicine, Mayo Clinic, Scottsdale, AZ, USA. 2Saint Louis University
School of Medicine, Saint Louis, MO, USA. 3Department of Pediatrics, Washington
University School of Medicine, Saint Louis, MO, USA.
*Corresponding author. Email: singh.vijay@mayo.edu
ceral fat composition as influenced by dietary fat composition (29).
Epidemiologically, all clinical reports up to 2017 associating AP severity
with BMI were grouped by cutoff BMI, and the dietary fat composi-
tion representative of the country from which the study originated
was studied. The dietary fat data were based on publicly available
information archived by the Food and Agriculture Organization
(FAO) and were classified as saturated or unsaturated based on the
presence of double bonds in long-chain (>12 carbon) NEFA com-
posing the dietary triglycerides (described in Methods). After analyz-
ing the human metadata, we tested the plausibility of fat composition
affecting the outcomes of pancreatitis by altering mouse visceral fat
composition to replicate human visceral fat (25, 26, 29). Unexpectedly,
the human and experimental models cumulatively showed that the
U.S. Food and Drug Administration (FDA)–recommended higher
unsaturated fat intake (https://health.gov/dietaryguidelines/2015/
resources/2015-2020_Dietary_Guidelines.pdf; https://www.fda.gov/
files/food/published/Food-Labeling-Guide-%28PDF%29.pdf, appen-
dix F) increased the risk of severe AP (SAP). We therefore investigated
the mechanistic basis of this finding and noted that saturation of a
triglyceride makes its interaction with PNLIP energetically unfavor-
able and reduces its lipolysis. Moreover, in general, unsaturated NEFAs
achieved higher monomeric concentrations in vivo and aqueous
systems, consequently eliciting stronger biological responses and
causing worse injury and organ failure. These features may explain
how obese populations with higher visceral fat saturation may
sometimes be protected from severe disease.
RESULTS
Higher saturated fat intake is associated with SAP at
a higher BMI
As seen in Fig. 1A and fig. S1, 20 reports from 11 countries used a
BMI cutoff of ≥30 to stratify SAP. Six of these (blue countries and
text in table) reported no increased risk (2, 3, 30–34), while the 14
(golden text and green countries) reported an increased risk of SAP
at BMI of ≥30 (1, 6, 35–44). Six reports used a cut off BMI of 25 or

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 1 of 16

SCIENCE ADVANCES | RESEARCH ARTICLE

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

Fig. 1. Relationship of human SAP to cutoff BMI and composition of dietary fat intake. (A) Studies using a BMI cutoff of ≤25 are shown in pink, those associating SAP
with a BMI of ≥30 are shown in gold, and studies showing a BMI of >30 was not associated with severe AP (SAP) are show shown in blue. The respective countries from
which the study originated are shown in pink, green, and blue, with a checkered pattern for countries with studies showing different outcomes. Box plot comparing the
per capita per year saturated fat (from dairy, cattle, and palm oil) consumption in countries with BMI cutoffs of >30 and those with ≤25 BMI (B) and %UFA in dietary fat
intake (C). (D) Meta-analysis showing OR for SAP for studies using a BMI cutoff of ≤25 (pink background) and those using a BMI cutoff of >30 (blue-yellow background).
The overall OR is shown at the bottom (orange background).

less (8, 9, 45–49). Adult AP typically occurs after the third decade
(50, 51). Since the composition of visceral fat necrosed during AP
may be influenced by the dietary fat composition over the preced-
ing years (26), we analyzed dietary fat composition of different
countries shown in Fig. 1A. The per capita fat consumption was
calculated by averaging the yearly data (1970 to 2011) for each
country. Countries with a BMI cutoff of ≥30 had higher per capita
saturated fat consumption (Fig. 1B and fig. S1). While the amount
of unsaturated fat consumption was the same (fig. S3A), unsatu-
rated fat comprised a higher percentage of fat intake in the coun-
tries with reports having a cutoff BMI of ≤25 (pink countries;
Fig. 1, A and C). Overall, there was a moderate correlation between
the percentage of patients with SAP and the percentage of unsatu-
rated fat intake (fig. S3B). On meta-­analysis (Fig. 1D), a significantly
increased risk of severity was noted for cutoff BMIs of ≤25 [pink
shade, odds ratio (OR) 2.8, CI 1.3 to P = 0.008] and also BMI of >30
(blue and greens, OR 2.7, CI 1.8 to 3.8, P < 0.001). Publication bias
was not detected on the basis of a Funnel plot (fig. S1C), an Egger’s
regression, or a Begg and Mazumdar rank correlation. There were
no differences in age, sex distribution, or etiology of AP between the
two groups (fig. S1). While the effect of genes and comorbidities on
%SAP cannot be commented on in these data, meta-regression
showed that %unsaturated fatty acid (UFA) was able to explain 33%
of the heterogeneity in the rate of SAP, and neither age, AP etiology,
nor per capita gross domestic product (GDP) correlated with SAP.
Furthermore, while per capita GDP of countries averaged for 5 years
preceding publication was significantly lower in countries reporting
SAP at BMIs of ≤25 ($15,768 ± 14,624 versus $32,272 ± 17,007,
P = 0.018), neither mortality (5.2 ± 6.9 versus 6.7 ± 4.3%, P = 0.698)
nor the proportion of patients with SAP (24 ± 12.5 versus 20.7 ±
8.4%, P = 0.892) was different in the two BMI cutoff groups. Therefore,
despite differences in income, the proportion of SAP and mortality
were unchanged. This implied that quality of care was not differ-
ent in the two groups of countries. Overall, these studies supported

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 2 of 16

SCIENCE ADVANCES | RESEARCH ARTICLE

that severe pancreatitis occurred at a lower BMI in countries with a
lower dietary saturated fatty acid (SFA) intake. Since we are not aware
of the literature on how diet would affect the genetic background of
a population over several generations to alter the severity of pan-
creatitis, we therefore went on to experimentally study whether and
how altering visceral fat composition affected the development of
SAP in the commonly used caerulein (CER) model of AP, which, in
lean C57BL6 normal chow-fed mice, remains mild, self-limited, and
without organ failure (27).
Dietary unsaturated fat results in unsaturated visceral fat
and worsens AP more than higher amounts of saturated
visceral fat
We first aimed at recapitulating the human studies showing that
dietary fat composition can change adipose tissue composition in
an animal model (26). Linoleic acid (LA; C18:2) is an unsaturated
essential fatty acid (i.e., only available through diet) present at high
concentrations in common FDA-recommended dietary fats (52)
(e.g., cooking oils) and comprises a high proportion in pancreatic
fat necrosis (FN) (5, 6). Being essential, LA’s concentrations in
visceral fat parallel dietary intake (29). We thus formulated a diet
enriched in LA (70% LA in a 45% fat diet) to represent high %UFA
intake in dietary fat (red box in fig. S4A and Fig. 2A1, red columns)
(26, 52). Similarly, a diet enriched in saturated fat was formulated,
which had palmitic acid (PA; C16:0, PA 68% of a 47% fat diet; fig.
S4B, green rectangle) replicating the 65 to 70% saturation of dairy
fat. These diets spanned the range of %UFA intake in humans (16% in
Australia and 79% in Japan), as shown in fig. S3A.
Mice fed these diets had a similar food intake (6.5 ± 1.4 g/day of
UFA diet or 6.3 ± 0.5 g/day of SFA diet). This altered their visceral
triglyceride composition, with LA and PA increasing to 40 to 45%
(Fig. 2A1 and detailed in fig. S5) in the UFA- and SFA-fed groups,
respectively. We previously showed that a normal chow diet with
5% fat diet (Purina 5053), which contains ≥70% UFA and <20% SFA

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

Fig. 2. Dietary and adipose fat composition and parameters of local, systemic severity of CER pancreatitis in UFA- and SFA-fed ob/ob mice. (A1) Table comparing
the fatty acid composition of fat pad triglycerides (TG) and diets of mice given the SFA- and UFA-enriched diets. Body weights (A2), body fat (A3), and body fat as a per-
centage of body weight (A4) in the SFA- and UFA-fed mice. Serum amylase (B1) and lipase (B2) in control (CON) mice and after 24 hours of AP (CER). Local pancreatic in-
jury seen histologically (C1) and quantified as acinar necrosis (C2) as a percentage of total parenchymal area and percentage of acinar necrosis adjacent to the FN (shown
in yellow rectangles), termed as “% peri-fat acinar necrosis” (C3). Systemic injury measured as renal injury [TUNEL staining showing brown nuclei in (D1) and serum BUN
in (D2)], lung TUNEL positivity highlighted with arrows in (D3), and shown as number per high-power field (HPF) in (D4), along with shock (as measured by a drop in
carotid pulse distention) in (E1), serum calcium (E2), and survival curve (E3) of mice with CER AP. NS, not significant.

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 3 of 16

SCIENCE ADVANCES | RESEARCH ARTICLE

consistent with FDA recommendations, results in fat pad tri-
glyceride to be composed of 18 ± 6% PA and 31 ± 14% LA, with
35 ± 6% oleic acid (OA), i.e., C18:1 in ob/ob mice (7). On feeding
the special diets, the LA concentrations in the fat pads of the UFA-
and SFA-fed ob/ob mice correspondingly matched reports of those
in human adipose tissue from Japan [≈40% (25)] and the United
States [5% (26)]. By 8 to 14 weeks, the mice averaged 45.5 ± 0.5 g in
both groups. CER AP reduced survival to 10% in the UFA group by
day 3 versus 90% in the SFA group (P < 0.02; fig. S6). We then sim-
ulated the lower cutoff BMI (≤25) associated with SAP in countries
with a higher %UFA intake. AP was initiated in UFA-fed mice
weighing 20 to 30% less and having 35 to 40% less adipose tissue
(Fig. 2, A2 to A4). AP increased serum amylase and lipase similarly
in both groups (Fig. 2, B1 and B2) but was worse in the leaner UFA
group. The SFA group has lesser pancreatic necrosis (5.8 ± 0.8 versus
16.8 ± 4.7%, P = 0.024; Fig. 2, C1 and C2) especially bordering FN,
termed peri-fat acinar necrosis (2.7 ± 0.4 versus 6.9 ± 1.6%, P = 0.03;
green outline, Fig. 2, C1 and C3). Consistent with SAP (53, 54), the
deoxynucleotidyl transferase–mediated deoxyuridine triphosphate
nick end labeling) positivity, higher serum blood urea nitrogen
(BUN) levels (Fig. 2, D1 to D4), a greater decrease in carotid pulse
distention (consistent with shock), and SAP-associated hypocalcemia
(55, 56) (Fig. 2, E1 and E2), resulting in 20% survival (P < 0.02 versus
90% in the SFA group; Fig. 2E3). The findings of SAP were replicated
in UFA-fed C57BL6 mice with diet-induced obesity (DIO), but not
the normal chow-fed mice (30.8 ± 0.7 g), which had mild pancreatitis
(fig. S7, A to F) (27). However, we did not use DIO as the primary
model since the time to weight gain (>5 months) was prolonged.
Preferential lipolysis of unsaturated long-chain triglycerides
worsens inflammation and systemic injury
It has recently been shown that PNLIP mediates FN and severity of
AP (7). To understand the worse outcomes in UFA-fed mice, we
compared the necrosed gonadal fat pads in both groups (Fig. 3A).
Grossly, the SFA group has reduced FN; however, the pancreatic
amylase, lipase activity, and PNLIP protein (6- to 14-fold control)

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

UFA-fed mice had greater lung and renal tubular TUNEL (terminal had a large increase in both groups (Fig. 3, B1 and B2, and fig. S8A).

Fig. 3. Effect of visceral fat composition on its lipolysis and associated inflammatory response. (A) Gross appearance of the FN in SFA-fed (top) and UFA-fed mice
with pancreatitis. (B1) PNLIP, IKB-, and -tubulin (-Tub) amounts on Western blotting (B2) in the fat pads of SFA- or UFA-fed control (Con) mice or those with CER
pancreatitis (CER). Cytokine mRNA levels in fat pads (C1) shown as an increase over control levels or serum levels of IL-6 (C2), MCP-1 (C3), or TNF- (C4) in control mice or
those with CER pancreatitis after they had been fed with UFA- or SFA-enriched diets. Serum NEFA showing OA (D1), LA (D2), total serum UFA (C3), total serum NEFA (D4),
PA (D5), palmitoleic acid (D6), total serum SFA (D7), and serum SFA as a percentage of total NEFA (D8) in control mice or those with CER pancreatitis after they had been
fed with UFA- or SFA-enriched diets. Photo credit: Krutika Patel, Mayo Clinic, AZ.

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 4 of 16

SCIENCE ADVANCES | RESEARCH ARTICLE

The UFA mice had a larger cytokine mRNA increase in the fat pads
during AP and interleukin-6 (IL-6), monocyte chemoattractant
protein-1 (MCP-1), and tumor necrosis factor– (TNF-) proteins
in the sera (Fig. 3, C1 to C4), which correlated with lower IB-
(42 ± 15% of controls, P < 0.05) in the necrosed UFA fat pads
(Fig. 3B2).
Since NEFA mediates cytokine increase (7), we measured serum
NEFA to understand the underlying mechanism. The UFA group
with AP had higher serum NEFA increase, especially UFAs, includ-
ing C18:1 (OA) and C18:2 (LA) (Fig. 3, D1 to D4). This pattern has
been noted during SAP-induced organ failure (7, 28, 53, 54) in both
rodents (5, 6, 27) and humans (4). However, inexplicably, the in-
crease in C18:1, C18:2, and C16:1 (Fig. 3, D1, D2, and D6) was muted
in the SFA-fed mice, despite C18:1 being equal and C16:1 being
higher in the fat pads of the SFA group (Fig. 2A1 and fig. S5). Simi-
larly, C16:0 and SFA overall (Fig. 3, D5 and D7, and fig. S8B) in-
creased more in the UFA group, despite the SFA group having more
of these in the visceral triglyceride, serum at baseline. The propor-
tion of SFA in general went down with AP (Fig. 3D8). This general-
ized reduction in SFA release suggested that SFAs in triglyceride are
an unfavorable substrate for PNLIP.
We next directly investigated whether visceral triglyceride
composition, irrespective of obesity or genetic background, affects
its lipolysis and consequent severity during AP. For this experiment,
the triglycerides of LA (C18:2), glyceryl trilinoleate (GTL), or PA
(C16:0), i.e., glyceryl tripalmitate (GTP), were intraperitoneally giv-
en to lean mice with a genetically dissimilar background than the
C57Bl6 mice, i.e., lean CD-1 mice. Two mechanistically distinct AP
models—i.e., CER and IL12/18, both of which are mild in lean ro-
dents (27, 57)—were then induced (Fig. 4 and fig. S9).
Mice given GTL or GTP alone exhibited normal behavior, with
no change in physiologic or biochemical parameters. During AP,
neither GTL nor GTP affected the initial serum amylase or lipase
increase (Fig. 4, A1 and A2, and fig. S9, A1 and A2), which is similar

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

Fig. 4. Effect of intraperitoneal GTL or GTP on IL12,18-induced pancreatitis in lean CD-1 mice. Serum amylase (A1), lipase (A2), and trends and survival (A3) during
IL12/18 pancreatitis alone (IL12,18), with GTL (IL12,18 + GTL) or GTP (IL12,18 + GTP). Note similar amylase and lipase at day 2 in all groups and progressive worsening in
the GTL group, which has reduced survival. (B1 to B3) Representative H&E-stained images of the pancreas of mice belonging to the groups mentioned below the image.
Note the increased necrosis at the periphery of the lobules in IL12,18 + GTL group (black arrows). Box plots showing serum glycerol (C1) and NEFA (C2) concentrations in
control (CON) mice and in the different pancreatitis groups. (D1 to D3) Representative TUNEL-stained images of the kidneys of mice belonging to the groups mentioned
below the image. Note the increased positivity in the tubules of the IL12,18 + GTL group (black arrows), which also has a significantly higher BUN than other groups on
ANOVA (*) (D4). Box plots of the serum resistin (E1), IL-6(E2), TNF- (E3), and MCP-1(E4) control (CON) mice and in the different pancreatitis groups. * indicates significant-
ly higher than control on ANOVA, and # indicates a significant reduction compared to the IL12,18 + GTL group.

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 5 of 16

SCIENCE ADVANCES | RESEARCH ARTICLE
to ob/ob mice given UFA or SFA diets (Fig. 2, B1 and B2). The AP
outcomes also paralleled the UFA and SFA groups (Figs. 2 and 3)
with 0% survival in the GTL groups versus 80 to 90% in the GTP
groups (P < 0.01; Fig. 4A3 and fig. S9A3). The GTL group had worse
pancreatic necrosis (Fig. 4, B1 to B3, and fig. S9, B1 and B2), pre-
dominantly at the periphery of the lobules exposed to the lipolytically
generated LA, resembling peri-fat acinar necrosis (Fig. 2, C1 to C3).
The higher serum glycerol and corresponding NEFA in the GTL
groups with AP (Fig. 4, C1 and C2, and fig. S9, C1 to C3) supported
preferential unsaturated triglyceride lipolysis. Serum BUN elevations,
renal tubular injury (Fig. 4, D1 to D4, and fig. S9, D1 and D2), lung
injury (fig. S10, A and B), and profound hypotension noted as shock
(fig. S10C) were only noted in the GTL groups with AP. Consistent
with the small adipose tissue mass and visceral FN in lean mice (fig.
S11, A1 and A2), there was no increase in serum resistin (Fig. 4E1
and fig. S9E1) and little or no increase in serum lactate dehydrogenase
(LDH) (fig. S11, B and E). However, consistent with NEFA-driven
inflammation (7), serum IL-6, TNF-, and MCP-1 were higher in
both AP models with GTL (Fig. 4, E2 to E4, and fig. S9, E2 to E4),
perhaps due to the systemic lipotoxicity of LA noted as higher
serum damage-associated molecular patterns (DAMPs), i.e., double-­
stranded DNA (dsDNA) and histone-complexed DNA fragments
in the GTL groups with pancreatitis (fig. S11, C, D, F, and G). These
findings suggested that unsaturated visceral triglyceride is hydrolyzed
more than saturated triglyceride and worsens systemic inflammation
and organ failure, consistent with what we note epidemiologically
(Fig. 1). We thus went on to study the mechanistic basis of this
in more detail.
Saturation reduces long-chain triglyceride lipolysis by
pancreatic lipases
To understand how visceral triglyceride composition influences
AP severity, we simulated the in vivo AP-associated lipase leak into
fat using an in vitro system (Fig. 3, B1 and B2) (6, 7, 27). Acini release
pancreatic enzymes into the surrounding medium under the basal
state in vitro (58). Thus, we measured the hydrolysis of triglycerides
added to the medium and biological responses of the NEFA generated.
Addition of pure triglycerides GTP, glyceryl trioleate (GTO; the
triglyceride of OA, C18:1), or GTL (300 M each) to the stirred ac-
inar medium in a quartz cuvette at 37°C resulted in 60 to 70% hy-
drolysis of GTL and GTO within 15 min but <10% of GTP (Fig. 5,
A1 and A2). Lipolysis of GTL and GTO, unlike GTP or GTLO (i.e.,
GTL + the lipase inhibitor orlistat at 50 M) which has <15% lipoly-
sis, also caused acinar mitochondrial depolarization (m) and cyto-
solic calcium (Cai) increase (Fig. 5, A3 and A4). Separately, on longer
incubation (6 hours), 80 to 100% of GTL was hydrolyzed to glycerol
(fig. S12A) versus 20 to 30% of GTP even at a 3× molar excess. GTL
hydrolysis caused cytochrome c leakage, decreased ATP (adenosine
5′-triphosphate) levels, and increased LDH leakage and propidium
iodide uptake (fig. S12, B to E). These studies showed that triglyceride
saturation reduces its lipolysis and consequent biological effects.
We then studied the effect of saturation on the hydrolysis of mixed
triglycerides in the acinar medium, since triglycerides in vivo are mostly
mixed. On the basis of pancreatic lipase lacking stereoselectivity for
the sn-1/sn-3 position (59), we chose mixed triglycerides (each at
100 M) of LA, i.e., 1,2-dilinoleoyl-3-palmitoyl-rac-glycerol (LLP)
(LA-LA-PA), 1,3-dipalmitoyl-2-linoleoylglycerol (PLP) (PA-LA-PA),
and 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol (LOP) (LA-OA-PA),
and compared these to GTL (LA-LA-LA). Palmitate disproportionately
Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021
reduced lipolysis over 15 min. For example, LLP generated <30% LA,
and PLP generated <9% LA versus GTL (Fig. 5B1). Both linoleate
(4.3 ± 1 M) and oleate (4.4 ± 1.2 M) generation were markedly
reduced on LOP. This was paralleled by reductions in m and Cai
increase (Fig. 5, B2 and B3).
Triglyceride saturation makes its lipolysis by PNLIP
energetically and structurally unfavorable
We then studied the interaction and hydrolysis of GTL, LLP, and
LOP focusing solely on triglyceride hydrolysis by human PNLIP in
phosphate-buffered saline (PBS; pH 7.4, 37°C, 150 mM Na). The
hydrolysis, i.e., GTL > LLP > LOP, paralleled those in the acinar cell
media (Fig. 5C1). We thus studied their interaction with PNLIP using
isothermal titration calorimetry (ITC).
A single injection of human PNLIP (0.7 nmol/s) into a stable,
sonicated, stirred, and 100 M suspension of these triglycerides in
PBS (at 300 s; fig. S13A) caused an endothermic interaction with a
magnitude paralleling lipolysis, i.e., GTL > LLP > LOP, the enthalpy of
Downloaded from http://advances.sciencemag.org/ on February 2, 2021
which paralleled the raw heat data (Fig. 5, C2 and C3, and fig. S13B).
The enzyme kinetics studied with multiple-injection experiments
(fig. S13E) mirrored the above findings with the reaction rate (V;
micromolars per second) of GTL > LLP > LOP (fig. S13, F and G).
The calculated kinetic parameters (fig. S13G) for GTL hydrolysis are
similar to those previously published (60) and are more favorable
than for LLP or LOP. Addition of orlistat (50 M) to PNLIP signifi-
cantly reduced the H and Vmax in both types of injections (fig. S13,
C, D, H, and I), supporting the relevance of the parameters to
lipolysis. The above experiments thus showed that increasing satu-
ration makes the interaction of unsaturated triglycerides with PNLIP
energetically unfavorable, resulting in reduced lipolysis.
To verify the experimental results in an independent unbiased
manner, we undertook docking simulations using the open lid con-
formation of the human PNLIP-procolipase complex (1LPA) (61)
and studied access of the relevant triglycerides into the PNLIP ligand-­
binding domain (LBD) via the oxyanion hole. Ser152, Asp176, and
His263 compose the catalytic triad of human PNLIP and are critical
in fatty acid liberation from triglycerides. The closed conformation
(1LBP) (62) was not used since our goal was not to study molecular
dynamics of pancreatic lipase activation by interfaces.
Previous in silico studies (63) found that orlistat, a potent lipase
inhibitor, docks to pancreatic lipase with a GlideScore of −6.90 kcal/mol
with root mean square deviation (RMSD) between 1.2 and 4.8 Å.
This value served as a threshold to quantify strong binding. To verify
this value, we docked orlistat to 1LPA with the induced fit protocol,
and a similar GlideScore value was produced. Orlistat docked with
a distance of 3.74 Å between the catalytic serine and the -lactone
ring that inhibits PNLIP hydrolysis. The induced fit protocol docked
GTL into the 1LPA LBD with a GlideScore of −7.16 and with a distance
of 4.04 Å between the hydroxyl group of Ser152 and the carbonyl C
atom of the triglyceride’s glycerol backbone (Fig. 5D1). LLP docked
with a GlideScore of −4.72 kcal/mol at a distance of 9.99 Å from
Ser152, and LOP and GTP respectively produced GlideScores of −1.33
and −1.58 while being 12.42 and 11.98 Å from the catalytic serine
(Fig. 5, D2 to D4). To verify the integrity of the docking simulation,
the ligand present in the 1LPA crystal structure (61), dilauryl phos-
phatidyl choline (DLPC), was removed and then docked with the
same induced fit docking protocol used for the three triglycerides.
DLPC docked with a GlideScore of −7.46 (fig. S14A) and at a dis-
tance of 3.59 Å from the hydroxyl group of the catalytic serine,
6 of 16
SCIENCE ADVANCES | RESEARCH ARTICLE

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

Fig. 5. Influence of triglyceride composition on their lipolytic interaction, lipolysis and resulting biological effects. Lipase activity (A1), NEFA generation (A2), cy-
tosolic calcium (A3), and mitochondrial depolarization (A4) of acini alone (control) or with 300 M GTL, GTL with 50 M orlistat (GTLO), GTP, or GTO. (B1 to B3) Effect of
substituting LA in GTL (red) with palmitate at Sn-3 (LLP; magenta) or at Sn-1 and Sn-3 (PLP; cyan) or oleate and palmitate at Sn-2 and Sn-3, respectively (LOP; light brown)
on NEFA generation over 15 min by acini (B1), acinar mitochondrial depolarization (B2), and cytosolic calcium increase (B3). (C1) Time course of NEFA generation from
GTL, LLP, and LOP (100 M) by PNLIP (12 nM). (C2 and C3) hPNLIP-triglyceride interaction, showing raw heat per second (Q/t) as a function of PNLIP/triglyceride ratio
(C3) and measured peaks (C2). (D1) GTL docked into 1LPA via induced fit protocol. PNLIP molecule (top) and zoomed in LBD (bottom) are shown with catalytic triad (dark
blue), distances, and GlideScores (kilocalories per mole). LA, OA, and PA are in red, cyan, and green, respectively, with receptor interaction surface in yellow and oxygen
atoms in orange. (D2 to D4) LLP, LOP, and GTP docked into 1LPA.

which is 0.39 Å from its location in the crystal structure (3.20 Å).
The resolution of 1LPA is 3.04 Å, so the variance seen is within the
margin of error. DLPC inhibited the lipolysis of GTL added simul-
taneously in a dose-dependent manner (fig. S14B). This inhibition by
DLPC, along with the redocking closely recapitulating the crystallo-
graphic findings, thus validates the docking simulation. Overall, these
studies show that long-chain SFAs like palmitate make a triglyceride’s
interaction with PNLIP structurally and energetically unfavorable,
thus reducing its hydrolysis. We lastly went on to study how the fatty
acids generated may result in cell injury and consequent organ failure.
Double bonds increase monomeric NEFA concentrations,
signaling, and resulting organ failure
We first compared the ability of NEFA to directly induce inflamma-
tion and organ failure. LA [0.3% body weight (28, 53, 54)], OA (7),
or PA (0.5%) body weight was instilled into the peritoneal cavity to
simulate NEFA generated from visceral fat (2 to 10% body weight)
lipolysis (64). Unlike LA and OA, there was no increase in circulating
cytokines or organ failure (BUN increase) induced by PA (Fig. 6,
A and B). The median survival for LA-treated mice was 18 hours
and for OA was 64 hours, while the PA group had no adverse out-
come over 3 days. Serum NEFA (which are predominantly albumin
bound) and unbound NEFA (uNEFA; Fig. 6, C and D) increased
significantly only in the LA and OA groups, along with levels of the
DAMP and dsDNA (Fig. 6E) and a drop in serum albumin consistent
with the hypoalbuminemia noted during experimental (fig. S15, A
to C) and clinical SAP (55). This suggested that, unlike PA, both LA
and OA with high uNEFA may directly injure cells, triggering DAMP
release and downstream inflammation. This would also be consistent
with the in vivo AP models (Figs. 3 and 4 and figs. S9 and S11, B to
G) where higher NEFA were associated with worse outcomes.
We thus compared the ability of the most prevalent long-chain
NEFA (C18:2, C18:1, and C16:0; Fig. 6, F1 to F3) to exist in a non-
micellar monomeric form in aqueous media. On ITC at 37°C, we

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 7 of 16

SCIENCE ADVANCES | RESEARCH ARTICLE

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

Fig. 6. Effects of NEFA unsaturation on their in vivo effects and unbound monomeric behavior. Serum MCP-1 (A), BUN (B), NEFA (C), uNEFA (D), and dsDNA (E) after
intraperitoneally administering C16:0 (green), C18:1 (blue), and C18:2 (red) as mentioned below the x axis. Values are at necropsy if moribund or euthanasia at 72 hours.
(F1) ITC thermograms of injecting NEFA or solvent (0.34% DMSO for C16:0 and PBS for C18:1 or C18:2) at concentrations mentioned below the NEFA into PBS (pH 7.4) at
37°C, and corresponding enthalpograms (F2) and CMCs (F3) shown as shaded rectangles in the areas where they intersect the x axis. Effect of exogenous albumin (final
concentrations of 0.5%) on the cytosolic calcium (Cai) (G) and mitochondrial depolarization (m) (H and I) induced by 100 M of the indicated NEFA added at 100 s. (J and
K) Comparison of the dose responses of increase in mitochondrial depolarization (m) after 60 s of addition of different concentration of C18:1 (blue) or C18:2 (red) in
acini (J) and HEK293 cells (K). * indicates significant difference (P < 0.05) from the previous lower concentration by paired t test. # indicates a significant difference between
C18:2 and C18:1 for that specific concentration on t test.

noted the critical micellar concentration (CMC) and therefore the
aqueous monomeric NEFA concentrations to increase with the number
of double bonds (Fig. 6, F1 to F3). The CMCs of PA (C16:0), OA
(C18:1), and LA (C18:2) were respectively <8, ≈40, and ≈160 M,
which paralleled the uNEFA levels in vivo (Fig. 6D). The calorimetric
results were then validated using ultracentrifugation. Both of these
methods could be performed at room temperature (23°C; fig. S16)
and showed that the nonmicellar NEFA concentrations in the in-
franatents of 500 M NEFA in PBS (pH 7.4), spun at 105g for 1 hour
(fig. S16, A and B), were similar to the CMCs on calorimetry (fig.
S16, C and D) and those shown previously using diphenyl hexatriene
fluorescence spectroscopy (65). Thus, the CMC noted on calorimet-
rically at 37°C (Fig. 6, F1 to F3) are accurate.
The role of uNEFA in causing biological effects was then studied
in cells. Addition of 100 M LA (below CMC) to pancreatic acini
caused a larger increase in Cai (Fig. 6G) and m (Fig. 6H) than
100 M OA (above CMC). PA caused no change in these. A total of
0.5% albumin (Alb) completely aborted the Cai increase by both LA
and OA and prevented m from progressing (Fig. 6, H and I). This
effect of albumin proves that the Cai and m increases were from
the uNEFA. It also shows that Cai and m changes result from
distinct mechanisms of these uNEFA. The sustained m elevation,
without progression, despite albumin (Fig. 6, H and I) signifying
irreversible uNEFA toxicity, is distinct from the complete reversal
of Cai by albumin (Fig. 6G) and explains the cell death noted from
LA or OA in previous studies (6). Therefore, the greater Cai and m
induced by LA than OA at 100 M (Fig. 6, G and H) may be due to
LA’s higher CMC (Fig. 6F3) and thus explains the higher uNEFA
levels and shorter survival noted in LA-treated mice. We tested this
by measuring the dose response of increase in m over baseline

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 8 of 16

SCIENCE ADVANCES | RESEARCH ARTICLE

(m) 60 s after the addition of LA or OA to acini (Fig. 6J) or human
embryonic kidney (HEK) 293 cells (Fig. 6K) to represent the
pancreatic and kidney injury in vivo (Fig. 4, D1 to D4, and fig. S9,
D1 and D2). Consistent with OA’s CMC (≈40 M; Fig. 6F3), m
increased over baseline at 50 M OA (*) and then plateaued (Fig. 6,
J and K). Similarly, LA’s m increased till 100 M and ceased at
200 M, consistent with its CMC of ≈160 M. While LA’s m
equaled OA’s at 50 M, it was more than OA’s (#) at concentrations
of ≥100 M. LA and OA (60 M), unlike PA, also reduced endothelial
barrier integrity (fig. S15, D and E), potentially explaining the hypo-
tension (Fig. 2E1 and fig. S10C) hypothesized to be from vascular leak
during SAP (66), along with explaining the hypoalbuminemia (fig. S15,
A to C) (55) in severe AP, and the uNEFA increase noted (Fig. 6D).
Overall, these studies cumulatively validate that double bonds in-
crease monomeric long-chain NEFA concentrations and signaling
in an aqueous environment, thus enhancing their lipotoxicity.
in aqueous media, unlike saturated NEFA, resulting in injurious sig-
naling, lipotoxic inflammation, and organ failure. This can potentially
explain why higher dietary UFAs may result in worse AP in leaner
animals and humans with lower BMIs compared to the more obese
ones who consume a diet with higher proportions of saturated fat
(Fig. 7), resulting in the obesity paradox.
We note that the obesity paradox in pancreatitis (10) holds true
when the data are grouped and analyzed by BMIs using the World
Health Organization (WHO) cutoffs (67) relevant to the countries
from which the study originated (Fig. 1), including those that use
BMI cutoffs of ≤25, which have a lower SFA and higher %UFA con-
sumption in diet (Fig. 1, B and C). Socioeconomic and quality-of-­
care issues, age, sex, and etiology of AP are unlikely to have influenced
our findings since the rate of SAP and mortality were the same in
the >30 versus ≤25 BMI cutoff groups. We note that the SAP rates
and %UFA intake have a moderate correlation (fig. S3B), and on
meta-regression, %UFA intake explains 33% of the heterogeneity in
SAP rates. Thus, other factors such as male sex, genetic background,

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

DISCUSSION
Here, we find that a higher proportion of dietary unsaturated fat can
worsen AP outcomes at a lower adiposity than seen in individuals
with a higher proportion of saturated fats in their diet. We show
that the higher likelihood of SAP associates with a higher unsaturated
triglyceride content in visceral fat. This occurs because the presence
of SFAs in triglycerides makes the interaction of the substrate with
PNLIP structurally and energetically unfavorable. Moreover, the un-
saturated NEFA generated by lipolysis can exist as monomers (65)
and comorbidities that are not accounted for in the studies may
contribute to the remaining heterogeneity in SAP rates.
The difference in BMI cutoffs set by the WHO is commonly
attributed to a higher percentage fat per body mass in Asian popu-
lations compared to Caucasian populations (68). However, this is
brought into question since visceral adiposity (3) that undergoes
visceral FN during SAP (69) differs by ethnic groups (70) and in
women versus men (71). We note that all visceral fat is not the same,
since a higher proportion of diet-derived UFAs like LA make smaller

Fig. 7. Schematic summarizing how dietary fat composition affects visceral fat necrosis and causes the obesity paradox. The impact of consuming a Western diet en-
riched in saturated fat from dairy and red meat (left) or one enriched in unsaturated fat from vegetable oil and fish (right) are shown. In the event of AP, the relatively more
saturated adipocyte triglyceride shown in blue ( , left) or unsaturated triglyceride shown in red ( , right) is exposed to pancreatic lipase, principal among which is PNLIP
(zoomed circles). Despite being lesser in amount, the more unsaturated triglyceride is hydrolyzed more into the lipotoxic NEFA ( ), which are stable as monomers at
higher concentrations than saturated NEFA, that form micelles ( ) at lower concentrations. This results in worse systemic inflammation, injury, and organ failure in
those with a higher unsaturated fat consumption, despite having less adiposity. This pathophysiology can explain the obesity paradox.

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 9 of 16

SCIENCE ADVANCES | RESEARCH ARTICLE
amounts of visceral fat more prone to lipolysis (Figs. 2 and 3), gen-
erating higher concentrations of monomeric NEFA (Fig. 6), thus
worsening outcomes in leaner populations (Fig. 1). The reverse holds
true for SFAs, and this is relevant to studies from Western countries
(72) that show AP severity to be independent of intra-abdominal fat
amounts even when this fat is above the range in studies from Asian
countries (71). This is exemplified by the six studies reporting a
BMI of >30 to not be associated with severe AP—all of which came
from countries with <40% UFA as dietary intake (Fig. 1C).
Our epidemiologic studies are limited by being retrospective, not
involving dietary history, visceral fat sampling, or genetic studies,
and by using nationwide dietary and economic data. We thus verified
the conclusions in mice, cell, and pure enzyme models and physical
chemistry. The use of two different strains of mice (i.e., C57bl/6 and
CD-1) and two modes of increasing visceral triglyceride (i.e., obesity
and intraperitoneal injection of triglyceride) experimentally support
that acute triglyceride lipolysis is crucial in the pathogenesis of organ
failure and weakens the argument in favor of the genetic or chronic
effects of obesity such as insulin resistance and atherosclerosis in
explaining these outcomes. The deleterious effects that we note with
GTL are also seen with the triglyceride of OA but not with GTP during
pancreatitis (5). These along with the deleterious effect of monomeric
unsaturated NEFA (Fig. 6), lipase (16–18), and NEFA elevation
(19, 20, 23), being associated with worse outcomes in other diseases
states including COVID-19 (22, 24), perhaps support the general
relevance of dietary and visceral fat unsaturation in causing the
obesity paradox (11–15). A palmitate-enriched diet in ob/ob mice
helped avoid the confounding effects of maldigestion of a dietary
saturated triglyceride by pancreatic lipases, which we note as reduced
lipolysis of GTP, LLP, and PLP (Fig. 5, A2 and B1). We used PNLIP
since recent studies show that adipocyte triglyceride lipase, PNLIPRP2,
and carboxyl ester lipase are unlikely to mediate lipotoxic systemic
inflammation (7, 53). Moreover, the similar lipolysis pattern in acinar
media, which contains all three lipases (Fig. 5, A1 to A4 and B1 to
B3) and PNLIP (Fig. 5, C1 to C3), lends credence of the concept to
other lipases.
Stearoyl-CoA (coenzyme A) desaturase-1(SCD-1) (73) is likely
responsible for converting the dietary palmitate (C16:0) to palmi-
toleate (C16:1), which comprised 17% of the visceral triglyceride of
SFA mice (Fig. 2A1 and fig. S5), despite palmitoleate being absent
in the diet. SCD-1 is highly expressed in adipose tissue and can put
a double bond in stearoyl-CoA or palmitoyl-CoA, thus forming
oleate (C18:1) and palmitoleate from the palmitate in the diet. This
may also explain the similar amounts of C18:1 noted in the visceral
triglyceride of the UFA- and SFA-fed mice. Despite the potential in-
fluence of SCD-1 on our hypothesis, the similarity of dietary and vis-
ceral fat composition that we note in mice, the previous studies note in
humans (26), along with the lack of known pathways to saturate C18:1,
and our mechanistic data cumulatively support our conclusions.
An additional limitation may lie in our inability to resolve the
lipolytic impact of stereochemical structure of the triglycerides (74),
which we used in the racemic form rather than the enantiopure form.
While this remains beyond our scope, the previous findings that
pancreatic lipase hydrolyzes triglyceride without any stereoselectivity
for the sn-1 or sn-3 position (59) suggest that the interference by
palmitate is independent of these locations on the glycerol backbone.
Last, the mechanisms underlying the exothermic changes (Fig. 5, C2
and C3) following the initial interaction of PNLIP and triglycerides
remain unclear. The relative magnitude of these, i.e., GTL > LLP > LOP,
Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021
mirror the initial interaction. Previous studies suggest that this may
be due to binding of the generated LA to aromatic residues on PNLIP
and further increasing its activity (75).
In summary, diet-induced visceral fat unsaturation increases
lipolytic generation of unsaturated monomeric NEFA that cause cell
injury, systemic inflammation, and organ failure. Long-chain SFAs
like palmitate, however, interfere with this lipolysis, generating lower
amounts of monomeric NEFA, making pancreatitis milder despite
excess adiposity, thus explaining the obesity paradox (Fig. 7). The
FDA guidelines to reduce saturated and increase unsaturated fat intake
(https://www.fda.gov/files/food/published/Food-Labeling-Guide-
%28PDF%29.pdf, appendix F) (26, 52) could therefore potentially
contribute to worsening systemic inflammation and organ failure.
METHODS
Human data analysis
Literature search
Downloaded from http://advances.sciencemag.org/ on February 2, 2021
To explore the impact of BMI on AP severity, we conducted a liter-
ature review by searching the PubMed database from inception to
January 2017). The terms “BMI” or “obese” and “acute pancreatitis”
as well as “severe” or “severity” were used. A total of 118 studies
were retrieved, as shown (fig. S2). Seventy-nine studies were excluded,
as they were not related to the subject of this review, as were 12 studies
that were reviews/meta-analyses/hypotheses. The remaining studies
were extensively reviewed for eligibility criteria, which were as fol-
lows: (i) clear BMI cutoff to define obesity, (ii) clear report of the
incidence of mild AP (MAP) and SAP in obese versus nonobese
groups, and (iii) clear and satisfactory definitions of MAP and SAP.
Twenty-seven studies met our eligibility criteria and were therefore
included in this review (fig. S1). These are shown in different colors
in Fig. 1A for descriptive purposes. The studies were then catego-
rized into those using a BMI of 30 as a cutoff (n = 20, the one with a
cutoff of >29 was included here) and those that used a cutoff BMI
of ≤25 (n = 7; with text and country in pink) to define obesity’s
association with SAP. The dietary fat consumption in the countries
from which these papers originated was then extracted as described
below and compared (fig. S1). The incidence of MAP and SAP in
both obese and nonobese patients was extracted for the purpose of
meta-analysis. Total number of patients was divided into MAP and
SAP and then into those that were obese versus nonobese. In cases
where this was not explicitly clear, the communicating author was
contacted (30, 45), and the numbers so provided were included in
the study. Papers that did not have adequate data or for reasons
mentioned in the last column of fig. S1 and detailed in fig. S2 were
excluded from the meta-analysis (n = 7). When more than one BMI
cutoff was mentioned, the BMI used was the one at which the SAP
risk increased.
Statistical analysis
The proportion of SAP in all pancreatitis was calculated and
compared between obese and nonobese groups. Subgroup analysis
was performed on the basis of the definition of obesity used in the
different studies included. Random effects model was used to calcu-
late pooled OR with 95% CIs. Heterogeneity was assessed using
the I2 measure and the Cochran Q statistic. Statistical analysis was
performed using Comprehensive Meta-Analysis Software version
3.3.070 (Biostat, Englewood, NJ, USA). Publication bias was as-
sessed for using Begg and Mazumdar’s test as well as Egger’s regres-
sion intercept.
10 of 16
SCIENCE ADVANCES | RESEARCH ARTICLE

Dietary fat consumption of country from which paper originated
The per capita per year dietary fat consumption of each country was col-
lected from the FAO website (http://www.fao.org/faostat/en/#data/FBSH),
which is reported as kilograms per person per year. For purposes of
simplicity, the chief sources of long-chain saturated fat (dairy, red meat,
and palm oil) were analyzed separately from those of unsaturated fat
(fish and vegetable oil). Fat from dietary sources were calculated
by percentage weight (3.25% for whole milk, 20% from red meat,
and 10% for fish) unless the food source was directly fat (e.g.,
cream, butter, ghee, cheese, vegetable oil, or palm oil). Data from
the country from which each paper originated were included in the
categories “BMI >30 not related to SAP,” “BMI >30 associated with
SAP,” and cutoff “BMI ≤25” (shown as blue, green with golden text
or symbols, and pink in Fig. 1, respectively). Each country was rep-
resented once in the category for a contributed paper (shown in
Fig. 1, B and C). Comparisons were done by grouping the BMI of
>30 not related to SAP and BMI of >30 associated with SAP into a
grouped BMI of >30 category and by comparing this to the BMI of
Committee of the Mayo Clinic (Scottsdale, AZ). Ex vivo studies were
done on pancreatic acini harvested from ICR mice (Charles River
Laboratories, Wilmington, MA). In vivo studies were done on
genetically obese male ob/ob (B6.V-lepob/J; the Jackson laboratory,
Bar Harbor, ME) mice given an experimental diet below or ICR mice.
The mice were 7 to 14 weeks of age at the time of studies. There
were six to eight mice in each group.
In vivo studies in mice
Experimental diets
These were started immediately in ob/ob [B6.Cg-Lepob/J; Jax Stock
#00632, the Jackson laboratory (Bar Harbor, ME)] or C57bl/6J (to
induce DIO) after weaning (age of 4 to 5 weeks), with an intent to
change visceral fat composition. The diets are detailed in fig. S4 and
were given ad libitum. Normal chow [Purina 5053 diet, LabDiet
(www.labsupplytx.com/wp-content/uploads/2012/10/5053.pdf),
which contains 5.0% of which ≈70% is UFA] and water were fed to
control mice ad libitum. Mice were weighed periodically (1 to 4 weeks,

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

≤25 category using a Mann-Whitney test. P < 0.05 was regarded depending on the rate of weight gain), and the body fat was mea-
as significant. sured using a Minispec Body Composition analyzer (Bruker Corpo-
ration, Billerica, MA) using nuclear magnetic resonance as previously
Reagents described (27).
Glyceryl trilinolein (GTL) and other triglycerides including triolein Experimental pancreatitis
(GTO), tripalmitin (GTP), and mixed triglycerides 1,2-dilinoleoyl-3-­ The models used were CER, and IL12 + IL-18 (IL12,18) induced
palmitoyl-rac-glycerol (LLP), 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol pancreatitis in ob/ob mice, as detailed below. CER pancreatitis was
(LOP), 1,3-dipalmitoyl-2-linoleoylglycerol (PLP), 1,2-dioleoyl-3-­ also induced in C57bl/6 mice given the experimental diets or normal
palmitoyl-rac-glycerol (OOP), 1,3-dioleoyl-2-palmitoylglycerol chow after 28 weeks. CER pancreatitis was induced by hourly intra-
(OPO), 1,3-dipalmitoyl-2-oleoylglycerol (POP), linoleic acid (LA), peritoneal injections of CER (50 g/kg; Bachem AG, Bubendorf,
palmitic acid (PA), oleic acid (OA), dimethyl sulfoxide (DMSO), and Switzerland) × 12 doses on two consecutive days as described before
glycerol reagent were purchased from Sigma-Aldrich (St. Louis, MO). (27). IL12 (150 ng per dose per mouse; PeproTech) and IL18 (750 ng
CER (Bachem AG, Bubendorf, Switzerland), IL12 (PeproTech, Rocky per dose per mouse; R&D Systems) pancreatitis was induced by giving
Hill, NJ), IL18 (R&D Systems, Minneapolis, MN), PNLIP, IB-, IL12,18 as two doses 24 hours apart to lean ICR mice (10 to 14 weeks)
cytochrome c antibody (Santa Cruz Biotechnology, Dallas, TX), as described previously (57). Both agents were dissolved in PBS and
-tubulin [Developmental Studies Hybridoma Bank (DSHB), Uni- given intraperitoneally. When studying the effect of intraperitoneal
versity of Iowa, Iowa], Cox-IV antibody, and TO-PRO-3 iodide GTL (150 l) or GTP (150 mg) in these models, either agent was
(Thermo Fisher Scientific, Waltham, MA) were used. given 4 hours after the first CER injection or second IL12,18 injec-
tions. NEFA were administered into the peritoneal cavity of mice as
Use of lipids described previously (7, 22, 28).
GTL, other triglycerides, and fatty acids were sonicated into the media Carotid pulse distension (MouseOx Oximeter, STARR Life Sci-
in a two-step fashion to ensure that the triglyceride stays in solution ences, Pittsburgh, PA) was used as a noninvasive measure of blood
as described previously (53). Briefly, pure GTL, GTO, LLP, LOP, pressure. Mice were observed four times a day and were electively
and OOP were first directly sonicated into the PBS (concentration, sacrificed at the end of the experiment using carbon dioxide, unless
3 mM) using three 10-s pulses. This sonicate was promptly added to they became moribund prematurely. Terminal blood and tissue
the medium (final concentration, 300 M). The only triglycerides harvest was done if the animal was moribund and used in the analyses,
requiring a solvent (DMSO) were those with two or more PA chains unless specified for a different time point. Tissues were collected and
(PLP, GTP, and POP). These were prepared as follows: triglyceride preserved for mRNA (RNA later), activity (−80°C), histology and
stock (60 mM) was prepared in 100% DMSO by heating. These stocks TUNEL staining (formalin), and Luminex and biochemical assays
were sonicated into PBS (concentration, 3 mM and 5% DMSO). (serum) as described previously (6, 7, 28).
These sonicates were quickly added to the medium (final concen- Histology and TUNEL staining
tration, 300 M and 0.5% DMSO). The experimental endpoints Pancreas, kidney, and lung tissue were fixed with 10% neutral buff-
were not affected by this 0.1 to 0.5% DMSO in our system, as shown ered formalin and processed for embedding in paraffin. Sections
previously (6). (5 m) were used for hematoxylin and eosin (H&E) staining and for
TUNEL staining as described previously (5, 6, 27). Digital images of
Animal experiments section were captured with a digital microscope Axio Imager M2 or
All animals were acclimatized for a minimum of 2 days before use. Axio Observer Z1 (Zeiss, Oberkochen, Germany) with a 20× objec-
These were housed with a 12-hour light/dark cycle at room tem- tive with 10 random images per section and quantified as number of
perature, fed normal laboratory chow (ICR mice) or an experimental TUNEL-positive nuclei/high field of lung tissue. Pancreatic necrosis
diet (see below), and were allowed to drink ad libitum. All experi- was quantified on whole-pancreas H&E-stained sections after being
ments were approved by the Institutional Animal Care and Use examined by a trained morphologist blinded to the sample. Briefly,

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 11 of 16

SCIENCE ADVANCES | RESEARCH ARTICLE
all pancreatic parenchymal area was imaged using the PathScan
Enabler IV slide scanner (Meyer Instruments, Houston, TX), and
images were evaluated for total acinar area, total acinar necrosis, and
acinar necrosis in direct proximity to the necrosed fat, i.e., peri-fat
acinar necrosis as described previously (6, 27) in pixels for each
pancreas. Percentage necrosis was reported as a percentage of the
total acinar area for each pancreas.
In vitro cell studies
All data shown are from a three to five independent experiments.
Use of lipids
Triglycerides (e.g., GTL, LLP, LOP, and GTO) or NEFA were soni-
cated at 10× concentrations into the medium, and the only solvent
used was 5% DMSO (as a 10× stock) for GTP and PLP or for palmitate,
since these otherwise were insoluble and precipitated out in the
medium. Solvents were otherwise avoided, since lipolysis in vivo takes
place without solvents. These agents were added at a 1× concentration.
Pancreatic acinar harvest and use
Eight- to 12-week-old ICR mice (Charles River Laboratories, Wilmington,
MA) were euthanized, and pancreatic acini were harvested as de-
scribed (7, 28).
Cell lines and use
HEK293 cells were maintained in Eagle’s minimum essential media
[American Type Culture Collection (ATCC), Manassas, VA] sup-
plemented with 10% fetal bovine serum (ATCC, Manassas, VA)
and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA).
All cell cultures were maintained in a humidified 5% CO2 atmosphere
in air at 37°C. Human endothelial cells (HUV-EC-C) were main-
tained in Kaighn’s modification of Ham’s F-12 medium (F-12K,
ATCC, Manassas, VA) supplemented with 10% fetal bovine serum
(ATCC, Manassas, VA), 1% penicillin/streptomycin (Life Technol-
ogies, Carlsbad, CA), heparin (0.1 mg/ml; Millipore Sigma, St. Louis,
MO), and Corning endothelial cell growth supplement (50 g/ml;
Thermo Fisher Scientific).
TEER and dextran permeability assay
HUV-EC-C cells were grown as monolayers on a 0.4-m polyester
Transwell permeable membrane (Corning, NY) precoated with
type IV collagen and then used for trans-endothelial electrical resistance
(TEER) measurement and permeability tracer flux assay. Before
studies, cells were suspended in Hepes buffer (pH 7.4) (20 mM
Hepes, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose,
10 mM sodium pyruvate, and 1 mM CaCl2), and exposed to NEFA
at the indicated concentration (60 M). TEER measurements were
made using EVOM2 Epithelial Voltohmmeter with an STX2 elec-
trode (World Precision Instruments, Sarasota, FL). Results were
presented as absolute values (Ω·cm2) after 2 hours of treatment.
To evaluate the paracellular permeability of cultured HUV-EC-C
cells, 10 kD of Dextran Alexa Fluor 647 (Thermo Fisher Scientific,
Waltham, MA) was added to the upper chamber, and the increase
in fluorescence was measured in the lower chamber 2 hours after
stimulation with NEFA.
For in vitro calcium ion flux and mitochondrial depolarization
studies, cells were loaded with fura-2AM (5 g/ml; Molecular Probes,
Invitrogen) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimid-
azolylcarbocyanine iodide (JC-1, 5 g/ml; Enzo Life Sciences,
Farmingdale, NY) at 37°C for 30 min in the media that they were
cultured or harvested in. After this, these were stored on ice till just
before use. Just before the experiment, the cells were washed and
suspended in Hepes buffer (pH 7.4) (20 mM Hepes, 120 mM NaCl,
5 mM KCl, 1 mM MgCl2, 10 mM glucose, and 10 mM sodium
Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021
pyruvate) as previously described (6, 7, 28). Triglycerides or NEFA
were added after 2 min of stirring at a 1× concentration to the
stirred cell suspensions in a quartz cuvette at 37°C, and changes in
intracellular calcium concentrations were determined by alternate
excitation at 340 and 380 nm, measuring emission at 510 nm. Mito-
chondrial inner membrane potential values (m) were determined
by excitation at 490 nm and alternate measuring emission at 530 and
590 nm using the F2100 Hitachi Fluorescence Spectrophotometer.
The data were collected every 10 s. The first 2 min of data were
averaged and subtracted, and the net increase was plotted as a func-
tion of time or NEFA concentrations. In some studies, excess (i.e.,
0.5%) fatty acid–free bovine serum albumin was added to neutralize
and thus determine the changes attributable to the uNEFA.
Assays
Biochemical assays
The assays (amylase, lipase, BUN, and calcium; Pointe Scientific,
Canton, MI), DNA damage: Quant-iT PicoGreen dsDNA reagent
Downloaded from http://advances.sciencemag.org/ on February 2, 2021
(Life Technologies, Carlsbad, CA), LDH leakage using the Cytotoxicity
Detection Kit (Roche, Mannheim, Germany), intracellular ATP mea-
surement using ATP assay kit (CellTiter-Glo 2.0 Assay, Promega,
Madison, USA), glycerol using free glycerol reagent, and standard
(Sigma-Aldrich, St. Louis, MO) and colorimetric total free fatty acids
using NEFA kit (FUJIFILM Wako Diagnostics, Mountain View, CA)
were done as per the manufacturer’s protocol (5, 6, 27).
uNEFA measurement by fluorescence
uNEFAs were measured by the emission at 550/457 nm upon exci-
tation at 375 nm and calculated as described in the ADIFAB2 kit
(FFA Sciences LLC, CA, USA) protocol version 1.0 6-9-04. Standard
concentration versus fluorescence ratio curves were made with known
concentrations of either >99.5% purity LA, OA, or PA (1, 2, 5, 10,
20, 30, 50, and 100 M) in DMSO to have these all in the pure
monomeric form. Then, the unknown (e.g., mouse serum) was mea-
sured and reported as uNEFA (micromolar concentrations), based
on the fatty acid standard curve.
Cytokine/chemokine assays
Serum cytokine/chemokine protein levels were assayed with a
MILLIPLEX MAP Mouse Adipokine Magnetic Bead Panel (Millipore,
Burlington, MA) according to the manufacturer’s recommendations
on a Luminex 200 System (Life Technologies) and analyzed using
the xPONENT software.
Triglyceride lipid composition and NEFA analysis
These were done at the Hormone Assay and Analytical Services Core
(Vanderbilt University Medical Center). For tissue triglyceride com-
position, the Folch method (76) was used, and the lipids recovered
in the chloroform phase were separated by thin-layer chromatography
using Silica Gel 60A plates. The triglyceride fraction was methylated
using the BF3/methanol method (77). These were analyzed using gas
chromatography (Agilent 7890A) using a capillary column (SP2380,
0.25 mm by 30 m, 0.25-m film; Supelco, Bellefonte, PA) and helium
as a carrier gas. Methyl esters of the fatty acids were identified by
comparing their retention times to those of known standard flame
ionization detection. Internal lipid standards for quantification in-
cluded dipentadecanoyl phosphatidylcholine (C15:0), diheptadecanoin
(C17:0), trieicosenoin (C20:1), and cholesteryl eicosenoate (C20:1).
Serum-free fatty acids were extracted as described previously (78) in
30:70 heptane:isopropanol. This was acidified with 0.033 N H2SO4,
and fatty acids were recovered in the heptane layer. Thin-layer
chromatography and gas chromatography were done as described
12 of 16
SCIENCE ADVANCES | RESEARCH ARTICLE
above. Dipentadecanoic acid (C15:0) was added as an internal stan-
dard. UFA amounts were calculated by adding individual C16:1, C18:1,
C18:2, C18:3, C20:4, and C20:5 fatty acids. SFA amounts were calcu-
lated by adding individual C12:0, C14:0, C16:0, and C18:0 fatty acids.
Western blotting
Western blotting was performed as described previously (54). Briefly,
cell lysate or fat pad lysate was prepared in radioimmunoprecipitation
assay lysis buffer containing various protease inhibitors (complete
and free of EDTA; Roche, Pleasanton, CA). Protein concentration
(1 mg/ml) of the lysate was adjusted, boiled (10 min) in Laemmli
buffer, and run on (10 mg per lane) 4 to 20% gradient polyacrylamide
gels. Then, proteins were transferred from the gel to polyvinylidene
difluoride membrane (Merck KGaA, Darmstadt, Germany) and
blocked with 5% nonfat dry milk incubated with primary antibody
of PNLIP (1:1000; Santa Cruz Biotechnology, Dallas, TX), IB-
(1:1000; Santa Cruz Biotechnology), -tubulin (1:500; DSHB, Uni-
versity of Iowa, Iowa), cytochrome c (1:1000; Santa Cruz Biotech-
nology), and Cox-IV (1:1000; Thermo Fisher Scientific, Waltham, MA)
separately. Then, blots were probed with horseradish peroxidase–
labeled corresponding secondary antibodies (1:10,000; Millipore,
Billerica, MA). Band intensity was visualized by chemiluminescence
using the Pierce ECL 2 Western Blotting Substrate Kit (Thermo Fisher
Scientific) and quantified by densitometry as described previously (79).
Isothermal titration calorimetry
The hydrolysis of triglycerides by hPTL was carried out on a Nano
ITC (TA Instruments-Waters LLC, New Castle, DE) instrument and
through Nano ITCRun Software v3.5.6. The experimental setup was
equipped with a computer-controlled micro-syringe injection device,
which allowed to inject small amounts of stock solution of even 0.1 l
to the sample chamber. Enzyme reaction was performed by two
methods: either incremental injection of substrate to enzyme or
continuous injection of enzyme to substrate.
Incremental injection
A total of 10 aliquots of freshly prepared, degassed GTL or LLP or
LOP (0.4 mM, 5 l per injection) were injected from a rotating
syringe of speed 350 rpm into the ITC sample chamber containing
hPNLIP (12 nM, ~500 U/liter activity) equilibrated at 37°C. The in-
terval between two injections was 300 s. Similarly, individual tri-
glyceride into PBS or PBS into hPNLIP was run as a control. To
confirm the specificity, LLL was injected into hPNLIP having orlistat
(50 M orlistat; Cayman Chemical Company, Ann Arbor, MI) in a
similar process.
Continuous injection
Degassed hPNLIP (45 l, 120 nM) was injected during 900 s from a
rotating syringe into the ITC sample chamber containing LLL or
LLP or LOP (0.1 mM) equilibrated at 37°C. The control experiment
was performed by injecting PBS into hPNLIP. hPNLIP-mediated
hydrolysis of LLL was confirmed using hPNLIP with orlistat in the
same experimental setup.
Calculation
The ITC raw data were analyzed using the NanoAnalyze v3.10.0
software. Maximum velocity at saturating substrate concentration
(Vmax) was determined. Michaelis constant (Km), the measure of
dissociation/affinity of enzyme-substrate complex, was calculated
from the substrate concentration at Vmax/2. Enzyme turnover rate
(Kcat) was calculated by Vmax/enzyme concentration. Substrate to
product formation rate (V) was calculated by the equation [Kcat ×
enzyme concentration × substrate concentration]/[Km + substrate
concentration]. For each experiment, we calculated Vmax, Km, Kcat,
Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021
and V values and summarized as means ± SEM from three indepen-
dent experiments.
From the raw data of continuous injection, enthalpy of the reac-
tion was calculated from the delta heat change per mole. Interaction
of triglyceride and hPNLIP followed by hydrolysis of triglyceride was
graphically presented from three independent experiments.
ITC for CMC measurement experiments
We followed the method described previously (54). A total of 20 ali-
quots of degassed LA (0.7 mM, 2.5 l per injection from PBS, pH 7.4),
OA (0.34 mM, 2.0 l per injection from PBS, pH 7.4), and PA (0.34 mM
in 0.34% DMSO, 2.0 l per injection from PBS, pH 7.4) were injected
(10 M per injection for LA and 4 M per injection for both OA and
PA) from a rotating syringe (350 rpm) into the ITC sample chamber
containing PBS equilibrated at 37°C. Double-distilled water was used
in a reference electrode chamber. The interval between two injections
was kept at 200 s. The control experiment was performed by injecting
PBS into PBS and DMSO (0.34%) into PBS.
Ultracentrifugation studies
Downloaded from http://advances.sciencemag.org/ on February 2, 2021
A total of 500 M C18:1, C18:2, and C18:3 were probe-sonicated
into PBS (pH 7.4) at room temperature. For C16:0 and C18:0, 150 mM
stocks were prepared in pure DMSO, and these were then serially
sonicated and diluted down to 500 M (0.34% DMSO) in PBS (pH 7.4)
at room temperature. A total of 4.8 ml of sample was loaded in
OptiSeal 4.9-ml tubes (Beckman Instruments Inc., Palo Alto, CA)
in a NVT90 fixed-angle rotor (Beckman Instruments Inc., Palo Alto,
CA) and spun at 105g for 1 hour at room temperature in a Beckman
Coulter Optima L-100 XP Ultracentrifuge (Beckman Coulter Inc.,
IN, USA). The bottom of the tubes was then pierced with a 25-gauge
needle, and 1.0 ml of the liquid was removed. NEFA concentrations
in these were measured using the colorimetric NEFA kit (FUJIFILM
Wako Diagnostics, Mountain View, CA).
Molecular docking
Protein Data Bank structure 1LPA (interfacial activation of the
lipase-procolipase complex by mixed micelles revealed by x-ray
crystallography) was downloaded from the RCSB protein data
bank (80) (rcsb.org) and then imported to the Schrödinger Maestro
(Schrödinger Release 2019-4; Maestro, Schrödinger LLC, New York,
NY, 2019). Using the protein preparation wizard (Schrödinger
Release 2019-4: Protein Preparation Wizard; Epik, Schrödinger LLC,
New York, NY, 2016; Impact, Schrödinger LLC, New York, NY,
2016; Prime, Schrödinger LLC, New York, NY, 2019), 1LPA was pre-
processed at pH 7.4, and a restrained minimization was completed
by converging when the RMSD between iterations was less than
0.30 Å. The processed 1LPA structure was rendered in an aqueous
environment at pH 7.4 in the presence of 150 mM sodium ions and
150 mM chloride ions by using the Desmond System Builder
(Schrödinger Release 2019-4: Desmond Molecular Dynamics System,
D. E. Shaw Research, New York, NY, 2019; Maestro-Desmond
Interoperability Tools, Schrödinger, New York, NY, 2019) within
Schrödinger Maestro to mimic the physiological environment that
the enzyme functions in.
All structures to be docked (GTL, GTP, LLP, LOP, and DLPC)
were downloaded as SDF files from PubChem (81) and imported to
Schrödinger Maestro. The LigPrep (Schrödinger Release 2019-4:
LigPrep, Schrödinger LLC, New York, NY, 2019) feature was used
to obtain the ionization states of the ligands at pH 7.4 and to gener-
ate tautomers of the ligands. An induced fit docking (Schrödinger
Release 2019-4: Induced Fit Docking protocol; Glide, Schrödinger
LLC, New York, NY, 2016; Prime, Schrödinger LLC, New York,
13 of 16
SCIENCE ADVANCES | RESEARCH ARTICLE
NY, 2019) was performed by selecting the four triglycerides as the
ligands to be docked and designating the 1,2-didodecanoyl-sn-glycero-­
3-phosphocholine ligand from the 1LPA crystal structure as the
centroid of the workspace ligand around which the receptor grid
was generated. The ligand diameter midpoint box was constructed
with dimensions of X, 10 Å; Y, 10 Å; and Z, 10 Å; while the receptor
grid was generated with dimensions of X, 30 Å; Y, 30 Å; and Z, 30 Å.
No constraints were imposed to ensure that the docking simulation was
unbiased. The induced fit docking used the OPLS3e force field (82).
GlideScore docking energies were recorded in kilocalories per mole.
Statistical analysis and graphical representation
Independent variables for in vivo studies are shown in box plots in
which the mean (dotted line), median (solid line), and in vitro data
are shown as bar graphs reported as means ± SEM. Line graphs were
used for continuous variables. All values are reported as means ± SD.
Significance levels were evaluated at P < 0.05. Data for multiple groups
were compared by one-way analysis of variance (ANOVA) versus
controls, and values significantly different from controls were shown
as (*) or with the P value mentioned above the corresponding con-
ditions. When comparing two groups, a t test or Mann-Whitney test
was used, depending on the normality of distribution and shown as
(*) when significantly different; alternately, P values are shown in
the graph. Graphing was done using SigmaPlot 12.5 (Systat Software
Inc., San Jose, CA).
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/7/5/eabd6449/DC1
View/request a protocol for this paper from Bio-protocol.
REFERENCES AND NOTES
1. I. C. Funnell, P. C. Bornman, S. P. Weakley, J. Terblanche, I. N. Marks, Obesity: An important
prognostic factor in acute pancreatitis. Br. J. Surg. 80, 484–486 (1993).
2. R. C. Turner, R. McDermott, Clinical predictors of severe acute pancreatitis: Value-adding
the view from the end of the bed. ANZ J. Surg. 84, 672–676 (2014).
3. O. Sadr-Azodi, N. Orsini, Å. Andrén-Sandberg, A. Wolk, Abdominal and total adiposity
and the risk of acute pancreatitis: A population-based prospective cohort study. Am.
J. Gastroenterol. 108, 133–139 (2013).
4. S. Domschke, P. Malfertheiner, W. Uhl, M. Büchler, W. Domschke, Free fatty acids in serum
of patients with acute necrotizing or edematous pancreatitis. Int. J. Pancreatol. 13,
105–110 (1993).
5. P. Noel, K. Patel, C. Durgampudi, R. N. Trivedi, C. de Oliveira, M. D. Crowell, R. Pannala,
K. Lee, R. Brand, J. Chennat, A. Slivka, G. I. Papachristou, A. Khalid, D. C. Whitcomb,
J. P. De Lany, R. A. Cline, C. Acharya, D. Jaligama, F. M. Murad, D. Yadav, S. Navina,
V. P. Singh, Peripancreatic fat necrosis worsens acute pancreatitis independent
of pancreatic necrosis via unsaturated fatty acids increased in human pancreatic necrosis
collections. Gut 65, 100–111 (2016).
6. S. Navina, C. Acharya, J. P. De Lany, L. S. Orlichenko, C. J. Baty, S. S. Shiva, C. Durgampudi,
J. M. Karlsson, K. Lee, K. T. Bae, A. Furlan, J. Behari, S. Liu, T. M. Hale, L. Nichols,
G. I. Papachristou, D. Yadav, V. P. Singh, Lipotoxicity causes multisystem organ failure
and exacerbates acute pancreatitis in obesity. Sci. Transl. Med. 3, 107ra110 (2011).
7. C. de Oliveira, B. Khatua, P. Noel, S. Kostenko, A. Bag, B. Balakrishnan, K. S. Patel,
A. A. Guerra, M. N. Martinez, S. Trivedi, A. M. Cullough, D. M. Lam-Himlin, S. Navina,
D. O. Faigel, N. Fukami, R. Pannala, A. E. Phillips, G. I. Papachristou, E. E. Kershaw,
M. E. Lowe, V. P. Singh, Pancreatic triglyceride lipase mediates lipotoxic systemic
inflammation. J. Clin. Invest. 130, 1931–1947 (2020).
8. R. B. Thandassery, S. Appasani, T. D. Yadav, U. Dutta, A. Indrajit, K. Singh, R. Kochhar,
Implementation of the Asia-Pacific guidelines of obesity classification on the APACHE-O
scoring system and its role in the prediction of outcomes of acute pancreatitis: A study
from India. Dig. Dis. Sci. 59, 1316–1321 (2014).
9. K. Y. Shin, W. S. Lee, D. W. Chung, J. Heo, M. K. Jung, W. Y. Tak, Y. O. Kweon, C. M. Cho,
Influence of obesity on the severity and clinical outcome of acute pancreatitis. Gut Liver 5,
335–339 (2011).
10. R. Premkumar, A. R. Phillips, M. S. Petrov, J. A. Windsor, The clinical relevance of obesity
in acute pancreatitis: Targeted systematic reviews. Pancreatology 15, 25–33 (2015).
Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021
11. K. Al-Tarrah, S. W. Jones, N. Moiemen, J. M. Lord, Potential role of adipose tissue and its
hormones in burns and critically iII patients. Burns, 259–266 (2020).
12. G. C. Fonarow, P. Srikanthan, M. R. Costanzo, G. B. Cintron, M. Lopatin; ADHERE Scientific
Advisory Committee and Investigators, An obesity paradox in acute heart failure:
Analysis of body mass index and inhospital mortality for 108,927 patients in the Acute
Decompensated Heart Failure National Registry. Am. Heart J. 153, 74–81 (2007).
13. P. S. Whiting, G. A. White-Dzuro, F. R. Avilucea, A. C. Dodd, N. Lakomkin, W. T. Obremskey,
C. A. Collinge, M. K. Sethi, Body mass index predicts perioperative complications
following orthopaedic trauma surgery: An ACS-NSQIP analysis. Eur. J. Trauma Emerg. Surg.
43, 255–264 (2017).
14. M. Hartrumpf, R.-U. Kuehnel, J. M. Albes, The obesity paradox is still there: A risk analysis
of over 15 000 cardiosurgical patients based on body mass index. Interact. Cardiovasc.
Thorac. Surg. 25, 18–24 (2017).
15. Y. Zhao, Z. Li, T. Yang, M. Wang, X. Xi, Is body mass index associated with outcomes
of mechanically ventilated adult patients in intensive critical units? A systematic review
and meta-analysis. PLOS ONE 13, e0198669 (2018).
16. A. Subramanian, V. Albert, B. Mishra, S. Sanoria, R. M. Pandey, Association between
the pancreatic enzyme level and organ failure in trauma patients. Trauma Mon. 21,
e20773 (2016).
17. J. Manjuck, J. Zein, C. Carpati, M. Astiz, Clinical significance of increased lipase levels
on admission to the ICU. Chest 127, 246–250 (2005).
18. D. W. Rattner, Z. Y. Gu, G. J. Vlahakes, A. L. Warshaw, Hyperamylasemia after cardiac
Downloaded from http://advances.sciencemag.org/ on February 2, 2021
surgery. Incidence, significance, and management. Ann. Surg. 209, 279–283 (1989).
19. P. E. Cogo, G. Giordano, T. Badon, A. Orzali, L. J. I. Zimmermann, F. Zacchello, P. J. J. Sauer,
V. P. Carnielli, Simultaneous measurement of the rates of appearance of palmitic
and linoleic acid in critically ill infants. Pediatr. Res. 41, 178–182 (1997).
20. K. Kosaka, K. Suzuki, M. Hayakawa, S. Sugiyama, T. Ozawa, Leukotoxin, a linoleate
epoxide: Its implication in the late death of patients with extensive burns. Mol. Cell.
Biochem. 139, 141–148 (1994).
21. F. Liu, X. Long, B. Zhang, W. Zhang, X. Chen, Z. Zhang, ACE2 expression in pancreas may
cause pancreatic damage after SARS-CoV-2 infection. Clin. Gastroenterol. Hepatol. 18,
2128–2130.e2 (2020).
22. B. El-Kurdi, B. Khatua, C. Rood, C. Snozek, R. Cartin-Ceba, V. P. Singh; Lipotoxicity in
COVID-19 Study Group, Mortality from coronavirus disease 2019 increases
with unsaturated fat and may be reduced by early calcium and albumin
supplementation. Gastroenterology 159, 1015–1018.e4 (2020).
23. T. Thomas, D. Stefanoni, J. A. Reisz, T. Nemkov, L. Bertolone, R. O. Francis, K. E. Hudson,
J. C. Zimring, K. C. Hansen, E. A. Hod, S. L. Spitalnik, A. D’Alessandro, COVID-19 infection
alters kynurenine and fatty acid metabolism, correlating with IL-6 levels and renal status.
JCI Insight 5, e140327 (2020).
24. P. Hegyi, Z. Szakács, M. Sahin-Tóth, Lipotoxicity and cytokine storm in severe acute
pancreatitis and COVID-19. Gastroenterology 159, 824–827 (2020).
25. C. Fujii, T. Kawai, K. Azuma, Y. Oguma, F. Katsukawa, H. Hirose, K. Tanaka, S. Meguro,
H. Matsumoto, H. Itoh, Relationships between composition of major fatty acids and fat
distribution and insulin resistance in Japanese. J. Diabetes Res. 2017, 1567467 (2017).
26. S. J. Guyenet, S. E. Carlson, Increase in adipose tissue linoleic acid of US adults in the last
half century. Adv. Nutr. 6, 660–664 (2015).
27. K. Patel, R. N. Trivedi, C. Durgampudi, P. Noel, R. A. Cline, J. P. DeLany, S. Navina,
V. P. Singh, Lipolysis of visceral adipocyte triglyceride by pancreatic lipases converts mild
acute pancreatitis to severe pancreatitis independent of necrosis and inflammation. Am.
J. Pathol. 185, 808–819 (2015).
28. B. Khatua, J. R. Yaron, B. El-Kurdi, S. Kostenko, G. I. Papachristou, V. P. Singh, Ringer’s
lactate prevents early organ failure by providing extracellular calcium. J. Clin. Med. 9, 263
(2020).
29. L. Hodson, C. M. Skeaff, B. A. Fielding, Fatty acid composition of adipose tissue and blood
in humans and its use as a biomarker of dietary intake. Prog. Lipid Res. 47, 348–380 (2008).
30. P. J. W. Lee, A. Bhatt, J. Holmes, A. Podugu, R. Lopez, M. Walsh, T. Stevens, Decreased severity
in recurrent versus initial episodes of acute pancreatitis. Pancreas 44, 896–900 (2015).
31. P. Mentula, M.-L. Kylänpää, E. Kemppainen, H. Repo, P. Puolakkainen, Early inflammatory
response in acute pancreatitis is little affected by body mass index. Scand.
J. Gastroenterol. 42, 1362–1368 (2007).
32. V. P. Deenadayalu, U. Blaut, J. L. Watkins, J. Barnett, M. Freeman, J. Geenen, M. Ryan,
H. Parker, J. T. Frakes, E. L. Fogel, W. B. Silverman, K. S. Dua, G. Aliperti, P. Yakshe, M. Uzer,
W. Jones, J. Goff, M. Temkit, G. A. Lehman, S. Sherman, Does obesity confer an increased
risk and/or more severe course of post-ERCP pancreatitis?: A retrospective, multicenter
study. J. Clin. Gastroenterol. 42, 1103–1109 (2008).
33. S. Sawalhi, H. Al-Maramhy, A. I. Abdelrahman, S. E. G. Allah, S. Al-Jubori, Does
the presence of obesity and/or metabolic syndrome affect the course of acute
pancreatitis?: A prospective study. Pancreas 43, 565–570 (2014).
34. A. Brown, T. James-Stevenson, T. Dyson, D. Grunkenmeier, The panc 3 score: A rapid
and accurate test for predicting severity on presentation in acute pancreatitis. J. Clin.
Gastroenterol. 41, 855–858 (2007).
14 of 16
SCIENCE ADVANCES | RESEARCH ARTICLE
35. K. A. Porter, P. A. Banks, Obesity as a predictor of severity in acute pancreatitis. Int.
J. Pancreatol. 10, 247–252 (1991).
36. C. J. Tsai, Is obesity a significant prognostic factor in acute pancreatitis? Dig. Dis. Sci. 43,
2251–2254 (1998).
37. J. Martinez, J. Sánchez-Payá, J. M. Palazón, J. R. Aparicio, A. Picó, M. Pérez-Mateo, Obesity:
A prognostic factor of severity in acute pancreatitis. Pancreas 19, 15–20 (1999).
38. C. D. Johnson, S. K. C. Toh, M. J. Campbell, Combination of APACHE-II score and an obesity
score (APACHE-O) for the prediction of severe acute pancreatitis. Pancreatology 4, 1–6
(2004).
39. G. I. Papachristou, D. J. Papachristou, H. Avula, A. Slivka, D. C. Whitcomb, Obesity
increases the severity of acute pancreatitis: Performance of APACHE-O score
and correlation with the inflammatory response. Pancreatology 6, 279–285 (2006).
40. B. De Waele, B. Vanmierlo, Y. Van Nieuwenhove, G. Delvaux, Impact of body overweight
and class I, II and III obesity on the outcome of acute biliary pancreatitis. Pancreas 32,
343–345 (2006).
41. L. Sempere, J. Martinez, E. de Madaria, B. Lozano, J. Sanchez-Paya, R. Jover, M. Perez-Mateo,
Obesity and fat distribution imply a greater systemic inflammatory response and a worse
prognosis in acute pancreatitis. Pancreatology 8, 257–264 (2008).
42. R. Hegazi, A. Raina, T. Graham, S. Rolniak, P. Centa, H. Kandil, S. J. O’Keefe, Early jejunal
feeding initiation and clinical outcomes in patients with severe acute pancreatitis. JPEN
J. Parenter. Enteral Nutr. 35, 91–96 (2011).
43. P. J. B. Davis, K. M. Eltawil, B. Abu-Wasel, M. J. Walsh, T. Topp, M. Molinari, Effect of obesity
and decompressive laparotomy on mortality in acute pancreatitis requiring intensive
care unit admission. World J. Surg. 37, 318–332 (2013).
44. J. Katuchova, J. Bober, P. Harbulak, A. Hudak, T. Gajdzik, R. Kalanin, J. Radonak, Obesity
as a risk factor for severe acute pancreatitis patients. Wien. Klin. Wochenschr. 126,
223–227 (2014).
45. E. Guzman Calderon, P. Montes Teves, E. Monge Salgado, Bisap-O: Obesity included
in score BISAP to improve prediction of severity in acute pancreatitis. Rev. Gastroenterol.
Peru 32, 251–256 (2012).
46. J. Suazo-Baráhona, R. Carmona-Sánchez, G. Robles-Díaz, P. Milke-García, F. Vargas-Vorácková,
L. Uscanga-Domínguez, M. Peláez-Luna, Obesity: A risk factor for severe acute biliary
and alcoholic pancreatitis. Am. J. Gastroenterol. 93, 1324–1328 (1998).
47. Y. P. Yeung, B. Y. Lam, A. W. Yip, APACHE system is better than Ranson system
in the prediction of severity of acute pancreatitis. Hepatobiliary Pancreat. Dis. Int. 5,
294–299 (2006).
48. Y. Yashima, H. Isayama, T. Tsujino, R. Nagano, K. Yamamoto, S. Mizuno, H. Yagioka,
K. Kawakubo, T. Sasaki, H. Kogure, Y. Nakai, K. Hirano, N. Sasahira, M. Tada, T. Kawabe,
K. Koike, M. Omata, A large volume of visceral adipose tissue leads to severe acute
pancreatitis. J. Gastroenterol. 46, 1213–1218 (2011).
49. F. Yang, H. Wu, Y. Li, Z. Li, C. Wang, J. Yang, B. Hu, Z. Huang, R. Ji, X. Zhan, H. Xie, L. Wang,
M. Zhang, C. Tang, Prevention of severe acute pancreatitis with octreotide in obese
patients: A prospective multi-center randomized controlled trial. Pancreas 41, 1206–1212
(2012).
50. S. R. Thomson, W. S. Hendry, G. A. McFarlane, A. I. Davidson, Epidemiology and outcome
of acute pancreatitis. Br. J. Surg. 74, 398–401 (1987).
51. D. Yadav, A. B. Lowenfels, The epidemiology of pancreatitis and pancreatic cancer.
Gastroenterology 144, 1252–1261 (2013).
52. T. L. Blasbalg, J. R. Hibbeln, C. E. Ramsden, S. F. Majchrzak, R. R. Rawlings, Changes
in consumption of omega-3 and omega-6 fatty acids in the United States during the 20th
century. Am. J. Clin. Nutr. 93, 950–962 (2011).
53. B. Khatua, R. N. Trivedi, P. Noel, K. Patel, R. Singh, C. de Oliveira, S. Trivedi, V. Mishra,
M. Lowe, V. P. Singh, Carboxyl ester lipase may not mediate lipotoxic injury during severe
acute pancreatitis. Am. J. Pathol. 189, 1226–1240 (2019).
54. C. de Oliveira, B. Khatua, A. Bag, B. El-Kurdi, K. Patel, V. Mishra, S. Navina, V. P. Singh,
Multimodal transgastric local pancreatic hypothermia reduces severity of acute
pancreatitis in rats and increases survival. Gastroenterology 156, 735–747.e10 (2019).
55. S. L. Blamey, C. W. Imrie, J. O'Neill, W. H. Gilmour, D. C. Carter, Prognostic factors in acute
pancreatitis. Gut 25, 1340–1346 (1984).
56. C. W. Imrie, I. S. Benjamin, J. C. Ferguson, A. J. McKay, I. Mackenzie, J. O'Neill,
L. H. Blumgart, A single-centre double-blind trial of trasylol therapy in primary acute
pancreatitis. Br. J. Surg. 65, 337–341 (1978).
57. M. Pini, J. A. Sennello, R. J. Cabay, G. Fantuzzi, Effect of diet-induced obesity on acute
pancreatitis induced by administration of interleukin-12 plus interleukin-18 in mice.
Obesity (Silver Spring) 18, 476–481 (2010).
58. J. A. Williams, M. Korc, R. L. Dormer, Action of secretagogues on a new preparation
of functionally intact, isolated pancreatic acini. Am. J. Physiol. 235, 517–524 (1978).
59. E. Rogalska, S. Ransac, R. Verger, Stereoselectivity of lipases. II. Stereoselective hydrolysis
of triglycerides by gastric and pancreatic lipases. J. Biol. Chem. 265, 20271–20276
(1990).
60. R. F. Oliveira, G. A. Gonçalves, F. D. Inácio, E. A. Koehnlein, C. G. M. De Souza, A. Bracht,
R. M. Peralta, Inhibition of pancreatic lipase and triacylglycerol intestinal absorption by
Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021
a Pinhão Coat (Araucaria angustifolia) extract rich in condensed tannin. Nutrients 7,
5601–5614 (2015).
61. H. van Tilbeurgh, M.-P. Egloff, C. Martinez, N. Rugani, R. Verger, C. Cambillau, Interfacial
activation of the lipase-procolipase complex by mixed micelles revealed by x-ray
crystallography. Nature 362, 814–820 (1993).
62. M.-P. Egloff, F. Marguet, G. Buono, R. Verger, C. Cambillau, H. van Tilbeurgh, The 2.46
A resolution structure of the pancreatic lipase-colipase complex inhibited by a C11 alkyl
phosphonate. Biochemistry 34, 2751–2762 (1995).
63. G. K. Veeramachaneni, K. K. Raj, L. M. Chalasani, J. S. Bondili, V. R. Talluri, High-throughput
virtual screening with e-pharmacophore and molecular simulations study
in the designing of pancreatic lipase inhibitors. Drug Des. Devel. Ther. 9, 4397–4412
(2015).
64. S. M. Camhi, G. A. Bray, C. Bouchard, F. L. Greenway, W. D. Johnson, R. L. Newton,
E. Ravussin, D. H. Ryan, S. R. Smith, P. T. Katzmarzyk, The relationship of waist
circumference and BMI to visceral, subcutaneous, and total body fat: Sex and race
differences. Obesity (Silver Spring) 19, 402–408 (2011).
65. J. Serth, A. Lautwein, M. Frech, A. Wittinghofer, A. Pingoud, The inhibition of the GTPase
activating protein-Ha-ras interaction by acidic lipids is due to physical association
of the C-terminal domain of the GTPase activating protein with micellar structures. EMBO
J. 10, 1325–1330 (1991).
66. P. Dumnicka, D. Maduzia, P. Ceranowicz, R. Olszanecki, R. Drożdż, B. Kuśnierz-Cabala, The
interplay between inflammation, coagulation and endothelial injury in the early phase
Downloaded from http://advances.sciencemag.org/ on February 2, 2021
of acute pancreatitis: Clinical implications. Int. J. Mol. Sci. 18, 354 (2017).
67. WHO Expert Consultation, Appropriate body-mass index for Asian populations and its
implications for policy and intervention strategies. Lancet 363, 157–163 (2004).
68. M. Deurenberg-Yap, G. Schmidt, W. A. van Staveren, P. Deurenberg, The paradox of low
body mass index and high body fat percentage among Chinese, Malays and Indians
in Singapore. Int. J. Obes. Relat. Metab. Disord. 24, 1011–1017 (2000).
69. E. J. Balthazar, D. L. Robinson, A. J. Megibow, J. H. Ranson, Acute pancreatitis: Value of CT
in establishing prognosis. Radiology 174, 331–336 (1990).
70. E. C. Rush, I. Freitas, L. D. Plank, Body size, body composition and fat distribution:
Comparative analysis of European, Maori, Pacific Island and Asian Indian adults. Br.
J. Nutr. 102, 632–641 (2009).
71. E. C. Rush, J. H. Goedecke, C. Jennings, L. Micklesfield, L. Dugas, E. V. Lambert, L. D. Plank,
BMI, fat and muscle differences in urban women of five ethnicities from two countries.
Int. J. Obes. (Lond) 31, 1232–1239 (2007).
72. J. van Grinsven, J. L. A. van Vugt, A. Gharbharan, T. L. Bollen, M. G. Besselink,
H. C. van Santvoort, C. H. J. van Eijck, D. Boerma; Dutch Pancreatitis Study Group, The
association of computed tomography-assessed body composition with mortality
in patients with necrotizing pancreatitis. J. Gastrointest. Surg. 21, 1000–1008 (2017).
73. P. Cohen, M. Miyazaki, N. D. Socci, A. Hagge-Greenberg, W. Liedtke, A. A. Soukas,
R. Sharma, L. C. Hudgins, J. M. Ntambi, J. M. Friedman, Role for stearoyl-CoA desaturase-1
in leptin-mediated weight loss. Science 297, 240–243 (2002).
74. R. J. Craven, R. W. Lencki, Crystallization, polymorphism, and binary phase behavior
of model enantiopure and racemic triacylglycerols. Cryst. Growth Des. 11, 1723–1732
(2011).
75. B. A. van Kuiken, W. D. Behnke, The activation of porcine pancreatic lipase by
cis-unsaturated fatty acids. Biochim. Biophys. Acta 1214, 148–160 (1994).
76. J. Folch, M. Lees, G. H. Sloane Stanley, A simple method for the isolation and purification
of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957).
77. W. R. Morrison, L. M. Smith, Preparation of fatty acid methyl esters and dimethylacetals
from lipids with boron fluoride–methanol. J. Lipid Res. 5, 600–608 (1964).
78. H. Ko, M. E. Royer, A gas-liquid chromatographic assay for plasma free fatty acids.
J. Chromatogr. 88, 253–263 (1974).
79. L. S. Orlichenko, J. Behari, T.-H. Yeh, S. Liu, D. B. Stolz, A. K. Saluja, V. P. Singh,
Transcriptional regulation of CXC-ELR chemokines KC and MIP-2 in mouse pancreatic
acini. Am. J. Physiol. Gastrointest. Liver Physiol. 299, G867–G876 (2010).
80. H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov,
P. E. Bourne, The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).
81. S. Kim, J. Chen, T. Cheng, A. Gindulyte, J. He, S. He, Q. Li, B. A. Shoemaker, P. A. Thiessen,
B. Yu, L. Zaslavsky, J. Zhang, E. E. Bolton, PubChem 2019 update: Improved access
to chemical data. Nucleic Acids Res. 47, D1102–D1109 (2019).
82. E. Harder, W. Damm, J. Maple, C. Wu, M. Reboul, J. Y. Xiang, L. Wang, D. Lupyan,
M. K. Dahlgren, J. L. Knight, J. W. Kaus, D. S. Cerutti, G. Krilov, W. L. Jorgensen, R. Abel,
R. A. Friesner, OPLS3: A force field providing broad coverage of drug-like small molecules
and proteins. J. Chem. Theory Comput. 12, 281–296 (2016).
Acknowledgments: We thank J. N. Singh and R. Cannon for working with us on the
illustration shown in Fig. 7. Funding: This project was supported by R01DK092460 and
R01DK119646 from the NIDDK and PR151612 and PR191945 from the DOD (to V.P.S.). Author
contributions: V.P.S. designed, supervised, and conceptualized the study. Acquisition and
15 of 16
SCIENCE ADVANCES | RESEARCH ARTICLE

analysis of data were facilitated and carried out by B.K., B.E.-K., K.P., C.R., P.N., M.C., J.R.Y., S.K., Submitted 2 July 2020
A.G., M.L., and V.P.S.; while B.K., D.O.F., M.L., and V.P.S. critically evaluated the manuscript. Accepted 11 December 2020
Interpretation of data was done by B.K., B.E.-K., K.P., C.R., M.L., and V.P.S. Manuscript was Published 29 January 2021
drafted by B.K., B.E.-K., K.P., C.R., A.G., and V.P.S. Statistical analysis was done by B.K., B.E.-K., K.P., 10.1126/sciadv.abd6449
M.C., and V.P.S. Funding was obtained by V.P.S. Competing interests: The authors declare
that they have no competing interests. Data and materials availability: All data needed to Citation: B. Khatua, B. El-Kurdi, K. Patel, C. Rood, P. Noel, M. Crowell, J. R. Yaron, S. Kostenko, A. Guerra,
evaluate the conclusions in the paper are present in the paper and/or the Supplementary D. O. Faigel, M. Lowe, V. P. Singh, Adipose saturation reduces lipotoxic systemic inflammation
Materials. Additional data related to this paper may be requested from the authors. and explains the obesity paradox. Sci. Adv. 7, eabd6449 (2021).

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

Khatua et al., Sci. Adv. 2021; 7 : eabd6449 29 January 2021 16 of 16

Adipose saturation reduces lipotoxic systemic inflammation and explains the obesity paradox
Biswajit Khatua, Bara El-Kurdi, Krutika Patel, Christopher Rood, Pawan Noel, Michael Crowell, Jordan R. Yaron, Sergiy
Kostenko, Andre Guerra, Douglas O. Faigel, Mark Lowe and Vijay P. Singh

Sci Adv 7 (5), eabd6449.

DOI: 10.1126/sciadv.abd6449

Downloaded from http://advances.sciencemag.org/ on February 2, 2021

ARTICLE TOOLS http://advances.sciencemag.org/content/7/5/eabd6449

SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2021/01/25/7.5.eabd6449.DC1
MATERIALS

REFERENCES This article cites 81 articles, 9 of which you can access for free
http://advances.sciencemag.org/content/7/5/eabd6449#BIBL

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Use of this article is subject to the Terms of Service

Science Advances (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 New
York Avenue NW, Washington, DC 20005. The title Science Advances is a registered trademark of AAAS.
Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of
Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial
License 4.0 (CC BY-NC).