The Journal of Nutrition

Obesity and Eating Disorders

Decreasing the Ratio of Dietary Linoleic to

α-Linolenic Acid from 10 to 4 by Changing
Only the Former Does Not Prevent Adiposity
or Bone Deterioration in Obese Mice
Jay J Cao, Brian R Gregoire, Kim G Michelsen, and Matthew J Picklo Sr

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Grand Forks Human Nutrition Research Center, Agricultural Research Service, USDA, Grand Forks, ND, USA

Background: Linoleic acid (LA; 18:2n–6) has been considered to promote low-grade chronic inflammation and adiposity.
Studies show adiposity and inflammation are inversely associated with bone mass.
Objectives: This study tested the hypothesis that decreasing the dietary ratio of LA to α-linolenic acid (ALA, 18:3n–3),
while keeping ALA constant, mitigates high-fat diet (HF)-induced adiposity and bone loss.
Methods: Male C57BL/6 mice at 6 wk old were assigned to 4 treatment groups and fed 1 of the following diets ad
libitum for 6 mo: a normal-fat diet (NF; 3.85 kcal/g and 10% energy as fat) with the ratio of the PUFAs LA to ALA at 6;
or HFs (4.73 kcal/g and 45% energy as fat) with the ratio of LA to ALA at 10:1, 7:1, or 4:1, respectively. ALA content in
the diets was kept the same for all groups at 1% energy. Bone structure, body composition, bone-related cytokines in
serum, and gene expression in bone were measured. Data were analyzed using 1-factor ANOVA.
Results: Compared with those fed the NF, mice fed the HFs had 19.6% higher fat mass (P < 0.01) and 13.5% higher
concentration of serum tartrate-resistant acid phosphatase (TRAP) (P < 0.05), a bone resorption cytokine. Mice fed the
HFs had 19.5% and 12.2% lower tibial and second lumbar vertebral bone mass, respectively (P < 0.01). Decreasing the
dietary ratio of LA to ALA from 10 to 4 did not affect body mass, fat mass, serum TRAP and TNF-α, or any bone structural
parameters.
Conclusions: These data indicate that decreasing the dietary ratio of LA to ALA from 10 to 4 by simply reducing LA
intake does not prevent adiposity or improve bone structure in obese mice. J Nutr 2020;150:1370–1378.

Keywords: linoleic acid, α-linolenic acid, ratio of LA to ALA, bone structure, obesity, high-fat diet

Introduction inflammation and adiposity. ALA can be converted to EPA

(20:5n–3) and DHA (22:6n–3) in humans but the conversion
Obesity, a major public health problem of growing prevalence
rate is usually <5% depending on the concentration of n–
in the United States (1), is associated with chronic low-
6 LC-PUFAs in the diet (7). ALA, EPA, and DHA are n–3
grade inflammation (2). Evidence suggests that the increased
PUFAs considered anti-inflammatory and may prevent obesity
production of proinflammatory cytokines is involved in the
by decreasing adipogenesis through inhibition of peroxisome
development of obesity-related bone deterioration by increasing
proliferator–activated receptor-γ (PPARγ ) (6, 8, 9).
bone resorption and osteoclast activity (3–5).
Based on data from NHANES 2015–2016, American diets
Modification of the content of dietary fatty acids, such as n–3
are high in n–6 PUFAs (mean LA intake: 17.10 g/d) and deficient
long-chain PUFAs (n–3 LC-PUFAs), n–6 PUFAs, and the ratio of
in n–3 PUFAs (mean ALA intake: 1.84 g/d) with a dietary ratio
n–6 to n–3 PUFAs, with the aim of decreasing inflammation and
of LA to ALA of ∼10:1 (10). The abundance of LA in the
reducing adiposity is an important research focus. Linoleic acid
American diet is due in part to the use of soybean oil which has
(LA; 18:2n–6) and α-linolenic acid (ALA; 18:3n–3) are essential
an LA to ALA ratio of 8:1 (11). Although essential for certain
PUFAs because all mammals cannot synthesize these fatty acids
functions, it is thought that high intakes of LA could inhibit the
and must obtain them from the diet. These n–6 and n–3 PUFAs
desaturation and elongation of ALA to n–3 LC-PUFAs which
are metabolized via a multitude of pathways yielding numerous
are beneficial to health (12, 13). Therefore, limiting the intake
biologically active compounds with distinct and often opposing
of LA in order to reduce the ratio of n–6 to n–3 PUFAs has
physiological functions (6). LA is a precursor for arachidonic
been suggested by some researchers to reduce inflammation and
acid (ARA; 20:4n–6) and the substrate for the production of
for prevention of obesity and obesity-related chronic diseases
proinflammatory eicosanoids, such as PGE2 ; therefore, some
(8, 14).
suggest that intake of n–6 PUFAs promotes low-grade chronic

Published by Oxford University Press on behalf of the American Society for Nutrition 2020. This work is written by (a) US Government employee(s) and is in the
public domain in the US.
Manuscript received October 16, 2019. Initial review completed November 18, 2019. Revision accepted February 7, 2020.
1370 First published online March 5, 2020; doi: https://doi.org/10.1093/jn/nxaa044.
 Contradictory evidence exists as to whether excessive intake
of n–6 PUFAs (mainly LA) and a high ratio of n–6 to
n–3 PUFAs are linked to the pathogenesis of cardiovascular
diseases or skeletal health (15–18). The inconsistent findings
could be due to many studies that not only modified the
amount of LA but also modified the amount of ALA and/or
EPA + DHA to achieve the desired ratio. However, in reviewing
30 prospective observational studies from 13 countries with
68,659 participants, Marklund et al. (19) concluded that higher
circulating and tissue concentrations of LA, and possibly ARA,
were associated with lower risk of cardiovascular disease, a
finding contrary to what would be hypothesized.
Whether the ratio of n–6 to n–3 PUFAs in the form of
LA:ALA affects bone health in diet-induced obesity has not been
investigated. We hypothesized that decreasing the dietary ratio
of LA to ALA would reduce adiposity and adiposity-induced
bone deterioration in an obese mouse model. In this study, we
evaluated the impact of varying ratios of LA to ALA from 10
to 4 on bone health in an obese mouse model by changing the
amount of LA in the diets. Because the total quantity of n–6
and n–3 PUFAs or the type of n–3 PUFAs may likely affect bone
health, we kept ALA amounts constant at 1% energy for all
obesogenic diets in this study.
Methods
Animals and treatments
Male C57BL/6 mice, 6 wk old, were obtained from Charles River
Laboratories (Wilmington, MA). Upon arrival, mice were individually
placed in Plexiglas® ventilated cages located in an environmentally
controlled pathogen-free facility with a 12-h light/12-h dark cycle.
The animal protocol for the study was approved by the USDA,
Agricultural Research Service, Grand Forks Human Nutrition Research
Center Institutional Animal Care and Use Committee. Animals were
maintained and processed in accordance with the NIH Guide for the
Care and Use of Laboratory Animals. Mice were fed Purina Rat Chow
#5012 (Ralston-Purina) while acclimating to the animal facility for 2 d.
Mice were randomly assigned by body weight to 4 groups (n = 12–
13/group) and fed ad libitum for 6 mo normal-fat purified diets (NF;
3.85 kcal/g, 10% energy as fat) based on AIN-93G (20) or high-
fat diets (HF; 4.73 kcal/g) with n–6 PUFAs at either 10%, 7%, or
4% energy (HF10, HF7, and HF4), respectively. Tibia trabecular bone
volume/total volume (BV/TV) was used as the primary outcome for
power calculation. It was estimated that 13 mice/group were needed
to have 90% power to detect a difference of 4% in tibia trabecular
BV/TV among the 3 dietary ratios of LA to ALA, assuming a within-
group SD of 3% and α = 0.05. These ratios were chosen because
Supported by USDA Agricultural Research Service grant #3062-51000-053-00D
(to JJC).
Author disclosures: The authors report no conflicts of interest.
USDA is an equal opportunity provider and employer. Mention of trade names
or commercial products in this publication is solely for the purpose of providing
specific information and does not imply recommendation or endorsement by
the USDA. The findings and conclusions in this article are those of the authors
and should not be construed to represent any official USDA or US Government
determination or policy.
Supplemental Tables 1 and 2 are available from the “Supplementary data” link
in the online posting of the article and from the same link in the online table of
contents at https://academic.oup.com/jn.
Address correspondence to JJC (e-mail: jay.cao@usda.gov).
Abbreviations used: ALA, α-linolenic acid; ARA, arachidonic acid; BALP, bone-
specific alkaline phosphatase; BMD, bone mineral density; BV, bone volume;
Fabp4, fatty acid binding protein 4; HF, high-fat diet; HF4, HF7, and HF10, high-
fat diet with 45% energy from fat and the ratio of LA to ALA at 4:1, 7:1,
and 10:1, respectively; LA, linoleic acid; LC-PUFA, long-chain PUFA; L2, second
lumbar vertebra; NF, normal-fat diet (10% energy from fat); PPARg, peroxisome
proliferator–activated receptor-γ ; TRAP, tartrate-resistant acid phosphatase; TV,
total volume; μCT, micro computed tomography.
typical Western diets have a ratio of LA to ALA at 10:1 based on data
from NHANES 2015–2016 (10) and decreasing the ratio of n–6 to
n–3 PUFAs is associated with lower bone mineral density (BMD) (17).
Diets were designed in such a way that ALA content in the diets was
kept the same for all diets at 1% energy, which is above the minimum
requirement (0.68% energy) for rodents (21) and similar to the intake
in humans (10). As a result, the ratios of LA to ALA for the 3 HFs were
10:1, 7:1, and 4:1, respectively, whereas the ratio was 6:1 for the NF
which is similar to the NF in our previous studies (3, 22). Diets were
formulated and prepared in-house with a combination of high-oleic
sunflower oil (Natural Oils International Inc.), palm oil (Natural Oils
International Inc.), flaxseed oil (Dyets Inc.), and safflower oil (Natural
Oils International Inc.) to achieve the desired ratios of n–6 to n–3
PUFAs. Oils were analyzed for their fatty acid compositions before being
used in diet formulation. The diets were formulated based on energy,
with essential nutrient concentrations being adjusted to be greater in
the HFs than in the NF (Supplemental Table 1). The ratios by analyses
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were 6.0:1, 10.0:1, 6.9:1, and 4.0:1 for the NF, HF10, HF7, and HF4,
respectively, which were very close to the original estimates.
Mice had free access to diets and water throughout the study. Body
mass was recorded once every 2 wk and food consumption was recorded
for 2 consecutive days every 2 wk.
Mice were killed via intraperitoneal injection of a cocktail of
ketamine:xylazine (Animal Health Co. and Phoenix Scientific, respec-
tively) at 100 and 10 mg/kg body weight for ketamine and xylazine,
respectively. Samples were collected and processed as previously
described (3).
Body composition
Body composition (fat compared with lean mass) was measured
1 d before killing by an EchoMRI-100 whole body composition
analyzer (Echo Medical Systems, LLC) according to the manufacturer’s
instructions and as described in detail previously (22).
Bone structure evaluation with micro computed
tomography
The tibia and the second lumbar vertebra (L2) of each mouse
were analyzed for bone structure using a Scanco micro computed
tomography (μCT) scanner 40 (Scanco Medical AG) as described
previously (3, 23). The recommended guidelines for μCT scanning (24)
and bone histomorphometry nomenclature were followed (25).
Measurements of serum biochemical markers
Serum concentrations of bone biochemical markers were determined
by using commercial anti-mouse ELISA kits according to the man-
ufacturers’ instructions: bone-specific alkaline phosphatase (BALP)
(Antibodies-online, Inc.), osteocalcin (Biomedical Technologies Inc.),
leptin (ALPCO Diagnostics), TNF-α (R&D Systems), and tartrate-
resistant acid phosphatase 5b (TRAP) (Immunodiagnostic System). The
same cytokine was measured for all samples in 1 batch preparation on
the same day to minimize variation.
Fatty acid determination
Bone samples were pulverized using a liquid nitrogen–cooled stainless
steel BioPulverizer (Biospec Products, Inc.). Lipids were extracted from
serum, pulverized bone samples (∼10 mg), or diet samples (∼50 mg)
using the method as described previously (3, 26).
Measurement of mRNA in bone
Total RNA was obtained from pulverized bone samples using Trizol
(Invitrogen), reverse transcribed, and amplified and quantified by using
a Sequence Detection System (SDS 7300) as previously described (3).
Relative mRNA expression was normalized to the expression of Gapdh
mRNA in the same sample. Supplemental Table 2 presents the sequence
information of the oligonucleotide primers. All oligonucleotide primers
for PCR amplification were designed using PrimerQuest software,
synthesized, and purified by HPLC by Integrated DNA Technologies.
Linoleic acid on bone structure of obese mice 1371
Statistical analyses
Data are expressed as mean ± SD. To test for differences between
groups, data were analyzed using 1-factor ANOVA (JMP version 12.1.0,
SAS Institute, Inc.). Data were tested for normality using the Shapiro–
Wilk test and homogeneity of variances using Levene’s test in JMP.
Tukey–Kramer post hoc contrasts were performed to compare the
4 groups if the main effect was significant. Planned comparison, or a
priori contrast, was performed to test whether the mean of the 3 HF
groups differed from that of the NF group. In all analyses, P < 0.05
and P ≥ 0.05 were considered statistically significant or nonsignificant,
respectively.
Results
Food and energy intake, body mass, and body
composition
Mean food intake (Figure 1A) by animals fed the HFs was less
than (P < 0.01) that by those fed the NF, but total energy intake
was similar (P ≥ 0.05), due to the higher energy content of the
HF (3.85 compared with 4.73 kcal/g for the NF and the HF,
respectively). Among the HF groups, the ratio of LA to ALA
did not affect either food or energy intake (P ≥ 0.05).
There were no differences in the initial body mass of mice
between groups (Figure 1B). Starting from day 14 to the end of
the study, body mass of the pooled HF groups was higher than
that of those fed the NF (P < 0.01). Among the HF groups, the
ratio of n–6 to n–3 PUFAs did not affect body mass throughout
the study (P ≥ 0.05). We further examined the extent to which
the HF and the ratio of LA to ALA affected body composition
with an EchoMRI-100 whole body composition analyzer. Mice
of the pooled HF groups had 19.6% and 7.2% greater fat
mass and lean mass, respectively (Figure 1C), than those fed
the NF. There were no differences in fat mass and lean mass
between the 3 HF groups with different ratios of LA to ALA
(P ≥ 0.05).
Fatty acid content in serum and bone
Compared with those fed the NF, mice of the pooled HF groups
had higher serum concentrations of palmitic acid (16:0), oleic
acid (18:1n–9), ARA, and DHA and lower LA, ALA, and EPA
(Table 1). The HF did not affect serum total SFA, n–6, and n–3
PUFA concentrations (P ≥ 0.05). Decreasing the ratio of LA to
ALA while keeping ALA constant in the HFs decreased serum
LA and increased EPA and DHA (P < 0.05) but did not affect
ALA or total n–3 PUFA concentrations (P ≥ 0.05). Compared
with the HF10, the HF4 had lower serum LA and higher EPA
and DHA. As expected, decreasing the ratio of LA to ALA in the
HFs also decreased the ratio of n–6 to n–3 PUFAs in serum with
HF10 (5.49 ± 0.60) > HF7 (4.79 ± 0.28) > HF4 (3.80 ± 0.79)
(all P < 0.05).
Mice fed the HFs had higher palmitic acid, 18:1n–9, and
LA, and lower ALA and EPA in bone (Table 2) than those
fed the NF (P < 0.01). Decreasing the dietary ratio of LA
to ALA while keeping ALA constant in the HFs decreased
LA and total n–6 PUFAs with HF10 (22.8 ± 1.9) > HF7
(18.5 ± 0.9) > HF4 (13.6 ± 1.8) (mean ± SD; g/100g total
fatty acids; all P < 0.05). Femur ALA and total n–3 PUFA
content were not affected by the dietary ratios. The total
content of EPA + DHA in bone was not affected by the HF
(P ≥ 0.05). Similarly, decreasing the ratio of LA to ALA in the
HFs decreased the ratio of n–6 to n–3 PUFAs in bone with HF10
(6.68 ± 1.63) > HF7 (4.42 ± 0.74) > HF4 (3.17 ± 0.58)
(all P < 0.05).
1372 Cao et al.
Bone structure parameters
Bone structure was scanned and analyzed by nondestructive
μCT to evaluate the extent to which dietary fat and the ratio
of n–6 to n–3 PUFAs affected distal and mid-diaphysis of the
tibia and L2 (Table 3). For tibial bone structure, mice fed the
HFs had lower trabecular BV/TV by 19.5%, trabecular number
by 15.3%, connectivity density by 27%, and BMD by 38%, and
higher trabecular separation by 23%, than those fed the NF. For
bone structure at L2, the HFs decreased BV/TV by 13.5% and
BMD by 9% while increasing Structure Model Index, compared
with the NF. The HFs did not affect cortical bone parameters of
the tibial mid-diaphysis.
Among the HF groups, the ratio of LA to ALA did not
affect any bone structure parameters of the tibia and L2
(P ≥ 0.05).
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Serum biochemical markers and gene expression in
bone
Mice of the pooled HF groups had 59%, 14.7%, and 13.8%
higher serum concentrations of TNF-α (P < 0.01) (Figure
2A), glucose (P < 0.01) (Figure 2B), and TRAP (P < 0.05)
(Figure 2C), respectively, than those of the NF group. Serum
osteocalcin (Figure 2D) was not affected by the HFs (P ≥ 0.05)
and was higher in mice fed the HF10 than in those fed the
HF4 (P < 0.05). Serum BALP concentration (Figure 2E) was
not affected by the HFs or the ratio of LA to ALA. Among
the HF groups, the ratio of LA to ALA did not affect serum
concentrations of TNF-α, glucose, and TRAP (P ≥ 0.05).
We measured the gene expression of peroxisome
proliferator–activated receptor-γ (Pparg) and fatty acid
binding protein 4 (Fabp4) in bone due to their role in adipocyte
differentiation. Pparg is a ligand-activated nuclear receptor
and is indispensable for adipocyte differentiation and Fabp4
is highly expressed in adipocytes and highly induced during
adipocyte differentiation (27). The HFs increased expression
of Pparg (P < 0.05) and Fabp4 (P < 0.01) in bone (Figure 3).
The HFs did not affect gene expression of leptin, Trap5, and
osteocalcin in femur. Among the HF groups, altering the
ratio of LA to ALA did not affect gene expression in femur
(P ≥ 0.05).
Discussion
In this study we investigated whether modifying the ratio of LA
to ALA by reducing LA intake, while keeping dietary ALA as
a percentage of energy intake constant, would reduce adiposity
and ameliorate adiposity-induced bone structure deterioration
in obese mice. We used a diet-induced obesity mouse model
with animals being fed diets with 45% energy as fat but varying
ratios of LA to ALA for 6 mo. With this animal model, we and
others have shown that obesity induced by an HF is detrimental
to bone by increasing bone resorption, tissue inflammation, and
osteoclast activity, and decreasing bone mass at various bone
sites (3, 4, 28, 29). As expected and consistent with previous
findings, in this study mice fed the HF for 6 mo had increased
body fat mass and serum concentrations of TNF-α and TRAP,
and lower bone mass at proximal tibia and L2, compared
with those fed the NF, despite significantly higher body mass,
indicative of elevated inflammation and bone resorption and
bone structural deterioration.
The major findings of this study were that after consuming
the respective diet for 6 mo, 1) LA content in serum and bone
decreased whereas ARA content in serum and bone did not
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FIGURE 1 Food and energy intake (A), body mass (B), and body composition (C) in male mice fed either an NF or an HF with the ratio of LA
to ALA at 10:1, 7:1, or 4:1 for 6 mo. Food intake was recorded for 2 consecutive days every 2 wk. Data are mean ± SD (n = 13 except n = 12
for HF10). Labeled means without a common letter differ, P < 0.05. The pooled HF data were compared with the NF data (a priori contrast).
∗∗ Different from pooled HF groups, P < 0.01. ALA, α-linolenic acid; HF4, HF7, and HF10, high-fat diet with 45% energy from fat and the ratio of

LA to ALA at 4:1, 7:1, and 10:1, respectively; LA, linoleic acid; NF, normal-fat diet with 10% energy from fat.

Linoleic acid on bone structure of obese mice 1373

TABLE 1 Major fatty acids in serum of male growing mice fed either a purified NF or an HF with the ratio of LA to ALA at 10:1, 7:1,
or 4:1 for 6 mo1

Fatty acid, g/100 g total A priori contrast (NF

fatty acids NF (n = 13) HF10 (n = 12) HF7 (n = 13) HF4 (n = 13) ANOVA P value vs. pooled HF) P value
16:0 21.0 ± 2.00b 22.2 ± 1.43a,b 21.8 ± 0.60a,b 22.6 ± 1.22a 0.043 0.013
18:0 8.75 ± 2.06 8.37 ± 0.51 8.09 ± 0.56 7.58 ± 0.61 0.095 0.060
20:0 0.12 ± 0.04 0.08 ± 0.03 0.10 ± 0.03 0.11 ± 0.05 0.858 0.444
22:0 0.46 ± 0.25b 0.32 ± 0.03b 0.37 ± 0.07b 0.66 ± 0.06a <0.001 0.820
16:1n–7 2.58 ± 0.55a 0.81 ± 0.19c 1.01 ± 0.12b,c 1.31 ± 0.23b <0.001 <0.001
18:1n–7 1.21 ± 0.38a 0.65 ± 0.07c 0.74 ± 0.09b,c 0.92 ± 0.17b <0.001 <0.001
18:1n–9 9.28 ± 1.29c 11.6 ± 1.36b 13.5 ± 0.83b 18.3 ± 3.69a <0.001 <0.001
18:2n–6 19.4 ± 2.67a 15.8 ± 1.96b 13.9 ± 0.88b 11.4 ± 1.23c <0.001 <0.001
20:3n–6 1.15 ± 0.31c 1.31 ± 0.29b,c 1.50 ± 0.17a,b 1.70 ± 0.37a <0.001 <0.001
20:4n–6 20.6 ± 6.68b 26.2 ± 3.64a 26.2 ± 1.97a 21.9 ± 3.87a,b 0.004 0.007
22:4n–6 1.14 ± 1.00 0.71 ± 0.24 0.51 ± 0.22 0.76 ± 0.42 0.127 0.036

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18:3n–3 0.35 ± 0.16a 0.12 ± 0.06b 0.15 ± 0.06b 0.15 ± 0.10b <0.001 <0.001
20:5n–3 0.70 ± 0.22a 0.30 ± 0.07b 0.41 ± 0.05b 0.62 ± 0.12a <0.001 <0.001
22:5n–3 0.34 ± 0.11 0.33 ± 0.19 0.26 ± 0.08 0.26 ± 0.10 0.238 0.204
22:6n–3 6.39 ± 0.90c 7.29 ± 1.11b,c 8.00 ± 0.56a,b 8.33 ± 0.74a <0.001 <0.001
1
Values are mean ± SD. Labeled means in a row without a common letter differ, P < 0.05. The pooled HF data were compared with the NF data (a priori contrast). ALA,
α-linolenic acid; HF, high-fat diet; HF4, HF7, and HF10, high-fat diet with 45% energy from fat and the ratio of LA to ALA at 4:1, 7:1, and 10:1, respectively; LA, linoleic acid; NF,
normal-fat diet with 10% energy from fat.

change in mice as the ratio of LA to ALA decreased from 10:1 to
4:1; 2) ALA concentrations in serum and bone were unaltered,
as expected by design; and 3) the changes in ratios of LA to ALA
in diets, and ratios of n–6 to n–3 PUFAs in serum and bone were
not accompanied by any changes in fat mass, proinflammatory
and bone resorption cytokines in serum, or bone structural
parameters in tibia or lumbar vertebrae.
The beneficial effects of n–3 LC-PUFAs, such as EPA
and DHA, on bone health and body composition and their
anti-inflammatory mechanisms have been well established
(12, 13, 30–34) in various animal models. We recently
also demonstrated that increasing n–3 LC-PUFA–rich fish
oil in the diet ≤3% energy reduces inflammation and bone
resorption, decreases adiposity, and mitigates HF-induced bone
deterioration in growing mice fed an HF but not in those fed an
NF (35).
However, the influence of the intake of n–6 PUFAs and
the ratio of n–6 to n–3 PUFAs on health outcomes has been
inconsistent and controversial (8, 36, 37). Overconsumption
of n–6 PUFAs, in addition to SFAs, and the resulting high
ratio of n–6 to n–3 PUFAs have conventionally been implicated
in the increased rates of obesity and other associated chronic
diseases such as cardiovascular disease and osteoporosis (8,
14, 37, 38). LA is of primary concern because it accounts
for >95% of dietary n–6 PUFAs. The average mean US
intake of LA, according to NHANES 2015–2016 data for
adults, is 17.1 g/d and the intake of ARA is 0.16 g/d
(10). The purported detrimental effects of LA are largely
based on the assumption that elevated intake of LA would
increase circulating and tissue concentrations of n–6 ARA
and systemic inflammation because ARA is the precursor for
some eicosanoids with proinflammatory properties and elevated

TABLE 2 Major fatty acids in femur of male growing mice fed either a purified NF or an HF with the ratio of LA to ALA at 4:1, 7:1, or
10:1 for 6 mo1

Fatty acid, g/100 g total A priori contrast (NF

fatty acids NF (n = 13) HF10 (n = 12) HF7 (n = 13) HF4 (n = 13) ANOVA P value vs. pooled HF) P value
16:0 17.0 ± 0.79b 18.6 ± 0.81a 19.2 ± 0.73a 18.8 ± 0.57a <0.001 <0.001
18:0 4.51 ± 0.81 4.35 ± 0.88 4.70 ± 0.66 4.60 ± 1.35 0.839 0.912
20:0 0.12 ± 0.04 0.08 ± 0.03 0.10 ± 0.03 0.11 ± 0.05 0.061 0.074
22:0 0.20 ± 0.05a,b 0.13 ± 0.03c 0.17 ± 0.04b,c 0.23 ± 0.08a <0.001 0.157
16:1n–7 11.5 ± 1.23a 4.18 ± 0.73c 4.83 ± 0.49b,c 5.22 ± 0.83b <0.001 <0.001
18:1n–7 3.00 ± 0.56a 1.33 ± 0.14b 1.54 ± 0.19b 1.68 ± 0.22b <0.001 <0.001
18:1n–9 30.8 ± 2.38c 41.9 ± 3.14b 43.5 ± 2.13b 48.0 ± 4.22a <0.001 <0.001
18:2n–6 18.2 ± 1.82a 17.1 ± 0.77a 12.5 ± 0.56b 8.34 ± 0.35c <0.001 <0.001
20:2n–6 0.17 ± 0.03a 0.17 ± 0.04a 0.16 ± 0.04a 0.10 ± 0.04b <0.001 0.066
20:4n–6 3.98 ± 0.78 4.55 ± 2.33 4.71 ± 0.99 4.23 ± 1.34 0.620 0.296
22:4n–6 0.49 ± 0.11 0.61 ± 0.16 0.63 ± 0.13 0.50 ± 0.15 0.029 0.062
18:3n–3 1.32 ± 0.23a 0.63 ± 0.08b 0.65 ± 0.05b 0.63 ± 0.11b <0.001 <0.001
20:5n–3 0.09 ± 0.03a 0.03 ± 0.02b 0.04 ± 0.02b 0.06 ± 0.04a,b <0.001 <0.001
22:5n–3 0.60 ± 0.13a 0.40 ± 0.10b 0.49 ± 0.09a,b 0.48 ± 0.12a,b 0.002 <0.001
22:6n–3 3.13 ± 1.12 2.54 ± 0.79 3.13 ± 0.85 3.30 ± 1.08 0.273 0.670
1
Values are mean ± SD. Labeled means in a row without a common letter differ, P < 0.05. The pooled HF data were compared with the NF data (a priori contrast). ALA,
α-linolenic acid; HF, high-fat diet; HF4, HF7, and HF10, high-fat diet with 45% energy from fat and the ratio of LA to ALA at 4:1, 7:1, and 10:1, respectively; LA, linoleic acid; NF,
normal-fat diet with 10% energy from fat.

1374 Cao et al.

TABLE 3 Bone structural properties of tibia and lumbar vertebrae from male mice fed either a purified NF or an HF with the ratio of
LA to ALA at 4:1, 7:1, or 10:1 for 6 mo1

A priori contrast (NF

Fatty acid NF (n = 13) HF10 (n = 12) HF7 (n = 13) HF4 (n = 13) ANOVA P value vs. pooled HF) P value
Proximal tibia (trabecular bone)
TV, mm3 2.60 ± 0.18 2.62 ± 0.20 2.55 ± 0.09 2.51 ± 0.17 0.359 0.497
BV, mm3 0.21 ± 0.04a 0.16 ± 0.03b 0.17 ± 0.05a,b 0.17 ± 0.04b 0.014 0.001
BV/TV, % 8.18 ± 1.13a 6.20 ± 1.29b 6.80 ± 2.06a,b 6.68 ± 0.16a,b 0.018 0.003
Tb.N, mm−1 3.52 ± 0.26a 2.81 ± 0.48b 3.08 ± 0.54a,b 3.04 ± 0.43b 0.002 <0.001
Tb.Th, mm 0.05 ± 0.002 0.05 ± 0.003 0.05 ± 0.001 0.05 ± 0.004 0.735 0.976
Tb.Sp, mm 0.28 ± 0.02b 0.37 ± 0.07a 0.34 ± 0.08a,b 0.33 ± 0.06a,b 0.013 0.003
Conn.Dn, mm−3 30.4 ± 9.2 20.4 ± 6.8 23.9 ± 13.8 22.1 ± 9.9 0.088 0.016
SMI 2.55 ± 0.21 2.58 ± 0.21 2.64 ± 0.20 2.59 ± 0.17 0.696 0.430
BMD, mg HA/cm3 101 ± 21a 68 ± 40a,b 65 ± 52a,b 52 ± 49b 0.038 0.006
Tibial mid-diaphysis (cortical bone)

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B.Ar, mm2 0.65 ± 0.03 0.66 ± 0.04 0.66 ± 0.03 0.67 ± 0.05 0.772 0.396
T.Ar, mm2 1.07 ± 0.05 1.08 ± 0.06 1.08 ± 0.06 1.11 ± 0.09 0.331 0.238
Me.Ar, mm2 0.42 ± 0.03 0.42 ± 0.03 0.42 ± 0.05 0.45 ± 0.05 0.245 0.271
Ct.Th, mm 0.212 ± 0.006 0.214 ± 0.010 0.214 ± 0.011 0.211 ± 0.012 0.839 0.793
Second lumbar vertebrae (trabecular bone)
TV, mm3 2.43 ± 0.17 2.59 ± 0.15 2.60 ± 0.42 2.59 ± 0.24 0.337 0.070
BV, mm3 0.47 ± 0.04 0.44 ± 0.04 0.46 ± 0.09 0.43 ± 0.07 0.342 0.160
BV/TV, % 19.5 ± 1.8a 17.2 ± 1.5b 17.6 ± 1.4a,b 16.6 ± 2.7b 0.003 <0.001
Tb.N, mm−1 5.09 ± 0.30 4.99 ± 0.17 5.06 ± 0.22 4.94 ± 0.27 0.373 0.204
Tb.Th, mm 0.05 ± 0.002 0.05 ± 0.002 0.05 ± 0.002 0.04 ± 0.002 0.268 0.067
Tb.Sp, mm 0.19 ± 0.01 0.22 ± 0.01 0.19 ± 0.01 0.19 ± 0.01 0.147 0.092
Conn.Dn, mm−3 168 ± 19.0 154 ± 12.2 166 ± 17.7 155 ± 26.2 0.178 0.131
SMI 1.27 ± 0.20b 1.52 ± 0.12a 1.52 ± 0.10a 1.59 ± 0.31a 0.001 <0.001
BMD, mg HA/cm3 220 ± 15a 196 ± 24b 205 ± 15a,b 201 ± 14b 0.006 <0.001
1
Values are mean ± SD. Labeled means in a row without a common letter differ, P < 0.05. The pooled HF data were compared with the NF data (a priori contrast). ALA,
α-linolenic acid; B.Ar, cross-sectional bone area; BMD, bone mineral density; BV, bone volume; Conn.Dn, connectivity density; Ct.Th, cortical thickness; HA, hydroxyapatite; HF,
high-fat diet; HF4, HF7, and HF10, high-fat diet with 45% energy from fat and the ratio of LA to ALA at 4:1, 7:1, and 10:1, respectively; LA, linoleic acid; Me.Ar, cross-sectional
medullary area; NF, normal-fat diet with 10% energy from fat; SMI, Structure Model Index; T.Ar, cross-sectional total area; Tb.N, trabecular number; Tb.Sp, trabecular
separation; Tb.Th, trabecular thickness; TV, total volume.

inflammation is considered a primary contributor for many
chronic diseases including osteoporosis (36, 37).
However, some eicosanoids from ARA metabolism such
as prostacyclin and lipoxin A4 are anti-inflammatory (36).
Based on available clinical evidence, a Science Advisory from
the American Heart Association concluded that reducing the
consumption of LA <5–10% of energy would be more likely
to increase rather than to decrease the risk of coronary
artery disease (36). A recent comprehensive review of a
global consortium of prospective studies concluded that higher
circulating and tissue LA concentrations are not associated with
higher risk of cardiovascular outcomes, and on the contrary,
that higher concentrations of circulating LA are associated
with lower risk of cardiovascular outcomes (19). In a review
of 36 adult human clinical trials in which changes in dietary
LA intake and blood ARA concentrations were evaluated,
Rett and Whelan (39) concluded that although elevated tissue
ARA concentrations are positively associated with eicosanoid
formation and the risk of various chronic diseases and increased
inflammation, increasing dietary LA does not modify tissue
ARA content in an adult population consuming a Western-type
diet. In this study, we found that in general the content of fatty
acids in serum and bone such as palmitic acid and 18:1n–9 was
reflective of fatty acid content in the diet. However, ARA content
in serum and bone among the 3 HFs did not change with the
dietary LA being decreased from 58 to 23 mg/g diet and the
ratio of LA to ALA decreased from 10:1 to 4:1, likely because
the conversion of LA to ARA is very low, ∼0.2% (40).
Few studies have specifically evaluated the impact of the ratio
of n–6 to n–3 PUFAs on bone in humans and animals. However,
the total amounts of n–6 PUFAs and/or other specific n–3 PUFAs
were modified to achieve the desired ratios in these studies (17,
18, 41–44). To our knowledge, no study has been conducted
on the impact of the ratio of LA to ALA on body composition
and bone of obese animals by changing LA intake while keeping
ALA constant. For example, Watkins et al. (18) investigated the
effects of dietary ratio of n–6 to n–3 PUFAs varying from 1:1
to 24:1 on bone formation in rats fed the respective diet for
42 d and reported that decreasing the ratio reduced bone PGE2
production and ARA content and increased the activity of the
serum bone formation marker, BALP. However, in that study the
LA and ALA contents of the diets were both modified and n–3
PUFAs from fish oil were also added in all 4 experimental diets
and bone structure was not evaluated (18). Although the ratio
of LA to ALA may have played a role in the observed changes in
that study, the results may also have been influenced by changes
in intake of ALA and/or EPA + DHA.
Longo et al. (43) compared the effects of diets with the
ratio of n–6 to n–3 PUFAs at 10:1 or 5:1 by modifying the
source of n–3 PUFAs using flaxseed or menhaden oil on bone in
growing or estrogen-deficient rats and found that the impact of
ratio on bone was minimal in that only 1 bone parameter, i.e.,
cortical area, was increased by the ratio of 5:1 with flaxseed
in 3-mo-old growing but not 6-mo-old ovariectomized rats.
Unlike our study, energy from n–6 PUFAs was similar for the
2 flaxseed diets with different ratios of n–6 to n–3 PUFAs

Linoleic acid on bone structure of obese mice 1375

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FIGURE 2 Serum concentrations of TNF-α (A), glucose (B), TRAP (C), osteocalcin (D), and BALP (E) in male mice fed either an NF or an HF
with the ratio of LA to ALA at 10:1, 7:1, or 4:1 for 6 mo. Data are mean ± SD (n = 13 except n = 12 for HF10). Labeled means without a common
letter differ, P < 0.05. The pooled HF data were compared with the NF data (a priori contrast). ALA, α-linolenic acid; BALP, bone-specific alkaline
phosphatase; HF4, HF7, and HF10, high-fat diet with 45% energy from fat and the ratio of LA to ALA at 4:1, 7:1, and 10:1, respectively; LA,
linoleic acid; NF, normal-fat diet with 10% energy from fat; TRAP, tartrate-resistant acid phosphatase.

(14.8% compared with 13.5%, for the ratios of 10:1 and
5:1, respectively) and energy from n–3 PUFAs was modified
(1.5% compared with 2.7% for the 2 diets). In another study
with piglets in which only plant oils were used in the diets,
Weiler and Fitzpatrick-Wong (44) found that decreasing the
ratio from 9:1 to 4.5:1 did not affect growth, bone mass, or
ARA content in plasma, adipose, and brain, a finding similar to
ours.
FIGURE 3 Expression of Pparg, Lep, Trap5, Bglap, and Fabp4 in
femur in male mice fed either an NF or an HF with the ratio of LA
to ALA at 10:1, 7:1, or 4:1 for 6 mo. Data are mean ± SD (n = 13
except n = 12 for HF10). Labeled means without a common letter
differ, P < 0.05. The pooled HF data were compared with the NF data
(a priori contrast). ALA, α-linolenic acid; Bglap, osteocalcin; Fabp4,
fatty acid binding protein 4; HF4, HF7, and HF10, high-fat diet with
45% energy from fat and the ratio of LA to ALA at 4:1, 7:1, and 10:1,
respectively; LA, linoleic acid; Lep, leptin; NF, normal-fat diet with 10%
energy from fat; Pparg, peroxisome proliferator–activated receptor-γ ;
Trap5, tartrate-resistant acid phosphatase 5.
1376 Cao et al.
There are few studies that have investigated the impact of
ratio of n–6 to n–3 PUFAs on bone in humans. Rajaram et al.
(41) reported that altering the dietary ratio of n–6 to n–3 PUFAs
from 10:1 to 2:1 by modification of ALA and/or EPA/DHA
did not change bone turnover in an 8-wk crossover study in
healthy humans. In a human study with similar design to this
animal experiment in that dietary ALA was kept constant at
1% energy and the effects of the ratio of LA to ALA on plasma
phospholipids in healthy men were evaluated, Liou et al. (45)
reported that varying the ratio from 8:1 to 2:1 did not influence
plasma ALA or ARA, but increased phospholipid EPA. We also
found that mice fed the HF4 had higher EPA in serum, but
not in bone, than those fed the HF10. This difference may
in part be the result of different tissue distribution kinetics.
However, the increase in serum EPA was not accompanied
by any changes in parameters regarding body composition or
bone structure. It is likely that the increase in concentration
of serum EPA was too small to have detectable physiological
effects. In this study, serum EPA concentrations increased from
0.30 to 0.62 g/100 fatty acids with the ratio of LA to ALA
being decreased from 10:1 to 4:1. Whereas, in our previous
study, serum EPA concentration was 3.25 g/100 fatty acids in
HF with 3% energy from Menhaden oil and bone mass was
increased at this fish oil content compared with a no-fish-oil
diet (35).
In our previous study with fish oil supplementation,
increasing n–3 PUFAs (with fish oil ≤3% energy) while keeping
the ratios of n–6 to n–3 PUFAs the same among the HFs (ranging
between 12.3:1 and 12.7:1) reduced adiposity and improved
the bone structure of HF-induced obese mice (35). The results
from that fish oil supplementation and the current study indicate
that the amount and type of n–3 PUFAs in the diets are more
important than the ratio of n–6 to n–3 PUFAs on adiposity and
bone metabolism.
In this study, the diets were designed in such a way that
ALA content was kept constant for all diets at 1% energy,
which is above the minimal requirement for rodents (21), and
SFAs were comparable across the 3 HFs. Although this design
limited our ability to investigate the effects of much higher
or lower ratios of LA to ALA on bone-related outcomes, we
consider decreasing the ratio of LA to ALA from 10:1 is valuable
because it is comparable with that consumed in humans (10).
Whereas a diet with a ratio of LA to ALA ≤4:1 without fish oil
supplementation may not be practical in humans, animal studies
with this ratio can provide scientific evidence and mechanisms
through which LA may affect bone health in an obese condition.
Ideally a pair-fed design should have been included within the
HF contents; however, this is not a concern because diets were
formulated (adjusted) based on energy density and the intake
of energy was the same for the NF and HF groups, therefore,
the intake of micronutrients was the same for the HF and NF
and not affected by the ratio of LA to ALA. Although the
effects of n–6 and n–3 PUFAs on bone would likely be found
in animals with elevated inflammatory status such as in aging
and estrogen deficiency (46–48), obesity is also associated with
a state of elevated chronic low-grade inflammation and bone
deterioration, a focus of the present study (3).
In summary, our data demonstrate that HF-induced obesity
is detrimental to bone microstructure in mice and decreasing
intake of LA while maintaining constant ALA does not alter
serum and bone content of ARA, adiposity, or bone structure
in obese mice. These findings suggest that the type and
the amount of n–3 PUFAs rather than the amount of n–6
PUFAs or the ratio of LA to ALA should be considered as a
preferred dietary strategy to reduce adiposity and improve bone
health.
Acknowledgments
We thank James Lindlauf for help in preparation of the
experimental diets, food intake measurement, and animal care
and Dr. Michael Bukowski for fatty acid analysis of samples.
The authors’ responsibilities were as follows—JJC: contributed
to implementation, data analysis, and data interpretation;
JJC and MJP: contributed to manuscript preparation; BRG
and KGM: contributed to the experiment execution, data
collection, and manuscript review; and all authors: con-
tributed to the study design and read and approved the final
manuscript.
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