Fuller et al. Biology of Sex Differences (2018) 9:41
https://doi.org/10.1186/s13293-018-0199-1
RESEARCH
Open Access
Potential adverse effects of botanical
supplementation in high-fat-fed female
mice
Scott Fuller1,2, Yongmei Yu1, Tamra Mendoza1, David M. Ribnicky3, William T. Cefalu1 and Z. Elizabeth Floyd1*
Abstract
Background: Insulin resistance underlies metabolic syndrome and is associated with excess adiposity and visceral
fat accumulation, which is more frequently observed in males than females. However, in young females, the prevalence
of metabolic syndrome is rising, mainly driven by accumulation of abdominal visceral fat. The degree to which
sex-related differences could influence the development of insulin resistance remains unclear, and studies of
potential therapeutic strategies to combat metabolic syndrome using rodent models have focused predominantly on
males. We therefore evaluated the effects of two nutritional supplements derived from botanical sources, an extract of
Artemisia dracunculus L. (termed PMI5011) and Momordica charantia (commonly known as bitter melon), on female
mice challenged with a high-fat diet in order to determine if dietary intake of these supplements could ameliorate
obesity-induced insulin resistance and metabolic inflexibility in skeletal muscle.
Methods: Body composition, physical activity and energy expenditure, fatty acid oxidation, insulin signaling, and gene
and protein expression of factors controlling lipid metabolism and ectopic lipid accumulation were evaluated in female
mice fed a high-fat diet supplemented with either PMI5011 or bitter melon. Statistical significance was assessed
by unpaired two-tailed t test and repeated measures ANOVA.
Results: PMI5011 supplementation resulted in increased body weight and adiposity, while bitter melon did not
induce changes in these parameters. Pyruvate tolerance testing indicated that both supplements increased hepatic
glucose production. Both supplements induced a significant suppression in fatty acid oxidation in skeletal muscle
homogenates treated with pyruvate, indicating enhanced metabolic flexibility. PMI5011 reduced lipid accumulation in
skeletal muscle, while bitter melon induced a downward trend in lipid accumulation in the skeletal muscle and liver.
This was accompanied by transcriptional regulation of autophagic genes by bitter melon in the liver.
Conclusions: Data from the current study indicates that dietary supplementation with PMI5011 and bitter melon
evokes a divergent, and generally less favorable, set of metabolic responses in female mice compared to effects
previously observed in males. Our findings underscore the importance of considering sex-related variations in
responses to dietary supplementation aimed at combating metabolic syndrome.
Keywords: Sex, Botanical, Skeletal muscle, Insulin, Metabolic syndrome, Liver, Obesity
* Correspondence: elizabeth.floyd@pbrc.edu
1
Pennington Biomedical Research Center, Louisiana State University System,
Baton Rouge, LA 70808, USA
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Fuller et al. Biology of Sex Differences (2018) 9:41
Background
The prevalence of metabolic syndrome (MetS) has risen
to epidemic proportions in recent decades and constitutes
an emergent threat to global public health [1–3]. MetS is
characterized by abdominal obesity, insulin resistance,
dyslipidemia, and hypertension; these factors predispose
individuals to greater risk for cardiovascular disease,
chronic kidney disease, and several types of cancer [4–7].
Although research effort focused on MetS continues to intensify as the disease burden associated with the condition
becomes ever more acute, an issue that remains relatively
unclear is the extent to which MetS differentially affects
males versus females [8, 9]. In light of evidence that there
are sex-related differences in the prevalence and pathophysiology of MetS, research specifically evaluating MetS
in females offers the potential for developing sex-specific
treatment modalities for chronic diseases including obesity, cardiovascular, and metabolic diseases [10, 11]. However, the research undertaken thus far in both human and
animal models has focused primarily on males [8]. Thus,
there remains a relatively underserved need for investigation into MetS specifically in females.
Current recommendations for the prevention and treatment of MetS emphasize lifestyle modifications that
include dietary changes, weight loss, and exercise [12].
However, despite persistent advice to the public emphasizing the importance of lifestyle factors in the prevention
and management of MetS, the rising prevalence of the
syndrome and its associated pathologies is a testament to
the difficulties encountered in successfully implementing
long-term behavioral changes. Pharmacotherapy has been
employed with varying degrees of success, although disadvantages associated with prolonged use of pharmaceuticals
include side effects, cost, and public access [13, 14], which
is a particularly acute problem in the developing world
where the incidence of MetS is escalating at an alarming
rate and availability of pharmaceuticals can be a limiting
factor [15]. Natural products derived from food sources
therefore represent an attractive complementary therapy
for the treatment of MetS due to their relative safety and
tolerability compared to several of the drugs currently
available, although vigilance remains necessary to ensure
proper safety standards for nutritional supplements in the
marketplace [16, 17].
Several species of the plant genus Artemisia have demonstrated potential for ameliorating MetS in laboratory
studies [18–20]. In particular, Artemisia dracunculus L., or
Russian tarragon, is a perennial herb with a documented
history of medicinal use as an anti-diabetic [21]. Previous
studies in our laboratory provide evidence that an ethanolic
extract of A. dracunculus L. termed PMI5011 favorably
modulates insulin signaling, lipid metabolism, and glucose
homeostasis primarily via effects on skeletal muscle both
in vitro and in obese male mice with established insulin
Page 2 of 14
resistance [22–25]. We recently extended these findings by
demonstrating that early dietary supplementation with
PMI5011 in male mice protects against the development of
insulin resistance and ectopic lipid accumulation in the
skeletal muscle and liver independent of any changes in
adiposity or body mass [26]. Momordica charantia, commonly known as bitter melon, has been demonstrated to
have anti-hyperglycemic and hypolipidemic [27, 28] effects
and is a staple of the traditional diet in Okinawa, where
rates of mortality and morbidity due to chronic diseases
are among the lowest in the world [29]. Evidence from
mechanistic studies in diabetic rodents indicates that bitter
melon enhances insulin sensitivity by decreasing serum
levels of the pro-inflammatory modulators tumor necrosis
factor-alpha (TNF-α) and interleukin-6 (IL-6), decreasing
expression of suppressor of cytokine signaling-3 (SOCS-3)
and c-Jun N-terminal kinase (JNK), and augmenting
insulin-stimulated tyrosine phosphorylation of the insulin
receptor substrate-1 (IRS-1) [27, 30]. Multiple studies in
male rodents from our laboratories and others demonstrate that bioactives in bitter melon improve insulin sensitivity, possibly via reduced skeletal muscle and hepatic lipid
accumulation [31–33]. The effect of bitter melon extract
on hepatic lipids is attributed to reduced glucose production and lipid synthesis in the liver [34].
While experimental evidence demonstrates that PMI5011
and bitter melon favorably modulate insulin responsiveness
and lipid metabolism in male rodents [26, 35], sex differences in the prevalence and pathogenesis of MetS raise the
possibility that females might respond differently to dietary
intervention. Given the current scarcity of data in females,
we sought to evaluate the effectiveness of PMI5011 and bitter melon in female mice in order to clarify whether sex
differences in the response to botanical dietary supplementation might be evident. In the present study, we evaluated
the hypothesis that dietary supplementation with PMI5011
or bitter melon prior to the onset of high-fat diet-induced
obesity prevents development of high-fat diet-related insulin resistance in female C57BL/6 mice. The novel results reported herein indicate that female mice respond in a
generally less favorable manner to supplementation with
PMI5011 and bitter melon compared to the pattern previously reported in males [26]. The findings of the present
study therefore indicate that sex is an important biological
variable that merits serious consideration when evaluating
the safety and efficacy of dietary interventions aimed at
combating metabolic syndrome.
Methods
Sourcing and characterization of PMI5011 extract
The PMI5011 botanical extract from Artemisia dracunculus L. was provided by the Botanical and Dietary Supplement Research Center at Pennington Biomedical Research
Center. Bitter melon was obtained from Verdure Sciences
Fuller et al. Biology of Sex Differences (2018) 9:41
(Noblesville, IN). Detailed information about quality control, preparation, and biochemical characterization of
PMI5011 has been previously reported [24, 25, 36–40].
Experimental animals
Reproductively intact female C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME). The
estrous cycle was not evaluated at the end of the
4-month study as evidence shows estrous cycle stage
does not significantly contribute to variability of molecular or metabolic outcomes measured in female mice
[41]. All animal experiments were approved by the Pennington Biomedical Research Center Animal Care and
Use Committee (protocol #922). The animals were singly
housed with a 12-h light-dark cycle at 24 °C. At 4 weeks
of age, mice of similar body weight were randomly
assigned (n = 14/group) to a defined low-fat diet (LFD;
10% kcal fat, Research Diets, #D12450H) or the low-fat
diet supplemented with 1% w/w PMI5011 or bitter
melon. After 4 weeks, the LFD only-fed mice were
switched to a high-fat diet (HFD; 45% kcal fat, Research
Diets, #D12451) and maintained as the control group.
The mice fed a LFD supplemented with either PMI5011
or bitter melon were switched to the HFD supplemented
with PMI5011 or bitter melon, formulated with the same
mass botanical extract/kcal (equivalent to 1.2% w/w
HFD) as contained in the LFD and were fed ad libitum
for 3 months thereafter. The 45% fat content is similar
to the fat intake (30–40% of energy intake) for adult
men and women in the USA [42]. Body weight and food
intake were measured weekly, and body composition
was measured bi-weekly by nuclear magnetic resonance
(Bruker, Billerica, MA). Activity, food intake, and indirect calorimetry were measured at 12 weeks on each diet
(TSE PhenoMaster). The mice were acclimated to the
TSE chambers for 2 days prior to data collection over
4 days. At the end of the study, the mice were euthanized between 7 and 11 AM. Human insulin (Humulin,
Eli Lilly, Indianapolis, IN) was administered to a subgroup of the control and botanical-supplemented mice
(7/group) at a dose of 1.5 U/kg 10 min prior to euthanasia to assay insulin signaling.
Glucose and insulin tolerance tests
For the glucose (GTT) and insulin (ITT) tolerance tests,
the amount of glucose or insulin administered was normalized to body weight, which did not vary significantly
among groups (23.0∓ 0.57 g body weight for females) at
10 weeks on the HFDs. Female mice were fasted 4 h
prior to administering 2 g/kg body weight of glucose/
mouse (GTT) or 1 U/kg body weight of insulin/mouse
(HumulinR) (ITT) by intraperitoneal injection.
Page 3 of 14
Blood chemistry
Fasting glucose levels were measured in whole blood using
a Breeze2 glucometer (Bayer, Leverkusen, Germany). Fasting insulin levels were assayed via ELISA (Crystal Chem,
Downers Grove, IL). Serum nonesterified fatty acids
(Abcam, Cambridge, MA), triglycerides (Eagle Diagnostics, Cedar Hill, TX), and total cholesterol (Cell Biolabs,
San Diego, CA) levels were assayed according to manufacturers’ instructions. The index of homeostasis model assessment for insulin resistance, e.g., HOMA-IR [insulin
(mU/L) × glucose (mM)/22.5], of each animal was calculated from fasting glucose and insulin levels [43]. Triglyceride levels in the skeletal muscle and liver were assayed
according to Folch et al. [44] and reported as milligrams
per deciliter. Visible fat was carefully removed from the
tissue before assaying triglyceride levels.
Immunohistochemistry
A portion of mixed gastrocnemius muscle and liver was
fixed in 10% formalin, embedded in paraffin, and sectioned onto slides. The sections were hematoxylin and
eosin (H&E) stained and scanned (NanoZoomer Digital
Pathology, Hamamatsu Corp., Bridgewater, NJ).
Fatty acid oxidation assay
Mixed gastrocnemius muscle homogenates were prepared as described [45]. Palmitate oxidation was assessed
in the whole muscle homogenates as described by Hulver et al [46] with 14CO2 collected over 60 min. When
present, pyruvate was added at a final concentration of
10 mM. CO2 levels were normalized to total protein and
palmitate oxidation reported as nmol CO2/mg protein/h.
Analysis of protein expression
Skeletal muscle and liver lysates were prepared from
powdered tissue by homogenizing in 25 mM HEPES,
pH 7.4, 1% Igepal CA630, 137 mM NaCl, 1 mM PMSF,
10 μg/ml aprotinin, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 10 mM Na4P2O7, 100 mM NaF, and 2 mM NaVO4
using a Sonifier 450 homogenizer (VWR, Radnor, PA).
The samples were centrifuged at 14,000×g for 10 min at
4 °C. Protein concentrations were determined using a
BCA assay (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s instructions. The tissue
supernatants (50 μg) were resolved by SDS-PAGE and
subjected to immunoblotting using chemiluminescence
detection (Thermo Fisher Scientific, Rockford, IL) and
quantified as described [47]. Nitrocellulose membranes
were incubated with antibodies for 1–2 h at room
temperature or overnight at 4 °C as indicated. An additional file provides detailed information about each antibody used (see Additional file 1).
Fuller et al. Biology of Sex Differences (2018) 9:41
Analysis of gene expression
Total RNA was purified from powdered the skeletal
muscle tissue or liver using Direct-zol RNA MiniPrep
(ZYMO Research, Irvine, CA). In each case, RNA
(500 ng) was reverse transcribed using Multiscribe Reverse Transcriptase (Applied Biosystems, Thermo Fisher
Scientific, Waltham, MA) with random primers at 37 °C
for 2 h. Real-time PCR was performed with PowerUP
SYBR Green Master Mix (Applied Biosystems) according
to the manufacturer’s instructions, using the 7900
Real-Time PCR system and universal cycling conditions
(50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for
15 s and 60 °C for 1 min; followed by 95 °C for 15 s, 60 °C
for 15 s, and 95 °C for 15 s). The assays were performed in triplicate, and the results were normalized
to Cyclophilin B mRNA and analyzed using the
2−ΔΔCTmethod with the control diet used as the calibrator. The gene list is provided in an additional file
(see Additional file 2).
Statistical analysis
Normal distribution of the data for glucose and insulin
levels, food intake, and body weight was determined
using the D’Agostino-Pearson omnibus K2 normality
test. Western blot data was quantified using Un-Scan-It
software (version 3, Silk Scientific). Statistical significance was determined using an unpaired two-tailed t
Page 4 of 14
test or repeated measures ANOVA. All statistical analysis was carried out using JMP Pro13 (SAS Institute)
and GraphPad Prism 5 software (GraphPad Software, La
Jolla, CA). Variability is expressed as the mean ± standard deviation.
Results
Effect of PMI5011 and bitter melon on body composition
in female mice
A number of previous studies established that the ethanolic extract from A. dracunculus termed PMI5011 enhances insulin signaling in skeletal muscle and improves
insulin sensitivity on a preexisting background of insulin
resistance in vitro and in vivo in male mice [22, 24, 25,
48, 49]. To determine if high-fat diet-related insulin resistance can be prevented in female mice by dietary supplementation with either PMI5011 or bitter melon, we
carried out a feeding study in female C56BL/6J mice
given a LFD alone or supplemented with PMI5011 or
bitter melon for 1 month beginning at 4 weeks of age.
After 1 month, the diet was switched to a 45% HFD
alone or supplemented with PMI5011 or bitter melon
and these diets were maintained for 3 months. As shown
in Fig. 1a, c, PMI5011 supplementation resulted in a statistically significant increase in body weight due to increased adiposity unrelated to food intake in female
mice. Bitter melon supplementation did not affect body
Fig. 1 Effects of PMI5011and bitter melon on body composition in female mice: PMI5011 supplementation resulted in a significant increase in
body weight due to increased adiposity unrelated to food intake, whereas bitter melon did not affect body weight (a). PMI5011 and bitter melon
resulted in a non-significant reduction in food intake on a high-fat diet (b). An increase in percent fat mass and corresponding decrease in fat-free
mass was observed in females supplemented with PMI5011, while bitter melon did not affect fat mass or fat-free mass (c). Statistical significance was
set at p < 0.05, as determined by repeated measures ANOVA. Variability is expressed as mean ± SD. LFD low-fat diet, HFD high-fat diet
Fuller et al. Biology of Sex Differences (2018) 9:41
weight. Both PMI5011 and bitter melon supplementation resulted in a non-significant reduction in food intake in females on the HFD (Fig. 1b). While PMI5011
supplementation resulted in a significant increase in percent fat mass in females (Fig. 1c), bitter melon supplementation in female mice did not change body weight or
body composition (Fig. 1a, c).
Effect of PMI5011 and bitter melon on energy
expenditure and substrate utilization in female mice
Our data on body weight and adiposity revealed that
the female mice did not become obese in response to
HFD, a finding that is consistent with other published
data [50–52]. We then evaluated activity and energy
expenditure to determine which of these factors could
account for the resistance to weight gain and adiposity observed in our female mice. PMI5011 did not
affect energy expenditure or activity, but we observed
decreased activity and energy expenditure in response
to bitter melon supplementation (Fig. 2). However,
these small decreases in already high levels of activity
and energy expenditure were not associated with body
weight gain or increased adiposity. Neither botanical
Page 5 of 14
supplement significantly altered the respiratory exchange ratio (Fig. 2).
Carbohydrate and lipid metabolism in response to
PMI5011 and bitter melon supplementation
We next assayed the effects of PMI5011 and bitter melon
on glucose homeostasis and insulin sensitivity in female
mice via glucose and insulin tolerance testing along with
fasting glucose and insulin levels. Although neither glucose
nor insulin tolerance testing shows any statistically significant effect on AUC for glucose for either botanical supplement (Fig. 3a, b), we observed that glucose levels were
somewhat higher at the later time points for both
PMI5011 and bitter melon in insulin tolerance testing
(Fig. 3b). This raised the possibility that the supplements
induced alterations in hepatic glucose production, which
we assessed by conducting pyruvate tolerance testing. Repeated measures ANOVA indicated that both supplements
significantly increased glucose production, consistent with
increased gluconeogenesis (Fig. 3c). However, fasting
serum glucose and insulin levels indicate that although neither supplement significantly altered blood glucose concentrations, PMI5011 induced an increase in fasting serum
Fig. 2 Effects of PMI5011 and bitter melon on energy expenditure and substrate utilization in high-fat-fed female mice: Bitter melon, but not
PMI5011, decreased energy expenditure and physical activity. RER was not altered by supplementation with bitter melon or PMI5011. Statistical
significance was set at p < 0.05, as determined by least squares means analysis. Variability is expressed as mean ± SD
Fuller et al. Biology of Sex Differences (2018) 9:41
Page 6 of 14
Fig. 3 PMI5011 and bitter melon alter carbohydrate and lipid metabolism in high-fat-fed female mice: PMI5011 and bitter melon supplementation do
not result in statistically significant alterations in AUC for GTT and ITT (a, b); however, there was a trend toward higher blood glucose levels at the later
time points in the ITT for both PMI5011 and bitter melon (b). Pyruvate tolerance tests showed that both bitter melon and PMI5011 increased hepatic
glucose production, consistent with increased gluconeogenesis (c). Neither PMI5011 nor bitter melon induced statistically significant changes in blood
glucose (d), although PMI5011 supplementation resulted in a trend (p = 0.06) toward elevated fasting insulin (e). Statistically significant modulations
were not detected in HOMA-IR (f), although PMI5011 appears to result in non-significant increase in this parameter. Statistical significance was set at
p < 0.05, as determined by repeated measures ANOVA or unpaired two-tailed t test. Variability is expressed as mean ± SD
insulin (Fig. 3e) that approached statistical significance (p
= 0.06). Collectively, these data suggest that neither botanical supplement favorably alters glucose homeostasis or insulin sensitivity in female mice challenged with a HFD,
with PMI5011 demonstrating a tendency to increase fasting insulin levels despite the lack of a commensurate reduction in fasting glucose. Furthermore, HOMA-IR data
suggests that PMI5011 negatively modulates whole-body
insulin sensitivity, whereas bitter melon supplementation
did not affect HOMA-IR (Fig. 3f).
Effect of PMI5011 and bitter melon on lipid metabolism
in female mice
To determine the effect of PMI5011 and bitter melon on
blood lipids in high-fat-fed female mice, we assayed total
cholesterol, serum triglycerides, and free fatty acids.
PMI5011 modestly increased total cholesterol while bitter
melon induced a statistically significant increase in total
cholesterol (Fig. 4a). Serum triglycerides were significantly
increased by PMI5011, whereas bitter melon did not appear
to have an effect (Fig. 4b). Both botanicals tended to increase
Fuller et al. Biology of Sex Differences (2018) 9:41
Page 7 of 14
Fig. 4 PMI5011 and bitter melon modulate lipid metabolism in high-fat-fed female mice: Bitter melon induced a statistically significant increase in
total cholesterol, while PMI5011 modestly increased total cholesterol (a). Serum triglycerides were significantly increased by PMI5011, but not by
bitter melon (b). Both supplements induced non-significant trends toward increased serum free fatty acids (c). Mixed gastrocnemius homogenates
exhibited an increase in the rate of fatty acid oxidation in response to PMI5011 and bitter melon supplementation (d). Fatty acid oxidation rates in
mixed gastrocnemius homogenates were suppressed when exposed to pyruvate in the female mice supplemented with PMI5011 or bitter melon,
indicating enhanced metabolic flexibility in response to changes in nutrient availability (d). Statistical significance was set at p < 0.05, as determined by
unpaired two-tailed t test. Variability is expressed as mean ± SD
serum free fatty acids, although neither effect was statistically significant (Fig. 4c). To further investigate the effects of
PMI5011 and bitter melon on lipid metabolism in females,
we assessed fatty acid oxidation rates in mixed gastrocnemius homogenates in response to treatment with these botanicals. Results indicate that both supplements increase the
capacity of skeletal muscle to utilize lipid as a metabolic fuel
source in response to a high-fat diet, as shown by significant
increases in fatty acid oxidation rates compared to controls
(Fig. 4d). Moreover, both PMI5011 and bitter melon robustly suppress fatty acid oxidation rates in mixed gastrocnemius homogenates exposed to pyruvate ex vivo as a
surrogate for glucose oxidation (Fig. 4d). Although carbohydrate metabolism is not assessed by pyruvate-mediated suppression of fatty acid oxidation, the results indicate that both
PMI5011 and bitter melon promote metabolic flexibility in
the context of high-fat diet consumption.
Enhanced metabolic flexibility in skeletal muscle in
response to PMI5011 and bitter melon supplementation
is not mediated by transcriptional regulation
In order to investigate the biochemical mechanisms
underlying the improved metabolic flexibility in skeletal
muscle observed in female mice, mRNA and protein
abundance of a range of factors controlling fatty acid metabolism were assayed by qRT-PCR and immunoblotting,
respectively. Overall, neither PMI5011 nor bitter melon
induced changes in the gene expression of transcription
factors (Pgc1a, Ppara, Ppard, Pparg) that regulate fatty
acid oxidation or mitochondrial function (Cpt1b, Cpt2,
Cs). However, PMI5011 increased mRNA and protein
levels of CD36 and bitter melon intake is associated with
increased CD36 protein levels, suggesting increased fatty
acid uptake in the skeletal muscle of the PMI5011 and bitter melon-supplemented females (Fig. 5a, c). Interestingly,
the increased fatty acid oxidation rates with botanical supplementation (Fig. 4d) are not associated with increased
activation of AMPK or increased insulin responsiveness as
measured by AKT phosphorylation (Fig. 5b, c).
Effects of PMI5011 and bitter melon on hepatic gene and
protein expression
The mild elevation in blood glucose in the HFD-fed females (Fig. 3d) and increased blood glucose observed with
the pyruvate tolerance test in the botanical-supplemented
females (Fig. 3c) prompted us to assay gene and protein
Fuller et al. Biology of Sex Differences (2018) 9:41
Page 8 of 14
Fig. 5 Botanical supplement-induced enhanced metabolic flexibility in skeletal muscle is not mediated by transcriptional regulation: Neither PMI5011
nor bitter melon alters overall patterns of mRNA expression (a) or protein abundance (b, c) in biochemical factors regulating fatty acid metabolism or
insulin signaling in high-fat-fed female mice. However, both mRNA and protein abundance of CD36 were increased in the skeletal muscle of mice
supplemented with PMI5011 (a, b). Statistical significance was set at p < 0.05, as determined by unpaired two-tailed t test. Variability is expressed
as mean ± SD
expression of factors controlling hepatic glucose and lipid
metabolism. Although Pgc1a increases, expression of the
lipogenic transcriptional regulator Pgc1b and markers of
mitochondrial function (Cpt1a, Cpt2, Cs) is unchanged
(Fig. 6a). De novo lipogenesis (DNL) does not appear to
be regulated by either botanical. Although Chrebp1 and
Foxo1 levels are modestly upregulated by PMI5011, the
botanicals do not alter srebp-1c expression. Moreover,
SREBP-1c target genes that regulate DNL (Scd1, Fasn) are
not increased by the botanicals or, in the case of Elovl6,
are suppressed (Fig. 6b). In contrast, the gene encoding
PEPCK (Pck1) is significantly upregulated, consistent with
increased gluconeogenesis. However, expression of two
other genes regulating gluconeogenesis, g6pc and pc, is
reduced.
To determine if the botanicals altered signaling events
controlling hepatic glucose and lipid metabolism, we
assayed the phosphorylation status of AMPK, ACC,
FoxO1, and AKT as well as the steady state levels of
SREBP-1c with PMI5011 (Fig. 6c) or bitter melon
(Fig. 6d) supplementation. Hepatic AMPK activity does
not increase in response to supplementation with either
botanical, as indicated by diminished AMPKα1 protein
expression and unaltered ACC phosphorylation. Additionally, acute insulin-dependent AKT phosphorylation
is not enhanced by the botanicals in the female mice.
Failure of the botanicals to enhance insulin responsiveness is also reflected in the absence of changes in
phosphorylation-dependent downregulation of FoxO1.
While SREBP-1c is not regulated by PMI5011, the levels
trend upward with bitter melon supplementation.
Effect of PMI5011 and bitter melon on lipid accumulation
We next assessed lipid content in the skeletal muscle and
liver by histological examination. Analysis by hematoxylin
and eosin staining indicated that both botanicals induced
changes in lipid accumulation in mixed gastrocnemius
muscle and liver (Fig. 7a, c). To assess lipid accumulation
Fuller et al. Biology of Sex Differences (2018) 9:41
Page 9 of 14
Fig. 6 Effects of PMI5011 and bitter melon on hepatic gene and protein expression in high-fat-fed female mice: Both PMI5011 and bitter melon
increased mRNA abundance of Pgc1a, but not other markers of lipid metabolism (a). Markers of de novo lipogenesis are not upregulated while
Cd36 and a marker of gluconeogenesis (Pck1) are upregulated (b). PMI5011 (c) and bitter melon (d) do not regulate the levels of proteins involved in
lipid metabolism or insulin signaling. Statistical significance was set at p < 0.05, as determined by unpaired two-tailed t test. Variability is expressed
as mean ± SD
quantitatively, we performed triglyceride assays on the liver
and mixed gastrocnemius samples. Results showed a reduction in lipid content in mixed gastrocnemius that was
statistically significant with PMI5011 but not bitter melon
(Fig. 7b). Bitter melon supplementation resulted in a
non-significant reduction (p = 0.06) in triglyceride content
in the liver whereas PMI5011 induced a non-significant increase in hepatic triglyceride levels (Fig. 7d) although lipid
accumulation was not readily apparent with H&E staining
on the females fed a high-fat diet alone (Fig. 7a, c).
Fuller et al. Biology of Sex Differences (2018) 9:41
Page 10 of 14
Fig. 7 PMI5011 and bitter melon modulate lipid accumulation in high-fat-fed female mice: H&E staining indicates that both PMI5011 and bitter
melon induce alterations in lipid accumulation in the mixed gastrocnemius muscle and liver (a, c). Triglyceride assays showed that PMI5011
induced statistically significant reductions in triglyceride content in mixed gastrocnemius muscle (b). Bitter melon resulted in a trend (p = 0.06)
toward reduction in liver triglyceride accumulation whereas PMI5011 induced a non-significant increase in hepatic triglyceride levels although
lipid accumulation was not readily apparent with H&E staining on the females fed a high-fat diet alone (d). PMI5011 supplementation induced
some transcriptional regulation in a set of autophagic genes in the skeletal muscle and liver, although this effect was not as pronounced as that
observed in response to bitter melon supplementation (e, f). Statistical significance was set at p < 0.05, as determined by unpaired two-tailed
t test. Variability is expressed as mean ± SD
Autophagy is an important cellular process that is engaged in response to challenges to energy homeostasis
such as fasting and exercise [53]. Moreover, evidence indicates that increased hepatic autophagy is positively associated with an improved serum lipid profile [54] and
there is compelling evidence that hepatic lipid storage is
regulated by autophagy [55]. Autophagic genes are transcriptionally regulated by nutrient conditions [56]. Thus,
we assessed the mRNA levels of a panel of autophagic
genes (atg5, atg7, beclin1, gabarapl1, sqstm1, ulk1, lc3a,
lc3b) in the liver and skeletal muscle. We observed a degree of transcriptional regulation in the muscle and liver
by PMI5011 (Fig. 7e, f ). However, bitter melon induced
robust upregulation in autophagic genes in the liver
(Fig. 7f ). These results are consistent with reduced lipid
accumulation in the liver of the bitter melon-fed female
mice compared to those supplemented with PMI5011.
Discussion
Approximately one third of adults in the USA have
obesity-related metabolic syndrome, defined by the presence of insulin resistance, dyslipidemia, hypertension,
and visceral obesity [57, 58]. Although metabolic syndrome is typically associated with central/visceral obesity
Fuller et al. Biology of Sex Differences (2018) 9:41
in men and post-menopausal women, it is becoming
more prevalent in premenopausal women with central
obesity associated with increased waist circumference
[58]. Thus, it is important to include fertile female rodents in preclinical animal studies designed to test potential therapeutic approaches to combat metabolic
syndrome.
In the current study, we sought to determine if dietary
supplementation with PMI5011 or bitter melon prior to
the onset of high-fat diet-induced obesity prevents development of high-fat diet-related insulin resistance in female
C57BL/6 mice. Although the botanicals increase fatty acid
oxidation capacity in skeletal muscle, lipid profiles are not
improved and early indications of hepatic insulin resistance related to glucose output are observed with botanical
supplementation in the female mice. Thus, our results indicate that dietary supplementation with PMI5011 and
bitter melon is likely more effective in males than females
in preventing high-fat diet-induced insulin resistance. In
part, this is due to the female C57BL/6 mouse resistance
to high-fat diet-induced obesity or insulin resistance, as
found in earlier studies [50–52]. We found that obesity resistance in the female mice is independent of botanical
supplementation although dietary intake of PMI5011 increases body weight and percent fat mass in the females
compared to the high-fat diet alone. PMI5011-mediated
fat accumulation occurs although energy intake and expenditure are comparable to the high-fat diet-fed females.
This surprising finding suggests PMI5011 supplementation may increase the efficiency of calorie absorption in
the gut, resulting in storage of excess energy in adipose
tissue. Interestingly, the rate of body weight gain decreased after 6 weeks on the high-fat diet in all groups
and is associated with a striking dissimilarity in energy expenditure due to higher physical activity levels of the female mice compared to previously reported data in male
mice [26]. Thus, elevated physical activity may account for
resistance to high-fat diet-induced obesity typically observed in the female C57BL/6 mice. It is important to
note, however, that in the present study the mice began
the high-fat diet at 8 weeks of age. The age of onset of
high-fat diet consumption in female C57BL6 mice can be
an important factor in determining weight gain [59], and
it is possible that the absence of weight gain observed in
the females is at least partially accounted for by the age
when the high-fat diet was introduced. Nonetheless, sex
differences in body weight gain, adiposity, activity, and energy expenditure is a notable finding in the present study
and carries implications for future studies aimed at
investigating sex as a biological variable in a nutritional context. The data reported herein indicate that
sex-related differences in physical activity and energy
expenditure can be a determining factor underlying
variable responses to dietary intervention and are
Page 11 of 14
therefore a major consideration in experimental design and interpretation of results.
Next, we employed several methods to investigate the
efficacy of the botanical supplements in modulating insulin sensitivity and glucose homeostasis both systemically and at the individual tissue level. Neither PMI5011
nor bitter melon induced any significant differences in
the responses to glucose or insulin tolerance testing,
supporting the notion that these botanicals do not enhance glucose disposal in high-fat-fed female mice. This
is consistent with the observation that PMI5011 supplementation was associated with a trend toward elevated
fasting serum insulin levels despite the absence of any
commensurate decrease in fasting glucose. Evaluation of
the HOMA-IR, coupled with no increase in skeletal
muscle protein kinase B (AKT) activity, further consolidates the interpretation that neither of the botanicals
improves whole-body glucose homeostasis in female
mice when challenged with a high-fat diet.
We also observed a general pattern of elevated plasma
lipid levels in the female mice associated with both
PMI5011 and bitter melon supplementation that was absent in previous studies of high-fat-fed male mice [26].
However, we note an absence of any correlation between
the botanical-mediated increase in plasma fatty acid
levels and adiposity or insulin sensitivity in females,
consistent with higher fatty acid levels in women independent of fat mass when compared to men [60]. Importantly, skeletal muscle contributes to whole-body
glucose disposal to a greater degree than any other tissue
type and insulin resistance related to dysregulation of
lipid metabolism in skeletal muscle is a defining characteristic of metabolic syndrome and type 2 diabetes [61].
The upward trend in lipid levels in response to the botanicals is met by an increased capacity in the females to
use fatty acids as a fuel source in skeletal muscle. The
botanicals promoted a “metabolically flexible” phenotype
in skeletal muscle that was reflected in the trend toward
a lower respiratory exchange ratio consistent with increased ability to use fatty acids as a fuel source while
consuming a high-fat diet. However, analysis of genes
and proteins involved in the regulation of lipid metabolism indicates enhanced metabolic flexibility is largely
unrelated to transcriptional regulation in skeletal muscle,
although PMI5011 induced a significant increase in cd36
mRNA expression. This finding raises the possibility that
CD36-mediated fatty acid uptake in skeletal muscle in
the females contributes to enhanced fatty acid oxidation
with dietary intake of PMI5011. This mechanism may
also influence fatty acid metabolism in skeletal muscle of
the bitter melon-supplemented females as CD36 protein
levels are increased with either botanical.
However, botanical supplementation in the females
may be associated with increased hepatic glucose output
Fuller et al. Biology of Sex Differences (2018) 9:41
as indicated by higher glucose levels with botanical supplementation at the late time points on the insulin tolerance test, elevated glucose with pyruvate tolerance testing
and increased mRNA levels of pck1 in the liver with botanical supplementation. It is possible that the increased
glucose levels at the later time points with botanical supplementation in the insulin tolerance test is due to counterregulatory mechanisms against hypoglycemia since
these mechanisms occur in male mice at 80 mg/dl glucose
[62], a level observed in the female mice. Even so, physiological adaptation to defend glucose levels seems an unlikely explanation for the increased glucose output upon
pyruvate tolerance testing or the elevated hepatic pck-1
mRNA levels at the end of study.
Insulin mediates hepatic glucose output by regulating
transcription of the rate-determining enzymes in gluconeogenesis: cytosolic phosphenolpyruvate carboxykinase,
encoded by Pck1 and the catalytic subunit of glucose6-phosphatase encoded by G6pc, which is also rate-limiting
in glycogenolysis (reviewed in [63]). Insulin-dependent
phosphorylation of AKT suppresses Pck1 and G6pc via
regulation of FoxO1 and PGC-1α activity. At the same
time, insulin signaling is associated with increased hepatic
de novo lipogenesis (DNL) via ChREBP and SREBP-1c
transcriptional activity [64]. Botanical supplementation does
not significantly affect expression of key transcriptional regulators of DNL at the gene or protein level, and expression
of the fatty acid elongase gene elovl6, a target of SREBP-1
that catalyzes long chain fatty acid formation, is suppressed, supporting decreased DNL. However, the effect
of PMI5011 and bitter melon on the balance between
glucose output and lipid synthesis in the liver appears
to be complex as triglyceride levels trend upward with
PMI5011 and are unchanged with bitter melon.
Pathway-selective hepatic insulin resistance is proposed to explain the failure of insulin to suppress glucose production but support lipogenesis and liver fat
accumulation [65]. However, hepatic triglycerides are
primarily derived from nonesterified fatty acids (NEFAs)
rather than DNL [66] and re-esterification of NEFAs is
driven by substrate availability independent of insulin signaling or transcriptional changes related to lipogenesis [67].
Our data is consistent with a model of botanical-mediated
early pathway-selective insulin resistance in the liver characterized by a failure of insulin signaling to suppress glucose output. Unlike previous studies in male mice, the
botanicals do not enhance insulin signaling in the females.
Thus, DNL is not increased with dietary botanical supplementation when compared to the HFD alone. Increased
uptake of NEFAs with the high-fat diet is supported by the
increased Cd36 expression and the modest upregulation of triglycerides, primarily for PMI5011 supplementation. Thus, increased hepatic glucose output in
response to the botanicals raises concerns about
Page 12 of 14
possible early adverse effects of the botanicals on glucose metabolism in the high-fat-fed females.
Despite evidence suggesting that supplementation with
PMI5011 and bitter melon may adversely affect hepatic
function, we found that there were some advantageous effects of the supplements on ectopic lipid accumulation in
the skeletal muscle and liver. In the skeletal muscle,
PMI5011 significantly reduced triglyceride accumulation
while bitter melon induced a downward trend, consistent
with enhanced fatty acid oxidation in the skeletal muscle.
In the liver, dietary intake of bitter melon decreased triglyceride content to a level that approached statistical significance (p = 0.06) while PMI5011 was associated with
increased hepatic triglycerides. Remarkably, modulation of
lipid accumulation in these tissues was closely associated
with transcriptional regulation of autophagic genes with
the most robust response elicited by bitter melon in the
liver. Transcriptional regulation of autophagy is regulated
by more than 20 transcription factors [56]. Our findings
are the first to identify bitter melon-mediated transcriptional regulation of hepatic autophagy. Interestingly, upregulation of gamma-aminobutyric acid receptor-associated
protein-1 (Gabarapl1), which encodes a ubiquitin-like protein associated with autophagic vesicles, was first identified
as an estrogen-regulated gene [68]. Gabarapl1 is most
robustly upregulated in skeletal muscle in the PMI5011supplemented females where estrogen plays an important
role in skeletal muscle quality in males [69] as well as
females.
Conclusion
The more pronounced effect of PMI5011 supplementation
on improving insulin sensitivity and reducing ectopic lipid
accumulation in the skeletal muscle and liver previously
reported in males on a high-fat diet compared to females
indicates there are sex-related differences in the potential
benefits of dietary intake of PMI5011 in preventing risk
factors for metabolic syndrome. Alternatively, the absence
of any evidence of high-fat diet-induced obesity or insulin
resistance in the females raises the possibly that PMI5011
and bitter melon are effective only in the presence of
obesity-related insulin resistance as observed in our previous animal studies in male mice [25, 26] as well as in vitro
models of insulin resistance in skeletal muscle [22, 24, 48].
In that case, PMI5011 and bitter melon may be effective
in females that develop insulin resistance with obesity. Although our current study does not differentiate between
these two possibilities, our data supporting early selective
hepatic insulin resistance related to glucose production
with PMI5011 or bitter melon dietary supplementation
underscores the potential for untoward effects of botanical
supplementation in females and the importance of considering sex-related variations in metabolism and metabolic
responses to dietary supplementation.
Fuller et al. Biology of Sex Differences (2018) 9:41
Page 13 of 14
Additional files
Additional file 1: Antibody Information. (PDF 13 kb)
5.
Additional file 2: Supporting Information Related to Gene Expression
Analysis. (PDF 81 kb)
6.
Abbreviations
ACC: Acetyl-CoA carboxylase; AKT: Protein kinase B; AMPK: AMP-activated
protein kinase; CPT-1: Carnitine palmitoyltransferase I; DNL: De novo
lipogenesis; HFD: High-fat diet; HOMA-IR: Homeostatic model assessment for
insulin resistance; IL-6: Interleukin-6; IRS-1: Insulin receptor substrate-1; JNK:
c-Jun N-terminal kinase; LFD: Low-fat diet; MetS: Metabolic syndrome;
PEPCK: Phosphoenolpyruvate carboxykinase; SOCS-3: Suppressor of cytokine
signaling-3; TNF-α: Tumor necrosis factor-alpha
Funding
This research is supported by P50AT002776 (NCCIH), NIH8 1P30GM118430-01
(NIGMS, COBRE), and P30 DK072476 (NIDDK, NORC).
Availability of data and materials
The data supporting the conclusions of this article are included within the
article and the additional files (Additional files 1 and 2).
Authors’ contributions
YY and TM contributed to the animal care, sample collection, data, and
statistical analysis. SF, DMR, and ZEF contributed to the data analysis and
interpretation. DMR, WTC, and ZEF contributed to the experimental design.
SF and ZEF wrote the manuscript. All authors read and approved the final
draft.
Ethics approval
All animal experiments were conducted in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory Animals (8th
edition) and approved by the Pennington Biomedical Research Center
Animal Care and Use Committee (protocol #922).
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Consent for publication
Not applicable.
18.
Competing interests
The authors declare that they have no competing interests.
19.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
Pennington Biomedical Research Center, Louisiana State University System,
Baton Rouge, LA 70808, USA. 2School of Kinesiology, University of Louisiana
at Lafayette, Lafayette, LA 70506, USA. 3Biotech Center, Rutgers University,
New Brunswick, NJ 08901, USA.
20.
1
21.
22.
Received: 31 May 2018 Accepted: 27 August 2018
23.
References
1. Aguilar M, Bhuket T, Torres S, Liu B, Wong RJ. Prevalence of the metabolic
syndrome in the United States, 2003–2012. JAMA. 2015;313(19):1973–4.
2. Moore JX, Chaudhary N, Akinyemiju T. Metabolic syndrome prevalence by
race/ethnicity and sex in the United States, National Health and Nutrition
Examination Survey, 1988-2012. Prev Chronic Dis. 2017;14:E24.
3. Park YW, Zhu S, Palaniappan L, Heshka S, Carnethon MR, Heymsfield SB. The
metabolic syndrome: prevalence and associated risk factor findings in the
US population from the Third National Health and Nutrition Examination
Survey, 1988-1994. Arch Intern Med. 2003;163(4):427–36.
4. Johansen D, Stocks T, Jonsson H, Lindkvist B, Bjorge T, Concin H, Almquist
M, Haggstrom C, Engeland A, Ulmer H, et al. Metabolic factors and the risk
of pancreatic cancer: a prospective analysis of almost 580,000 men and
24.
25.
26.
women in the Metabolic Syndrome and Cancer Project. Cancer Epidemiol
Biomark Prev. 2010;19(9):2307–17.
Mottillo S, Filion KB, Genest J, Joseph L, Pilote L, Poirier P, Rinfret S, Schiffrin
EL, Eisenberg MJ. The metabolic syndrome and cardiovascular risk a
systematic review and meta-analysis. J Am Coll Cardiol. 2010;56(14):
1113–32.
Singh AK, Kari JA. Metabolic syndrome and chronic kidney disease. Curr
Opin Nephrol Hypertens. 2013;22(2):198–203.
Stocks T, Bjorge T, Ulmer H, Manjer J, Haggstrom C, Nagel G, Engeland A,
Johansen D, Hallmans G, Selmer R, et al. Metabolic risk score and cancer
risk: pooled analysis of seven cohorts. Int J Epidemiol. 2015;44(4):1353–63.
Iyer A, Kauter K, Brown L. Gender differences in metabolic syndrome: a key
research issue? Endocr Metab Immune Disord Drug Targets. 2011;11(3):
182–8.
Rochlani Y, Pothineni NV, Mehta JL. Metabolic syndrome: does it differ
between women and men? Cardiovasc Drugs Ther. 2015;29(4):329–38.
Dallongeville J, Cottel D, Arveiler D, Tauber JP, Bingham A, Wagner A,
Fauvel J, Ferrieres J, Ducimetiere P, Amouyel P. The association of metabolic
disorders with the metabolic syndrome is different in men and women.
Ann Nutr Metab. 2004;48(1):43–50.
Chen CH, Lin KC, Tsai ST, Chou P. Different association of hypertension and
insulin-related metabolic syndrome between men and women in 8437
nondiabetic Chinese. Am J Hypertens. 2000;13(7):846–53.
Dalle Grave R, Calugi S, Centis E, Marzocchi R, El Ghoch M, Marchesini G.
Lifestyle modification in the management of the metabolic syndrome:
achievements and challenges. Diab Metab Syndr Obes. 2010;3:373–85.
Pladevall M, Riera-Guardia N, Margulis AV, Varas-Lorenzo C, Calingaert B,
Perez-Gutthann S. Cardiovascular risk associated with the use of glitazones,
metformin and sufonylureas: meta-analysis of published observational
studies. BMC Cardiovasc Disord. 2016;16:14.
Herman WH. The global agenda for the prevention of type 2 diabetes. Nutr
Rev. 2017;75(suppl 1):13–8.
Ranasinghe P, Mathangasinghe Y, Jayawardena R, Hills AP, Misra A.
Prevalence and trends of metabolic syndrome among adults in the asiapacific region: a systematic review. BMC Public Health. 2017;17(1):101.
Cohen PA. American roulette--contaminated dietary supplements. N Engl J
Med. 2009;361(16):1523–5.
Cohen PA. Hazards of hindsight--monitoring the safety of nutritional
supplements. N Engl J Med. 2014;370(14):1277–80.
Yamamoto N, Kanemoto Y, Ueda M, Kawasaki K, Fukuda I, Ashida H. Antiobesity and anti-diabetic effects of ethanol extract of Artemisia princeps in
C57BL/6 mice fed a high-fat diet. Food Funct. 2011;2(1):45–52.
Choi Y, Yanagawa Y, Kim S, Whang WK, Park T. Artemisia iwayomogi extract
attenuates high-fat diet-induced obesity by decreasing the expression of
genes associated with adipogenesis in mice. Evid Based Complement
Alternat Med. 2013;2013:915953.
Wang ZQ, Zhang XH, Yu Y, Tipton RC, Raskin I, Ribnicky D, Johnson W,
Cefalu WT. Artemisia scoparia extract attenuates non-alcoholic fatty liver
disease in diet-induced obesity mice by enhancing hepatic insulin and
AMPK signaling independently of FGF21 pathway. Metab Clin Exp. 2013;
62(9):1239–49.
Swanston-Flatt SK, Flatt PR, Day C, Bailey CJ. Traditional dietary adjuncts for
the treatment of diabetes mellitus. Proc Nutr Soc. 1991;50(3):641–51.
Kheterpal I, Coleman L, Ku G, Wang ZQ, Ribnicky D, Cefalu WT. Regulation
of insulin action by an extract of Artemisia dracunculus L. in primary human
skeletal muscle culture: a proteomics approach. Phytother Res. 2010;24(9):
1278–84.
Obanda DN, Hernandez A, Ribnicky D, Yu Y, Zhang XH, Wang ZQ, Cefalu
WT. Bioactives of Artemisia dracunculus L. mitigate the role of ceramides in
attenuating insulin signaling in rat skeletal muscle cells. Diabetes. 2012;61(3):
597–605.
Wang ZQ, Ribnicky D, Zhang XH, Raskin I, Yu Y, Cefalu WT. Bioactives of
Artemisia dracunculus L enhance cellular insulin signaling in primary human
skeletal muscle culture. Metabolism. 2008;57(7 Suppl 1):S58–64.
Wang ZQ, Ribnicky D, Zhang XH, Zuberi A, Raskin I, Yu Y, Cefalu WT. An
extract of Artemisia dracunculus L. enhances insulin receptor signaling and
modulates gene expression in skeletal muscle in KK-A(y) mice. J Nutr
Biochem. 2011;22(1):71–8.
Yu Y, Mendoza T, Ribnicky D, Poulev A, Noland RC, Mynatt RL, Raskin I,
Cefalu WT, Floyd ZE. An extract of Russian tarragon prevents obesity-related
ectopic lipid accumulation. Mol Nutr Food Res. 2018;62(8):e1700856.
Fuller et al. Biology of Sex Differences (2018) 9:41
27. Ma C, Yu H, Xiao Y, Wang H. Momordica charantia extracts ameliorate
insulin resistance by regulating the expression of SOCS-3 and JNK in type 2
diabetes mellitus rats. Pharm Biol. 2017;55(1):2170–7.
28. Mahwish SF, Arshad MS, Nisa MU, Nadeem MT, Arshad MU. Hypoglycemic
and hypolipidemic effects of different parts and formulations of bitter
gourd (Momordica Charantia). Lipids Health Dis. 2017;16(1):211.
29. Willcox DC, Scapagnini G, Willcox BJ. Healthy aging diets other than the
Mediterranean: a focus on the Okinawan diet. Mech Ageing Dev. 2014;136-137:
148–62.
30. Sridhar MG, Vinayagamoorthi R, Arul Suyambunathan V, Bobby Z, Selvaraj N.
Bitter gourd (Momordica charantia) improves insulin sensitivity by
increasing skeletal muscle insulin-stimulated IRS-1 tyrosine phosphorylation
in high-fat-fed rats. Br J Nutr. 2008;99(4):806–12.
31. Han JH, Tuan NQ, Park MH, Quan KT, Oh J, Heo KS, Na M, Myung CS.
Cucurbitane triterpenoids from the fruits of Momordica charantia improve
insulin sensitivity and glucose homeostasis in streptozotocin-induced
diabetic mice. Mol Nutr Food Res. 2018;62(7):e1700769.
32. Bai J, Zhu Y, Dong Y. Bitter melon powder protects against obesityassociated fatty liver disease by improving colonic microenvironment in rats
with high-fat diet-induced obesity. Biomed Environ Sci. 2017;30(8):611–5.
33. Xu J, Cao K, Li Y, Zou X, Chen C, Szeto IM, Dong Z, Zhao Y, Shi Y, Wang J, et
al. Bitter gourd inhibits the development of obesity-associated fatty liver in
C57BL/6 mice fed a high-fat diet. J Nutr. 2014;144(4):475–83.
34. Shih CC, Shlau MT, Lin CH, Wu JB. Momordica charantia ameliorates insulin
resistance and dyslipidemia with altered hepatic glucose production and
fatty acid synthesis and AMPK phosphorylation in high-fat-fed mice.
Phytother Res. 2014;28(3):363–71.
35. Matsui S, Yamane T, Takita T, Oishi Y, Kobayashi-Hattori K. The
hypocholesterolemic activity of Momordica charantia fruit is mediated by
the altered cholesterol- and bile acid-regulating gene expression in rat liver.
Nutr Res. 2013;33(7):580–5.
36. Schmidt B, Ribnicky DM, Poulev A, Logendra S, Cefalu WT, Raskin I. A natural
history of botanical therapeutics. Metabolism. 2008;57(7 Suppl 1):S3–9.
37. Ribnicky DM, Poulev A, Watford M, Cefalu WT, Raskin I. Antihyperglycemic
activity of Tarralin, an ethanolic extract of Artemisia dracunculus L.
Phytomedicine. 2006;13(8):550–7.
38. Ribnicky DM, Kuhn P, Poulev A, Logendra S, Zuberi A, Cefalu WT, Raskin I.
Improved absorption and bioactivity of active compounds from an antidiabetic extract of Artemisia dracunculus L. Int J Pharm. 2009;370(1–2):87–92.
39. Zuberi AR. Strategies for assessment of botanical action on metabolic
syndrome in the mouse and evidence for a genotype-specific effect of
Russian tarragon in the regulation of insulin sensitivity. Metabolism. 2008;
57(7 Suppl 1):S10–5.
40. Logendra S, Ribnicky DM, Yang H, Poulev A, Ma J, Kennelly EJ, Raskin I.
Bioassay-guided isolation of aldose reductase inhibitors from Artemisia
dracunculus. Phytochemistry. 2006;67(14):1539–46.
41. Prendergast BJ, Onishi KG, Zucker I. Female mice liberated for inclusion in
neuroscience and biomedical research. Neurosci Biobehav Rev. 2014;40:1–5.
42. Service USDoAAR: Nutrient intakes from food: mean amounts of consumed
per individual, by Gender and Age, What We Eat in America, NHANES 2009–
2010. 2012.
43. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC.
Homeostasis model assessment: insulin resistance and beta-cell function
from fasting plasma glucose and insulin concentrations in man.
Diabetologia. 1985;28(7):412–9.
44. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and
purification of total lipides from animal tissues. J Biol Chem. 1957;226(1):497–509.
45. Noland RC, Woodlief TL, Whitfield BR, Manning SM, Evans JR, Dudek RW,
Lust RM, Cortright RN. Peroxisomal-mitochondrial oxidation in a rodent
model of obesity-associated insulin resistance. Am J Physiol Endocrinol
Metab. 2007;293(4):E986–E1001.
46. Hulver MW, Berggren JR, Cortright RN, Dudek RW, Thompson RP, Pories WJ,
MacDonald KG, Cline GW, Shulman GI, Dohm GL, et al. Skeletal muscle lipid
metabolism with obesity. Am J Physiol Endocrinol Metab. 2003;284(4):E741–7.
47. Wang ZQ, Floyd ZE, Qin J, Liu X, Yu Y, Zhang XH, Wagner JD, Cefalu WT.
Modulation of skeletal muscle insulin signaling with chronic caloric
restriction in cynomolgus monkeys. Diabetes. 2009;58(7):1488–98.
48. Kheterpal I, Scherp P, Kelley L, Wang Z, Johnson W, Ribnicky D, Cefalu WT.
Bioactives from Artemisia dracunculus L. enhance insulin sensitivity via
modulation of skeletal muscle protein phosphorylation. Nutrition. 2014;
30(7–8 Suppl):S43–51.
Page 14 of 14
49. Vandanmagsar B, Haynie KR, Wicks SE, Bermudez EM, Mendoza TM, Ribnicky
D, Cefalu WT, Mynatt RL. Artemisia dracunculus L. extract ameliorates insulin
sensitivity by attenuating inflammatory signalling in human skeletal muscle
culture. Diabetes Obes Metab. 2014;16(8):728–38.
50. Benz V, Bloch M, Wardat S, Bohm C, Maurer L, Mahmoodzadeh S, Wiedmer
P, Spranger J, Foryst-Ludwig A, Kintscher U. Sexual dimorphic regulation of
body weight dynamics and adipose tissue lipolysis. PLoS One. 2012;7(5):e37794.
51. Pettersson US, Walden TB, Carlsson PO, Jansson L, Phillipson M. Female
mice are protected against high-fat diet induced metabolic syndrome and
increase the regulatory T cell population in adipose tissue. PLoS One. 2012;
7(9):e46057.
52. Yang Y, Smith DL Jr, Keating KD, Allison DB, Nagy TR. Variations in body
weight, food intake and body composition after long-term high-fat diet
feeding in C57BL/6J mice. Obesity (Silver Spring). 2014;22(10):2147–55.
53. Smith BK, Marcinko K, Desjardins EM, Lally JS, Ford RJ, Steinberg GR.
Treatment of nonalcoholic fatty liver disease: role of AMPK. Am J Physiol
Endocrinol Metab. 2016;311(4):E730–e740.
54. Sun L, Zhang S, Yu C, Pan Z, Liu Y, Zhao J, Wang X, Yun F, Zhao H, Yan S, et
al. Hydrogen sulfide reduces serum triglyceride by activating liver
autophagy via the AMPK-mTOR pathway. Am J Physiol Endocrinol Metab.
2015;309(11):E925–35.
55. Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, Tanaka K, Cuervo
AM, Czaja MJ. Autophagy regulates lipid metabolism. Nature. 2009;
458(7242):1131–5.
56. Fullgrabe J, Ghislat G, Cho DH, Rubinsztein DC. Transcriptional regulation of
mammalian autophagy at a glance. J Cell Sci. 2016;129(16):3059–66.
57. Beltran-Sanchez H, Harhay MO, Harhay MM, McElligott S. Prevalence and
trends of metabolic syndrome in the adult U.S. population, 1999-2010. J Am
Coll Cardiol. 2013;62(8):697–703.
58. Kuk JL, Ardern CI. Age and sex differences in the clustering of metabolic
syndrome factors: association with mortality risk. Diabetes Care. 2010;33(11):
2457–61.
59. Salinero AE, Anderson BM, Zuloaga KL. Sex differences in the metabolic
effects of diet-induced obesity vary by age of onset. Int J Obes. 2018;42(5):
1088–91.
60. Karpe F, Dickmann JR, Frayn KN. Fatty acids, obesity, and insulin resistance:
time for a reevaluation. Diabetes. 2011;60(10):2441–9.
61. DeFronzo RA, Tripathy D. Skeletal muscle insulin resistance is the primary
defect in type 2 diabetes. Diabetes Care. 2009;32(Suppl 2):S157–63.
62. McGuinness OP, Ayala JE, Laughlin MR, Wasserman DH. NIH experiment in
centralized mouse phenotyping: the Vanderbilt experience and
recommendations for evaluating glucose homeostasis in the mouse. Am J
Physiol Endocrinol Metab. 2009;297(4):E849–55.
63. Lin HV, Accili D. Hormonal regulation of hepatic glucose production in
health and disease. Cell Metab. 2011;14(1):9–19.
64. Sanders FW, Griffin JL. De novo lipogenesis in the liver in health and
disease: more than just a shunting yard for glucose. Biol Rev Camb Philos
Soc. 2016;91(2):452–68.
65. Otero YF, Stafford JM, McGuinness OP. Pathway-selective insulin resistance
and metabolic disease: the importance of nutrient flux. J Biol Chem. 2014;
289(30):20462–9.
66. Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ.
Sources of fatty acids stored in liver and secreted via lipoproteins in patients
with nonalcoholic fatty liver disease. J Clin Invest. 2005;115(5):1343–51.
67. Vatner DF, Majumdar SK, Kumashiro N, Petersen MC, Rahimi Y, Gattu AK,
Bears M, Camporez JP, Cline GW, Jurczak MJ, et al. Insulin-independent
regulation of hepatic triglyceride synthesis by fatty acids. Proc Natl Acad Sci
U S A. 2015;112(4):1143–8.
68. Vernier-Magnin S, Muller S, Sallot M, Radom J, Musard JF, Adami P, Dulieu P,
Remy-Martin JP, Jouvenot M, Fraichard A. A novel early estrogen-regulated
gene gec1 encodes a protein related to GABARAP. Biochem Biophys Res
Commun. 2001;284(1):118–25.
69. Cooke PS, Nanjappa MK, Ko C, Prins GS, Hess RA. Estrogens in male
physiology. Physiol Rev. 2017;97(3):995–1043.