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OPEN Saturated fatty acid concentrations

are predictive of insulin sensitivity
and beta cell compensation in dogs
Matthew Peloquin 1*, Ashley Tovar 1, Jessica L. Graves 1, Darko Stefanovski 2, Katya Tucker 1,
Entonio Marietti 1, Karen Greenwood 1, Celine‑Lea Halioua‑Haubold 1 & Dina Juarez‑Salinas 1

Chronic feeding of a high fat diet (HFD) in preclinical species induces broad metabolic dysfunction
characterized by body weight gain, hyperinsulinemia, dyslipidemia and impaired insulin sensitivity.
The plasma lipidome is not well characterized in dogs with HFD-induced metabolic dysfunction.
We therefore aimed to describe the alterations that occur in the plasma lipid composition of dogs
that are fed a HFD and examine the association of these changes with the clinical signs of metabolic
dysfunction. Dogs were fed a normal diet (ND) or HFD for 12 weeks. Insulin sensitivity ­(SI) and beta cell
compensation ­(AIRG) were assessed through an intravenous glucose tolerance test (IVGTT) and serum
biochemistry was analyzed before the introduction of HFD and again after 12 weeks of continued
ND or HFD feeding. Plasma lipidomics were conducted prior to the introduction of HFD and again at
week 8 in both ND and HFD-fed dogs. 12 weeks of HFD feeding resulted in impaired insulin sensitivity
and increased beta cell compensation measured by ­SI (ND mean: 11.5 [mU/l]–1 ­min–1, HFD mean:
4.7 [mU/l]–1 ­min–1) and ­AIRG (ND mean: 167.0 [mU/l]min, HFD mean: 260.2 [mU/l]min), respectively,
compared to dogs fed ND over the same duration. Chronic HFD feeding increased concentrations
of plasma lipid species and deleterious fatty acids compared to dogs fed a ND. Saturated fatty acid
(SFA) concentrations were significantly associated with fasting insulin ­(R2 = 0.29), ­SI ­(R2 = 0.49) and
­AIRG ­(R2 = 0.37) in all dogs after 12 weeks, irrespective of diet. Our results demonstrate that chronic
HFD feeding leads to significant changes in plasma lipid composition and fatty acid concentrations
associated with metabolic dysfunction. High SFA concentrations may be predictive of deteriorated
insulin sensitivity in dogs.

Insulin sensitivity is the ability of multiple organ systems to respond to insulin and effectuate physiological pro-
cesses efficiently, such as initiating glucose uptake into muscle or inhibiting lipolysis in adipose tissue. Failure
to maintain adequate insulin sensitivity and action, commonly referred to as insulin resistance, is a hallmark of
various metabolic diseases in humans, such as metabolic s­ yndrome1 (MetS), type II d ­ iabetes2 (T2D) and non-
alcoholic fatty liver ­disease1. Moreover, advancing chronological age also results in broad metabolic decline
that is often associated with the onset of disease, marked by decreased insulin sensitivity and impaired insulin
clearance in r­ odents3,4, ­dogs5,6 and ­humans7,8.
Preclinical rodent models that induce metabolic dysfunction, such as chronic high fat diet (HFD) feeding, are
commonly used to investigate aspects of various metabolic diseases. In mice, HFD feeding reproducibly leads
to body weight gain, adipose expansion, dyslipidemia, hyperglycemia and h ­ yperinsulinemia9. Consequently,
excess circulating insulin and fatty acids contribute to lipid deposition in the liver and muscle, systemic and
local inflammation and impairments to insulin sensitivity in peripheral ­tissues10,11. The use of these rodent
models has been integral to the mechanistic understanding and subsequent therapeutic development for treat-
ing metabolic disease.
Although large animal models of metabolic dysfunction are less commonly employed, dogs are an excellent
model of human disease due to their complex physiology, exposure to the same environmental factors and simi-
larities in their timeline of disease onset. Many outcomes from studies using dogs have yielded similar pheno-
types to that seen in rodent studies and naturally occuring metabolic disease. Feeding dogs a HFD leads to body
weight gain, primarily driven by the expansion of adipose tissue, with accompanying fasting hyperinsulinemia
and impaired insulin ­sensitivity12–14. This adipose expansion in dogs, specifically the hypertrophy of the visceral
adipocytes, has been shown to correlate with deteriorated insulin sensitivity—A phenotype previously described

1
Cellular Longevity Inc., San Francisco, CA, USA. 2Department of Clinical Studies ‑ NBC, University of Pennsylvania
School of Veterinary Medicine, Kennett Square, PA, USA. *email: matt@loyalfordogs.com

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in human p ­ opulations12,15. Moreover, HFD-fed dogs experience elevations in clinically measured lipids, such as
total triglycerides and cholesterol, producing a biochemical profile indicative of broad metabolic ­dysfunction16.
However, much less is known about changes in plasma lipid composition in metabolically stressed ­dogs17,18.
Investigations in young, healthy dogs have revealed a potentially causal role of certain fatty acids in deteriorating
insulin sensitivity. While not all fatty acids and lipid species negatively impact insulin sensitivity, co-infusing a
neutral lipid and fatty acid cocktail comprised of linoleic, oleic, palmitic, linolenic and stearic acid at various time
points during a hyperinsulinemic euglycemic clamp resulted in blunted glucose uptake into the muscle, altered
endogenous glucose production by the liver and impaired whole body insulin a­ ction19. Although infusing these
fatty acids is sufficient to produce an insulin resistant phenotype, it is still unclear what effect HFD feeding has
on the plasma lipidome and if these changes are related to insulin sensitivity and action in dogs.
In this investigation, we aimed to further characterize several aspects of the HFD dog model: First, we aimed
to characterize any HFD-induced changes in plasma lipid composition, specifically focusing on fatty acid species.
Second, we aimed to recapitulate previous studies which demonstrate deteriorated insulin sensitivity and hyper-
insulinemic beta cell compensation resulting from HFD feeding. Lastly, we aimed to determine if any associa-
tion exists between the changes in lipid composition and insulin sensitivity and beta cell compensation in dogs.

Methods
Dogs
Male mixed-breed dogs and beagles (7–8 mixed-breed/group + 3 beagles/group) aged 4–7 years old, between
13 and 26 kg in weight, a BCS score of 4–6, and of mixed castration status (4–5 castrated/per group) were
supplied by ClinVet of ClinGlobal (South Africa). Descriptive statistics of the groups pre-study enrollment is
reported in Supplemental Table 1. All animal studies were submitted to and approved by the ClinVet (South
Africa) Institutional Animal Care and Use Committee. Certificates of approval were issued. An ethics approval
certificate was issued under National Health Research Ethics Council Reg No. AREC-230817–020. The study
plans were designed to allow the use of the study animals in compliance with the ClinVet policy on the ethical
use of animals, using the most recent version South African National Standard SANS 10386: The care and use
of animals for scientific purposes, as a r­ eference20. All methods were performed in accordance with the relevant
guidelines and regulations. Dogs enrolled in this study received regular blood draws and examinations. No other
procedures were conducted during this study. All dogs recovered and were returned to the ClinVet colony upon
study completion. This study is reported in accordance with ARRIVE guidelines.

Study design
Prior to study start, all dogs were fed a dry normal diet (ND) which contained 55% of total calories derived
from fat (VetsBrands Premium adult maintenance dog food, manufactured by VetsBrands; Reg No.: V 24369).
The daily food ration for each dog was determined by body weight at study start and following the Vetbrands
recommended guidelines for grams of food per kilogram of body weight. Dogs were randomized into ND or
HFD groups balanced across age and body weight. On Day 0, dogs were randomized into the HFD group (n = 11)
were transitioned from the aforementioned ND to a 66% (total calories) fat diet for 7 days and then given a 74%
(total calories) fat diet (HFD) through the remainder of the study, for a total duration of 12 weeks. Increased
dietary fat content was derived from the incorporation and thorough mixing of wet food (Husky Adult [Chunky],
manufactured by Purina; Reg No.: V13339) and pork lard to the ND. Dogs in the ND group (n = 10) were con-
tinued on the dry ND through the conclusion of the study. Dogs assigned to a HFD were offered double the daily
ration of food as the ND to maximize opportunity for consumption. Food intake was quantified by weighing the
amount of diet presented subtracted from the amount of diet remaining, with lard pre-incorporated, if applica-
ble. Body weights were assessed at study enrollment and again weekly over the 12-week duration. Study design
details are described in Fig. 1. Macronutrient composition and dietary details for all diets used are reported in
Supplement Tables 2, 3.

IVGTT​
Intravenous glucose tolerance tests (IVGTT) were performed at week 0 (prior to HFD initiation) and week 12,
and were carried out as previously ­described15. Briefly, dogs were fasted overnight prior to conducting the pro-
cedure. Blood was sampled and serum and plasma separated at − 10 and − 1 min pre-glucose bolus to establish
fasting glucose and insulin values. At 0 min (t = 0), a glucose bolus (0.3 g/kg, i.v.) was delivered. At t = 20 min a
bolus of insulin (0.03 U/kg, i.v.) was delivered. Blood was collected at 2, 4, 6, 10, 14, 19, 22, 30, 40, 50, 60, 80, 100,
120, 150, and 180 min post-glucose bolus. Serum was then analyzed for glucose and plasma was analyzed for
insulin levels at each time point. In a blinded fashion, glucose and insulin values were entered into the MINMOD
Millenium 6.02 software to generate parameter estimates for insulin sensitivity (­ SI,), and glucose effectiveness
­(SG) for each animal. Acute insulin response to glucose ­(AIRG) was calculated from the AUC of insulin from t = 0
to t = 10 and the disposition index (DI) was calculated by the multiplication of A ­ IRG and ­SI.
A single dog in the ND group was removed as an outlier due to a supraphysiological S­ I value (44.2 [mU/
l]–1 ­min–1) at week 12.

Serum biochemistry
Measures of leptin, non-esterified fatty acids (NEFA), triglycerides, cholesterol, aspartate aminotransferase (AST),
alanine transaminase (ALT), alkaline phosphatase (ALP), creatine kinase (CK) were derived from fasting blood
samples which were serum separated for analysis at week 0 (prior to diet initiation) and week 12. Serum canine
leptin and adiponectin concentrations were measured by single-analyte enzyme-linked immunosorbent assay
(ELISA) (Canine Leptin ELISA, Millipore EZCL-31 K and Adiponectin ELISA (MBL International CY-8052:

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Figure 1.  Experimental design. Serum biochemistry and IVGTT were assessed in dogs at week 0 (pre-HFD)
and again after 12 weeks of the HFD or ND feeding (Week 12). Lipidomics were assessed at week 0 and after
8 weeks of the HFD or ND feeding (Week 8).

CircuLex Dog, respectively). Serum NEFA concentrations were measured using the Cobas 6000 Chemistry
Analyzer (Charles River Laboratories, Spencerville, Ohio). Serum clinical chemistry, fasting glucose, and fasting
insulin quantification was performed and analyzed using the Abbott Alinity chemistry analyzer by PathCare
(South Africa).

Lipidomics
Targeted lipidomic profiling was conducted at Metabolon Inc (Complex Lipids Targeted Panel; Morrisville,
NC) on canine plasma prior to study start (week 0) and again at 8 weeks post diet intervention (week 8). Lipids
were extracted from plasma in the presence of deuterated internal standards using an automated BUME extrac-
tion according to the method of Löfgren et al.21. The extracts were dried under nitrogen and reconstituted in
ammonium acetate dichloromethane:methanol. The extracts were transferred to vials for infusion-MS analysis,
performed on a Shimadzu (Pleasanton, CA) LC with nano PEEK tubing and the Sciex (Toronto, Canada) Selex-
Ion-5500 QTRAP. The samples were analyzed via both positive and negative mode electrospray. The 5500 QTRAP
was operated in MRM mode with a total of more than 1100 MRMs. Individual lipid species were quantified by
taking the ratio of the signal intensity of each target compound to that of its assigned internal standard, then
multiplying by the concentration of internal standard added to the sample.
Lipid class concentrations were calculated from the sum of all molecular species within a class, and fatty acid
compositions were determined by calculating the proportion of each class comprised by individual fatty acids.
Lipid classes quantified are: Ceramides (CER), cholesteryl esters (CE), diacylglycerol (DAG), dihydroceramide
(DCER), hexosylceramide (HCER), lactosylceramide (LCER), lysophosphatidylcholine (LPC), lysophosphati-
dylethanolamine (LPE), monoacylglycerol (MAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylinositol (PI), sphingomyelin (SM), triacylglycerol (TAG). All lipid classes and species measured by
the Complex Lipids Targeted Panel from Metabolon are reported in Supplemental Table 4.

Statistical analysis
Descriptive analyses included computation of means, standard deviations, medians, interquartile ranges (IQR)
of continuous variables and tabulation of categorical variables. Tests of normal distribution (Shapiro–Wilk)
were performed to determine the extent of skewness of the data. Frequency counts and percentages were used
for summarizing categorical data.
Differences in body weight and food consumption were analyzed using mixed effects models using diet and
week as main and interaction effects. Post-hoc group comparisons were performed to estimate group differences
at each timepoint. Group differences in percent change in body weight and food consumption were analyzed
using Two-sample t-tests at each timepoint. IVGTT parameters were estimated using statistical package STATA
17MP (StataCorp; College Station,TX) as previously ­described18. Two-sample t-tests were used to test group
differences in lipidomics, serum biochemistry, and IVGTT parameters. Data are expressed as mean ± standard
deviation. Pearson’s correlations were used to explore simple correlations between fatty acid species, fasting
insulin, ­SI and ­AIRG at the conclusion of the study across both diet types. All statistical tests were two-sided with
an alpha-level of 0.05 and were performed in R version 4.1.2. All subjects were included in analyses (ND = 10,

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HFD = 11) with the exception of data pertaining to the IVGTT (N = 9, HFD = 11) as described in the IVGTT
methods section.

Results
Body weight and food intake after 12 weeks of ND or HFD
At the start of the study (week 0), dogs in both ND and HFD groups were similar in body weight and food intake
(Fig. 2a,b). After the 1st week, dogs in the HFD group consumed significantly more food per week on average
than dogs fed ND (5315.9 ± 859.5 g, 2729.6 ± 801.8 g, p < 0.001), which continued through all 12 weeks of the
study (Fig. 2b). The average body weights of ND-fed dogs remained stable throughout the duration of the study.
Similarly, by week 1, HFD-fed dogs had a significantly greater percent change in food intake compared to
dogs fed ND (HFD: 44.6 ± 21.2%, ND: − 20.5 ± 3.0%, p < 0.001), which continued to week 8 (Fig. 2d). By week 9,
the body weight of dogs fed HFD tended to increase as compared to ND-fed dogs, though this change was not
significant (HFD: 20.9 ± 5.5 kg, ND: 17.5 ± 3.6 kg, p = 0.09) (Fig. 2a). However, when assessed by percent change
from week 0, HFD-fed dogs showed a significant increase in body weight compared to ND-fed dogs starting at
week 3 (HFD: 7.5 ± 9.3%, ND: − 0.5 ± 2.1%, p = 0.018), which continued for the duration of the study (Fig. 2c).

Serum biochemistry after 12 weeks of ND or HFD

Chronic HFD feeding resulted in significantly higher cholesterol (235.7 ± 53.4 mg/dL, p = 0.019) and leptin
levels (8.6 ± 7.6 ng/mL, p = 0.012) than ND feeding (187.9 ± 27.0 mg/dL, 1.6 ± 1.7 ng/mL, respectively, Table 1).
Mean triglycerides appeared to be elevated in the HFD group (77.5 ± 71.5 mg/dL) compared to the ND group
(54.1 ± 12.7 mg/dL), however this difference did not reach statistical significance (p = 0.310, Table 1). There was
no effect of diet on ALP, ALT, AST, CK, adiponectin or NEFA levels (p > 0.05, Table 1).

Insulin sensitivity and beta cell compensation after 12 weeks of ND or HFD

After 12 weeks, fasting glucose remained stable in both the ND and HFD-fed groups. In contrast, fasting insulin
was higher in HFD-fed dogs (10.3 ± 3.8 mIU/L) than ND-fed dogs (7.6 ± 2.9, p = 0.10, Fig. 3a,b), however this
did not reach statistical significance.

Figure 2.  (a) Body weight and (b) food intake in dogs fed a ND (n = 10) or HFD (n = 11) for 12 weeks. Percent
change from week 0 in (c) body weight and (d) food intake, in dogs fed a ND (n = 10) or HFD (n = 11) for
12 weeks. All values shown are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

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Serum biochemistry
Week 0 Week 12
Parameter ND HFD p-value ND HFD p-value
ALP (IU/L) 58.9 ± 36.9 61.2 ± 23.2 0.869 63.6 ± 73.0 39.5 ± 16.4 0.333
ALT (IU/L) 53.2 ± 34.7 38.2 ± 11.5 0.219 50.7 ± 19.7 44.9 ± 14.9 0.461
AST (IU/L) 42.9 ± 12.6 39.5 ± 15.9 0.597 43.3 ± 8.9 41.0 ± 9.6 0.575
CK (IU/L) 258.5 ± 144.7 217.4 ± 120.3 0.490 213.1 ± 73.0 280.1 ± 168.1 0.249
Cholesterol (mg/dL) 214.8 ± 38.0 200.3 ± 28.8 0.341 187.9 ± 27.0 235.7 ± 53.4 0.019*
Triglycerides (mg/dL) 48.9 ± 14.7 49.9 ± 14.7 0.877 54.1 ± 12.7 77.5 ± 71.5 0.310
Leptin (ng/mL) 1.7 ± 1.0 5.1 ± 6.1 0.104 1.6 ± 1.7 8.6 ± 7.6 0.012*
Adiponectin (ug/mL) 8.7 ± 2.8 9.9 ± 3.7 0.429 11.3 ± 5.6 10.0 ± 4.8 0.570
NEFA (mmol/L) 0.7 ± 0.3 0.9 ± 0.5 0.378 0.9 ± 0.6 0.6 ± 0.2 0.156

Table 1.  Serum biochemistry in dogs fed a ND (n = 10) or HFD (n = 11) for 12 weeks. All values shown are
mean ± SD. ALP alkaline phosphatase, ALT alanine transaminase, AST aspartate aminotransferase, CK creatine
kinase, NEFA non-esterified fatty acids. *p < 0.05; **p < 0.01; ***p < 0.001.

At week 0, both ND and HFD groups had similar ­SI values. However, by week 12, the HFD-fed group had sig-
nificantly lower S­ I values than dogs fed the ND (7.8 ± 2.6 [mU/l]–1 ­min–1 vs. 4.7 ± 2.3 [mU/l]–1 ­min–1, respectively,
p = 0.012). The ­SI values of the HFD-fed group decreased by 47% from week 0 levels (from 8.9 ± 3.0 [mU/l]–1 ­min–1
at week 0 to 4.7 ± 2.3 [mU/l]–1 ­min–1 at week 12), whereas it remained unchanged in the ND-fed group (Fig. 3c).
At week 0, both ND and HFD groups had similar ­AIRG values. However, by week 12, A ­ IRG was significantly
elevated in HFD-fed dogs relative to ND-fed dogs (260.2 ± 111.4 [mU/l]min vs. 170.7 ± 65.3 [mU/l]min, respec-
tively; p = 0.039). The ­AIRG of the HFD-fed group increased by 41% from week 0 levels (187.4 ± 78.7 [mU/l]min
at week 0 vs. 260.2 ± 111.4 [mU/l]min at week 12) (Fig. 3d). DI and S­ G were unchanged after 12 weeks of HFD
feeding (Fig. 3e,f).

Plasma lipid composition after 8 weeks of ND or HFD

Both dogs in the ND and HFD groups had similar plasma lipid composition profiles for all lipid species meas-
ured prior to study start at week 0 (p > 0.05, Table 2). Chronic HFD feeding resulted in elevations in plasma CE
(p = 0.024), HCER (p < 0.001), LPC (p = 0.035), PC (p = 0.036), PE (p = 0.002) and SM (p = 0.001) compared to
dogs in the ND group (Table 2).

Fatty acid profiles after 12 weeks of ND or HFD

Both dogs in the ND and HFD groups had similar plasma fatty acid profiles in all fatty acid species measured
prior to study start at week 0 (p > 0.05, Table 3). Chronic HFD feeding resulted in elevated total plasma fatty acids
species 15:0, 16:0, 17:0, 18:0, 18:1, 18:2, 20:0, 20:1, 20:2, 20:3, 20:4, 22:0, 22:4 and aggregated FFA and SFA com-
pared to dogs in the ND group (all p < 0.05, see Table 3 for p-values). Dogs in the HFD group also demonstrated
significant reductions in plasma fatty acids 22:5 and 22:6 (all p < 0.05) and trending decrease in 20:5 compared
to dogs in the ND group (p = 0.076, Table 3).

Association between SFA levels, fasting insulin, insulin sensitivity and beta cell compensation
Associations were assessed using the SFA values generated at week 8 and IVGTT parameters generated at week
12 in both ND and HFD dogs (n = 20). Pearson’s correlations demonstrated that fasting insulin is negatively
associated with S­ I ­(R2 = 0.40) and positively associated with A ­ IRG ­(R2 = 0.47) and aggregated SFA concentrations
­(R2 = 0.29) (all p ≤ 0.015, Fig. 4a,b,e). Aggregated SFA concentrations were also negatively associated with ­SI
­(R2 = 0.49) and positively associated with A ­ IRG ­(R2 = 0.37) in all dogs (all p < 0.01, Fig. 4c,d).

Discussion
In this investigation we aimed to characterize the changes in the plasma lipidome resulting from chronic HFD
feeding and determine if these changes were associated with the deterioration of insulin sensitivity and beta cell
compensation. Increased concentrations of deleterious lipid species and fatty acids, such as sphingolipids and
long chain saturated fatty acids, have been associated with visceral adipose mass and the development of meta-
bolic disease in h­ umans22,23, while the accumulation of certain PUFA species has been shown to be metabolically
­beneficial24. The impact of these lipid species are well described in metabolically stressed rodents and humans,
however little is known about the plasma lipidome in dogs presented with a metabolic stressor such as HFD.
Recapitulating previous investigations, chronic HFD feeding in dogs led to hyperphagia and increased body
weight, likely due to an increase in total adiposity. This apparent adipose expansion is likely due to the increased
levels of leptin, an adipokine which has a known positive relationship with increased abdominal adipose tissue
mass and a hallmark of metabolic ­disease25. Additionally, this study demonstrated HFD feeding dogs induces
elevations in clinically measured total triglycerides and cholesterol, suggesting the presence of dyslipidemia,
which has been previously r­ eported16. The serum biochemistry of dogs fed a HFD in this study is similar to
previous investigations in rodents, while mirroring aspects of metabolic disease in human p ­ opulations26–29.

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a. b.
p=0.10
130 20 ND
ND

Fasting glucose (mg/dL)

Fasting insulin (mU/L)

120 HFD HFD
15
110

100 10

90
5
80

70 0
Week 0 Week 12 Week 0 Week 12

c. d.
20 ND
600 ND

HFD HFD

AIRg (mU/l x min)

SI (mU-1I-1min-1)

15
400

10

200
5

0 0
Week 0 Week 12 Week 0 Week 12

e. f.

4000 ND
0.08 ND

HFD HFD
3000 0.06
Sg (%/min)
DI Value

2000 0.04

1000 0.02

0 0.00
Week 0 Week 12 Week 0 Week 12

Figure 3.  Chronic HFD feeding induces beta cell compensation and reduced insulin sensitivity. (a) Fasting
glucose and (b) insulin concentrations prior to IVGTT in dogs fed a ND (n = 9) or HFD (n = 11) for 12 weeks.
IVGTT measurements of (c) ­SI,, (d) ­AIRG, (e) DI and (f) ­SG in dogs fed a ND (n = 9) or HFD (n = 11) for
12 weeks. All values shown are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Our study is also consistent with previous investigations describing the effects of HFD feeding on insulin
sensitivity and beta cell compensation. Here we show a similar phenotype, albeit using a different macronutrient
composition HFD, whereby insulin sensitivity is compromised and hyperinsulinemic compensation from the
beta cells is required to maintain glycemic ­control12,14,30. This phenotype is analogous to HFD models in rodents
and characteristic of metabolic disease and insulin resistance in h ­ umans31,32.
However, unlike rodents and humans, the hyperinsulinemic compensation and reduced insulin sensitivity in
the HFD-fed group occurs without impacting glycemia. This innate ability for dogs to compensate for blunted
peripheral insulin sensitivity by continually producing additional insulin in order to maintain euglycemia, even
in the presence of metabolic stressors, has previously been reported in several models of metabolic dysfunction in
­dogs33. Although glycemia in dogs is highly regulated, the presence of continual hyperinsulinemic compensation
and peripheral insulin resistant phenotypes may have deleterious effects as seen in other ­species34,35. Most notably,
metabolically stressed obese dogs have reduced lifespans, a phenotype also present in human p ­ opulations36,37.
Additional studies are needed to further define the clinical impact of hyperinsulinemia and impaired insulin
sensitivity in dogs.
In healthy dogs, fasting hyperinsulinemia has been associated with impaired insulin sensitivity when meas-
ured by hyperinsulinemic euglycemic c­ lamp38. Our data recapitulate the association between fasting insulin and
insulin sensitivity in all dogs assessed, regardless of diet. Interestingly, we further identify that fasting hyperinsu-
linemia is associated with commensurate beta cell compensation, augmenting the previous findings that fasting
insulin, not fasting glycemia, is indicative of the insulin sensitivity state in d­ ogs33.

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Week 0 Week 8
Lipid species (µM) ND HFD p-value ND HFD p-value
CER 4.3 ± 1.1 4.3 ± 0.6 0.961 4.6 ± 1.1 5.1 ± 0.9 0.214
CE 1545.9 ± 109.6 1545.3 ± 182.7 0.992 1486.8 ± 173.7 1771.2 ± 331.2 0.024*
DAG 7.9 ± 2.3 8.0 ± 2.9 0.935 7.3 ± 1.8 7.8 ± 2.7 0.585
DCER 1.7 ± 0.2 1.6 ± 0.2 0.528 1.6 ± 0.3 1.7 ± 0.2 0.430
HCER 1.2 ± 0.1 1.2 ± 0.2 0.682 1.1 ± 0.2 1.6 ± 0.3 < 0.001***
LCER 2.2 ± 0.5 2.1 ± 0.4 0.587 3.4 ± 0.7 3.9 ± 0.7 0.115
LPC 232.9 ± 16.4 228.8 ± 28.1 0.686 216.8 ± 22.9 245.2 ± 33.6 0.035*
LPE 7.1 ± 1.3 6.9 ± 1.1 0.699 6.6 ± 1.0 7.2 ± 1.1 0.205
MAG 2.4 ± 2.3 3.8 ± 2.9 0.225 5.4 ± 7.7 3.5 ± 3.1 0.488
PC 3650.3 ± 332.3 3742.3 ± 324.3 0.529 3513.0 ± 537.2 4141.2 ± 731.0 0.036*
PE 59.4 ± 8.1 61.9 ± 8.8 0.506 55.5 ± 6.3 70.0 ± 11.5 0.002**
PI 3.0 ± 1.2 2.6 ± 1.1 0.424 2.4 ± 0.7 3.1 ± 1.1 0.119
SM 325.1 ± 25.5 328.4 ± 32.7 0.799 313.6 ± 41.1 400.0 ± 62.3 0.001**
TAG​ 295.6 ± 85.4 292.5 ± 106.3 0.942 211.5 ± 39.8 252.1 ± 61.6 0.088

Table 2.  Plasma lipid species composition in dogs fed a ND (n = 10) or HFD (n = 11) for 8 weeks.
All values shown are mean ± SD. CER Ceramides, CE cholesteryl esters, DAG diacylglycerol, DCER
dihydroceramide, HCER hexosylceramide, LCER lactosylceramide, LPC lysophosphatidylcholine,
LPE lysophosphatidylethanolamine, MAG monoacylglycerol, PC phosphatidylcholine, PE
phosphatidylethanolamine, PI phosphatidylinositol, SM sphingomyelin, TAG​triacylglycerol. *p < 0.05;
**p < 0.01; ***p < 0.001.

While the effects of HFD on body weight, fasting biochemistry and insulin sensitivity have been previously
described in ­dogs13, a considerable knowledge gap exists regarding the composition of circulating lipid species
and how they impact metabolic function. Our data reveal that chronic consumption of HFD leads to significant
elevations in the plasma lipid species CE, HCER, LPC, PE, PC and SM. The elevation of many of these lipids are
notable, namely HCER, PC and SM, due to their roles as biological intermediates in the formation of ceramides
and other sphingolipids. The increased abundance of these lipid species has been previously implicated as del-
eterious actors in the development of tissue lipotoxicity and have been associated with impaired insulin action
and ­sensitivity39. Bidirectionality has also been established by Warshauser et al. wherein reducing levels of HCER
and other ceramides pharmacologically improved HbA1C, insulin sensitivity and beta cell function in patients
with existing ­MetS40. More studies are needed to determine if a simple correlative or causal relationship exists
between these lipid species and impaired insulin sensitivity in both dogs and humans.
Plasma fatty acid concentrations and their physiological role in the development of insulin resistance, MetS
and T2D have been investigated for decades in humans and r­ odents17,41. Of note, Kang et al. demonstrate that
increases in visceral adipose mass, not subcutaneous adipose mass, are associated with elevated fatty acid con-
centrations in h­ umans42. Our analysis of fatty acid profiles in dogs revealed a significant elevation in fatty acid
species that have been directly implicated in the deterioration of insulin sensitivity, such as palmitic and stearic
­acid43,44. Furthermore, HFD feeding elevated nearly all SFA species and associated 18:1 (oleic acid) and 18:2
(linoleic acid), while significantly reducing the abundance of DPA and DHA—2 fatty acids associated with
improved insulin s­ ensitivity45.
Two groups of aggregated fatty acids, FFA and SFA, were also elevated with HFD feeding while no change
was observed to aggregated MUFA and PUFA pools. The presence of persistently elevated FFA and SFA concen-
trations has been linked to the development of ectopic lipid deposits, inflammation, lipotoxicity and impaired
insulin signaling in other s­ pecies17. These changes in fatty acids, along with the previously described changes in
plasma lipid species composition, suggests that HFD induces deleterious changes in the lipidome which may
elicit metabolic consequences on peripheral tissues, such as the skeletal muscle and l­ iver46,47.
As previously mentioned, nearly every species of SFA was significantly elevated as a result of the HFD feeding
and has been associated with obesity and T2D in ­humans17,19. Therefore, we examined the relationship between
SFA concentrations, insulin and insulin sensitivity metrics to determine if an association could be established.
We show that as SFA concentrations increase, insulin sensitivity is deteriorated, while ­AIRG is increased to
compensate. This finding is supported by previous studies which infused a fatty acid cocktail consisting of the
most abundant fatty acids present in our HFD model and caused impairments in the whole body and peripheral
insulin sensitivity of d­ ogs19.
Interestingly, the association between SFA concentrations and insulin sensitivity was stronger than its associa-
tion with fasting insulin (SFA ­R2: 0.49; fasting insulin ­R2: 0.40). Conversely, beta cell compensation is associated
more strongly with fasting insulin than SFA concentrations. These data suggest that SFA concentrations may be
a useful predictor of insulin sensitivity, regardless of dietary status in dogs. Further investigations are needed to
determine if SFA concentrations are predictive of metabolic health, specifically insulin sensitivity, in real world
companion dog populations.
The present study had several limitations. Dogs in the HFD group were presented with a double daily ration
of food, meanwhile dogs in the ND group were presented with their normal single ration. Due the ND and

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Fatty acid quantification

Week 0 Week 8
Fatty acid (µM) ND HFD p-value ND HFD p-value
Total [FA12:0] 0.8 ± 0.3 0.9 ± 0.5 0.669 1.6 ± 0.5 1.2 ± 0.5 0.092
Total [FA14:0] 24.6 ± 5.0 23.5 ± 6.4 0.661 30.1 ± 4.2 26.7 ± 6.0 0.140
Total [FA14:1] 2.3 ± 0.7 2.3 ± 1.2 0.959 2.2 ± 0.8 2.9 ± 1.8 0.254
Total [FA15:0] 9.1 ± 1.8 8.6 ± 3.0 0.655 9.5 ± 1.2 7.0 ± 1.2 < 0.001***
Total [FA16:0] 1742.1 ± 123.4 1760.3 ± 178.9 0.788 1643.8 ± 198.7 2211.1 ± 347.8 < 0.001***
Total [FA16:1] 139.9 ± 34.8 135.0 ± 25.8 0.716 134.3 ± 21.9 120.0 ± 22.9 0.160
Total [FA17:0] 36.2 ± 5.8 34.5 ± 3.3 0.412 41.4 ± 4.8 28.7 ± 4.2 < 0.001***
Total [FA18:0] 2438.3 ± 287.4 2525.2 ± 295.1 0.503 2325.1 ± 418.9 2796.7 ± 529.7 0.035*
Total [FA18:1] 1238.9 ± 121.2 1238.1 ± 125.5 0.989 1137.4 ± 106.7 1295.7 ± 181.9 0.025*
Total [FA18:2] 2135.1 ± 195.6 2173.7 ± 215.0 0.671 1867.1 ± 191.8 2268.6 ± 378.1 0.007**
Total [FA18:3] 66.5 ± 10.1 61.4 ± 16.6 0.401 51.1 ± 5.4 49.1 ± 10.9 0.600
Total [FA18:4] 0.4 ± 0.1 0.4 ± 0.1 0.149 1.8 ± 1.3 1.1 ± 0.7 0.157
Total [FA20:0] 28.3 ± 2.2 28.9 ± 1.6 0.554 26.1 ± 2.7 31.8 ± 5.0 0.004**
Total [FA20:1] 11.0 ± 1.1 11.3 ± 1.6 0.669 10.0 ± 1.3 12.0 ± 1.2 0.002**
Total [FA20:2] 23.7 ± 3.5 23.9 ± 2.4 0.867 20.0 ± 2.7 24.8 ± 3.4 0.002**
Total [FA20:3] 172.0 ± 43.3 169.5 ± 31.6 0.879 136.6 ± 37.3 181.9 ± 33.4 0.009**
Total [FA20:4] 1949.9 ± 258.9 1995.4 ± 264.3 0.695 1422.6 ± 293.5 1863.7 ± 412.5 0.011*
Total [FA20:5] 35.2 ± 6.3 30.7 ± 8.0 0.165 355.4 ± 199.8 213.8 ± 131.0 0.076
Total [FA22:0] 47.8 ± 5.1 49.2 ± 4.2 0.498 41.9 ± 6.6 54.3 ± 10.4 0.004**
Total [FA22:1] 12.6 ± 1.1 12.6 ± 1.0 0.965 11.0 ± 1.4 11.0 ± 1.9 0.970
Total [FA22:2] 0.8 ± 0.2 0.8 ± 0.2 0.747 0.7 ± 0.2 0.7 ± 0.1 0.939
Total [FA22:4] 77.4 ± 18.1 82.6 ± 22.1 0.561 25.9 ± 5.3 36.6 ± 10.6 0.010**
Total [FA22:5] 143.3 ± 21.5 144.3 ± 22.4 0.917 242.9 ± 47.1 170.4 ± 38.8 0.001**
Total [FA22:6] 51.5 ± 14.3 54.6 ± 12.8 0.615 231.2 ± 75.0 169.5 ± 54.6 0.048*
Total [FA24:0] 17.7 ± 1.8 17.3 ± 1.8 0.591 15.8 ± 2.0 15.6 ± 2.3 0.834
Total [FA24:1] 44.3 ± 4.0 43.8 ± 5.9 0.853 44.4 ± 6.5 43.6 ± 6.9 0.808
Total [FA26:0] 0.5 ± 0.0 0.5 ± 0.1 0.341 0.4 ± 0.1 0.5 ± 0.1 0.177
Total [FA26:1] 0.9 ± 0.1 0.9 ± 0.1 0.587 0.7 ± 0.1 0.8 ± 0.2 0.215
Σ SFA 4345.3 ± 396.4 4448.7 ± 401.4 0.560 4135.7 ± 583.0 5173.6 ± 884.1 0.005**
Σ MUFA 1449.9 ± 153.7 1444.0 ± 149.9 0.930 1340.0 ± 130.2 1486.1 ± 201.3 0.062
Σ PUFA 4655.9 ± 467.4 4737.2 ± 436.8 0.686 4355.2 ± 594.1 4980.3 ± 885.5 0.072
Σ FFA 7797.0 ± 696.1 7928.2 ± 689.6 0.670 7200.2 ± 836.5 8770.0 ± 1401.8 0.006**

Table 3.  Plasma fatty acid species composition in dogs fed a ND (n = 10) or HFD (n = 11) for 8 weeks. All
values shown are mean ± SD. FFA Free fatty acids, SFA saturated fatty acids, MUFA monounsaturated fatty
acids, PUFA polyunsaturated fatty acids. *p < 0.05; **p < 0.01; ***p < 0.001.

HFD groups seeing different total quantities of food, it is difficult to decouple the metabolic effects of elevated
fat content in the HFD from simple hyperphagic overnutrition. Additionally, tissue specific gene expression and
protein changes, which greatly impact substrate utilization, were not investigated and therefore we are unable to
make conclusions about the molecular impact to peripheral tissue.

Conclusion
These results demonstrate that chronic HFD feeding leads to deleterious changes in the plasma lipidome, fatty
acid species composition, hyperinsulinemic compensation and impaired insulin sensitivity in dogs. Furthermore,
we demonstrated that HFD feeding elevated nearly every SFA species while reducing the abundance of meta-
bolically beneficial species DPA and DHA. Elevations in SFA concentrations were associated with deteriorated
insulin sensitivity, which identifies, for the first time, the detrimental changes in plasma fatty acid species and
the development of metabolic dysfunction in dogs.

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a. b.

15 600

AIRG (mIU/l x min)

R2 = 0.40 R2 = 0.47

SI (mIU-1min-1)
p = 0.003 p < 0.001
10 400

5 200

0 0

0 5 10 15 20 0 5 10 15 20
Fasting Insulin (mIU/L) Fasting Insulin (mIU/L)

c. d.

15 600

AIRG (mIU/l x min)

R2 = 0.49 R2 = 0.37
SI (mIU-1min-1)

p < 0.001 p = 0.004

10 400

5 200

0 0

2000 4000 6000 8000 2000 4000 6000 8000

SFA (mM) SFA (mM)

e.
20 • ND
Insulin (mIU/L)

HFD
15

10
R2 = 0.29
5 p = 0.015

0
2000 4000 6000 8000
SFA (mM)

Figure 4.  Fasting insulin and SFA are associated with insulin sensitivity. Correlations of (a) fasting insulin to
insulin sensitivity ­SI, (b) fasting insulin to A
­ IRG, (c) SFA to ­SI, (d) SFA to ­AIRG, and (e) SFA to fasting insulin in
dogs after 12 weeks of HFD or ND feeding (n = 20).

Data availability
All data available by request from authors.

Received: 22 January 2024; Accepted: 28 May 2024

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Acknowledgements
The authors would like to thank James McMahon for comments on the manuscript and Dr. Josiane Broussard
for guidance with implementation of the HFD dog model.

Author contributions
DJS designed and managed the study. MP, AT and JG drafted the manuscript and carried out the analysis of the
data. DJS, MP, KT and EM were involved in study operations and data acquisition. DS and JG were involved
with IVGTT modeling and statistical analyses. CLHH, DJS and KG provided intellectual support. DJS, MP, AT,
JG, and KG were involved in writing the manuscript. All authors had final approval.

Competing interests
MP, AT, JG, KT, EM, KG, CLHH and DJS are employees of Cellular Longevity Inc. DS is an external consultant
for Cellular Longevity Inc.

Additional information
Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​024-​63373-5.
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