Partial Replacement of High-Fat Diet with Beef Tallow Attenuates Dyslipidemia and Endoplasmic Reticulum Stress in db / db Mice

Abstract

High-fat diet (HFD) consumption is closely associated with an increased risk of metabolic syndromes (MetS), such as obesity, type 2 diabetes, and cardiovascular diseases (CVDs). Therefore, the consumption of alternative and functional fatty acids to replace saturated fatty acids and/or trans-fatty acids with polyunsaturated fatty acids has become an important dietary strategy for the prevention of MetS. Consumption of omega-3 fatty acids (n-3) reduces various physiological complications, including CVDs, nonalcoholic fatty liver disease, and insulin resistance, related to inflammatory responses. In this study, we investigated the partial replacement effects of HFD with beef tallow (BT) on dyslipidemia and endoplasmic reticulum (ER) stress in male db/db mice. The animals were grouped to one of four dietary intervention groups (n = 16 per group): (1) normal diet, (2) HFD, (3) HFD partially replaced with regular beef tallow (HFD+BT1), or (4) HFD partially replaced with beef tallow containing a relatively reduced omega-6 fatty acid (n-6)/n-3 ratio (HFD+BT2) than HFD+BT1. After 6 weeks of dietary intervention, 1 mg/kg of phosphate-buffered saline or tunicamycin (TM) was injected intraperitoneally. HFD+BT2 significantly suppressed the serum total cholesterol and non-high-density lipoprotein cholesterol levels more than HFD and HFD+BT1, and triglyceride levels in the epididymal adipose tissue (EAT) were remarkably decreased. Mice that received HFD+BT2 had elevated protein expressions of phospho-AMP-activated protein kinase (p-AMPK). Moreover, HFD+BT2 effectively inhibited ER stress in the liver and EAT. Consistent with our hypothesis, HFD+BT2 remarkably alleviated dyslipidemia and TM-inducible ER stress, while activating p-AMPK.

INTRODUCTION

Diabetes is a metabolic complication characterized by prolonged systemic hyperglycemia caused by impaired insulin secretion and/or other relevant pathological impairments. It is classified as type 1 diabetes (T1D) and type 2 diabetes (T2D). T1D is characterized by destruction of pancreatic islet beta cells resulting in insufficient insulin production.¹ T2D is the most common form of the disease (over 90%) and is related to beta cell dysfunction and insulin resistance (IR) often caused by inadequate dietary consumption.^2,3 According to a global report by the World Health Organization (WHO), the total number of diabetic patients has dramatically increased in the past three decades and is expected to rise to 642 million by 2040.^4,5 In 2017, the estimated cost of diagnosed diabetes in the United States accounted for $327 billion,⁶ and the global health care expenditure on diabetes was estimated to be $850 billion.⁷ Metabolic syndrome (MetS) described as a combination of glucose intolerance, hypertension, dyslipidemia, and central obesity with IR is a critical risk factor for T2D, cardiovascular diseases (CVDs), and all-cause mortality.^8,9 Thus, lifestyle modification (diet and exercise) is the most important strategy to prevent or delay the onset of T2D and its complications.

Individuals consuming a high-fat diet (HFD) are at high risk of MetS.^10,11 HFD (often referred to as a Western diet)¹² is characterized by a relatively higher intake of saturated fatty acids (SFAs) and sucrose and/or lower intake of fiber.¹³ Continuous exposure of pancreatic beta cells to SFAs further promotes inflammation and endoplasmic reticulum (ER) stress, which can ultimately lead to pancreatic apoptosis.^14
–16 The WHO has suggested that total fatty acid intake should not exceed 30% of the total calorie intake to prevent overweight.^17
–19 Moreover, it has recommended to reduce the intake of SFAs to less than 10% and trans-fats to less than 1% of total calorie intake to lower the risk of diabetes, CVDs, cancer, and stroke.¹⁹ Therefore, consumption of alternative and functional fatty acids by replacing SFAs and trans-fatty acids with polyunsaturated fatty acids (PUFAs) has become an important dietary strategy for MetS prevention.¹⁴

PUFAs are considered essential fatty acids that must be consumed through the diet, as they are not able to be synthesized by humans or other mammals. Previous studies have demonstrated that increased consumption of omega-3 fatty acids (n-3) reduces the risk of CVDs, nonalcoholic fatty liver disease, and adverse complications related to IR and/or ER stress.^20,21 In contrast, excess intake of omega-6 fatty acids (n-6) exerts proinflammatory responses.²² The reference daily intake of total n-3 varies according to different international organizations; however, consuming more than 250 mg is highly preferable and recommended.^23,24 The ratio of n-6/n-3 is another key factor in PUFA consumption. However, the Western diet contains lower n-3, and the estimated ratio of n-6/n-3 is 20 or higher.^25,26 Therefore, reducing the n-6/n-3 ratio is important to prevent overweight, IR, inflammation, and hyperlipidemia.^27

–30 Based on the evidence to date, whether a low n-6/n-3 ratio suppresses ER stress in diabetic patients remains largely unknown.

Beef is a widely and frequently consumed meat in the United States. According to the 2019 USDA Food Availability data, the average American consumption is ∼55 pounds of beef per year.³¹ Beef is a great source of key nutrients, especially essential amino acids, B-vitamins, and minerals³²; thus, consumption of beef may provide multiple health benefits. However, evidence from the late 1970s indicated that beef consumption may elevate the risk of chronic diseases, such as CVDs and/or cancer, due to the relatively high content of SFAs,³³ resulting in a steady decline in the average amount of beef consumption.^34
–36 To compensate for these adverse effects of beef tallow (BT) previously reported, in our study, we partially replaced SFAs with PUFAs. In this study, we aimed to investigate the effects of isocaloric HFD prepared by partial replacement of lard with either a regular BT (HFD+BT1) or a BT containing lower n-6/n-3 ratio (HFD+BT2) on dyslipidemia, phospho-AMP-activated protein kinase (p-AMPK) expression, and tunicamycin (TM)-induced ER stress in diabetic (db/db) mice. TM, an antibiotic used to activate ER stress inducers, can activate the unfolded protein responses (UPRs) to induce cell death.³⁷ As a potent inducer of ER stress and the UPRs, TM has been reported to induce metabolic disorders in multiple reports.^37
–39 Therefore, in this study, we used TM to investigate the effects of severe ER stress on metabolic changes in db/db mice.

MATERIALS AND METHODS

Animal experiments and dietary interventions

All experimental mice studies were approved by the Institutional Animal Care Use Committee (IACUC) of Dankook University (No. DKU-20-028). A total of 64, 5-week-old, male, db/db mice (Cg-Dock7 ^m +/+ Lepr^db /J strain) were purchased from DooYeol Biotech, Inc. (Seoul, Korea). The mice were maintained in polypropylene cages under a controlled temperature of 20°C ± 2°C and humidity of 50–55% with a 12-h light/12-h dark cycle. The experimental mice were assigned at random to one of the eight groups (n = 8 per group) based on dietary intervention and TM injection. The experimental groups were as follows: (1) normal diet (ND), based on the AIN-93G formulation (15.75% of energy from fat); (2) HFD (44.57% of energy from fat); (3) HFD partially replaced with regular beef tallow (HFD+BT1, 44.24% of energy from fat); (4) HFD partially replaced with beef tallow containing a lower n-6/n-3 ratio (HFD+BT2, 44.33% of energy from fat); (5) ND with TM injection; (6) HFD with TM injection; (7) HFD+BT1 with TM injection; and (8) HFD+BT2 with TM injection. The composition of fatty acids and experimental diets is presented in Tables 1 and 2, respectively. BT1 and BT2 used in this study were derived from the retroperitoneal white adipose tissue (WAT) of the Hanwoo and perilla oil-fed black Angus, respectively. The n-3 content of BT2 was ∼2.7-fold higher compared with BT1 (Table 1). The n-6/n-3 ratio of BT1 and BT2 was 0.9 and 0.4, respectively. In the experimental diets, the absolute content of n-3 in HFD+BT1 and HFD+BT2 was formulated to match. Despite the equivalent composition of n-3, the total amount and n-6/n-3 ratio of PUFAs were remarkably higher in the HFD+BT1 group than in the HFD+BT2 group. After 6 weeks of animal experimental periods, the mice were injected with either 1 mg/kg of phosphate-buffered saline (PBS) or TM to induce ER stress and then fasted for 12 h. Subsequently, the mice were sacrificed by thoracotomy after CO₂ narcosis. Whole blood was collected by cardiac puncture, and the serum was separated by centrifugation (3000 g, at 4°C). The liver and WATs (epididymal, mesenteric, retroperitoneal, and perirenal tissues) were harvested, immediately weighed, and then stored at −80°C until further analysis.

Table 1.

Fatty Acid Composition of Beef Tallow

Fatty acids (%)	Diet
Fatty acids (%)	BT1	BT2
n-6	1.211	1.453
n-3	1.343	3.585
n-6/n-3	0.9	0.4

BT, beef tallow; n-3, omega-3 fatty acids; n-6, omega-6 fatty acids.

Table 2.

Composition of the Experimental Diets

Ingredients (g)	Diet
Ingredients (g)	ND	HFD	HFD+BT1	HFD+BT2
Casein	200	245	220	220
l-Cysteine	3	3.5	3.5	3.5
Sucrose	100	88	110	100
Cornstarch	397.486	115	132	130
Dextrose	132	190	180	191
t-Butylhydroquinone	0.014	0.034	0.034	0.034
Cellulose	50	58	56	56
Mineral mix^a	35	43	43	43
Vitamin mix^b	10	19	19	19
Choline bitartrate	2.5	3	3	3
Soybean oil	70	40	40	40
Lard	—	196	176	188
BT1	—	—	18.35	—
BT2	—	—	—	6.85
Percent kcal from fat	15.75	44.57	44.24	44.33
kcal/g diet	4.00	4.76	4.76	4.77

AIN-93-GX mineral mixture.

AIN-93-VX vitamin mixture.

HFD, high-fat diet; HFD+BT1, high-fat diet+regular beef tallow; HFD+BT2, high-fat diet+BT containing a lower n-6/n-3 ratio; ND, normal diet.

Biochemical analysis of serum

To measure the insulin sensitivity in experimental animals, insulin (Mercodia AB, Uppsala, Sweden) and serum glucose (Crystal Chem, Downers Grove, IL, USA) levels were assessed according to the manufacturer's instructions. The homeostasis model assessment of insulin resistance (HOMA-IR) values was processed using the following formula: HOMA-IR = serum insulin (μU/L) × serum glucose (mg/dL)/405.⁴⁰ Serum levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) were measured using commercial kits (MBL, Gunpo, Korea). To obtain the low-density lipoprotein cholesterol (LDL-C) levels, HDL-C and TG values were subtracted from the TC levels.⁴¹ The cardiac risk factor (CRF = TC/HDL-C) was calculated.^42,43 Hepatic biological function was determined by aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels using commercial kits (MBL).

Lipid contents in the adipose tissue

Lipids in epididymal adipose tissue (EAT) were extracted as previously described by Bligh and Dyer,⁴⁴ with slight modifications.⁴⁵ Briefly, ∼0.1 g tissues were added to chloroform:methanol (1:2, v/v) solution and then centrifuged at 805 g for 15 min. Subsequently, the lower phase was separated, and 1 mL of n-hexane: isopropanol (3:2, v/v) was added to dissolve the lipids. TG and TC concentrations were measured using commercial kits (MBL) and normalized by the premeasured tissue weights.

Western blotting analysis

Proteins from the liver and EAT were isolated using ice-cold radioimmune precipitation assay (RIPA) lysis buffer (ATTO, Tokyo, Japan) with protease and phosphatase inhibitors (Thermo Fisher Scientific). Equal amounts (30 μg) of proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk (BD Difco^™, Franklin Lakes, NJ, USA) and incubated with antibodies against phospho-AMP-activated protein kinase (p-AMPK), total AMP-activated protein kinase (t-AMPK), binding immunoglobulin protein (BiP), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), X-box binding protein 1 (XBP-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An enhanced chemiluminescence solution (Thermo Fisher Scientific) was used to detect the membrane using a Davinch Chemi Fluoro Imager (Davinch-K, Seoul, Korea). The protein bands were then quantitatively analyzed using Image J Software (v.1.8 National Institutes of Health, Bethesda, MD, USA), using GAPDH as an internal control. Details of the antibody information are available in Table 3.

Table 3.

List of Antibodies Used for Western Blotting Analysis

Antibody	Company	Catalog No.	Dilution
p-AMPK	Cell Signaling	2535	1:1000
t-AMPK	Cell Signaling	2532	1:1000
BiP	Cell Signaling	3183	1:1000
ATF4	Cell Signaling	11815	1:1000
CHOP	Cell Signaling	2895	1:1000
XBP-1	Cell Signaling	40435	1:1000
GAPDH	Santa Cruz	365062	1:2000
Anti-rabbit IgG	Cell Signaling	7074	1:3000
Anti-mouse IgG	Cell Signaling	7076	1:1000

ATF4, activating transcription factor 4; BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-AMPK, phosphorylated AMP-activated protein kinase; t-AMPK, total AMP-activated protein kinase; XBP-1, X-box binding protein 1.

Statistical analysis

Results are presented as the means and standard deviations or Box-and-Whisker plots. All figures were plotted using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Data were statistically analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test using SPSS 26.0 (Statistical Package for Social Science, SPSS, Inc., Chicago, IL, USA) to test the significance of the dietary intervention. Two-way ANOVA with Tukey's post hoc test was performed using XLAST 2012 (Addinsoft, Inc., Paris, France) to test the effects of diet and ER stress or their interactions. Statistical significance was set at P < .05. A summary of the two-way ANOVA results from the main effects and interactions is provided in Table 4.

Table 4.

Summary of Statistical Analyses by Two-Way Analysis of Variance for Main Effects and Interactions

Parameter	Factor, P values
Parameter	TM	Diet	TM × diet
Relative tissue weights
Liver	<.01^**	.087^ns	.343^ns
WATs	.070^ns	<.0001^****	.590^ns
EAT	.083^ns	<.0001^****	<.05^*
MAT	.269^ns	<.01^**	.334^ns
RAT	.817^ns	.097^ns	.641^ns
PAT	.400^ns	.125^ns	.667^ns
Serum glucose and insulin levels
Glucose	<.0001^****	<.0001^****	<.0001^****
Insulin	.377^ns	.202^ns	<.0001^****
HOMA-IR	<.0001^****	<.05^*	<.0001^****
Serum lipid profile
TC	<.0001^****	<.0001^****	<.0001^****
HDL-cholesterol	<.0001^****	<.0001^****	.647^ns
LDL-cholesterol	<.0001^****	<.0001^****	<.0001^***
TG	<.0001^****	<.0001^****	<.0001^****
Cardiac risk factor	<.0001^****	<.0001^****	<.0001^****
Hepatic function parameters
Serum ALT	<.0001^****	<.0001^****	<.0001^****
Serum AST	<.0001^****	<.0001^****	<.0001^****
Serum ALP	<.0001^****	<.0001^****	.066^ns
Lipid contents in the EAT
EAT TG	<.01^**	<.01^**	<.0001^****
EAT TC	<.0001^***	<.0001^****	<.0001^****

P < .05; ^** P < .01; ^*** P < .001; ^**** P < .0001.

ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; EAT, epididymal adipose tissue; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment of insulin resistance; LDL, low-density lipoprotein; MAT, mesenteric adipose tissue; ns, not significant; PAT, perirenal adipose tissue; RAT, retroperitoneal adipose tissue; TC, total cholesterol; TG, triglyceride; TM, tunicamycin; WATs, white adipose tissues.

RESULTS

Weight gain and food efficiency

The body weight (BW) change (ΔBW, the final BW minus the initial BW) was remarkably higher in the HFD, HFD+BT1, and HFD+BT2 groups than in the ND group (Fig. 1A). Daily food intake was significantly higher in the HFD group than in the ND group. However, HFD + BT supplementation had a lower daily food intake than HFD (HFD > HFD+BT1 > HFD+BT2 or ND; Fig. 1B). Daily energy intake showed a trend similar to that of daily food intake (Fig. 1C). According to the food efficiency ratio (FER) assessment, ND feeding resulted in the lowest FER, whereas the HFD+BT2 diet resulted in the highest FER (P < .05; Fig. 1D).

FIG. 1.

Effects of the partial replacement of HFD with BT on the body weight, daily food intake, daily energy intake, and FER of mice. Five-week-old male diabetic (db/db) mice were fed an ND, HFD, HFD partially replaced with regular beef tallow (HFD+BT1), or HFD partially replaced with BT containing a lower n-6/n-3 ratio (HFD+BT2) for 6 weeks. (A) Delta body weight gain (the final body weight after dietary feeding − the initial body weight), (B) daily food intake, (C) daily energy intake, and (D) FER were measured. Values are presented as the mean ± SD; n = 16 per individual group. Data were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test; labeled means without a common letter differ significantly, P < .05. ANOVA, analysis of variance; BT, beef tallow; FER, food efficiency ratio; HFD, high-fat diet; HFD+BT1, high-fat diet+regular beef tallow; HFD+BT2, high-fat diet+BT containing a lower n-6/n-3 ratio; n-3, omega-3 fatty acids; n-6, omega-6 fatty acids; ND, normal diet; SD, standard deviation.

Weights of the liver and WATs

To examine the weight changes in central metabolic organs, the liver and WATs (i.e., EAT, mesenteric adipose tissue [MAT], retroperitoneal adipose tissue [RAT], and perirenal adipose tissue [PAT]) were weighed and expressed as a percentage of BW (Table 5). In TM-injected mice, liver weight was significantly increased by 13.05%, regardless of the dietary intervention. Among the WATs, the relative weight of the EAT showed a decreasing trend in the TM-treated group compared to that in the PBS-treated group (P = .083); however, there were no statistical differences observed in the weights of WATs, MAT, RAT, and PAT after TM injection. Before TM treatment, the HFD-fed groups (HFD, HFD+BT1, and HFD+BT2) showed a significant increase in EAT compared to the ND group. After TM treatment, the weight of EAT was lower in the HFD+BT2 group than in the HFD+BT1-fed mice (4.46% vs 5.27% of BW, P < .05). However, there were no significant differences in the weight of the liver and MAT, RAT, and PAT following TM challenge.

Table 5.

Relative Weights of the Liver and White Adipose Tissues

	TM (−)				TM (+)
	ND	HFD	HFD+BT1	HFD+BT2	ND	HFD	HFD+BT1	HFD+BT2
Liver (% of BW)	6.15 ± 0.86	5.38 ± 0.64	6.16 ± 0.42	5.43 ± 0.96	6.42 ± 0.71	6.05 ± 0.61	6.34 ± 0.90	6.80 ± 0.69
WATs (% of BW)	9.83 ± 0.71	11.12 ± 1.11	11.15 ± 0.65	11.63 ± 0.69	9.12 ± 1.16	10.76 ± 0.88	11.22 ± 1.03	10.86 ± 1.26
EAT (% of BW)	4.38 ± 0.47^b	4.82 ± 0.78^a	5.01 ± 0.45^a	5.36 ± 0.57^a	4.05 ± 0.35^c	4.79 ± 0.62^b	5.27 ± 0.62^a	4.46 ± 0.39^b
MAT (% of BW)	3.18 ± 0.35	3.70 ± 0.57	3.82 ± 0.49	3.51 ± 0.37	2.91 ± 0.39	3.53 ± 0.34	3.49 ± 0.53	3.74 ± 0.67
RAT (% of BW)	1.38 ± 0.30	1.54 ± 0.45	1.30 ± 0.18	1.73 ± 0.55	1.27 ± 0.49	1.50 ± 0.40	1.49 ± 0.15	1.59 ± 0.46
PAT (% of BW)	0.89 ± 0.16	1.07 ± 0.18	1.02 ± 0.24	1.03 ± 0.23	0.89 ± 0.12	0.94 ± 0.14	0.97 ± 0.19	1.06 ± 0.17

Values are presented as the mean ± SD; n = 16 per individual group. Data were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test; labeled means without a common letter differ significantly, P < .05.

BW, body weight.

Serum glucose and insulin levels

Since replacement of HFD with BTs enhanced FER, we investigated whether BT could also alter insulin sensitivity at the end of the animal experimental periods. Following TM injection, serum glucose and HOMA-IR were remarkably reduced (P < .0001; Fig. 2A, C) compared to the PBS treatment group. In the absence of TM, serum glucose levels were similar in all groups, except in the HFD+BT1 group, where the levels were significantly decreased. After TM administration, the serum glucose level in the ND group was significantly lower than that in other HFD groups; however, BT replacement further reduced these levels in the HFD groups (P < .0001; Fig. 2A). In the absence of TM, serum insulin and HOMA-IR were significantly higher in the HFD+BT2 group than in the HFD group. In the presence of TM, serum insulin and HOMA-IR were remarkably lower in the HFD+BT1 and HFD+BT2 groups than in the HFD-fed mice (P < .0001; Fig. 2B, C). These results suggest that consumption of HFD+BT1 and HFD+BT2 may enhance insulin sensitivity in the ER stress induction.

FIG. 2.

Effects of the partial replacement of HFD with BT on the serum glucose and insulin levels. Five-week-old male db/db mice were fed the ND, HFD, HFD partially replaced with regular beef tallow (HFD+BT1), or HFD partially replaced with BT containing a lower n-6/n-3 ratio (HFD+BT2) for 6 weeks, followed by injection with PBS or TM (1 mg/kg) for 12 h. (A) Serum glucose, (B) serum insulin, and (C) HOMA-IR levels. Values are presented as Box-and-Whisker plots representing eight mice per individual group. Data were analyzed using two-way ANOVA followed by Tukey's multiple comparisons test; labeled means without a common letter differ significantly, P < .05. HOMA-IR, homeostasis model assessment-estimated insulin resistance; PBS, phosphate-buffered saline; TM, tunicamycin.

Serum lipid panels

To investigate the effects of HFD replacement with BTs on lipid-lowering effects and CVD risk factors in diet-induced dyslipidemia, we assessed lipid panels and determined CRF from serum analyses. TM injection significantly induced dyslipidemia by increasing the levels of TC, LDL-C, and CRF by 15.37%, 98.72%, and 143.09%, respectively, and decreased HDL-C level by 47.97% compared to the PBS-injected groups (Fig. 3). TC levels were attenuated in the HFD+BT2 group than in the HFD and HFD+BT1 groups in the absence of TM; however, after TM injection, TC levels were increased in the HFD+BT2 group compared to those in other groups in the presence of TM because of increased HDL-C levels, as shown in Figure 3B (Fig. 3A).

FIG. 3.

Effects of the partial replacement of HFD with BT on serum lipid panels. Five-week-old male db/db mice were fed the ND, HFD, HFD partially replaced with regular beef tallow (HFD+BT1), or HFD partially replaced with BT containing a lower n-6/n-3 ratio (HFD+BT2) for 6 weeks, followed by injection with PBS or TM (1 mg/kg) for 12 h. (A) Serum TC, (B) HDL-cholesterol, (C) LDL-cholesterol, (D) TG levels, and (E) CRF were measured. Values are presented as Box-and-Whisker plots representing eight mice per individual group. Data were analyzed using two-way ANOVA followed by Tukey's multiple comparisons test; labeled means without a common letter differ significantly, P < .05. CRF, cardiac risk factor; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TC, total cholesterol; TG, triglyceride.

The serum HDL-C levels were increased in the HFD+BT1 or HFD+BT2 groups than in the HFD group, regardless of TM stimulation (Fig. 3B). In the PBS (vehicle)-injected group, serum LDL-C levels were decreased in the HFD+BT1- or HFD+BT2-fed mice than in the HFD group by 24.40% and 57.04%, respectively, whereas the levels in the TM treatment group were similar between the HFD+BT1 and HFD+BT2 groups (Fig. 3C). As expected, the TG levels were significantly lower in the HFD+BT1 or HFD+BT2 groups than in the ND and HFD groups following TM injection (Fig. 3D).

Based on the TC and HDL-C levels, we calculated the CRF. The CRF was higher in the HFD group than in the ND group; however, HFD+BT2 significantly inhibited HFD-induced (P < .0001) and TM-injected (P < .0001) CVD-related indices (Fig. 3E). In summary, our findings indicate that replacing HFD with BT consumption could significantly improve lipid panels; in particular, HFD+BT2 had a protective effect against CVDs and reduced dyslipidemia effects regardless of TM injection.

Hepatic function tests

To evaluate the effects of partial replacement of HFD with BTs on enzymatic function in the liver, the ALT, AST, and ALP activities were examined in serum (Fig. 4). TM injection significantly increased ALT, AST, and ALP activities by 2.64-, 2.99-, and 1.26-fold, respectively, compared to the vehicle group (P < .0001; Fig. 4). The ALT, AST, and ALP activities were notably higher in the HFD, HFD+BT1, and HFD+BT2 groups than in the ND group regardless of TM treatment (P < .0001); however, there was no remarkable difference among the HFD-fed groups. The HFD+BT1 group had decreased ALT and AST levels compared to the HFD and HFD+BT2 groups following TM challenge. Unexpectedly, HFD+BT2 consumption significantly increased serum ALT and AST activities compared to HFD consumption (P < .0001; Fig. 4A, B). ALP activity did not alter dietary interventions between groups (Fig. 4C).

FIG. 4.

Effects of the partial replacement of HFD with BT on the enzyme levels related to hepatic function. Five-week-old-male db/db mice were fed the ND, HFD, HFD partially replaced with regular beef tallow (HFD+BT1), or HFD partially replaced with BT containing a lower n-6/n-3 ratio (HFD+BT2) for 6 weeks, followed by injection with PBS or TM (1 mg/kg) for 12 h. (A) ALT, (B) AST, and (C) ALP activities were measured. Values are presented as Box-and-Whisker plots representing eight mice per individual group. Data were analyzed using two-way ANOVA followed by Tukey's multiple comparisons test; labeled means without a common letter differ significantly, P < .05. ALP, alkaline phosphate; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Lipid accumulation in EAT

To measure lipid accumulation in the EAT, we measured the TG and TC levels. EAT TG and TC levels were increased by 15.97% and 27.26%, respectively, in the TM-treated groups compared to those in the PBS group. The HFD group had significantly higher levels of TG (Fig. 5A) and TC (Fig. 5B) than the ND group, regardless of TM injection. However, before TM treatment, TG and TC levels were decreased in HFD+BT1 and HFD+BT2 compared to those in the HFD group (P < .0001). Interestingly, after TM treatment, the HFD+BT1 and HFD+BT2 groups significantly attenuated TC levels by 43.89% and 51.57%, respectively, compared to the HFD group (P < .0001; Fig. 5B). These results indicate that replacement of HFD with BTs could improve lipid accumulation in adipose tissues.

FIG. 5.

Effects of the partial replacement of HFD with BT on the lipid contents in the EAT. Five-week-old male db/db mice were fed the ND, HFD, HFD partially replaced with regular beef tallow (HFD+BT1), or HFD partially replaced with BT containing a lower n-6/n-3 ratio (HFD+BT2) for 6 weeks, followed by injection with PBS or TM (1 mg/kg) for 12 h. (A) EAT TG and (B) TC levels were measured. Values are presented as Box-and-Whisker plots representing eight mice per individual group. Data were analyzed using two-way ANOVA followed by Tukey's multiple comparisons test; labeled means without a common letter differ significantly, P < .05. EAT, epididymal adipose tissue.

AMPK and ER stress-related protein expression in the liver

AMPK activation has been shown to exert protective effects against IR⁴⁶ and may function as an ER regulator.⁴⁷ We examined the endeavors of replacing HFD with PUFAs containing BTs on AMPK activation. In the liver, HFD supplementation remarkably reduced p-AMPK levels by 9.12-fold. The HFD+BT2 group showed significantly increased p-AMPK levels compared to the HFD group, but not the HFD+BT1 group (Fig. 6A, B). To understand ER stress in TM-induced db/db mice, we measured ER stress-relevant protein expressions. In the liver, the HFD group showed considerably elevated levels of ER stress indicators, such as ATF4, CHOP, and XBP-1 compared to the ND group by 0.80-, 0.33-, and 0.82-fold, respectively (Fig. 6A, D–F). The HFD+BT1 group did not significantly lower ER stress indicators from the HFD group (Fig. 6). However, the BiP, CHOP, and XBP-1 protein expressions in the HFD+BT2-fed mice were markedly lower than those in HFD-fed mice by 2.40-, 2.06-, and 3.06-fold, respectively (Fig. 6A, C, E, F).

FIG. 6.

Effects of the partial replacement of HFD with BT on the levels of proteins related to the AMPK and ER stress in the liver. Five-week-old male db/db mice were fed the ND, HFD, HFD partially replaced with regular beef tallow (HFD+BT1), or HFD partially replaced with BT containing a lower n-6/n-3 ratio (HFD+BT2) for 6 weeks, followed by injection with PBS or TM (1 mg/kg) for 12 h. (A) Representative Western blotting images, (B) p-AMPK, (C) BiP, (D) ATF4, (E) CHOP, and (F) XBP-1 levels were measured. The expression of each protein was normalized to that of GAPDH, the internal control of protein content. Values are presented as Box-and-Whisker plots representing 8 mice per individual group. Data were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test; labeled means without a common letter differ significantly, P < .05. AMPK, AMP-activated protein kinase; ATF4, activating transcription factor 4; BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-AMPK, phospho-AMP-activated protein kinase; XBP-1, X-box binding protein 1.

AMPK and ER stress-related protein expression in EAT

BT2 consumption significantly attenuated hepatic ER stress by increasing the p-AMPK levels. Therefore, we postulated that BT replacement may attenuate TM-inducible ER stress and p-AMPK in the peripheral tissue, the EAT. The partial replacement of HFD with BTs increased p-AMPK protein expression compared to ND (Fig. 7A, B). However, the HFD+BT1 and HFD+BT2 groups showed remarkably enhanced p-AMPK levels compared to the ND and HFD groups (Fig. 7A, B). Next, we examined the ER stress markers in the EAT. HFD consumption increased the expression of BiP (Fig. 7A, C) and XBP-1 (Fig. 7A, E) compared to ND. In contrast, the BiP, CHOP, and XBP-1 protein expressions were remarkably reduced in the HFD+BT1 and HFD+BT2 groups compared to those in the HFD group (Fig. 7A, C–E), and HFD+BT2 had greatly decreased BiP levels compared to the HFD and HFD+BT1 groups (Fig. 7A, C).

FIG. 7.

Effects of the partial replacement of HFD with BT on the levels of proteins related to AMPK and ER stress in EAT. Five-week-old male db/db mice were fed the ND, HFD, HFD partially replaced with regular beef tallow (HFD+BT1), or HFD partially replaced with BT containing a lower n-6/n-3 ratio (HFD+BT2) for 6 weeks, followed by injection with PBS or TM (1 mg/kg) for 12 h. (A) Representative Western blotting images, (B) p-AMPK, (C) BiP, (D) CHOP, and (E) XBP-1 levels were measured. The expression of each protein was normalized to that of GAPDH, the internal control of protein content. Values are presented as Box-and-Whisker plots representing eight mice per individual group. Data were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test; labeled means without a common letter differ significantly, P < .05.

DISCUSSION

In this study, we examined the effects of partial replacement of SFAs with BTs containing a relatively lower n-6/n-3 ratio on dyslipidemia, ER stress, and AMPK activation in db/db mice. db/db mice are genetically vulnerable to metabolically complicated conditions, such as increased IR associated with the pancreatic beta cell dysfunction, morbid obesity with polyphagic behavior, and diabetic dyslipidemia. Therefore, db/db mice may be an excellent murine model to explore the beneficial effects of replacing SFAs with functional fatty acids from BTs. Fatty acid composition of the diet, particularly from animal fats, has been suggested to be linked with BW gain and fat accumulation.^48,49 In this experiment, we raised the research question of whether the absolute content of n-3 or the ratio of n-6/n-3 in BTs is of more importance in PUFA consumption; therefore, the absolute amount of n-3 in the HFD+BT1 and the HFD+BT2 groups was matched. Diet-induced obesity and overweight are closely associated with peripheral IR and hyperinsulinemia, which may eventually progress to T2D.⁵⁰ In this study, all animals fed HFD had greater BW gain than those that received ND (Fig. 1A). Although we did not observe noticeable changes in BW gain among the HFD groups during the dietary intervention, HFD supplemented with BTs resulted in elevated FER (Fig. 1D). Furthermore, BT replacement reduced serum insulin and HOMA-IR levels in the presence of TM (Fig. 2B, C). Therefore, replacing HFD with BTs may enhance systemic glucose utilization in the presence of TM owing to n-3 consumption (Fig. 2).⁵¹ However, in the absence of TM, HOMA-IR levels were significantly increased in the HFD+BT2 group (Fig. 2C). Furthermore, the ALT and AST levels associated with hepatic function were significantly increased by HFD+BT2 supplementation (Fig. 4A, B). Our findings refuted previous rodent studies that reported a significant reduction in HOMA-IR, ALT, and AST by n-3 supplementation.^52
–54 Discrepancies between our findings and those of previous studies^52
–54 may be due to differences in the biological oxidative demands since n-3 consumption may elevate oxidative stress by depleting α-tocopherol⁵⁵ due to the inherent structural instability of n-3,⁵⁶ which acts as an antioxidant that protects against lipid peroxidations.^57
–59 Findings suggest that α-tocopherol may have beneficial effects on IR and hepatic injury. Emam et al. reported that n-3 supplementation with α-tocopherol significantly lowered the ALT and AST levels.⁵⁷ We assumed that the elevation of HOMA-IR and hepatic functional markers (AST and ALT) in HFD+BT2 without TM injection is presumably caused by increased demand for antioxidant activity; thus, we carefully suggest including a sufficient dose of antioxidants in a diet high in PUFAs.

Dyslipidemia is characterized by high concentrations of TG, TC, LDL-C, and low HDL-C in the serum, and these conditions can greatly influence CVD risk⁶⁰; therefore, we assessed lipid panels in the serum and calculated CRF levels to explain the modulation of lipid profiles by consumption of HFD supplemented with BTs. In a previous study, HFD-fed db/db mice exhibited dyslipidemia by presenting elevated levels of serum TG, TC, and LDL-C compared to the ND group.⁶¹ Likewise, in this study, HFD consumption increased serum TC and LDL-C levels and decreased HDL-C levels (Fig. 3A–C). Consequently, CRF significantly increased (Fig. 3E). Nevertheless, BT replacement attenuated HFD-induced dyslipidemia. In particular, HFD+BT2 remarkably decreased TC and LDL-C levels, yet increased HDL-C values compared to HFD and HFD+BT1 (Fig. 3A–C). Similarly, previous clinical reports have shown that replacement of SFAs with PUFAs results in a decreased incidence of CVDs.^62,63 In addition to replacing SFAs with PUFAs, the n-6/n-3 ratio may play an important role in preventing CVDs and MetS.^26,64 In accordance with this, Lee et al. demonstrated that a diet low in n-6/n-3 ratio (<4) decreased the percentage of MetS and CVD risks in obese individuals.⁶⁵ Consequently, these data suggest that the replacement of HFD with BTs may improve lipid metabolism in the serum. However, HFD+BT2 was more capable of exhibiting beneficial effects, particularly on lipid metabolism and CVD expectancy. Next, the effects of BT replacement on lipid metabolism in WATs were elucidated. HFD consumption increased the mass of WATs, EAT, and MAT, which are effective indicators of obesity (Table 5). PUFA consumption prevented total fatty acid expansion compared with SFAs.⁶⁶ However, in our experiment, HFD + BTs did not significantly alter WAT weights (Table 5). This result may be correlated with the unchanged BW data discussed earlier. Hence, the exposure time or composition of BTs might have been inadequate to exert robust effects. In the EAT, the HFD+BT1 and HFD+BT2 groups showed lower TG and TC levels when compared to the HFD group (Fig. 5). These outcomes suggest that HFD replaced with BTs may have protective effects against high lipid levels in the EAT.

Obesity and T2D are closely associated with conditions of chronic systemic inflammation and cellular stress.^67
–69 In particular, ER stress plays an important role in the network of stress signals involved in IR and diabetes by disrupting systemic glucose homeostasis.^70,71 As a key nutrient and energy sensor, AMPK plays an important role in maintaining energy homeostasis and controlling glucose and lipid metabolism.^46,72 Activation of AMPK inhibits ER stress responses induced by metabolic stress, especially in obesity and T2D.^73,74 Evidence has shown that administration of AMPK activators to obese mammals reduces plasma glucose levels and enhances insulin sensitivity.^73,75 It has been demonstrated that both HFD-fed and genetically obese animal models decreased p-AMPK.^76,77 In this study, HFD+BT2 supplementation protected against HFD-induced decrease in p-AMPK levels in the liver and EAT (Figs. 6 and 7A, B). AMPK activation inhibits ER stress^21,78,79; however, the ER stress level is elevated in the development of T2D.⁸⁰ To modulate ER stress in metabolically complicated conditions, such as diabetes, the UPR signaling pathways are activated.⁸¹ The UPRs are initiated by three signaling pathways present in the ER membrane: PERK and its downstream targets ATF4 and CHOP; IRE1 and its downstream target BiP; and ATF6 and its downstream targets BiP, CHOP, and XBP-1.^82

–86 As a pharmacologic ER stress inducer,³⁷ TM was injected into db/db mice to observe the effects of BTs on attenuating ER stress. In our experimental settings, dietary interventions did not significantly alter UPRs in all PBS-injected groups; therefore, we scrutinized the UPRs in both liver and EAT (data not shown) in TM-injected groups. In our study, TM injection significantly elevated messenger RNA (mRNA) and protein expression of BiP and CHOP in both liver and EAT (data not shown), which is consistent with a previous study.⁸⁷ These results may indicate that TM induced ER stress in the liver and EAT by upregulating BiP and CHOP. Numerous studies have shown the association between fat as a source of energy and ER stress. Different types of fatty acids exhibit varying effects on ER stress. Consistent with our results, excessive intake of SFAs was shown to induce ER stress.^14,88 Hepatic ER stress strongly stimulates hepatic gluconeogenesis and enhances insulin sensitivity.^89
–91 Furthermore, ER stress in adipose tissues has been recognized as an important mechanism for IR. ER stress not only triggers apoptosis in adipocytes but also induces adipose tissue dysfunction, characterized by altered secretion of adipokines.⁸⁹ In this study, we found that HFD+BT2 diets suppressed TM-induced ER stress in the liver, as indicated by the reduced protein expression of ATF4, BiP, CHOP, and XBP-1 (Fig. 6A, C–F). In addition, in the EAT, the effects of HFD+BT2 on BiP, CHOP, and XBP-1 protein levels showed that HFD+BT2 may alleviate ER stress (Fig. 7A, C–E). These results were consistent with a study by Wang et al., who reported that n-3 decreased the ER stress response to improve pancreatic beta cell damage.⁹² Furthermore, a previous study by Yang et al. reported that n-3 protects against HFD-induced ER stress by AMPK activation.²¹ Therefore, our findings suggest that HFD+BT2 may promote AMPK activation and contribute to the inhibition of ER stress. These findings agree with those of previous studies reporting that AMPK activation can prevent ER stress induction.⁹³

In conclusion, we have shown that replacing HFD with BT exhibits antidyslipidemic effects, as indicated by the improved lipid panels in the serum and EAT. Furthermore, HFD+BT2 supplementation activated AMPK and protected against TM-induced ER stress in the liver and EAT. These results suggest that HFD+BT2 supplementation may be utilized as a replacement for HFD to ameliorate the metabolic dysregulation caused by T2D. The results of this study can be used as a basis for future clinical trials, and the effects of BT containing a lower n-6/n-3 ratio in regulating the physiological activity may be further elucidated through additional clinical studies.

ETHICS APPROVAL

Approval was received from the IACUC of Dankook University (No. DKU-20-028) before conducting this study.

Footnotes

ACKNOWLEDGMENT

This dissertational work was conducted for J.L.'s Master's degree.

AUTHORs' CONTRIBUTIONS

Conceptualization: J.-J.L. and J.-H.H.; methodology: J.L., S.P., S.J., and J.-H.H.; software: J.L., J.K.L., S.P., S.J., and J.-H.H.; validation: J.L., J.K.L., S.P., and J.-H.H.; formal analysis: J.L., S.P., and J.-H.H.; investigation: J.K.L., H.-J.L., and J.-H.H.; resources: H.-J.L. and J.-H.H.; data curation: J.L., J.K.L., S.P., and J.-H.H.; writing—original draft preparation: J.L., J.K.L., J.-J.L., S.P., S.J., H.-J.L., and J.-H.H.; writing—review and editing: J.L., J.K.L., J.-J.L., S.P., S.J., H.-J.L., and J.-H.H.; visualization: J.L. and S.P.; supervision: J.-H.H.; project administration: J.-H.H.; and funding acquisition: J.-H.H. All authors have read and agreed to the published version of the article.

AUTHOR DISCLOSURE STATEMENT

The authors declare no conflict of interests.

FUNDING INFORMATION

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Innovative Food Product and Natural Food Materials Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA 319045-3).

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