Distinct metabolic pathways trigger adipocyte fat accumulation induced by high-carbohydrate and high-fat diets

Accepted Manuscript Distinct metabolic pathways trigger adipocyte fat accumulation induced by highcarbohydrate and high-fat diets Adaliene Versiani Matos Ferreira, Ph.D. Zélia Menezes-Garcia, M.Sc. Jonas Baeta Viana, M.Sc. Érica Guilhen Mário, Ph.D. Leida Maria Botion, Ph.D. PII:

S0899-9007(14)00122-1

DOI:

10.1016/j.nut.2014.02.017

Reference:

NUT 9235

To appear in:

Nutrition

Received Date: 30 September 2013 Revised Date:

13 February 2014

Accepted Date: 14 February 2014

Please cite this article as: Matos Ferreira AV, Menezes-Garcia Z, Viana JB, Mário ÉG, Botion LM, Distinct metabolic pathways trigger adipocyte fat accumulation induced by high-carbohydrate and highfat diets, Nutrition (2014), doi: 10.1016/j.nut.2014.02.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Distinct metabolic pathways trigger adipocyte fat accumulation induced by highcarbohydrate and high-fat diets Running head: Fat accumulation induced by carbohydrates and fat diets. Adaliene Versiani Matos Ferreira1, Ph.D.; Zélia Menezes-Garcia2, M.Sc.; Jonas

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Baeta Viana3, M.Sc.; Érica Guilhen Mário3, Ph.D.; Leida Maria Botion3, Ph.D.

1. Department of Nutrition, Nursing School, Universidade Federal de Minas

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Gerais, Belo Horizonte, Minas Gerais, Brazil.

2. Department of Microbiology, Biological Sciences Institute, Universidade

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Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. 3. Department of Physiology and Biophysics, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais,

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Brazil.

Correspondence: Adaliene Versiani Matos Ferreira, Av. Alfredo Balena, 190, 30130-100, Departamento de Nutrição, Escola de Enfermagem, Universidade

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Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil. Tel and fax: +55 31

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3409-8028. E-mail: [email protected]

Total word count: 4.495 Number of tables and figures: 05

ACKNOWLEDGMENTS Authors’ Contributions: JBV was responsible for PCR. AVMF, ZMG and EGM were responsible for other experiments. AVMF and LMB designed the experiment. AVMF, ZMG, EMG and LMB were responsible for results, discussion and 1

ACCEPTED MANUSCRIPT manuscript redaction. All authors read and approved the final manuscript. We are grateful for the financial assistance from Pró-Reitoria de Pesquisa da UFMG,

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Capes, FAPEMIG and CNPq.

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ACCEPTED MANUSCRIPT ABSTRACT Objective: Several metabolic pathways may be associated with obesity development and may be differentially modulated by dietary constituents. The aim of the present work was to access the adipocyte metabolic pathways that lead to lipid accumulation in

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fat cells of mice fed a high-carbohydrate or high-fat diet.

Research Methods & Procedures: Male Swiss mice aged 7–8 weeks were fed a standard laboratory rodent diet - chow diet (C), a high-carbohydrate (HC) diet or a high-

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fat (HF) diet for 8 weeks.

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Results: Animals fed the HC diet exhibited a high lipogenesis rate and lipoprotein lipase activity even in a fasted state. PPAR-gamma and SREBP-1 mRNAs were increased in adipose tissue. The fat-rich diet did not promote the lipogenic pathways but attenuated lipolytic activity and glucose uptake in adipocytes.

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Conclusion: The carbohydrate-rich diets induced constitutive expression of lipogenic transcription factors during fasting, whereas fat-rich diets decreased lipolysis in adipocytes, enhanced fat stores and maintained an obese state.

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Keywords: carbohydrate; fat; metabolic pathway; adipocyte; lipid accumulation.

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ACCEPTED MANUSCRIPT INTRODUCTION Excessive caloric consumption is associated with adipose tissue expansion, obesity and several comorbidities [1-3]. Carbohydrates and fats are two important

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caloric sources, and the intake of diets rich in such macronutrients has largely increased in recent years, mainly in the Western world [3]. Carbohydrates and lipids may be the sources of fatty acid synthesis and/or modulate the expression of genes involved in fat accumulation. However, these nutrients participate in distinct points of the metabolic

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pathways and can modulate similar or distinct genes [4-5]. Thus, several metabolic

modulated by dietary constituents.

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pathways may be associated with obesity development and may be differentially

The fat pad mass is governed by highly regulated pathways, whereby a balance between stored and delivered triglycerides maintains the lipid content in adipocytes. The

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triglycerides carried by lipoproteins are hydrolyzed by lipoprotein lipase (LPL), and the free fatty acids (FFA) become available to be uptake by adipocyte [5]. The de novo lipogenesis (DNL) also contributes to fat storage once the glucose is available for fatty

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acids synthesis [5-6]. Conversely, lipolysis plays a pivotal role in controlling the triglyceride quantity in fat deposits [7-8]. The adipose triglyceride lipase (ATGL),

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hormone sensitive lipase (HSL) and monoglyceride lipase (MGL) hydrolyze triglyceride stored within the adipocyte for FFA release into the plasma. Previous studies have demonstrated that multiple transcription factors regulate

fat deposition as they modulate the synthesis of enzymes involved in fat metabolism [912]. Carbohydrates and lipids may activate or inhibit lipogenic transcription factors, such as sterol regulatory element-binding proteins (SREBP) and peroxisome proliferator-activated receptor gamma (PPARγ). SREBP and PPARγ upregulate genes

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ACCEPTED MANUSCRIPT involved in sterol and fatty biosynthesis, which increases lipogenesis, glucose uptake and LPL activity [9-12]. In the present study, we hypothesized that even if the body weight and adiposity

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were similar between mice fed high-carbohydrate (HC) or high-fat (HF) diets, as previously demonstrated by our group [13], lipids and carbohydrates may modulate fat accumulation via distinct metabolic pathways. Therefore, the aim of the present work was to assess the adipocyte metabolic pathways that promote lipid accumulation in the

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adipocytes of mice fed HC or HF diets. Elucidation of the role nutritional regulation

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plays in fat deposition may lead to promising therapeutic interventions against obesity.

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ACCEPTED MANUSCRIPT METHODS Animals and Treatment Male Swiss mice aged 7–8 weeks were obtained from the Centro de Bioterismo

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da Universidade Federal de Minas Gerais, maintained in a controlled environment at a 14 h/10 h light/dark cycle, and allowed free access to tap water and food. The animals were age matched and maintained according to the ethical guidelines of our institution,

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and the experimental protocol was approved by the Ethics Committee in Animal Experimentation of the Federal University of Minas Gerais. During the 8 weeks

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preceding the experiment, the control (C) group was fed a standard laboratory rodent diet (the chow diet (Nuvital, Colombo, PR) at 3.41 Kcal/g), and the two remaining groups received a high-carbohydrate (HC) diet (64% carbohydrate, 19% protein, and 11% fat (corn oil) without fiber) at 4.3 Kcal/g or a high-fat (HF) diet (45%

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carbohydrate, 17% protein, and 38% fat (corn oil) without fiber) at 5.9 Kcal/g. HC diet carbohydrates sources were sucrose and starch. Fatty acid contents per 100 g of HF diet included 5.78g of saturated fatty acids, 12.69g of monounsaturated and 19.34g of

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polyunsaturated fatty acids. The proportion of polyunsaturated fatty acid present in corn oil is about 35% of ω-9 and 44% ω-6 and less than 2% is ω-3. After an overnight

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starvation, the mice were anesthetized with ketamine (130 mg/kg) and xylazine (0.3 mg/kg) and euthanized by exsanguination. Adipose tissue lipogenesis measurements. The rate of epididymal adipose tissue lipogenesis was determined by measuring the amount of newly synthesized fatty acid present in portions of the adipose tissue (100 mg). The fat pads were incubated at 37°C for 2 h in a buffer containing 5 mM glucose and 150 µCi/mL3 H2O as a radioisotopic tracer. The tissue was then homogenized, and

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ACCEPTED MANUSCRIPT the3H-labeled fatty acids were extracted with chloroform-methanol in a 2:1 ratio. The isolated fatty acid was added to toluene-diphenyloxazole scintillation fluid (5 g/L) and measured in a liquid scintillation counter. Lipogenesis was calculated as micromoles of H2O incorporated into the total lipid synthesis/g.h of epididymal adipose tissue.

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Reverse transcription and real-time PCR

The total RNA from epididymal adipose tissue was prepared using the Tri-

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Phasis reagent (BioAgency, São Paulo, SP, Brazil) and treated with DNAse. The cDNA was generated from 2 µg of RNA using M-MuLV Reverse Transcriptase (Fermentas, Fisher

Scientific

Inc.,

USA).

The

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Thermo

hypoxanthine-guanine

phosphoribosyltransferase (HPRT – endogenous control), peroxisome proliferatoractivated receptor gamma (PPARγ), sterol regulatory element-binding protein (SREBP) and acetyl CoA carboxylase (ACC) cDNA were amplified using specific primers

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(Invitrogen) and the SYBR Green reagent (Applied BioSystems) in an ABI Prism 7000 platform (Applied BioSystems). The following primer pairs were used: HPRT reverse 5’-gattcaacttgcgctcatcttaggc-3’; HPRT forward 5’-gttggatacaggccagactttgtt-3’; SREBP

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reverse 5’-gcccacaatgccattgaga-3’; SREBP forward 5’-gcccacaatgccattgaga-3’; ACC reverse 5′-aatccactcgaagaccactg-3′; ACC forward 5′-cggcttgcacctagtaaaac-3′; PPARγ 5’-aggaactccctggtcatgaatcct-3’;

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reverse

and

PPARγ

forward

5’-

agatcatctacaccatgctggcct-3’.

Adipocyte isolation

Adipocytes were isolated from epididymal fat pads. Briefly, digestion with collagenase (1 mg/mL), from Clostridium histolyticum type II (Sigma-C6885), was performed at 37o C with constant shaking (140 cycles/min) for 40 min. The cells were filtered through a nylon mesh and washed three times with buffer containing 137 mM 7

ACCEPTED MANUSCRIPT NaCl, 5 mM KCl, 4.2 mM NaHCO3, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.5 mM MgSO4, 0.5 mM KH2PO4, 20 mM HEPES (pH 7.4), and 1% bovine fatty acid free serum albumin. After centrifugation, the adipocytes were isolated from the supernatant phase. Glucose uptake.

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Adipocytes from mice were isolated from epididymal fat pads. After isolation, adipocytes were incubated for 45 min at 37°C in the presence or absence of insulin (25 ng/mL). The uptake of 2-deoxy-[3H]glucose (2DOG) was used to determine the rate of

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glucose transport as previously described [15]. Briefly, glucose uptake was initiated by the addition of 2DOG (0.2 µCi/tube) for 3 min. Thereafter, the cells were separated by

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centrifugation through silicone oil, and the cell-associated radioactivity was determined by scintillation counting. The nonspecific association of 2DOG was determined by performing parallel incubations in the presence of 15 mM phloretin, and this value was subtracted from the glucose transport activity in each condition.

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Lipoprotein lipase activity

The adipose tissue samples (50 mg) were homogenized in buffer containing heparin and detergents [16], and the total LPL activity was measured using a [9,10-3H]

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triolein-containing substrate emulsified with lecithin [17] and contained 24 hour fasted rat plasma as a source of apo CII. The reaction was stopped with an extraction mixture

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[18], and the liberated

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H-free fatty acids (FFAs) were quantified using liquid

scintillation. The enzyme activity was expressed as nanomoles of fatty acids (FA) released per minute.

Lipolysis measurements in vitro Lipolysis was measured as the rate of glycerol release by adipocytes, as previously described [14]. After the washes, the isolated adipocytes were incubated at 37°C in a water bath for 1 hour in basal conditions or in the presence of 0.1 µM

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ACCEPTED MANUSCRIPT isoproterenol (ISO), a non-selective beta-adrenoceptor agonist. The effects of 25 ng/mL insulin on isoproterenol-stimulated lipolysis were also determined. At the end of the incubation period, an aliquot of the infranatant was removed for the enzymatic determination of glycerol released into the incubation medium (KATAL, Belo

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Horizonte, MG, Brazil). Statistical Analysis

All results are reported as the mean ± SD. The statistical analyses were performed using

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an analysis of variance followed by the Newman–Keuls test. P < 0.05 was considered

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statistically significant.

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ACCEPTED MANUSCRIPT RESULTS We analyzed the adipose tissue lipogenesis rate and ACC mRNA expression (Figure 1), a key enzyme of lipogenesis. Lipid synthesis was measured in vitro as the

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incorporation of 3H2O into epididymal adipose tissue of mice fed a HC or HF diet under basal conditions. The basal lipogenesis increased in mice fed the HC diet and decreased in animals fed the HF diet when compared to mice fed a chow diet (Figure 1A). Akin to

fed a HC diet compared to the control group (Figure 1B).

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what was observed for the lipogenesis, ACC mRNA expression increased only in mice

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We evaluated glucose uptake by isolated adipocytes in basal and insulin stimulated conditions. The basal and insulin-stimulated glucose uptake in mice fed the HC diet was similar in the animals fed a chow diet (Figure 2). However, mice fed a HF diet displayed a decreased glucose uptake in both basal and insulin-stimulated

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conditions compared to the C and HC groups.

The lipolysis of circulating triglyceride-rich lipoprotein mediated by the lipoprotein lipase enzyme can also increase adiposity. Mice fed the HC and HF diet

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exhibited an increased lipoprotein lipase activity compared to mice fed the control diet

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(Figure 3A). However, mice fed the HF diet exhibited lower enzymatic activity than mice fed the HC diet.

Lipolysis in adipocytes, mediated by ATGL/HSL/MGL, was measured in

isolated adipocytes from mice fed the chow, HC and HF diets at the basal state and after stimulation with isoproterenol in the presence or absence of insulin. In adipocytes from mice fed the chow diet, the isoproterenol enhanced the glycerol release in the medium (Figure 3B). The high lipolysis mediated by isoproterenol was completely blunted by insulin in the control group. The lipolytic stimulus induced by the ISO was similar 10

ACCEPTED MANUSCRIPT between the chow and HC diet-fed mice. However, mice fed the HF diet exhibited reduced lipolysis stimulated by isoproterenol when compared with mice fed C or HC diets. The anti-lipolytic effect of insulin was reduced by 27% and 18% in mice fed HC or HF diets, respectively, compared with control adipocytes. These findings agree with

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previously published data from our group that demonstrated low insulin sensitivity in animals fed HC or HF diets [13].

The genes activated by PPAR-γ stimulate lipid uptake and synthesis. Therefore,

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PPAR-γ mRNA expression was measured by RT-PCR (Figure 4A). We demonstrated

the

chow

diet.

Additionally,

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that the HC and HF diets increased PPAR-γ mRNA expression compared to mice fed we

assessed

SREBP

mRNA

expression,

another transcription factor involved in sterol and lipid biosynthesis [10-11]. We observed that only mice fed the HC diet exhibited increased SREBP mRNA expression

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(Figure 4B) compared to control mice.

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ACCEPTED MANUSCRIPT DISCUSSION

Although diets rich in carbohydrates and lipids lead to an increased fad pad mass, the metabolic pathways and transcription factors that drive the expansion of the adipose

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tissue vary. This study demonstrated that carbohydrate-rich diets increased fat accumulation predominantly by increasing the expression of SREBP-1 and PPARγ, lipogenesis, LPL activity and glucose utilization in adipocytes, even in a fasting state.

the lipolytic activity in adipocytes (see Figure 5).

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Conversely, fat-rich diets did not promote the lipogenic pathways but rather decreased

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It has been well established that carbohydrate-rich diets induce the hepatic lipogenesis that contributes to increased fat accumulation in adipocytes [11; 19-21]. However, due to insulin signaling, these events occur mainly in a postprandial state [22-23]. Here, we also revealed that, even in a fasting state, mice fed a HC diet exhibited an increased

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adipocyte lipogenesis rate and high expression of two important transcription factors that drive fat synthesis and adipogenesis, SREBP-1 and PPARγ. Hudgins and colleagues (1996) [21] have demonstrated that a diet high in simple carbohydrates fed to

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mice for 25 days led to a constitutive elevation of de novo lipogenesis during fasting. In

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fact, because the half-life of the lipogenic enzymes is ~36-48 h, our observation of a prolonged stimulatory effect on fatty acid synthesis by the high-carbohydrate diets is consistent with the increased expression of PPARγ and SREBP-1, the main transcription factors controlling ACC expression [23].

SREBP-1 plays a crucial role in the induction of lipogenesis in the liver when an energy excess due to carbohydrates is consumed [24-25]. The hepatic SREBP-1 has also been demonstrated to coordinately regulate the expression of fatty acid synthase (FAS) and ACC [26]. Despite the extensive knowledge about the role of SREBP-1 in the liver, its 12

ACCEPTED MANUSCRIPT role in adipose tissue remains elusive. Sekiya and colleagues (2007) [27] proposed that lipogenic gene regulation is primarily independent of SREBP-1 in adipocytes. Conversely, overexpression of this transcription factor is associated with adipocyte hypertrophy [28], and during fasting and refeeding, its expression increased in adipose

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tissue [29]. In this study, we demonstrated that the diet composition modulated the expression of SREBP-1 in adipose tissue, as mice fed a HC diet exhibited high levels,

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whereas fat-rich diets did not elicit such an increase.

The genes that are under the transcriptional control of PPARγ in the adipose tissue

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include the codifiers of enzymes involved in fatty acid metabolism, such as LPL, acylCoA synthase and fatty acid transport proteins. This list suggests that PPARγ plays an important role in lipid capture by adipocytes [9, 30]. In fact, its high expression in mice fed the HC diet was associated with an increased LPL activity and fat synthesis.

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Although we did not evaluate the direct effect of HC and HF diet on fatty acid uptake by visceral adipocytes we hypothesize that the higher LPL activity in mice fed HC and HF diets increase the availability of fatty acids to be uptake by adipocytes. However, it

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is noteworthy that HC diet prompted a more accentuated LPL activity. Previous studies have shown that a higher LPL activity in adipose tissue is accompanied by a higher

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uptake of FFA [38, 39]. Similarly, mice that showed lower LPL activity also have less rate of FFA uptake [40].

Unlike diets rich in carbohydrates, the high-fat diet did not increase neither the lipogenesis rate nor glucose uptake in adipocytes. Conversely, the lipolytic rate was lower compared with the HC or control diets. These findings are consistent with previous published results that indicated that high-fat diets decreased de novo lipogenesis in the liver and the expression of transcription factors that trigger fat

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ACCEPTED MANUSCRIPT synthesis in rodents [25]. Otherwise, de novo lipogenesis in adipocytes, especially with high-fat feeding, is poorly understood. Recently, Lodhi and colleagues (2012) [31] demonstrated that depletion of fatty acid synthase (FAS), a key enzyme required for fat synthesis, in adipocytes suppressed high-fat diet induced obesity. This result suggested

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that, despite the fact that mice that were fed a HF diet displayed a lower lipogenesis rate, this pathway is dispensable for promoting lipid accumulation following the intake of a fat-rich diet. In support of this notion, Morgan and colleagues (2008) [32] have

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demonstrated that mice fed a HF diet (20% of calories from fat) displayed an increased expression of key transcriptions factors and enzymes involved in the lipogenesis

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pathways in adipose tissue in a fasted state. The discrepancy between our data and such studies may be due to the elderly age of the mice and the methods utilized. The DNL also depends on the basal and insulin-stimulated glucose uptake in adipocytes once glucose is available for synthesizing glycero-3-phosphate [23; 33-35]. According to the

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low lipogenesis rate, the adipocyte glucose uptake at basal and insulin-stimulated conditions from mice fed the HF diet decreased.

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Despite the low fat synthesis in adipocytes following a high-fat diet, the mice exhibit a high adiposity and body weight, as previously demonstrated [13]. However,

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the adipocyte lipolytic rate exhibited a low activity. Modifications in the lipolysis rate at basal and stimulated conditions are associated with obesity [7; 23]. In fact, human studies have demonstrated that decreased catecholamine-induced lipolysis in subcutaneous adipose tissue is an early, possibly primary, defect that is linked to a decreased protein expression of hormone sensitive lipase [36] and low cAMP levels [37]. Additionally, a growing body of evidence indicates that the dietary composition can regulate lipolysis [23]. Accordingly, our data indicate a low lipolytic rate in adipocytes from mice fed the HF diet, whereas mice fed the HC diet displayed similar 14

ACCEPTED MANUSCRIPT lipolysis rate as the controls. Inversely, previous studies showed that rats fed a lowprotein, high-carbohydrate diet presented with reduced lipolysis stimulated by norepinephrine [41, 42]. The discrepancy between our data and others may be explained by different protein content from the experimental diets and distinct animal models used

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in the studies. Importantly, such data confirms the relevance of diet composition in the regulation of metabolic pathways related to fat storage.

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Recent rodent studies proposed that the adipose tissue lipogenesis is downregulated in obesity, whereas the liver lipogenesis is increased [23; 28]. The low

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adipose lipogenesis rate in the obese state led to a more pronounced metabolic dysfunction [23; 28]. In our study, we demonstrated that the adipose tissue lipogenesis was likely related to the diet composition instead of the body fat mass. In fact, adipocyte fat accumulation is a dynamic process that responds to dietary conditions. Herein, we

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have demonstrated that carbohydrate-rich diets induced a persistent expression of lipogenic transcription factors during fasting, whereas fat-rich diets decreased lipolysis in adipocytes to enhance fat stores (Figure 5). Although, we have evaluated the main

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intermediary metabolic pathways that trigger fat accumulation in adipocytes it would be important to account for other possible pathways, such as glucose and FFA uptake.

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Furthermore, it is important to consider the inflammatory response in adipose tissue following the consumption of different diet composition. As we have previously showed, the magnitude of the inflammatory response can also determine the extension of fat accumulation [13; 43; 44]. An understanding of the processes that mediate adipocyte fat accumulation driven by diet composition is necessary to develop strategies for its prevention and management.

CONCLUSIONS

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ACCEPTED MANUSCRIPT High-carbohydrate and high-fat diets stimulate distinct metabolic pathways to trigger fat accumulation in mice adipocytes. Although carbohydrate-rich diets increased fat accumulation by increasing lipogenesis, lipoprotein lipase activity and glucose

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utilization, fat-rich diets decreased the lipolytic activity in adipocytes.

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ACCEPTED MANUSCRIPT carbohydrate-containing diet in mice. Obesity (Silver Spring), 2013; 21:E396406. 44. Menezes-Garcia Z, Oliveira MC, Lima RL, Soriani FM, Cisalpino D, Botion LM, Teixeira MM, Souza DG, Ferreira AV. Lack of platelet-activating factor

resistance

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receptor protects mice against diet-induced adipose inflammation and insulinObesity

Spring),

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ACCEPTED MANUSCRIPT LEGENDS Figure 1: (A) Rates of 3H incorporation from tritiated water into the total lipid content of epididymal adipose tissue of mice fed the HC or HF diet. (B) ACC mRNA expression in epididymal adipose tissue of mice fed the HC or HF diet. The data represent the mean

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± SEM of 3-6 mice per group. *HC or HF vs C, P<0.05. # HF vs HC, P<0.05.

Figure 2: Glucose uptake from adipocytes from mice fed the HC or HF diet. The glucose transport rates were determined by measuring the 2DOG uptake. The data

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represent the mean ± SEM of 3-7 animals per group. *Insulin vs Basal, P<0.05. # HFInsulin vs HC-Insulin and C-insulin, P<0.05. &HF- Basal vs C-Basal and HC-Basal,

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Figure 3: (A) Effect of the HC and HF diet on lipoprotein lipase activity of epididymal fat pads. The data represent the mean ± SEM of 3-4 mice per group. *HC or HF vs C, P<0.05. #HF vs HC, P<0.05. (B) Glycerol levels released by primary adipocytes in

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culture from mice fed the HC or HF diet at basal conditions or incubated with 0.1 µM isoproterenol (ISO), a non-selective beta-adrenoceptor agonist. The effects of 25 ng/mL insulin on isoproterenol-stimulated lipolysis were also determined. The data represent

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the mean ± SEM of 3-4 mice per group. *ISO vs Basal, P<0.05. #Insulin vs ISO,

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P<0.05. &ISO-C vs ISO-HF, P<0.05. Figure 4: (A) PPAR-γ and (B) SREBP mRNA expression in epididymal adipose tissue of mice fed the HC or HF diet. The data represent the mean ± SEM of 3-5 animals per group. *HC or HF vs C, P<0.05. #HF vs HC, P<0.05. Figure 5: Metabolic pathways of adipose tissue fat storage and mobilization of mice fed the HC or HF diet. Carbohydrate-rich diets increased fat accumulation by increasing lipogenesis, ACC mRNA expression, LPL activity and glucose uptake (left panel). The HC diet increased the expression of SREBP-1 and PPARγ, key transcription factors

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prompted a low lipolytic activity in adipocytes (right panel).

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