Polyunsaturated (n-3) Fatty Acids Reduce Risk of Heart Disease

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Polyunsaturated Fatty Acid Regulation of Gene Transcription: A

Molecular Mechanism to Improve the Metabolic Syndrome1 ,2

Steven D. Clarke3

 

Graduate Program of Nutrition and the Institute of Cell and Molecular

Biology, The University of Texas at Austin, Austin, Texas 78712

 

 

 

3To whom correspondence should be addressed at 115 Gearing Building,

The University of Texas, Austin, TX 78712. E-mail:

stevedclarke

 

 

 

 

ABSTRACT

INTRODUCTION

PUFA Induction of Lipid...

PUFA Suppression of Lipogenesis:...

Summary.

REFERENCES

 

 

This review addresses the hypothesis that polyunsaturated fatty acids

(PUFA), particularly those of the (n-3) family, play pivotal roles

as " fuel partitioners "

 

in that they direct fatty acids away from

triglyceride storage and toward oxidation,

 

and that they enhance

glucose flux to glycogen.

 

In doing this, PUFA protects against the adverse symptoms of the

metabolic syndrome and

 

reduce the risk of heart disease.

 

PUFA exert their beneficial effects by up-regulating the expression

of genes encoding proteins involved in

 

fatty acid oxidation while

simultaneously down-regulating genes encoding proteins of lipid

synthesis.

 

PUFA govern oxidative gene expression by activating the

transcription factor peroxisome proliferator-activated receptor .

 

PUFA suppress lipogenic gene expression by reducing the nuclear

abundance and DNA-binding affinity of transcription factors

responsible for imparting insulin and carbohydrate control to

lipogenic and glycolytic genes.

 

In particular, PUFA suppress the nuclear abundance and expression of

sterol regulatory element binding protein-1 and reduce the DNA-

binding activities of nuclear factor Y, Sp1 and possibly hepatic

nuclear factor-4.

 

Collectively, the studies discussed suggest that

the fuel " repartitioning " and gene expression actions of PUFA should

be considered among criteria used in defining the dietary needs of (n-

6) and (n-3) and in establishing the dietary ratio of (n-6) to (n-3)

needed for optimum health benefit.

 

 

ABSTRACT

 

 

Dietary (n-6) and (n-3) polyunsaturated fatty acids (PUFA)4

 

reduce triglyceride accumulation

 

in skeletal muscle and potentially in

cardiomyocytes and ß cells (1 ,2) .

 

Lower tissue lipids are associated with improvements in the

metabolic syndrome, such as increased insulin sensitivity (1 ,3) .

 

PUFA elicit their effects by coordinately suppressing lipid synthesis

in the liver,

 

up-regulating fatty acid oxidation in liver and

skeletal muscle

 

and increasing total body glycogen storage (3 4 5 6 7

8) . -6

 

Desaturation of 18:2(n-6) and 18:3(n-3) is required for

this " repartitioning " of metabolic fuel (9) , and on a carbon-for-

carbon basis,

 

(n-3) fatty acids are more potent than (n-6) fatty

acids.

The repartitioning activity of PUFA, particularly (n-3) PUFA,

has been observed in humans as well as various animal models (3 ,10

11 12 13) . Unfortunately, the amount of (n-6) and (n-3) and the best

(n-6)-to-(n-3) ratio required for optimum metabolic benefit are

unknown.

 

However, as little as 2–5 g of 18:3(n-3) or 20:5 and 22:6(n-3)

 

lower blood triglyceride concentrations

 

and reduce the risk of fatal

ischemic heart disease (12 ,13) .

 

Some of the beneficial effects of PUFA are due to changes in

membrane fatty acid composition and subsequent alterations in

hormonal signaling (1) .

 

However, fatty acids themselves exert a

direct, membrane-independent influence on molecular events that

governs gene expression.

 

It is the regulation of gene expression by

dietary fats that we believe has the greatest impact on the

development of insulin resistance and its related pathophysiologies

(i.e., the metabolic syndrome). More importantly, determination of

the cellular and molecular mechanisms regulated by PUFA may identify

novel sites for pharmacological intervention.

 

 

PUFA Induction of Lipid Oxidation: The Role of Peroxisome

Proliferator-activated Receptor .

 

PUFA Induction of Lipid...

PUFA Suppression of Lipogenesis:...

Summary.

REFERENCES

 

 

One of the first steps in the PUFA-dependent repartitioning of

metabolic fuels involves an immediate reduction in the production of

hepatic malonyl coenzyme A (CoA) (14) . Malonyl-CoA is a negative

metabolite effector of carnitine palmitoyltransferase (15) .

 

Consequently, a PUFA-mediated decrease in hepatic malonyl-CoA favors

fatty acid entry into the mitochondria and peroxisomes and leads to

enhanced fatty acid oxidation (15) .

 

Whether PUFA suppress malonyl-CoA levels in skeletal muscle and

heart remains to be determined, but such a mechanism would be

consistent with the higher rates of fatty acid oxidation observed in

humans and animals fed diets rich in PUFA (10 ,11) .

 

The reduction in hepatic malonyl-CoA is paralleled by a PUFA-

dependent induction of genes encoding proteins involved in fatty acid

oxidation and ketogenesis (3 ,4 ,7) .

These changes in gene transcription occur too quickly to be explained

simply by altered hormone signaling resulting from modifications of

the membrane lipid environment.

 

Rather, the changes are more consistent with the idea that PUFA

directly (e.g., ligand binding) regulate the activity or abundance of

a nuclear transcription factor. In 1990, PPAR, a novel lipid-

activated transcription factor, was cloned (16) . PPAR is a member of

the steroid receptor superfamily, and like other steroid receptors,

it possesses a DNA-binding domain and a ligand-binding domain

(7 ,8 ,16) . The interaction of PPAR with its DNA recognition site is

markedly enhanced by ligands such as the hypotriglyceridemic fibrate

drugs, conjugated linoleic acid and PUFA (17 ,18) . In general, PPAR

activation leads to the induction of several hepatic, cardiac and

skeletal muscle genes encoding proteins involved in lipid transport,

oxidation and thermogenesis, including carnitine

palmitoyltransferase, peroxisomal acyl-CoA oxidase and uncoupling

protein-3 (3 ,19 ,20) . The (n-3) PUFA are more potent than the (n-6)

PUFA as in vivo activators of PPAR (10 11 12 13) , but neither family

of PUFA is a particularly strong PPAR activator. However, PUFA

metabolites such as eicosanoids or oxidized fatty acids have one to

two orders of magnitude greater affinity for PPAR and are

consequently far more potent transcriptional activators of PPAR-

dependent genes (21) .

 

The importance of PPAR to overall glucose and fatty acid homeostasis

has been clearly demonstrated in PPAR knockout mice (4 ,22) . Because

PPAR-/- mice lack the ability to increase rates of fatty acid

oxidation during periods of food deprivation, they develop

characteristics of adult-onset diabetes, including fatty livers,

elevated blood triglyceride concentrations and hyperglycemia (22) .

The essentiality of PPAR to lipid oxidation was further underscored

when hyperglycemia was found to suppress PPAR expression, induce PPAR

expression, increase ß-cell and cardiomyocyte lipids and accelerate

cell death (23) . Such " lipotoxicity " may be a contributing factor to

the complications of non–insulin-dependent diabetes (23) . Clearly,

PPAR is emerging as a pivotal player in both fatty acid and glucose

metabolism.

More important, its regulation by PUFA, particularly (n-

3) PUFA and possibly conjugated linoleic acid (18) , may offer an

explanation for the reported benefits of these fatty acids in

protecting individuals from developing the detrimental

characteristics of non–insulin-dependent diabetes.

 

 

PUFA Suppression of Lipogenesis: The Roles of Sterol Regulatory

Element Binding Protein-1, Nuclear Factor Y and Hepatic Nuclear

Factor-4.

TOP

ABSTRACT

INTRODUCTION

PUFA Induction of Lipid...

PUFA Suppression of Lipogenesis:...

Summary.

REFERENCES

 

 

Dietary PUFA inhibit hepatic lipogenesis by suppressing the

expression of a number of hepatic enzymes involved in glucose

metabolism and fatty acid biosynthesis, including glucokinase,

pyruvate kinase, glucose-6-phosphate dehydrogenase, citrate lyase,

acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase

and the -6 and -5 desaturases (4 5 6 ,24 ,25) . The discovery of PPAR

led quickly to the idea that PPAR was a " master switch " transcription

factor that was targeted by PUFA to coordinately suppress genes

encoding proteins of lipid synthesis and to induce genes encoding

proteins of lipid oxidation. This attractive hypothesis was

strengthened by reports that potent pharmacological activators of

PPAR modestly reduced lipogenic gene transcription (4 ,20) . However,

PPAR does not interact with PUFA response regions identified in four

different genes (3 ,4 ,6 ,9) . Moreover, PUFA continue to suppress

the transcription of hepatic lipogenic genes in PPAR-/- mice (26) .

Thus, the inhibition of lipogenic gene transcription associated with

PPAR activation is indirect and may simply reflect the PPAR-dependent

induction of the -6 desaturase pathway (9 ,27) .

 

PUFA response sequences have been well characterized in only three

genes: fatty acid synthase, S14 and L-type pyruvate kinase

(3 ,4 ,20 ,28 ,29) . The rat fatty acid synthase gene contains two

independent PUFA regulatory sequences that are located between -118

and -43 and between -7250 and -7035 (M. Teran-Garcia and S. D.

Clarke, unpublished data). Approximately 65 and 35% of the PUFA

control can be attributed to the proximal and distal elements,

respectively. Interestingly, the proximal PUFA response region of the

fatty acid synthase gene has characteristics that are very similar to

the PUFA response region of the S14 gene (-220 to -80), whereas the

distal PUFA response region of the fatty acid synthase has

similarities to the L-type pyruvate kinase PUFA response region (-160

to -97) (4) .

 

The proximal PUFA response region of the fatty acid synthase gene

imparts insulin responsiveness to the gene and contains DNA-binding

sites for sterol regulatory element binding protein-1 (SREBP-1),

upstream stimulatory factor (USF), Sp1 and nuclear factor Y (NF-Y)

(20 ,29) . The nuclear abundance of USF and its DNA-binding activity

is unaffected by dietary PUFA (20) . In contrast, PUFA rapidly reduce

the nuclear content of hepatic SREBP-1, and this is associated with a

decrease in the rate of fatty acid synthase and S14 gene

transcription (20 ,29 30 31) . SREBP are a family of transcription

factors (i.e., SREBP-1a, -1c and -2) that were first isolated as a

result of their properties for binding to the sterol regulatory

element (32 ,33) . SREBP-2 is a regulator of genes encoding proteins

involved in cholesterol metabolism (32 ,33) . SREBP-1 exists in two

forms: 1a and 1c. SREBP-1a is the dominant form in cell lines and is

a regulator of genes encoding proteins involved in both lipogenesis

and cholesterogenesis. SREBP-1c constitutes 90% of the SREBP-1 found

in vivo and is a determinant of lipogenic gene transcription

(32 ,33) .

 

SREBP-1 is synthesized as a 125-kDa precursor protein that is

anchored in the endoplasmic reticulum membrane (32 ,33) . Proteolytic

release of the 68-kDa mature SREBP-1 occurs in the Golgi system, and

movement of SREBP-1 from the endoplasmic reticulum to the Golgi

requires the trafficking protein SREBP cleavage-activating protein

(33) . Once released, mature SREBP-1 translocates to the nucleus and

binds to the classic sterol response element and/or to a palindrome

CATG sequence. In the case of fatty acid synthase, SREBP-1 interacts

with a CATG palindrome that also functions as an insulin response

element (32) . Overexpression of mature SREBP-1a in liver is

associated with high rates of fatty acid biosynthesis, and ablation

of the SREBP-1 gene results in low expression of lipogenic genes

(32 ,33) . These observations led us to the hypothesis that PUFA

inhibit lipogenic gene transcription by impairing the proteolytic

release of SREBP-1c and/or by suppressing SREBP-1c gene expression.

Diets rich in 18:2(n-6) or 20:5 and 22:6(n-3) were found to reduce

the hepatic nuclear and precursor content of mature SREBP-1 by 65 and

90% and by 60 and 75%, respectively (20) . The decrease in SREBP-1

was accompanied by a comparable decrease in the transcription rate of

hepatic fatty acid synthase (20) . Unlike PUFA, saturated and

monounsaturated fatty acids had no effect on the nuclear content or

precursor content of SREBP-1 or on lipogenic gene expression (20 ,29

30 31 ,34) . The PUFA-dependent reduction in hepatic content of SREBP-

1 may explain how PUFA inhibit the transcription of several genes

encoding proteins involved in hepatic glucose metabolism and fatty

acid biosynthesis, including glucokinase, acetyl-CoA carboxylase and

stearoyl-CoA desaturase (4) . Interestingly, the inhibition of

lipogenic gene expression that reportedly occurs in adipose tissue

with the ingestion of fish oil does not involve an SREBP-1–dependent

mechanism (30) .

 

PUFA reduce the nuclear content of SREBP-1 via a two-phase mechanism.

The first phase is a rapid (<60-min) inhibition of the proteolytic

release process (34) . The second phase involves an adaptive (48-h)

reduction in the hepatic content of SREBP-1 mRNA that is subsequently

followed by a reduction in the amount of precursor SREBP-1 protein

(20 ,35) . The mechanism by which PUFA acutely inhibit the

proteolytic processes is unknown. However, nuclear run-on assays

suggested that PUFA reduce the hepatic content of SREBP-1 mRNA

through post-transcriptional mechanisms (20 ,35) . Using rat liver

cells in primary culture, we determined that PUFA reduced the half-

life of SREBP-1c mRNA from 11 h to <5 h (35) . The mechanism by which

PUFA control the half-life of SREBP-1 is unknown but may require the

synthesis of a rapidly turning over PUFA-dependent protein (35) .

 

SREBP-1c by itself possesses weak trans-activating power, but the

binding of SREBP-1c to its recognition sequence enhances the upstream

DNA binding of NF-Y and Sp1, which in turn amplifies the trans-

activating activities of the three transcription factors (32 ,36) .

NF-Y is a heterotrimeric nuclear protein that reportedly plays a role

in regulating chromatin structure by way of its interaction with

histone acetyl transferases (4) . The binding sites for NF-Y are

essential for fatty acid synthase (M. Teran-Garcia and S. D. Clarke,

unpublished data) and S14 promoter activity (4) . Mutations within

the Y-box region of -104 to -99 of the S14 gene eliminated promoter

activity by preventing NF-Y from interacting with upstream T3 (-2800

to -2500) and carbohydrate response (-1600 to -1400) regions (4) .

Similarly mutating the Y-box motif between -90 and -80 of the rat

fatty acid synthase gene eliminated 80% of the promoter activity, and

mutating the adjacent Sp1 site (-80) reduced promoter activity by

>90% (M. Teran-Garcia and S. D. Clarke, unpublished data). In

contrast, eliminating the SREBP-1 site (-67 to -53) reduced fatty

acid synthase promoter activity by only 40%. More important, only 35%

of the PUFA inhibition of fatty acid synthase promoter activity was

lost with the SREBP-1 mutation. On the other hand, mutating the NF-Y

site eliminated nearly 70% of the PUFA suppression of fatty acid

synthase promoter activity. Moreover, the near 90% inhibition in

hepatic fatty acid synthase gene transcription associated with the

ingestion of a diet rich in fish oil was accompanied by a 50–60%

reduction in DNA-binding affinity for NF-Y and Sp1 (M. Teran-Garcia

and S. D. Clarke, unpublished data).

 

The insulin response region and its associated transcription factors

(i.e., SREBP-1, NF-Y and Sp1) are not the only nuclear factors

regulated by PUFA. Transfection-reporter analyses indicate that PUFA

exert a negative influence on the carbohydrate response element of

the L-type pyruvate kinase (4) and fatty acid synthase genes (M.

Teran-Garcia and S. D. Clarke, unpublished data). The nature of the

transcription factors and the mechanism by which PUFA regulate them

are not well defined. One hepatic protein that may be a PUFA target

is hepatic nuclear factor-4 (HNF-4). HNF-4 is a member of the steroid

receptor superfamily. HNF-4 enhances the glucose/insulin induction of

L-type pyruvate kinase transcription by binding as a homodimer to a

direct repeat-1 motif (4) . Like PPAR, HNF-4 has a ligand binding

domain that interacts with acyl-CoA esters, but unlike PPAR, fatty

acyl-CoA binding to HNF-4 decreases its DNA-binding activity (37) .

This suggests that PUFA may exert part of its negative influence on

gene transcription by reducing HNF-4 DNA-binding activity. Linker

scanner mutations through the carbohydrate response region of the L-

type pyruvate kinase promoter (i.e., -183 to -97) did in fact reveal

that the HNF-4 recognition elements were essential for PUFA

suppression of the promoter (4) . Recently, we found that sequences

between -7242 and -7150 of the fatty acid synthase gene were required

for glucose to induce fatty acid synthase gene transcription (38) .

Subsequent studies have demonstrated that the -7242 to -7150 sequence

contains DNA recognition sites for HNF-4 and a novel carbohydrate

response factor (38) . Moreover, deleting this sequence eliminated 30–

40% of the total PUFA suppression of the fatty acid synthase promoter

(M. Teran-Garcia and S. D. Clarke, unpublished data). Thus, PUFA may

exert part of their suppressive effects on gene transcription by

interfering with the glucose-mediated trans-activation processes that

in part involve reducing HNF-4 DNA-binding activity.

 

 

Summary.

TOP

ABSTRACT

INTRODUCTION

PUFA Induction of Lipid...

PUFA Suppression of Lipogenesis:...

Summary.

REFERENCES

 

 

For nearly 40 y, PUFA have been known to uniquely suppress lipid

synthesis. PUFA, particularly (n-3), accomplish this by coordinating

an up-regulation of lipid oxidation and a down-regulation of lipid

synthesis.

 

In other words, PUFA function as metabolic fuel

repartitioners. The outcome is an improvement in the symptoms of the

metabolic syndrome and a reduced risk of heart disease.

 

PUFA control

these metabolic pathways by governing the DNA-binding activity and

nuclear abundance of select transcription factors responsible for

regulating the expression of genes encoding key regulatory proteins

of lipid and glucose metabolism.

 

PUFA increase the fatty acid

oxidative capacity of tissues through their ability to function as

ligand activators of PPAR and thereby induce the transcription of

several genes encoding proteins affiliated with fatty acid oxidation.

 

PUFA suppress lipid synthesis by inhibiting transcription factors

that mediate the insulin and carbohydrate control of lipogenic and

glycolytic genes. With respect to the insulin response element, PUFA

rapidly generate an intracellular signal that immediately suppresses

the proteolytic release of mature SREBP-1 from its membrane-anchored

precursor and simultaneously reduces the DNA-binding activities of NF-

Y and Sp1. Within a matter of minutes after PUFA treatment, the

nuclear content of SREBP-1c is greatly reduced. The drop in nuclear

content of SREBP-1c further contributes to the reduction in DNA

binding of NF-Y and Sp1. Continued ingestion of PUFA subsequently

lowers SREBP-1 mRNA levels by accelerating transcript decay, which in

turn results in a lower hepatic content of precursor, endoplasmic

reticulum–anchored SREBP-1. With regard to the carbohydrate response

element, PUFA may also mediate reductions in the DNA-binding activity

of pivotal transcription factors (e.g., HNF-4), but the nature of the

affected transcription factors remains to be unequivocally

established. Without question, the missing final chapter in the

entire PUFA-regulatory story is the nature of the intracellular

signal responsible for regulating the various affected transcription

factors.

 

 

 

 

 

 

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Figure 1. Nuclear mechanism for polyunsaturated fatty acids (PUFA)

regulation of gene expression. FA, fatty acids; NF-Y, nuclear factor

Y; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome

proliferator-activated receptor response element; Sp1, stimulatory

protein 1; SREBP

 

http://www.nutrition.org/cgi/content/full/131/4/1129