The Coordination Of Lipid Metabolism In The Body

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Several genes involved in lipid metabolism are themselves regulated by lipid or lipid-related molecules, and adipose tissue itself secretes proteins that regulate food intake and fat storage.

The flux of fatty acids through the pathways described in this chapter can vary enormously. The fat content of the diet varies in different parts of the world, between different people within one country, and even from day to day in one individual. The rate at which we transport fatty acids through the plasma varies considerably during a normal day. For non-esterified fatty acids it is high at night during fasting, suppressed after meals, and stimulated to very high levels during aerobic exercise; for triacylglycerol-fatty acids it is roughly the converse. This requires co-ordination between metabolic pathways in different organs, in relation both to the influx of dietary lipids and the body's requirement for lipids. In Chapters 4 and 5 we have seen how this co-ordination can be brought about by hormones, and in particular by insulin whose secretion from the pancreas is stimulated in the 'fed7 state. Insulin acts both by affecting the activity of enzymes on a short-term basis (e.g. by reversible phosphorylation/dephosphorylation) and on a longer term basis by regulation of gene expression. However, the main stimulus for insulin secretion is a rise in the plasma glucose concentration. It would seem sensible that the body should also have means for homeostasis of lipid metabolism that do not depend upon the simultaneous ingestion of carbohydrate in appropriate amounts.

Over the past few years there have been great advances in our understanding of how this is achieved. There are several, possibly interrelated, systems that regulate gene expression to control the flux through various pathways of lipid metabolism, and whose regulators are themselves lipids or products derived from lipids.

5.3.1 The sterol regulatory element binding protein (SREBP) system controls pathways of cholesterol accumulation in cells and may also control fatty acid synthesis

The American biochemists and 1985 Nobel Prize winners, Mike Brown and Joseph Goldstein, showed that cellular cholesterol content is regulated by two parallel mechanisms. When the content of unesterified cholesterol in cells increases, the expression of the LDL-receptor protein decreases. In addition, the key enzymes of cholesterol biosynthesis (hydroxymethylglutaryl(HMG)-CoA synthase, HMG-CoA reductase, squalene synthase, farnesyl diphosphate synthase; Section 7.5.1) are repressed. Thus, any further increase in cellular cholesterol is minimized. Conversely, if the cellular unesterified cholesterol content falls, these pathways are activated (Fig. 5.13). The molecular basis for this regulation is now clear. The genes for all these proteins contain an upstream sequence known as the sterol regulatory element-1 (SRE-1). The protein that binds to SRE-1, and activates expression of the gene, is part of a larger protein called sterol regulatory element binding protein (SREBP). SREBP is normally localized in the endoplasmic reticulum. In the absence of cholesterol a specific protease releases a peptide from SREBP, which migrates to the nucleus and binds to SRE-1,

Unesterified Cholesterol
Fig. 5.13 Regulation of the cellular cholesterol content. When the content of unesterified cholesterol in cells increases, the expression of the LDL-receptor protein and the enzymes of cholesterol biosynthesis is repressed.

Box 5.1 Methods for lipoprotein analysis

It may be useful to analyse various aspects of the lipoprotein system. This might include separation of the various classes of lipoprotein particle, analysis of the apolipoproteins present in plasma, or analysis of the chemical species; or, of course, a combination of any of these.

Lipoprotein separation

There are various methods for separation of lipoproteins. They are mostly based on physical properties. It must be remembered that physical properties and biological function are closely but not absolutely related. Thus, separation of a so-called chylomicron fraction on the basis of flotation of large, triacylglycerol-rich particles will produce a population of particles that contain some large particles secreted by the liver (which, on biological function, should be called VLDL). Similarly, the next population to be harvested, which will be mainly VLDL, will also contain small chylomicrons and chylomicron remnants. The main physical methods are flotation in the ultracentrifuge, electrophoresis, gel filtration and precipitation. They are compared in the table below.

The principles of sequential flotation were developed by the Californian pioneer in lipid research, Richard Havel, in the 1950s (he is still active in the field today). Essentially, the plasma is laid in a tube under a salt solution of known density, which, on ultracentrifugation, allows one species of particle to be 'floated'. The particles are harvested and the density re-adjusted to prepare the next fraction. An alternative is to prepare a density gradient in which all types of particle will be separated in one centrifugation (usually faster but giving less clear separation). An example is shown in the figure right.

The figure shows the distribution of components after a single density-gradient centrifugation for 90 min at 65 000 rpm. The components were pumped from the bottom, out of the top of the tube. The density initially was from 1.006 (top) to 1.30 (bottom) g ml"1. The major peaks eluting are (leftto right) VLDL (major component triacylglycerol, •), LDL (major component cholesterol, ▲), HDL (both triacylglycerol and cholesterol) and finally albumin from the bottom of the tube. Reproduced from Chung, B.H., Segrest, J.P., Ray, M.J., Brunzell, J.D., Hokanson, J.E., Krauss, R.M., Beaudrie, K. & Cone, J.T. (1986) Single vertical

Method

Advantages

Disadvantages

Ultracentrifugal flotation

Electrophoresis (usually on agarose gel)

Gel filtration

Precipitation

Regarded as the 'reference method'.

Can be used to prepare large amounts of material for further analytical work.

Quick to perform on large numbers of samples.

Appears to cause least alteration to the particles.

Can be applied to large numbers of samples.

Relatively low capacity (usually performed in a swinging-bucket rotor with only 4 or 6 buckets).

Alterations in particle composition are known to occur during isolation.

Semi-quantitative. Not preparative.

Very limited capacity (usually performed in FPLC mode, running one sample at a time).

Separation is often based upon empirical findings and the basis is not always understood: possibility for artefacts therefore exists.

Continued

Box 5.1 Continued

spin density gradient ultracentrifugation. In: Methods in Enzymology, Vol. 128 Plasma Lipoproteins Part A. Preparation, Structure, and Molecular Biology (eds. J.P. Segrest & J.J. Albers), pp. 181-209. Academic Press, Orlando, FL.

Because of the problem of lack of absolute separation of particles according to biological function, other techniques have been developed. For instance, chylomicrons and VLDL differ in that the chylomicrons carry the shorter apolipoprotein apoB48, whereas VLDL carry apoB100. Specific antibodies have been prepared that bind to the C-terminal region of apoB100, and not to apoB48. These may be attached to a support such as Sepharose gel. A fraction containing the triacylglycerol-rich lipoproteins is prepared. This is passed through the gel, and VLDL particles are then bound and retained whilst chylomicron particles pass through and may be collected. Such methods are as yet only used in specialized research applications.

Lipoprotein classes may also be separated by various precipitation techniques. The precise physico-chemical basis for these is not always known. For instance, a combination of heparin and MnCl2 will precipitate particles containing apoB (chylomicrons, VLDL, LDL); after centrifugation to remove these, the supernatant contains HDL whose cholesterol content may be measured. When fasting plasma (i.e. with no chylomicrons present) is incubated with the detergent sodium dodecyl sulphate (SDS), VLDL particles separate (they float to the top). Analysis of the infranatant gives the concentration of (for instance) cholesterol in LDL+HDL. If the HDL-cholesterol concentration is measured by heparin-MnCl2 precipitation as above, subtraction will give cholesterol concentrations in VLDL and LDL. The more subtractions that are performed, the less reliable is the result.

Analysis of apolipoproteins

It is often useful to measure the concentrations of specific apolipoproteins in plasma. The usual methods are electrophoretic, or immunological. Electrophoresis is usually carried out on denaturing SDS-polyacrylamide gels and separates the proteins according to their size. ApoB100, because of its very large size, remains near the origin. The separated apolipoproteins may be quantified by staining and measurement of absorbance. There is a great variety of immunological techniques, each based on the availability of an antibody or antiserum specifically directed against one apolipoprotein. For instance, the technique of immunoturbidimetry relies on the solubilization of the apolipoproteins in detergents. They are then allowed to bind to a specific antibody (e.g. anti-apoAl), forming complexes that make the solution turbid in proportion to the amount of the specific protein present. The turbidity is measured in a spectrophotometer or similar instrument. Such techniques are easily automated and hence can have high throughput.

Analysis of lipid components

The analysis of concentrations of triacylglycerol, cholesterol, etc. in plasma or in isolated lipoprotein fractions is usually based upon enzymic methods and is readily automated for high throughput. These methods are described in textbooks of clinical chemistry and will not be given in more detail here.

activating transcription. When cellular cholesterol levels are high, this protease is inhibited and gene expression is repressed. The natural regulator of this system appears not to be cholesterol itself but a hydroxylated derivative of cholesterol, an oxy-sterol. The working of the system is illustrated in Fig. 5.14.

High Cellular Low Cellular

Sterol Levels Sterol Levels

SREBP SREBP

High Cellular Low Cellular

Sterol Levels Sterol Levels

SREBP SREBP

Fig. 5.14 The Sterol Regulatory Element Binding Protein (SREBP) system. The precursor protein (125 kDa) is associated with the rough endoplasmic reticulum (RER) (left). When cell cholesterol content is low, a protease (scissors) cleaves this to release the transcriptionally active 68 kDa N-terminal portion, which binds to specific promoter sequences in target genes (e.g. LDL-receptor, enzymes of cholesterol synthesis; see Fig. 5.13). When cell cholesterol content is high, this pathway does not operate and transcription of the target genes is low. Reproduced from Edwards, P.A. & Davis, R. (1996) in Biochemistry of Lipids, Lipoproteins and Membranes (eds. D.E. Vance & J. Vance), pp. 341-362. Elsevier, Amsterdam.

Fig. 5.14 The Sterol Regulatory Element Binding Protein (SREBP) system. The precursor protein (125 kDa) is associated with the rough endoplasmic reticulum (RER) (left). When cell cholesterol content is low, a protease (scissors) cleaves this to release the transcriptionally active 68 kDa N-terminal portion, which binds to specific promoter sequences in target genes (e.g. LDL-receptor, enzymes of cholesterol synthesis; see Fig. 5.13). When cell cholesterol content is high, this pathway does not operate and transcription of the target genes is low. Reproduced from Edwards, P.A. & Davis, R. (1996) in Biochemistry of Lipids, Lipoproteins and Membranes (eds. D.E. Vance & J. Vance), pp. 341-362. Elsevier, Amsterdam.

There are three known isoforms of SREBP: SREBP-1a, SREBP-1c and SREBP-2. The first two arise from differential splicing of the transcript from one gene, whereas SREBP-2 is the product of a separate gene. It seems that SREBP-1a and 1c are concerned more with regulation of genes involved in fatty acid metabolism including fatty acid syn-thase, acetyl-CoA carboxylase and stearoyl-CoA (A9) desaturase (Section 2.2.8), whereas SREBP-2 regulates transcription of genes involved in cholesterol metabolism (Section 7.5.7). SREBP-1c is also expressed in adipose tissue (where it is also known as adipocyte determination and differentiation factor-1, ADD-1), and has been shown to regulate fatty acid synthesis by increasing the expression of fatty acid synthase in adipose tissue as well as liver. Leptin production and secretion (Section 5.3.4) are also increased. Expression of SREBP-1c is itself under regulation by insulin. Thus, when insulin levels are high, more SREBP-1c is produced and fatty acid synthesis is up-regulated. Since this also requires the involvement of cholesterol (or a derivative), it might be that the regulation really works more in the negative way: in times of fasting (low insulin), SREBP-1c expression is severely reduced, and fatty acid synthesis cannot be activated by the SREBP pathway. Some of the genes whose expression is regulated by the SREBP system are shown in Table 5.3.

The SREBP system has been manipulated to alter cholesterol metabolism. Certain fungal metabolites, compactin from Penicillium spp. and mevinolin from Aspergillus terreus, were found in the 1970s to inhibit HMG-CoA reductase. Synthetic derivatives of these molecules are now widely used as drugs (the 'statins7) to lower plasma cholesterol concentrations. The main mode of action is that inhibition of hepatic cholesterol synthesis reduces hepatocyte unesterified cholesterol concentrations, and this results in up-regulation of hepatic LDL-receptor expression: hence more LDL particles are removed from the circulation.

5.3.2 The peroxisome proliferator activated receptor (PPAR) system regulates fatty acid metabolism in liver and adipose tissue

A number of apparently unrelated chemicals can induce the proliferation of the subcellular oxidative organelles called peroxisomes, especially in the livers of rodents. These agents were suggested to work through a common receptor called the per-oxisome proliferator activated receptor (PPAR). It is now realized that there is a family of PPARs and that a major role is to regulate fatty acid metabolism. Like the SREBP system, the PPARs are activated by lipids - in this case derivatives of fatty acids - and act upon specific response elements, peroxisome proliferator response elements (PPRE), in the regulatory region of genes concerned with lipid metabolism (Table 5.4).

PPAR-a is expressed mainly in the liver. It is activated by long-chain fatty acids, amongst other factors. The actual ligand is thought to be a meta-

Table 5.3 Genes whose expression is increased by the sterol regulatory element binding protein (SREBP) system

Gene Function in cell

LDL-receptor

HMG-CoA synthase

Squalene synthase

Farnesyl diphosphate synthase

Lanosterol synthase

Acetyl-CoA carboxylase

Fatty acid synthase

Stearoyl-CoA desaturase

Glycerol 3-phosphate acyltransferase

Lipoprotein lipase

PPAR-y

Leptin

Import of LDL particles de novo cholesterol synthesis de novo cholesterol synthesis de novo cholesterol synthesis de novo cholesterol synthesis de novo fatty acid synthesis de novo fatty acid synthesis Synthesis of oleate

Triacylglycerol and phospholipid synthesis Import of lipoprotein-triacylglycerol fatty acids Induction of adipocyte differentiation Secretion from adipocyte to signal fat store size

Stimulation occurs in response to low cellular cholesterol content. These functions have been shown in hepatocytes and adipocytes. Based on Sul, H.S. & Wang, D. (1998) Nutritional and hormonal regulation of enzymes in fat synthesis: studies of fatty acid synthase and glycerol 3-phosphate acyltransferase gene transcription. Annual Review of Nutrition, 18, 331-351; Worgall, T.S. & Deckelbaum, R.J. (1999) Fatty acids: links between genes involved in fatty acid and cholesterol metabolism. Current Opinion in Clinical Nutrition and Metabolic Care, 2, 127-133.

bolic derivative of a fatty acid such as a member of the prostaglandin family. One candidate is 15-deoxy-(A)12,14-prostaglandin J2, which has been shown to be particularly active in cellular systems. The activated PPAR-a then dimerizes with another receptor, the retinoid-X receptor, which must itself be activated by binding 9-cis-retinoic acid (Section 4.2.4.1). The heterodimer migrates to the nucleus where it binds to the PPRE and activates gene expression. Some genes whose regulation is altered by PPAR-a are listed in Table 5.4. In general PPAR-a activation can be seen to up-regulate the secretion of the apolipoproteins forming HDL, and to increase hepatic fatty acid oxidation.

Another isoform, PPAR-y, is expressed mainly in adipose tissue and its major role in that tissue is the regulation of fat storage. Activation of PPAR-y occurs in a similar way through the binding of a fatty acid or a fatty acid derivative, and after dimerization with the activated retinoid-X receptor, PPAR-y binds to PPREs in the adipocyte nucleus to activate expression of specific genes. In this case the genes include those of adipocyte differentiation from precursor cells (immature adipocytes), so activation of PPAR-y causes the recruitment of new adipocytes, as well as increased fatty acid deposition by up-regulation of the expression of genes regulating fat storage. These include components of the pathway of glucose uptake and synthesis, glucose transporter-4 (GLUT4) and phosphoenolpyr-uvate carboxykinase (an enzyme of gluconeogenesis). Presumably their function is to increase availability of glycerol 3-phosphate for triacylglycerol synthesis. Therefore, in times of excess fatty acid availability, this system ensures the co-ordinated storage of the excess fatty acids in adipose tissue. The co-ordinated working of the PPAR-a and PPAR-y systems is illustrated in Fig. 5.15, p. 197.

There is cross-talk between the SREBP and PPAR pathways. One of the SREBP isoforms, SREBP-1c (ADD-1), is expressed in adipocytes. As described above, SREBP-1c activation in adipose tissue increases expression of fatty acid synthase, and thus potentially the generation of ligands for PPAR-y. But there is a more direct link in that SREBP-1 activation directly increases PPAR-y expression.

Just as the SREBP system has been manipulated pharmacologically to alter cholesterol metabolism, so the PPAR system is the target for drug inter-

Table 5.4 Peroxisome proliterator activated receptors (PPARs): tissue distribution and effects of activation

Receptor Other names Main tissue Genes whose expression Genes whose distribution is increased by expression is

PPAR activation suppressed by

PPAR activation

PPAR-a

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