Insulin regulation of triacylglycerol-rich lipoprotein synthesis and secretion

BiochimicaL et Bioplwsica Acta ELSEVIER

Biochimica

et Biophysics

Acta 1215 (1994) 9-32

Review

Insulin regulation of triacylglycerol-rich secretion

lipoprotein synthesis and

Janet D. Sparks *, Charles E. Sparks Department of Pathology and Laboratory Medicine, Universiy of Rochester, School of Medicine and Dentistry, Box 608, 601 Elmwood Avenue, Rochester, NY 14642, USA Received 2 March 1994, revised 16 June 1994 Keywords:

Triacylglycerol-rich

lipoprotein;

VLDL, Apolipoprotein

B; Liver; Hepatocyte;

Intestine; Insulin; Diabetes; Fatty acid

Contents ...................................................

1. Introduction.

9

2. Triacylglycerol-rich lipoprotein components. .................................. 2.1. Apolipoprotein B .............................................. 2.2. Phosphatidylcholine ............................................. 2.3. Cholesterol and cholesteryl ester ...................................... 2.4. Triacylglycerol and fatty acids .......................................

10 10 13 14 14

3. Triacylglycerol-rich lipoprotein assembly and degradation ........................... 3.1. Lipid assembly with apolipoprotein B ................................... 3.2. Fatty acid effects on lipoprotein assembly ................................. 3.3. Insulin-stimulated apolipoprotein B degradation .............................. 3.4. Mechanisms of intracellular protein degradation .............................. 3.5. Post-secretory lipoprotein catabolism ....................................

16 16 18 19 20 21

4. Insulin regulation of triacylglycerol-rich lipoprotein production ........................ 4.1. Long-term hypoinsulinemia ......................................... 4.2. Short-term hyperinsulinemia ........................................ 4.3. Long-term hyperinsulinemia ........................................ 4.4. Apolipoprotein B phosphorylation .....................................

22 22 24 25 26

5. Summary

.....................................................

Acknowledgements. References..

27

.................................................

28

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1. Introduction Abbreviations: ALLN, N-acetylleucyl-leucyl-norleucinal; Apo, apolipoprotein(s); CE, cholesteryl ester(s); CM, chylomicrons; DG, diacylglycerol; DGAT, diacylglycerol:acyltransferase; ER, endoplasmic reticulum; FA, fatty acid(s); HDL, high-density lipoprotein; HMG-CoA reductase, hydroxymethylglutaryl-CoA reductase; IRS-l, insulin receptor substrate-l; LDL, low-density lipoprotein; MTP, microsomal triacylglycerol transfer protein; PC, phosphatidylcholine; STZ, streptozotocin; TG, triacylglycerol; TRL, triacylglycerol-rich lipoprotein; VLDL, very-low-density lipoprotein. * Corresponding author. Fax: + 1 (716) 273 1027. 00052760/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSD10005-2760(94)00133-2

Lipoproteins containing apolipoprotein B (apo B) include the triacylglycerol-rich lipoproteins (TRL), and cholesteryl ester-rich low-density lipoprotein (LDL). Endothelial bound triacylglycerol lipases act on TRL to release fatty acids (FA) and create remnant TRL that are primarily hepatically cleared. Plasma TRL fractions contain hepatically synthesized very-low-density lipoproteins

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J.D. Sparks, C.E. Spark.s/Biochimica

(VLDL) and intestinally synthesized chylomicrons (CM). In plasma, a portion of VLDL is converted to LDL which delivers cholesterol to peripheral tissues following binding to cell surface receptors including the LDL receptor. Under specific conditions VLDL component lipid or apo B can become rate-limiting for VLDL biogenesis. Increased entry of VLDL in excess of clearance leads to hyperlipidemia and increased plasma LDL-apo B levels are associated with increased risk of premature atherosclerosis. Recognition of factors that control the proportion of newly synthesized apo B that enters the plasma compartment as TRL becomes critical to our understanding of human dyslipoproteinemias and their relationship to cardiovascular disease. A single apo B molecule serves as an integral structural protein for each TRL (and LDL) and is one of the largest single chain mammalian polypeptides. Apo B structure is unusual compared with other apolipoproteins having amphipathic P-strand structures that may be important for lipid binding. Lipids, destined for transport, arise in the cytoplasm of liver and intestinal cells and apo B by virtue of its structure provides an acceptor for lipid translocation into the secretory compartment making apo B and apo B-dependent processes obligatory for neutral lipid transport [l]. In a betalipoproteinemia, patients have undetectable levels of plasma apo B-containing lipoproteins, fat malabsorption, acanthocytosis, progressive spinocerebellar degeneration and retinopathy. This disorder is not a result of a defect in the apo B gene or in the ability to transcribe the apo B gene [2-41 suggesting that other gene products are required for lipoprotein assembly and secretion. Microsomal triacylglycerol transfer protein (MTP) may be one of these obligatory gene products [5,6]. Insulin reduces rat hepatocyte VLDL triacylglycerol (TG) secretion while increasing cellular TG [7-lo]. The effect is mediated by the insulin receptor [ll] and is accompanied by a reduction in apo B secretion [8,9,12]. Apo B intracellular degradation has been described in hepatocytes and is a mechanism for insulin mediated effects [12] as freshly translated apo B is unable to complete formation of stable VLDL. Insulin has also been shown to play a permissive role in hepatic apo B biosynthesis [13,14] in addition to a role in regulation of known lipid synthetic pathways. These and other recent studies suggest that insulin and its counterregulation play a central role in TRL metabolism [15]. The role of insulin in lipoprotein metabolism and defects of insulin action in dyslipoproteinemia in diabetes mellitus is under active investigation. This review focuses on formation and regulation of hepatic TRL summarizing insulin regulation and proposing mechanisms of insulin action on TRL and apo B. Hyperlipoproteinemia which occurs in diabetes is discussed in the context of current knowledge of insulin regulation of TRL metabolism. Understanding the role of insulin in the regulation of lipoprotein metabolism may provide insight as to why diabetic humans have accelerated atherosclerosis [16].

et Biophysics Acta 1215 (1994) 9-32

2. Triacylglycerol-rich lipoprotein components Excellent recent reviews have been published concerning the assembly and secretion of TRL [17-241 which have focused on specific components of TRL including apo B [15,25-291 phospholipids [18,30-321, TG [33] and cholesteryl esters (CE) [34]. TRL consists of a non-polar lipid core (TG and CE) surrounded by a monolayer of lipids embedded with apolipoproteins. CM and VLDL which enter the circulation are heterogeneous in size. TRLs produced by the intestine have diameters as large as 1000 nm while those produced by the liver are smaller with diameters between 30-90 nm. Despite this size heterogeneity, each CM and VLDL particle contains a single apo B molecule [35] which is present during initial formation until clearance of the particle. As water-insoluble core components are synthesized within the cytoplasm of enterocytes and hepatocytes, the process of translocation into the secretory pathway and assembly with apo B requires a transition from cytoplasmic lipid to secretory lipoprotein. Although a great deal is known about regulation of individual TRL components less is understood about coordination of the complex series of events and specific cellular locations required to form mature TRL. While secreted TRL contain other apolipoproteins, apo B is required in order to form TRL.

2.1. Apolipoprotein

B

Apo B is the product of a single copy gene located on chromosome 2 and the gene has been sequenced [36-391. Apo B is encoded in a 14 kb mRNA which produces a 4563 amino acid protein (including the 27 amino acid signal peptide) having a molecular weight of about 513 kDa. Apo B contains frequent alternating hydrophilic and hydrophobic sequences as well as unique, proline-rich, hydrophobic sequences. In lipoproteins apo B is both surface oriented as well as interactive with the hydrophobic core suggesting full integration in TRL. Apo B is a glycoprotein that contains 8-10010 by weight carbohydrate which includes galactose, mannose, N-acetylglucosamine and sialic acid residues [40]. Apo B carbohydrate chains are composed of ‘high-mannose’ as well as ‘complex oligosaccharide’ chains. Apo B protein plus carbohydrate yields a molecular weight of 550 kDa by gel electrophoresis of the mature, native protein. Apo B secreted by HepG2 cells is fatty acylated with stearic and palmitic acids [41] through thioester bonds with cysteine [42]. The function of fatty acylation is unknown but might function to allow proper apo B folding, interaction with lipid or intracellular membranes or to facilitate translocation into the secretory pathway (reviewed in Ref. [43]). Apo B is also a phosphoprotein [44,45]. Two forms of apo B are synthesized in mammals: a higher (B-100) and a lower (B-48) molecular weight form

J.D. Sparks, C.E. Sparks/Biochimica

[46-491. B-100 contains the full length protein sequence encoded by the apo B gene. B-48 is produced as a result of a novel, post-transcriptional mechanism termed ‘editing’, involving the introduction of a translational ‘stop’ site into the apo B mRNA [50-521. A site-specific deamination (editing) of cytidine 6666 to a uridine by a cytidine deaminase changes the sense of codon 2153 from a glutamine to a ‘stop’ codon. The stop codon terminates translation at about half of the fully encoded protein and B-48 derived from the edited mRNA has a molecular weight approx. 48% of B-100 [49]. Apo B mRNA editing and its regulation have been reviewed [29,53-551. A 27 kDa protein (~27) has recently been identified and its cDNA cloned [56]. Studies indicate that p27 is the catalytic subunit of the editing enzyme [57] which requires zinc as part of its catalytic domain [57,58]. Full length human B-100 has now been expressed in transgenic mice [59] and studies indicate that the human transcript undergoes editing similar to the endogenous transcript suggesting lack of species specificity for the editing process. Apo B is synthesized on polyribosomes bound to the cytoplasmic surface of the endoplasmic reticulum (ER). B-100 synthesis requires 14-17 min [60] and B-48 requires about half as long [14]. Targeting of apo B ribosomes to the ER is initiated by emergence of its 27 amino acid signal sequence from the ribosome which is then recognized by the signal recognition particle (SRP) [61,62]. Binding of the signal sequence to the SRP slows elongation allowing the complex (SRP-nascent chain-ribosomemRNA) to diffuse to the ER membrane and encounter the SRP receptor. After docking to the SRP receptor, an integral membrane protein, there is binding of the ribosome to the ER membrane. The subsequent release of SRP and SRP receptor allows for resumption of peptide chain elongation and initiation of translocation through a protein conducting channel [63]. GTP binding and hydrolysis occurs in conjunction with assembly of the translocation channel [64]. A mathematical model has been constructed to evaluate the potential for regulation of apo B synthesis at the level of interaction between the apo B mRNA-ribosome complex and its translocational channel [65]. The channel opened by binding of the signal sequence occurs at specific sites in the ER membrane termed ‘translocons’ [66] which are assemblies of specific proteins and cofactors required for translocation and may include lumenal proteins such as binding protein (BiP), the signal peptidase and oligosaccharyl transferase complexes. It has recently been demonstrated that the signal sequence is sealed off from the cytoplasm by a tight ribosome-membrane junction and that nascent polypeptides pass through an aqueous tunnel in the ribosome to reach the ER membrane [67]. After the signal sequence has translocated into the ER lumen it is cleaved from the nascent chain by the signal peptidase complex on the lumenal side of the membrane. Targeting of nascent apo B to the ER membrane and translocation across the ER membrane are the first events

et Biophysics Acta 1215 (1994) 9-32

11

in apo B intracellular sorting and current knowledge of these processes has recently been summarized [64,681. Maturation of nascent polypeptides involves protein folding mechanisms (reviewed in Ref. [69]) and protein folding on the lumenal side of the ER may be an important force in translocation [70]. Following cleavage of the signal sequence, neither forward movement into the ER lumen nor backwards progress is favored and it has been suggested that the net transfer of peptides into the lumen is driven by biased random thermal motion or by a so-called ‘Brownian ratchet’ mechanism [71]. Backwards translocation is prevented by protein folding within the lumen and interaction of the protein with lumenal chaperones, protein glycosylation and disulfide bond formation [72]. In the case of apo B translocation, tissue specific factors may also be required particularly for apo B sequences greater than B-15 (amino-terminal 15% of B-100). In CHO cells, B15 is efficiently translocated and secreted but B-53 is not and is degraded [73]. ER ‘chaperone’ proteins including BiP (GRP-78) and enzymes which catalyze isomerization steps such as protein disulfide isomerase (PDI) are required for proper protein folding and correct disulfide bond formation. Binding of BiP to nascent peptides stimulates its ATPase activity which results in peptide dissociation and entry into the lumen. PDI is a multifunctional protein and is also a subunit of MTP [74]. MTP catalyzes the transfer of CE, TG and phosphatidylcholine (PC) between membranes [75] and requires PDI to be catalytically active [76]. If apo B translation and translocation are tightly linked [77,78], newly synthesized apo B sequences (30-40 amino acid residues long) are not exposed to the cytoplasm and remain buried within the ribosome tunnel which is tightly sealed with the ER membrane and continuous with the translocation channel (Fig. la). Since the nascent chain fills the restricted space within the ribosome tunnel, elongation of the nascent chain at the peptidyltransferase end of the tunnel is accompanied by vectoral movement of the peptide out of the tunnel, through the channel and into the ER lumen [67]. Apo B translocation has been suggested to involve specific multiple ‘pause’ and ‘restart’ topogenic sites [79] based on in vitro observations of discrete, transient, translocational apo B intermediates [SO,Sl]. Slowing or pausing of apo B translocation is suggested to favor lipid binding to critical stretches of newly synthesized hydrophobic sequences of apo B as well as proper disulfide bond formation within the lumen and possibly posttranslational modification. Alterations in apo B conformation induced by these modification reactions might be important in ‘restarting’ translocation. Pauses in translocation might also affect protein translation as continued apo B synthesis during pauses might ‘fill-up’ the channel and slow peptide elongation. Polysomes bearing apo B mRNA demonstrate a specific apo B banding pattern [14] suggesting discrete pauses during apo B mRNA translation. This finding supports the occurrence of favored ‘stop’ sites

12

J.D. Sparks, C.E. Sparks/Biochimica ROUGHER LUMEN

(4

Fig. 1. Potential relationships between apo B translation and translocation. (a) Coordinated apo B translation and translocation. (b) Temporal unlinking of translation and translocation with formation of apo B sequence accessible to the cytoplasm. (c) Dissociation of apo B translation and translocation with the formation of apo B sequence associated with the cytoplasmic side of ER membranes.

during translation and stops in translation would effectively cause delay in apo B translocation [77,78]. Recent studies, however, suggest that pause transfer sequences act independently of each other and of translation and that the observed transient, transmembrane intermediates of apo B arise from sequential action of multiple, independent pause transfer sequences [79]. The question of whether apo B undergoes pauses during translocation due to specific apo B protein sequence or whether delays in translocation are related to ribosomal stalling due to apo B mRNA sequence remains unresolved. Apo B has been demonstrated on the cytoplasmic side of liver microsomes in rat, [82], rabbit [83,84], chicken [85] and in HepG2 cells [86,87]. Cytoplasmic apo B in rabbit microsomes contains both amino- and carboxy-terminal residues [83]. This finding raises the question of the mechanism by which apo B attains a cytoplasmic orientation. Is apo B sequence that is found on the cytoplasmic side of the ER a result of synthesis within the cytoplasm or is it a result of a slower step in translocation peculiar to apo B? Recent studies using digitonin-permeabilized HepG2 cells suggest a large portion of B-100 molecules have a cytoplasmic orientation early in the secretory pathway due to partial translocation [88]. The duration of time an B-100 molecule spends between compartments may determine whether apo B forms a lipoprotein or becomes integrated into the membrane [89]. One possibility to explain these findings is that translation and translocation which usually keep pace with each other become temporally unlinked resulting in entry of apo B sequence into the cytoplasm

et Biophysics Acta 1215 (1994) 9-32

through either ‘tilting’ of the ribosome-mRNA complex or dissociation of the complex from the ER membrane (Fig. lb). The presence of ‘pause-transfer’ translocational sequences might favor this unlinking of translation and translocation [79]. Continued translation during pauses in translocation would allow apo B to enter the cytoplasm possibly to be bound by cytoplasmic chaperone proteins and/or to associate with the cytoplasmic side of ER membranes. Such a spatially extended structure as depicted in Fig. lb would be consistent with the unusual sedimentation velocity characteristics of polysomes bearing edited and unedited apo B mRNA [90]. As B-42 polysomes fail to have altered sedimentation, the unusual features of B-48 and B-100 polysomes appear to depend upon apo B mRNA sequences 3’ to B-42 in the full length mRNA [90]. Whether apo B synthesized within the cytoplasm and remaining associated with ribosomes can reengage with the ER membrane and channel and subsequently translocate is not known and factors stabilizing lumenal vs. cytoplasmic apo B domains may become critical determinants for this occurrence [71]. Whether completed apo B chains released by ribosomes on the cytosolic side of the membrane can undergo translocation post-translationally (Fig. lc) is not known although this phenomenon has been observed for other large proteins [70]. It remains a possibility that the abundance of apo B found on the cytoplasmic side of the ER is magnified by isolation methods. Disruption of ribosomes, either mechanically or enzymatically by cellular RNAses, might release nascent apo B chains undergoing translocation. Movement out of the translocation channel back into the cytoplasm might then occur until a stable transmembrane configuration can be attained possibly through interaction of apo B with cytoplasmic or lumenal factors or components of the translocation channel itself. ER Ca” may regulate movement of specific proteins out of the ER as CaZf may be required for folding of newly synthesized proteins and proper folding then becomes a prerequisite for subsequent movement to the Golgi. ER Ca2+ may favor retention by stabilizing interactions of newly synthesized proteins with chaperone proteins such as BiP [91]. The interaction between ER Ca2+ and apo B is likely as the ER is not only a major cellular Ca*+ storage compartment but also a site of cellular apo B [92,93] and apo B is a known Ca2+-binding protein [94]. A role for Ca*+ in the regulation of TRL apo B secretion is supported by studies using prostaglandins (PGE, and PGD,), calcium antagonists and ionophores all of which are able to inhibit apo B secretion 1951. In HepG2 cells, Ca*’ ionophores (A23187 and ionomycin) block secretion and result in the accumulation of newly synthesized proteins in the rough ER [96]. The subsequent fate of ER-retained proteins may depend on inherent susceptibility to ER proteinases and Ca*+ may also be involved in proteinase activation for some ER degraded proteins [97,98]. The calcium channel blockers, verapamil and diltiazem,

J.D. Sparks, C.E. Sparks/Biochimica

inhibit TRL and apo B secretion by rat hepatocytes [99,100]. The effect of diltiazem on inhibition of secreted apo B is greater for B-100 than B-48. As both calcium channel blockers and calcium ionophores inhibit apo B and TRL TG secretion, an optimal level of ER Ca2+ may be necessary for proper apo B folding and subsequent movement out of the ER [95]. As apo B does not accumulate in cells, a reasonable hypothesis is that retention of apo B in the ER leads to accelerated degradation. 2.2. Phosphatidylcholine Regulation and biosynthesis of PC and TG in liver have recently been reviewed [18,31-33,101]. PC is the major component of the TRL surface monolayer and TG is the major core component. The biosynthesis of PC and TG occurs through a common intermediate, diacylglycerol (DG) formed from phosphatidate (1,2-diacylglycerol phosphate). Phosphatidate is formed by sequential fatty acylation of sn-glycerol 3-phosphate. The first step is mediated though the enzyme glycerolphosphate acyltransferase on the cytosolic side of the rough and smooth ER which is unselective for a particular FA. The second acylation reaction is mediated through monoacylglycerol phosphate acyltransferase and is relatively specific for unsaturated FAs. From phosphatidate is formed CDP-diacylglycerol, the precursor of acidic phospholipids, and diacylglycerol (DG), the precursor of PC, phosphatidylethanolamine (PE) and TG. Phosphatidate phosphohydrolase (PPH) is the enzyme that converts phosphatidate to DG. PPH exists both in the cytosol and in ER membranes. Compounds which inhibit the association of PPH to ER membranes inhibit synthesis of TG and PC [102]. Cytosolic PPH represents an inactive potential enzyme activity that becomes expressed when the enzyme associates with ER membranes and is activated [33,102]. Association of PPH with ER membranes is favored by membrane-bound fatty acyl-CoA esters and phosphatidate. Cyclic AMP analogues cause displacement of PPH from ER membranes reducing TG and PC synthesis but the effect of CAMP can be overcome by high concentrations of FAs [103]. Although glucagon increases levels of CAMP, combined actions of catecholamines and glucocorticoids as occurs in situations of stress [104], can stimulate hepatic TG synthesis by increasing the supply of FAs from extrahepatic tissues favoring association of active enzyme with membranes [ 1051. Glucagon and catecholamines also increase /3-oxidation of FAs balancing FAs available for TG and PC synthesis. The production of membrane DG acts as a signal for the association and activation of CTP:phosphocholine:cytidylytransferase (CT) which catalyzes the ratelimiting step in the synthesis of CDP-choline from phosphocholine and CTP (reviewed in Ref. [30]). The final step in the formation of PC is the transfer of CDP-choline to DG catalyzed by choline phosphotransferase. In the liver,

et Biophysics Acta I215 (1994) 9-32

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PC can be formed from the CT pathway and also by the methylation of PE utilizing S-adenoprogressive sylmethionine as the methyl donor by the enzyme PE methyltransferase. For lipoprotein synthesis it has been suggested that 60-80% of PC is derived from the CDPcholine pathway regulated by the activity of CT and 20-40% is derived by methylation of PE. The diet provides necessary choline for the formation of CDP-choline which enters cells via a specific, high affinity transporter which may be localized near enzymes required for PC synthesis leading to functional linkage of steps in PC synthesis [106]. Evidence suggests that newly formed DG is preferentially used for PC biosynthesis in part because DG is a precursor and in part because DG enhances CT binding to ER membranes and activation [30]. A decrease in PC synthesis in hepatocytes with increased phosphorylation of cytosolic CT has been demonstrated [107]. Cyclic AMPmediated inhibition of PC synthesis however is not due to an effect on phosphorylation of CT and altered subcellular distribution but is due instead to a decreased level of DG substrate [108]. Long chain FAs increase the rate of PC biosynthesis by enhancing CT binding to microsomes. The increase in biosynthesis of PC in hepatocytes incubated with oleic acid is postulated to be due to an increase in translocation of CT from cytosol to membranes and a reduction in phosphorylation state of cytosolic CT [107]. The binding of CT to membranes appears to be sensitive to the ratio of bilayer- to non-bilayer forming lipids [109]. Hepatocytes derived from choline-deficient rats have a specific impairment in VLDL-apo B secretion [llO]. Reduction in PC synthesis by choline deficiency does not affect secretion of other proteins suggesting that active, ongoing PC synthesis appears to be a requirement for maintenance of VLDL-apo B secretion. Incorporation of radiolabeled leucine into apo B is similar in control and choline-deficient rats even though PC synthesis is markedly reduced and compared with controls, a similar of number of TRL are present within the ER [ill]. In choline-deficient rats, however, the number of TRL within the Golgi is significantly reduced suggesting post-ER degradation of lipoprotein apo B occurs with choline deficiency. TRL secretion can be restored to control levels by addition to culture media of choline, methionine, or lyso PC suggesting the biosynthetic origin of the PC is not a critical factor in TRL production 11121. Ethanolamine and monomethylethanolamine (MME) additions are unable to restore VLDL secretion rates [113] whereas dimethylethanolamine leads to partial restoration of TRL B-48 secretion but not TRL B-100. Interestingly, MME addition to media causes a rapid and dose-dependent decrease in VLDL lipid and apolipoprotein secretion by rat hepatocytes [114]. The mechanism for this effect does not appear to be related to lipid component biosynthesis and recent evidence suggests that the resultant increase in microsomal PMME diminishes the ability of apo B to translocate possibly by

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J.D. Sparks,C.E. Sparks/Biochimica et BiophysicsActa 1215 (1994) 9-32

affecting the translocation channel through disruption of the bilayer structure [115]. However, as other secretory proteins are not affected another possibility is that PMME affects the ability to supply lipids for assembly with apo B [115]. 2.3. Cholesterol and cholesteryl ester Cholesterol is an important component of the surface monolayer of TRL. Cholesterol in most cells is highly regulated through coordinated control of key enzymes especially hydroxymethylglutaryl-CoA reductase (HMGCoA reductase), a microsomal enzyme catalyzing the ratelimiting step in cholesterol biosynthesis (reviewed in Ref. [116]). Cholesterol is present in membranes and in the cytoplasm in the form of neutral lipid droplets when esterified with FAs to form CE through the action of acyl-CoA:cholesterol acyltransferase (ACAT), an enzyme located predominantly in the ER. The coordinate regulation of FA and cholesterol synthesis by an AMP-activated protein kinase (AMP-PK) has recently been reviewed [ 1171. AMP-PK through phosphorylation inactivates acetyl-CoA carboxylase and HMG-CoA reductase and appears to play a central role in regulating lipid synthesis in cells when cellular ATP levels fall and ADP and AMP levels rise. AMP-PK is phosphorylated by a kinase to an active form and dephosphorylated to an inactive form by protein phosphatase 2C [117]. Under most circumstances, there is enough cholesterol synthesized for TRL assembly. When cholesterol synthesis is inhibited by addition of the HMG-CoA reductase inhibitor, lovastatin, reduced secretion of TRL is observed [118,119]. Lipoproteins that are secreted under these conditions contain newly synthesized cholesterol supporting the idea that there is a metabolically active pool of cholesterol required for lipoprotein assembly. Other HMG-CoA reductase inhibitors including simvastatin and pravastatin yield contradictory results. Simvastatin, which inhibits cholesterol synthesis in rat hepatocytes by 60%, when used in the presence of insulin, dexamethasone and oleic acid, enhances apo B synthesis and secretion without altering TG secretion whereas apo A-I synthesis and secretion is reduced [120]. In contrast, apo B secretion by rabbit hepatocytes appears to parallel de novo synthesis of cholesterol and both are inhibited by pravastatin [121]. In HepG2 cells chylomicron remnants and /3-VLDL additions stimulate secretion of B-100 [122]. As FA addition alone fails to duplicate this effect it has been suggested that increased cholesterol supply to the cells via lipoprotein uptake is the stimulus for secretion. Incubation of HepG2 cells with 25-hydroxycholesterol causing a marked increase in cellular CE also increases apo B secretion and results in a 55% increase in apo B mRNA [123]. In rabbit hepatocytes, cellular CE content, raised by addition of LDL to the media or lowered by pravastatin correlates well with changes in B-100 secretion without alterations in apo B

mRNA abundance [121] and decreased secretion of B-100 is associated with increased degradation of newly synthesized B-100. In HepG2 cells under conditions of high FA delivery, the rate of CE synthesis by ACAT in the ER may become a determinant for B-100 secretion suggesting that newly synthesized CE favors entry of freshly translated apo B into the ER [124] (reviewed in Ref. [34]). B-100 secretion, however, is not reduced in HepG2 cells incubated with an ACAT inhibitor even with increased lipid delivery [125]. When TG synthesis is inhibited using a fatty acyl-CoA synthase inhibitor, B-100 secretion is significantly decreased suggesting a critical role for newly synthesized TG [125]. Fluvastatin, an HMG-CoA reductase inhibitor which reduces CE production and 25-hydroxycholesterol which stimulates CE production do not affect B-100 production or kinetics in HepG2 cells [126]. Although some level of cholesterol synthesis is probably necessary for TRL assembly, cholesterol synthesis is likely to be coordinated with availability of neutral lipid core components (CE, TG) in TRL formation. The ER bilayer is relatively deficient in cholesterol having a similar cholesterol to phospholipid molar ratio as the VLDL surface monolayer. ER membranes maybe ‘cholesterol-poor’ so that proteins can be more easily inserted. Plasma membranes in contrast are rich in cholesterol and sphingolipids making them thicker, stiffer and less permeable to small molecules. Since cholesterol is synthesized in the ER, it must be continuously removed and concentrated as vesicles travel to the plasma membrane through the Golgi apparatus in order to maintain the cholesterol gradient. It has been suggested that the changing thickness of the hydrocarbon region of the bilayer due to differences in cholesterol content creates a self-sorting mechanism for ER and Golgi membrane proteins based on the length of each protein’s transmembrane region [127,128]. Pseudotransmembrane domains as may occur in apo B during biogenesis in the ER might delay entry of apo B into cholesterol-rich transport vesicles until membrane apo B has formed lumenal lipoproteins. 2.4. Triacylglycerol

and fatty acids

Conversion of DG, formed by PPH action on phosphatidate, into TG by esterification of FA at position 3 is catalyzed by the enzyme, diacylglycerol:acyltransferase (DGAT) which constitutes the last step in TG formation [129,130]. DGAT competes with choline phosphotransferase for DG substrate. DGAT is tightly bound to the ER membrane and its activity may be subject to regulation by reversible phosphorylation [131]. Reduced DGAT activity diverts DG to phospholipid formation in conditions such as starvation, hypoinsulinemic diabetes and stress [101,132]. Purification of DGAT from rat liver microsomes has recently been reported giving a molecular weight for the protein of 180 kDa [133]. Reduced activity of DGAT can be overcome by increased availability of FA, however,

J.D. Sparks, C.E. Sparks/Biochimica

certain FA inhibit formation of TG by inhibiting the enzyme [134]. Hepatocytes conditioned in media containing palmitic acid synthesize and secrete more TG than those incubated with eicosapentaenoic acid and the suppression of TG production is due, in part, to inhibition of DGAT [134]. Eicosapentaenoic acid not only is a poor substrate for DGAT but it decreases incorporation of other FA into TG [135]. Eicosapentaenoic acid also decreases cholesterol esterification in rat hepatocytes by reducing ACAT activity which subsequently reduces secretion of VLDL cholesteryl ester [136]. Effects of n - 3 FAs such as eicosapentaenoic acid may be due to their inability to ‘load’ hepatocytes with TG compared with oleic acid [135] or they may not assemble into TG of nascent lipoproteins as readily as those derived from oleic acid [137]. In rat liver perfusions, there are dose-dependent effects of IZ- 3 FAs on VLDL TG secretion [138]. At low perfusate concentrations, eicosapentaenoic acid inhibits TG secretion whereas at higher concentrations TG secretion is increased. In rat hepatocytes n - 3 FAs stimulate TG synthesis but compared with oleic acid, n - 3 FAs promote intracellular apo B degradation suggesting that an alternative mechanism of action of IZ- 3 FAs is interference either at the level of apo B availability or stability of the nascent lipoprotein formed [139]. The effect of it - 3 FA in stimulating TG synthesis while favoring apo B degradation is similar to the effect of insulin [12]. In the liver the mass of TG and secreted TRL depends upon the nutritional status of the animal [140]. The correspondence between nutritional state and TRL production is independent of availability of extracellular FA suggesting that the capacity for hepatic TRL TG secretion relates to changes in lipogenic enzymes. An important factor in TRL secretion is the size of the intracellular TG pool [141,142]. Rat hepatocytes ‘loaded’ with TG by overnight incubation with media containing oleic acid secrete significantly more TG than those incubated without FA. Evidence supports the idea that for most exogenous FAs to gain entry into the secretory pathway, they must first be esterified into TG and enter a cytoplasmic pool [141]. FA released from this pool by lipolysis are re-esterified in the ER for lipoprotein assembly. In the absence of extracellular FA, the proportion of secreted TRL TG arising from lipolysis and reesterification is at least 70% [141] and this proportion remains relatively constant even when hepatocytes are exposed to insulin or glucagon which produce marked reductions in TRL TG secretion. Intracellular lipolysis supplies 2 to 3 times more FA than required for secretion of TRL TG and excess FA are re-esterified and return to the cytoplasmic storage pool. Under conditions favoring suppression of intracellular lipolysis using tetrahydrolipstatin there is a dramatic decrease in secretion of TG [143]. In the presence of insulin which also reduces TG secretion, lipolysis of intracellular TG is unchanged and reesterification of FAs is favored [lo] leading to accumulation of cellular TG. When insulin is removed from the medium

et Biophysics Acta 1215 (1994) 9-32

15

there is mobilization of the cytosolic pool of TG which is reformed into TRL and secreted. Regulation of FA synthesis has recently been reviewed [144]. The rate-limiting step in FA synthesis is the formation of malonyl-CoA from acetyl-CoA and bicarbonate mediated by acetyl-CoA carboxylase (ACC). Acetyl-CoA is formed from citrate by action of ATP:citrate lyase. Citrate originates from mitochondria entering the cytoplasm via the tricarboxylate transporter in exchange for malate and high levels of citrate formation in mitochondria favor lipogenesis. De novo synthesis of FA is balanced by FA oxidation whose regulation by camitine palmitoyltransferases and malonyl-CoA has recently been reviewed 11451. ACC is regulated in the short-term by reversible phosphorylation and the allosteric regulators, citrate and long-chain fatty acyl-CoA. ACC is regulated through a protein phosphorylation cascade involving AMP-activated PK [ 1171. In rat hepatocytes, ACC is activated by insulin within minutes of addition, however, the mechanism for this effect is not clear. The requirement for FA synthesis for lipoprotein assembly and secretion was recently studied in primary cultures of hamster hepatocytes 11461. Using an inhibitor of ACC, de novo FA synthesis is almost completely inhibited and TRL TG secretion is decreased by 90% whereas synthesis and secretion of B-100 is reduced by 50%. Addition of exogenous FAs to the media restored B-100 secretion to control levels. As inhibition of ACC and FA synthesis would affect the supply of fatty acyl-CoA needed for synthesis of TG, CE as well as phospholipids it is not clear at what level the inhibitor acts. Fatty acid synthase (FAS) is a cytosolic enzyme containing seven enzyme activities within a single polypeptide encoded by a single gene. Nutritional state is the main factor controlling the rate of FA synthesis as both FAS and ACC are enzymes which undergo adaptive changes in mRNA and enzyme protein. FAS is increased in animals fed high carbohydrate diets and insulin increases de novo FA synthesis in the long-term by induction of gene transcription. In HepG2 cells, glucose also plays a regulatory role as glucose increases FAS mRNA stability [147]. FA chain elongation in liver occurs mainly in the ER by addition of two carbon fragments to palmitoyl-CoA using malonyl-CoA as the acetyl donor and NADPH as the more active electron donor. Chain elongation of newly synthesized and exogenous FA and formation of unsaturated FA has recently been reviewed [148]. ER chain elongation is the major source of FA greater than 16 carbons when dietary long chain FA are not available. Monounsaturated FA are formed by oxidative desaturation by enzymes associated with the ER. The predominant desaturase for saturated FA (14 to 18 carbons) is the Ag-desaturase system whose activity is dependent upon the presence of PC. The A’-desaturase system is composed of three proteins: NADH-cytochrome b5 reductase, cytochrome b5; and the terminal desaturase component. The A’-desaturase system is regulated by diet and hormones. The A’-de-

16

J.D. Sparks, C.E. Sparks / Biochimica et Biophysics Acta 1215 (1994) 9-32

saturase is decreased by fasting to less than 5% of control levels and is dramatically increased with refeeding fat-free, carbohydrate-rich or protein-rich diets. The marked increase in activity is due to increases in translationally active mRNA. Insulin induces the A’-desaturase system in vivo. In streptozotocin-induced diabetes, A’-desaturase activity is depressed and normalizes after in vivo insulin treatment but not insulin treatment in vitro. As the A’-desaturase mRNA is relatively stable, the mechanism of insulin action is unclear. Pyruvate stimulates hormonal induction of lipogenic enzymes [149] and TRL TG secretion in primary rat hepatocytes [150]. The effect of pyruvate and lactate on TRL TG secretion in young cultures is more pronounced in the presence of dexamethasone [151]. Addition of pyruvate to hepatocytes specifically increases the activities of malic enzyme, glucose-6-phosphate dehydrogenase, FAS and ATP-citrate lyase due to increases in the amount and translation of corresponding mRNAs [149]. Sodium butyrate, a short chain saturated FA, stimulates secretion of B-100 and apo A-I by HepG2-cells [152] and significantly increases apo A-I mRNA levels but not apo B mRNA. For B-100, the lack of correspondence of secretion with mRNA levels suggests that the stimulatory effect of butyrate on secretion of B-100 is mediated by a translational or posttranslational mechanism. The requirements for newly synthesized cholesterol and choline for PC synthesis for TRL formation by liver are largely overcome in the intestine through absorption of preformed intermediates (lysoPC, 2-monoacylglycerols). The component that most closely correlates with TRL secretion by enterocytes is the quantity of fat ingested. Digestion of fat leads mainly to formation of FA and 2-monoacylglycerol which are absorbed and rapidly reesterified in enterocytes. Only a minor portion of intestinal TRL TG are formed from the phosphatidate pathway. Approx. 30-40% of dietary PC is absorbed as 1ysoPC which is re-esterified to form PC. Free cholesterol is absorbed through the brush border and becomes diluted with endogenous cellular cholesterol which can become reesterified and transported in CM. Dietary n - 3 and n - 6 FA products of the A6-desaturase system can alter hepatic TG synthesis and secretion by suppressing gene transcription of lipogenic enzymes including FAS [153], malic enzyme, A’-desaturase and S14 genes (reviewed in Ref. [154]). Saturated and monounsaturated FAs have no inhibitory capability. It is hypothesized that n - 3 and n - 6 FAs are transported though the plasma membrane, bind to cytosolic FA-binding proteins which shuttle them first to the A6-desaturase system and then to the nucleus where the product is transferred to a specific nuclear binding protein. The activated nuclear complex can either bind to a specific cis-element which suppresses gene transcription or may function as an auxiliary protein which interacts with a DNA binding protein and modulates other truns-acting proteins govern-

ing transcription. Dietary n - 3 FA also causes enrichment of membrane phospholipids with polyenic FAs which increases membrane fluidity and may alter responsiveness of hepatocytes to hormones such as glucagon and insulin [155] (reviewed in Ref. [156]).

3. Triacylglycerol-rich lipoprotein assembly and degra-

dation 3.1. Lipid assembly with apolipoprotein B Lipoprotein formation is a major function for liver and intestine and there could be specialized areas or complexes in these cell types dedicated to lipoprotein synthesis and assembly. This hypothesis is consistent with the general finding that by limiting the synthesis or availability of any single lipoprotein component, the production of the whole particle can be inhibited. Lipoprotein assembly complexes might be in areas of the ER where there is localization of apo B polysomes, enzymes for PC synthesis [30], cholesteryl ester formation [34], lipolysis of cytosolic TG [141], re-esterification of FA by DGAT [24] and lipid transfer proteins such as MTP/PDI [74,157]. Additional protein components acting as apo B chaperones may also be required to aid in translocation and possibly intracellular transport. In HepG2 cells, lipids derived from lipoprotein uptake can serve as alternate lipid sources for lipoprotein assembly [122] and in intestinal cells, lysoPC, cholesterol and 1-monoacylglycerol may be absorbed and directly substitute for those requiring de novo synthesis. If such a lipoprotein assembly complex exists there may be additional potential control sites for metabolic regulation such as proper localization of apo B mRNA, membrane association of PC and TG synthesizing enzymes as well as proper targeting of apo B to specific translocation channels associated with appropriate intralumenal chaperones. Furthermore, if apo B translocation involves multiple pause transfer events [SO] there may be additional regulation at the level of restarting translocation possibly through posttranslational modifications of apo B. The cellular location where TG assembles with apo B for formation of TRL remains controversial. Early studies in rat liver provide evidence for the importance of lipid transfer to apo B at the junction of the rough and smooth ER [158]. The similarity in size and lipid composition of lumenal lipoproteins derived from ER fractions, Golgi vesicles and plasma [159] supports the hypothesis that the full complement of lipids associate with apo B in the ER although some remodeling and lipid exchange reactions may occur during intracellular transport (Fig. 2a). Kinetic studies of apo B in rat hepatocytes support the importance of the ER in that movement out of the ER determines the overall rate of TRL secretion [160]. In rat liver while most B-100 particles are found in VLDL densities roughly half of B-48-containing particles are found in LDL and HDL

J.D. Sparks, C.E. Sparks/Biochimica ER LUMEN

(4

GOLGI

-

Fig. 2. Mechanisms of triacylglycerol-rich lipoprotein assembly. (a) Nascent triacylglycerol-rich lipoprotein forms cotranslationally producing very-low-density lipoprotein within the ER lumen. (b) Nascent triacylglycerol-rich lipoprotein formation requires two steps. In the first step, apo B forms a microemulsion during translation. In the second step, there is coalescing of the apo B microemulsion with triacylglycerol droplets. (cl Small nascent apo B-containing lipoproteins are formed within the ER and during passage through the secretory pathway enlarge into triacylglycerol-rich lipoproteins by lipid transfers within the ER and Golgi apparatus.

densities throughout the secretory pathway [159]. HDL B-48 particles have also been shown to be secreted in rat liver perfusions [161,162] and by rat hepatocytes [113] and contain roughly equal amounts of TG and CE. The finding of no progressive increase in size, lipid content or decrease in density of apo B-containing particles upon isolation of lumenal lipoproteins in sequential compartments of the secretory pathway argues against the idea that smaller particles gradually become larger TRL during transport and favors the concept that HDL and VLDL sized particles are directly formed within the ER [159]. A two step process for TRL assembly has recently been proposed [163,164] which takes place entirely within the ER (Fig. 2b). In the first step there is transfer of phospholipid, TG and CE to newly synthesized apo B in the rough ER dependent on MTP activity [164] to form a microemulsion which is released into the lumen as an apo B-containing primordial particle. The second step is fusion of the primordial particle with lumenal TG-enriched globules made by the smooth ER. HepG2 cells which lack extensive smooth ER are suggested to be deficient in the second step, the formation of lipid globules, which limits the formation of true VLDL [164]. Pulse-chase studies in HepG2 cells suggest that the first step occurs cotranslationally and that B-100 is assembled with lipid during translocation in a smooth membrane compartment proximal to the cis-Golgi which is enriched in DGAT [165]. For B-100 only LDL size particles become potential precursors to

et Biophysics Acta 1215 (1994) 9-32

17

VLDL as HDL-size B-100 is degraded [86]. In rat liver evidence suggests that HDL B-48 can serve as a precursor to VLDL B-48 as labeled B-48 appears first in secretory HDL and later in TRL fractions in radiolabeling experiments [166]. In rat liver HDL B-48 particles are stable as they are found within the secretory pathway [159,167] and are secreted by primary hepatocytes and rat liver during perfusions [161,162]. Perhaps LDL B-100 secreted by HepG2 cells and HDL B-48 secreted by rat hepatocytes are stabilized equivalents of nascent primordial particles which had the potential to become TRL in the ER under the appropriate circumstances. The two step hypothesis of TRL formation is supported by studies of erotic acid fed rats [167,168]. Orotic acid feeding of rats results in a selective defect in secretion of apo B-containing VLDL. Lipid-rich lipoproteins are present within the ER but are not secreted suggesting that blockage occurs in the transfer of nascent particles from the ER to cis-Golgi [167,168]. The ER is enriched in HDL apo B [168] and contains newly synthesized B-48 which continues to be secreted despite the defect in VLDL secretion [167]. In human abetalipoproteinemia, B-100 synthesis occurs [4] in the absence of functional MTP [5] and lack of MTP activity may interfere with microemulsion formation leading to the complete inability to assembly apo B-containing lipoproteins. Overall, the data supports that formation of TRL apo B is likely to occur in the ER. The main difference between the hypotheses outlined in Fig. 2a and Fig. 2b is the separation of the formation of TG-rich droplets in terms of localization. Unresolved issues in the two step hypothesis are the mechanism for intralumenal TG globule formation and how TRL containing 2 or more apo B molecules per particle are prevented from forming during the fusion process. Results of studies in estrogen-induced chick hepatocytes [169,170] and in rat liver [171] suggest that a sizable proportion of lipid assembles with apo B in the Golgi and that transport through the Golgi is rate-limiting in TRL secretion [169]. In rabbit hepatocytes a major site of assembly of apo B with TG and phospholipid is also in the Golgi [172] and Golgi membranes have been shown to synthesize phospholipid [ 1731. Phosphatidylethanolamine enrichment appears to be a characteristic of nascent TRL [174]. It is possible that lipoprotein remodeling and transfer of lipid to lipoproteins in exchange for phospholipid takes place throughout intracellular transport both at the level of the ER and Golgi [175] and this idea is supported by rat liver studies suggesting lipid addition to apo B is progressive [92] (Fig. 2~). However, as gradual enlargement of lipoprotein particles during transport from the ER to the Golgi and secretory vesicles has not been clearly demonstrated and the presence of immature precursors of VLDL within the Golgi has been disputed [176] it is likely that observed ‘lipid additions’ occur through lipid exchange mechanisms which do not result in enlargement of particles. Hence, variability in the rate and magnitude of

18

J.D. Sparks, C.E. Sparks/Biochimica

lipid transfers during apo B translation and the size of coalescing lipid droplets during assembly within the ER must be responsible for final TRL size heterogeneity. It is of interest that intravascular lipolysis of TRL B-100 produces a relatively homogenous population of LDL-sized particles suggesting that the B-100 apolipoprotein commands a minimal lipid core the size of LDL for stability. This critical size is achieved twice during the lifetime of the particle: first, during lipoprotein biogenesis [163,177] and second, during TRL catabolism. Underlying differences in assembly of B-100 and B-48 TRL may explain reported variances in cell transit and differences in conclusions regarding sites of lipoprotein assembly for rat vs. rabbit hepatocytes and HepG2 cells. In order for full understanding it will be necessary to determine the nature of the differences in B-100 and B-48 TRL formation, the role of each compartment in TRL modifications and resolution of the mechanism(s) for neutral lipid addition [164]. There is, however, an apparent absolute requirement for MTP early in TRL assembly [51 and therefore MTP gene expression might play a regulatory role in TRL production. MTP mRNA has recently been demonstrated to be increased in liver and intestine of hamsters fed a high fat diet but relatively unaffected by sucrose-feeding or fasting [178]. 3.2. Fatty acid effects on lipoprotein assembly In HepG2 cells B-100 is present either within the ER lumen as lipoproteins of LDL-VLDL and HDL densities or as partially translocated apo B tightly bound to ER membranes [165]. Lumenal lipoproteins are formed when B-100 is cotranslationally associated with lipids [177], a process which may occur simultaneously with translocation. Apo B that fails to fully translocate becomes integrated into membranes where it is sorted and degraded [86,165] and similar conclusions have been drawn in rat liver [82]. It has recently been suggested that partial cotranslational translocation of apo B through the ER membrane may be a discrete step in normal lipoprotein assembly [88]. When full translocation is accomplished lumenal lipoproteins are formed whereas when translocation is delayed too long, integration of apo B with the membrane is favored and degradation ensues [88]. Apo B translocation, therefore, may be the first determinant in regulating the rate of VLDL secretion by hepatocytes. Lumenal B-lOO-containing lipoproteins can progress through the secretory pathway if they have attained a minimum lipid core size relative to the length of the polypeptide chain (i.e., LDLVLDL size) [163,177]. Smaller lumenal lipoproteins of higher density fail to be transported through the secretory pathway [19] and disappear with time from the cell [86,177]. VLDL movement out of the ER therefore is a second determinant in regulating the rate of VLDL secretion by hepatocytes. Regulation at the level of movement into the secretory pathway is supported by studies of

et Biophysics Acta 1215 (1994) 9-32

choline deficiency [Ill] and erotic acid feeding in rats [168]. Mechanisms for retention of apo B within the ER and degradation of unstable lumenal lipoproteins are currently unknown. Oleic acid stimulation of B-100 secretion in HepG2 cells was first described by Rash et al. [179] and has been confirmed [180-1841. Stimulation of B-100 secretion is not a result of an increase in apo B mRNA abundance [185,186] but due to increased utilization of B-100 for lipoprotein assembly [87,89] decreasing intracellular degradation [87,126,184,187]. In HepG2 cells incubated in the absence of oleic acid as much as 40-60% of freshly synthesized B-100 is degraded within the first 60 min of chase [87,89,184,188]. Using monensin, which prevents protein secretion at the level of the trans-Golgi network, early degradation of nascent B-100 still occurs with more apo B reaching the Golgi in the presence of oleic acid suggesting that there is not protection of apo B from ER degradation but rather there is reduced accessibility to endogenous proteolytic enzymes during transport [87]. The oleic acid effect might be due to alteration of apo B conformation associated with stimulated TG assembly [126] and this is consistent with the finding that more nascent B-100 is assembled into mature LDL-VLDL lipoproteins than in cultures without added fatty acid [86]. Little difference is observed in the amount of intracellular turnover of B-100 bound to the ER membrane with oleic acid [86] suggesting that treatment diverts the cell from forming unstable HDL-sized B-100 to formation of more stable LDL-VLDL which are then able to move forward in the secretory pathway [86]. Recent studies indicate that ALLN (N-acetylleucyl-leucyl-norleucinal), a calpain inhibitor, can protect B-100 from intracellular degradation suggesting a calpain-like proteinase may be involved [89]. ALLN, however, does not by itself stimulate B-100 secretion unlike oleic acid indicating that B-100 degradation and lipidfacilitated intracellular transport are distinct but competitive processes [89]. Although oleic acid addition to HepG2 cells stimulates biosynthesis of TG and PC [189] it is likely that at least with respect to its effects on B-100 secretion that oleic acid is acting through stimulation of the synthesis of neutral lipid core components [86]. The theory suggested is that if neutral lipid core availability (TG or CE) is augmented to more closely balance apo B synthesis, apo B secretion can be stimulated. HepG2 cells which lack extensive smooth ER for de novo lipid synthesis have a large imbalance in lipid vs. apo B synthesis, therefore, provision of core precursors such as extracellular FA or induction of CE synthesis leads to substantial increases in apo B production [34,122-124,187]. The magnitude of stimulation of apo B secretion depends on the extent of the initial imbalance which in transformed cells may be high. In fed rats oleic acid addition to primary hepatocytes or liver perfusions does not stimulate secretion of apo B suggesting that the imbalance of apo B and neutral lipid

J.D. Sparks, C.E. Sparks/Biochimica

synthesis is not as large as in HepG2 cells [190-1921. It has been suggested that the ability to ‘lipid load’ apo B and form lipoproteins within the ER may be retained in rat liver without the need of lipid precursors and this ability is limited in HepG2 cells [86]. In contrast to the fed rat, apo B secretion by isolated perfused liver of fasted rats is increased when oleic acid is added to perfusates [192] suggesting that at least in the fasting state an imbalance in apo B and neutral lipid synthesis may develop. Rat VLDL and B-48 secretion is reduced by 50% in fasted rats [193,194] whereas LDL and freshly synthesized B-100 is not [194] or only modestly reduced [193] indicating that fasting affects primarily the production of TRL by liver. Fasted rats have an increased proportion of B-100 mRNA (unedited) [195-1971 which limits translation and secretion to predominantly B-lOO-containing lipoproteins [193,194]. As observed for B-100 in HepG2 cells, there may be more stringent requirements for cotranslational translocation, assembly into VLDL and efficient intracellular transport than for B-48 [73]. Fasting reduces neutral lipid synthesis due to decreased DGAT activity [101,132] and FA synthesis [144] and related enzyme activities [148] and under these circumstances rat B-100 may be more susceptible to intracellular degradation when there are concurrent deficits in neutral lipid synthesis. 3.3. Insulin-stimulated

apolipoprotein

B degradation

An imbalance of apo B synthesis with lipid synthesis is not the only situation which leads to decreased apo B secretion by liver. The presence of insulin in the media of primary rat hepatocytes [8,9,11,12,198] while stimulating TG synthesis [7,9,10,199] inhibits apo B secretion. The reduction in apo B secretion is attributable, in part, to enhanced intracellular degradation of freshly translated apo B, a process which favors degradation of B-100 (50-60% degraded) over B-48 (25-30%) [12]. Some B-48 is always secreted by rat hepatocytes over a longer period during

Table 1 Insulin regulation

1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11. 12.

of hepatic apo B and triacylglycerol-rich

lipoprotein

19

et Biophysics Acta 1215 (1994) 9-32

chase periods because of a larger intracellular pool and movement from this pool appears to be independent of insulin action. Enterocytes which synthesize mostly B-48, therefore, would exhibit less insulin-dependent apo B inhibition than hepatocytes which secrete B-100. The outcome of insulin action in rat hepatocytes is a net reduction in TRL secretion (about 40% of total apo B) and a shift in the proportion of TG carried by B-48. Ample evidence exists in the literature for insulin regulation of hepatic apo B secretion (Table 1). In primary rat hepatocytes incubated with insulin, apo B fragments are present in ER but not Golgi membranes [207] suggesting that fragmentation of apo B occurs in the ER and either the fragments produced are fully degraded in the ER or are sorted and completely degraded before reaching the Golgi. In addition to B-100 and B-48, two distinct apo B peptides are labeled in pulse-chase studies: a 120 kDa and a 60 kDa peptide. The 120 kDa peptide disappears during the chase period at a rate comparable to that seen with intact apo B possibly by elongation with unlabeled amino acid followed by assembly and secretion. The 60 kDa peptide (amino-terminal?) shows little reduction in radioactivity during the chase period suggesting that it is an intermediate of some kind produced either by proteolysis or by delayed translation [207]. Insulin also inhibits apo B secretion by HepG2 cells and, as in rat hepatocytes, there is accumulation of intracellular TG [181,185,186,205]. The magnitude of the insulin effect on inhibition of apo B secretion does not correlate with apo B mRNA abundance [185,186] supporting the idea that the effect of insulin occurs at a post-transcriptional level. In both HepG2 cells and rat hepatocytes insulin effects occur even in the presence of an extracellular supply of oleic acid [11,185,199] suggesting that insulin and oleic acid effects are distinct processes and that insulin-stimulated apo B degradation is not a consequence of reduced FA or TG availability. Moreover, in HepG2 cells, oleic acid treatment actually increases by 3-fold the amount of apo B subject to insulin effects [185].

secretion

Evidence for the pathway

Refs.

VLDL triacylglycerol secretion is inhibited by insulin in the short-term Insulin inhibits apo B secretion by primary hepatocytes The effect on apo B is insulin receptor-mediated and subject to down-regulation Insulin inhibits apo B production by rat liver in perfusion Insulin stimulates degradation of freshly synthesized apo B Insulin inhibits apo B secretion in the presence of oleate and dexamethasone Triacylglycerol production is inhibited by first-phase insulin release Insulin inhibits apo B secretion by HepG2 cells with or without oleic acid addition Apo B mRNA changes do not correlate with changes in B-100 secretion Short-term hyperinsulinemia inhibits production of B-100 in humans Short-term effects of insulin on apo B are lost in long-term cultures in vitro Short-term effects of insulin on apo B are attenuated in long-term hyperinsulinemia

[7,8,200,201] [8,9,12,198,202,203] [ill 11981

1121 [1991 [2041 [181,185,205] [185,1861

in vivo

[2061 [11,186,199] [206] (Sparks, J.D. and Sparks, C.E., unpublished data)

20

J.D. Sparks, C.E. Sparks/Biochimica

3.4. Mechanisms of intracellular protein degradation Proteins that are not folded correctly or fail to assemble into appropriate oligomers are not transported to the cisGolgi and are degraded by a distinct and highly selective, non-lysosomal proteolytic system located in the early secretory pathway. Degradation of intracellular proteins by this pathway is referred to as ‘pre-Golgi’ or ‘ER’ degradation (for reviews see Refs. [208,209]) and degradation can occur with both membrane-bound proteins as well as translocated proteins. Characteristics of ER degradation include a variable lag period (lo-30 min); proteins degraded lack Golgi-associated carbohydrate processing and degradation is temperature and may be energy dependent. At least two species of apo B have been described which undergo intracellular degradation: apo B which is tightly bound to ER membranes and fails to completely translocate [82,165,177] and apo B which forms unstable apo B-containing lipoproteins within the ER lumen [86,177,210]. The mechanism of targeting apo B for degradation and the location and nature of the proteinases involved (cytoplasmic vs. lumenal) are not known. B-100 degradation in HepG2 cells is believed to occur within the ER compartment based on studies using brefeldin A, a fungal metabolite, which blocks ER-to-Golgi transport (reviewed in Ref. [211]) and fails to protect B-100 from intracellular degradation [87,188]. Degradation of B-100 is not related to mixing of Golgi proteins with ER proteins as co-treatment with nocodazole, which prevents retrograde transport of Golgi proteins into the ER, does not prevent degradation [87]. Brefeldin A, however, does inhibit the disappearance of cellular apo B stimulated by insulin suggesting that insulin-dependent apo B degradation may differ from ER degradation observed in HepG2 cells (Sparks, J.D. and Sparks, C.E., unpublished data). Targeting of apo B for intracellular degradation may be mediated by a determinant of apo B which binds to a proteolytic complex or apo B may carry conditional degradation signals that allow sorting to the site of degradative enzymes when activated. Candidate proteinases involved in non-lysosomal, intracellular degradation include: ubiquitin/ATP-dependent proteinase (26S, 1500 kDa also termed ubiquitin-conjugate-degrading enzyme), multicatalytic proteinase complex (MPC, 2OS, 650 kDa, an ATP independent proteinase also termed prosome, proteasome or papain-like promacropain), calpain (Ca2+ -dependent, teinase) and proline endopeptidase. Ubiquitin-dependent proteinase, MPC and calpain are sulfhydryl proteinases whereas proline endopeptidase is a serine proteinase. The major degradation pathway for abnormal, cytosolic proteins is ubiquitin-dependent and targeting involves conjugation of ubiquitin to a protein prior to its degradation (see reviews in Refs. [212,213]). Recent studies have shown that MPC is a subunit of ubiquitin-dependent proteinase [214,215]. MPC (reviewed in Ref. [216]) is associated with nuclei and polyribosomes and has been suggested to be

et Biophysics Acta 1215 (1994) 9-32

involved in post-translational protein modifications, processing and/or protein degradation. MPC exhibits trypsin-like, chymotrypsin-like and peptidylglutamyl-hydrolyzing activities which are able to rapidly degrade targeted proteins to small peptides and amino acids. Both the 265 and 20s proteinases are inhibited by SDS, chymostatin and some thiol reagents. The state of assembly of a protein is an important determinant for degradation as proper assembly may mask conditional degradation signals [217]. The T cell receptor (TCR) is composed of at least seven chains which must be assembled within the ER for transport to the cell surface and unassembled chains are selectively degraded. For the cx and /3 chain of the TCR, the single transmembrane region and the presence of basic amino acids within this region determines retention and rapid degradation in the ER [218,219]. How these charged residues promote retention and favor degradation is not known however, it is suggested that they form charge pairs with the transmembrane domain of a putative recognition protein that is part of the ER retention degradation apparatus [219]. Although apo B does not contain transmembrane domains, two similar degradation targeting sequences on pseudotransmembrane regions of apo B have been identified: one on the amino terminal side of the B-48 junction and one on the carboxyl terminal side [73,82]. Recent studies suggest that the cytoplasmic tail of the TCR (Y chain is also important as the cytoplasmic tail may induce proper conformation of the transmembrane region allowing the targeting sequence to escape recognition [220]. Perhaps the cytoplasmic tail of apo B which fails to completely translocate favors recognition of an intralumenal targeting sequence which leads to degradation. Apo B also contains PEST sequences [15] which are believed to act in other proteins as conditional degradation signals [221,222]. PEST sequences are rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues. PEST sequences are surface loops, potential phosphorylation sites with critical proline residues embedded in uncharged or hydrophobic pockets. Human and rat B-100 contain two PEST sequences [15] located using the PEST-FIND program [222] between residues 22-44 and 3968-3994 surrounding prolines 38 and 3977. PEST sequences may be activated by changes in protein conformation through alterations in lipid environment, protein fatty acylation or phosphorylation and may be revealed by dissociation of masking subunits [221,222] Many PEST sequences including the two contained in B-100 are potential phosphorylation sites for casein kinases as recently defined [223]. It has been suggested that phosphorylation of proteins in PEST regions produce Ca2+ binding sites which increase Ca2+ concentration locally thereby activating calpains [222]. Proline endopeptidase can directly cleave exposed proline residues of PEST sequences. A number of proteins other than TCR chains and apo B have been shown to be selectively degraded and overall

J.D. Sparks, C.E. Sparks/Biochimica

studies suggest that for each protein there are distinctive characteristics for degradation such as energy dependence, proteinases involved and Ca2+ requirements. HMG-CoA reductase, the H2a subunit of the human asialoglycoprotein (ASGP) receptor, secretory immunoglobulin M (sIgM) and apo E all undergo specific intracellular degradation. Regulated degradation of HMG-CoA reductase occurs within the ER when cholesterol or mevalonate levels in cells are high and an important determinant for selectivity is the seventh transmembrane domain [224]. Although degradation of HMG-CoA reductase does not appear to involve ubiquitin [225] MPC may be involved [97]. Complete degradation of the H2a subunit of the ASGR requires two steps [226,227]. After a 30 min lag period, fragmentation of the H2a subunit occurs next to the transmembrane domain within the rough ER [228] which generates a soluble, 35 kDa intermediate within the lumen corresponding to the end of the H2a subunit. Initial cleavage is followed by complete degradation which may entail a membrane trafficking event 12261. Membrane IgM is expressed on the surface of 38C B lymphocytes whereas secretory IgM (sIgM) is rapidly degraded intracellularly [229]. Brefeldin A inhibits the degradation of sIgM indicating that functional export from the ER is a prerequisite for degradation. Calpain inhibitor I strongly inhibits the degradation of sIgM (sIgM) suggesting that the post-ER degradation of sIgM is mediated by a cysteine proteinase and/or a Cazf-dependent proteinase [229]. Recent studies indicate that a portion of newly synthesized apo E is degraded by HepG2 cells [230] and, in contrast to the above mentioned proteins, apo E degradation takes place in a post-Golgi compartment through the action of both cytosolic Cazf-dependent cysteine proteinases and possibly lysosomes [231]. Apo B degradation stimulated by insulin is not prevented by incubation of hepatocytes with leupeptin which inhibits in isolated hepatocytes lysosomal protein degradation by 80-85% [232,233]. Degradation of apo B, however, can be partially inhibited by chymostatiu (38%) which inhibits non-lysosomal degradation by more than 50% [233,234] and lysosomal degradation by about 45% [233]. Chymostatin has also been shown to inhibit the activity of MPC [216] and calpain. Consistent with the possible involvement of cysteine proteinases and/or calpain in insulinstimulated degradation is the finding that degradation is partially inhibited by ALLN (48%) and is almost completely inhibited by EST (92%) (Sparks, J.D. and Sparks, C.E., unpublished data). EST is a membrane-permeant form of E-64 [235,236]. Interestingly, ALLN also inhibits B-100 degradation in HepG2 cells, a process which is thought to occur in the ER compartment [89]. Results of inhibitor studies indicate that insulin-stimulated intracellular apo B degradation most likely involves cysteine proteinases, possibly MPC and/or calpain however, the lack of specificity of inhibitors for non-lysosomal vs. lysosomal enzymes and the observation that brefeldin A attenuates insulin-stimulated degradation of apo B suggests

21

et Biophysics Acta 1215 (1994) 9-32

that involvement of lysosomes entirely ruled out. 3.5. Post-secretory

lipoprotein

in the process

cannot

be

catabolism

Movement of secretory proteins from the ER is governed by the budding and fusion of transport vesicles mediated by different ‘coat’ proteins and the role of GTPbinding proteins in regulating specific events in vesicle formation, budding and targeting has recently been reviewed [237]. In the trans-Golgi network, vesicles are sorted to lysosomes or exit for secretion and in hepatocytes, vesicles containing TRL and HDL move to the sinusoidal surface where they are discharged into the space of Disse by fusion with plasma membranes. In the intestine, secretory vesicles containing CM fuse with enterocyte plasma membranes and are secreted into the spaces between intestinal cells making their way into the lymphatic system. The composition of TRL upon entry into the plasma compartment plays a significant role in its ultimate catabolic fate (reviewed in Ref. [2381X Apo C and E, which are critical to catabolism of TRL, transfer onto the TRL surface from HDL and possibly from hepatocyte plasma membranes. Apo C-II is a required cofactor for the insulin-dependent enzyme, lipoprotein lipase (LPL) which is bound to the endothelium and progressively hydrolyzes TRL core TG. A portion of released FA returns to the liver bound by serum albumin, while the bulk of FA enters tissue to be utilized. Excess FA is re-esterified to TG for storage mostly in adipose tissue. As a consequence of LPL activity, TRL shrink in size and lose apo C protein and phospholipid which move to HDL whereas apo E is retained on the particle. The resulting TRL remnant is smaller and enriched in cholesterol in part because of selective loss of TG. Formation of intermediate density lipoproteins (IDL) (remnants) and TRL remnants which contain B-48 are more rapidly taken up by the liver [166]. The relative role of the LDL receptor vs. other receptors (CM remnant receptor, LDL-receptor related protein) in remnant uptake is probably determined by remnant composition and regulated receptor expression. Factors important to TRL catabolism include apo E and apo C composition, the size of the TRL particle and whether the particle bears B-48. Recent studies using HepG2 cells suggest that reuptake of newly secreted B-100 lipoproteins may regulate net B-100 production by liver [239]. While the extent of post-secretory catabolism of newly secreted TRL particles by hepatocytes is considered to be small, the re-uptake of LDL by HepG2 cells may be more significant and it is possible that part of the stimulatory effect of oleic acid on B-100 secretion may be attributable to secretion of TG-enriched particles which are less ‘recognizable’ by hepatocyte lipoprotein receptors. Editing of apo B mRNA with the resultant secretion of TRL containing B-48 has significant metabolic conse-

22

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quences as TRL B-48 remnants are cleared more rapidly than TRL B-100 and fail to become a precursor of plasma LDL [166]. TRL B-48 does not contain the domain of apo B responsible for binding to the LDL apo B, E-receptor. In humans, edited apo B mRNA is present mainly in intestine although small amounts have been detected in liver [240]. Human liver therefore produces mainly TRL B-100 [179,241] and a significant portion of secreted particles become LDL after entry into the plasma. In contrast, rat liver which edits apo B mRNA, secretes both TRL B-48 and TRL B-100 and consequently as much as two-thirds of secreted TRL (the portion containing B-48) is diverted into lipoproteins having very short plasma half-lives. The finding that hepatic editing corresponds with low levels of plasma apo B-containing lipoproteins [242] suggests that if editing could be stimulated in human liver, lower plasma LDL levels might result.

4. Insulin regulation of triacylglycerol-rich lipoprotein

production Cellular effects of insulin action are diverse including regulation of membrane transport (glucose, amino acids, ions), activation and inactivation of key enzymes in intermediary metabolism, regulation of gene expression, control of levels of cyclic nucleotides, protein synthesis and degradation, and regulation of growth. Many of these effects are believed to involve stimulation of serine kinases and phosphatases which alter phosphorylation of key cellular substrates [243]. This global control by insulin over cellular metabolism is balanced by a variety of other hormones including glucagon, glucocorticoids, growth hormone, catecholamines and vasopressin which provide the environment for hormone signaling by other receptors including the EGF receptor involved in cell growth and differentiation. In terms of lipid metabolism, these interactive regulatory mechanisms allow for rapid modulation of enzymatic activities leading to short-term changes as well as to induction of enzymes producing long-term changes in FA and lipid substrates which impact on lipoprotein synthetic capability. Short-term effects in hepatic lipid metabolism are influenced by the peak of post-prandial portal insulin during first phase insulin release following ingestion of food whereas long-term effects in hepatic lipid metabolism are influenced by insulin balanced by adaptive mechanisms by the cell in response to the metabolic milieu created by hormones and other growth factors. After release by the p-cells of the pancreas, insulin binds to cell surface insulin receptors (IR). The IR is composed of two (Y and two P-subunit glycoproteins linked by disulfide bonds in a heterotetrameric structure. The a-subunits which bind insulin are entirely extracellular (125-135 kDa) while the P-subunits (95 kDa) are transmembrane containing a small extracellular domain, a membrane spanning domain and a carboxyl-terminal intra-

et Biophysics Acta 1215 (1994) 9-32

cellular tail. Within the midportion of each P-subunit is a tyrosine protein kinase activity. Insulin binding by the extracellular a-subunit, through a conformational change, activates the tyrosine kinase in the /?-subunit which phosphorylates itself on tyrosine residues near the membrane, within the catalytic domain and on the carboxyl-terminal tail. The ability of insulin to activate the IR tyrosine kinase is believed to be the critical event for initiation of insulin action [244]. Internalized IR remain catalytically active within cells [245] and may continue to elicit signals during intracellular transport. A recently proposed mechanism suggests that IR signal transduction occurs by multiple tyrosine phosphorylations by the IR of a specific cellular substrate (reviewed in Refs. [245,246]). The tyrosine phosphorylated substrate, (insulin receptor substrate-l, IRS-l) through protein-protein interactions forms specific high affinity physical complexes with cytoplasmic signaling molecules containing Src homology 2 (SH2) domains including phosphatidylinositol 3-kinase, SH-PTP2 (tyrosine phosphatase pathway) and GRB-2 (~21”’ pathway) which then become activated [245,247]. The proposed role of tyrosine phosphorylation is to reveal recognition sites on IRS-l that bind ‘insulin-specific’ cellular signaling proteins and IRS-l is believed essential to insulin action in cells 12451. Dephosphorylation of the IR and IRS-l via phosphotyrosine phosphatases can, in turn, modulate insulin and post-receptor signaling (reviewed in Ref. [248]). In cells the balance between IR, IRS-l and phosphotyrosine phosphatases as well as the presence of the signaling molecules themselves is believed to be important for appropriate insulin sensitivity and imbalances may explain certain insulin resistant states at a molecular level [249]. IRS-l protein levels are differentially expressed in target tissues such as muscle and liver. Both IRS-l and IR can be regulated at the level of transcription, translation and posttranslational levels [249,250]. Insulin action in carbohydrate metabolism has been studied extensively, however, the central role of insulin in regulating lipoprotein metabolism is only beginning to be appreciated [251,252]. Insulin dysregulation of lipoprotein metabolism results in hypertriglyceridemia associated with diabetes mellitus which has been reviewed [253-2551. Insulin action may be divided into short-term (acute) and long-term (chronic) effects and long-term effects can operate in states of hypoinsulinemia as well as hyperinsulinemia in an environment of counterregulation and resistance of tissues to insulin action (insulin resistance). Much of our current understanding of long-term and short-term insulin effects on TRL metabolism is derived from studies of animal models of insulin-dependent and non-insulin-dependent diabetes (reviewed in Ref. [256]). 4.1. Long-term

hypoinsulinemia

Chronic insulin deficiency can be induced in animals by injection of anti-insulin antibodies or by destruction of the

J.D. Sparks, C.E. Sparks/Biochimica

p-cells of the pancreas by specific toxins such as alloxan or streptozotocin (STZ). STZ produces low plasma levels of insulin in rats and livers from chronically insulin-deficient rats secrete less VLDL TG [257-2591. The secretion of TG and incorporation of [1-‘4C]oleic acid into liver perfusates and hepatic TG is also reduced in spontaneously diabetic BB Wistar rats [260]. Consistent with reduced TG secretion is the finding that apo B secretion by liver is also reduced in STZ-treated rats [13,14,198]. Despite reduced hepatic TG production, rats are hypertriglyceridemic and studies suggest that as the duration of insulin deficiency increases, lipoprotein removal by the liver declines and the liver loses its ability to secrete VLDL-TG [257]. Enterocytes are not as severely impaired compared with hepatocytes and the level of hypertriglyceridemia can be accentuated by dietary fat [257]. This suggests that the hyperlipoproteinemia associated with chronic hypoinsulinemia relates to increased intestinal TRL production coupled with defective catabolism of both hepatic and intestinal TRL by liver [13] and by peripheral tissues [261] with accumulation in plasma of TRL and its remnants. The clearance defect has been attributed to insulin-dependent decreases in tissue lipoprotein lipase and altered activity of TRL apo C-II for lipoprotein lipase activation. In STZ rats there is also decreased apo E production by liver [14,262,263] which affects both TRL and CM remnant clearance by hepatic receptors [264-2661. In STZ diabetes the increase in plasma apo B is due mainly to increased B-48 [13,14,261] which can be derived from either liver or intestine of rats. The impairment in hepatic apo B synthesis and secretion in STZ diabetes was recently evaluated to determine at what level the defect occurred. No measurable change in liver apo B mRNA abundance was observed in diabetic liver [14] suggesting that the reduced ability of the liver to synthesize TRL apo B was not due to alterations in gene transcription and/or mRNA stability. Because hepatocytes derived from STZ diabetic rats synthesized predominantly B-48, mRNA editing might have been responsible, however, assay of edited apo B mRNA in liver indicated a similar proportion as that found in control animals. Pulsechase and recovery studies of labeled apo B suggested that increased intracellular apo B degradation was not a major factor in reduced hepatic apo B output [14] however, as virtually no B-100 was synthesized during the 10 min pulse, B-100 recovery and hence degradation could not readily be assessed. Results suggest that the impairment in hepatic apo B production in long-term hypoinsulinemia occurs at the level of apo B translation. Although apo B mRNA polysomes from control and diabetic rats show retarded sedimentation rate [90], polysome profiles were similar indicating that defective apo B mRNA initiation was an unlikely cause for the impairment in apo B synthesis. Apo B peptide elongation, however, was markedly delayed [14] pointing to a defect at this level in long term hypoinsulinemia. Considering that in diabetic rat liver 50%

et Biophysics Acta 1215 (1994) 9-32

23

of the apo B mRNA encodes for B-100 and initiation of B-100 and B-48 synthesis were similar to controls, the finding of low levels of labeled B-100 was surprising suggesting that the deficit in B-100 synthesis is almost complete. It is possible however that B-48-like peptides may be forming from B-100 mRNA as mechanisms besides editing have been demonstrated to produce B-48-like proteins in rat hepatoma cells [267]. Together results suggest that structural features of the apo B mRNA near the editing site may require stabilization for complete ‘read through’ for B-100 synthesis. There may also be deficiencies of critical translation or translocation factors which lead to disruption of the mRNA-ribosome complex resulting in premature release of B-48-like proteins. Proteins have recently been demonstrated to bind apo B mRNA sequences in the immediate vicinity of the editing site [268,269] and the unusual structure of apo B polysomes related to mRNA sequences within this same region [90] suggest the importance of this site for B-100 synthesis. The critical nature of the B-48-B-100 junction for B-100 synthesis is also suggested by studies of human normotriglyceridemic abetalipoproteinemia where only B-48 can be secreted [270]. Studies of long-term hypoinsulinemic diabetic rats suggest that there are insulin-dependent factors which contribute to efficient apo B synthesis and lipoprotein formation. As hepatic FA, PC and TG synthesis are significantly impaired in STZ diabetic rat livers, it is possible that B-100 peptide elongation may be closely coordinated with lipid synthesis and assembly. Intestinal TRL production, however, is not as markedly impaired when rats are fed fat-containing diets [257] possibly because enterocytes obtain required TRL lipid components (lysoPC, FA and monoglycerides) from the diet. Stabilization of nascent B-100 with critical lipid components may favor peptide elongation and translocation of apo B into the ER lumen. Deficiences could delay translation or fail to ‘restart’ apo B translocation to such an extent there is disruption of the polyribosome-mRNA-translocation channel complex. Factors important to reversal of the impairment in hepatic TRL production in diabetes were recently investigated [271]. Three days of culture of hepatocytes derived from diabetic rats were necessary in insulin-free medium containing oleic acid, pyruvate, lactate and dexamethasone for restoration of apo B and TG secretion to control levels. In contrast, only 4 h of culture were required with fully supplemented media plus insulin suggesting insulin-dependent factors are critical and these results support the idea that lipid synthesis and apo B translation are closely coordinated. Whether the rate of apo B peptide elongation is restored first or subsequent to restoration of TG or PC synthesis will be important to investigate. In addition to being hypoinsulinemic, STZ diabetic rats are hyperglucagonemic and it is possible that some or all of the observed effects on hepatic apo B synthesis in STZ diabetes may be mediated by an imbalance of insulin with

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counterregulatory hormones with consequent effects on lipid synthetic pathways. Glucagon inhibiton of TG and PC synthesis can be overcome when FA availability is high, which favors translocation of PPH and CT to ER membranes. As FAs levels are increased in STZ rats due to mobilization of adipose stores, the partitioning by the liver of incoming FAs to P-oxidation and ketone formation vs. reesterification becomes an important determining factor in hepatic TG production. In vivo insulin treatment of STZ diabetic rats restores apo B secretion rates in corresponding hepatocytes and in liver perfusions, however, short-term in vitro insulin treatment alone of hepatocytes or liver is ineffectual [198]. As suggested by recent studies [271] additional in vivo factors may be necessary to reverse the physiological changes observed on hepatic apo B translation and TRL formation in hypoinsulinemic diabetes. The role of glucagon in regulating hepatic TRL secretion and its relationship with insulin and glucocorticoid in lipid metabolism is not completely understood. Glucagon has been demonstrated to inhibit TRL and apo B secretion by hepatocyte cultures [150,199]. The ability of glucagon to suppress TRL secretion persists even after 24 and 48 h of incubation with glucagon [199]. While glucagon inhibits de novo fatty acid synthesis, there is little effect on cholesterol synthesis and esterification of labeled oleic acid into TG. Studies of glucagon action suggest that the transient increase in CAMP elicited by glucagon in hepatocytes activates a mechanism responsible for down-regulating VLDL secretion which persists well after cellular CAMP levels have returned to baseline [199,272]. The glucagon effect on TRL secretion requires activation of the CAMP-dependent protein kinase as Rp-CAMPS, which specifically binds to the regulatory subunit and prevents dissociation and activation of the kinase, reverses the inhibitory effect [272]. Glucagon not only causes intracellular CAMP transients but also causes a rise in cytosolic Ca2+ [273-2761 suggesting the possibility that glucagon effects on TRL secretion might be a combination of both CAMP and Ca2+-dependent processes. The Ca2+-dependent effect on apo B could also be related to depletion of ER Ca2+ stores. Both insulin and glucagon suppress apo B secretion when used in media of primary cultures of rat hepatocytes [199]. This is unusual in that many actions of glucagon are antagonized by insulin and the balance of the two hormones is used to explain many aspects of regulation of key intracellular metabolic pathways. Insulin and glucagon, however, are both growth-potentiating hormones for hepatocytes and act to ‘condition’ cells for division. Some of the observed hormonal effects on hepatic apo B and TRL production may therefore relate to stimulation of hepatocyte growth. In rats subjected to two-thirds partial hepatectomy (PH) to induce regulated hepatocyte regrowth, glucagon and insulin act as comitogens. Hepatocytes derived from rats 3 days following PH secrete significantly less apo B than sham-operated controls with apo B secre-

tory rates returning to control levels when the original size of the liver is restored (B-10 days) [277]. In regrowing hepatocytes insulin inhibits apo B secretion by as much 65% suggesting that insulin-stimulated apo B degradation is functionally active during hepatocyte regrowth. Balance of TRL output with intracellular lipid utilization mediated by insulin and glucagon may be necessary under conditions that require energy and lipid substrates for cell replication and for maintenance of required hepatic functions. 4.2. Short-term

hyperinsulinemia

Insulin release into the portal vein is biphasic with an early burst followed by a progressive increase in insulin secretion whose duration is as long as the stimulus. The liver is a major target for first phase insulin release and resultant high portal insulin levels play an important role in priming the liver for maintenance of carbohydrate and lipid homeostasis. Early studies using rat liver perfusions suggested that insulin stimulated VLDL secretion [278,279] however, the stimulatory effect observed on TG secretion may have related more to glucocorticoid effects [202]. Contrary results have been obtained in humans where insulin administration was demonstrated to decrease hepatic TRL production [200,201]. The short-term effect of high levels of insulin to inhibit hepatic TG secretion has been directly demonstrated using primary rat hepatocyte cultures [7] and this effect (Table 1) has been confirmed by others [8,9,150,151,280]. Short-term inhibition of hepatic TG secretion by insulin has now been demonstrated in vivo in humans [206] and in rats [204]. Insulin also inhibits secretion of apo B by primary rat hepatocytes [8,9,12] in the presence of dexamethasone plus oleic acid [199], in intact rat liver perfusions in the presence of cortisol [198] and in human hepatocytes [203] and in humans in vivo [206]. The effect of insulin is receptor-mediated and subject to control by insulin-receptor down-regulation [ll]. The effect of insulin on lipid and apo B is biphasic [9] and at low levels of insulin, there is stimulation of lipid and apo B synthesis whereas at higher levels of insulin, consistent with post-prandial, portal levels, hepatic apo B and TRL secretion is depressed while lipid synthesis continues to be stimulated. TG accumulates within hepatocytes and cellular apo B is reduced [12] suggesting that insulin does not block secretion of already assembled TRL but occurs earlier in the process of assembly. In insulin-stimulated states, newly synthesized TRL lipid components are readily available for lipoprotein assembly and therefore are unlikely to limit TRL secretion and it is apo B availability that becomes rate determining [12]. Short-term high levels of insulin not only stimulate the degradation of freshly synthesized apo B, but in 16-18 h cultures, insulin reduces apo B synthesis by about 50% [ 121. Recent in vitro cell-free translation studies using HepG2 cells suggest that the translational activity of apo B mRNA is reduced in the

J.D. Sparks, C.E. Sparks/Biochimica et Biophysics Acta I215 (1994) 9-32

presence of insulin [281,282]. Reduced apo B secretion by liver elicited by insulin therefore may be a combination of effects on inhibition of apo B translation and increased apo B degradation [12]. High portal levels of insulin may act to reduce hepatic lipoprotein secretion during periods of peak intestinal fat absorption and formation of intestinal CMs [8,12,15,17] making the liver a temporary storage pool for newly synthesized lipids [ 142,254]. Hepatic lipid storage pools are further expanded during the post-prandial period as there is hepatic uptake of intestinal lipoprotein remnants. In an analogous fashion, high portal insulin levels during first-phase insulin release are hypothesized to signal the ‘fed state’ to the liver, interrupt hepatic gluconeogenesis and to stimulate glycogen storage from incoming nutrients. Overall, the role of short-term high levels of insulin may represent a regulatory mechanism which modulates lipemia during periods of increased hepatic lipogenesis [7,150]. 4.3. Long-term hyperinsulinemia Long-term hyperinsulinemia may result in stimulation of hepatic TRL secretion by altering normal regulatory pathways and it has been demonstrated that the ability of FA to stimulate TG secretion by perfused rat liver depends on ambient plasma insulin levels of the donor rat [283]. In long-term cultures of rat hepatocytes incubated with insulin ( > 24 h) the secretion of TRL TG increases [151] correspondent with the extent of stimulation of TG synthesis [17,284]. When rat hepatocytes are incubated with high levels of insulin plus dexamethasone and oleic acid for 72 h they no longer respond to insulin by inhibiting VLDL

.f

350 -

3 h z 0 F

325 300 -

p

250-

25

apo B secretion [199] suggesting the development of insulin resistance with respect to this pathway. Development of an attenuated response to insulin has also been observed in HepG2 cells [186]. In rat hepatocytes sustained high levels of insulin result in increased secretion of TRL apo B [199]. Insulin also has long-term positive effects on gene transcription of lipogenic enzymes and induces FAS, ACC and the A’-desaturase system. Moreover, insulin has an overall positive influence on general protein synthesis and stimulates the rate of ribosome biogenesis while reducing ribosome degradation [285]. Resistance to the acute inhibitory action of insulin on hepatic apo B secretion may be important in chronic hyperinsulinemic states in vivo as can occur in obesity. The Zucker fatty rat (fa/fa> is hypertriglyceridemic, hyperinsulinemic and insulin resistant with impaired glucose tolerance compensated for by stable endogenous insulin production by the pancreas without hyperglycemia (reviewed in Ref. [256]). Zucker rats are resistant to insulin’s inhibitory effect on hepatic glucose production [286-2881. Insulin resistance at the level of the adipocyte results in the inability to suppress the release of free FA [289] and high FA levels favor increased production and secretion of hepatic VLDL TG. Hepatic TRL production is increased in Zucker fatty rats compared with lean controls [290-2931. While TG secretion by perfused livers derived from Zucker rats is markedly increased, apo B mass secretion per g liver is similar [293] or somewhat reduced compared with lean controls [291]. Differences in reported apo B secretion rates have been attributed to the variable degree of hyperlipidemia of donor Zucker rats [293]. An additional consideration is the shift in production by livers derived from

275 -

125,-

Fig. 3. Effect of insulin on cellular (left panel) and total apo B (right panel) of primary rat hepatocytes derived from lean control and obese Zucker (fa/fa) rats. Primary cultures of hepatocytes were incubated in Waymouth’s medium containing 0.2%, w/v, bovine serum albumin and various insulin concentrations. After 12-14 h the apo B content of cells and media were quantitated by monoclonal radioimmunoassay. Results are the average cellular apo B and total apo B (cells plus media) of hepatocytes from 5-9 lean control (open squares) and 5 Zucker fatty rats (open circles) plotted against the insulin concentration present in the medium at the beginning of the incubation period.

26

J.D. Spark,

C.E. Sparks / Biochimica et Biophysics Acta 1215 (I 994) 9-32

Zucker rats to B-48 containing lipoproteins [293] and the associated problems encountered in apo B mass vs. molar quantitation for B-100 and B-48. Using a monoclonal based radioimmunoassay which measures apo B on a molar basis [294], the effect of insulin on apo B was examined in primary hepatocytes derived from Zucker obese and lean control rats. In control hepatocytes there is a dose-dependent decline of total and cellular apo B with insulin. In hepatocytes derived from Zucker obese rats, insulin does not inhibit apo B secretion or reduce apo B in cells plus media to any great extent (Fig. 3). This result suggests that the insulin inhibitory action on apo B is attenuated in insulin-resistant states (Sparks, J.D. and Sparks, C.E., unpublished data). The attenuation of insulin action on hepatic B-100 production in chronic hyperinsulinemia has recently been demonstrated in humans [206]. Using short-term hyperinsulinemia produced by euglycemic hyperinsulinemic clamps, hepatic B-100 production is decreased by 50% in controls but not in obese individuals with chronic hyperinsulinemia [206]. Analysis of insulin dose-response curves of Zucker fatty rats with respect to apo B inhibition is most consistent with combined receptor and postreceptor defects (decreased insulin sensitivity and responsiveness). It has been suggested that hyperinsulinemia may be involved in regulation of insulin receptors and post-receptor cellular desensitization [295]. The mechanism of IR and postreceptor alterations in several models of altered insulin responsiveness has recently been examined by assessing levels of IRS-l and the phosphorylation state in liver of the receptor and IRS-l after insulin stimulation [249]. In insulin resistant obese mice IR binding, IRS-1 phosphorylation and IRS-l protein levels are reduced and of the models studied, the level of IRS-l protein was inversely related to insulin levels suggesting insulin may play a role in IRS-l expression [249]. The consequences of altered IRS-l function on insulin-mediated post-receptor events related to apo B remain unexplored. Recent studies suggest that insulin may also play a role in stimulating hepatic apo B mRNA editing in rats, and changes in ambient insulin levels correlate with changes in edited apo B message. Hepatic apo B mRNA editing is reduced by fasting [195-1971 and is increased during refeeding high carbohydrate diets [195,197]. In insulintreated STZ diabetic rats there is a 2-fold increase in hepatic apo B mRNA due mainly to an increase in edited message (Sparks, J.D., Sparks, C.E. and Smith, H.C. unpublished data). In support of this hypothesis are studies of Zucker obese rats demonstrating that the ratio of B-48 to B-100 secreted in liver perfusions is 2.8 to 1 in obese rats compared with 1 to 1 in lean controls [293]. There are at least two potential effects of the loss of the acute insulin inhibitory pathway on hepatic TRL apo B production. First, the ability to regulate hepatic TRL during the fed state when insulin levels peak may be compromised leading to hypertriglyceridemia of both hepatic and

intestinal origin during the postprandial period. Catabolism of TRL may become saturated as there is a common removal mechanism for CM and VLDL [296] and this would result in prolonged residence of plasma TRL and its remnants [292,297]. Second, that portion of apo B that would have been degraded by the liver during first phase insulin release is not degraded and the release of TRL by the liver is no longer limited by availability of apo B. TRL apo B secretion then becomes dependent on factors such as the size of the hepatic neutral lipid storage pool [141,142]. Overall, the loss of insulin inhibition results in a net increase in apo B-containing lipoproteins secreted by the liver. This suggestion is supported by the finding that chronically high levels of insulin result in increased TRL production by primary rat hepatocytes [199] and by livers of Zucker obese rats [290,292]. Chronic hyperinsulinemia may also affect liver apo B mRNA levels in vivo. Insulin treatment of STZ diabetic rats which may also produce hyperinsulinemia on a long-term basis has been shown to double apo B mRNA and halve apo A-I mRNA in the liver [14] thus the ability to synthesize apo B-containing lipoproteins may be further augmented whereas that for apo A-I containing lipoproteins is diminished. Hypertriglyceridemia may have additional consequences in carbohydrate metabolism and altered fat metabolism may play a role in development of insulin resistance states and diabetes [251]. The Randle glucose-FA cycle suggests that there is a reciprocal relationship between glucose and FA oxidation [298]. Increased FA oxidation in muscle decreases glucose entry and utilization. The importance of this pathway is supported by the finding that insulin resistance is directly related to accumulation of TG in skeletal muscle [299] and insulin sensitivity improves as muscle TG stores are depleted. As muscle TG is derived from intravascular lipolysis of TRL, increased levels of TRL produced by either liver or intestine or delayed clearance of TRL during the post-prandial period raises plasma free FA levels and favors TG deposition in tissues such as muscle in addition to adipose tissue. The consequent decrease in glucose utilization by muscle results in incremental increases in blood glucose which in turn stimulates increased levels of insulin production by the pancreas and ultimately results in hyperinsulinemia and insulin resistance. FA present in the portal blood can also directly affect insulin clearance by the liver [300] and it is postulated that attenuated hepatic insulin clearance elevates peripheral insulin which may subsequently lead to insulin resistance in peripheral tissues. FAs in physiological concentrations also inhibit insulin binding, degradation and function in isolated hepatocytes [301] and in perfused rat liver [300]. 4.4. Apolipoprotein

B phosphorylation

Apo B phosphorylation has been proposed as a mechanism for short-term effects of insulin on apo B secretion

J.D. Sparks, C.E. Sparks/Biochimica

by hepatocytes [4.5,302]. Phosphorylation of apo B as with other proteins may stabilize a critical protein conformation which can alter biological properties of the protein such as membrane association. For proteins such as HMG-CoA reductase, increased phosphorylation has been associated with accelerated loss of activity and may lead to increased susceptibility to proteolysis [303]. Phosphorylation of hepatocyte secretory proteins such as apo B may also be related to intracellular transport as described for avian vitellogenin [304]. Vitellogenin synthesis is sequentially followed by glycosylation and phosphorylation in a compartment containing high kinase activity immediately prior to secretion. The presence of low levels of intracellular phosphorylated vitellogenins compared with highly phosphorylated secretory vitellogenins suggests that phosphorylation is associated with rapid secretion from hepatocytes. Rat B-48 is secreted from hepatocytes as a phosphoserine-containing protein [44,45] which is rapidly dephosphorylated after entry into plasma. In studies employing hepatocytes derived from control and diabetic rats, phosphorylation of B-100 in addition to B-48 has been demonstrated and apo B was shown to contain both phosphoserine and phosphotyrosine residues [45]. Initial studies showing only B-48 phosphorylation [44] used hepatocytes cultured in medium containing high levels of insulin in which almost two-thirds of B-100 were later demonstrated to be degraded [160]. As incubation of hepatocytes with insulin increases intracellular degradation of apo B and cellular apo B levels are reduced [12], apo B phosphorylation was hypothesized to increase apo B susceptibility to intracellular degradation [302]. In hepatocytes cultured under basal insulin conditions, both B-48 and B-100 phosphorylated forms could be demonstrated [45]. The effect of protein phosphorylation on the synthesis and secretion of apo B by Caco-2 cells has recently been investigated [305] using okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A. B-48 was phosphorylated in Caco-2 cells and incorporation of 32P was significantly increased by okadaic acid indicating serine phosphorylation of apo B also occurs in the intestinal secretory pathway. The effect of insulin on phosphorylation of apo B has been preliminarily evaluated in primary rat hepatocyte cultures [302]. Insulin enhances the phosphorylation state of intracellular apo B and apo B-related peptides with molecular weights smaller than 160 kDa as well as secretory B-48 and results suggest that phosphorylated cellular apo B peptides smaller than intact apo B may be translational intermediates or intermediates of apo B fragmentation. Insulin has recently been shown to decrease 32Plabeled cellular B-100 in rat hepatocyte suspensions with less of an effect on B-48 [306]. These results are consistent with the proposed hypothesis that 32P-labeled B-100 degradation is stimulated with insulin [302] however apo B specific activity in cells was not measured nor were phos-

et Biophysics Acta 1215 (1994) 9-32

27

phorylated apo B peptides analyzed. The role of apo B phosphorylation in insulin action remains to be established, however, two potential effects are suggested for B-100 [302,306] and for B-48 [44,45,305]. Early in the secretory pathway preferential B-100 degradation may be favored by phosphorylation whereas later, presecretory phosphorylation of B-48 occurs immediately prior to movement of TRL B-48 into the plasma compartment as occurs with vitellogenin. The number of phosphorylation sites on apo B, the specific kinases involved and the nature of hormonal effects on apo B phosphorylation remain to be explored. 5. Summary This review has considered a number of observations obtained from studies of insulin in perfused liver, hepatocytes, transformed liver cells and in vivo and each of the experimental systems offers advantages. The evaluation of insulin effects on component lipid synthesis suggests that overall, lipid synthesis is positively influenced by insulin. Short-term high levels of insulin through stimulation of intracellular degradation of freshly translated apo B and effects on synthesis limit the ability of hepatocytes to form and secrete TRL. The intracellular site of apo B degradation may involve membrane-bound apo B, cytoplasmic apo B and apo B which has entered the ER lumen. How insulin favors intracellular apo B degradation is not known. An area of recent investigation is in insulin-stimulated phosphorylation of intracellular substrates such as IRS-l which activates insulin specific cellular signaling molecules [245]. Candidate molecules to study insulin action on apo B include IRS-l and SHZcontaining signaling molecules. Insulin dysregulation in carbohydrate metabolism occurs in non-insulin-dependent diabetes mellitus due to an imbalance between insulin sensitivity of tissue and pancreatic insulin secretion (reviewed in Refs. [307,308]). Insulin resistance in the liver results in the inability to suppress hepatic glucose production; in muscle, in impaired glucose uptake and oxidation and in adipose tissue, in the inability to suppress release of free FA. This lack of appropriate sensitivity towards insulin action leads to hyperglycemia which in turn stimulates compensatory insulin secretion by the pancreas leading to hyperinsulinemia. Ultimately, there may be failure of the pancreas to fully compensate, hyperglycemia worsens and diabetes develops. The etiology of insulin resistance is being intensively studied for the primary defect may be over secretion of insulin by the pancreas or tissue insulin resistance and both of these defects may be genetically predetermined. We suggest that, in addition to effects in carbohydrate metabolism, insulin resistance in liver results in the inability of first phase insulin to suppress hepatic TRL production which results in hypertriglyceridemia leading to high levels of plasma FA which accentuate insulin resistance in other target organs.

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As recently reviewed [17,254] the role of insulin as a stimulator of hepatic lipogenesis and TRL production has been long established. Several lines of evidence support that insulin is stimulatory to the production of hepatic TRL in vivo. First, population based studies support a positive relationship between plasma insulin and total TG and VLDL [253]. Second, there is a strong association between chronic hyperinsulinemia and VLDL overproduction [309]. Third, insulin stimulates hepatic lipogenic enzymes both in terms of activity as well as in the long-term through induction of enzyme synthesis (reviewed in Ref. [310]). Consistent with the idea insulin stimulates TRL production are studies of insulin deficiency states which clearly demonstrate a necessary role of insulin for efficient apo B translation and TRL formation [13,14,198]. These lines of evidence contrast with other studies indicating that shortterm, hyperinsulinemia inhibits hepatic TRL production by making apo B rate-determining for TRL assembly (Table 1). In humans and in rats chronic hyperinsulinemia results in loss of the acute inhibitory pathway [199,206] and the loss of this pathway can lead to abnormalities in hepatic TRL during the post-prandial period [311]. Hypertriglyceridemia and increased FA in plasma may influence insulin sensitivity of target organs and may be important in development of insulin resistance and diabetes in humans [251]. If chronic hyperinsulinemia can alter hepatic apo B gene transcription [14] TRL production by the liver might be further augmented. Insulin dysregulation of hepatic TRL production may be a possible explanation for the positive relationship between hyperinsulinemia and hypertriglyceridemia observed in humans. Insulin resistance as it affects lipid metabolism is proposed to result from the disruption of coordination of postprandial lipid metabolism by insulin [289]. The resultant increased plasma TRL TG and free FA further affect hepatic and nonhepatic metabolic pathways [251]. Hypertriglyceridemia can also alter HDL and LDL (small dense LDL) creating, in combination with hypertension, Syndrome X (insulin resistance syndrome) [312]. Syndrome X is a highly atherogenic state associated with insulin resistance and with non-insulin-dependent diabetes. The relationship of postprandial hypertriglyceridemia and atherogenesis was first proposed by Zilversmit [313] and this hypothesis may have particular application in the area of diabetes-related accelerated atherosclerosis. This review has attempted to place some recent studies and preliminary findings into perspective and heavily emphasizes the need for further research into the inter-relationships of insulin, apo B, lipid and lipoprotein pathways.

Acknowledgements Research in the authors’ laboratories was supported by HL29837 from the National Heart, Lung and Blood Institute of the National Institutes of Health and the Council For Tobacco Research-USA. The authors would like to

acknowledge G.F. Gibbons, D.N. Brindley, Z. Yao, D.E. Vance and J.E. Vance for helpful comments and suggestions during preparation of this manuscript.

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