The clinical potential of adipogenesis and obesity-related microRNAs.

Obesity is a growing health problem commonly associated with numerous metabolic disorders including type 2 diabetes, hypertension, cardiovascular disease, and some forms of cancer. The burden of obesity and associated cardiometabolic diseases are believed to arise through complex interplay between genetics and epigenetics predisposition, nutrition, environment, and lifestyle. However, the molecular basis and the repertoire of obesity-affecting factors are still unknown. Emerging evidence is connecting microRNAs (miRNAs) dysregulation with adipogenesis and obesity. Alteration in miRNAs expression could result in changes in the pattern of genes controlling a range of biological processes including inflammation, lipid metabolism, insulin resistance and adipogenesis. Hence, understanding exact roles of miRNAs as well as the degree of their contribution to the regulation of adipogenesis and fat cell development in obesity would provide new therapeutic targets for the development of novel and effective anti-obesity drugs. The objective of the current review is to: (i) discuss some of the latest development on relevant miRNAs dysregulation mainly in human adipogenesis and obesity, (ii) emphasize the role of circulating miRNAs as new promising therapeutics and attractive potential biomarkers for treating obesity and associated risk factor diseases, (iii) describe how dietary factors may influence obesity through modulation of miRNAs expression, (iv) highlight some of the actual limitations to the promise of miRNAs as novel therapeutics as well as to their translation for the benefit of patients, and finally (v) provide recommendations for future research on miRNA-based therapeutics that could lead to a breakthrough in the treatment of obesity and its associated pathologies.

Stimulating effect of growth hormone on type IV collagen production by endothelial cells cultured in normal and high glucose.

Collagen IV accumulation is characteristic of diabetic angiopathy. To test the possible contribution of GH, we studied its effects on collagen IV production by human umbilical vein endothelial cells at 5.5 and 16.7 mmol/l glucose. GH (100 ng/ml) markedly increased collagen IV level in the culture supernatant and in the insoluble extracellular matrix and cell fraction at both glucose concentrations. This stimulating effect of GH was additional to that of high glucose. It was more pronounced on collagen IV than on total protein synthesis. GH increased free latent gelatinase activity slightly at normal and markedly at high glucose. Using GF109203X, a PKC inhibitor, we observed that high glucose, but not GH, activated PKC. These two factors stimulating collagen IV production appear to work through different pathways, favoring an additivity of their effects. This supports the contribution of high plasma GH in diabetic vascular basement membrane thickening.

Binding of microsomal triglyceride transfer protein to lipids results in increased affinity for apolipoprotein B: evidence for stable microsomal MTP-lipid complexes.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are known to interact with each other. We evaluated the effect of different lipids on the protein-protein interactions between MTP and apoB100 or its C-terminally truncated forms. Negatively charged lipids decreased protein-protein interactions between apoB and MTP. In contrast, zwitterionic phospholipids enhanced (2-4-fold) the binding of apoB100 to MTP by increasing affinity (1.5-3-fold) between these proteins without affecting the number of binding sites. Similarly, phospholipids augmented (1.5-4-fold) the binding of various C-terminally truncated apoB peptides to MTP. The increased binding was greater for apoB peptides containing lipid-binding domains, such as apoB28 and apoB42. Surprisingly, preincubation of apoB28 with lipid vesicles had no effect on MTP binding. In contrast, incubation of MTP with lipid vesicles resulted in a stable association of MTP with vesicles, and MTP-lipid vesicles bound better (5-fold increase) to LDL than did lipid-free MTP. To determine whether MTP exists stably associated with lipids in cells, microsomal contents from COS cells expressing MTP, HepG2 cells, and mouse liver were ultracentrifuged, and MTP was visualized in different density fractions. MTP was found associated and unassociated with lipids. In contrast, apoB17 and apoB:270-570 were present unassociated with lipids in COS cells. These studies show that the binding of MTP to lipids results in increased affinity for apoB and that stable MTP-lipid complexes exist in the lumen of the endoplasmic reticulum. Protein-protein interactions between apoB and MTP may juxtapose lipids associated with MTP to lipid-binding domains of apoB and facilitate hydrophobic interactions leading to enhance affinity. We speculate that MTP-lipid complexes may serve as nuclei to form "primordial lipoproteins" and may also play a role in the bulk addition of lipids during the "core expansion" of these lipoproteins.

Decreased secretion of ApoB follows inhibition of ApoB-MTP binding by a novel antagonist.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly of triglyceride-rich lipoproteins. Evidence has been presented for physical interactions between these proteins. To study the importance of apoB-MTP binding in apoB secretion, we have identified a compound, AGI-S17, that inhibited (60-70% at 40 microM) the binding of various apoB peptides to MTP but not to an anti-apoB monoclonal antibody, 1D1, whose epitope overlaps with an MTP binding site in apoB. AGI-S17 had no significant effect on the lipid transfer activity of the purified MTP. In contrast, another antagonist, BMS-200150, did not affect apoB-MTP binding but inhibited MTP's lipid transfer activity. The differential effects of these inhibitors suggest two functionally independent, apoB binding and lipid transfer, domains in MTP. AGI-S17 was then used to study its effect on the lipid transfer and apoB binding activities of MTP in HepG2 cells. AGI-S17 had no effect on cellular lipid transfer activities, but it inhibited coimmunoprecipitation of apoB with MTP. These studies indicate that AGI-S17 inhibits apoB-MTP binding but has no effect on MTP's lipid transfer activity. Experiments were then performed to study the effect of inhibition of apoB-MTP binding on apoB secretion in HepG2 cells. AGI-S17 (40 microM) did not affect cell protein levels but decreased the total mass of apoB secreted by 70-85%. Similarly, AGI-S17 inhibited the secretion of nascent apoB by 60-80%, but did not affect albumin secretion. These studies indicate that AGI-S17 decreases apoB secretion most likely by inhibiting apoB-MTP interactions. Thus, the binding of MTP to apoB may be important for the assembly and secretion of apoB-containing lipoproteins and can be a potential target for the development of lipid-lowering drugs. It is proposed that the apoB binding may represent MTP's chaperone activity that assists in the transfer from the membrane to the lumen of the endoplasmic reticulum and in the net lipidation of nascent apoB, and may be essential for lipoprotein assembly and secretion.

Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B.

The microsomal triglyceride transfer protein (MTP) is essential for the hepatic secretion of apolipoprotein (apo) B-containing lipoproteins. Previous studies have indicated that inhibition of MTP results in decreased apoB plasma levels and decreased hepatic triglyceride secretion. However, the metabolic effects of overexpression of MTP have not been investigated. We constructed a recombinant adenovirus expressing MTP (AdhMTP) and used it to assess the effects of hepatic overexpression of MTP in mice. Injection of AdhMTP into C57BL/6 mice resulted in a 3-fold increase in hepatic microsomal triglyceride transfer activity compared to mice injected with Adnull. On day 4 after virus injection, AdhMTP-injected mice had significantly elevated plasma TG levels as compared to control virus (Adnull)-injected mice. Hepatic TG secretion rates were significantly greater in AdhMTP-injected mice (184 +/- 12 mg/kg/h) compared with Adnull-injected mice (65 +/- 9 mg/kg/h, P < 0.001). In addition, hepatic very low density lipoprotein (VLDL) apoB secretion in the AdhMTP-injected group was 74% higher than in the control virus group. Hepatic secretion of apoB-48 and apoB-100 contributed equally to this increase. These results provide the first data that hepatic overexpression of MTP results in increased secretion of VLDL-triglycerides as well as VLDL-apoB in vivo. These results suggest that MTP is rate-limiting for VLDL apoB secretion in wild-type mice under basal chow-fed conditions.

The mammalian low-density lipoprotein receptor family.

The low-density lipoprotein (LDL) receptor (LDL-R) family consists of cell-surface receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. The LDL-R is the prototype of this family, which also contains very-low-density lipoprotein receptors (VLDL-R), apolipoprotein E receptor 2, LRP, and megalin. The family members contain four major structural modules: the cysteine-rich complement-type repeats, epidermal growth factor precursor-like repeats, a transmembrane domain, and a cytoplasmic domain. Each structural module serves distinct and important functions. These receptors bind several structurally dissimilar ligands. It is proposed that instead of a primary sequence, positive electrostatic potential in different ligands constitutes a receptor binding domain. This family of receptors plays crucial roles in various physiologic functions. LDL-R plays an important role in cholesterol homeostasis. Mutations cause familial hypercholesterolemia and premature coronary artery disease. LDL-R-related protein plays an important role in the clearance of plasma-activated alpha 2-macroglobulin and apolipoprotein E-enriched lipoproteins. It is essential for fetal development and has been associated with Alzheimer's disease. Megalin is the major receptor in absorptive epithelial cells of the proximal tubules and an antigenic determinant for Heymann nephritis in rats. Mutations in a chicken homolog of VLDL-R cause female sterility and premature atherosclerosis. This receptor is not expressed in liver tissue; however, transgenic expression of VLDL-R in liver corrects hypercholesterolemia in experiment animals, which suggests that it can be a candidate for gene therapy for various hyperlipidemias. The functional importance of individual receptors may lie in their differential tissue expression. The regulation of expression of these receptors occurs at the transcriptional level. Expression of the LDL-R is regulated by intracellular sterol levels involving novel membrane-bound transcription factors. Other members of the family are not regulated by sterols. All the members are, however, regulated by hormones and growth factors, but the mechanisms of regulation by hormones have not been elucidated. Studies of these receptors have provided important insights into receptor structure-function and mechanisms of ligand removal and catabolism. It is anticipated that increased knowledge about the LDL-R family members will open new avenues for the treatment of many disorders.

Tetradecylthioacetic acid (a 3-thia fatty acid) impairs secretion of oleic acid-induced triacylglycerol-rich lipoproteins in CaCo-2 cells.

The fatty acid analogue tetradecylthioacetic acid (TTA) has previously been shown to decrease triacylglycerol secretion in CaCo-2 cells (Gedde-Dahl et al., J. Lipid Res. 36 (1995) 535-543). The present study was designed to further elucidate the effect of TTA on lipoprotein production in CaCo-2 cells. TTA did not affect oleic acid-induced triacylglycerol synthesis, but it significantly decreased secretion of newly synthesized triacylglycerol when compared to cells incubated with oleic acid alone or oleic acid in combination with palmitic acid. In contrast, pulse-chase experiments showed no difference in the amount of labeled triacylglycerol secreted from cells exposed to either fatty acid combination during the chase period, indicating that TTA did not affect the secretory process in general. Cells incubated with TTA alone secreted triacylglycerol present at 1.025

Amino acids 430-570 in apolipoprotein B are critical for its binding to microsomal triglyceride transfer protein.

Several studies have demonstrated protein-protein interactions between microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). However, the binding sites involved in these interactions have not been elucidated. To identify an MTP binding site in apoB, we have expressed several apoB sequences as fusion proteins with the eight-amino acid FLAG peptide. The chimeras were transiently expressed in COS cells, and conditioned media were used to study the binding of these sequences to either immobilized or soluble MTP. A polypeptide containing amino acids 270-570 (B:270-570), but not 1-300, bound to MTP. AGI-S17, an antagonist of apoB-MTP binding, inhibited the binding of B:270-570 to MTP but not to M2, a monoclonal antibody that recognizes the FLAG peptide. These data indicated that B:270-570 contains an MTP binding site. Next, sequences within 270-570 were subjected to C-terminal truncations at natural proline residues. B:270-509 bound less efficiently than B:270-570, whereas, B:270-430 and other shorter chimeras did not bind to MTP. Furthermore, truncations at amino acids 502 and 509 decreased MTP binding by 73 and 42%, respectively. These data indicate that B:430-570 in the alpha1-globular domain of apoB plays a crucial role in MTP binding and presumably in the initiation and maturation of apoB-containing lipoproteins.

Lysophosphatidylcholine increases apolipoprotein B secretion by enhancing lipid synthesis and decreasing its intracellular degradation in HepG2 cells.

Free fatty acids and lysophosphatidylcholine (lysoPC) are the major lipids bound to human plasma albumin. The effects of fatty acids on the hepatic production of Apolipoprotein B (apo B) have been studied but those of lysoPC have not. In HepG2 cells, lysoPC increased apo B secretion in different experiments by 50-120%, but did not affect the flotation properties of secreted lipoproteins. LysoPC affected neither the cellular protein levels nor apo A-I secretion suggesting that its effect was specific to apo B. Apo B secretion was maximum after incubating cells for 6 h with 0.2 mM lysoPC as equimolar fatty acid free bovine serum albumin (BSA) complexes. LysoPC was metabolized by cells and its fatty acids were used for the synthesis of phosphatidylcholine and triglycerides (TG). Experiments were performed to understand the mechanism of lysoPC action. LysoPC increased the incorporation of 3H-glycerol into newly synthesized cellular (3-fold) and secreted (4-fold) triglycerides, and increased the synthesis (40%) and secretion (4-fold) of phospholipids. LysoPC did not affect apo B synthesis, but inhibited the intracellular degradation of apo B and increased its secretion. Triacsin C (5 microM), an inhibitor of long chain acyl-CoA synthase, completely inhibited the induction of lipid synthesis and abolished the effect of lysoPC on apo B secretion. These studies indicated that lysoPC increased apo B secretion by inducing lipid synthesis; newly synthesized lipids probably protected apo B from intracellular degradation and enhanced secretion. These studies are consistent with the hypothesis that physiologic concentrations of lysoPC can be an important modulator for hepatic apo B secretion.

Lysine and arginine residues in the N-terminal 18% of apolipoprotein B are critical for its binding to microsomal triglyceride transfer protein.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly and secretion of triglyceride-rich lipoproteins. We have presented evidence for a high-affinity interaction between these proteins [Hussain, M. M., et al. (1997) Biochemistry 36, 13060-13067]. In this study, we used chemically modified low-density lipoproteins (LDL) and recombinant human apoB18 to identify amino acid residues in apoB that are critical for its interactions with MTP. Acetoacetylation of 74% of lysine residues and cyclohexanedione modification of 54% of arginine residues completely abolished the interactions between LDL and MTP. Regeneration of lysine and arginine residues by hydroxylamine treatment completely restored the binding of modified LDL to MTP. Carboxyethylation of all the histidine residues decreased, but did not abolish, apoB-MTP interactions. In contrast, glycine methyl ester modifications of aspartic and glutamic acid residues, up to 38-44%, had no effect on LDL-MTP interactions. Furthermore, modification of lysine and arginine, but not the aspartic and glutamic acid, residues in apoB18 also completely abolished its interactions with MTP. These studies indicated that lysine and arginine, but not aspartic and glutamic acid, residues are critical for apoB-MTP interactions, whereas histidine residues are not as critical. Since lysine and arginine residues in apoB are known to interact with the LDL receptors and heparin, we studied the effect of different glycosaminoglycans on apoB-MTP interactions. Glycosaminoglycans had no significant inhibitory effect on apoB-MTP interactions, suggesting that the lysine and arginine residues crucial for apoB-MTP interactions are different from those that interact with the LDL receptor and heparin. The lysine and arginine residues in apoB18 may directly interact with negatively charged residues in the MTP molecule, or they may function to maintain the conformation of the recognition site.

Assembly and secretion of VLDL in nondifferentiated Caco-2 cells stably transfected with human recombinant ApoB48 cDNA.

Intestinal cells secrete apoB48-containing very low density lipoproteins (VLDLs) and chylomicrons for the transport of biliary and dietary lipids. The molecular mechanisms regulating the assembly of intestinal lipoproteins are not known due to a lack of reliable and specific cell culture models. Caco-2 (a human colon carcinoma) cells have been used to study intestinal lipid metabolism. These cells have been shown to secrete both apoB100- and apoB48-containing triglyceride (TG)-rich lipoproteins only after differentiation into enterocyte-like cells. To study lipoprotein assembly in nondifferentiated Caco-2 cells, we stably expressed human recombinant apoB48 cDNA under the control of a constitutive cytomegalovirus promoter. Pulse-chase analysis revealed that the majority (> 50%) of apoB48 synthesized was degraded intracellularly in the presence or absence of oleic acid. Transfected nondifferentiated cells secreted lipoproteins with flotation densities similar to those of plasma HDL or LDL when cultured in serum-free or serum-containing media, respectively. Incubation of cells with media containing serum and oleic acid resulted in the secretion of VLDL-like particles. Secretion of VLDL was inhibited (> 80%) by triacsin C due to > 60% inhibition of oleate-induced TG synthesis. However, inhibition of cholesteryl ester synthesis by 70% with an acyl coenzyme A:cholesterol acyltransferase inhibitor did not affect VLDL secretion. Efficient assembly of lipoproteins usually requires the microsomal TG transfer protein (MTP). The presence of MTP in transfected Caco-2 cells was investigated by measuring TG transfer activity in microsomal fractions. Microsomal fractions had 0.2% TG transfer activity per hour per microgram of protein, which corresponds to 30% to 60% of the MTP activity present in liver-derived cells. To determine whether MTP activity was required for lipoprotein assembly, transfected cells were incubated in the presence of the MTP inhibitor CP-10,447. This compound completely abolished the secretion of apoB. These data show that the transfected cell lines secrete lipoproteins of different densities under different culture conditions and that the assembly of larger VLDL particles requires active TG synthesis and MTP activity. Thus, in nondifferentiated Caco-2 cells, the amount of apoB secreted and not the MTP activity is the limiting factor for lipoprotein assembly.

Measurement of apolipoprotein B in various cell lines: correlation between intracellular levels and rates of secretion.

We have standardized simple but sensitive enzyme-linked immunoassays to understand a relationship between intracellular levels and secretion rates of apoB. The assays were based on commercially available antibodies and were specific to human apoB. A monoclonal antibody, 1D1, was immobilized on microtiter wells and incubated with different amounts of low density lipoproteins to obtain a standard curve. Conditioned media were added to other wells in parallel, and the amount of apoB was quantitated from a linear regression curve. To standardize conditions for the measurement of intracellular apoB, cells were homogenized and solubilized with different concentrations of taurocholate. We found that 0.5% taurocholate was sufficient to solubilize all the apoB in HepG2, Caco-2, and McA-RH7777 cells. Next, a standard curve was prepared in the presence of taurocholate and used to determine intracellular levels of apoB in different cell lines. The intracellular levels (pmol/mg cell protein) and the rates of secretion (pmol/mg/h) of apoB100 were positively correlated (r2 = 0.81, P = 0.0009) in HepG2 cells. Furthermore, a positive correlation (r2 = 0.88, P < 0.0001) was found between intracellular and secreted apoB42 in stably transfected McA-RH7777 cells. In contrast, no correlation was observed for human apoB28 and apoB18 in stably transfected cells that were secreted either partially associated or completely unassociated with lipoproteins. These studies indicated that the rate of secretion of lipid-associated apoB, but not the lipid-free apoB, was tightly controlled.

Apolipoprotein B binding to microsomal triglyceride transfer protein decreases with increases in length and lipidation: implications in lipoprotein biosynthesis.

Microsomal triglyceride transfer protein (MTP), a heterodimer of 97 kDa and protein disulfide isomerase, is required for the assembly of apolipoprotein B (apoB)-containing triglyceride-rich lipoproteins. These proteins have been shown to interact with each other during early stages of lipoprotein biosynthesis. Our studies indicated that binding between apoB and heterodimeric MTP was of high affinity (Kd 10-30 nM) due to ionic interactions. In contrast to MTP, protein disulfide isomerase alone interacted very poorly with lipoproteins, indicating the importance of the heterodimer in these bindings. Preincubation of lipoproteins with detergents enhanced their interaction with MTP. Native VLDL bound poorly to MTP, but its preincubation with Tween-20 resulted in significantly increased binding to MTP. Furthermore, binding of LDL was enhanced by preincubation with taurocholate, indicating that partial delipidation of apoB-containing lipoproteins results in increased binding to MTP. Subsequently, attempts were made to study interactions between C-terminally truncated apoB polypeptides and MTP. Binding of all the polypeptides to MTP was enhanced in the presence of taurocholate. Comparisons revealed that the binding of different apoB polypeptides to MTP was in the order of apoB18 > apoB28 > apoB42 > apoB100. These studies indicated that optimum interactions occur between apoB18 and MTP, and that the increase in apoB length beyond apoB18 has a negative effect on these interactions. Since apoB18 does not assemble triglyceride-rich lipoproteins, these studies suggest that apoB may interact with MTP before its lipidation. It is proposed that steps in lipoprotein biosynthesis may be dictated by the sequential display of different functional domains on the apoB polypeptide.

Effect of an aldose reductase inhibitor on type IV collagen production by human endothelial cells cultured in high glucose.

Diabetic microangiopathy is characterized by a thickening of capillary basement membranes associated with type IV collagen accumulation. An increase in type IV collagen content of the aortic wall is also observed in macroangiopathy. In order to analyse the importance of the polyol pathway in the development of the collagen metabolism alterations seen in diabetic angiopathy and their prevention by aldose reductase inhibitors, we have studied the effects of sorbinil on the high glucose-induced stimulation of type IV collagen biosynthesis in human umbilical vein endothelial cells. Primary cultures were exposed to high glucose (16.7 mmol/l), with and without 0.11 mmol/l sorbinil, for 3 or 6 days after beginning of confluence. We measured the soluble type IV collagen secreted into the culture medium and the insoluble type IV collagen accumulated in the extracellular matrix and cells, by ELISA. We also studied [14C]proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. High glucose decreased the number of cells and increased the amount of type IV collagen in the culture supernatant and in the extracellular matrix and cell fraction. It also increased proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. Sorbinil corrected all these high glucose-induced alterations. The corrective effects of sorbinil on the proliferation and on type IV collagen metabolism of endothelial cells cultured in high glucose may be attributed to prevention of polyol pathway dysregulation.

Chylomicron assembly and catabolism: role of apolipoproteins and receptors.

Chylomicrons are lipoproteins synthesized exclusively by the intestine to transport dietary fat and fat-soluble vitamins. Synthesis of apoB48, a translational product of the apob gene, is required for the assembly of chylomicrons. The apob gene transcription in the intestine results in 14 and 7 kb mRNAs. These mRNAs are post-transcriptionally edited creating a stop codon. The edited mRNAs chylomicrons from the shorter apoB48 peptide remains to be elucidated. In addition, the roles of proteins involved in the assembly pathway, e.g. apobec-1, MTP and apoA-IV, needs to be studied. Cloning of enzymes involved in the intestinal biosynthesis of triglycerides will be crucial to fully appreciate the assembly of chylomicrons. There is a need for cell culture and transgenic animal models that can be used for intestinal lipoprotein assembly. The catabolism of chylomicrons is far more complex and efficient than the catabolism of VLDL. Even though the major steps involved in the catabolism of chylomicrons are now known, the determinants for apolipoprotein exchange, processing of remnants in the space of Disse, as well as the mechanism of uptake of these particles by extra-hepatic tissue needs further exploration.

Production of type IV collagen and 72-kDa gelatinase by human endothelial cells cultured in high glucose. Effects of a protein kinase C inhibitor, GF 109203X.

Since diabetic microangiopathy and macroangiopathy are characterized by type IV collagen accumulation in vascular basement membranes, it was of interest to study type IV collagen production and type IV collagenase secretion by endothelial cells (EC) cultured in high glucose and to evaluate the role of protein kinase C (PKC) activation in the alterations induced by high glucose. Primary cultures of human umbilical vein EC were exposed to high glucose concentration for 3 days at the beginning of confluence. The number of EC decreased with glucose concentration from 5 to 50 mM. At 16.7 mM glucose concentration, the amount of type IV collagen, determined by a two-step ELISA, increased in the culture supernatant and in the insoluble fraction associated with the extracellular matrix and cells; proline incorporation was more markedly elevated in the collagenous than in the total proteins of the culture supernatant and of the extracellular matrix and cell extracts. Gelatin zymography of the culture supernatant showed that EC mainly produce a 72-kDa gelatinase known to degrade type IV collagen. At 16.7 mM glucose concentration, total gelatinase activity per millilitre of culture supernatant was reduced and the 72-kDa gelatinase activity measured on the zymogram scan was lowered. When EC were exposed to 16.7 mM glucose, the specific PKC inhibitor GF 109203X corrected the increases in type IV collagen concentration and in proline incorporation into the collagenous or total proteins present in he culture supernatant or in the extract of the insoluble fraction, including the extracellular matrix and cells. Our results show that soluble and insoluble type IV collagen accumulation by EC cultured at high glucose concentration is not only associated with increased synthesis of the collagenous and total proteins but also with decreased total 72-kDa gelatinase activity in the extracellular fluid. The observed effects of GF 109203X are in favor of the involvement of PKC activation in the type IV collagen accumulation.

[Changes in collagen type IV metabolism in diabetes]. / Etude des altérations du métabolisme du collagène de type IV au cours du diabète.

In Diabetes Mellitus, type IV collagen biosynthesis is increased: the alpha 1(IV) procollagen specific mRNA concentration is elevated, particularly in the kidney, and the type IV collagen protein is accumulating is the thickened basement membranes. Aldose reductase inhibitors like sorbinil do prevent basement membrane thickening and type IV collagen overproduction. The latter seems related to intracellular sorbitol accumulation and also to protein kinase C activation. Autocrine or paracrine TGF beta may be involved in the type IV collagen oversecretion. The secreted type IV collagen is subject to posttranslational alterations, especially glycation which leads to advanced glycation end-products and covalent crosslinks. This decreases collagen extractability and susceptibility to collagenases and favours basement membrane thickening. Disaccharide unit-specific alpha-glucosidase activity is inhibited by glucose (Kp = 7.5 mM). Type IV collagenase activity secreted by endothelial cells cultured at high glucose concentrations appears to be diminished. Therefore type IV collagen catabolism may be decreased in Diabetes Mellitus.