Differentiation of human adipose stromal cells in vitro into insulin-sensitive adipocytes.

Adipose tissue-related diseases such as obesity and type 2 diabetes are worldwide epidemics. In order to develop adipose tissue cultures in vitro that mimic more faithfully the in vivo physiology, new well-characterized and publicly accepted differentiation methods of human adipose stem cells are needed. The aims of this study are (1) to improve the existing natural adipose tissue extract (ATE)-based induction method and (2) to study the effects of a differentiation method on insulin responsiveness of the resulting adipocytes. Different induction media were applied on human adipose stromal cell (hASC) monocultures to study the differentiation capacity of the induction media and the functionality of the differentiated adipocytes. Cells were differentiated for 14 days to assess triglyceride accumulation per cell and adipocyte-specific gene expression (PPARγ, adiponectin, AP2, leptin, Glut4, Prdm16, CIDEA, PGC1-α, RIP140, UCP and ADCY5). Insulin response was studied by measuring glucose uptake and inhibition of lipolysis after incubation with 100 or 500 nM insulin. The selected differentiation method included a 3-day induction with ATE, 6 days in serum-free medium supplemented with 1.15 µM insulin and 9.06 µM Troglitazone, followed by 4 days in a defined serum- and insulin-free stimulation medium. This protocol induced prominent general adipocyte gene expression, including markers for both brown and white adipocytes and triglyceride accumulation. Moreover, the cells were sensitive to insulin as observed from increased glucose uptake and inhibition of lipolysis. This differentiation protocol provides a promising approach for the induction of hASC adipogenesis to obtain functional and mature human adipocytes.

Adipose tissue is inflamed in NAFLD due to obesity but not in NAFLD due to genetic variation in PNPLA3.

AIMS/HYPOTHESIS: The rs738409 C>G single-nucleotide polymorphism in PNPLA3 leads to a missense mutation (I148M) which increases liver fat but does not cause insulin resistance. We hypothesised that patients with non-alcoholic fatty liver disease (NAFLD) due to the PNPLA3 variant ('PNPLA3 NAFLD' = PNPLA3-148MM) do not have adipose tissue (AT) inflammation in contrast with those with NAFLD due to obesity ('obese NAFLD'). METHODS: Biopsy specimens of AT were taken, and PNPLA3 genotype and liver fat ((1)H-magnetic resonance spectroscopy) were determined in 82 volunteers, who were divided into groups based on either median BMI (obese 36.2 ± 0.7 kg/m(2); non-obese 26.0 ± 0.4 kg/m(2)) or PNPLA3 genotype. All groups were similar with respect to age and sex. The PNPLA3 subgroups were equally obese (PNPLA3-148MM, 31.1 ± 1.3 kg/m(2); PNPLA3-148II, 31.2 ± 0.8 kg/m(2)), while the obese and non-obese subgroups had similar PNPLA3 genotype distribution. Gene expression of proinflammatory (MCP-1, CD68) and anti-inflammatory (Twist1, ADIPOQ) markers was measured using quantitative real-time RT-PCR. RESULTS: Liver fat was similarly increased in obese NAFLD (9.5 ± 1.3% vs 5.1 ± 0.9%, obese vs non-obese, p = 0.007) and PNPLA3 NAFLD (11.4 ± 1.7% vs 5.3 ± 0.8%, PNPLA3-148MM vs PNPLA3-148II, p < 0.001). Fasting serum insulin was higher in the obese than the non-obese group (76 ± 6 vs 47 ± 6 pmol/l, p < 0.001), but similar in PNPLA3-148MM and PNPLA3-148II (60 ± 8 vs 62 ± 5 pmol/l, NS). In obese vs non-obese, MCP-1 and CD68 mRNAs were upregulated, whereas those of Twist1 and ADIPOQ were significantly downregulated. AT gene expression of MCP-1, CD68, Twist1 and ADIPOQ was similar in PNPLA3-148MM and PNPLA3-148II groups. CONCLUSIONS/INTERPRETATION: PNPLA3 NAFLD is characterised by an increase in liver fat but no insulin resistance or AT inflammation, while obese NAFLD has all three of these features.

Acyl chain and headgroup specificity of human plasma phospholipid transfer protein.

Phospholipid transfer protein (PLTP) is a plasma protein with two reported in vitro activities: transfer of phospholipids and modulation of HDL particle size. The mechanism of PLTP-mediated phospholipid transfer was studied by determining the acyl chain and headgroup specificity and comparing the results with those obtained with the non-specific lipid transfer protein (ns-LTP), a previously characterised intracellular transfer protein. To verify the results obtained with purified plasma PLTP, recombinant PLTP produced in COS-1 cells was used. The transfer rates were determined by monitoring the transfer of fluorescent, pyrene-labeled phospholipids from quenched donor phospholipid vesicles to HDL3 particles. When the length of the pyrene-labeled acyl chain was varied from 6 to 14 carbons, a fairly monotonous decrease in the transfer rate was observed. No difference in rate was observed for the isomers having the pyrene-labeled and unlabeled acyl chains in reversed positions. PLTP mediated equally the transfer of the various headgroup derivatives except phosphatidylethanolamine (PE), which was transferred 2-3-fold more slowly. In all experiments the plasma and recombinant PLTP behaved identically. The specificity patterns observed for PLTP and ns-LTP were very similar. No PLTP-phospholipid intermediate could be observed, indicating that PLTP, like ns-LTP, does not form a tight complex with the lipid substrate and may thus mediate the transfer of phospholipid via another, yet unspecified mechanism.

Oxidative modification of HDL3 in vitro and its effect on PLTP-mediated phospholipid transfer.

The oxidation of HDL3 by Cu(II) and its effect on the ability of these particles to act as phospholipid acceptors in human plasma phospholipid transfer protein (PLTP)-mediated lipid transfer were investigated. Oxidation of HDL3 was monitored by measuring the following parameters: (i) formation of conjugated dienes, (ii) production of thiobarbituric acid reactive substances (TBARS), (iii) decrease in reactive lysine and (iv) tryptophan residues, (v) change in particle charge and (vi) diameter, and (vii) oligomerisation of apoA-I and apoA-II. Formation of conjugated dienes was the parameter responding to the oxidative treatment with the fastest kinetics. The appearance of TBARS and modification of apolipoprotein tryptophan residues were detected simultaneously but required higher Cu(II) concentrations for maximal kinetics. Cross-linking of the major protein constituents of HDL3, apoA-I and apoA-II, represented later steps of the oxidation process. Further, the oxidative modification was accompanied by a progressive change in HDL3 particle charge and a minor increase in particle diameter. PLTP-mediated phospholipid transfer to the oxidized particles was investigated using an assay measuring the transfer of fluorescent, pyrene-labeled PC. The transfer was significantly inhibited, but only after extensive modification of the HDL proteins, suggesting that the HDL oxidative modifications occurring in vivo do not essentially impair its phospholipid acceptor function. A similar but less pronounced inhibition was observed when two other phospholipid transfer proteins, the nonspecific lipid transfer protein (ns-LTP) and the phosphatidylcholine transfer protein (PC-TP), were studied in parallel. This indicates that the inhibition was partly due to unspecific effects of the modification on acceptor particle surface properties, but included an aspect specific for PLTP.

Role of Rab GTPases in membrane traffic.

Small GTPases of the Rab subfamily have been known to be key regulators of intracellular membrane traffic since the late 1980s. Today this protein group amounts to more than 40 members in mammalian cells which localize to distinct membrane compartments and exert functions in different trafficking steps on the biosynthetic and endocytic pathways. Recent studies indicate that cycles of GTP binding and hydrolysis by the Rab proteins are linked to the recruitment of specific effector molecules on cellular membranes, which in turn impact on membrane docking/fusion processes. Different Rabs may, nevertheless, have slightly different principles of action. Studies performed in yeast suggest that connections between the Rabs and the SNARE machinery play a central role in membrane docking/fusion. Further elucidation of this linkage is required in order to fully understand the functional mechanisms of Rab GTPases in membrane traffic.

Generation of infectious nucleocapsids by in vitro assembly of the shell protein on to the polymerase complex of the dsRNA bacteriophage phi 6.

A method for the in vitro uncoating of the phi 6 nucleocapsid (NC) was developed. The resulting particle, designated as the NC core, containing the genomic double-stranded (ds) RNA segments and the proteins P1, P2, P4 and P7, was not infectious but had a highly enhanced in vitro transcriptase activity compared to that of the intact NC. The NC shell protein P8 was purified by immunoaffinity chromatography, and it was shown to self-assemble to shell-like structures upon addition of calcium ions. The conditions for the self-assembly of the shell were optimized. Shell reassembly on to the NC cores restored the infectivity but resulted in a decrease of transcriptase activity. No reassembly of the shell on to RNA-less cores (procapsids) produced from a cDNA construction in Escherichia coli was observed. Our results suggest that the intracellular uncoating of the NC is the event activating the phi 6 dsRNA transcriptase and that the NC shell is necessary for infectivity, probably for the passage of the NC through the host cytoplasmic membrane. Packaging of the dsRNA segments into the procapsid appears to be a prerequisite for NC shell assembly.

Vesicular transport and kidney development.

Vesicular transport processes play crucial roles in the biogenesis of cellular membranes and in the polarized transport functions of epithelial cells. During the 1990's we have witnessed major progress in elucidation of the machineries responsible for the intracellular membrane trafficking. The components of these machineries are abundant in tissues with a high content of epithelial cells, such as the kidney. However, the developmental role of the membrane trafficking apparatus in higher eukaryotes has been addressed hardly at all. We summarize here data on the presence and the functional role of vesicle transport proteins in the kidney, and describe work addressing the developmentally regulated expression and localization of three molecules suggested to be involved in polarized trafficking in kidney epithelia, Rab17, syntaxin 3, and Munc-18-2. The results show that specialized transport machinery is induced during differentiation of renal epithelia. However, the expression levels of the components under study are highest in the mature structures, indicating that the proteins are predominantly required for the function of mature epithelia and possibly for the maintenance of the polarized phenotype of specific epithelial cells. The proteins are, however, detected at low levels already in earlier, differentiating structures, and could thus also be involved in the differentiation of kidney epithelia.

HMG-17, a chromosomal non-histone protein, shows developmental regulation during organogenesis.

We used the differential hybridization technique for isolating developmentally regulated genes from the mouse metanephric kidney. In this screening, we identified the cDNA encoding high-mobility-group protein 17 (HMG-17), a chromosomal non-histone protein which modulates the conformation of transcriptionally active chromatin. Using Northern blot analysis, the HMG-17 mRNA was strongly expressed during embryogenesis and downregulated in various adult murine organs. At the histological level, the transcript localized to differentiating tissue regions and was apparently downregulated in mature structures indicating that HMG-17 expression is linked to cell differentiation. HMG-17 can thus be regarded as a general marker for tissues or cells undergoing differentiation during organogenesis.

Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes.

In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.

Sequence of a canine cDNA clone encoding a Ran/TC4 GTP-binding protein.

We report the isolation and characterization of a canine cDNA encoding a 216-amino acid GTP-binding protein of the Ras superfamily. The protein is almost identical to the human TC4 [Drivas et al., Mol. Cell. Biol. 10 (1990) 1793-1798] and Ran [Bischoff and Ponstingl, Proc. Natl. Acad. Sci. USA 88 (1991) 10830-10834; Nature 354 (1991) 80-82] proteins, the latter of which has been found to be involved in cell cycle control. Furthermore, the protein is highly similar to the fission yeast spi1 gene product [Matsumoto and Beach, Cell 66 (1991) 347-360]. The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.

Isolation of a mouse cDNA encoding Rab23, a small novel GTPase expressed predominantly in the brain.

The full-length cDNA encoding Rab23, a novel Ras-related small GTPase, was isolated using the sequence of a previously described [Chavrier et al., Gene 112 (1992) 261-264] short cDNA fragment and the rapid amplification of cDNA ends (RACE) PCR techniques. The deduced amino acid sequence was not very closely related to any previously described small GTPase, but was within the Rab subfamily. A Northern analysis revealed that the rab23 mRNA is predominantly expressed in the brain, which places the protein, together with Rab3a and Rab15, in the group of small GTPases characteristic of the nervous system.

Isolation of a murine cDNA clone encoding Rab19, a novel tissue-specific small GTPase.

Using a rapid amplification of cDNA ends (RACE) cloning approach, we have isolated a cDNA clone encoding Rab19, a novel small GTPase of the Rab subfamily contained within partial sequences previously described [Chavrier et al., Gene 112 (1992) 261-264]. Northern blot analysis of the distribution of the rab19 mRNA in various adult mouse tissues and NIH 3T3 fibroblasts revealed that rab19 is expressed in a tissue-specific manner. The rab19 transcript was detected at high levels in intestine, lung and spleen, and at a lower level in kidney. In contrast, liver, brain, heart and NIH 3T3 fibroblasts contain only very little or no detectable rab19 mRNA. Therefore, Rab19 is likely to represent a novel tissue- or cell type-specific small GTPase.

Overproduction, purification, and characterization of DNA-binding protein P19 of bacteriophage PRD1.

The early protein, P19, of bacteriophage PRD1 was purified after overexpression of the cloned gene, XIX, in Escherichia coli DH5 alpha cells. The purified protein binds as multimers to single-stranded DNA (ssDNA), and with a lower affinity to double-stranded DNA (dsDNA), without sequence-specificity. Two distinct P19-ssDNA complexes were discovered in gel- mobility-shift assays at different protein:DNA ratios. P19 was capable of fully protecting ssDNA against nuclease P1. Electron microscopy of protein P19-ssDNA complexes showed DNA molecules which were extensively coated with protein and whose contour length was clearly reduced by P19 binding. The results suggest that P19 binds to ssDNA with moderate cooperativity and are consistent with the DNA being wrapped around the P19 multimers.

Co-operative regulation of endocytosis by three Rab5 isoforms.

Rab proteins are small GTPases involved in the regulation of membrane traffic. Rab5a has been shown to regulate transport in the early endocytic pathway. Here we report the isolation of cDNA clones encoding two highly related isoforms, Rab5b and Rab5c. The two proteins share with Rab5a all the structural features required for regulation of endocytosis. Rab5b and Rab5c colocalize with the both transferrin receptor and Rab5a, stimulate the homotypic fusion between early endosomes in vitro and increase the rate of endocytosis when overexpressed in vivo. These data demonstrate that three Rab5 isoforms cooperate in the regulation of endocytosis in eukaryotic cells.

The impact of phospholipid transfer protein (PLTP) on HDL metabolism.

High-density lipoproteins (HDL) play a major protective role against the development of coronary artery disease. Phospholipid transfer protein (PLTP) is a main factor regulating the size and composition of HDL in the circulation and plays an important role in controlling plasma HDL levels. This is achieved via both the phospholipid transfer activity of PLTP and its capability to cause HDL conversion. The present review focuses on the impact of PLTP on HDL metabolism. The basic characteristics and structure of the PLTP protein are described. The two main functions of PLTP, PLTP-mediated phospholipid transfer and HDL conversion are reviewed, and the mechanisms and control, as well as the physiological significance of these processes are discussed. The relationship between PLTP and the related cholesteryl ester transfer protein (CETP) is reviewed. Thereafter other functions of PLTP are recapitulated: the ability of PLTP to transfer cholesterol, alpha-tocopherol and lipopolysaccharide (LPS), and the suggested involvement of PLTP in cellular cholesterol traffic. The discussion on PLTP activity and mass in (patho)physiological settings includes new data on the presence of two forms of PLTP in the circulation, one catalytically active and the other inactive. Finally, future directions for PLTP research are outlined.

Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity.

A sensitive sandwich-type enzyme-linked immunosorbent assay (ELISA) for human plasma phospholipid transfer protein (PLTP) has been developed using a monoclonal capture antibody and a polyclonal detection antibody. The ELISA allows for the accurate quantification of PLTP in the range of 25-250 ng PLTP/assay. Using the ELISA, the mean plasma PLTP concentration in a Finnish population sample (n = 159) was determined to be 15.6 +/- 5.1 mg/l, the values ranging from 2.30 to 33.4 mg/l. PLTP mass correlated positively with HDL-cholesterol (r = 0.36, P < 0.001), apoA-I (r = 0.37, P < 0.001), apoA-II (r = 0.20, P < 0.05), Lp(A-I) (r=0.26, P=0.001) and Lp(A-I/A-II) particles (r=0.34, P<0.001), and negatively with body mass index (BMI) (r = -0.28, P < 0.001) and serum triacylglycerol (TG) concentration (r = -0.34, P < 0.001). PLTP mass did not correlate with phospholipid transfer activity as measured with a radiometric assay. The specific activity of PLTP, i.e. phospholipid transfer activity divided by PLTP mass, correlated positively with plasma TG concentration (r=0.568, P<0.001), BMI (r=0.45, P<0.001), apoB (r = 0.45, P < 0.001). total cholesterol (r=0.42, P < 0.001), LDL-cholesterol (r = 0.34, P < 0.001) and age (r = 0.36, P < 0.001), and negatively with HDL-cholesterol (r= -0.33, P < 0.001), Lp(A-I) (r= -0.21, P < 0.01) as well as Lp(A-I/A-II) particles (r = -0.32, P < 0.001). When both PLTP mass and phospholipid transfer activity were adjusted for plasma TG concentration, a significant positive correlation was revealed (partial correlation, r = 0.31, P < 0.001). The results suggest that PLTP mass and phospholipid transfer activity are strongly modulated by plasma lipoprotein composition: PLTP mass correlates positively with parameters reflecting plasma high density lipoprotein (HDL) levels, but the protein appears to be most active in subjects displaying high TG concentration.

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in prebeta-HDL generation.

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce pre(beta)-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.

Sequence of a human cDNA encoding Cab45, a Ca2+-binding protein with six EF-hand motifs.

We report the sequence of a human cDNA encoding a deduced 362 amino acid protein with six EF-hand calcium-binding motifs. The protein is a likely human counterpart of the Cab45 protein recently identified in the 3T3-L1 mouse adipocyte cell line [Scherer et al. (1996), J. Cell Biol. 133, 257-268], displaying 87% aa and 83% nt identity with this sequence. The mRNA for human Cab45 is detected ubiquitously in tissue Northern blots.

The OSBP-related proteins (ORPs): global sterol sensors for co-ordination of cellular lipid metabolism, membrane trafficking and signalling processes?

Protein families related to OSBP (oxysterol-binding protein) are present in eukaryotes from yeast to human. The functions of the ORPs (OSBP-related proteins) have remained largely enigmatic. Even though they have been implicated in the function of ERJs (endoplasmic reticulum junctions), it is evident that any single model for their mechanism of action is insufficient. The existing evidence points in many different directions, such as integration of sterol and sphingomyelin metabolism, regulation of neutral lipid metabolism, control of signalling cascades, regulation of secretory vesicle generation, and function in the microtubule-based motility of endo/lysosomes. Some of these functions could involve ERJ and non-vesicular transport of lipids, but this is unlikely to be the unifying feature. We believe, rather, that the common denominator for ORP function is acting as sterol sensors that relay information to a spectrum of cellular processes.

Quantitation of the adsorption and penetration stages of bacteriophage phi 6 infection.

The enveloped dsRNA bacteriophage phi 6 uses the pilus of Pseudomonas syringae as its receptor. It enters the host cell by fusion of the virus envelope with the host outer membrane, followed by penetration of the cytoplasmic membrane by the phage nucleocapsid. In this investigation we quantitated the adsorption and penetration of phi 6wt and a host range mutant, phi 6h 1s, to five bacterial strains. Adsorption rate constants were measured for the different phage-host combinations, the constant for phi 6wt with the standard host was 3.3 X 10(10) ml/min. Infections with 14C-labeled phage at different phage/cell ratios were used to measure the numbers of adsorbing and entering virions/sensitive cell. At high phage/cell ratios (200-250) the standard host adsorbed on the average 35-40 wild-type virions/cell, the saturation level being somewhat higher. It was shown that at phage/host cell ratios of 0.1-1 practically every virion produces an infectious center. The average number of entering phage particles per infectious center reached saturation around the phage/cell ratio of 50 and did not exceed 3 for the standard host. The phi 6 preparations used in this study had a specific infectivity of 0.7-0.9.