Ulf Lidberg

SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity.

The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), α-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of α-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.

Influence of systematically varied nanoscale topography on the morphology of epithelial cells

With the knowledge that cells can react to lithographically manufactured nanometer-sized surface objects, our interest concerned whether cells would respond to surface structures of systematically increasing size. Our approach to answer this question was to fabricate surfaces with the same surface chemistry and similar surface roughness but increasing size of structural features. To fabricate large areas of patterned surfaces, required for cell culture studies, we used colloidal lithography utilizing colloidal particles as a template for surface nanostructuring. The fabricated surfaces contained hemispherical nanopillars with diameters ranging from 60 to 170 nm. Changes in cell morphology of a pancreatic epithelial cell line (AR4-2J) were studied by evaluating cell area and cell shape. The latter was studied by applying the cell shape classification method using three shape descriptors. The pancreatic cells responded in a systematic way to the surface nanostructures. The cells spread more and became more nonround when cultured on surfaces with increasing size of the topographic features.

The VLDL receptor promotes lipotoxicity and increases mortality in mice following an acute myocardial infarction

Impaired cardiac function is associated with myocardial triglyceride accumulation, but it is not clear how the lipids accumulate or whether this accumulation is detrimental. Here we show that hypoxia/ischemia-induced accumulation of lipids in HL-1 cardiomyocytes and mouse hearts is dependent on expression of the VLDL receptor (VLDLR). Hypoxia-induced VLDLR expression in HL-1 cells was dependent on HIF-1α through its interaction with a hypoxia-responsive element in the Vldlr promoter, and VLDLR promoted the endocytosis of lipoproteins. Furthermore, VLDLR expression was higher in ischemic compared with nonischemic left ventricles from human hearts and was correlated with the total lipid droplet area in the cardiomyocytes. Importantly, Vldlr-/- mice showed improved survival and decreased infarct area following an induced myocardial infarction. ER stress, which leads to apoptosis, is known to be involved in ischemic heart disease. We found that ischemia-induced ER stress and apoptosis in mouse hearts were reduced in Vldlr-/- mice and in mice treated with antibodies specific for VLDLR. These findings suggest that VLDLR-induced lipid accumulation in the ischemic heart worsens survival by increasing ER stress and apoptosis.

Allele-specific regulation of MTTP expression influences the risk of ischemic heart disease

Promoter polymorphisms in microsomal triglyceride transfer protein (MTTP) have been associated with decreased plasma lipids but an increased risk for ischemic heart disease (IHD), indicating that MTTP influences the susceptibility for IHD independent of plasma lipids. The objective of this study was to characterize the functional promoter polymorphism in MTTP predisposing to IHD and its underlying mechanism. Use of pyrosequencing technology revealed that presence of the minor alleles of the promoter polymorphisms -493G>T and -164T>C result in lower transcription of MTTP in vivo in the heart, liver, and macrophages. In vitro experiments indicated that the minor -164C allele mediates the lower gene expression and that C/EBP binds to the polymorphic region in an allele-specific manner. Furthermore, homozygous carriers of the -164C were found to have increased risk for IHD as shown in a case-control study including a total of 544 IHD patients and 544 healthy control subjects. We concluded that carriers of the minor -164C allele have lower expression of MTTP in the heart, mediated at least partly by the transcription factor CCAAT/enhancer binding protein, and that reduced concentration of MTTP in the myocardium may contribute to IHD upon ischemic damage.

Evaluation of macrophage-specific promoters using lentiviral delivery in mice

In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. Fluorescence activated cell sorting analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150-bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.

Structure and organization of the human carboxyl ester lipase locus

We have earlier reported the sequence of the human carboxyl ester lipase (CEL) gene, which is constitutively expressed in pancreas and during lactation in the mammary gland (Lidberg et al. 1992; Nilsson et al. 1990). It was shown that CEL is encoded by a single gene that is 9850 bp long, consisting of 11 exons. During the screening procedure, another gene with striking similarity to the CEL gene was found, hence called the carboxyl ester lipase-like (CEL-like) gene. This new gene proved to span 4846 bp. Data reveal that this gene, compared with the CEL gene, is missing a 5-kb large segment, including the CEL gene exons 2-7, and that the remaining sequence shows a high degree of similarity (97%). The two genes were localized at Chromosome (Chr) 9q34-qter. This result is in agreement with the work by Taylor et al. (1991), who localized the CEL gene to 9q34.3. Further studies of the CEL-like gene have shown that this is a transcribed pseudogene that has lost both the expression level and the tissue specificity (Nilsson et al. 1993) and may have arisen through a gene duplication of the CEL gene. The degree of nucleotide identity suggests that the emergence of the CEL gene and its pseudogene was a comparatively recent event. This is supported by the fact that neither the rat nor the mouse genome seem to include a CEL pseudogene (Fontaine et al. 1991; Lidmer et al. 1995). Gene duplication plays an important role in the evolution process of organisms because duplication followed by alteration in either or both of the coding and regulatory sequences gives rise to new genes with new functions (Hood et al. 1975). It is, therefore, of great interest to understand the mechanism of gene duplication, and also the nature and possible order of events that subsequently alter the structure and pattern of expression of the corresponding gene products. In human, numerous genes and gene families have been found to originate by duplication, but there are few examples in the literature where the molecular mechanism of the gene duplication is described. The problem in identifying the molecular mechanisms involved in the duplication event is that the sequences utilized in the rearrangement are no longer identifiable. As mentioned, the nucleotide identity between the CEL gene and its pseudogene predicts that the duplication of the CEL gene seems to have taken place during primate evolution. Hence, the possible identification of sequences utilized in the gene duplication would provide an opportunity to determine the mechanisms involved in the process. Some of the examples of gene duplication described in the literature have been shown to be generated by unequal homologous recombination between repetitive sequences; for example, in the human globin cluster there are duplications mediated by Alu sequences (Nicholls et al. 1987). Also, diseases associated with partial gene duplication are known to be the result of mispairing and unequal crossing over between repeated elements. Enlarged LDL receptors have been found in patients with familial hypercholesterolemia. The partial duplication was found to be the result

Characterization of Transcriptional Control Elements in the Mammary Gland and Pancreas: Studies of the Carboxyl Ester Lipase Gene

The human lactating mammary gland synthesizes and secretes with the milk a bile saltstimulated lipase (BSSL) that, after specific activation by primary bile salts, contributes to the breast-fed infant’s endogenous capacity for intestinal fat digestion. This enzyme, which accounts for approximately 1% of total milk protein, is a very nonspecific lipase. BSSL is not degraded during passage with the milk through the stomach. It is, however, inactivated when the milk is pasteurized. Our group has isolated a 2359 nucleotide (nt) cDNA clone from human lactating mammary gland and deduced from this, the BSSL enzyme consists of 745 amino acid (aa) residues including a 23 aa leader peptide. We have also isolated and analysed the human BSSL gene, which proved to span a region of 9832 nt and contains 11 exons, see Figure 1. The carboxyl ester lipase (CEL) of human pancreatic juice is a product of the same gene. Because of this, the BSSL gene now is called CEL.