Aims/hypothesisApolipoprotein A-I (apoA-I), the main protein constituent of HDL, has a central role in the reverse cholesterol-transport pathway, which together with the anti-inflammatory properties of apoA-I/HDL provide cardioprotection. Recent findings of direct stimulation of glucose uptake in muscle by apoA-I/HDL suggest that altered apoA-I and HDL functionality may be a contributing factor to the development of diabetes. We have studied the in vivo effects of short treatments with human apoA-I in a high-fat diet fed mouse model. In addition to native apoA-I, we investigated the effects of the cardioprotective Milano variant (Arg173Cys).MethodsMale C57Bl6 mice on a high-fat diet for 2 weeks that received a single injection of human apoA-I proteins (wild-type and Milano) were analysed for blood glucose and insulin levels during a 3 h incubation followed by glucose tolerance tests. Incorporation of injected human apoA-I protein into HDLs was analysed by native gel electrophoresis.ResultsApoA-I treatment significantly improved insulin secretion and blood glucose clearance in the glucose tolerance test, with an efficiency exceeding that of lean control animals, and led to decreased basal glucose during the 3 h incubation. Notably, the two apoA-I variants triggered insulin secretion and glucose clearance to the same extent.Conclusions/interpretationApoA-I treatment leads to insulin- and non-insulin-dependent effects on glucose homeostasis. The experimental model of short-term (2 weeks) feeding of a high-fat diet to C57Bl6 mice provides a suitable and time-efficient system to unravel the resulting tissue-specific mechanisms of acute apoA-I treatment that lead to improved glucose homeostasis.

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Lipid uptake can be facilitated via caveolae, specific plasma membrane invaginations abundantly expressed in adipocytes. The dynamin-related protein EH domain-containing 2 (EHD2) stabilizes caveolae at the cell surface. Here, we have examined the importance of EHD2 for lipid handling using primary adipocytes isolated from EHD2 knockout (Ehd2−/−) C57BL6/N mice. Following high-fat diet (HFD) feeding, we found a clear impairment of epididymal, but not inguinal, adipose tissue expansion in Ehd2−/− compared with Ehd2+/+ (WT) mice. Cell size distribution analysis revealed that Ehd2−/− mice had a lower proportion of small adipocytes, and an accumulation of medium-sized adipocytes in both epididymal and inguinal adipose tissue. Further, PPARγ activity, FABP4 and caveolin-1 expression were decreased in adipocytes isolated from Ehd2−/− mice. Inguinal adipocytes isolated from Ehd2−/− mice displayed reduced lipolysis in response to beta adrenergic receptor agonist, which was associated with reduced phosphorylation of perilipin-1 and hormone sensitive lipase (HSL). This impairment could not be rescued using a cAMP analog, indicating that impaired lipolysis in Ehd2−/− primary adipocytes likely occurs at the level of, or downstream of, protein kinase A (PKA). Altogether, these findings pinpoint the importance of EHD2 for maintained intracellular lipid metabolism, and emphasize differences in mechanisms regulating lipid handling in various adipose-tissue depots.

Myocardin related transcription factors (MRTFs: MYOCD/myocardin, MRTF-A, and MRTF-B) play a key role in smooth muscle cell differentiation by activating contractile genes. In atherosclerosis, MRTF levels change, and most notable is a fall of MYOCD. Previous work described anti-inflammatory properties of MRTF-A and MYOCD, occurring through RelA binding, suggesting that MYOCD reduction could contribute to vascular inflammation. Recent studies have muddled this picture showing that MRTFs may show both anti- and pro-inflammatory properties, but the basis of these discrepancies remain unclear. Moreover, the impact of MRTFs on inflammatory signaling pathways in tissues relevant to human arterial disease is uncertain. The current work aimed to address these issues. RNA-sequencing after forced expression of myocardin in human coronary artery smooth muscle cells (hCASMCs) showed reduction of pro-inflammatory transcripts, including CCL2, CXCL8, IL6, and IL1B. Side-by-side comparison of MYOCD, MRTF-A, and MRTF-B in hCASMCs, showed that the anti-inflammatory impact was shared among MRTFs. Correlation analyses using human arterial transcriptomic datasets revealed negative correlations between MYOCD, MRTFA, and SRF, on the one hand, and the inflammatory transcripts, on the other. A pro-inflammatory drive from lipopolysaccharide, did not change the size of the suppressive effect of MRTF-A in hCASMCs on either mRNA or protein levels. To examine cell type-dependence, we compared the anti-inflammatory impact in hCASMCs, with that in human bladder SMCs, in endothelial cells, and in monocytes (THP-1 cells). Surprisingly, little anti-inflammatory activity was seen in endothelial cells and monocytes, and in bladder SMCs, MRTF-A was pro-inflammatory. CXCL8, IL6, and IL1B were increased by the MRTF-SRF inhibitor CCG-1423 and by MRTF-A silencing in hCASMCs, but depolymerization of actin, known to inhibit MRTF activity, had no stimulatory effect, an exception being IL1B. Co-immunoprecipitation supported binding of MRTF-A to RelA, supporting sequestration of this important pro-inflammatory mediator as a mechanism. Dexamethasone treatment and silencing of RelA (by 76 ± 1%) however only eliminated a fraction of the MRTF-A effect (≈25%), suggesting mechanisms beyond RelA binding. Indeed, SRF silencing suggested that MRTF-A suppression of IL1B and CXCL8 depends on SRF. This work thus supports an anti-inflammatory impact of MRTF-SRF signaling in hCASMCs and in intact human arteries, but not in several other cell types.

The present work addressed the hypothesis that NG2/CSPG4, CD146/MCAM, and VAP1/AOC3 are target genes of myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MKL1, MRTF-B/MKL2) and serum response factor (SRF). Using a bioinformatics approach, we found that CSPG4, MCAM, and AOC3 correlate with MYOCD, MRTF-A/MKL1, and SRF across human tissues. No other transcription factor correlated as strongly with these transcripts as SRF. Overexpression of MRTFs increased both mRNA and protein levels of CSPG4, MCAM, and AOC3 in cultured human smooth muscle cells (SMCs). Imaging confirmed increased staining for CSPG4, MCAM, and AOC3 in MRTF-A/MKL1-transduced cells. MRTFs exert their effects through SRF, and the MCAM and AOC3 gene loci contained binding sites for SRF. SRF silencing reduced the transcript levels of these genes, and time-courses of induction paralleled the direct target ACTA2. MRTF-A/MKL1 increased the activity of promoter reporters for MCAM and AOC3, and transcriptional activation further depended on the chromatin remodeling enzyme KDM3A. CSPG4, MCAM, and AOC3 responded to the MRTF-SRF inhibitor CCG-1423, to actin dynamics, and to ternary complex factors. Coincidental detection of these proteins should reflect MRTF-SRF activity, and beyond SMCs, we observed co-expression of CD146/MCAM, NG2/CSPG4, and VAP1/AOC3 in pericytes and endothelial cells in the human brain. This work identifies highly responsive vascular target genes of MRTF-SRF signaling that are regulated via a mechanism involving KDM3A.