Jens O. Lagerstedt

Context: Statin use is associated with increased risk of type 2 diabetes (T2D) and mild hyperglycemia. The underlying mechanisms are not well studied, and the effect of statin treatment on glucagon-like peptide 1 (GLP-1) secretion or production is unknown. Objective: This work aimed to assess the effects of rosuvastatin on GLP-1 secretion and production. Methods: We performed association studies in the Malmö Diet and Cancer study cardiovascular cohort (MDCS-CC) reexamination cohort, in vitro investigations using GLUTag cells and acute and chronic studies in female, normoglycemic C57Bl/6j mice. Results: Studies in the MDCS-CC reexamination cohort (n = 3734) revealed that in individuals without T2D, statin usage was associated with higher fasting glucose-dependent insulinotropic peptide (GIP), insulin, glucose, glucagon, and homeostatic model assessment of insulin resistance, but not GLP-1. However, in patients with T2D, statin usage was associated with higher fasting GLP-1 levels. Rosuvastatin treatment or 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (Hmgcr) knockdown (KD) reduced GLP-1 secretion and increased Gcg messenger RNA in GLUTag cells. Rosuvastatin acutely reduced postprandial GLP-1 secretion, whereas chronic rosuvastatin treatment in mice caused hyperglycemia and increased postprandial GLP-1 levels. The acute effect of Hmgcr KD on GLP-1 secretion could be mimicked by targeting intracellular cholesterol using a PCSK9 inhibitor. Finally, transcriptomic alterations induced by rosuvastatin were limited to genes involved in cholesterol biosynthesis. Conclusion: We have established HMGCR as a regulator of GLP-1 secretion and provide a plausible explanation for the clinically observed mild hyperglycemia associated with statin use. Given the negative acute effect on GLP-1 secretion, monitoring of blood glucose levels is recommended after prescribing rosuvastatin.

Apolipoprotein A-I (ApoA-I), the primary component of high-density lipoprotein (HDL) cholesterol primes β-cells to increase insulin secretion, however, the mechanisms involved are not fully defined. Here, we aimed to confirm ApoA-I receptors in β-cells and delineate ApoA-I-receptor pathways in β-cell insulin output. An LRC-TriCEPS experiment was performed using the INS-1E rat β-cell model and ApoA-I for unbiased identification of ApoA-I receptors. Identified targets, alongside ATP binding cassette transporter A1 (ABCA1) (included control) were silenced in the same cells, and insulin secretion (ELISA) and mitochondrial metabolism (seahorse) were assessed with/without ApoA-I priming. Human β-cell expression data was used to investigate ApoA-I receptor pathways in type 2 diabetes (T2D). Scavenger receptor B1 (SR-BI) and regulator of microtubule dynamics 1 were identified as ApoA-I targets. SR-BI or ABCA1 silencing abolished ApoA-I induced increases in insulin secretion. ApoA-I priming increased mitochondrial OXPHOS, however this was greatly attenuated with SR-BI or ABCA1 silencing. Supporting this, human β-cell expression data investigations found SR-BI and ABCA1 to be correlated with genes associated with mitochondrial pathways. In all, SR-BI and ABCA1 correlated with 73 and 3 genes differentially expressed in T2D, respectively. We confirm that SR-BI and ABCA1 are the primary β-cell ApoA-I receptors and demonstrate that ApoA-I priming enhances β-cell insulin secretion via the upregulation of mitochondrial metabolism through ApoA-I-SR-BI and ApoA-I-ABCA1 pathways. We propose that SR-BI relies on mitochondrial and exocytotic pathways, while ABCA1 depends solely on mitochondrial pathways. Our findings uncover new targets in ApoA-I β-cell mechanism for T2D therapies.

High‐density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A‐I (ApoA‐I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA‐I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA‐I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA‐I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small‐angle x‐ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA‐I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid–protein interactions and their behavior.

Aims/hypothesis: Roux-en-Y gastric bypass surgery (RYGB) frequently results in remission of type 2 diabetes as well as exaggerated secretion of glucagon-like peptide-1 (GLP-1). Here, we assessed RYGB-induced transcriptomic alterations in the small intestine and investigated how they were related to the regulation of GLP-1 production and secretion in vitro and in vivo. Methods: Human jejunal samples taken perisurgically and 1 year post RYGB (n=13) were analysed by RNA-seq. Guided by bioinformatics analysis we targeted four genes involved in cholesterol biosynthesis, which we confirmed to be expressed in human L cells, for potential involvement in GLP-1 regulation using siRNAs in GLUTag and STC-1 cells. Gene expression analyses, GLP-1 secretion measurements, intracellular calcium imaging and RNA-seq were performed in vitro. OGTTs were performed in C57BL/6j and iScd1^−/− mice and immunohistochemistry and gene expression analyses were performed ex vivo. Results: Gene Ontology (GO) analysis identified cholesterol biosynthesis as being most affected by RYGB. Silencing or chemical inhibition of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in the synthesis of monounsaturated fatty acids, was found to reduce Gcg expression and secretion of GLP-1 by GLUTag and STC-1 cells. Scd1 knockdown also reduced intracellular Ca²⁺ signalling and membrane depolarisation. Furthermore, Scd1 mRNA expression was found to be regulated by NEFAs but not glucose. RNA-seq of SCD1 inhibitor-treated GLUTag cells identified altered expression of genes implicated in ATP generation and glycolysis. Finally, gene expression and immunohistochemical analysis of the jejunum of the intestine-specific Scd1 knockout mouse model, iScd1^−/−, revealed a twofold higher L cell density and a twofold increase in Gcg mRNA expression. Conclusions/interpretation: RYGB caused robust alterations in the jejunal transcriptome, with genes involved in cholesterol biosynthesis being most affected. Our data highlight SCD as an RYGB-regulated L cell constituent that regulates the production and secretion of GLP-1. Graphical

Apolipoprotein A-I (ApoA-I) amyloidosis is a rare protein misfolding disease where fibrils of the N-terminal domain of the protein accumulate in several organs, leading to their failure. Although ApoA-I amyloidosis is systemic, the different amyloidogenic variants show a preferential tissue accumulation that appears to correlate with the location of the mutation in the protein sequence and with the local extracellular microenvironment. However, the factors leading to cell/tissues damage, as well as the mechanisms behind the observed organ specificity are mostly unknown. Therefore, we investigated the impact of ApoA-I variants on cell physiology and the mechanisms driving the observed tissue specificity. We focused on four ApoA-I amyloidogenic variants and analyzed their cytotoxicity as well as their ability to alter redox homeostasis in cell lines from different tissues (liver, kidney, heart, skin). Moreover, variant-specific interactions with extracellular matrix (ECM) components were measured by synchrotron radiation circular dichroism and enzyme-linked immunosorbent assay. Data indicated that ApoA-I variants exerted a cytotoxic effect in a time and cell-type-specific manner that seems to be due to protein accumulation in lysosomes. Interestingly, the ApoA-I variants exhibited specific preferential binding to the ECM components, reflecting their tissue accumulation pattern in vivo. While the binding did not to appear to affect protein conformations in solution, extended incubation of the amyloidogenic variants in the presence of different ECM components resulted in different aggregation propensity and aggregation patterns.

Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) induces glucose uptake by muscle tissues and stimulates pancreatic insulin secretion, and also facilitates cholesterol transport in circulation, and is explored for anti-diabetic and anti-atherosclerotic treatments. As the better alternative to complex protein–lipid formulations it was recently established that the C-terminal region of the ApoA-I protein singly improves the metabolic control and prevents formation of atherosclerotic plaques. Additional investigations of peptides based on the ApoA-I structure may lead to novel anti-diabetic drugs. We here investigate a short peptide (33mer, RG33) that corresponds to the two last helical segments (aa 209–241) of the ApoA-I structure (so-called class Y-helices which forms amphipathic helices) for stability and solubility in serum, for in vitro cholesterol efflux capability, and for providing in vivo glucose control in an insulin resistant mouse model. The RG33 peptide efficiently solubilizes lipid-vesicles, and promotes the efflux of cholesterol from cultured macrophages. The efflux capacity is significantly increased in the presence of lipids compared to non-lipidated RG33. Finally, acute treatment with the RG33 peptide significantly improves the glucose clearance capacity of insulin resistant mice. The impact of the RG33 peptide on glucose control and cholesterol transport, as well as the physicochemical properties, makes it a good candidate for translational exploration of its therapeutic potential in diabetes treatment.

Specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are responsible for a late-onset systemic amyloidosis. Carriers do not exhibit increased cardiovascular disease risk despite reduced levels of ApoA-I/ HDL-cholesterol. To explain this paradox, we show that the HDL particle profile of L75P and L174S patients presents a higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered secondary structure composition and display a more flexible binding to lipids compared to their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles and better cholesterol efflux due to altered, region-specific protein structure dynamics.