Human gain-of-function variants in HNF1A confer protection from diabetes but independently increase hepatic secretion of atherogenic lipoproteins

Loss-of-function mutations in hepatocyte nuclear factor 1A (HNF1A) are known to cause rare forms of diabetes and alter hepatic physiology through unclear mechanisms. In the general population, 1:100 individuals carry a rare, protein-coding HNF1A variant, most of unknown functional consequence. To characterize the full allelic series, we performed deep mutational scanning of 11,970 protein-coding HNF1A variants in human hepatocytes and clinical correlation with 553,246 exome-sequenced individuals. Surprisingly, we found that ∼1:5 rare protein-coding HNF1A variants in the general population cause molecular gain of function (GOF), increasing the transcriptional activity of HNF1A by up to 50% and conferring protection from type 2 diabetes (odds ratio [OR] = 0.77, p = 0.007). Increased hepatic expression of HNF1A promoted a pro-atherogenic serum profile mediated in part by enhanced transcription of risk genes including ANGPTL3 and PCSK9. In summary, ∼1:300 individuals carry a GOF variant in HNF1A that protects carriers from diabetes but enhances hepatic secretion of atherogenic lipoproteins.publishedVersio

Efficient Synthesis of Heparinoid Bioconjugates for Tailoring FGF2 Activity at the Stem Cell-Matrix Interface

Heparan sulfate glycosaminoglycans (HS GAGs) attached to proteoglycans harbor high affinity binding sites for various growth factors (GFs) and direct their organization and activity across the cell-matrix interface. Here, we describe a mild and efficient method for generating HS-protein conjugates. The two-step process utilizes a “copper-free” click coupling between differentially sulfated heparinoids primed at their reducing end with an azide handle and a bovine serum albumin protein modified with complementary cyclooctyne functionality. When adsorbed on tissue culture substrates, the glycoconjugates served as extracellular matrix proteoglycan models with the ability to sequester FGF2 and influence mesenchymal stem cell proliferation based on the structure of their HS GAG component

Analyses of total cholesterol, triglycerides and lipoprotein profiles in ApoE^−/− (open bars or Δ) and ApoE^−/−LRP1^n2/n2 (filled bars or ▪) mice.

<p>A–C, Plasma lipid levels (A), immunoblot analysis of plasma apolipoproteins (B) and their relative expression levels (C) (n = 8 per genotype). D–G, Lipoprotein profiles in fasted and postprandial state (pooled plasma from six mice per genotype). Plasma lipoprotein distribution of cholesterol (D–E) and triglyceride (F–G) levels in 5 hour fasted apoE^−/− and apoE^−/−LRP1^n2/n2 mice just before (D & F) or 2 hours after receiving a gastric olive oil load (E & G). Data are mean±SEM. *<i>P</i><0.05.</p

Reduced atherosclerosis development in ApoE^−/−LRP1^n2/n2 mice.

<p>A–B, Spontaneous atherosclerosis development in 26- (A) and 52-week (A–B) old mice. C–E, Different cholesterol levels in lipoprotein fractions (VLDL, LDL and HDL) separated via sequential ultracentrifugation in mice at 52-weeks of age (C), total triglyceride levels (D) and correlation plot between atherogenesis and total cholesterol for individual mice (E). Statistical analysis via determination of the Pearson’s correlation coefficient (Rp) revealed a significant positive correlation between atherosclerosis load in the aorta and circulating cholesterol levels. F, Total plasma cholesterol levels in mice at 12-, 26- or 52-weeks of age. G, Immunoblot analyses of hepatic LDLR and β-actin expression levels in 8- and 52-week old mice. ApoE^−/− (□ or ○) and apoE^−/−LRP1^n2/n2 (▪ or •) mice, n = 7–12 on a chow diet, data are mean±SEM. *<i>P</i><0.005, **<i>P</i><0.0005.</p

VLDL production, postprandial triglyceride response, intestinal lipid absorption and TRL clearance in apoE^−/− (Δ or □) and ApoE^−/−LRP1^n2/n2 (▪) mice.

<p>A–C, VLDL production after a Tyloxapol injection to inhibit lipolysis (A), postprandial triglyceride response after gastric olive oil load (B) and lipid absorption and chylomicron production after a combined gastric olive oil load and Tyloxapol injection (C) (n = 6–10 per genotype). D, Postprandial accumulation of ³H-Triolein in liver, white adipose tissue (WAT) and proximal (Prox.), medial (Med.) and distal (Dist.) intestine 2 h after a gastric load with olive oil mixed with ³H-Triolein (E) (n = 5 per genotype). Data are mean±SEM. *<i>P</i><0.05, **<i>P</i><0.001.</p

Compensatory LDLR up-regulation associated with an increased chylomicron remnant clearance.

<p>A–C, Internalization of ¹²⁵I-CR (A) and ¹²⁵I-CR-K1 (B) in primary hepatocytes and LDLR immunofluorescence staining in primary hepatocytes (bars are 20 µm) (C). D, Immunoblot analyses of hepatocytes for LDLR and β-actin protein levels after a 16 h incubation period with either 10% FBS or 10% Lipoprotein Deficient FBS (LPDS). E, Immunoblot analysis of microsomal liver extracts for LDLR and β-actin protein levels (n  = 6 per genotype). ApoE^−/− (□) and apoE^−/−LRP1^n2/n2 (▪) mice, data are mean±SEM. *<i>P</i><0.05, **<i>P</i><0.005.</p

Inefficient insulin-mediated LRP1 translocation and impaired slow recycling.

<p>A–B, LRP1 staining in primary hepatocytes before and after insulin stimulation (A) and immunoblot analysis (B) in liver plasma membrane (PM) extracts [ApoE^−/− (□) and apoE^−/−LRP1^n2/n2 (▪)] before and after insulin injection (n = 3–4; bars are 20 µm). C, Cell Fractionation analysis of LRP1^+/+ and LRP1^n2/n2 MEFs. D–F, Steady-state internalization or binding (4°C) of FITC-α₂M in mouse embryonic fibroblasts (MEFs) (D), steady-state internalization of FITC-α₂M in MEFs in the absence (−) or presence (+) of either a lysosomal inhibitor, chloroquine (E), or a proteasomal inhibitor, MG123 (F) [twice in triplicate, LRP1^+/+ (□) and LRP1^n2/n2 (▪)].G–H, Fast (G) and slow (H) recycling kinetics of LRP1 in MEFs at the indicated time intervals [twice in triplicate, LRP1^+/+ (□) and LRP1^n2/n2 (▪)]. Data are mean±SEM. *<i>P</i><0.05, **<i>P</i><0.005, ***<i>P</i><0.001.</p

Hepatic Remnant Lipoprotein Clearance by Heparan Sulfate Proteoglycans and Low-Density Lipoprotein Receptors Depend on Dietary Conditions in Mice

OBJECTIVE: Chylomicron and very low-density lipoprotein remnants are cleared from the circulation in the liver by heparan sulfate proteoglycan (HSPG) receptors (syndecan-1), the low-density lipoprotein receptor (LDLR), and LDLR-related protein-1 (LRP1), but the relative contribution of each class of receptors under different dietary conditions remains unclear. APPROACH AND RESULTS: Triglyceride-rich lipoprotein clearance was measured in AlbCre(+)Ndst1(f/f), Ldlr(−/−), and AlbCre(+)Lrp1(f/f) mice and mice containing combinations of these mutations. Triglyceride measurements in single and double mutant mice showed that HSPGs and LDLR dominate clearance under fasting conditions and postprandial conditions, but LRP1 contributes significantly when LDLR is absent. Mice lacking hepatic expression of all three receptors (AlbCre(+)Ndst1(f/f) Lrp1(f/f) Ldlr(−/−)) displayed dramatic hyperlipidemia (870 ± 270 mg triglyceride/dL; 1300 ± 350 mg of total cholesterol/dL) and exhibited persistent elevated postprandial triglyceride levels due to reduced hepatic clearance. Analysis of the particles accumulating in mutants showed that HSPGs preferentially clear a subset of small triglyceride-rich lipoproteins (~20-40 nm diameter), while LDLR and LRP1 clear larger particles (~40-60 nm diameter). Finally, we show that HSPGs play a major role in clearance of TRLs in mice fed normal chow or under postprandial conditions but appear to play a less significant role on a high fat diet. CONCLUSION: These data show that HSPGs, LDLR, and LRP1 clear distinct subsets of particles, that HSPGs work independently from LDLR and LRP1, and that HSPGs, LDLR, and LRP1 are the three major hepatic TRL clearance receptors in mice