Human islets expressing HNF1A variant have defective β cell transcriptional regulatory networks

Abstract

Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained β cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.

Keywords: Diabetes; Endocrinology; Insulin; Islet cells.

Figure 1. Histological and functional analysis of HNF1A^+/T260M pancreas and islets.

(A) Example of expression of insulin (INS), glucagon (GCG), and somatostatin (SOM) in donor’s native pancreatic tissue compared with control. Scale bar: 50 μm. (B) β and α cell mass (grams) in HNF1A^+/T260M pancreas compared with controls (n = 7 donors; ages 10–55 years). Each data point represents the average mass across the combined pancreatic head, body, and tail regions. (C) Insulin secretion in islets isolated from the HNF1A^+/T260M pancreas compared with controls (n = 6 donors; ages 8–55 years) and normalized to overall islet cell volume (expressed as islet equivalents, IEQs). Islets were teated with 5.6 mM glucose (G 5.6); 16.7 mM glucose (G 16.7); 16.7 mM glucose plus 100 μM isobutylmethylxanthine (G 16.7 + IBMX 100); 1.7 mM glucose plus 1 μM epinephrine (G 1.7 + Epi 1); or 20 mM potassium chloride (KCl 20) at the indicated times. Insets show average insulin response of controls and HNF1A^+/T260M donor to 30-minute stimulation with 16.7 mM glucose. (D) Integrated insulin secretion was calculated as the area under the curve (AUC) for the indicated secretagogue (shaded to correspond to color-matched regions of perifusion trace in panel C). Results of control samples are expressed as mean ± SEM.

Figure 2. Expression and functional characterization of HNF1A^T260M variant.

(A) Analysis of donor’s native pancreatic tissue for HNF1A compared with controls (n = 4 donors; ages 10–55 years) revealed HNF1A protein in donor β cells. Scale bar: 50 μm. (B) Electrophoretic mobility shift assay (EMSA) shows that the HNF1A^T260M variant has impaired DNA binding, with loss of the HNF1A-specific DNA binding complex (arrow) in Myc-tagged HNF1A^T260M–transfected HeLa cells compared with Myc-tagged HNF1A^WT. Specificity of this complex (arrow) was shown by exclusive elimination of these species by adding either Myc antibody (Myc-Ab) or unlabeled oligonucleotide (WT Oligo) containing the HNF1A consensus recognition motif, but not a mutated form of this oligonucleotide (Comp Oligo). Moreover, HNF1A antibody (HNF1A-Ab) only supershifted (s.s.) this complex. All samples in B include oligonucleotide labeled with ³²P as described in the supplemental material. Asterisk indicates nonspecific complexes. NT, nontransfected HeLa cells. One representative experiment of 3 is shown. (C) Molecular modeling of the HNF1A^T260 variant in PyMOL predicts that the hydroxyl group (red) on threonine 260 (Thr-260) stabilizes arginine 263 (Arg-263) by hydrogen bonding to nitrogen (blue). Arg-263 H-bonds to the DNA backbone of the fifth adenosine of the HNF1A consensus recognition motif (5′-CTTGGTTAATAATTCACCAGA-3′) in control conditions (18). A missense mutation from threonine to methionine at position 260 is predicted to result in the loss of this interaction by destabilizing Arg-263 and subsequently DNA binding. Results of control samples are expressed as mean ± SEM. See complete unedited blots in the supplemental material.

Figure 3. Transcriptomic analysis of HNF1A^+/T260M β cells.

(A) Principal component analysis (PCA) plot depicts clustering of control β cells (n = 5 donors; ages 26–55 years) (31) separate from HNF1A^+/T260M β cells. (B) The volcano plot demonstrates transcripts differentially expressed between control and HNF1A^+/T260M β cells (red, upregulated gene expression; green, downregulated gene expression). Differential expression was calculated based on fold change (FC) ≥1.5 with a P-value cutoff of <0.05 for calculated Z score. (C) Genes of interest and HNF1A targets are significantly downregulated in HNF1A^+/T260M β cells. The vertical dotted line represents FC = 1.5 times the threshold; P < 0.05 for all values shown. (D) Significant processes identified by Gene Ontology (GO) term-enrichment analysis are grouped and displayed by their P value on a log₂ scale.

Figure 4. Model of HNF1A dysfunction in human β cells.

From these results, we propose that dysfunction of HNF1A leads to decreased expression of direct targets, which encompass both enzymatic and gene regulatory products, producing broad changes in transcriptional regulation, glucose metabolism, and hormone secretion. These processes ultimately lead to β cell dysfunction and result in clinical manifestation of insulin-insufficient diabetes.