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The discovery of insulin in 1921 has been one of greatest scientific achievements of the 20th century. Since then, the availability of insulin has shifted the focus of diabetes treatment from trying to keep patients alive to saving and improving the life of millions. Throughout this time, basic and clinical research has advanced our understanding of insulin synthesis and action, both in healthy and pathological conditions. Yet, multiple aspects of insulin production remain unknown. In this review, we focus on the most recent findings on insulin synthesis, highlighting their relevance in diabetes.

In this phase 2 trial, children and young adults with newly diagnosed overt type 1 diabetes were randomly assigned to receive golimumab, a human monoclonal antibody to tumor necrosis factor α , or placebo. Golimumab resulted in better endogenous insulin production and less exogenous insulin use than placebo.

Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell-derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcsk1 expression were reduced in hypothalami of fasted Snord116 paternal knockout (Snord116p-/m+) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p-/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that Snord116p-/m+ mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH-releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency.

The production of recombinant insulin remains challenging, particularly in enhancing refolding efficiency and bioactivity. Mini-proinsulin analogs, which involve reducing the length of the C-peptide, offer potential improvements in insulin production. This study aims to evaluate mini-proinsulin analogs’ design and receptor binding dynamics to optimize recombinant insulin production in E. coli . Mini-proinsulin analogs were engineered by replacing the 33-residue C-peptide with a pentapeptide sequence to improve refolding. The three-dimensional structure of mini-proinsulin was predicted using AlphaFold and performed docking analysis of mini-proinsulin analogs to the insulin receptor using AutoDock Tools, with comparisons made to previously available NMR-determined analog and the native insulin-insulin receptor complex. Normal Mode Analyses (GNM and ANM) were performed in detail to assess binding dynamics. In silico analyses revealed that mini-proinsulin analogs closely replicate the structural features of native insulin and display receptor binding dynamics similar to native insulin, though they follow distinct receptor interaction paths. All analysis suggests that C-peptide mobility may contribute to the allosteric behavior observed in mini-proinsulin analogs during receptor interaction.

In pancreatic β cells, misfolded proinsulin is a substrate for ER-associated protein degradation (ERAD) via HRD1/SEL1L. Alternately, β cell HRD1 activity is reported to improve, or impair, insulin biogenesis. Further, while β cell SEL1L deficiency causes HRD1 hypofunction and diminishes islet insulin content, reports conflict as to whether β cell ERAD deficiency increases or decreases proinsulin levels. Here, we examined β cell-specific Hrd1-KO mice (chronic deficiency) and rodent (and human islet) β cells treated acutely with HRD1 inhibitor. β-Hrd1-KO mice developed diabetes with decreased islet proinsulin, yet a relative increase of misfolded proinsulin redistributed to the ER. They also showed upregulated biochemical markers of β cell ER stress and autophagy, electron microscopy evidence of ER enlargement and decreased insulin granule content, and increased glucagon-positive islet cells. Misfolded proinsulin was also increased in islets treated with inhibitors of lysosomal degradation. Preceding any loss of total proinsulin, acute HRD1 inhibition triggered increased nonnative proinsulin, increased phospho-eIF2α with inhibited proinsulin synthesis, and increased LC3b-II (the abundance of which requires expression of ΣR1). We posit a subset of proinsulin molecules undergo HRD1-mediated disposal. When HRD1 is unavailable, misfolded proinsulin accumulates, accompanied by increased phospho-eIF2α that limits further proinsulin synthesis, plus ΣR1-dependent autophagy activation, ultimately lowering steady-state β cell proinsulin (and insulin) levels and triggering diabetes.

Biosynthesis of insulin – critical to metabolic homeostasis – begins with folding of the proinsulin precursor, including formation of three evolutionarily conserved intramolecular disulfide bonds. Remarkably, normal pancreatic islets contain a subset of proinsulin molecules bearing at least one free cysteine thiol. In human (or rodent) islets with a perturbed endoplasmic reticulum folding environment, non-native proinsulin enters intermolecular disulfide-linked complexes. In genetically obese mice with otherwise wild-type islets, disulfide-linked complexes of proinsulin are more abundant, and leptin receptor-deficient mice, the further increase of such complexes tracks with the onset of islet insulin deficiency and diabetes. Proinsulin-Cys(B19) and Cys(A20) are necessary and sufficient for the formation of proinsulin disulfide-linked complexes; indeed, proinsulin Cys(B19)-Cys(B19) covalent homodimers resist reductive dissociation, highlighting a structural basis for aberrant proinsulin complex formation. We conclude that increased proinsulin misfolding via disulfide-linked complexes is an early event associated with prediabetes that worsens with ß-cell dysfunction in type two diabetes. Our body fine-tunes the amount of sugar in our blood thanks to specialized ‘beta cells’ in the pancreas, which can release a hormone called insulin. To produce insulin, the beta cells first need to build an early version of the molecule – known as proinsulin – inside a cellular compartment called the endoplasmic reticulum. This process involves the formation of internal staples that keep the molecule of proinsulin folded correctly. Individuals developing type 2 diabetes have spikes of sugar in their blood, and so their bodies often respond by trying to make large amounts of insulin. After a while, the beta cells can fail to keep up, which brings on the full-blown disease. However, scientists have discovered that early in type 2 diabetes, the endoplasmic reticulum of beta cells can already show signs of stress; yet, the exact causes of this early damage are still unknown. To investigate this, Arunagiri et al. looked into whether proinsulin folds correctly during the earliest stages of type 2 diabetes. Biochemical experiments showed that even healthy beta cells contained some misfolded proinsulin molecules, where the molecular staples that should fold proinsulin internally were instead abnormally linking proinsulin molecules together. Further work revealed that the misfolded proinsulin was accumulating inside the endoplasmic reticulum. Finally, obese mice that were in the earliest stages of type 2 diabetes had the highest levels of abnormal proinsulin in their beta cells. Overall, the work by Arunagiri et al. suggests that large amounts of proinsulin molecules stapling themselves to each other in the endoplasmic reticulum of beta cells could be an early hallmark of the disease, and could make it get worse. A separate study by Jang et al. also shows that a protein that limits the misfolding of proinsulin is key to maintain successful insulin production in animals eating a Western-style, high fat diet. Hundreds of millions of people around the world have type 2 diabetes, and this number is rising quickly. Detecting and then fixing early problems associated with the condition may help to stop the disease in its track.

Aims/hypothesisIt is unclear whether type 1 diabetes is a single disease or if endotypes exist. Our aim was to use a unique collection of pancreas samples recovered soon after disease onset to resolve this issue.MethodsImmunohistological analysis was used to determine the distribution of proinsulin and insulin in the islets of pancreas samples recovered soon after type 1 diabetes onset (<2 years) from young people diagnosed at age <7 years, 7–12 years and ≥13 years. The patterns were correlated with the insulitis profiles in the inflamed islets of the same groups of individuals. C-peptide levels and the proinsulin:C-peptide ratio were measured in the circulation of a cohort of living patients with longer duration of disease but who were diagnosed in these same age ranges.ResultsDistinct patterns of proinsulin localisation were seen in the islets of people with recent-onset type 1 diabetes, which differed markedly between children diagnosed at <7 years and those diagnosed at ≥13 years. Proinsulin processing was aberrant in most residual insulin-containing islets of the younger group but this was much less evident in the group ≥13 years (p < 0.0001). Among all individuals (including children in the middle [7–12 years] range) aberrant proinsulin processing correlated with the assigned immune cell profiles defined by analysis of the lymphocyte composition of islet infiltrates. C-peptide levels were much lower in individuals diagnosed at <7 years than in those diagnosed at ≥13 years (median <3 pmol/l, IQR <3 to <3 vs 34.5 pmol/l, IQR <3–151; p < 0.0001), while the median proinsulin:C-peptide ratio was increased in those with age of onset <7 years compared with people diagnosed aged ≥13 years (0.18, IQR 0.10–0.31) vs 0.01, IQR 0.009–0.10 pmol/l; p < 0.0001).Conclusions/interpretationAmong those with type 1 diabetes diagnosed under the age of 30 years, there are histologically distinct endotypes that correlate with age at diagnosis. Recognition of such differences should inform the design of future immunotherapeutic interventions designed to arrest disease progression.

As a tool to explore proinsulin (PI) trafficking, a human PI cDNA has been constructed with GFP fused within the C peptide. In regulated secretory cells containing appropriate prohormone convertases, the hProCpepGFP construct undergoes endoproteolytic processing to CpepGFP and native human insulin, which are specifically detected and cosecreted in parallel with endogenous insulin. Expression of C(A7)Y mutant PI results in autosomal dominant diabetes in Akita mice. We directly identify the misfolded PI in Akita islets and also show that C(A7)Y mutant PI, either in the context of the hProCpepGFP chimera or not, engages directly in protein complexes with nonmutant PI, impairing the trafficking and recovery of nonmutant PI. This trapping mechanism decreases insulin production in β cells. Thereafter we observe a loss of β cell viability. The data imply that PI misfolding leading to impaired endoplasmic reticulum exit of nonmutant PI may be a key early step in a chain reaction of β cell dysfunction and demise leading to onset and progression of diabetes.

To date, structural analysis has demonstrated a highly consistent binding pattern of the TCR to the MHC molecule. Rossjohn and colleagues reveal the first structures of two human T reg cell TCRs and show that they bind with a reversed polarity to the MHC. Central to adaptive immunity is the interaction between the αβ T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT reg ) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT reg TCR α-chain and β-chain are overlaid with the α-chain and β-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.

A precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)–Cys(A20) “interchain” disulfide bond, facilitating formation of the Cys(B7)–Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)–Cys(A11), is not required for anterograde trafficking, i.e., a “lose-A6/A11” mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)–Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.