Commemorating insulin's centennial: engineering insulin pharmacology towards physiology
Authors: Peter Kurtzhals, Erica Nishimura, Hanne Haahr, Thomas Høeg-Jensen, Eva Johansson, Peter Madsen, Jeppe Sturis, Thomas Kjeldsen
Journal: Trends in Pharmacological Sciences (2021)

Pharmacokinetically tailored insulin has been rationally designed by molecular structural engineering and/or pharmaceutical formulation to improve glucose control and convenience for people with diabetes.Novel mechanisms for protracted insulin action based on reduced plasma clearance hold promise to enable once-weekly subcutaneous and once-daily oral basal insulin delivery.Hepato-preferential insulin analogs can be designed by tailoring molecular size and receptor affinity to provide more physiological insulin replacement.Successful oral insulin delivery has been achieved in early clinical trials but is still hampered by low bioavailability.Clinical trials have been initiated with glucose-sensitive analogs chemically engineered to switch between active and inactive conformations or to undergo glucose-dependent clearance, thus giving hope that a 'smart insulin' is within reach. The life-saving discovery of insulin in Toronto in 1921 is one of the most impactful achievements in medical history, at the time being hailed as a miracle treatment for diabetes. The insulin molecule itself, however, is poorly amenable as a pharmacological intervention, and the formidable challenge of optimizing insulin therapy has been ongoing for a century. We review early academic insights into insulin structure and its relation to self-association and receptor binding, as well as recombinant biotechnology, which have all been seminal for drug design. Recent developments have focused on combining genetic and chemical engineering with pharmaceutical optimization to generate ultra-rapid and ultra-long-acting, tissue-selective, or orally delivered insulin analogs. We further discuss these developments and propose that future scientific efforts in molecular engineering include realizing the dream of glucose-responsive insulin delivery. The life-saving discovery of insulin in Toronto in 1921 is one of the most impactful achievements in medical history, at the time being hailed as a miracle treatment for diabetes. The insulin molecule itself, however, is poorly amenable as a pharmacological intervention, and the formidable challenge of optimizing insulin therapy has been ongoing for a century. We review early academic insights into insulin structure and its relation to self-association and receptor binding, as well as recombinant biotechnology, which have all been seminal for drug design. Recent developments have focused on combining genetic and chemical engineering with pharmaceutical optimization to generate ultra-rapid and ultra-long-acting, tissue-selective, or orally delivered insulin analogs. We further discuss these developments and propose that future scientific efforts in molecular engineering include realizing the dream of glucose-responsive insulin delivery. The year 2021 marks the centennial of the discovery of insulin in Toronto in 1921, a momentous event in modern medicine. Over the past 100 years we have come to know insulin not only as a life-saving medicine for people with diabetes but also as a complex pharmacological agent with a narrow therapeutic window (see Glossary). Every decade has had its innovative highlights, testifying to the scientific efforts and achievements across disciplines that collectively have brought us to the current state of insulin therapy (Figure 1). These improvements have been driven by the inherent challenge of dosing subcutaneous (s.c.) insulin to precisely match the constantly changing insulin requirement over the day, where rapid insulin action is required during meals whereas slow but steady basal insulin action is necessary between meals – and especially during the night. Therefore, the overarching goal over the past century has been to apply pharmaceutical formulation, chemical engineering, biotechnology, and delivery technology to make it possible to mimic the natural patterns of insulin release from the pancreatic β cells as closely as possible and to reduce the time spent either in hyper- or hypoglycemia. Nevertheless, despite these insulin innovations, the overall level of glycemic control in type 1 diabetes (T1D) and type 2 diabetes (T2D) is far from normalized (Box 1). This calls for continued research focus aiming to further optimize insulin treatment options for people with diabetes.Box 1FAQs and facts about diabetes and insulin therapyWho is in need of insulin therapy?As of 2019, an estimated 463 million people are living with diabetes [101.International Diabetes Federation IDF Diabetes Atlas.9th edn. IDF, 2019Google Scholar]. T1D is a condition that is brought about by autoimmune destruction of insulin-producing pancreatic β cells. Therefore, all people with T1D require insulin treatment. The vast majority (~90%) of people with diabetes have T2D due to failing β cell function, often linked to insulin resistance. T2D can initially be managed by lifestyle adaptations, oral antidiabetic agents, and injectable GLP-1 analogs. However, because of the progressive nature of T2D, many people with T2D will eventually require insulin therapy [2.Basu S. et al.Estimation of global insulin use for type 2 diabetes, 2018–30: a microsimulation analysis.Lancet Diabetes Endocrinol. 2019; 7: 25-33Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar]. The 'rule of halves' [102.Hart J.T. Rule of halves: implications of increasing diagnosis and reducing dropout for future workload and prescribing costs in primary care.Br. J. Gen. Pract. 1992; 42: 116-119PubMed Google Scholar] illustrates that the treatment of T2D is not optimal because only a small fraction of people diagnosed with T2D achieve adequate glycemic control (Figure IA) [48.Bain S.C. et al.Evaluating the burden of poor glycemic control associated with therapeutic inertia in patients with type 2 diabetes in the UK.J. Med. Econ. 2020; 23: 98-105Crossref PubMed Scopus (5) Google Scholar].Why is insulin therapy challenging?Physiological insulin secretion from the pancreas is exquisitely regulated such that the right amount of insulin is present exactly when it is needed (Figure IB). S.c. administration of native human insulin is unable to match the fast appearance of insulin required at meals, nor is it sufficiently flat and long to provide 24 h basal insulin coverage. Therefore, modified insulin analogs and formulations have been engineered to give fast or slow absorption from the injection site for mealtime or basal insulin coverage, respectively. However, mismatches in dosing can occur, and thus many opt to reduce or miss their insulin dose to avoid hypoglycemia, leading to less than optimal glucose control. In addition, many people with T2D who are in need of insulin often delay initiation of insulin therapy (clinical inertia) owing to the perceived complexity, fear of hypoglycemia, and concerns about weight gain associated with daily insulin treatment [47.Russell-Jones D. et al.Identification of barriers to insulin therapy and approaches to overcoming them.Diabetes Obes. Metab. 2018; 20: 488-496Crossref PubMed Scopus (87) Google Scholar].What are the consequences of poor glycemic and metabolic control?Whether it is the result of being undiagnosed, not receiving diabetes care, delaying treatment intensification, or not adhering to prescribed medications, being in poor glycemic and metabolic control leads to long-term complications and a risk of early death. Poor glycemic control in diabetes leads to microvascular complications that affect many organs, notably kidneys, eyes, and nerves, while the overall poor metabolic regulation, including dyslipidemia, associated with diabetes results in damage to the cardiovascular system and macrovascular complications (Figure IC) [101.International Diabetes Federation IDF Diabetes Atlas.9th edn. IDF, 2019Google Scholar,103.American Diabetes Association Cardiovascular disease and risk management: standards of medical care in diabetes – 2021.Diabetes Care. 2021; 44: S125-S150Crossref PubMed Scopus (16) Google Scholar,104.American Diabetes Association Microvascular complications and foot care: standards of medical care in diabetes – 2021.Diabetes Care. 2021; 44: S151-S167Crossref PubMed Scopus (16) Google Scholar].Figure IComplexity of diabetes treatment.Show full captionAbbreviation: T2D, type 2 diabetes. Panel B adapted, with permission, from [105.Polonski K.S. et al.Twenty-four-hour profiles and pulsatile patterns of insulin secretion in normal and obese subjects.J. Clin. Invest. 1988; 81: 442-448Crossref PubMed Google Scholar].View Large Image Figure ViewerDownload Hi-res image Download (PPT) Who is in need of insulin therapy? As of 2019, an estimated 463 million people are living with diabetes [101.International Diabetes Federation IDF Diabetes Atlas.9th edn. IDF, 2019Google Scholar]. T1D is a condition that is brought about by autoimmune destruction of insulin-producing pancreatic β cells. Therefore, all people with T1D require insulin treatment. The vast majority (~90%) of people with diabetes have T2D due to failing β cell function, often linked to insulin resistance. T2D can initially be managed by lifestyle adaptations, oral antidiabetic agents, and injectable GLP-1 analogs. However, because of the progressive nature of T2D, many people with T2D will eventually require insulin therapy [2.Basu S. et al.Estimation of global insulin use for type 2 diabetes, 2018–30: a microsimulation analysis.Lancet Diabetes Endocrinol. 2019; 7: 25-33Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar]. The 'rule of halves' [102.Hart J.T. Rule of halves: implications of increasing diagnosis and reducing dropout for future workload and prescribing costs in primary care.Br. J. Gen. Pract. 1992; 42: 116-119PubMed Google Scholar] illustrates that the treatment of T2D is not optimal because only a small fraction of people diagnosed with T2D achieve adequate glycemic control (Figure IA) [48.Bain S.C. et al.Evaluating the burden of poor glycemic control associated with therapeutic inertia in patients with type 2 diabetes in the UK.J. Med. Econ. 2020; 23: 98-105Crossref PubMed Scopus (5) Google Scholar]. Why is insulin therapy challenging? Physiological insulin secretion from the pancreas is exquisitely regulated such that the right amount of insulin is present exactly when it is needed (Figure IB). S.c. administration of native human insulin is unable to match the fast appearance of insulin required at meals, nor is it sufficiently flat and long to provide 24 h basal insulin coverage. Therefore, modified insulin analogs and formulations have been engineered to give fast or slow absorption from the injection site for mealtime or basal insulin coverage, respectively. However, mismatches in dosing can occur, and thus many opt to reduce or miss their insulin dose to avoid hypoglycemia, leading to less than optimal glucose control. In addition, many people with T2D who are in need of insulin often delay initiation of insulin therapy (clinical inertia) owing to the perceived complexity, fear of hypoglycemia, and concerns about weight gain associated with daily insulin treatment [47.Russell-Jones D. et al.Identification of barriers to insulin therapy and approaches to overcoming them.Diabetes Obes. Metab. 2018; 20: 488-496Crossref PubMed Scopus (87) Google Scholar]. What are the consequences of poor glycemic and metabolic control? Whether it is the result of being undiagnosed, not receiving diabetes care, delaying treatment intensification, or not adhering to prescribed medications, being in poor glycemic and metabolic control leads to long-term complications and a risk of early death. Poor glycemic control in diabetes leads to microvascular complications that affect many organs, notably kidneys, eyes, and nerves, while the overall poor metabolic regulation, including dyslipidemia, associated with diabetes results in damage to the cardiovascular system and macrovascular complications (Figure IC) [101.International Diabetes Federation IDF Diabetes Atlas.9th edn. IDF, 2019Google Scholar,103.American Diabetes Association Cardiovascular disease and risk management: standards of medical care in diabetes – 2021.Diabetes Care. 2021; 44: S125-S150Crossref PubMed Scopus (16) Google Scholar,104.American Diabetes Association Microvascular complications and foot care: standards of medical care in diabetes – 2021.Diabetes Care. 2021; 44: S151-S167Crossref PubMed Scopus (16) Google Scholar]. The insulin structure has revealed physiological mechanisms for insulin storage and secretion and has at the same time been applied to modify insulin pharmacokinetics and pharmacodynamics. Hence, today's rationally designed insulin analogs used by millions of people with diabetes [1.Mobasseri M. et al.Prevalence and incidence of type 1 diabetes in the world: a systematic review and meta-analysis.Health Promot. Perspect. 2020; 10: 98-115Crossref PubMed Scopus (17) Google Scholar,2.Basu S. et al.Estimation of global insulin use for type 2 diabetes, 2018–30: a microsimulation analysis.Lancet Diabetes Endocrinol. 2019; 7: 25-33Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar] provide a unique example of the advances in drug discovery that can be accomplished when basic science meets translational biotechnology. With a focus on the structural engineering and molecular pharmacology of key past, current, and emerging insulin analog concepts, the current review chronicles the progress of insulin molecular design from 1921 until today, followed by a discussion of the coming generation of insulin analogs that have the potential to address the limitations of today's insulin products. The structure of the insulin molecule and how it interacts with its receptor form the foundation of drug design. Native human insulin consists of two chains of amino acids, the A-chain with 21 residues and the B-chain with 30 residues (Figure 2A ). Two disulfide bridges crosslink the chains, namely A7–B7 and A20–B19, and a third (A6–A11) is internal within the A-chain. The amino acid sequence (i.e., the primary structure) of insulin was deduced by Sanger, who received the 1958 Nobel prize for this work [3.Sanger F. Tuppy H. The amino-acid sequence in the phenylalanyl chain of insulin. I. The identification of lower peptides from partial hydrolysates.Biochem. J. 1951; 49: 463-481Crossref PubMed Google Scholar, 4.Sanger F. Thompson E.O. The amino-acid sequence in the glycyl chain of insulin. I. The identification of lower peptides from partial hydrolysates.Biochem. J. 1953; 53: 353-366Crossref PubMed Google Scholar, 5.Ryle A.P. et al.The disulphide bonds of insulin.Biochem. J. 1955; 60: 541-556Crossref PubMed Google Scholar]. The amino acid sequence shows a high degree of homology across vertebrates. The secondary, tertiary, and quaternary structures of insulin were revealed when Nobel laureate Hodgkin et al. solved the crystal structure of the hormone in 1969 [6.Adams M.J. et al.Structure of rhombohedral 2 zinc insulin crystals.Nature. 1969; 224: 491-495Crossref Scopus (397) Google Scholar]. The structure of insulin showed that the molecule folds into three α-helices and a β-strand (Figure 3A ) [6.Adams M.J. et al.Structure of rhombohedral 2 zinc insulin crystals.Nature. 1969; 224: 491-495Crossref Scopus (397) Google Scholar]. The crystal structure also revealed how the insulin hexamer is built from three insulin dimers, each formed by hydrophobic interactions between two monomers with antiparallel facing of the B-chain C-terminal β-strands. Intriguingly, the insulin hexamer can exist in two forms, depending on the conformation of the monomers [7.Derewenda U. et al.Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer.Nature. 1989; 338: 594-596Crossref PubMed Google Scholar] (Figure 3A). Thus, binding of phenol to the hexamer induces the insulin molecules to adapt the so-called relaxed or R-state in which the B-chain helix is extended from B7–B18 towards the N terminus (B1–B6). This fold is described as an R6 hexamer. In the absence of phenol, the N-terminal part of the B-chain adopts a non-helical fold, termed the tense or T-state, and the hexamer is described as T6. An intermediate hexamer has also been described, namely T3R3, that comprises two trimers, one in the R-state and the other in the T-state. Insulin exerts its biological activity via binding to its cognate receptor in target tissues (Box 2). The insulin receptor (IR) is dimeric, and its 3D structure as well as its interaction with insulin have been partially determined by X-ray analysis and cryo-electron microscopy [8.Menting J.G. et al.How insulin engages its primary binding site on the insulin receptor.Nature. 2013; 493: 241-245Crossref PubMed Scopus (223) Google Scholar,9.Uchikawa E. et al.Activation mechanism of the insulin receptor revealed by cryo-EM structure of the fully liganded receptor–ligand complex.eLife. 2019; 8e48630Crossref PubMed Scopus (0) Google Scholar]. Mutagenesis studies have also provided insight into the receptor-binding domains of the hormone [10.Kristensen C. et al.Alanine scanning mutagenesis of insulin.J. Biol. Chem. 1997; 272: 12978-12983Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar]. The binding of insulin to the IR has been described as occurring via two mainly hydrophobic patches on the insulin monomer surface, termed site 1 and site 2 (Figure 3B) [11.Ward C.W. Lawrence M.C. Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor.Bioessays. 2009; 31: 422-434Crossref PubMed Scopus (100) Google Scholar].Box 2Insulin molecular pharmacology applied to drug designThe principles underlying insulin molecular pharmacology are central to the design of insulin analogs to achieve modified properties. Insulin exerts its biological activity through binding to the IR, a transmembrane tyrosine kinase receptor [106.Ullrich A. et al.Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes.Nature. 1985; 313: 756-761Crossref PubMed Scopus (1457) Google Scholar]. Two major surfaces, termed site 1 and site 2, composed of amino acid residues from both the A- and B-chains of the insulin molecule, are recognized by the IR resulting in high-affinity receptor binding (Figure IA) [9.Uchikawa E. et al.Activation mechanism of the insulin receptor revealed by cryo-EM structure of the fully liganded receptor–ligand complex.eLife. 2019; 8e48630Crossref PubMed Scopus (0) Google Scholar,11.Ward C.W. Lawrence M.C. Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor.Bioessays. 2009; 31: 422-434Crossref PubMed Scopus (100) Google Scholar]. The classical binding site (site 1) comprises residues A1–A3, A5, A8, A19, A21, B12, B16, and B23–B26. Evidently, there is a large overlap between this site and the dimer-forming surface (Figure 3); hence, the species recognized by the IR is the insulin monomer. A second receptor-binding surface (site 2) involves residues A12, A13, A17, B10, B13, and B17 from the hexamer-forming face of the molecule [11.Ward C.W. Lawrence M.C. Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor.Bioessays. 2009; 31: 422-434Crossref PubMed Scopus (100) Google Scholar]. Insulin binding leads to a conformational change and phosphorylation of tyrosine residues in the intracellular part of the receptor to initiate a signaling cascade with two main pathways: one through protein kinase B (AKT) that leads to metabolic effects in target tissues including glucose uptake, glycogen synthesis, lipid synthesis, and protein synthesis, and another pathway through extracellular signal-regulated kinase (ERK) that leads to cell proliferation (Figure IB) [107.Haeusler R.A. et al.Biochemical and cellular properties of insulin receptor signalling.Nat. Rev. Mol. Cell Biol. 2018; 19: 31-44Crossref PubMed Scopus (162) Google Scholar,108.Boucher J. et al.Insulin receptor signaling in normal and insulin-resistant states.Cold Spring Harb. Perspect. Biol. 2014; 6a009191Crossref PubMed Scopus (534) Google Scholar]. IR binding is important in drug design not only because of the obvious link to pharmacodynamics but also for pharmacokinetics because the main elimination route for insulin is via IR-mediated clearance [109.Tokarz V.L. et al.The cell biology of systemic insulin function.J. Cell Biol. 2018; 217: 2273-2289Crossref PubMed Scopus (83) Google Scholar]. Thus, following receptor binding, the IR complex is internalized and insulin undergoes lysosomal degradation, while the receptor is recycled to the cell surface (Figure IC) [109.Tokarz V.L. et al.The cell biology of systemic insulin function.J. Cell Biol. 2018; 217: 2273-2289Crossref PubMed Scopus (83) Google Scholar]. Insulin analogs with reduced affinity for the IR will be cleared more slowly by IRs and will be prone to renal elimination [110.Ribel U. et al.Equivalent in vivo biological activity of insulin analogues and human insulin despite different in vitro potencies.Diabetes. 1990; 39: 1033-1039Crossref PubMed Google Scholar]. However, analogs designed to escape renal elimination will retain full biological efficacy despite lower receptor affinity because the only remaining elimination route is through receptor binding and activation. Moreover, because of the lower receptor affinity, such analogs are cleared more slowly and hence have a longer half-life. Insulin analogs have been engineered by strategies that modify pharmacokinetics and/or distribution, but preserve the molecular pharmacology of human insulin by activating the IR and downstream signaling elements similarly to human insulin. The insulin analogs approved for clinical use generally fulfill this criterion [20.Gammeltoft S. et al.Insulin aspart: a novel rapid-acting human insulin analogue.Expert Opin. Investig. Drugs. 1999; 8: 1431-1442Crossref PubMed Scopus (0) Google Scholar,111.Kurtzhals P. et al.Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use.Diabetes. 2000; 49: 999-1005Crossref PubMed Google Scholar, 112.Hennige A.M. et al.Insulin glulisine: insulin receptor signaling characteristics in vivo.Diabetes. 2005; 54: 361-366Crossref PubMed Scopus (0) Google Scholar, 113.Sommerfeld M.A. et al.In vitro metabolic and mitogenic signaling of insulin glargine and its metabolites.PLoS One. 2010; 5e9540Crossref PubMed Scopus (102) Google Scholar]. The principles underlying insulin molecular pharmacology are central to the design of insulin analogs to achieve modified properties. Insulin exerts its biological activity through binding to the IR, a transmembrane tyrosine kinase receptor [106.Ullrich A. et al.Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes.Nature. 1985; 313: 756-761Crossref PubMed Scopus (1457) Google Scholar]. Two major surfaces, termed site 1 and site 2, composed of amino acid residues from both the A- and B-chains of the insulin molecule, are recognized by the IR resulting in high-affinity receptor binding (Figure IA) [9.Uchikawa E. et al.Activation mechanism of the insulin receptor revealed by cryo-EM structure of the fully liganded receptor–ligand complex.eLife. 2019; 8e48630Crossref PubMed Scopus (0) Google Scholar,11.Ward C.W. Lawrence M.C. Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor.Bioessays. 2009; 31: 422-434Crossref PubMed Scopus (100) Google Scholar]. The classical binding site (site 1) comprises residues A1–A3, A5, A8, A19, A21, B12, B16, and B23–B26. Evidently, there is a large overlap between this site and the dimer-forming surface (Figure 3); hence, the species recognized by the IR is the insulin monomer. A second receptor-binding surface (site 2) involves residues A12, A13, A17, B10, B13, and B17 from the hexamer-forming face of the molecule [11.Ward C.W. Lawrence M.C. Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor.Bioessays. 2009; 31: 422-434Crossref PubMed Scopus (100) Google Scholar]. Insulin binding leads to a conformational change and phosphorylation of tyrosine residues in the intracellular part of the receptor to initiate a signaling cascade with two main pathways: one through protein kinase B (AKT) that leads to metabolic effects in target tissues including glucose uptake, glycogen synthesis, lipid synthesis, and protein synthesis, and another pathway through extracellular signal-regulated kinase (ERK) that leads to cell proliferation (Figure IB) [107.Haeusler R.A. et al.Biochemical and cellular properties of insulin receptor signalling.Nat. Rev. Mol. Cell Biol. 2018; 19: 31-44Crossref PubMed Scopus (162) Google Scholar,108.Boucher J. et al.Insulin receptor signaling in normal and insulin-resistant states.Cold Spring Harb. Perspect. Biol. 2014; 6a009191Crossref PubMed Scopus (534) Google Scholar]. IR binding is important in drug design not only because of the obvious link to pharmacodynamics but also for pharmacokinetics because the main elimination route for insulin is via IR-mediated clearance [109.Tokarz V.L. et al.The cell biology of systemic insulin function.J. Cell Biol. 2018; 217: 2273-2289Crossref PubMed Scopus (83) Google Scholar]. Thus, following receptor binding, the IR complex is internalized and insulin undergoes lysosomal degradation, while the receptor is recycled to the cell surface (Figure IC) [109.Tokarz V.L. et al.The cell biology of systemic insulin function.J. Cell Biol. 2018; 217: 2273-2289Crossref PubMed Scopus (83) Google Scholar]. Insulin analogs with reduced affinity for the IR will be cleared more slowly by IRs and will be prone to renal elimination [110.Ribel U. et al.Equivalent in vivo biological activity of insulin analogues and human insulin despite different in vitro potencies.Diabetes. 1990; 39: 1033-1039Crossref PubMed Google Scholar]. However, analogs designed to escape renal elimination will retain full biological efficacy despite lower receptor affinity because the only remaining elimination route is through receptor binding and activation. Moreover, because of the lower receptor affinity, such analogs are cleared more slowly and hence have a longer half-life. Insulin analogs have been engineered by strategies that modify pharmacokinetics and/or distribution, but preserve the molecular pharmacology of human insulin by activating the IR and downstream signaling elements similarly to human insulin. The insulin analogs approved for clinical use generally fulfill this criterion [20.Gammeltoft S. et al.Insulin aspart: a novel rapid-acting human insulin analogue.Expert Opin. Investig. Drugs. 1999; 8: 1431-1442Crossref PubMed Scopus (0) Google Scholar,111.Kurtzhals P. et al.Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use.Diabetes. 2000; 49: 999-1005Crossref PubMed Google Scholar, 112.Hennige A.M. et al.Insulin glulisine: insulin receptor signaling characteristics in vivo.Diabetes. 2005; 54: 361-366Crossref PubMed Scopus (0) Google Scholar, 113.Sommerfeld M.A. et al.In vitro metabolic and mitogenic signaling of insulin glargine and its metabolites.PLoS One. 2010; 5e9540Crossref PubMed Scopus (102) Google Scholar]. The observation that insulin is naturally stored in the pancreatic β cells as stable Zn(II) hexamers [12.Emdin S.O. et al.Role of zinc in insulin biosynthesis. Some possible zinc insulin interactions in the pancreatic B-cell.Diabetologia. 1980; 19: 174-182Crossref PubMed Scopus (0) Google Scholar], which spontaneously dissociate into the biologically active monomers upon secretion into the blood, has been inspirational in the design of particularly fast-acting insulin analogs. Insulin self-association has been an important parameter in understanding and ultimately manipulating the absorption rate of insulin from the s.c. injection site [13.Brange J. et al.Monomeric insulins obtained by protein engineering and their medical implications.Nature. 1988; 333: 679-682Crossref PubMed Google Scholar]. This is because molecular size has a major role in governing the rate of diffusion in the tissue and the rate of passage through the capillary wall. Hence, the absorption rate of an insulin preparation depends on its self-association state, where monomers are absorbed faster and dimers and hexamers more slowly [14.Hildebrandt P. et al.Diffusion and polymerization determines the insulin absorption from subcutaneous tissue in diabetic patients.Scand. J. Clin. Lab. Invest. 1985; 45: 685-690Crossref PubMed Scopus (60) Google Scholar,15.Mosekilde E. et al.Modeling absorption kinetics of subcutaneous injected soluble insulin.J. Pharmacokinet. Biopharm. 1989; 17: 67-87Crossref PubMed Google Scholar]. Molecular strategies for weakening the insulin hexamer to more rapidly dissociate after injection have included rational substitutions in the monomer–monomer interaction surface as well as removal of the zinc-binding site [13.Brange J. et al.Monomeric insulins obtained by protein engineering and their medical implications.Nature. 1988; 333: 679-682Crossref PubMed Google Scholar]. Brange et al. were the first to successfully demonstrate a faster absorption and action of a rationally engineered insulin analog (B28Asp insulin, aspart; Figure 2B) as compared to native human insulin [13.Brange J. et al.Monomeric insulins obtained by protein engineering and their medical implications.Nature. 1988; 333: 679-682Crossref PubMed Google Scholar]. In insulin aspart, dimer fo