Proteomic Changes to the Updated Discovery of Engineered Insulin and Its Analogs: Pros and Cons

The destruction of β-cells of the pancreas leads to either insulin shortage or the complete absence of insulin, which in turn causes diabetes Mellitus. For treating diabetes, many trials have been conducted since the 19th century until now. In ancient times, insulin from an animal’s extract was taken to treat human beings. However, this resulted in some serious allergic reactions. Therefore, scientists and researchers have tried their best to find alternative ways for managing diabetes with progressive advancements in biotechnology. However, a lot of research trials have been conducted, and they discovered more progressed strategies and approaches to treat type I and II diabetes with satisfaction. Still, investigators are finding more appropriate ways to treat diabetes accurately. They formulated insulin analogs that mimic the naturally produced human insulin through recombinant DNA technology and devised many methods for appropriate delivery of insulin. This review will address the following questions: What is insulin preparation? How were these devised and what are the impacts (both positive and negative) of such insulin analogs against TIDM (type-I diabetes mellitus) and TIIDM (type-II diabetes mellitus)? This review article will also demonstrate approaches for the delivery of insulin analogs into the human body and some future directions for further improvement of insulin treatment.     

Nasal insulin gel as an alternate to parenteral insulin: Formulation,preclinical, and clinical studies

The objective of the present study was to formulate insulin gel for intranasal administration and to evaluate with respect to in vitro release studies and hypoglycemic activity in animal model and healthy human volunteers. The insulin gel was formulated using the combination of carbopol and hydroxypropyl methylcellulose as gelling agent. The in vivo efficacy of insulin gel administered intranasally was assessed by measuring the blood glucose levels and serum insulin levels at specified time intervals in rats and humans. The use of bioadhesive nasal gel containing insulin not only promoted the prolonged contact between the drug and the absorptive sites in the nasal cavity but also facilitated direct absorption of medicament through the nasal mucosa. Absorption of the drug through the nasal mucosa was high in the first 0.5 to 1.5 hours of the study with a sharp decline in blood sugar and rise in insulin values corresponding to that decline in blood sugar. This study further demonstrates that administration of insulin intranasally in gel form is a pleasant and painless alternative to injectable insulin. Published: September 30, 2005.     

Effect of polyethylenimine on recombinant adeno-associated virus mediated insulin gene therapy

BACKGROUND: Recombinant adeno-associated virus (rAAV) is becoming a promising vector for gene therapy for type I diabetes. The objective of this study was to investigate the effect of incorporation of polyethylenimine (PEI) on rAAV-mediated insulin gene therapy in vitro and in vivo. METHODS: Recombinant AAV vector, harboring the furin-mutated human insulin and enhanced green fluorescent protein (EGFP) genes, was constructed. The effect of complexation with PEI on rAAV-mediated gene transfer was examined in Huh7 human hepatoma cells. The transgene expression was also examined in streptozotocin (STZ)-induced diabetic C57BL/6J mice by direct administration of rAAV into the livers of the animals, followed by monitoring changes in body weight and blood glucose levels. Secretion of human insulin was determined by radioimmunoassay (RIA) and immunohistochemical staining in the livers. RESULTS: Complexation with PEI was shown to enhance rAAV-mediated transgene expression in Huh7 cells, resulting in higher transduction efficiency and enhanced production of immunoreactive human insulin. Heparin competition assay demonstrated that endocytosis of rAAV-PEI was partially inhibited by heparin. The enhancement of rAAV-mediated transgene expression was also demonstrated in the animals, showing lowering of blood glucose and longer duration of normoglycemia. Immunofluorescent staining of the liver sections demonstrated that PEI increased the uptake of rAAV and enhanced insulin secretion. The enhancement of PEI on rAAV-mediated insulin gene therapy was further confirmed by glucose challenge and a 10-h fasting blood glucose test. CONCLUSIONS: Results obtained in this study demonstrated that incorporation of PEI augmented rAAV-mediated insulin gene transfer and enhanced amelioration of hyperglycemia in the STZ-induced diabetic animals.     

胰岛素受体变异性剪接与肿瘤和2型糖尿病

    胰岛素受体基因第11号外显子因为变异性剪接而形成两种胰岛素受体,两者与配体胰岛素、胰岛素样生长因子的结合力以及分别诱导的信号传导通路、发挥的生物学效应存在显著差异。这种差异不仅可能是导致胰岛素抵抗、2型糖尿病的重要原因,也会影响肿瘤细胞的生长、增殖、抗凋亡。虽然具体的调节机制尚不明确,但高胰岛素血症及高血糖等代谢因素是影响胰岛素受体变异性剪接的重要原因,同时基因序列敲除试验证实,胰岛素受体基因水平的改变会影响胰岛素受体的变异性剪接。           

The use of Fmoc-Lys(Pac)-OH and penicillin G acylase in the preparation of novel semisynthetic insulin analogs.

In this paper, we present the detailed synthetic protocol and characterization of Fmoc-Lys(Pac)-OH, its use for the preparation of octapeptides H-Gly-Phe-Tyr-N-MePhe-Thr-Lys(Pac)-Pro-Thr-OH and H-Gly-Phe-Phe-His-Thr-Pro-Lys(Pac)-Thr-OH by solid-phase synthesis, trypsin-catalyzed condensation of these octapeptides with desoctapeptide(B23-B30)-insulin, and penicillin G acylase catalyzed cleavage of phenylacetyl (Pac) group from Nepsilon-amino group of lysine to give novel insulin analogs [TyrB25, N-MePheB26,LysB28,ProB29]-insulin and [HisB26]-insulin. These new analogs display 4 and 78% binding affinity respectively to insulin receptor in rat adipose membranes.     

Insulin receptor binding from mid-term and full-term placentas of patients with gestational diabetes mellitus and normal pregnant women

Insulin receptor binding was examined in the microvillous membranes of mid-term (20–22 weeks of gestation, MT) and full-term (FT) placentas from patients with gestational diabetes mellitus (GDM) and in normal pregnant control (N). Mid-term placentas were obtained from patients who have had spontaneous abortion. The maximum per cent specific binding (%SB) in MT placenta for GDM was significantly lower (4.8%) compared with the FT placenta (22%, p<0.001), while in the N group the maximum per cent specific binding for MT placenta was 14.1% compared with 26% for the FT placneta (p<0.001). Binding data from FT placenta of well-controlled GDM patients were similar with the FT placenta from N group (22%SB for GDM VS 26% SB for N). Even as there were similarities in the binding characteristics of FT placentas from both groups the placental membrane protein content in the GDM group was lower by 50% compared with the N control (2.5±0.11 VS 4.8±0.15 mg protein/g placenta respectively, p<0.001) suggesting that in the GDM group achieving a tight glycemic control could improve receptor affinities. Data from the competitive binding assay of GDM patients showed that the insulin necessary to achieve 50% inhibition (ID₅₀) was significantly lower in MT compared with the FT placenta (0.9×10^–9 M VS 3.8×10^–9 M, p<0.001) but in the N placenta there was no alteration in the ID₅₀ of MT and FT placentas (3.1×10^–9 M VS 4×10^–9 M, p<0.01, respectively). The present study demonstrated that in GDM the placental insulin receptor binding was significantly lower in spontaneously aborted placenta compared with placentas collected at full-term. Furthermore, these data suggest that the objective to achieve a tight glycemic control in GDM patients could optimize insulin receptor function similar to that of a normal pregnancy. Thus a full term placenta from GDM patients under a well managed glycemic control throughout the entire duration of pregnancy would result in an optimum insulin receptor function.     

Insulin resistance: Review of the underlying molecular mechanisms

Most human cells utilize glucose as the primary substrate, cellular uptake requiring insulin. Insulin signaling is therefore critical for these tissues. However, decrease in insulin sensitivity due to the disruption of various molecular pathways causes insulin resistance (IR). IR underpins many metabolic disorders such as type 2 diabetes and metabolic syndrome, impairments in insulin signaling disrupting entry of glucose into the adipocytes, and skeletal muscle cells. Although the exact underlying cause of IR has not been fully elucidated, a number of major mechanisms, including oxidative stress, inflammation, insulin receptor mutations, endoplasmic reticulum stress, and mitochondrial dysfunction have been suggested. In this review, we consider the role these cellular mechanisms play in the development of IR.     

Fasting-based estimates of insulin sensitivity in overweight and obesity: a critical appraisal

Objective: To identify simple methods to estimate the degree of insulin resistance. Research Methods and Procedures: The performance of a wide range of fasting‐based index estimates of insulin sensitivity was compared by receiver operating characteristic analysis (area under curves and their 95% confidence intervals) against the M value from euglycemic insulin clamp studies collected in the San Antonio (non‐Hispanic whites and Hispanic residents of San Antonio, TX) and European Group for the Study of Insulin Resistance (non‐diabetic white Europeans) databases (n = 638). Results: Insulin resistance differed substantially between lean (BMI < 25 kg/m²), overweight or obese (BMI ≥ 25 kg/m²), and type 2 diabetic individuals. Estimates of insulin resistance were, therefore, assessed in each group separately. In the overweight and obese subgroup (n = 302), the receiver operating characteristic performance of fasting‐based indices varied from 0.72 (0.62 to 0.82), in the case of the insulin/glucose ratio, to 0.80 (0.72 to 0.88) in the case of Belfiore free fatty acids. One superior method could not be identified; the confidence intervals overlapped, and no statistically significant differences emerged. All indices performed better when using the whole study population, with fasting plasma insulin, homeostatic model assessment, insulin/glucose ratio, quantitative insulin sensitivity check index, glucose/insulin ratio, Belfiore glycemia, revised quantitative insulin sensitivity check index, McAuley index, and Belfiore free fatty acids showing area under curves of 0.83, 0.90, 0.66, 0.90, 0.66, 0.90, 0.85, 0.83, and 0.86, respectively, because of the inclusion of very insulin sensitive (lean) and very insulin resistant cases (diabetic subjects). Discussion: In conclusion, a superior fasting‐based index estimate to distinguish between the presence and absence of insulin resistance in overweight and obesity could not be identified despite the use of the large datasets.     

Vanadium and tungsten derivatives as antidiabetic agents: a review of their toxic effects

Tungstate is an oxyanion that has biological similarities to vanadate. In recent years, a number of studies have shown the antidiabetic effects of oral tungstate in animal models of diabetes. However, because of the tissue accumulation and potential toxicity derived from chronic administration of vanadium and tungsten compounds, the pharmacological use of vanadate or tungstate in the treatment of diabetes is not necessarily exempt from concern. In the context of a potential use in the treatment of human diabetes mellitus, the most relevant toxic effects of vanadium derivatives are reviewed and compared with those reported for tungsten. Hematological and biochemical alterations, loss of body weight, nephrotoxicity, immunotoxicity, reproductive and developmental toxicity, and behavioral toxicity have been reported to occur following exposure to vanadium compounds. Moreover, vanadium also has a mitogenic activity affecting the distribution of chromosomes during mitosis and inducing aneuploidyrelated end points. In contrast to vanadate, studies about the toxic effects of tungstate are very scant. Early investigations in cats, rabbits, dogs, mice, and rats showed that tungstate was less toxic than vanadate when given intravenously. Although in vitro investigations showed a direct effect of tungstate on the embryo and fetus of mice at concentrations similar to those causing effects in vivo, information on the potential cellular toxicity of tungstate is particularly scarce. Taking into account the recent interest of tungstate as a new potential oral antidiabetic agent, an exhaustive evaluation of its toxicity in mammals is clearly necessary.     

Non-standard insulin design: structure-activity relationships at the periphery of the insulin receptor.

The design of insulin analogues has emphasized stabilization or destabilization of structural elements according to established principles of protein folding. To this end, solvent-exposed side-chains extrinsic to the receptor-binding surface provide convenient sites of modification. An example is provided by an unfavorable helical C-cap (Thr(A8)) whose substitution by favorable amino acids (His(A8) or Arg(A8)) has yielded analogues of improved stability. Remarkably, these analogues also exhibit enhanced activity, suggesting that activity may correlate with stability. Here, we test this hypothesis by substitution of diaminobutyric acid (Dab(A8)), like threonine an amino acid of low helical propensity. The crystal structure of Dab(A8)-insulin is similar to those of native insulin and the related analogue Lys(A8)-insulin. Although no more stable than native insulin, the non-standard analogue is twice as active. Stability and affinity can therefore be uncoupled. To investigate alternative mechanisms by which A8 substitutions enhance activity, multiple substitutions were introduced. Surprisingly, diverse aliphatic, aromatic and polar side-chains enhance receptor binding and biological activity. Because no relationship is observed between activity and helical propensity, we propose that local interactions between the A8 side-chain and an edge of the hormone-receptor interface modulate affinity. Dab(A8)-insulin illustrates the utility of non-standard amino acids in hypothesis-driven protein design.     

Increased tissue kallikrein amidase activity in urine of patients with type 1 diabetes under insulin therapy, and in those with gestational diabetes mellitus not under insulin therapy

Human tissue kallikrein (hK1) is reduced in hypertension, cardiovascular and renal diseases. There is little information on the participation of hK1 in type 1 diabetes mellitus (DM), type 2 DM, and gestational diabetes mellitus (GDM), respectively. The aim of this study was to evaluate the roles of insulin and hyperglycemia on urinary hK1 activity in type 1 DM and in GDM. Forty-three type 1 DM patients (5–35 years, disease duration ?5 years, receiving insulin, HbA_1c > 7.6%) were selected. Forty-three healthy individuals, paired according to gender and age, were used as controls. Thirty GDM patients (18–42 years, between the 24th and 37th week of pregnancy, recently diagnosed, not under insulin therapy) were also selected. Thirty healthy pregnant (18–42 years, between the 24th and 37th week of pregnancy) and 30 healthy non-pregnant women (18–42 years) were selected as controls. Random midstream urine was used. hK1 amidase activity was estimated with D-Val-Leu-Arg-Nan substrate. Creatinine was determined by Jaffe’s method. hK1 specific amidase activity was expressed as μM/(min mg creatinine) to correct for differences in urine flow rate. hK1 specific amidase activity was significantly higher in the urine of type 1 DM than in controls, and in the urine of GDM patients than in healthy pregnant women and healthy non-pregnant women, respectively. The data suggest that hyperglycemia, rather than insulin, is involved in the mechanism of increased hK1 specific amidase activity in both type 1 DM and GDM patients, respectively.     

5-Hydroxytryptophan: An absorption and fluorescence probe which is a conservative replacement for [A14 tyrosine] in insulin

Use of insulin's intrinsic tyrosine absorption and fluorescence to monitor its interaction with the insulin receptor is limited because the spectral properties of the receptor tryptophan residues mask the spectral properties of the hormone tyrosine residues. We describe the synthesis of an insulin analog where A14 tyrosine is replaced by a tryptophan analog, 5-hydroxytryptophan. This insulin is spectrally enhanced since 5-hydroxytryptophan has an absorption band above 300 nm which is at lower energies than the absorption of tryptophan. Steady-state and time-resolved fluorescence parameters indicate that 5-hydroxytryptophan reports the same information about the environment of the A14 side chain as does the corresponding tryptophan-containing insulin. The synthetic hormone is a full agonist compared to porcine insulin, but has slightly reduced specific activity. Consequently, this spectrally enhanced insulin analog will be useful for hormone-receptor interaction studies since it can be observed by both absorption and fluorescence even in the presence of the tryptophan-containing receptor.     

Improvement of insulin response in the streptozotocin model of insulin-dependent diabetes mellitus. Insulin response with and without a long-acting insulin treatment

Streptozotocin-induced diabetes mellitus (STZ-DM) in rats is a model of type 1 diabetes, which is commonly used to study diabetes, but differs from human diabetic pathophysiology in its insulin resistance. An STZ-DM rat can be administered five times the dose of insulin compared to that of a diabetic patient. Thus, attaining normoglycaemia in STZ-DM rats with insulin injections is complicated, and it involves an obvious risk of overdosing before getting a response. This study was designed to investigate whether suboptimal treatment with long-acting insulin restores insulin sensitivity in the STZ-DM rat, and thus an approach to more closely mimic the human condition. Male Sprague-Dawley rats were made diabetic by means of a single intravenous injection of STZ (55 mg/kg body weight (BW)), resulting in an increase in blood glucose (BG) from 6.5 ± 0.2 to 22.5 ± 1.0 mmol/l (P 0.05) within 24 h. After treating the STZ-DM rats with vehicle for 14 days, BG was 26.1 ± 1.1 mmol/l, and the response to a single injection of fast-acting insulin (Humalog, 5 IE/kg BW) was a 23% reduction in BG. Thereafter, the rats were treated daily with a suboptimal dose of long-acting insulin for a total of 7 days (Insulatard, 5 IE/kg per day), which resulted in a BG level of 19.4 ± 2.7. The response to fast-acting insulin after the suboptimal treatment was a 61% reduction in BG. Thereafter, the animals were vehicle-treated for another 7 days, which resulted in a response to fast-acting insulin similar to the initial values (-34%). Furthermore, the group treated with suboptimal doses of long-acting insulin had a longer duration of the reduction in BG (150 min, as opposed to 90 min in the vehicle-treated groups). We conclude that the development of a decreased insulin response occurs rapidly within the first 2 weeks after the onset of diabetes in STZ-DM rats. This leads to a brief and significantly reduced decrease in BG when fast-acting insulin is administered. The insulin response is increased by treatment with suboptimal doses of long-acting insulin, but rapidly decreases again when treatment is withdrawn. Regular administration of suboptimal insulin doses may provide an approach to eliminate the effects of a lowered insulin response.     

Possible implications of insulin resistance and glucose metabolism in Alzheimer's disease pathogenesis

Type 2 diabetes mellitus (DM) appears to be a significant risk factor for Alzheimer disease (AD). Insulin and insulin-like growth factor-1 (IGF-1) also have intense effects in the central nervous system (CNS), regulating key processes such as neuronal survival and longevity, as well as learning and memory. Hyperglycaemia induces increased peripheral utilization of insulin, resulting in reduced insulin transport into the brain. Whereas the density of brain insulin receptor decreases during age, IGF-1 receptor increases, suggesting that specific insulin-mediated signals is involved in aging and possibly in cognitive decline. Molecular mechanisms that protect CNS neurons against β-amyloid-derived-diffusible ligands (ADDL), responsible for synaptic deterioration underlying AD memory failure, have been identified. The protection mechanism does not involve simple competition between ADDLs and insulin, but rather it is signalling dependent down-regulation of ADDL-binding sites. Defective insulin signalling make neurons energy deficient and vulnerable to oxidizing or other metabolic insults and impairs synaptic plasticity. In fact, destruction of mitochondria, by oxidation of a dynamic-like transporter protein, may cause synapse loss in AD. Moreover, interaction between Aβ and τ proteins could be cause of neuronal loss. Hyperinsulinaemia as well as complete lack of insulin result in increased τ phosphorylation, leading to an imbalance of insulin-regulated τ kinases and phosphatates. However, amyloid peptides accumulation is currently seen as a key step in the pathogenesis of AD. Inflammation interacts with processing and deposit of β-amyloid. Chronic hyperinsulinemia may exacerbate inflammatory responses and increase markers of oxidative stress. In addition, insulin appears to act as 'neuromodulator', influencing release and reuptake of neurotransmitters, and improving learning and memory. Thus, experimental and clinical evidence show that insulin action influences cerebral functions. In this paper, we reviewed several mechanisms by which insulin may affect pathophysiology in AD.     

Vanadium Salts as Insulin Substitutes: Mechanisms of Action,a Scientific and Therapeutic Tool in Diabetes Mellitus Research

Abstract

Vanadium and its compounds exhibit a wide variety of insulin-like effects. In this review, these effects are discussed with respect to the treatment of type I and type II diabetes in animal models, in vitro actions, antineoplastic role, treatment of IDDM and NIDDM patients, toxicity, and the possible mechanism(s) involved. Newly established CytPTK plays a major role in the bioresponses of vanadium. It has a molecular weight of approximately 53 kDa and is active in the presence of Co²⁺ rather than Mn²⁺. Among the protein-tyrosine kinase blockers, staurosporine is found to be a potent inhibitor of Cyt PTK but a poor inhibitor of InsRTK. Vanadium inhibits PTPase activity, and this in turn enhances the activity of protein tyrosine kinases. Our data show that inhibition of PTPase and protein tyrosine kinase activation has a major role in the therapeutic efficacy of vanadium in treating diabetes mellitus.     

Beta-cell dysfunctions and insulin resistance in subjects with increased risk for type 2 diabetes mellitus

The aim of this study was to test if a beta-cell defect is associated to deterioration of glucose tolerance early during the natural history of the type 2 diabetes mellitus . In 41 overweight women, with macrosomic infants in their antecedent deliveries, measures of insulin response and insulin sensitivity were derived from a short (45 min) iv glucose test. The early (EIR) and the late (LIR) phase insulin responses and the insulin sensitivity index (Si) were calculated. According the response to 75 g oral glucose test the subjects were divided into two groups: Imparired glucose tolerance (IGT;n = 12), and normal glucose tolerance (NGT; n = 29). EIR was reduced in IGT group (14.9 ± 3.6 vs 37.0 ± 4.0; p< 0.002). Glucose tolerance during oral glucose tolerance test (OGTT), correlated inversly to EIR (r=-0.45; n=41; p< 0.01). A strong correlation of EIR to LIR (r=0.88; n = 41; p< 0.001) but no correlation between glucose tolerance and Si was found.     

Genetically engineered insulin and its pharmaceutical analogues

Studies of replacement therapy of diabetes mellitus resulted not only in introduction of series of forms of insulin available at pharmaceutical market but also in new insulin analogues, which exhibit better control of blood glucose level. The present paper deals with basic tendencies in this field.     

Endothelial Dysfunction in Obesity and Insulin Resistance: A Road to Diabetes and Heart Disease

Obesity, insulin resistance, and endothelial dysfunction closely coexist throughout the natural history of type 2 diabetes. They all can be identified not only in people with type 2 diabetes, but also in various groups at risk for the disease, such as individuals with impaired glucose tolerance, family history of type 2 diabetes, hypertension, dyslipidemia, prior gestational diabetes, or polycystic ovary syndrome. Whereas their evident association cannot fully establish a cause‐effect relationship, fascinating mechanisms that bring them closer together than ever before are rapidly emerging. Central or abdominal obesity leads to insulin resistance and endothelial dysfunction through fat‐derived metabolic products, hormones, and cytokines. Insulin resistance leads to endothelial dysfunction through the frequent association with traditional cardiovascular risk factors and through some more direct novel mechanisms. Some specific and shared insulin signaling abnormalities in muscle, fat, and endothelial cells, as well as some new genetic and nontraditional factors, may contribute to this interesting association. Some recent clinical studies demonstrate that nonpharmacological and pharmacological strategies targeting obesity and/or insulin resistance ameliorate endothelial function and low‐grade inflammation. All these findings have added a new dimension to the association of obesity, insulin resistance, and endothelial dysfunction that may become a key target in the prevention of type 2 diabetes and cardiovascular disease.