11061-68-0

Gene, mRNA, and precursor

The human preproINS gene, INS, is located at 11p15.5 and consists of three exons. In mice, it is located on chromosome 7. There are many regulatory elements, such as A-box, GG box, and the cAMP response element, in the promoter region. The coding region of human and tilapia preproINS mRNAs comprises 330 and 339 bases, respectively. In humans, INS forms a gene cluster?with IGF2 and H19, which is a gene for a long noncoding RNA.

Regulation of synthesis

INS gene transcription is stimulated by glucose in mammals, and glucose treatment results in a 2- to 5-fold elevation within 60–90min. Long-term exposure to physiological concentrations of estradiol-17β increases the β-cell content, INS gene expression, and INS release in?mice. Fasting reduces the expression levels of two INS genes in the trout.

Receptors

INS receptors (INSRs) are heterotetrameric glycoproteins containing many phosphorylated or glycosylated sites. INS binds to the two extracellular α-subunits linked by a disulfide bond. The two β-subunits are connected to the α-subunits by a disulfide bond and contain a tyrosine kinase domain in their intracellular region. Human INSR is encoded by a single gene, INSR, location 19p13.3-p13.2, comprising 22 exons.INSRencodes 1382 aa residues of the ISNR precursor, which contains a signal peptide and α- and β-subunits. Human INSR generates two alternative splicing variants (INSR-A and -B) that differ at the carboxyl terminus of their α-subunit. INSR-A lacks the terminus encoded by exon 11. INSR-B has not been detected in the chicken or Xenopus.? There are three types of hybrid receptors among these INSRs and the IGF-I receptor (IGF-IR), and?their expression ratio influences the INS action at the target organs. Dysregulation of the INSR-A/INSR-B ratio is associated with INS resistance, aging, and increased proliferative activity of normal and neoplastic tissues. The dissociation constants (Kd) of human INS to human INSR and IGF-1R are 0.23 and 16nM, respectively. IGF-I binds to hybrid receptors with at least a 50-fold higher affinity than INS, irrespective of the splicing variant. Guinea pig INSR shows an exceptionally higher affinity (Kd= 0.083nM). Several fish species have multiple INSR genes that show different expression patterns among tissues. Human INS binds to fish INSR at a Kd of 0.67 and 0.15nM in the trout and carp, respectively.

Biological functions

INS is a key regulator of glucose homeostasis in mammals because the peptide is the only hormone that lowers blood glucose levels.? INSR-A mainly enhances the effects of IGF-II during embryogenesis and fetal development, whereas INSR-B is predominantly expressed in adults and enhances the metabolic effects of INS. ProINS binds to INSR-A, which is present in the nervous system, to elicit antiapoptotic and neuroprotective effects in the developing and postnatal nervous system. In the pancreas, the exocrine and endocrine components stimulate each other via intrapancreatic axes of communication. The insular-acinar axis is involved in the regulation of pancreatic digestive enzyme production in acinar cells located in the blood downstream of β-cells, and the acini-insular axis is involved in the regulation of INS release by pancreatic enzymes.

Clinical implications

Diabetes is classified as type 1 or type 2. Type 1 diabetes (INS-dependent diabetes mellitus) results from the autoimmune destruction of β-cells. Type 2 diabetes (noninsulin-dependent diabetes mellitus) is a metabolic disorder characterized by hyperglycemia due to an absolute or relative lack of INS or cellular resistance to INS. In mammals and fish, the administration of alloxan or streptozotocin selectively destroys β-cells, producing a type 2 diabetes model.

Biochem/physiol Actions

Insulin is responsible for two types of actions- excitatory and inhibitory. In its excitatory role, it increases the uptake of glucose and lipid synthesis, and in its inhibitory role it inhibits glycogenolysis, gluconeogenesis, lipolysis, proteolysis and ketogenesis. Aberrant insulin secretion leads to various disorders such as diabetes, hyperglycemia or hypoglycaemia. Type I diabetes is a result of autoimmune destruction of β cells of pancreas, which leads to depletion of insulin. Mutant INS-gene Induced Diabetes of Youth (MIDY) syndrome is an autosomal dominant disorder caused by missense mutations, which lead to aberrant proinsulin folding. Impaired glucose tolerance (IGT) or non-insulin-dependent diabetes mellitus (NIDDM) is caused by resistance to insulin-stimulated glucose uptake.

in vitro

methotrexate (mtx) was found to be linked to insulin covalently. as effectively as insulin, insulin-mtx complex competed with 125i-insulin for insulin receptor. it was found that ic50 and ki for insulin-mtx were 93.82 nm and 91.88 nm, respectively, whereas those for insulin were 5.01 nm and 4.85 nm, respectively [1].

in vivo

previous study showed that insulin-stimulated glucose uptake in extensor muscles from sjl mice was reduced, but the basal uptake rates were not different. in another mouse study, knockdown of tc10α but not tc10β in 3t3-l1 adipocytes resulted in a inhibition of both insulin-stimulated glucose uptake and glut4 translocation [2].

IC 50

5.01 nmol/l for insulin receptor

Structure and conformation

Human preproINS consists of 110 aa residues: 24 in the signal peptide, 30 in the B-chain, 31 in the C-peptide, and 21 in the A-chain. The signal peptide and C-peptide are excised from the preproINS to produce the mature peptide, consisting of heterodimeric A- and B-chains. Six cysteine residues are conserved throughout evolution from agnathans to mammals. Furthermore, the N-terminal seven aa residues of the A-chain and the receptor binding region of the B-chain (Gly-Phe-Phe-Tyr) are highly conserved. Mr 5808, pI 5.3. INS is soluble under acidic and alkaline conditions and almost insoluble at neutral pH. INS can be stored for up to 6 months at 4°C in 1M acetic acid. Long-term storage at an alkaline pH increases the rate of deamidation and aggregation. One IU is equivalent to 0.0347mg human INS.

references

[1] ou x,kuang a,liang z,peng x,zhong y. the binding characteristics of insulin-mtx to insulin receptor. hua xi yi ke da xue xue bao.2001 dec;32(4):538-40.[2] leney se,tavaré jm. the molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets. j endocrinol.2009 oct;203(1):1-18.

Agonists and Antagonists

IGFs bind to INSR, but INS binds to IGF-IR with low affinity. Several synthetic peptides (S519 and RB539) show agonistic effects with a high affinity to INSR. Fish INSs bind mammalian INSR with high affinity and vice versa.?RB537, S661, and S961 are INSR antagonists with high affinity. S961 shows an agonistic effect only on 3H-thymidine incorporation

Brand name

Humulin (Lilly); Novolin (Novo Nordisk); Velosulin (Novo Nordisk).

General Description

The INS gene encodes for preproinsulin, which is enzymatically converted into insulin. Insulin is produced in the insulin-producing pancreatic β cells. Preproinsulin is converted to proinsulin in ER and proinsulin is then proteolytically processed to form insulin in newly-forming insulin secretory granules. Insulin production is tightly regulated by specific DNA elements present within ～400 bp in the proximal region of the INS promoter.