Insulin Resistance. Biol 405 Molecular Medicine

Transcription

1 Insulin Resistance Biol 405 Molecular Medicine

2 Insulin resistance: a subnormal biological response to insulin. Defects of either insulin secretion or insulin action can cause diabetes mellitus. Insulin-dependent diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic -cells with a consequent deficiency in the secretion of insulin. In contrast, non-insulin-dependent diabetes mellitus (NIDDM) is more complex. Defects may occur in both insulin action and insulin secretion.

3 The earliest detectable defect in patients at risk from developing NIDDM is insulin resistance. In these patients the pancreatic -cell may compensate by increased secretion of insulin to prevent fasting hyperglycaemia. If insulin resistance is complicated by development of a deficiency in insulin secretion, then the patient develops overt NIDDM. Insulin resistance may also play a major role in the pathogenesis of other disorders e.g. obesity, hypertension.

4 Type A extreme insulin resistance Mutations in the insulin receptor (or other proteins involved in the transduction of insulin-generated intracellular signals). This is the prototype inherited syndrome of severe insulin resistance and is characterised by marked endogenous hyperinsulinemia with or without glucose intolerance. Type B extreme insulin resistance An autoimmune syndrome caused by the generation of autoantibodies to the insulin receptor. The antibodies (typically IgG) may cause steric inhibition of insulin binding although certain antibodies may bind the receptor and affect function without directly blocking insulin binding. The antibodies may also accelerate the degradation of the receptor and cause desensitization.

5 The Human Insulin Receptor The gene for the human insulin receptor is on chromosome 19, spans approximately 120,000 base pairs of genomic DNA and contains 22 exons. The 11 exons encoding the subunit are dispersed over more 90,000 base pairs of DNA whereas the 11 exons encoding the - subunit are located in a region of ~ 30,000 base pairs of DNA. The and subunits are derived by proteolytic processing of a common 1382 amino acid preproreceptor.

6 The 731 amino acid subunit (Mr 135,000) is external to the plasma membrane and contains the insulin binding region. It is linked, by interchain disulphide bonds, to the 620 amino acid -subunit (Mr 95,000). Exon Function 1 Signal peptide 2 Putative insulin binding domain 3 Cysteine rich region 4-10? 11 Alternatively spliced mini-exon The -subunit includes a 194 amino acid extracellular domain, a 23 amino acid membrane spanning segment and a 403 amino acid cytoplasmic segment that has intrinsic tyrosine kinase activity ? 15 Transmembrane domain 16? Tyrosine kinase domain 22 COOH-hydrophilic tail

7 ligand binding domain phe 382 alternative splice site Cell Membrane ala 1134 met 1153 trp 1200 Cytosol tyrosine kinase activity The human insulin receptor

8 Mutations in the Insulin Receptor Gene. Multiple different mutations have been identified in the insulin receptor genes of patients with genetic syndromes associated with extreme insulin resistance. Some patients are homozygous for a single mutation whereas others are heterozygotes, having inherited different mutant alleles of the insulin receptor gene from each parent. Other patients, with less severe insulin resistance, appear to be heterozygotes with a mutation in one allele while the other allele appears to be normal.

9 Mutations causing insulin resistance fall into at least 5 different classes: decreased rate of receptor biosynthesis inhibition of the intracellular transport of receptors to the cell surface (e.g. ala 1135 mutation to glu prevents processing of the proreceptor and transport to the cell surface) reduction in the affinity of the receptor for insulin inhibition of the insulin receptor tyrosine kinase accelerated receptor degradation

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11 Target cell defects could also be due to mutations affecting any protein between the receptor and the final insulin-regulated proteins. To date over 20 different receptor mutations have been identified in insulin resistant patients. Most of the mutations so far detected are in the structural gene rather than in the promoter or other noncoding regions. Some mutations are dominant-negative i.e. they impair the function of the receptor encoded by the normal allele. This relates to the fact that the receptor is an oligomeric protein ( 2 2 ) that may contain products of both normal and mutant genes.

12 met--> ile at position 1153 in the subunit An insulin resistant patient has been identified who is heterozygous for the mutation of a met for ile at position 1153 in the tyrosine kinase domain of the subunit (codon ATG --> ATA). The mutation has been investigated by cloning and expression of the mutant receptor in NIH-3T3 cells. It impairs receptor tyrosine kinase activity and inhibits the ability of insulin to stimulate both the uptake of 2-deoxyglucose and the incorporation of thymidine into DNA. This mutation exerts a dominant negative effect. The presence of met at position 1153 is conserved in all sequenced insulin receptors. It may be that mutation alters the conformation of the receptor in this region so that tyrosines at positions 1158, 1162, 1163 are not available to be autophosphorylated.

13 trp --> ser at position 1200 and ala --> thr at position 1134 In cell transfectants that express either of these mutant receptors there is no detectable insulin-stimulated receptor autophosphorylation or tyrosine kinase activity with either endogenous or exogenous substrates. In additon the cells showed no increase in the incorporation of thymidine into DNA or GLUT - 1(glucose transport protein) mrna accumulation. Cells expressing the trp --> ser mutant receptor show insulindependent stimulation of the uptake of 2-deoxyglucose and glucose incorporation into glycogen. In contrast the ala --> thr mutant receptors failed to signal these metabolic responses.

14 These results are important. They indicate that a possible divergence of the insulin signalling pathway (metabolic and mitogenic arms) may occur and that activation of the receptor tyrosine kinase may not be necessary for certain important biological actions.

15 phe --> val at position 382 of the subunit This mutation is associated with decreased transport of the receptor to the plasma membrane. In addition, it impairs the ability of insulin to stimulate receptor autophosphorylation. i.e. the conformational change brought about by substitution of val for phe results in a diminution in the ability of the receptor to transmit a signal across the plasma membrane from the to the subunit. The mutant receptor can still be phosphorylated by the wild type receptor i.e. transphosphorylation may occur.

16 Insulin signalling through IRS1-PI3K-Akt. After binding to its receptor, insulin stimulates tyrosine phosphorylation of both the receptor -subunit and of IRS-1 (insulin receptor substrate 1). Then, PI3K and Akt (also known as protein kinase B), are activated. Several negative modulators of insulin signalling have been described as major determinants of in vivo human insulin resistance; these include TRIB3 which exerts its inhibitory effect by interacting with Akt. A prevalent Q84R TRIB3 missense single nucleotide polymorphism may be a gain of function mutation with the potential to affect insulin signalling and thus, to increase the risk of insulin resistance.

17 Toll-like Receptors and Insulin Resistance There is growing evidence that inflammatory processes are responsible for insulin resistance associated with obesity. Toll-like receptors (TLRs) initiate immune responses following the binding of bacterial glycolipids such as lipoarabinomannan (LAM) and lipopolysaccharide (LPS). They may also initiate inflammatory responses to dietary lipids, thereby promoting insulin resistance.

18 Activation of TLRs leads to the induction and release of the inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor-a (TNF-a). Cytokine induction is dependent upon the nature of dietary lipid. Saturated fatty acids are the most potentunsaturated acids less so. Omega-3 polyunsaturated fatty acids have no effect.

19 In mice, lipid infusion activates TLR4 signalling and results in an inflammatory response. Furthermore, insulin resistance in muscles occurs - as measured by decreased IRS-1 phosphorylation. Insulin resistance does not occur, in response to lipid infusion, in TLR4-deficient mice. Increased ser307 phosphorylation (an indicator for insulin resistance) and decreased insulin-induced tyrosine phosphorylation of IRS-1 seen in obese mice are reversed in mice treated with TLR2 antisense oligonucleotides. i.e. mutations in the TLR4 receptor may affect the susceptibility to insulin resistance.

20 Summary Multiple different mutations have been identified in the insulin receptor genes of patients with genetic syndromes associated with extreme insulin resistance. Some mutations are dominant-negative This relates to the fact that the receptor is an oligomeric protein ( 2 2 ) that may contain products of both normal and mutant genes. In many mutant receptors there is no detectable insulinstimulated receptor autophosphorylation or tyrosine kinase activity with either endogenous or exogenous substrates. Some mutations suggest a possible divergence of the insulin signalling pathway (metabolic and mitogenic arms) may occur and that activation of the receptor tyrosine kinase may not be necessary for certain important biological actions.

21 References Takahashi, I. et al., (2010) Endocrine J. 57, phenotypic variability and mutation of the insulin receptor. Caricilli, A. M. et al., (2008) J. Endocrinol. 199, Toll-like receptors and insulin resistance. Prudente S. et al., (2009) Acta Diabetol. 46, TRIB3 as a gene affecting human insulin resistance. Jiang, S. et al., (2011) Endocrine J. 58, Functional characterisation of insulin receptor gene mutations.