Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing.

A point mutation in the human insulin receptor gene in a patient with type A insulin resistance alters the amino acid sequence within the tetrabasic processing site of the proreceptor molecule from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser. Epstein-Barr virus-transformed lymphocytes from this patient synthesize an insulin receptor precursor that is normally glycosylated and inserted into the plasma membrane but is not cleaved to mature alpha and beta subunits. Insulin binding to these cells is severely reduced but can be increased about fivefold by gentle treatment with trypsin, accompanied by the appearance of normal alpha subunits. These results indicate that proteolysis of the proreceptor is necessary for its normal full insulin-binding sensitivity and signal-transducing activity and that a cellular protease that is more stringent in its specificity than trypsin is required to process the receptor precursor.

Defective processing of insulin-receptor precursor in cultured lymphocytes from a patient with extreme insulin resistance.

We have studied a patient with extreme insulin resistance, acanthosis nigricans, and decreased erythrocyte insulin binding. EBV-transformed lymphocytes from this patient exhibited markedly reduced binding of 125I-insulin. Radioiodination of cell surface receptors followed by immunoprecipitation with anti-receptor antibodies revealed the presence of increased amounts of a 210-kD protein but no detectable alpha or beta subunits. Continuous labeling with 2-[3H]mannose revealed the synthesis of a 190-kD precursor and a 210-kD protein. The 210-kD protein was phosphorylated in an insulin-dependent manner at high insulin concentrations. These results suggest that in this patient the biosynthesis of 190-kD receptor precursor, its terminal glycosylation, and intracellular transport to the cell surface proceed normally, while proteolytic maturation to alpha and beta subunits does not occur. We postulate that this defect either results from mutation(s) within the insulin-receptor gene, which render the precursor resistant to cleavage, or from a defect in the receptor processing enzyme.

Regulation of glucose-transporter gene expression by insulin in cultured human fibroblasts.

To clarify the effect of insulin on glucose-transporter (GT) biosynthesis, we determined GT mRNA levels in human cultured skin fibroblasts, using HepG2 GT cDNA as a probe. Insulin specifically increased the GT mRNA level in a time- and dose-dependent manner. Time-course study demonstrated that the mRNA level peaked within 3 h of insulin (1 x 10(-7) M) addition. After remaining elevated for several hours, mRNA decreased and returned to the basal level after 24 h. In the cell strains from seven normal subjects, the mean (+/- SE) GT mRNA level determined after 3 h of treatment with 1 x 10(-7) M insulin was 164.3 +/- 8.5% of the level found in untreated control cells. The insulin dose-response curve of GT mRNA levels showed that the maximum stimulation was elicited at 1 x 10(-7) M, and the half-maximum stimulation occurred at approximately 5 x 10(-10) M. Degradation rates of GT mRNA determined in the presence of actinomycin D were not different between insulin-treated and untreated cells. These results suggest that insulin increases GT gene expression in cultured human fibroblasts.

Regulation of glucose transporter synthesis in cultured human skin fibroblasts.

The present study was designed to see the effects of glucose on glucose transporter expression and glucose transport activity using cultured human skin fibroblasts. When the cells were incubated with various concentrations of glucose (11.1-44.4 mM), no differences were found in the HepG2 glucose transporter mRNA, protein levels and basal and insulin-stimulated 2-deoxyglucose uptake. Glucose deprivation, however, resulted in approximately 4-fold increases in the mRNA and 3-fold increases in the protein and the basal 2-deoxyglucose uptake. Chronic exposure to insulin increased the glucose transporter protein levels to similar degrees in the cells incubated with 11.1, 22.2 and 44.4 mM glucose accompanied by increases in the glucose transport activity. Effects of insulin on the glucose transporter mRNA and protein levels, however, were not evident in the glucose-deprived cells. It is concluded that glucose transport activity correlates closely with HepG2 glucose transporter expression in cultured human fibroblasts and that glucose (11.1-44.4 mM) does not affect the glucose transporter expression and glucose transport activity.

Binding and tyrosine kinase activities of the insulin receptor on Epstein-Barr virus transformed lymphocytes from patients with Werner's syndrome.

To assess the abnormalities of insulin receptor in patients with Werner's syndrome, we established Epstein-Barr virus transformed lymphocytes and studied the binding as well as kinase activities of insulin receptor. The insulin binding to both intact cells and WGA-purified insulin receptor preparations was within normal range. Autophosphorylation of the beta-subunit was not altered in patients with Werner's syndrome, and the receptors of these patients phosphorylated an exogenous substrate to a degree comparable to that of the normal participants. Taken together, these findings indicate that insulin resistance in Werner's syndrome likely is caused by a defect that cannot be detected by means used in the present study.

An ultrastructural study of the interaction of liposomes with plant protoplasts.

A one-step procedure using a mixture of glutaraldehyde and osmium tetroxide was devised to fix in situ large unilamellar liposomes of phosphatidylserine for transmission electron microscopy (TEM), since the conventional fixation method was found to be inadequate in this respect. The new fixation procedure enabled us to visualize the sequence of events in the interaction of liposomes with protoplasts from Vinca rosea suspension cultures in the presence of polyethylene glycol. Liposomes were thus found adhering to the surface of protoplasts, in association with invaginating plasmalemma, and within intracellular vesicles. These observations showed that liposomes enter plant protoplasts via endocytosis. Ultrastructural profiles indicating fusion of liposomes with protoplasts were not observed.

Extensive RNA editing and possible double-stranded structures determining editing sites in the atpB transcripts of hornwort chloroplasts.

Three nonsense codons and an unusual initiation codon were located within the putative coding region of the atpB gene of chloroplast DNA of the hornwort Anthoceros formosae. Nucleotide sequencing of cDNA prepared from transcripts revealed extensive RNA editing. The unusual initiation codon ACG was changed to AUG and three nonsense codons were converted into sense codons. In total 15 C residues of the genomic DNA were replaced by U residues in the mRNA sequences, while 14 U residues were replaced by C residues. This is the highest number of editing events for a chloroplast mRNA reported so far. Partial editing was also shown in a cDNA clone where 23 sites were edited but six sites remained unedited, representing the existence of premature mRNA. The expected two-dimensional structure of the mRNA shows the existence of a sequence complementary to every editing site, which can produce continuous base pairing longer than 5 bp, suggesting that mispairing in the double strand is the site determinant for RNA editing in Anthoceros chloroplasts. Comparison of the cDNA sequence with other chloroplast genes suggests that the mechanism arose in the first land plants and has been reduced during evolution.

Change of insulin action with aging in conscious rats determined by euglycemic clamp.

To clarify the mechanism responsible for age-related changes in insulin action, the euglycemic clamp technique was performed with graded doses of insulin in conscious rats aged 2, 4, 10, and 20 mo. Insulin binding (IB) to muscle membranes was also studied. Maximal response of insulin-induced glucose disappearance rate (Rd) was decreased significantly between 2 and 4 mo of age. Dose-response curves shifted to the right progressively up to 20 mo of age. However, IB to the muscle membrane diminished between 1 and 4 mo of age without a decrease thereafter. When Rd was plotted against insulin bound to the membranes, the resulting curves shifted to the right with aging, suggesting a coupling defect between the binding and effector unit. In conclusion, insulin action alters in rats between 2 and 20 mo of age. The most pronounced impairment in IB and maximal response of insulin-induced Rd occurs during early life stage (through maturation) and then a coupling defect seems to be superimposed with further aging. However, we cannot exclude the possibility that these changes may be secondary to obesity or reduced physical activity, rather than aging per se.

Postreceptor defect in insulin action in streptozotocin-induced diabetic rats.

To clarify the mechanism(s) responsible for the insulin resistance in streptozotocin (STZ)-treated diabetic rats, we studied insulin-induced glucose disposal by using the glucose clamp technique and measured insulin receptor and glucose transporter of muscles. The insulin dose-response curve of the metabolic clearance rate (MCR) of glucose revealed a decrease of the maximal response without a rightward shift in STZ rats. Maximal MCR was even lower when clamped at 300 rather than 150 mg/dl of blood glucose levels. Insulin binding to the crude plasma membrane of muscles from STZ rats was increased compared with controls. The number of glucose transporter of the plasma and microsomal membranes were significantly decreased in STZ rats. These in vivo and in vitro studies using skeletal muscles suggest that in STZ-treated diabetic rats 1) a defect or defects exist in the signal transduction mechanism of insulin in postbinding steps, 2) the decreased maximal MCR is related at least partly to the decrease of glucose transporter numbers, and 3) a defect in glucose metabolism (postglucose transport defect) is also present.

Insulin resistance in a boy with congenital generalized lipodystrophy.

We have studied insulin resistance in a 12-year-old Japanese boy who presented with congenital generalized lipodystrophy. Oral glucose tolerance test exhibited a diabetic pattern with normal fasting plasma glucose. Results from euglycemic glucose clamp study showed decreases in both insulin sensitivity and responsiveness. Both the patient's erythrocytes and Epstein-Barr virus transformed lymphocytes showed low-normal insulin binding with a slight reduction in binding affinity in the latter. Insulin binding to the cultured fibroblasts was decreased due to a lowered affinity. In addition, they displayed a rightward shift of the insulin dose-response curve for D-14C-glucose uptake with no decrease in the maximum uptake. Insulin-stimulated autophosphorylation and kinase activity of the wheat germ agglutinin purified receptors from the Epstein-Barr virus-transformed lymphocytes appeared normal. The reason for some discrepancies in insulin binding among the cells remains unknown, and we cannot formulate a conclusion as to whether or not a primary binding defect of insulin receptors exists and contributes to insulin resistance in the patient. The decrease in insulin responsiveness demonstrated in the glucose clamp study may result from a defect at the rate-limiting step in the postbinding process of insulin action, presumably a defect in the glucose transport system in muscle tissues. The defect may be secondary to changes in in vivo circumstances.

Binding activity and autophosphorylation of the insulin receptor from patients with myotonic dystrophy.

To clarify a mechanism of insulin resistance associated with myotonic dystrophy, we studied the insulin receptor by using three different types of cells--circulating erythrocytes, cultured skin fibroblasts, and Epstein-Barr virus(EBV)-transformed lymphocytes. In myotonic dystrophy, insulin binding to erythrocytes and fibroblasts was significantly decreased as a result of a reduction of the binding affinity. Insulin binding to EBV-transformed lymphocytes was normal. When the receptors were purified from fibroblasts with wheat germ agglutinin, we could not find a decrease in the binding affinity seen in the intact cells. No difference was observed in the magnitude of basal and insulin-stimulated autophosphorylation of insulin receptors from EBV-transformed lymphocytes between the control and myotonic dystrophy. Southern blot analysis of the insulin receptor gene revealed no restriction fragment length polymorphism associated with myotonic dystrophy. These findings suggest that there is no primary defect of the insulin receptor per se in terms of insulin binding and autophosphorylation in myotonic dystrophy. The reduction of the insulin binding to erythrocytes and fibroblasts may be caused by the plasma membrane abnormality that affects the binding affinity of the receptor.

Changes in insulin receptor kinase with aging in rat skeletal muscle and liver.

To see if insulin receptor kinase activity alters with aging, the activity of wheat germ agglutinin-purified receptor preparations from liver and skeletal muscle was compared in 2-, 4-, 10-, and 20-mo-old rats. Basal and insulin-stimulated autophosphorylation of liver insulin receptor and its kinase activities toward histone 2b and poly(Glu4Tyr1) did not alter with aging. On the other hand, the muscle insulin receptor showed different results. Insulin-stimulated increases of autophosphorylation and the kinase activity toward histone 2b above basal were comparable in the four groups. However, insulin-stimulated phosphorylation of poly(Glu4Tyr1) was decreased in 20-mo-old rats compared with 10- and 4-mo-old rats. These results indicate that insulin receptor kinase activity could vary under certain conditions, depending on the substrate used to measure the activity. It is concluded that insulin receptor kinase activity does not change markedly during the process of aging, although subtle changes seem to exist.