Paulos Berhanu

Effects of pioglitazone on lipid and lipoprotein profiles in patients with type 2 diabetes and dyslipidaemia after treatment conversion from rosiglitazone while continuing stable statin therapy

4The aim of this study was to evaluate changes in lipid profiles in patients with type 2 diabetes after treatment conversion from rosiglitazone to pioglitazone while maintaining stable statin and other lipid-altering therapies. A total of 305 patients were enrolled in this open-label study. Patients had been taking stable dosages of rosiglitazone and statins for > 90 days. At baseline, patients discontinued rosiglitazone and started pioglitazone 30 mg/day, but continued statins and other lipid-altering therapies. The primary end point was change from baseline in fasting triglyceride levels. At 17 weeks after treatment conversion, patients had significant reductions in triglycerides (-15.2%), total cholesterol (-9.0%), and low-density lipoprotein (LDL) particle concentration (-189 nmol/L), and increases in LDL cholesterol (+2.2%), high-density lipoprotein (HDL) cholesterol (+1.8%), and LDL particle diameter (+0.23 nm). In conclusion, after treatment conversion from rosiglitazone to pioglitazone while maintaining stable statin therapy, patients with type 2 diabetes had marked improvements in lipid profiles along with stable glycaemic control.

Effects of Insulin and Insulin-like Agents on the Glucose Transport System of Cultured Human Fibroblasts

We have studied the effects of insulin and insulin-like agents on glucose transport by cultured human fibro-blast monolayers. Initial rates of glucose transport were determined by measurement of 2-deoxy-D-glu-cose uptake. At physiologic concentrations, insulin stimulates 2-deoxy-D-glucose transport (average of 50% over basal) with a half-maximally effective insulin concentration of 3.3 ± 0.9 ng/ml. This effect of insulin is rapid and is half-maximal at 10 min and becomes maximal by about 30 min. Kinetic analyses showed that insulin increased the transport Vmax from 7.4 ± 0.9 nmol/min/106 cells to 11.0 ± 1.5 nmol/min/106 cells and had no effect on the Km value (2.5 ± 0.3 mM). While glucose starvation led to a higher overall rate of 2-deoxy-D-glucose transport, the relative stimulation by insulin was the same as in non-glucose-starved cells. Insulin mimickers [insulin-like growth factor (IGF), anti-insulin receptor antibody, and concanavalin A] also stimulate 2-deoxy-D-glucose transport by human fibroblast monolayers in a dose-dependent manner and the maximal effects of IGF and anti-insulin receptor antibody were the same as that of insulin, while the maximal effect of concanavalin A was only 78% of that of insulin. The maximal effects of either insulin and IGF or insulin and anti-insulin receptor antibody were not additive, suggesting that these agents all act via the same glucose transport effector system in human fibroblasts. In conclusion, human fibroblasts possess an insulin-sensitive glucose transport system that displays many of the characteristics common to other more well studied transport systems. Thus, cultured human fibroblasts can serve as an important model for physiologic studies of insulin action and glucose transport, and for studies of pathophysiologic abnormalities of these processes in cells obtained from patients with various disease states.

Degradation of Insulin Receptors in Rat Adipocytes

Insulin receptors on viable rat adipocytes were affinity-labeled using a biologically active and photosensitive analogue of insulin, 125I-B2(2-nitro, 4 azidophenylacetyl)-des-PheB1-insulin (125I-NAPA-DP-insulin). The radio-labeled proteins were identified by SDS polyacrylamide gel electrophoresis and autoradiography. Binding of 125I-NAPA-DP-insulin (40 ng/ml) to rat adipocytes at 16°C, followed by photolysis, resulted in the specific labeling of essentially one protein with an apparent molecular weight of 430–450,000 daltons. When this radiolabeled protein was treated with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a 125,000-dalton protein and to a lesser extent in a 90,000-dalton protein. In addition, there was a minimal amount of specific labeling of a 115,000-dalton protein. Under certain experimental conditions, the nonreduced form of the photoaffinity-labeled receptor appeared as a heterogeneous population of proteins having apparent molecular weights of 430,000, 350,000, and 270,000 daltons. Subsequent to photoaffinity labeling of insulin receptors at 16°C, adipocytes were incubated at 37°C for various periods of time to allow for internalization. This resulted in an initial rapid loss of radioactivity in the 430,000- and 125,000-dalton bands. At 60 min the amount of radioactivity in each of these bands was approximately 50% of that present before incubation at 37°C and stayed constant for 120 min. A first-order plot of the decline in receptor-associated radioactivity was biphasic with the initial phase having a half-life of 1.4 h. This loss of radiolabeled receptors was not observed at 16°C and was inhibited by chloroquine (200μM) and cycloheximide (3.5 μM). Under conditions in which approximately 6% of the receptors were photoaffinity-labeled, occupancy of the remaining receptors with insulin had no effect on the degradation of photolabeled receptors. The results suggest that photoaffinity-labeled insulin receptors on viable adipocytes are initially degraded via an endocytotic lysosomal pathway. This pathway of degradation is very rapid and appears to be different from that mediating the basal turnover of unoccupied receptors.

Exercise capacity is a predictor of cardiovascular events in patients with type 2 diabetes mellitus.

Peak exercise oxygen consumption (peak VO2), as measured by expired gas analysis, is an accurate, reproducible and reliable method for determining exercise capacity. In this study, a cohort of 468 patients with type 2 diabetes underwent graded exercise testing to measure peak VO2 at baseline; the cohort was followed for five years for the occurrence of cardiovascular disease (CVD) events. Patients who developed CVD events during the five-year follow-up period were found to have significantly lower baseline peak VO2, as compared to those who did not (p=0.02). Analysis by gender showed that the mean peak VO2 in male patients who developed CVD events was significantly lower than the peak VO2 in those who did not (p<0.03). Multiple Cox regression analysis also showed low peak VO2 to be an independent factor. In conclusion, patients with type 2 diabetes mellitus with reduced peak VO2 during exercise have a greater tendency to develop future CVD events.

Insulin receptors in isolated human adipocytes. Characterization by photoaffinity labeling and evidence for internalization and cellular processing.

We photolabeled and characterized insulin receptors in isolated adipocytes from normal human subjects and then studied the cellular fate of the labeled insulin-receptor complexes at physiologic temperatures. The biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)des-PheB1-insulin (NAPA-DP-insulin) was used to photoaffinity label the insulin receptors, and the specifically labeled cellular proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. At saturating concentrations, the binding of 125I-NAPA-DP-insulin to the isolated adipocytes at 16 degrees C was rapid (half-maximal in approximately 1 min and maximal in approximately 10 min) and approximately 25% of the specifically bound ligand was covalently linked to the cells by a 3-min exposure to long-wave (366 nm) ultraviolet light. Analysis of the photolabeled cellular proteins by PAGE in the absence of disulfide reductants revealed the specific labeling of a major protein band of Mr 330,000 and two less intense bands of Mr 295,000 and 260,000. Upon reduction of disulfide bonds with dithiothreitol, all three unreduced forms of the insulin receptor were converted into a major labeled Mr-125,000 band and a less intensely labeled Mr-90,000 band. The labeling of the Mr-125,000 receptor subunit was saturable and native porcine insulin effectively inhibited (half-maximal inhibition at 12 ng/ml) the photolabeling of this binding subunit by NAPA-DP insulin. When intact adipocytes photolabeled at 16 degrees C (a temperature that inhibits endocytosis) were immediately trypsinized, all of the labeled receptor bands were converted into small molecular weight tryptic fragments, indicating that at 16 degrees C all of the labeled insulin-receptor complexes remained on the cell surface. However, when the photolabeled cells were further incubated at 37 degrees C and then trypsinized, a proportion of the labeled receptors became trypsin insensitive, indicating that this fraction has been translocated to the cell interior and thus was inaccessible to the trypsin in the incubation medium. The intracellular translocation of the labeled receptors was observed within 2 min, became half-maximal by 10 min, and maximal by approximately 30 min of incubation at 37 degrees C. Cellular processing of the internalized insulin-receptor complexes also occurred, since incubation at 37 degrees C (but not 16 degrees C) resulted in the generation of a Mr-115,000 component from the labeled receptors. Inclusion of chloroquine, a drug with lysosomotropic properties, in the incubation media caused a time-dependent increase (maximal increase of 50% above control by 2 h at 37 degrees C) in the intracellular pool of labeled receptors. In contrast to these findings in human adipocytes, no appreciable internalization of insulin-receptor complexes and no chloroquine effect was observed in cultures human IM-9 lymphocytes during a 1-h incubation at 37 degrees C. We concluded that in isolated human adipocytes: (a) the subunit structure of insulin receptors is the same as that reported for several other tissues, (b) insulin-receptor complexes are rapidly internalized and processed at physiologic temperatures, and (c) the cellular processing of insulin-receptor complexes occurs at one or more chloroquine-sensitive intracellular site(s).

Insulin Signal Transduction by a Mutant Human Insulin Receptor Lacking the NPEY Sequence EVIDENCE FOR AN ALTERNATE MITOGENIC SIGNALING PATHWAY THAT IS INDEPENDENT OF Shc PHOSPHORYLATION

The cytoplasmic juxtamembrane domain of the human insulin receptor (hIR) contains a single copy of the tetrameric amino acid sequence Asn-Pro-Glu-Tyr (NPEY) (residues 969–972 in the exon 11-containing B-isoform), which exists in the context of NPXY. In this study, we examined the role of NPEY972 in mediating insulin signal transduction and cellular biological effects. Transfected Chinese hamster ovary cell lines expressing either the wild-type hIR-B isoform (hIR·WT) or a mutant receptor lacking the NPEY972 sequence (hIRΔNPEY) and control Chinese hamster ovary·Neo cells were used to comparatively analyze the following insulin effects: in vivo receptor tyrosine autophosphorylation and kinase activity, signal transduction to downstream signaling molecules, and stimulation of glycogen and DNA synthesis. The results showed that in comparison to hIR·WT, the hIRΔNPEY mutant demonstrated the following: (a) normal insulin-mediated receptor tyrosine phosphorylation, but ∼50% reduction in phosphorylation of p185-(insulin receptor substrate-1) and binding of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase), (b) an enhanced stimulation of PI 3-kinase enzymatic activity, (c) a complete inability to phosphorylate Shc, (d) minimal impairment of insulin sensitivity for glycogen synthesis, and (e) an augmented response to insulin-stimulated DNA synthesis via a high capacity, low sensitivity pathway. These results demonstrate the following: 1) the NPEY972 sequence is important but not absolutely essential for coupling of hIR kinase to insulin receptor substrate-1 and p85 or for mediating insulin’s metabolic and mitogenic effects, 2) the NPEY972 sequence is necessary for Shc phosphorylation, and 3) the absence of Shc phosphorylation releases the constraints on maximal insulin-stimulated mitogenic response, thus indicating that alternate signaling pathway(s) exist for this insulin action. This alternate pathway appears to be associated with enhanced activation of PI 3-kinase and is of high capacity and low sensitivity.

Insulin-Stimulated Glucose Transport and Insulin Internalization Share a Common Postbinding Step in Adipocytes

We recently demonstrated that chymotrypsin substrate analogues inhibit receptor-mediated insulin internalization in isolated rat adipocytes. In this study, the effect on glucose transport of inhibiting insulin internalization with these agents was examined. Glucose transport was assayed by measuring [3H]-2-deoxyglucose uptake, and internalized insulin was measured after rapidly dissociating surface-bound insulin with an acidic buffer. The chymotrypsin substrate analogue N-acetyl-Tyr ethyl ester inhibited insulin internalization by 85% while increasing surface-bound insulin by 80-110%. Under these conditions, ATP levels were minimally altered, and basal glucose transport was unchanged; however, insulin-stimulated glucose transport was decreased by 86%. The inhibition of insulinstimulated glucose transport was not overcome by supramaximal concentrations (400 ng/ml) of insulin. When insulin internalization and insulin-stimulated glucose transport were measured in the presence of increasing concentrations of N-acetyl-Tyr ethyl ester (0.1-1 mM), a strong and highly significant correlation (r = .97, P < .001) was found between inhibition of insulin internalization and inhibition of insulin-stimulated glucose uptake. Fragments of N-acetyl-Tyr ethyl ester that do not inhibit insulin internalization were also without effect on insulin-stimulated glucose transport. In addition to N-acetyl-Tyr ethyl ester, four other chymotrypsin substrate analogues that are effective inhibitors of insulin internalization also markedly inhibited insulin-stimulated glucose transport. These results indicate that insulin internalization and insulin-stimulated glucose transport share a common postbinding step in adipocytes and that this step is inhibitable by chymotrypsin substrate analogues.

Insulin downregulates the steady-state level of its receptor's messenger ribonucleic acid

The effects of insulin on the steady-state level of human insulin receptor (hIR) mRNA were examined in the HepG2 human liver cell line using Northern blot analysis of either total cellular or poly(A)+ RNA. In control cells, up to six (4.5, 5.2, 7.4, 8.5, 9.4 and 10.8 kb) hybridizable species of hIR mRNA were identified, with the 8.5 and 10.8 kb species being most prominent. Incubation for 18 hrs with 1 microM insulin resulted in a similar decrease (to approximately 35% of control) of all the hIR mRNA species. The insulin effect was dose-dependent and was half-maximal by 2-3 hrs and maximal by 4-6 hrs of incubation at 37 degrees C. The hIR mRNA levels remained maximally insulin suppressed for up to 18 hrs but thereafter the effect became attenuated. These results indicate that insulin downregulates the level of hIR mRNA with a biphasic time-course and that this process is most likely part of the general mechanism by which insulin maintains the homeostatic control of its cellular receptor levels.

Idiopathic Hypoparathyroidism Presenting With Severe Hypocalcemia and Asymptomatic Basal Ganglia Calcification Followed by Acute Intracerebral Bleed

OBJECTIVE To report a case of idiopathic hypoparathyroidism presenting with severe hypocalcemia and intracerebral calcifications that resulted in a spontaneous intracerebral bleed. METHODS We present the clinical, laboratory, and radiologic findings in a woman with idiopathic hypoparathyroidism who developed spontaneous intracerebral bleed in the setting of chronic intracerebral calcifications. RESULTS A 37-year-old woman presented with vague symptoms of hypocalcemia. Clinical evaluation revealed brisk deep tendon reflexes and positive Chvosteks and Trousseaus signs. The serum calcium level was 3.7 mg/dL (reference range, 8.0 to 10.6 mg/dL) and the phosphorus level was 8.2 mg/dL (reference range, 2.3 to 5.0 mg/dL). Serum intact parathyroid hormone was undetectable. Computed tomography of the head showed extensive bilateral symmetrical calcification of basal ganglia and dentate nucleus in the cerebellum and centrum semiovale. Fluid and electrolytes were replaced appropriately, and calcium and calcitriol were prescribed. While in the hospital, the patient developed an acute intracerebral bleed confirmed by computed tomography. The patient recovered without neurologic sequelae and was discharged from the hospital on calcium supplementation and calcitriol. Repeated computed tomography of the head 3 years later demonstrated complete resolution of the bleed. CONCLUSION This case suggests that patients with severe hypoparathyroidism and intracerebral calcification may be at risk for spontaneous intracerebral bleed and should be monitored accordingly.

Effects of metalloendoprotease inhibitors on insulin binding, internalization and processing in adipocytes

The effects of metalloendoprotease inhibitors on insulin binding, internalization, and processing were studied in isolated rat adipocytes. The metalloendoprotease inhibitor phosphoramidon caused a marked (threefold) increase in intracellular insulin accumulation without affecting surface binding. The dipeptide metalloendoprotease substrate analogues benzyloxycarbonyl-Gly-Phe-NH2 and benzyloxycarbonyl-Gly-Leu-NH2 caused similar large increases in intracellular insulin but also caused a doubling of cell surface bound insulin. The effect on surface binding was due to increased insulin receptor affinity as demonstrated by Scatchard analysis and the benzyloxycarbonyl-Gly-Phe NH2 induced inhibition of the dissociation of prebound insulin from the cell surface. These results suggest a role for endogenous metalloendoprotease-like enzymes in insulin processing by rat adipocytes.