Alan C. Moses

Receptors and growth-promoting effects of insulin and insulinlike growth factors on cells from bovine retinal capillaries and aorta.

It has been suggested that elevated levels of insulin or insulin-like growth factors (IGFs) play a role in the development of diabetic vascular complications. Previously, we have shown a differential response to insulin between vascular cells from retinal capillaries and large arteries with the former being much more insulin responsive. In the present study, we have characterized the receptors and the growth-promoting effect of insulinlike growth factor I (IGF-I) and multiplication-stimulating activity (MSA, an IGF-II) on endothelial cells and pericytes from calf retinal capillaries and on endothelial and smooth muscle cells from calf aorta. We found single and separate populations of high affinity receptors for IGF-I and MSA with respective affinity constants of 1 X 10(-9) M-1 and 10(-8) M-1 in all four cell types studied. Specific binding of IGF-I was between 7.2 and 7.9% per milligram of protein in endothelial cells and 9.1 and 10.4% in the vascular supporting cells. For 125I-MSA, retinal endothelial cells bound only 1.7-2.5%, whereas the aortic endothelial cells and the vascular supporting cells bound between 5.6 and 8.5% per milligram of protein. The specificity of the receptors for IGF-I and MSA differed, as insulin and MSA was able to compete with 125I-IGF-I for binding to the IGF-I receptors with 0.01-0.1, the potency of unlabeled IGF-I, whereas even 1 X 10(-6) M, insulin did not significantly compete with 125I-MSA for binding to the receptors for MSA. For growth-promoting effects, as measured by the incorporation of [3H]thymidine into DNA, confluent retinal endothelial cells responded to IGF-I and MSA by up to threefold increase in the rate of DNA synthesis, whereas confluent aortic endothelial cells did not respond at all. A similar differential of response to insulin between micro- and macrovascular endothelial cells was reported by us previously. In the retinal endothelium, insulin was more potent than IGF-I and IGF-I was more potent that MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In addition, insulin and IGF-I at 1 X 10(-6) and 1 X 10(-8) M, respectively, stimulated these cells to grow by doubling the number of cells as well. In all responsive tissues, the combination of insulin and IGFs were added together, no further increase in effect was seen. These data showed that vascular cells have insulin and IGF receptors, but have a differential response to these hormones. These differences in biological response between cells from retinal capillaries and large arteries could provide clues to understanding the pathogenesis of diabetic micro- and macroangiopathy.

Differential Cardiac Effects of Growth Hormone and Insulin-like Growth Factor1 in the Rat A Combined In Vivo and In Vitro Evaluation

BACKGROUND Despite their increasing clinical use and recent evidence that growth hormone (GH) and insulin-like growth factor-1 (IGF-1) target the heart, there has been no systematic investigation of the effects of GH and IGF-1 on the cardiovascular system. METHODS AND RESULTS Sixty normal but growing adult female rats were randomized to receive 4 weeks of treatment with GH (3.5 mg.kg-1.d-1), IGF-1 (3 mg.kg-1.d-1), a combination of the two, or placebo. Transthoracic echocardiograms were performed at baseline and at 2 weeks and 4 weeks of treatment. After the final echocardiography, rats underwent either closed-chest left ventricular (LV) catheterization or Langendorff perfusion studies. Myocyte diameter and interstitial tissue fraction were assessed by morphometric histology. Echocardiographic and ex vivo data demonstrated a LV hypertrophic response in all three groups of treated animals that was most marked in the GH group, which alone exhibited a concentric growth pattern (relative wall thickness, 0.52 versus 0.42 to 0.44 in the other groups; P < .001). At 4 weeks, cardiac index was significantly higher and total systemic vascular resistance was lower in all groups of treated animals than in control animals (both P < .001), whereas arterial blood pressure did not differ significantly. All indexes of in vivo and in vitro cardiac function were higher in GH- and IGF-1-treated rats than in control animals, whereas combination therapy yielded a blunted effect. Myocyte diameter was increased in all three treated groups without an increase in interstitial tissue. CONCLUSIONS Exogenous administration of GH and IGF-1 in the normal adult rat induces a cardiac hypertrophic response without development of significant fibrosis. Cardiac performance is increased both in vivo and in the isolated heart.

Recombinant Human Insulin-Like Growth Factor I Increases Insulin Sensitivity and Improves Glycemic Control in Type II Diabetes

Insulin resistance is a major factor in the pathophysiology of type II diabetes and a major impediment to successful therapy. The identification of treatments that specifically target insulin resistance could improve diabetes management significantly. Since IGFs exert insulin-like actions and increase insulin sensitivity when administered at supraphysiological doses, we determined the effect of 6 weeks of recombinant human IGF-I (rhIGF-I) administration on insulin resistance and glycemic control in obese insulin-resistant patients with type II diabetes. A total of 12 patients with type II diabetes were recruited for the study. Subcutaneous administration of rhIGF-I (100 μg/kg b.i.d.) significantly lowered blood glucose. Fructosamine declined from 369 to 299 µmol/l by 3 weeks of administration and then declined further to 271 at the end of 5 weeks. Glycosylated hemoglobin, which was 10.4% pretreatment, declined to 8.1% at the end of therapy. Mean 24-h blood glucose during a modal day was 14.71 ± 4.5 mmol/l pretreatment and declined to 9.1 ± 3.21 mmol/l by the end of treatment. These improvements in glycemia were associated with a decrease in serum insulin levels. Mean insulin concentrations declined from 108.0 to 57.0 pmol/l during the modal day measurements and from 97.2 to 72.0 pmol/l during the mixed-meal tolerance test. Changes in glycemia were accompanied by a marked increase in insulin sensitivity. The insulin sensitivity index (SI) calculated from a frequently sampled intravenous glucose tolerance test (FSIVGTT) after the method of Bergman et al. (Bergman RN, Finegold DT, Ader M: Assessment of insulin sensitivity in vivo. Endocr Rev 6:45–86, 1985) increased 3.4-fold. Furthermore, the improvement in glycemic control was accompanied by a change in body composition with a 2.1% loss in body fat as calculated by dual energy x-ray absorptiometry without change in total body weight. Significant side effects were present in some subjects, although nine subjects were able to complete at least 4.5 weeks of the protocol and six subjects completed the entire 6 weeks. Supraphysiological IGF-I concentrations were maintained throughout the study, increasing from 206¼g/l in the control period to 849 ¼g/l at the end of 6 weeks of rhIGF-I treatment. The increase in IGF-I levels was accompanied by a significant increase in IGF binding protein-2 levels, a slight reduction in IGF binding protein-3 levels, and an increase in levels of IGF binding protein-1. In summary, IGF-I significantly lowered blood glucose as reflected by short-term and long-term indexes of glycemic control and increased insulin sensitivity. It remains to be determined whether a dosage can be administered that avoids significant side effects and still achieves reasonable glycemic control.

Serum CTX: a new marker of bone resorption that shows treatment effect more often than other markers because of low coefficient of variability and large changes with bisphosphonate therapy.

Abstract: Serum CrossLaps is a new assay for measuring carboxy-terminal collagen crosslinks (CTX) in serum. This measurement is reported to be more specific to bone resorption than other measurements. However, the utility of this and other markers in monitoring patients on antiresorptive therapy depends on how often changes anticipated with therapy exceed changes attributable to random variability. In a study where subjects received either placebo or pamidronate, we calculated the minimum significant change (MSC), that is, the change that was sufficiently large that it was unlikely to be due to spontaneous variability. We also examined the changes in markers of bone turnover in subjects treated with pamidronate (APD) (30 mg I.V. in 500 ml D5W over 4 hours) to see how often observed changes in turnover after treatment exceeded the MSC. The MSC for serum CTX was 30.2%, and was significantly (P < 0.05) lower than the MSC for urinary NTX (54.0%), and not significantly different from the MSC of urinary DPD (20.6%). Ninety percent of subjects treated with APD had a decline in serum CTX that exceeded the MSC, compared with 74% for bone-specific alkaline phophatase (BSAP), 57% for urinary N-telopeptide cross-links (NTX), and 48% for free deoxypyridinoline. Changes in serum CTX correlated reasonably well with changes in spine BMD after 2 years (r = 0.47), but this correlation did not quite reach statistical significance because of the small number of subjects. In conclusion, the serum CTX assay shows greater utility for assessing efficacy of antiresorptive treatment than some previously described markers.

Insulin Administered Intranasally as an Insulin-Bile Salt Aerosol: Effectiveness and Reproducibility in Normal and Diabetic Subjects

Efficacy and reproducibility of insulin administered intranasally as an insulin-deoxycholate 1% (w/v) aerosol to normal and diabetic subjects were assessed by measurements of blood glucose and serum insulin levels. Following administration of 0.5 U insulin/kg with the unconjugated bile salt to fasting volunteers (N = 29), peak serum insulin levels of 103 ± 49 μM/ml above baseline were observed at 10 min. Blood glucose concentration began to fall by 10 min, reaching 54 ± 14% of control levels by 30 min, and returning to baseline by 60–80 min. Blood glucose response and peak serum insulin levels were reproducible when the same aerosol dose was repeatedly administered to the same subjects; however, intersubject variations were noted. By comparing serum insulin levels after i.v. and nasal routes of administration, nasal insulin absorption was approximately 10% as efficient as intravenous insulin. Dose response studies revealed that peak serum insulin concentrations were a linear function of the administered dose. In subjects with type I and type II diabetes mellitus, serum insulin levels increased in a manner similar to controls, and resulted in a prompt reduction of blood glucose concentration. However, in contrast to normal subjects, the duration of the glucose response was more prolonged, lasting as long as 5 h. Nasal administration of insulin as an aerosol with bile salts or bile salt analogs should be further evaluated as a possible nonparenteral approach to insulin therapy.

Specificity of urinary excretion of cross-linked N-telopeptides of type I collagen as a marker of bone turnover.

Urinary excretion of cross-linked N-telopeptide of type I collagen (NTX) has been reported to be a specific indicator of bone resorption. We studied the utility of a new immunoassay for NTX as an indicator of changes in bone resorption caused by treatment with pamidronate (APD) followed by T3. Twenty-two male subjects received either placebo (Group 1) or APD on study days 1–2 (Group 2). One week later all subjects received T3 100 μg/day (days 8–15). Urinary NTX, pyridinoline (PYD), hydroxyproline (HYP), and creatinine (cr) were measured on 2-hour fasting urine samples at baseline (day 1), after APD/placebo (day 8), after T3 (day 16), and at days 30 and 58. NTX/cr excretion fell 85% after treatment with APD (P<0.001 versus baseline), but not after placebo. The fall in mean urinary NTX after receiving APD was greater than the fall in PYD (25%) or HYP (31%) (P<0.001 NTX versus PYD and HYP). After treatment with APD, NTX excretion remained suppressed below baseline until day 58, whereas PYD and HYP excretion returned to baseline by study day 16. Persistence of APDs effect on bone until day 58 was suggested by the fact that serum calcium and parathyroid hormone levels had not returned to baseline by day 58. On day 16, after all subjects were treated with T3, urinary NTX/cr rose significantly (P<0.01) in Group 1 (-bisphosphonate) but not in Group 2 (+bisphosphonate). We conclude that urinary NTX is responsive to acute thyroid hormone-induced increases and bisphosphonate-induced decreases in bone resorption, and may reflect these changes more accurately than PYD or HYP.

Utility of biochemical markers of bone turnover in the follow-up of patients treated with bisphosphonates.

Abstract. Biochemical markers of bone turnover are often measured in patients treated with antiresorptive agents to monitor the effects of therapy. In order for a change in these markers to clearly indicate treatment effect, the change in the markers must exceed the amount of spontaneous variation typically seen with no treatment. Based on the measured long-term variability of markers in untreated patients, we defined a minimum significant change (MSC), that is, a change that was sufficiently large that it was unlikely to be due to spontaneous variability. We also examined the changes in markers of bone turnover in subjects treated with pamidronate to see how often observed changes in turnover after treatment exceeded the MSC. We found that urinary markers of bone resorption are best measured on 2-hour fasting samples, because results on random urine showed poor precision and less decline with therapy. We also found that of all the markers, urinary N-telopeptide cross-links (NTX) had the greatest decline after therapy (58%), although it also had the highest long-term variability (29.5%). The marker that most often showed a decline with treatment that exceeded the MSC was serum bone-specific alkaline phosphatase where 74% of observed changes exceeded the MSC. Other markers that often showed a decline with treatment that exceeded the MSC were 2-hour fasting urine NTX and free deoxypyridinoline, where 57% and 48%, respectively, of changes in therapy exceeded the MSC. The ideal marker would combine the large decline after treatment characteristic of NTX (60–70%) with the good precision of bone-specific alkaline phosphatase.

Familial Euthyroid Hyperthyroxinemia Resulting from Increased Thyroxine Binding to Thyroxine-Binding Prealbumin

MORE than 99 per cent of the thyroxine (T4) in blood is normally bound to specific plasma T4-binding proteins, including T4-binding globulin (TBG), T4-binding prealbumin (TBPA), and albumin.1 Howev...

Characterization of insulin-like growth factor binding to human granulosa cells obtained during in vitro fertilization.

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I. Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 +/- 0.30%/10(5) cells (SEM, n = 9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (alpha IR-3) inhibited 125I-IGF-I but not 125I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the alpha subunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays. IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.

Improvement in Highly Active Antiretroviral Therapy—Induced Metabolic Syndrome by Treatment with Pioglitazone but Not with Fenofibrate: A 2 × 2 Factorial, Randomized, Double-Blinded, Placebo-Controlled Trial

We designed a 2 x 2 factorial, randomized, double-blinded, placebo-controlled trial to evaluate the effects of treatment with pioglitazone and/or fenofibrate in patients with highly active antiretroviral therapy (HAART)-induced metabolic syndrome. We found that the administration of pioglitazone, but not fenofibrate, improved insulin resistance, blood pressure, and lipid profile over a 12-month period.