Effect of pioglitazone on body composition and bone density in subjects with prediabetes in the ACT NOW trial.

AIMS: This study examined the effects of pioglitazone on body weight and bone mineral density (BMD) prospectively in patients with impaired glucose tolerance as pioglitazone (TZD) increases body weight and body fat in diabetic patients and increases the risk of bone fractures. METHODS: A total of 71 men and 163 women aged 49.3 (10.7) years [mean (s.d.)]; body mass index (BMI), 34.5 (5.9) kg/m(2) were recruited at five sites for measurements of body composition by dual energy X-ray absorptiometry at baseline and at conversion to diabetes or study end, if they had not converted. RESULTS: Mean follow-up was 33.6 months in the pioglitazone group and 32.1 months in the placebo group. Body weight increased 4.63 ± 0.60 (m ± s.e.) kg in the pioglitazone group compared to 0.98 ± 0.62 kg in the PIO group (p < 0.0001). Body fat rose 4.89 ± 0.42 kg in the pioglitazone group compared to 1.41 ± 0.44 kg, (p < 0.0001) in placebo-treated subjects. The increase in fat was greater in legs and trunk than in the arms. BMD was higher in all regions in men and significantly so in most. PIO decreased BMD significantly in the pelvis in men and women, decreased BMD in the thoracic spine and ribs of women and the lumbar spine and legs of men. Bone mineral content also decreased significantly in arms, legs, trunk and in the total body. CONCLUSIONS: Pioglitazone increased peripheral fat more than truncal fat and decreased BMD in several regions of the body.

Postprandial lipoprotein responses in hypertriglyceridemic subjects with and without cardiovascular disease.

Three groups of age- and weight-matched men (aged 40 to 70 years) without diabetes were studied: controls (n = 10), plasma triglycerides (TG) less than 180 mg/dL and no cardiovascular disease (CVD); HTG-CVD (n = 11), hypertriglyceridemic (HTG) (TG > 240 mg/dL) without CVD; and HTG+CVD (n = 10), HTG (TG > 240 mg/dL) with documented CVD. HTG+CVD subjects had higher fasting and post-oral glucose tolerance test insulin levels than the other two groups, respectively. Very-low-density lipoprotein (VLDL)+chylomicrons (CMs), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and three high-density lipoprotein (HDL) subfractions (HDL-L, HDL-M, and HDL-D, from least to most dense) were isolated by gradient ultracentrifugation. Fasting lipoproteins were similar in HTG groups, except for higher VLDL lipid to apolipoprotein (apo) B ratios (P < .04) in the HTG+CVD group. Subjects were fed a high-fat mixed meal, and lipoprotein composition was determined at 3, 6, 9, and 12 hours postprandially. Postprandial responses of the core lipids (TG and cholesterol esters [CE]) in all of the lipoprotein subfractions were similar in the two HTG groups at each time point. However, both controls and HTG-CVD subjects had increases in HDL-M phospholipid (PL) at 9 and 12 hours with no change in HDL-D PL. The HTG+CVD group, on the other hand, had no increase in HDL-M PL and had a substantial reduction in HDL-D PL. These changes resulted in significant increases in HDL-M and HDL-D PL to apo A-I ratios in both controls and HTG-CVD subjects between 6 and 12 hours, whereas there was no increase seen in the HTG+CVD group. The HTG-CVD group also had a significantly greater increase in the VLDL+CM PL to apo B ratio (P = .038) at 3 hours than the HTG+CVD group. This diminished amount of surface lipid per VLDL particle may account for the late decrease in the HDL-D PL to apo A-I ratio seen in HTG+CVD patients. There were no other postprandial lipid or apolipoprotein differences between the two HTG groups. We conclude therefore that the major postprandial lipoprotein abnormality in these HTG+CVD patients was a failure to increase the PL content per particle in VLDL+CM, HDL-M, and HDL-D. This abnormality could prevent the usual increase in reverse cholesterol transport seen in postprandial plasma and therefore contribute to their increased incidence of CVD. The greater insulin resistance seen in these patients also appears to contribute significantly to their CVD.

Replacement of dehydroepiandrosterone enhances T-lymphocyte insulin binding in postmenopausal women.

OBJECTIVE: To demonstrate bioavailability of 3 weeks of oral micronized DHEA and to delineate changes induced on insulin sensitivity, morphometric indexes, and lipoprotein profiles. DESIGN: Oral micronized DHEa (50 mg/d) was administered in 3-week treatments to 11 postmenopausal women in a prospective, placebo-controlled, randomized, blinded, crossover trial with an interarm washout. After dose (23 hour) serum DHEA, DHEAS, T, and cortisol levels were measured, as were fasting lipoproteins, oral glucose tolerance tests (OGTT), T-lymphocyte insulin binding and degradation, and urine collagen cross-links. Morphometric changes were determined by hydrostatic weighing. RESULTS: Dehydroepiandrosterone sulfate, DHEA, T, and free T increased up to two times premenopausal levels with treatment. Fasting triglycerides declined; no change in collagen cross-links or morphometric indexes was noted. Oral glucose tolerance test parameters did not change, but both T-lymphocyte insulin binding and degradation increased with DHEA. CONCLUSION: Fifty milligrams per day of oral DHEA gives suprahysiologic androgen levels; 25 mg/d may be more appropriate. Dehydroepiandrosterone enhanced tissue insulin sensitivity and lowered serum triglycerides. Rationale is provided for postmenopausal replacement therapy with this androgen.

Oral dehydroepiandrosterone in physiologic doses modulates immune function in postmenopausal women.

OBJECTIVE: This study tests the hypothesis that dehydroepiandrosterone or its metabolic products are immunomodulatory in postmenopausal women with relative adrenal androgen deficiency. STUDY DESIGN: A prospective, randomized, double-blind, crossover study of 11 subjects with 3-week treatment arms separated by a 2-week washout period was performed. Immunologic evaluation at the beginning and end of the treatment arms consisted of flow cytometry to delineate T-cell populations, in vitro T-cell mitogenic response and cytokine production, and natural killer cell cytotoxicity. Statistical analysis was based on a split-plot design with analysis of variance with repeated measures. RESULTS: Dehydroepiandrosterone supplementation decreased CD4+ (helper) T cells and increased CD8+/CD56+ (natural killer) cells. Although T-cell mitogenic and interleukin-6 responses were inhibited, natural killer cell cytotoxicity increased dramatically. CONCLUSIONS: These data provide the first in vivo evidence in human for an immunomodulatory effect of dehydroepiandrosterone. The salutary immune changes could account for clinical and experimental evidence of antioncogenic effects of this steroid. This study provides a strong rationale for further clinical studies on dehydroepiandrosterone supplementation in adrenal androgen-deficient states.

Identification of insulin intermediates and sites of cleavage of native insulin by insulin protease from human fibroblasts.

We have studied the time sequence degradation of native insulin by insulin protease from human fibroblast using multiple steps involving purification of the products by high performance liquid chromatography, determination of peak composition by amino acid sequence analysis, and confirmation of structure by mass spectrometry and thus elucidated the sites of cleavage of insulin by human insulin protease. We observed that as early as 0.5 min of incubation, three major new peptide peaks, intact insulin, and four smaller peptide peaks can be detected. The major peptides are portions of the insulin molecule, with the amino ends of the A and B chains or the carboxyl ends of the A and B chains still connected by disulfide bonds. Peptide peak I is A1-13-B1-9. Peptide peak II is A1-14-B1-9. Peptide peak III is A14-21-B14-30. The smaller peptide peaks are A14-21-B17-30, A15-21-B14-30, A15-21-B10-30, and A14-21-B10-30. The major peptide bond cleavage sites therefore consist of A13-14, A14-15, B9-10, B13-14, and B10-17. With longer incubation times, peptide peak II appears to lose the A14 tyrosine to form peptide peak I. This peptide I, which is the amino end of the A and B chains, is not further degraded even after 1.5 h of incubation. With longer incubation times, the peptides containing the carboxyl ends of the A and B chains are further degraded to form products from cleavage at the A18-19, B14-15, B25-26, and a small amount of A19-20, B10-11, and B24-25 cleavage and the emergence of 2-5-amino acid peptide chains, tyrosine, alanine, histidine, and leucine-tyrosine. We conclude, based on the three-dimensional structure of insulin, that human insulin protease recognizes the alpha-helical regions around leucine-tyrosine bonds and that final degradation steps to small peptides do not require lysosomal involvement.

Cyclosporine disposition in the dog. Comparison of radioimmunoassay with high-performance liquid chromatographic assay and pharmacokinetics following intravenous administration.

Radioimmunoassay (RIA) and high performance liquid chromatography (HPLC) with ultraviolet absorbance detection have been compared as potential tools for cyclosporine pharmacokinetic studies in dogs. RIA clearly affords greater assay sensitivity, although crossreactivity with cyclosporine metabolites causes an over-estimation of parent drug concentrations with a subsequent reduction in the apparent values of clearance and volume of distribution. HPLC appears to be specific for parent cyclosporine. Thus, with the sacrifice of some sensitivity, HPLC-measured time-course data afford more reliable estimates of cyclosporine pharmacokinetic parameters. After the selection of a dosage regimen from preliminary studies, the pharmacokinetics of i.v.-administered cyclosporine were studied in six adult male mongrel dogs. Following administration of 20 mg/kg by constant-rate 30-min i.v. infusion the time courses of cyclosporine were studied in plasma and urine. Concentrations were measured by reversed-phase HPLC with ultraviolet absorbance detection. Data were fitted to triexponential equations using a digital computer with the CSTRIP and NONLIN programs, and pharmacokinetic parameters were calculated. Present findings suggest that cyclosporine is slowly yet extensively distributed into peripheral body regions that might serve as slowly releasing storage areas. Large volumes of distribution along with moderately slow clearances resulted in long half-lives for the disposition of cyclosporine. Less than 1% of the administered dose was recovered as parent cyclosporine in the urine, suggesting that renal clearance of cyclosporine was negligible. The potential relevance of present findings to cyclosporine therapy of transplant patients is discussed.

The effect of inhibitors of insulin processing on generation of insulin intermediate products from human fibroblast as detected by high performance liquid chromatography (HPLC).

To assess the role of various modulators of insulin processing on cell-associated A14-125I-insulin intermediates in human fibroblasts, we have studied the effect of N-ethylmaleimide (NEM), chloroquine, bacitracin, dansylcadavarine, and phenylarsine oxide on generation of these intermediate products with the use of HPLC. NEM completely inhibited generation of intermediate peaks or iodotyrosine. Chloroquine inhibited conversion of A14-125I-insulin to iodotyrosine by about 75 percent and the remaining A14-125I-insulin was not susceptible to acid wash. Bacitracin, dansylcadavarine, and phenylarsine oxide, on the other hand, stimulated formation of intermediate products with concomitant inhibition of iodotyrosine formation. We conclude that there are at least three components of insulin degradation in human fibroblasts. These include the sulfhydryl group inhibitor-sensitive, the intracellular chloroquine-sensitive, and membrane site inhibitor-sensitive components.

Characterization of insulin-degrading activity of intact and subcellular components of human fibroblasts.

We have studied insulin degrading activity (IDA) in cultured human fibroblasts and assessed the effect of various inhibitors of insulin processing on IDA. To evaluate the role of three enzymes of insulin degradation (neutral protease, microsomal glutathione insulin transhydrogenase, and lysosomal acid protease), we subfractionated homogenized fibroblasts into membrane (and nuclei) cytosol, mitochondria, microsomes, and lysosomes. Greater than 90% of IDA was found to be present in the cytosolar fraction containing neutral protease. IDA in intact fibroblasts was completely inhibited by 1 mM N-ethylmaleimide and partially by 0.5 mM dansylcadaverine (75%), 0.5 mM chloroquine (48%), 1 mg/ml bacitracin (32%) and Trasylol (30%). Lidocaine (5 mM) and glucagon (10(-6)M) exhibited about 15% inhibition with minimal inhibition (7%) by nonsuppressible insulin-like activity. Study of similar inhibitors on subfractionated components indicated inhibition of cytosolar enzyme by N-ethylmaleimide (100%), glucagon (30%), chloroquine (41%), nonsuppressible insulin-like activity (30%), Lidocaine (25%), dansylcadaverine (16%), and bacitracin (11%). Incubation of ammonium sulfate-fractionated cytosolar enzyme at 37 C with A14-125I-insulin resulted in generation of two intermediate peaks as early as 1 min. These peaks could be identified by HPLC but not by molecular sieve chromatography. These intermediates exhibited less immunoprecipitability with antiinsulin antibody and receptor binding with liver membrane preparations than intact insulin. Further incubation of A14-125I-insulin with the cytosolar enzyme(s) resulted in reduction of these peaks as well as insulin and formation of 125Iodotyrosine peak. We conclude that human fibroblast is capable of metabolizing cell-associated A14-125I-insulin in a time- and temperature-dependent manner. This process is inhibited by various inhibitors of insulin processing. The bulk of IDA consists of soluble neutral protease(s) with properties similar to other more purified neutral insulin protease preparations. This fraction, similar to the intact fibroblast degrades insulin to two intermediates with similar molecular weight to that of intact insulin but with more hydrophilicity and less binding affinity to antiinsulin antibody and liver membrane than intact insulin.

In vivo chloroquine-induced inhibition of insulin degradation in a diabetic patient with severe insulin resistance.

We report on a 26-yr-old patient with an 11-yr history of insulin-dependent diabetes mellitus who exhibited insulin resistance with a requirement of up to 15,000 U of intravenous (i.v.) insulin/day. Attempts to diminish her insulin requirement by administration of sulfated insulin or Trasylol were unsuccessful, with the patient remaining resistant to subcutaneous (s.c.) and i.v. administration of pure pork insulin. Chloroquine phosphate therapy (500 mg twice a day) resulted in a decreased requirement for i.v. insulin (700 U/day as compared with the pretreatment requirement of 8400 U/day). Accelerated insulin degradation in s.c. fat tissue of the patient before treatment with chloroquine was demonstrated. This activity was decreased by 64% during chloroquine therapy. Inhibition of insulin degrading activity (IDA) during chloroquine therapy was associated with reductions in the leukocyte lysosomal enzymes alpha-galactosidase and hexosaminidase-A but not hexosaminidase-B and beta-glucuronidase. This study constitutes the first reported use of chloroquine for treatment of insulin resistance as a result of accelerated insulin degradation, and it provides evidence of the effectiveness of this agent in this rare condition.

Activation of pyruvate dehydrogenase complex by porcine and biosynthetic human insulin in cultured human fibroblasts.

Cultured human fibroblasts represent an appropriate model for studying both insulin receptor interaction and hormone responsiveness. We have investigated the properties of the pyruvate dehydrogenase multi-enzyme complex (PDC) and have studied the effects of various concentrations of porcine and biosynthetic human insulin (BHI) on the activity of the enzyme. Under optimal conditions of the assay, both BHI and porcine insulin activated PDC in a dose-dependent fashion in which full activation of the enzyme was achieved with 10(-8) M insulin. The half-maximal concentration for porcine and human insulin was similar, occurring at the level of 5 X 10(-9) M for activation of the PDC of human fibroblasts. We conclude that the PDC of cultured human fibroblasts is activated by both human and porcine insulin at a comparable physiologic concentration. Human fibroblasts may therefore serve as a useful model to study insulin action in isolated human tissue.

Somatostatin metabolism: differences in clearance of N-terminal and central portions of molecule during perfusion of rat liver.

Clearance of cyclic somatostatin (SRIF) from a plasma-free recirculating medium containing human erythrocytes and a bovine albumin fraction was measured with site-specific N-terminal (sheep B) and central core-directed (R101) radioimmunoassays during perfusion of the isolated rat liver (3-4 g). With the N-terminal radioimmunoassay (RIA), the t 1/2, hepatic clearance, and extraction of somatostatinlike immunoreactivity (SLI) were 20.9 +/- 2.0 (SE) min, 2.82 +/- 0.27 ml/min, and 35.2 +/- 3.4%. Corresponding values for the centrally directed assay were 51.0 +/- 6.3 min, 1.16 +/- 0.14 ml/min, and 14.4 +/- 1.8%. Clearances of immunoprecipitable 125I-Tyr-SRIF and [125I-Tyr11]SRIF were 6.56 and 1.06 ml/min, respectively, and were not saturable by 1 microM Tyr-SRIF and SRIF, respectively. SRIF (1.26 +/- 0.09 nM) and SRIF-28 (1.34 +/- 0.14 nM) clearances determined by R101 RIA were similar. After SRIF-28 perfusion, high-performance liquid chromatographic analysis of SLI showed 86% to be retained with the SRIF-28 peak and 14% with the SRIF peak, suggesting no major conversion of SRIF-28 to SRIF. Des-(Ala1,Gly2)-N3-Ac-SRIF and dihydrosomatostatin were cleared more rapidly than SRIF. Clearance of SLI by the perfusate without the liver was 12-43% of liver clearance, depending on the peptide examined. These results support the hypothesis that aminopeptidase and endopeptidase activities are involved in SRIF clearance by the intact liver. The activities appear to function independently. The intrachain disulfide bond of SRIF may confer relative stability during its hepatic metabolism.

Early detection of degraded A14-125I-insulin in human fibroblasts by the use of high performance liquid chromatography.

We studied the metabolism of A14-125I-insulin in intact human fibroblasts using high performance liquid chromatography (HPLC) to detect and separate its early degradation products. The high resolving power of HPLC enabled us to separate what has been considered "intact insulin" by Sephadex G-50 chromatography or TCA precipitability into two additional peaks that had decreased biochemical properties with respect to immunoprecipitability and receptor binding but not decreased TCA precipitability. We conclude that human fibroblast is capable of metabolizing insulin within 2 min at 37 degrees C into intermediate molecules that can be detected by HPLC but not by TCA precipitability or molecular sieve chromatography.

A rapid means of separating A14-125I-insulin from heterogeneously labeled insulin molecules for biologic studies.

We have used two methods for the preparation of a highly homogeneous insulin with high specific activity. After iodination with chloramine T, the labeled peptides were retained on a disposable Sep Pak cartridge and subsequently eluted. The eluted labeled insulins were further purified by either DEAE cellulose or high performance liquid chromatography (HPLC) to separate A14-125I- from A19-125I-insulin. Both methods of chromatography were effective, but HPLC offered the advantage of better resolution in less time and higher yields of A14-125I-insulin, which is suitable for biologic studies in various target tissues.

Accelerated insulin degradation: an alternate mechanism for insulin resistance.

We have examined insulin and glucagon degrading activities of muscle and fat tissues in 11 subjects (4 lean controls, 3 insulin-resistant obese subjects, 2 non-insulin-dependent diabetic subjects, and 2 insulin-treated diabetic subjects) and correlated degrading activity with (1) basal insulin level and (2) state of insulin resistance. We found hyperinsulinemia and insulin resistance to be significantly correlated with accelerated insulin and glucagon degrading activity. Weight reduction in an insulin-resistant obese patients results in parallel reduction in both basal insulin level and insulin-glucagon degrading activity. These data are consistent with the hypothesis that an alternative mechanism for insulin resistance may be an accelerated insulin degradation at the level of target tissues.

Initial site of insulin cleavage by insulin protease.

Exposure of insulin to insulin protease (insulinase, EC 3.4.22.11), a degradative enzyme with considerable specificity toward insulin, results in alterations in the properties of the insulin molecule. Limited degradation by the enzyme results in a decrease in the ability of insulin to bind to membrane receptors with less change in the immunoprecipitability or trichloracetic acid precipitability of the hormone. Limited degradation by insulin protease also alters insulin so that the molecule becomes susceptible to attack by nonspecific endopeptidases which have no effect on unaltered insulin. These data demonstrate the production of an intermediate in the proteolytic degradation of insulin. By labeling with [14C]dansyl chloride, an insulin intermediate with three amino-terminal residues, glycine, phenylalanine, and leucine, was identified. Analysis of this intermediate demonstrated that it was composed of an intact A chain and a B chain cleaved between residues B16 and B17, with the three peptide chains held together by disulfide bonds. Based on these findings, we hypothesize that a stepwise degradation of insulin occurs in vivo and that an early step in the process is the cleavage between B16 and B17 that renders the molecule sucseptible to further degradation by nonspecific proteases.