Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site.

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose-basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.

mRNA of the pro-apoptotic gene BBC3 serves as a molecular marker for TNF-α-induced islet damage in humans.

AIMS/HYPOTHESIS: TNF-α plays important roles in the pathogenesis of type 1 and type 2 diabetes mellitus. In light of this, we examined the involvement of a pro-apoptotic gene, BBC3 (also known as PUMA), in TNF-α-mediated beta cell dysfunction and destruction in human islets. METHODS: Human islets were exposed in vitro to TNF-α alone or in combination with IFN-γ. Gene expression was assessed by RT-PCR using a set of single islets. Protein abundance and cellular localisation of BBC3 were assessed by immunoblot and immunohistochemistry. A marginal number of islets were transplanted into diabetic NODscid mice to correlate in vivo islet function with BBC3 expression. RESULTS: BBC3 and IL8 mRNA were upregulated in TNF-α-stimulated islets in a dose-dependent manner and enhanced through addition of IFN-γ, but not upregulated by IFN-γ alone. Immunohistochemistry revealed that TNF-α in combination with IFN-γ upregulated basal BBC3 abundance in the cytoplasm of beta cells along with the perinuclear clustering of mitochondria partially co-localised with BBC3. TNF-α alone did not induce beta cell death, but did abrogate preproinsulin precursor mRNA synthesis in response to high glucose stimulation, which was inversely associated with upregulation of BBC3 mRNA expression by TNF-α. Higher BBC3 mRNA expression in islets correlated with decreased graft function in vivo. CONCLUSIONS/INTERPRETATION: These results suggest that BBC3 mRNA can serve as a molecular marker to detect early TNF-α-induced beta cell stress and may help identify islet-protective compounds for the treatment of diabetes.

Validation of methodologies for quantifying isolated human islets: an Islet Cell Resources study.

BACKGROUND: Quantification of islet mass is a crucial criterion for defining the quality of the islet product ensuring a potent islet transplant when used as a therapeutic intervention for select patients with type I diabetes. METHODS: This multi-center study involved all eight member institutions of the National Institutes of Health-supported Islet Cell Resources Consortium. The study was designed to validate the standard counting procedure for quantifying isolated, dithizone-stained human islets as a reliable methodology by ascertaining the accuracy, repeatability (intra-observer variability), and intermediate precision (inter-observer variability). The secondary aim of the study was to evaluate a new software-assisted digital image analysis method as a supplement for islet quantification. RESULTS: The study demonstrated the accuracy, repeatability and intermediate precision of the standard counting procedure for isolated human islets. This study also demonstrated that software-assisted digital image analysis as a supplemental method for islet quantification was more accurate and consistent than the standard manual counting method. CONCLUSIONS: Standard counting procedures for enumerating isolated stained human islets is a valid methodology, but computer-assisted digital image analysis assessment of islet mass has the added benefit of providing a permanent record of the isolated islet product being evaluated that improves quality assurance operations of current good manufacturing practice.

Comprehensive analysis of human pancreatic islets using flow and laser scanning cytometry.

Assessing islet cellular composition and beta cell viability using Flow Cytometry (FC) and Laser Scanning Cytometry (LSC) may aid in determining the transplant quality of islets. Human islets (2500 IEQ, n = 44, purity >or=80%) dissociated into a single cell suspension were stained with ductal marker CA19, with Newport Green (NG) and FluoZin3 (FL3) for beta-cell identification, with TMRE to assess mitochondrial membrane potential, with DAPI to identify live vs. dead cells, and with Annexin-V/DAPI to differentiate apoptotic and necrotic cells. For LSC, cell preparations (n = 9) were stained for insulin (beta-cells), glucagon (alpha-cells), somatostatin (delta cells), and pancreatic polypeptide (ppp cells). Fluorescence microscopy (EtBr/FDA) and insulin response were also measured. DAPI- staining was 73.78% +/- 1.37, while EtBr/FDA was 96% +/- 0.48. 52.5% +/- 3.73 of all cells were NG+, of which 58.08% +/- 2.61 were NG+/TMRE+. Annexin-V/DAPI staining (n = 26) showed 13.8% +/- 0.89 apoptotic, 27.2% +/- 2.0 necrotic, and 51.9% +/- 2.22 live cells. 26.0% +/- 5.19 of cells were CA19 positive (n = 17), of which 45.5% +/- 4.37 were also TMRE+, and 5.2% +/- 1.2 of the TMRE+ were also NG+/CA19+. NG and FL3 showed similar staining (n = 8). Comparison of short-term (or=3 days) culture showed similar TMRE+/NG+ averages, albeit lower percentages of live (36.4% vs 51.9%), and higher percentages of apoptotic (19.2% vs 13.8%) and necrotic cells (37.4% vs 27.2%) for long-term, as determined by Annexin-V staining. LSC resulted in 54.17% +/- 4.62 beta-cells, 33.33% +/- 4.16 alpha-cells, 8.75% +/- 2.5 delta-cells, and 3.75% +/- 0.79 ppp cells. There is no significant difference between insulin positive cells and NG positive cells (P

Endogenous pancreatic protease activity and methods for impeding their function.

Pefabloc and Trasylol are serine protease inhibitors that have been used during islet isolation to inhibit endogenous proteases (trypsin, chymotrypsin, and elastase) and improve islet yields. Using in vitro studies, we evaluated the effects of Pefabloc and Trasylol on the activities of these proteases and the effect of Pefabloc on islet function. In addition, it has been reported that Protector Solution (PS) enhances the efficiency of Pefabloc. Thus, we evaluated the efficacy of Pefabloc in the presence or absence of PS. EnzCheck protease assay was used to measure the activities of trypsin, chymotrypsin, elastase, liberase, and thermolysin in the presence or absence of 0.4 mmol/L Pefabloc (with or without PS) or 0.43 micromol/L Trasylol. We also tested switch samples containing the highest concentration of enzymes. Pefabloc significantly inhibited trypsin, chymotrypsin, elastase, and switch, but not liberase or thermolysin. Trasylol significantly inhibited all enzymes except for elastase and switch sample. Unexpectedly, the potency of Pefabloc was abrogated when diluted first in PS. Insulin release was diminished when islets were incubated or perifused with Pefabloc. In conclusion, Pefabloc when added appropriately significantly blocked in vitro protease activity. Unfortunately, Pefabloc also decreased islet insulin secretion, making it unsuitable for islet isolation. Trasylol cannot be used with collagenase because it impaired both liberase and thermolysin.

Ulinastatin is a novel protease inhibitor and neutral protease activator.

Pancreata preserved in a solution containing ulinastatin may improve islet quality and quantity. This in vitro study was performed to investigate the efficacy of this agent to inhibit endogenous proteases (trypsin, chymotrypsin, and elastase) as well as its effects on thermolysin, liberase, neutral protease, and pancreas digestion switch samples. The EnzCheck Protease Assay Kit was used to measure the activities of these enzymes in the presence of drug at various concentrations (10, 25, 50, 100, and 200 U/mL) to determine the optimal conditions for inhibition/activation. The percentage of inhibition or activation was determined based on a comparison to controls using standard curves. At 100 U/mL the drug significantly inhibited trypsin (91%; P = .001), chymotrypsin (97%; P = .002), and elastase (43%; P = .01); however, inhibition of the switch samples was not significant (13%; P = .7). Serendipitously, ulinastatin at 10, 25, 50, 100, and 200 U/mL increased thermolysin activity by 9%, 123%, 149%, 172%, and 311%, respectively, and liberase activity by 35%, 27%, 44%, 51%, and 63%, respectively. In conclusion, ulinastatin displays dual functions to inhibit endogenous proteases and to increase neutral protease activity, possibly through allosteric effects. This activation of neutral proteases may significantly enhance collagenase activity, thereby resulting in higher islet yields.

Testing combinations of protease inhibitor and preservation solution to improve islet quality and yield.

UNLABELLED: Pancreas preservation using an oxygenated two-layer method (TLM) has been reported to improve islet yields, as has supplementation of Liberase with Pefabloc. We hypothesized that using both TLM and Pefabloc could enhance islet yield as compared with preservation in University of Wisconsin (UW) or Histidine-Tryptophan Ketoglutarate (HTK) solution. METHODS: Ninety-eight pancreata with no significant differences of age, body mass index, or cold ischemia time preserved randomly with UW (n = 40), TLM (n = 48), or HTK (n = 10) were processed with (n = 36) or without (n = 66) Pefabloc. RESULTS: The total islet equivalent (IEQ) from TLM-preserved pancreata processed with Pefabloc (n = 12) showed lower yields versus those processed without Pefabloc (n = 36): 216,120 +/- 27,906 vs. 301,427 +/- 21,447 IEQ (P < .05). Islets from 1 of 12 (8.33%) pancreata processed with Pefabloc in TLM were transplanted, in contrast with 15/36 TLM (41.67%) pancreata processed without it. Islet yields were not significantly different among pancreata preserved in UW and processed with Pefabloc (n = 17) versus without Pefabloc (n = 23): 342,693 +/- 45,588 versus 266,609 +/- 29,006 IEQ (P = .149). The number of transplants from UW-preserved pancreata was 3/17 (17.65%) when processed with Pefabloc and 4/23 (17.39%) without. Among the HTK group, there was no significant difference in islet yields between pancreata processed with (n = 7) versus without Pefabloc (n = 3): 248,227 +/- 65,294 versus 483,555 +/- 144,070 IEQ (P = .118). CONCLUSIONS: Pefabloc showed no benefit to improve islet yields. Pancreata preserved in TLM provided better transplant quality islets when processed in the absence of Pefabloc.

Evaluation of human islet-specific functional quality cultured on different gas-permeable membranes.

The aim of this study was to investigate different gas-permeable membranes for culturing human islets. Dynamic insulin release was used to assess islet functional quality. Islets isolated from cadaveric pancreata (n = 8) using standard isolation methods were stained with dithizone, counted, and cultured on five different commercially available medical-grade membranes reported to have high permeability to O2, CO2, and other gases. Fraction 1 (20,000 islet equivalents [IEQ] purity >70%; viability >85%) was cultured using serum-free medium in nonadherence tissue culture flasks (group I) and custom-made chambers with membranes (group II). Each vessel contained 5000 IEQ at a density of 30 IEQ/cm2 and 69 IEQ/cm2 for groups I and II, respectively. Islets were cultured for 48 to 90 hours at 37 degrees C in 5% CO2. In vitro dynamic insulin response to low glucose (3 mmol/L), high glucose (16.7 mmol/L), and 25 mmol/L KCI was measured. Stimulation indices were calculated by dividing average of initial response over basal insulin release; basal insulin release defined as average of the first seven values. Islets cultured on MG7 (n = 3) showed a higher stimulation index (3.49 +/- 0.37) compared with flasks (2.44 +/- 0.22), indicating better specific functional quality. Islets cultured on other membranes proved to show similar or worse functional quality than those cultured in flasks. In fact, islets cultured on MG6 (n = 2) were not tested owing to complete disintegration. Islet functional quality was improved when cultured on selected biocompatible gas-permeable membranes; however, finding the best membrane requires further investigation before clinical application.

Long-term survival and function of intraportal porcine and human islet xenografts in nondiabetic nude mice.

Instant blood-mediated inflammatory reaction (IBMIR) is a serious obstacle to both clinical islet allotransplantation and future islet xenotransplantation via the portal vein. We have previously observed uniform long-term tilapia (fish) islet xenograft survival when islets were transplanted intraportally into nondiabetic nude mice (nDNM), but not in diabetic nude mice (DNM). In this study, we examined whether human islets (HI) and adult porcine islets (API) can tolerate intraportal transplantation into nDNM like tilapia islets. HI and API were transplanted intraportally into nDNM. Recipients were humanely killed either 14 or 28 days after transplantation and livers were processed for histology. Human insulin and human C-peptide were measured in the terminal serum samples of HI recipients. In six of seven HI and seven of seven API recipients, liver histology showed insulin-positive islet xenografts. In recipients with HI, the numbers of islets/ductal structures seen histologically correlated well with serum sample results. These results show that HI and API can survive and function long term after intraportal transplantation into nDNM recipients. Our previous and present data indicated that DNM and nDNM could be useful models to study "glucose toxicity" and the role of IBMIR in the fate of intraportal islet grafts.

Glucose-stimulated increment in oxygen consumption rate as a standardized test of human islet quality.

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (DeltaOCR(glc)) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of DeltaOCR(glc) obtained at both institutions were compared. Islet specificity was determined by comparing DeltaOCR(glc) in islet and nonislet tissue. The DeltaOCR(glc) ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (DeltaOCR(glc) was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The DeltaOCR(glc) was statistically independent of percent beta cells, demonstrating that DeltaOCR(glc) is governed more by islet quality than by islet composition. The DeltaOCR(glc), but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing DeltaOCR(glc) as a measurement of islet quality for human islet transplantation.

Improvement of human islet cryopreservation by a p38 MAPK inhibitor.

The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum-free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS-p38IH and islet viability was not significantly different in the three groups. beta Cell numbers and function were the highest in islets cryopreserved with ICS-p38IH. Glucose-stimulated human C-peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

Mesenchymal stem cells facilitate the induction of mixed hematopoietic chimerism and islet allograft tolerance without GVHD in the rat.

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.

Multipotent progenitor cells isolated from adult human pancreatic tissue.

The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.

Inhibition of p38 mitogen-activated protein kinase protects human islets from cryoinjury and improves the yield, viability, and quality of frozen-thawed islets.

The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.

Almonds vs complex carbohydrates in a weight reduction program.

OBJECTIVE: To evaluate the effect of an almond-enriched (high monounsaturated fat, MUFA) or complex carbohydrate-enriched (high carbohydrate) formula-based low-calorie diet (LCD) on anthropometric, body composition and metabolic parameters in a weight reduction program. DESIGN: A randomized, prospective 24-week trial in a free-living population evaluating two distinct macronutrient interventions on obesity and metabolic syndrome-related parameters during weight reduction. SUBJECTS: In total, 65 overweight and obese adults (age: 27-79 y, body mass index (BMI): 27-55 kg/m(2)). INTERVENTION: A formula-based LCD enriched with 84 g/day of almonds (almond-LCD; 39% total fat, 25% MUFA and 32% carbohydrate as percent of dietary energy) or self-selected complex carbohydrates (CHO-LCD; 18% total fat, 5% MUFA and 53% carbohydrate as percent of dietary energy) featuring equivalent calories and protein. MAIN OUTCOME MEASUREMENTS: Various anthropometric, body composition and metabolic parameters at baseline, during and after 24 weeks of dietary intervention. RESULTS: LCD supplementation with almonds, in contrast to complex carbohydrates, was associated with greater reductions in weight/BMI (-18 vs -11%), waist circumference (WC) (-14 vs -9%), fat mass (FM) (-30 vs -20%), total body water (-8 vs -1%) and systolic blood pressure (-11 vs 0%), P=0.0001-0.05. A 62% greater reduction in weight/BMI, 50% greater reduction in WC and 56% greater reduction in FM were observed in the almond-LCD as compared to the CHO-LCD intervention. Ketone levels increased only in the almond-LCD group (+260 vs 0%, P<0.02). High-density lipoprotein cholesterol (HDL-C) increased in the CHO-LCD group and decreased in the almond-LCD group (+15 vs -6%, P=0.05). Glucose, insulin, diastolic blood pressure, total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C) and LDL-C to HDL-C ratio decreased significantly to a similar extent in both dietary interventions. Homeostasis model analysis of insulin resistance (HOMA-IR) decreased in both study groups over time (almond-LCD: -66% and CHO-LCD: -35%, P<0.0001). Among subjects with type 2 diabetes, diabetes medication reductions were sustained or further reduced in a greater proportion of almond-LCD as compared to CHO-LCD subjects (96 vs 50%, respectively) [correction]. CONCLUSION: Our findings suggest that an almond-enriched LCD improves a preponderance of the abnormalities associated with the metabolic syndrome. Both dietary interventions were effective in decreasing body weight beyond the weight loss observed during long-term pharmacological interventions; however, the almond-LCD group experienced a sustained and greater weight reduction for the duration of the 24-week intervention. Almond supplementation of a formula-based LCD is a novel alternative to self-selected complex carbohydrates and has a potential role in reducing the public health implications of obesity.

Male sexual function and its disorders: physiology, pathophysiology, clinical investigation, and treatment.

This review is designed to help the reproductive endocrinologist integrate his or her professional activity with those of other disciplines including urology, radiology, neurology, and psychology in order to successfully manage all of the inseparable aspects of male sexual and reproductive functioning. Significant advances in the field of male sexual physiology and pathophysiology and new methods of investigation and treatment of male sexual disorders are outlined. The review synthesizes available data on the following: norms of sexual organs, aging and sexuality, role of central and peripheral neurochemicals in each stage of the sexual cycle, role of corporeal smooth muscles in the hemodynamic control of erection and detumescence, influence of psychological factors, drugs, and disease on all aspects of sexual functioning, and use of nocturnal penile tumescence monitoring, imaging investigations, and neurophysiologic studies in the diagnostic workup of males with sexual dysfunction. Clinical algorithms are presented where appropriate. Extensive discussions on newly developed strategies in psychological and behavioral counseling, drug therapy, tissue engineering, nonsurgical devices, and surgical treatments for all forms of sexual disorders are also provided. Lastly, the effect of sexual dysfunction and its treatment on quality of life in affected men is addressed, along with recommendations for future research endeavors.

Effects of allogeneic bone marrow transplantation on recipient bone mineral density: A prospective study.

Allogeneic bone marrow transplant (BMT) recipients have many known risk factors for developing decreased bone mineral density (BMD) after transplantation. We performed a prospective sequential evaluation of BMD in the lumbar spine and nondominant hip using dual-energy x-ray absorptiometry (DEXA) in a cohort of 47 adult patients (median age, 43 years) who were undergoing radiation-based BMT for hematologic malignancies. Baseline DEXA studies were performed before BMT and repeated at 3 to 4 months, 6 to 8 months, and 12 to 14 months after BMT. The majority of patients (60%) had been minimally treated with combination cytotoxic chemotherapy, having received no more than 1 treatment regimen before BMT. Graft-versus-host disease prophylaxis consisted of cyclosporine in combination with either methotrexate or prednisone, or both. Mean lumbar spine and hip BMD were normal before BMT (spine: 1.01 g/cm2, z score = 96%; hip: 0.86 g/cm2, z score = 100%) and gradually decreased (spine: 0.98 g/cm2, z score = 94%; hip: 0.76 g/cm2, z score = 91%) at 12 to 14 months. These declines were statistically significant (P < .006 and < .002 for lumbar spine; P < .001 and < .001 for hip). In addition, the sharpest decline occurred during the first 6 months after BMT and was more marked in the hip than the lumbar spine. These data suggest that BMT adversely affects BMD in this patient population.

The interaction between beta-endorphin and gonadal steroids in regulation of luteinizing hormone (LH) secretion and sex steroid regulation of LH and proopiomelanocortin peptide secretion by individual pituitary cells.

Reported studies using the conventional pituitary cell culture technique suggest that beta-endorphin (B-EP) produced locally in the pituitary or reaching it from the hypothalamus acts in conjunction with estradiol (E2) to initiate and in conjunction with progesterone (P4) to terminate the midcycle surge of LH. In addition, the reverse hemolytic plaque assay (RHPA) was used to investigate the effects of E2 and P4 on the secretory activity of individual pituitary cells. The results of these experiments indicate that 1) E2 enhances the secretion of LH, ACTH, and B-EP by individual pituitary cells; 2) E2 increases the number of secreting cells for each of the three hormones; 3) the rise in B-EP and ACTH secretion antecede that of LH; 4) P4 augments ACTH and B-EP secretion by individual pituitary cells; and 5) P4 has dual effects, acutely (1 h) potentiating LH secretion from already active cells and subsequently (8 h) recruiting cells that formerly had little or no secretory activities. Collectively, the above studies support a role for steroid hormones in regulation of midcycle LH secretion at the pituitary level. The results also suggest that intrapituitary (paracrine/autocrine) and extrapituitary (endocrine) B-EP modulates gonadal steroid effects on LH secretion by pituitary gonadotrophs.

Magnesium deficiency and glucose metabolism in rat adipocytes.

We examined the effect of reducing ambient and intracellular free Mg ion ([Mg]i) concentrations on insulin action in epididymal adipocytes from male Sprague-Dawley rats in terms of (1) cellular transport of nonmetabolizable 2-deoxyglucose, (2) [U-14C]glucose oxidation to CO2, and (3) D-[3H]glucose incorporation into triglycerides. There were no significant differences in basal or insulin-stimulated transport of 2-deoxyglucose between adipocytes cultured in physiologic (1.24 mmol) or low (0.16 mmol) Mg for up to 24 hours. In contrast, insulin-stimulated but not basal [U-14C]glucose oxidation to CO2 was significantly reduced in adipocytes cultured in low versus physiologic Mg (P < .05 to .01). Similarly, there were no differences in basal glucose incorporation into triglycerides between cells cultured in low or physiologic Mg media for up to 24 hours. However, long-term (24-hour) but not short-term (2-hour) exposure of cells to low Mg was associated with a significant 30% reduction in insulin-stimulated D-[3H]glucose incorporation into triglycerides. When adipocytes incubated in low Mg were reincubated in high Mg (1.24 or 5 mmol) for 30 minutes, normal insulin-stimulated D-[3H]glucose incorporation into triglycerides was restored. Incubation of adipocytes in low Mg (0.16 mmol) for 24 hours resulted in a significant decrease in [Mg]i (264 +/- 89 v 437 +/- 125 micromol/cell [mean +/- SEM]) as compared with cells incubated in physiologic Mg (1.24 mmol; P < .01). These data support a role for intracellular Mg deficiency in the development of insulin resistance and suggest that the effect occurs at a site(s) distal to glucose entry into the cell. The effect of Mg deficiency on insulin action appears to be reversible.

Evaluation of the infertile couple.

The evaluation of the infertile couple is usually a lengthy investigation in which all possible etiologic factors in both partners have to be considered. Optimal and cost-effective investigation requires adequate recognition of significant historical data and physical findings. Males without stigmata of endocrinopathies or general medical illnesses require an analysis of their semen as the minimum initial step of evaluation. Those suspected of deficient androgen production and/or action and those with abnormal sperm counts, motility, and/or morphology need assessment of their serum concentrations of selected reproductive hormones. When these initial investigations are negative and there are no demonstrable etiologic female factors underlying the state of infertility, specialized sperm function and sperm allergy testing needs to be performed. The initial investigation of the female partner is best served by assessing the frequency of ovulation and adequacy of corpus luteum function. Women without ovulatory defects should be assessed for the presence of the hostile cervical mucus and structural anomalies of the reproductive tract. Investigations of patients with menstrual dysfunctions should be based upon the presence or absence of hirsutism, changes in body weight, and evidence of other endocrinopathies or medical illnesses. Following the identification and normalization of causes of anovulation, further work-up of patients who remain infertile is similar to those with regular menstrual cycles. The diagnosis of idiopathic infertility is essentially by exclusion of all other causes. Algorithms for the diagnostic evaluation of most infertile couples are provided.