Leptin resistance following over-expression of protein tyrosine phosphatase 1B in liver.

Obesity is typically associated with resistance to leptin, yet the mechanism by which leptin signaling becomes impaired is poorly understood. Here we sought to determine if the development of obesity and leptin resistance correlates with increased expression of protein tyrosine phosphatase 1B (PTP1B) in peripheral tissues and whether over-expression of this phosphatase, specifically in liver, could alter the leptin-mediated effects on feeding and glucose metabolism. Obesity was induced in mice through a high-fat diet that resulted in hyperglycemia, hyperinsulinemia and hyperleptinemia. Resistance to leptin was confirmed as exogenous leptin administration reduced food intake in animals on low-fat, but not high-fat diets. Diet-induced resistance to leptin and insulin was associated with increased hepatic levels of PTP1B. Intriguingly, hepatic adenoviral over-expression of PTP1B in ob/ob mice attenuated the ability of exogenous leptin to reduce both plasma glucose levels and food intake. These findings suggest that leptin reduces both plasma glucose and food intake in part through actions on the liver, and hepatic leptin resistance resulting from over-expression of PTP1B may contribute to the development of both diabetes and obesity.

Glucose-dependent insulin release from genetically engineered K cells.

Genetic engineering of non-beta cells to release insulin upon feeding could be a therapeutic modality for patients with diabetes. A tumor-derived K-cell line was induced to produce human insulin by providing the cells with the human insulin gene linked to the 5'-regulatory region of the gene encoding glucose-dependent insulinotropic polypeptide (GIP). Mice expressing this transgene produced human insulin specifically in gut K cells. This insulin protected the mice from developing diabetes and maintained glucose tolerance after destruction of the native insulin-producing beta cells.

Glucose-dependent insulinotropic polypeptide confers early phase insulin release to oral glucose in rats: demonstration by a receptor antagonist.

A novel GIP receptor antagonist was developed to evaluate the acute role of glucose-dependent insulinotropic polypeptide (GIP) in the insulin response to oral glucose in rats. Antisera to an extracellular epitope of the GIP receptor (GIPR) detected immunoreactive GIPR on rat pancreatic beta-cells. Purified GIPR antibody (GIPR Ab) specifically displaced GIP binding to the receptor and blocked GIP-mediated increases in intracellular cAMP. When delivered to rats by ip injection, GIPR Ab had a half-life of approximately 4 days. Treatment with GIPR Ab (1 microg/g BW) blocked the potentiation of glucose-stimulated insulin secretion by GIP (60 pmol) but not glucagon-like peptide-1 (GLP-1, 60 pmol) in anesthetized rats. The insulin response to oral glucose was delayed in conscious unrestrained rats that were pretreated with GIPR Ab. Plasma insulin levels were approximately 35% lower at 10 min in GIPR Ab treated animals compared with controls. As a result, the glucose excursion was greater in the GIPR Ab treated group. Fasting plasma glucose levels were not altered by GIPR Ab. We conclude that release of GIP following oral glucose may act as an anticipatory signal to pancreatic beta-cells to promote rapid release of insulin for glucose disposal.

An ultrasonic approach to localization of fiducial markers for interactive, image-guided neurosurgery--Part I: Principles.

Fiducial markers are reference points used in the registration of image space(s) with physical (patient) space. As applied to interactive, image-guided surgery, the registration of image space with physical space allows the current location of a surgical tool to be indicated on a computer display of patient-specific preoperative images. This intrasurgical guidance information is particularly valuable in surgery within the brain, where visual feedback is limited. The accuracy of the mapping between physical and image space depends upon the accuracy with which the fiducial markers were located in each coordinate system. To effect accurate space registration for interactive, image-guided neurosurgery, the use of permanent fiducial markers implanted into the surface of the skull is proposed in this paper. These small cylindrical markers are composed of materials that make them visible in the image sets. The challenge lies in locating the subcutaneous markers in physical space. This paper presents an ultrasonic technique for transcutaneously detecting the location of these markers. The technique incorporates an algorithm based on detection of characteristic properties of the reflected A-mode ultrasonic waveform. The results demonstrate that ultrasound is an appropriate technique for accurate transcutaneous marker localization. The companion paper to this article describes an automatic, enhanced implementation of the marker-localization theory described in this article.

An ultrasonic approach to localization of fiducial markers for interactive, image-guided neurosurgery--Part II: Implementation and automation.

Registration of image space and physical space lies at the heart of any interactive, image-guided neurosurgery system. This paper, in conjunction with the previous companion paper [1], describes a localization technique that enables bone-implanted fiducial markers to be used for the registration of these spaces. The nature of these subcutaneous markers allows for their long-term use for registration which is desirable for surgical follow-up, monitoring of therapy efficacy, and performing fractionated stereotactic radiosurgery. The major challenge to using implanted markers is determining the location of the markers in physical space after implantation. The A-mode ultrasonic technique described here is capable of determining the three-dimensional (3-D) location of small implanted cylindrical markers. Accuracy tests were conducted on a phantom representing a human head. The accuracy of the system was characterized by comparing the location of a marker analogue as determined with an optically tracked pointer and the location as determined with the ultrasonic localization. Analyzing the phantom in several orientations revealed a mean system accuracy of 0.5 mm with a +/- 0.1-mm 95% confidence interval. These tests indicate that transcutaneous localization of implanted fiducial markers is possible with a high degree of accuracy.

Insulin action in previously diabetic rats receiving graded numbers of islets of Langerhans.

We characterized insulin sensitivity in islet transplanted rats receiving from 500 to 3000 islets. Male Wistar Furth rats made previously diabetic with streptozotocin (55 mg/kg) were infused intraportally with islets of Langerhans (500 islets: n = 8; 1000: n = 6; 2000: n = 6; 3000: n = 5) from syngeneic donors and compared with sham-operated controls (n = 7). At four weeks after islet transplantation, fasting blood glucose was not significantly different between groups (500: 5.1 +/- 0.3; 1000: 4.8 +/- 0.3; 2000: 5.1 +/- 0.3; 3000: 4.6 +/- 0.1; control: 4.7 +/- 0.2 mM; P = 0.6146), and fasting plasma insulin was also not different (P = 0.28). The acute insulin response to glucose (0.3 g/kg i.v.) was correlated with islet equivalent mass (r = 0.63, P = 0.004; transplant rats only); islet transplant animals presented a range of acute insulin secretion from 3 to 90% of control values. Insulin action was measured in vivo in fasted, conscious animals during a hyperinsulinemic-euglycemic glucose clamp with insulin infused at 29 and 72 nmol/kg/min. Despite a wide range of islet mass and insulin secretory capacity, there was no significant difference in the glucose infusion rate between islet groups at either insulin level (P = 0.8211, P = 0.8021). There was also no difference in the glucose infusion rate normalized to the prevailing insulin level (P = 0.1638, P = 0.2302). Thus, our results demonstrate that the islet transplanted rat is consistent with other animal studies and human studies illustrating that a diminished insulin secretion does not necessarily precipitate insulin resistance.

Insulin secretory function in relation to transplanted islet mass in STZ-induced diabetic rats.

In vivo insulin secretion was quantified as the AIRg or AIRa in islet-transplanted rats. Male Wistar-Furth rats previously made diabetic by STZ administration (55 mg/kg) were transplanted with 500, 1000, 2000, or 3000 islets infused into the portal vein (n = 12-14 per group) and were compared with sham-treated controls (CN, n = 16). At 4-5 wk posttransplantation, no significant differences were noted in the FPG or fasting plasma insulin of the experimental groups (P > 0.05). Body weight, however, was 10% less (P < 0.05) in rats receiving 500 islets than in controls, indicating an effect of beta-cell deficiency on growth rates. To determine the relationship between islet mass and insulin secretion, we measured AIRg after a 0.3 g/kg glucose bolus in fasted conscious animals. A significant correlation was observed between the AIRg and islet number (r = 0.61, P = 0.0001), and both 500- and 1000-islet groups could be differentiated from controls by ANOVA (500: 8%; 1000: 12% of controls; P < 0.05). During a glycemic potentiation protocol, AIRa was measured at basal and elevated blood glucose (approximately 16 mM). At neither basal nor elevated blood glucose was AIRa correlated with islet number (basal r = 0.0622, P = 0.7834; elevated r = 0.3133, P = 0.1667). None of the groups could be differentiated by ANOVA (elevated 500: 37%; 1000, 68% of controls; P > 0.05). Although this study illustrates that AIRa may be better preserved in islet-transplanted rats, AIRg is the better correlate of islet number.(ABSTRACT TRUNCATED AT 250 WORDS)

Markedly reduced beta-cell function does not result in insulin resistance in islet autografted dogs.

Autotransplantation of islets of Langerhans has resulted in long-term normoglycemia in pancreatectomized dogs. This canine model is useful in evaluating both the progress of islet transplantation and the effect of a reduced islet mass upon the determinants of glucose tolerance: i.e., insulin secretion, insulin sensitivity, and glucose effectiveness. To determine the effect of a reduced islet mass on these factors, we measured the acute insulin response to arginine (AIRa) and glucose (AIRg), the slope of glycemic potentiation of AIRa (SP), insulin sensitivity (Sl), and glucose effectiveness (SG) in control (CN), diabetic (DM), and pancreatectomized dogs rendered normoglycemic with transplanted autografts of islets of Langerhans (TX). Normal fasting plasma glucose (FPG) (TX 4.7 +/- 0.2 mM; CN 4.9 +/- 0.1 mM; P greater than 0.05) was maintained despite a markedly reduced insulin secretion in TX (AIRa 24%, AIRg 15%, and SP 11% of CN). All measures of insulin secretion were significantly correlated (SP vs. AIRg, r = 0.80, P less than 0.0001; AIRa vs. AIRg, r = 0.92, P less than 0.0001) across all animals, but none of the measures of secretion were significantly correlated with either the number of islets transplanted or time posttransplant (P greater than 0.10). Insulin sensitivity was normal in islet autografted dogs (TX: 136 +/- 12 min-1/(nmol/ml); CN: 101 +/- 11 min-1/(nmol/ml), P greater than 0.05) but SG was reduced (TX: 1.93 +/- 0.28 x 100 min-1; CN: 3.53 +/- 0.35 x 100 min-1, P less than 0.05), as determined by the minimal-model method. In diabetic animals (FPG = 16.1 +/- 1.3 mM), insulin secretion was negligible by all measures (P greater than 0.05), and was associated with insulin resistance (Sl = 28 +/- 8 min-1/(nmol/ml)) and reduced SG (1.72 +/- 0.11 x 100 min-1). These studies indicate that across a range of insulin secretion in dogs, the secretagogues arginine and glucose provide similar estimates of beta-cell function. This markedly reduced beta-cell function does not result in insulin resistance when fasting normoglycemia is maintained, but is associated with a decrease in glucose action at basal insulin.

Dynamics of glycemic normalization following transplantation of incremental islet masses in streptozotocin-diabetic rats.

We examined the dynamics of glycemic normalization following intraportal infusion of an incremental number of islets of Langerhans in male Wistar-Furth rats. Non-fasted plasma glucose, 24-hr urine volume, and body weight were determined weekly during three weeks of streptozotocin-induced diabetes and for 5 weeks following transplantation of 250-3000 freshly isolated islets. At one week following transplantation, urine volume was inversely proportional to the mass of islets transplanted, but by 5 weeks posttransplantation urine volume was near-normal except in rats receiving only 250 islets. On the basis of the mean data, the nonfasted plasma glucose fell linearly at a rate of 66 mg/dl per week in rats receiving 500-1000 islets, with normoglycemia (147 +/- 9 mg/dl) being obtained 5 weeks posttransplantation. Examination of the individual time courses for nonfasted plasma glucose revealed a different pattern of glycemic normalization, which consisted of sustained hyperglycemia followed by a rapid fall in the plasma glucose level. During the week prior to normalization glucose fell at a rate of 170 mg/dl per week and normoglycemia was obtained from 1 to 5 weeks following transplantation. Examination of the frequency distribution of nonfasted glucose levels suggested a threshold of 300 mg/dl for glycemic normalization. We conclude that the dynamics of glycemic normalization following transplantation of a suboptimal islet mass include sustained hyperglycemia of variable duration, followed by a rapid fall in the nonfasted plasma glucose level. The contributions of changes in insulin secretion and insulin action underlying this dynamic behavior remain to be determined.

Preparation of target antigens specifically recognized by human natural killer cells.

In an attempt to identify target cell membrane molecules recognized by natural killer (NK) cells, artificial membranes were prepared from detergent-solubilized plasma membranes of NK target cells and synthetic lipids. Such reconstituted membranes from human and rat NK target cells were shown to inhibit both human and rat NK-target cell conjugates in a species-specific fashion; these reconstituted membranes failed to inhibit NK cytotoxicity. The detergent-solubilized material from the human NK target K562 was subjected to various procedures prior to reconstitution and the conjugate inhibition assay. Conjugate inhibitory activity was lost upon trypsin digestion and incubation at 65 degrees C. This inhibition activity was absorbed to concanavalin A agarose and could subsequently be eluted with alpha-methylmannoside, resulting in approximately 20-fold purification. Gel filtration of this material on an AcA-34 column in detergent gave a broad activity peak with maximal activity in the molecular weight range of 30,000-165,000. Gel electrophoresis of purified membranes demonstrated multiple molecular weight bands in lipid membranes. The K562 membrane material, purified by concanavalin A agarose and gel filtration, inhibited conjugates between human NK cells and any of four human target cells, but not of conjugates with (1) human large granular lymphocytes and antibody-coated mouse tumor cells nor (2) rat NK cells and their target cells. Thus the purified glycoproteins from K562 retain the property of specific inhibition of human NK-target conjugates.

Fusion and biochemical expression of membrane receptors in foreign living cells.

Successful transplantation of cell surface molecules from the membranes of one cell type to recipient cells of a different type is described. Plasma membranes purified from donor cells were fluoresceinated and fused to recipient cells using poly(ethylene glycol) and the fate of the transplanted membrane components was followed by fluorescence microscopy. In approximately 100 min the 'foreign' membrane components were seen to cluster and internalise. During this time, judged by the criteria of hormonal stimulation and immune cytotoxic killing, the cell surface of the recipient cell mimicked the cell surface phenotype of the donor cell.

Triggering of the macrophage and neutrophil respiratory burst by antibody bound to a spin-label phospholipid hapten in model lipid bilayer membranes.

The specific antibody-dependent stimulation of the respiratory burst (cyanide-insensitive oxygen consumption, 1-C-glucose oxidation) of RAW264 macrophage cell line by haptenated lipid vesicles depends strongly on the physical properties of the lipid membrane, as well as the surface density of antibodies on the vesicles. Lipid membranes that are "solid" at 37 degrees C (dipalmitoylphosphatidylcholine, DPPC) are much more effective, per vesicle bound, than are "fluid" membranes (dimyristoylphosphatidylcholine, DMPC). Vesicle membranes that have both fluid and solid regions (DPPC containing < 20 mol % cholesterol) show both enhanced binding rates (due to the fluid regions) and enhanced respiratory rates (due to the solid regions). In contrast to these results, the specific antibody-dependent respiratory burst of neutrophils due to haptenated vesicles parallels the antibody-dependent vesicle binding and shows no significant difference between fluid and solid target membranes.

Freeze-fracture of reconstituted model membranes used as targets for cell-mediated cytotoxicity.

In a recent communication (Hollander, N., Mehdi, S.Q., Weissmann, I.L., McConnell, H.M. and Kriss, J.P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4042-4045) we reported that reconstituted model membranes containing murine tumor cell membrane proteins can be substituted for living cells as targets for cell-mediated cytolysis by allosensitized T-lymphocytes. The specificity of the lytic process was governed by the appropriate histocompatibility antigen (H-2). It was stressed, however, that although a standard protocol was faithfully followed for the reconstitution of the target membrane vesicle, the system was not uniformly reproducible. Some experiments showed high levels of specific vesicle killing while no lysis was observed in others. This work extends our description of the structural requirements of reconstituted membrane vesicles.

A study of the physiologic relationship of lower and upper airways with 133Xe.

In order to test the hypothesis that there is air streaming through the upper airways to the ipsilateral lung, and that reflex hypoventilation of the ipsilateral lung may occur in unilateral nasal obstruction, 11 volunteers and three patients were examined for these phenomena, employing Xenon-133 (133Xe) and a special mask permitting inhalation of the gas through one nostril at a time. Patients with deviated nasal septa were also examined, one of these before and after surgery. There was no difference in 133Xe distribution between right and left lungs when gas was inhaled through either normal nostril as compared to inhalation through the mouth, in patients with deviated septa or with cotton obstruction of the contralateral nostril. Furthermore, there was no evidence of delayed washout of 133Xe from either lung or any segment thereof under conditions of nasal obstruction. Thus acute or chronic nasal obstruction does not alter the distribution of inpired gas to the lungs. Neither air streaming nor reflex bronchoconstriction occurs with nasal obstruction.

Collimator evaluation for Tl-201 myocardial imaging.

Three collimators--high-resolutions, converging, and pinhole--were evaluated for Tl-201 myocardial imaging. Line spread function, sensitivity measurements, and phantom and animal studies were used. Features common to all the collimators were: a) better resolution at a closer distance with higher count density, and b) higher infarct detection rate in the tangenital projection than in the en face view relative to the lesion. Furthermore, an infarct in the epicardial location was better visualized than one in the endocardial location. In terms of resolution and sensitivity, the high-resolution collimator was found to be satisfactory in most clinical imagings, but for visualization of an infarct, its size by weight must be over 10--12 g. The pinhole collimator could resolve an infarct as small as 7 g, and use of the pinhole yielded a diagnostic accuracy of over 90%, compared with 75-80% for the high-resolution collimator. Although the low sensitivity of the pinhole collimator precludes its routine clinical use, the selected view would increase diagnostic accuracy. The converging collimator performed poorly in terms of lesion detectability, and its routine clinical use is not encouraged. The conclusion drawn here is valid in the system we have studied, but the variety of converging collimators must be evaluated further for their specific purposes.

The effect of photon energy on tests of field uniformity in scintillation cameras: concise communication.

Although Co-57 is generally used for testing the field uniformity of scintillation cameras, the various photon energies of other radionuclides require uniform response throughout the entire range of energies to which a scintillation camera can respond. The use of Co-57, however, may not adequately demonstrate the field response, which may be uniform at 122 keV but not at other energies. Two scintillation camera systems were investigated in this regard by storing field-flood images, obtained at several photon energies, in a minicomputer. The stored data were analyzed using the Kolmogorov-Smirnov test, revealing that field uniformity may change with photon energy. One of the scintillation cameras showed a variation in field response with photon energy, whereas the other camera did not. These results, however, should not be extrapolated to other cameras of the same type. If a particular scintillation camera is to be used routinely with several energies, its performance should be tested with each one to provide assurance that valid information is being obtained. The effects of dynamic uniformity field correction remain to be evaluated.

Intercomparison of myocardial imaging agents: 201Ti, 129Cs, 43K, and 81Rb.

Thallium-201, 129Cs, 43K, and 81Rb were evaluated as "static" myocardial-imaging agents. Optimal settings of the scintillation camera were determined for each agent. Accumulation for good-quality images can be started as early as 5 min after the dose with 43K, 10 min with 201T1, and 30 min with 129Cs. Imaging times were comparable for 43K, 129Cs, and 201T1 (using the 80-keV x-rays). High-energy photons from the 81Rb preparation, largely from 82mRb contaminant, made it impossible to obtain an interpretable image without the addition of more shielding. Absorbed radiation dose from 81Rb is lower than that from 43K, 129Cs, and 201T1. The highest background activity was observed with 81Rb, followed by 43K, 129Cs, and 201T1 in that order. Overall, 201T1 was best suited for imaging acute myocardial infarction with currently available equipment, and 129Cs was next best. However, because of instrument setting and commercially obtained preparations, 81Rb could not be properly compared with the other radionuclides in our study.