Low insulin content of large islet population is present in situ and in isolated islets

The existence of morphologically distinct populations of islets in the pancreas was described over 60 years ago. Unfortunately, little attention has been paid to possible functional differences between islet subpopulations until recently. We demonstrated that one population, the small islets, were superior to large islets in a number of functional aspects. However, that work did not determine whether these differences were inherent, or whether they arose because of the challenge of isolation procedures. Nor, were there data to explain the differences in insulin secretion. We utilized immunohistochemistry, immunofluorescence, ELISA, and transmission electron microscopy to compare the unique characteristics found in isolated rat islet populations in situ and after isolation. Insulin secretion of small isolated islets was significantly higher compared to large islets, which correlated with higher insulin content/area in small islets (in situ), a higher density of insulin secretory granules, and greater insulin content/volume in isolated islets. Specifically, the core b-cells of the large islets contained less insulin/cell with a lower insulin granule density than peripheral b-cells. When insulin secretion was normalized for total insulin content, large and small islets released the same percentage of total insulin. Small islets had a higher density of cells/area than large islets in vitro and in situ. The data provide a possible explanation for the inferior insulin secretion from large islets, as they have a lower total cell density and the b-cells of the core contain less insulin/cell.     

Hypercytolipidemia-induced nuclear lipoapoptosis: cytochemical analysis and integrated review of hypogonadal, diabetes-obesity syndrome-induced female reproductive axis disruption

Expression of the diabetes (db/db) mutation (i.e., leptin receptor defect) in C57BL/KsJ mice results in the functional suppression of the female pituitary-gonadal axis accompanied by premature utero-ovarian lipocytoatrophy. The current studies define the cytostructural, metabolic and endocrine disturbances associated with hypercytolipidemia and coincident nuclear lipoapoptosis following expression of the db/db-mutation. Adult, female C57BL/KsJ control (+/+ and +/? genotypes) and db/db mutant littermates were monitored for systemic alterations in blood glucose, insulin, luteinizing hormone (LH) and 17-B-estradiol (E2) concentrations associated with db/db-enhanced cytolipid depositions and TUNEL-labeled 3'-DNA fragmentation indexed nuclear lipoapoptosis. Obesity, hyperglycemia and hyperinsulinemia, in addition to depressed LH and E2 concentrations, characterized all db/db-mutants relative to control indices. Structural and cytochemical analysis of basophilic gonadotroph cells, ovarian follicular granulosa cells and uterine endometrial epithelial layers indicated that db/db mutants demonstrated prominent hypercytolipidemia relative to control cytoarchitecture profiles. Vasolipidemia and interstitial cytoadiposity were prominent in all db/db tissue compartments. In each affected cell type within the db/db pituitary-reproductive tract axis, hypercytolipidemia was localized with pronounced nuclear lipo-infiltration and 3'-DNA TUNEL-labeled fragmentation. These data indicate that coincident cytostructural, endocrine and metabolic disturbances associated with hypogonadal pituitary-reproductive tract hypercytolipidemia are functional manifestations of the expressed diabetes-obesity syndrome in db/db-mutants. The progressive vaso-, interstitial-, and cyto-lipidemic alterations in cytoarchitecture correlated with the coincident nuclear lipoapoptotic dissolution and pronounced organo-involution, alterations which contributed to the functional disruption of the pituitary-hypogonadal axis in C57BL/KsJ-db/db mice.     

Urinary C-peptide creatinine ratio to detect absolute insulin deficiency in type 2 diabetes

BackgroundNational Institute for Health and Clinical Excellence guidelines (CG87) recommend neutral protamine hagedorn (NPH) insulin for the provision of basal insulin in type 2 diabetes, but use of analogue insulin is as much as 40%. Where residual endogenous insulin secretory capacity is present there is no evidence that analogue insulins provide any additional benefit over human insulins, and they come at an expensive premium. Anecdotally, however, there is a reluctance to switch people back to NPH insulin, partly because of a perceived risk of pancreatic failure and potential ketosis. Urinary C-peptide creatinine ratio (UCPCR) has been validated as a method for evaluating residual endogenous insulin secretion in type 1 and type 2 diabetes, with a UCPCR of no more than 0·2 nmol/mmol suggestive of absolute insulin deficiency. We aimed to evaluate the prevalence of true insulin deficiency among patients with type 2 diabetes with UCPCR, and confirm findings with the gold standard mixed meal tolerance test (MMTT).Methods191 insulin-treated patients with a clinical diagnosis of type 2 diabetes (diagnosed at or after age 45 years and who did not start insulin within the first year of diagnosis) collected a 2-h post-prandial urine sample for UCPCR measurement. Nine patients from two subgroups (UCPCR ≤0·2 nmol/mmol and UCPCR >0·2) completed a standard MMTT.Findings11 (5·8%) of 191 patients had two consistent UCPCRs of less than or equal to 0·2 nmol/mmol. Nine were able to do the MMTT, of whom five were confirmed to have absolute insulin deficiency (stimulated serum c-peptide <0·2 nmol/L). Three of these five patients were glutamic acid decarboxylase antibody-negative. Nine of nine patients with UCPCR of more than 0·2 nmol/L had confirmed endogenous insulin secretion in their MMTT. Those with insulin deficiency had a shorter time to starting insulin (median 2·5 years [IQR 1·5–3·0] vs 6·0 [3·0–10·75], p=0·005) and lower body-mass index (25 kg/m² vs 29, p=0·04) but no other significant differences in clinical characteristics.InterpretationWe have demonstrated a very low prevalence of true pancreatic failure in this population of insulin-treated patients with type 2 diabetes. This requires further exploration by comparison of a population being treated with NPH insulin with one on analogue insulin, and then determining whether UCPCR could act as a clinical decision support tool to safely switch from analogue insulin to NPH insulin.FundingNational Institute for Health Research.     

Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation

The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals.The assembly and budding of retroviruses involve a series of regulated steps, driven primarily by the viral Gag protein (reviewed in refs. 1 and 2). In the case of HIV-1, assembly and budding occur predominantly at the plasma membrane. The HIV-1 Gag protein is expressed as a 55-kDa polyprotein, comprising four major domains and two spacer peptides (SPs). The major domains are matrix (MA), capsid (CA), nucleocapsid (NC), and p6; the spacer peptides are known as SP1 and SP2 and are located between CA-NC and NC-p6, respectively. Assembly and budding from the host cell are driven by the full-length Gag protein; concomitant with, or shortly after budding, viral particles undergo maturation, wherein the viral protease (PR) cleaves Gag in an ordered cascade to release the mature proteins (3).In addition to Gag, the other major structural component of retroviral particles is the envelope glycoprotein (Env). HIV-1 Env is synthesized as a 160-kDa precursor that traffics to the plasma membrane via the Golgi apparatus, where it is processed to form the surface glycoprotein gp120 and the transmembrane glycoprotein gp41 (reviewed in ref. 4). The processed Env glycoproteins remain noncovalently associated as a heterodimer; the Env spike is a homotrimer of these dimers (5, 6). On the surface of the viral particle, gp120 binds the viral receptor CD4 and the chemokine coreceptors CXCR4 or CCR5. Binding of gp120 to receptor and coreceptor triggers structural changes in gp41 that lead to fusion of the viral and target cell membranes. The fusion activity of gp41 is conferred by the ecto- and transmembrane domains of the protein (7). A third domain, the cytoplasmic tail (CT), is dispensable for fusion but plays important roles in Env trafficking and cell signaling. Like most lentiviruses, HIV-1 encodes an Env bearing a very long CT composed of ∼150 amino acids. In contrast, most other retroviruses encode Env CTs that are ∼25–35 amino acids in length (4). The reasons for the greater length of lentivirus Env CTs are not fully understood. For HIV-1, the CT is required for Env incorporation into particles in physiologically relevant cell types, such as peripheral blood mononuclear cells, monocyte-derived macrophages, and most T-cell lines (8, 9). Recent work has shown that the CT mediates an interaction with Rab11 family-interacting protein 1c (FIP1c), which in turn interacts with Rab14 and appears to direct trafficking of HIV-1 Env to sites of assembly; it is likely that interactions with FIP1c contribute to the observed requirement for the CT in Env incorporation (10, 11). It is, however, unclear why lentiviral CTs are so large, because far smaller Env CTs also contain essential functional trafficking and signaling motifs (12).A consequence of the large lentiviral CT is the potential for, or inevitability of, interactions with the MA domain of Gag during particle assembly. The Gag protein forms a hexameric lattice, driven primarily by CA–CA interactions. Whereas the structure of CA in mature particles has been studied extensively, with many high-resolution structures now available (13–16), and recently progress has been made in determining the structure of the immature Gag lattice (17), the organization of MA in particles has proven difficult to address directly, with no long-range order discernable for the MA shell. The structure of HIV-1 MA has been solved in vitro, using both NMR and crystallography approaches (18, 19). The structure of the monomer is very similar using either approach; however, crystallography suggests a trimeric arrangement for both HIV-1 and simian immunodeficiency virus (SIV) MA proteins (19, 20), whereas NMR reveals only structures for the monomer, with no evidence for higher-order interactions (18). A third approach visualized 2D lattices of MA or MA-CA, using myristylated proteins on a synthetic lipid bilayer with a composition intended to mimic that of the plasma membrane at sites of assembly (21). Under these conditions, MA was seen to arrange as hexamers of trimers, although the low resolution of this approach precluded more-detailed structural analysis. A hexamer-of-trimers arrangement for MA would be compatible with the most recently suggested model for the immature CA lattice, which proposes a hexamer-of-trimers arrangement for the CA amino-terminal domain (CA-NTD), which lies immediately below MA (17).The available structures of MA can be used to place the data acquired through molecular and genetic approaches into context. Mutations have been identified in MA that prevent the incorporation of Env into particles (22–26). The majority of these mutations map to the tips of the MA trimer (27), a region of the protein that lies around the central aperture of the hexamer of trimers. These findings implicate this central aperture as the site of Env incorporation in the particle. This idea is further supported by the observation that, in cell lines permissive for packaging of CT-truncated Env, removal of the CT relieves the inhibition of Env incorporation imposed by the MA mutations (22, 25, 28). The ability to rescue Env incorporation by removing the CT suggests that steric hindrance of Env incorporation may be a key mechanism for the loss of Env incorporation imposed by mutations in MA. This view is consistent with our recent data showing that a mutation at the trimer interface was able to rescue the Env incorporation defects imposed by several MA mutations and a deletion in the Env CT (24). These data suggest that the MA trimer interface regulates the ability to rescue mutants that are defective for Env incorporation.In this study, we sought to develop a system that would allow us to directly determine whether MA forms trimers in virions and, if so, whether MA trimerization plays a role in Env incorporation. By using a combination of biochemical, genetic, and virological approaches, we demonstrate the presence of MA trimers in replication-competent HIV-1 particles, and show a strong correlation between loss of MA trimerization and impaired Env incorporation. These MA trimerization-defective mutants could be rescued by removal of the long Env CT, demonstrating that the requirement for MA trimerization in Env incorporation is linked to the presence of the long gp41 CT. This report both demonstrates the existence of MA trimers in infectious HIV-1 particles and establishes the importance of this structure for Env incorporation.     

Induction of immunologic tolerance to thymus-dependent antigen (sheep's red blood cells) in the absence of T cells

The role of T cells in the induction of tolerance of B cells to sheep's red blood cells (SRBC) by means of cyclophosphamide (CP) was investigated. Tolerance was obtained in adult intact mice, in mice irradiated lethally and protected with syngeneic embryonic liver cells and thymocytes (TB mice), and in mice deprived of T cells—either thymectomized or lethally irradiated and protected with embryonic liver cells (B mice). This form of tolerance was shown to be due to specific elimination of T lymphocytes and, to some extent also, of B lymphocytes. Tolerogenic treatment of B mice, as also of TB mice, led to depression of their immunoreactivity. Spleen cells of tolerant B mice did not suppress the immune response of intact spleen cells. It is concluded that under the conditions investigated, tolerance of B cells can be formed without the participation of T lymphocytes.Laboratory of Immunologic Tolerance, N. F. Gamaleya Institute of Epidemiology and Microogy, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academican of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 87, No. 1, pp. 27–30, January, 1979.     

Reconstruction of soft tissue defects of the hand using the shape-modified radial forearm flap.

We have reconstructed soft tissue defects in 121 hands with radial forearm flaps. So that the flap perfectly fitted the defect, and to minimise the size of the donor site, we divided the flap into two or three components in each case. We call this the shape-modified radial forearm flap. Of the 118 patients, 113 had complete survival of the flap. The follow-up time was 1 to 15 years. The donor sites were closed primarily in all patients, giving good aesthetic results. The shape-modified radial forearm flap seems to be reliable, and makes it possible to adjust the flap according to the defect. The donor area can be closed primarily in all cases.     

A peptide agonist of the neural cell adhesion molecule (NCAM), C3, protects against developmental defects induced by a teratogen pyrimethamine.

The neural cell adhesion molecule, NCAM, not only plays an important role in neuronal migration, differentiation and formation of connections in the developing nervous system, but also in the condensation of the mesodermal mesenchyme of the limb bud. Therefore, NCAM may be regarded as a target molecule for preventive strategies aimed at minimizing the effects of teratogens affecting the prenatal development of the nervous system and the skeleton. Treatment of fetuses with the teratogen pyrimethamine results in a reduced body weight, microcephaly and malformations of the hind limbs and forelimbs, e.g. micromelia, brachydactyly and adactyly. We here show that a peptide agonist of NCAM, C3, partly prevented the defects induced by this treatment. Although intra-amniotic administration of C3 at gestational day 14 had no effect on the pyrimethamine-induced reduction in body weight, it rescued the deficit in brain weight (microcephaly), partly reversed a decrease in thickness of the cortical plate, and significantly reduced the number of malformed fetuses. In vitro, C3 promoted survival of PC12-E2 cells treated with pyrimethamine. Since C3 is a peptide mimetic of NCAM, our data strongly suggest that stimulating of NCAM results in neuroprotection in vivo and in vitro.     

Immunological aspects of food intolerance in children during first years of life]

A study was made of the morphofunctional status and local defence of the gastrointestinal tract in 122 children aged 4 months to 6 years, suffering from food intolerance showed up by atopic dermatitis in 52 children and by chronic diarrhea in 70 children. Based on the allergological anamnesis, scarification cutaneous tests with food allergens, detection of antibodies to food antigens (RAST, HAIT) food allergy was revealed in all the children. Chronic gastroduodenitis was identified in all the children suffering from atopic dermatitis and in 95% of the children with chronic diarrhea. It should be mentioned that one-third of that group had a graver illness--diffuse duodenitis with sub-atrophy of the villi. The allergic genesis of the impairment of the gastroduodenal mucosa was confirmed. It was more remarkable in atopic dermatitis (tissue eosinophilia and high content of IgE-plasmacytes in the duodenal mucosa). The decrease of local immune defence of the mucous membrane, lactase deficiency, elevated growth of microorganisms in the duodenal contents promote the rise of intestinal barrier permeability for food antigens and enhancement of sensitization.