Richard A. Zuellig

Superiority of Small Islets in Human Islet Transplantation

Many factors influence the outcome of islet transplantation. As islets in the early posttransplant setting are supplied with oxygen by diffusion only and are in a hypoxic state in the portal system, we tested whether small human islets are superior to large islets both in vitro and in vivo. We assessed insulin secretion of large and small islets and quantified cell death during hypoxic conditions simulating the intraportal transplant environment. In the clinical setting, we analyzed the influence of transplanted islet size on insulin production in patients with type 1 diabetes. Our results provide evidence that small islets are superior to large islets with regard to in vitro insulin secretion and show a higher survival rate during both normoxic and hypoxic culture. Islet volume after 48 h of hypoxic culture decreased to 25% compared with normoxic culture at 24 h due to a preferential loss of large islets. In human islet transplantation, the isolation index (islet volume as expressed in islet equivalents/islet number), or more simply the islet number, proved to be more reliable to predict stimulated C-peptide response compared with islet volume. Thus, islet size seems to be a key factor determining human islet transplantation outcome.

Altered synaptic clustering of GABAA receptors in mice lacking dystrophin (mdx mice)

Dystrophin is selectively localized in the postsynaptic density of neurons in cerebral cortex, hippocampus and cerebellum. Here, we show by double‐immunofluorescence staining that dystrophin is extensively colocalized with GABAA receptor subunit clusters in these brain regions. To determine the relevance of this observation, we investigated in mdx mice, which provide a model of Duchenne muscular dystrophy, whether the absence of dystrophin affects the synaptic clustering of GABAA receptors. A marked reduction in the number of clusters immunoreactive for the α1 and α2 subunits was observed in, respectively, cerebellum and hippocampus of mdx mice, but not in striatum, which is normally devoid of dystrophin. Furthermore, these alterations were not accompanied by a change in gephyrin staining, although gephyrin is colocalized with the majority of GABAA receptor clusters in these regions. These results indicate that dystrophin may play an important role in the clustering or stabilization of GABAA receptors in a subset of central inhibitory synapses. These deficits may underlie the cognitive impairment seen in Duchenne patients.

Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms

Cardiac hypertrophy is an adaptive response that normalizes wall stress and compensates for increased workload. It is accompanied by distinct qualitative and quantitative changes in the expression of protein isoforms concerning contractility, intracellular Ca(2+)-homeostasis and metabolism. Changes in the myosin subunit isoform expression improves contractility by an increase in force generation at a given Ca(2+)-concentration (increased Ca(2+)-sensitivity) and by improving the economy of the chemo-mechanical transduction process per amount of utilised ATP (increased duty ratio). In the human atrium this is achieved by partial replacement of the endogenous fast myosin by the ventricular slow-type heavy and light chains. In the hypertrophic human ventricle the slow-type beta-myosin heavy chains remain unchanged, but the ectopic expression of the atrial myosin essential light chain (ALC1) partially replaces the endogenous ventricular isoform (VLC1). The ventricular contractile apparatus with myosin containing ALC1 is characterised by faster cross-bridge kinetics, a higher Ca(2+)-sensitivity of force generation and an increased duty ratio. The mechanism for cross-bridge modulation relies on the extended Ala-Pro-rich N-terminus of the essential light chains of which the first eleven residues interact with the C-terminus of actin. A change in charge in this region between ALC1 and VLC1 explains their functional difference. The intracellular Ca(2+)-handling may be impaired in heart failure, resulting in either higher or lower cytosolic Ca(2+)-levels. Thus the state of the cardiomyocyte determines whether this hypertrophic adaptation remains beneficial or becomes detrimental during failure. Also discussed are the effects on contractility of long-term changes in isoform expression of other sarcomeric proteins. Positive and negative modulation of contractility by short-term phosphorylation reactions at multiple sites in the myosin regulatory light chain, troponin-I, troponin-T, alpha-tropomyosin and myosin binding protein-C are considered in detail.

Purification of axonin-1, a protein that is secreted from axons during neurogenesis.

Using selective metabolic labelling in a compartmental cell culture system two proteins, denoted axonin‐1 and axonin‐2, were found to be secreted by axons of dorsal root ganglia neurons from chicken embryos. Based on its characteristic coordinates and spot morphology in two‐dimensional gel electrophoresis, axonin‐1 was detected in the cerebrospinal fluid and the vitreous fluid, axonin‐1 was purified 476‐fold to homogeneity by a four‐step chromatographic procedure. The identity of the purified protein as axonin‐1 was confirmed by immunological methods. Axonin‐1 is a glycoprotein that subdivides into at least 16 immunologically similar isoelectric variants; their molecular weight range extends from 132 to 140 kd and their pI range from 5.3 to 6.2. In the vitreous fluid of the embryo, axonin‐1 could first be detected on the embryonic day 5 and highest concentrations were measured during the second half of embryonic life; in the vitreous fluid of the adult chicken, concentrations were approximately 20 times lower. The early onset of secretion and the time course of expression suggest a role for axonin‐1 in the development of the nervous system.

Alterations in dystrophin and utrophin expression parallel the reorganization of GABAergic synapses in a mouse model of temporal lobe epilepsy

Dystrophin and its autosomal homologue utrophin are coexpressed in muscle cells, and utrophin is functionally able to replace dystrophin in models of Duchenne muscular dystrophy. In brain, the two proteins are expressed differentially, suggesting distinct functional roles. Dystrophin is associated with postsynaptic GABAA receptors in hippocampus, cortex and cerebellum, whereas utrophin is present extrasynaptically, notably in large brainstem neurons. Here, the regulation of dystrophin and utrophin was investigated in a model of temporal lobe epilepsy. Adult mice were injected unilaterally with kainic acid into the dorsal hippocampus to induce loss of pyramidal cells and hypertrophy of dentate gyrus (DG) granule cells, as described ( Suzuki, F., Junier, M.P., Guilhem, D., Sorensen, J.C. & Onteniente, B. (1995)Neuroscience, 64, 665–674.). These morphological changes were associated with an increase in postsynaptic GABAA‐receptors in the ipsilateral DG, as demonstrated by a parallel increase in punctate immunoreactivity to GABAA‐receptor α2 subunit, gephyrin and dystrophin in the molecular layer. Thus, both dystrophin and gephyrin were involved in postsynaptic clustering of GABAA receptors. A transient induction of utrophin was seen at the onset of degeneration in CA1 and CA3 pyramidal cells and in the hilus. Most strikingly, however, utrophin immunoreactivity appeared in the granule cell layer of the DG and became very strong in hypertrophic granule cells 1–2 months post‐kainate treatment. These results suggest that utrophin provides structural support of neuronal membranes, whereas dystrophin is a component of GABAergic synapses.

DIFFERENTIAL EXPRESSION OF UTROPHIN AND DYSTROPHIN IN CNS NEURONS: AN IN SITU HYBRIDIZATION AND IMMUNOHISTOCHEMICAL STUDY

The cellular distribution of utrophin, the autosomal homologue of dystrophin, was investigated in developing and adult rat and mouse brain by in situ hybridization and immunohistochemistry. Digoxigenin‐labeled cRNA probes complementary to N‐terminal, rod‐domain, and C‐terminal encoding sequences of utrophin were used to differentiate between full‐length and short C‐terminal isoforms. Largely overlapping distribution patterns were seen for the three probes in neurons of cerebral cortex, accessory olfactory bulb, and several sensory and motor brainstem nuclei as well as in blood vessels, pia mater, and choroid plexus. The C‐terminal probe was detected in addition in the main olfactory bulb, striatum, thalamic reticular nucleus, and hypothalamus, suggesting a selective expression of G‐utrophin in these neurons. Western blot analysis with isoform‐specific antisera confirmed the expression of both full‐length and G‐utrophin in brain. Immunohistochemically, only full‐length utrophin was detected in neurons, in close association with the plasma membrane. In addition, intense staining was seen in blood vessels, meninges, and choroid plexus, selectively localized in the basolateral membrane of immunopositive epithelial cells. The expression pattern of utrophin was already established at early postnatal stages and did not change thereafter. Double‐labeling analysis revealed that utrophin and dystrophin are differentially expressed on the cellular and subcellular levels in juvenile and adult brain. Likewise, in mice lacking full‐length dystrophin isoforms (mdx mice), no change in utrophin expression and distribution could be detected in brain, although utrophin was markedly up‐regulated in muscle cells. These results suggest that utrophin and dystrophin are independently regulated and have distinct functional roles in CNS neurons. J. Comp. Neurol. 422:594–611, 2000.

Differential Effects of Protein Kinase B/Akt Isoforms on Glucose Homeostasis and Islet Mass

ABSTRACT Protein kinase B (PKB)/Akt is considered to be a key target downstream of insulin receptor substrate 2 (IRS2) in the regulation of β-cell mass. However, while deficiency of IRS2 in mice results in diabetes with insulin resistance and severe failure of β-cell mass and function, only loss of the PKBβ isoform leads to a mild metabolic phenotype with insulin resistance. Other isoforms were reported not to be required for metabolic regulation. To clarify the roles of the three PKB isoforms in the regulation of islet mass and glucose homeostasis, we assessed the metabolic and pancreatic phenotypes of Pkbα, Pkbβ, and Pkbγ-deficient mice. Our study uncovered a novel role for PKBα in the regulation of glucose homeostasis, whereas it confirmed that Pkbβ−/− mice are insulin resistant with compensatory increase of islet mass. Pkbα−/− mice displayed an opposite phenotype with improved insulin sensitivity, lower blood glucose, and higher serum glucagon concentrations. Pkbγ−/− mice did not show metabolic abnormalities. Additionally, our signaling analyses revealed that PKBα, but not PKBβ or PKBγ, is specifically activated by overexpression of IRS2 in β-cells and is required for IRS2 action in the islets.

Glycemic Control in Simultaneous Islet-Kidney Versus Pancreas-Kidney Transplantation in Type 1 Diabetes: A Prospective 13-Year Follow-up

OBJECTIVE In patients with type 1 diabetes and end-stage renal disease, combined transplantation of a kidney together with a pancreas or isolated pancreatic islets are options to improve glycemic control. The aim of this study was to compare their long-term outcome with regard to metabolic control and surgical complication rate, as well as function of the transplanted kidney. RESEARCH DESIGN AND METHODS We conducted a prospective cohort study in consecutive patients receiving either a pancreas or islet transplant simultaneously with or after kidney transplantation (simultaneous pancreas-kidney [SPK]/pancreas-after-kidney [PAK] or simultaneous islet-kidney [SIK]/islet-after-kidney [IAK] transplantation). RESULTS Ninety-four patients who had undergone SPK/PAK transplantation were compared with 38 patients who had undergone SIK/IAK transplantation over a period of up to 13 years. HbA1c levels declined from 7.8 ± 1.3% (62 ± 14 mmol/mol) to 5.9 ± 1.1% (41 ± 12 mmol/mol), and from 8.0 ± 1.3% (64 ± 14 mmol/mol) to 6.5 ± 1.1% (48 ± 12 mmol/mol), respectively, in the SPK/PAK and SIK/IAK groups (P < 0.001 for both) and remained stable during follow-up, despite a reduction in the rate of severe hypoglycemia by >90%. The 5-year insulin independence rate was higher in the SPK/PAK group (73.6 vs. 9.3% in the SIK/IAK group), as was the rate of relaparotomy after transplantation (41.5 vs. 10.5% in the SIK/IAK group). There was no difference in the rate of kidney function decline. CONCLUSIONS During a long-term follow-up, SPK/PAK transplantation as well as SIK/IAK transplantation resulted in a sustained improvement of glycemic control with a slightly higher glycated hemoglobin level in the SIK/IAK group. While insulin independence is more common in whole-organ pancreas recipients, islet transplantation can be conducted with a much lower surgical complication rate and no difference in kidney function decline.

Deoxysphingolipids, Novel Biomarkers for Type 2 Diabetes, Are Cytotoxic for Insulin- Producing Cells

Irreversible failure of pancreatic β-cells is the main culprit in the pathophysiology of diabetes, a disease that is now a global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as a novel biomarker for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of insulin-producing Ins-1 cells and primary islets. Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic, and apoptotic characteristics and compromised glucose-stimulated insulin secretion. In addition, 1-deoxysphinganine altered cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the Rho family GTPase Rac1. Moreover, 1-deoxysphinganine selectively upregulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified Jun N-terminal kinase and p38 mitogen-activated protein kinase as antagonistic effectors of cellular senescence. The results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic β-cells. Thus, targeting deoxysphingolipid synthesis may complement the currently available therapies for diabetes.

Identification and characterisation of transcript and protein of a new short N-terminal utrophin isoform.

Dystrophin and utrophin are known to link the intracellular cytoskeleton to the extracellular matrix via a transmembraneous glycoprotein complex. Four short C‐terminal isoforms (Dp71, Dp116, Dp140, and Dp260) are described for dystrophin and three for utrophin (Up71, Up113, and Up140). We describe here for the first time the existence of a 3.7‐kb transcript and a 62‐kDa protein in C6 glioma cells representing a short N‐terminal isoform unique for utrophin (N‐utrophin). More than 20 clones covering the entire coding region of utrophin were isolated from a rat C6 glioma cell cDNA library. Two clones were found to code for a protein with 539 amino acids. Its sequence is identical to that of the full‐length utrophin, except for the last residue where Cys is replaced by Val. This isoform contains the actin binding domain (consisting of two calponin homology subdomains), followed by two spectrin‐like repeats. A recombinant fragment corresponding to N‐utrophin binds to F‐actin in vitro with an equilibrium constant (affinity) K of 4.5 × 105 M−1 and a stoichiometry of one fragment per around five actin monomers. Immunocytochemical staining of C6 glioma cells with antisera specific for different utrophin regions localised full‐length utrophin in the submembraneous cortical actin layer as revealed by confocal microscopy. A distinct staining pattern for the N‐utrophin was not detectable, although it was expected to localise at the actin stress fibers. It is assumed that it co‐localises via the two spectrin‐like repeats with the full‐length utrophin at the cell membrane. J. Cell. Biochem. 77:418–431, 2000.