Derrel W. Clarke

Maturity-Onset Diabetes of Youth in Black Americans

Twelve of 129 black patients with youth-onset diabetes were identified as having an unusual clinical course, with apparent insulin dependence at the time of presentation followed by absence of dependence months to years later. This atypical form of diabetes was found in at least two generations in 9 of the 12 families of the propositi. Fourteen of the diabetic relatives, as well as the 12 propositi, were studied. Islet-cell autoantibodies were not found in any of the patients, and thyroid microsomal auto-antibodies were found in only one. The frequencies of the insulin-dependent-diabetes-associated antigens HLA-DR3 and DR4 were not increased among the propositi, and diabetes did not cosegregate with HLA haplotypes in the informative families. Insulin secretion, as measured by C-peptide responses to a liquid mixed meal (Sustacal), was intermediate between secretion in nondiabetic controls and that in patients with classic insulin-dependent diabetes. Peripheral-blood monocytes expressed increased numbers of insulin receptors as well as decreased empty-site affinities. The atypical form of diabetes in black Americans can be distinguished from classically defined insulin-dependent diabetes and may be best classified as a form of maturity-onset diabetes of youth.

Insulin is released from rat brain neuronal cells in culture.

Abstract: Depolarization of neuronal cells in primary culture from the rat brain by potassium ions in the presence of calcium or by veratridine caused a greater than threefold stimulation of release of immunoreactive insulin. HPLC of the released insulin immunoreactivity from the neuronal cultures comigrated with the two rat insulins. The depolarization‐induced release of insulin was inhibited by cycloheximide and was specific for neuronal cultures since potassium ions failed to cause the release in comparably prepared astrocytic glial cells from the rat brain. Prelabelling of neuronal cultures with [3H]leucine followed by depolarization resulted in the release of radioactivity that immunoprecipitated with insulin antibody. The release of [3H]insulin was biphasic. These observations suggest that neuronal cells from the brain have the capacity to synthesize insulin that could be released under depolarization conditions.

Insulin receptors in the brain: structural and physiological characterization.

The present study was conducted to characterize insulin receptors and to determine the effects of insulin in synaptosomes prepared from adult rat brains. Binding of125I-insulin to synaptosome insulin receptors was highly specific and time dependent: equilibrium binding was obtained within 60 minutes, and a t1/2 of dissociation of 26 minutes. Cross-linking of125I-insulin to its receptor followed by SDS-PAGE demonstrated that the apparent molecular weight of the alpha subunit of the receptor was 122,000 compared with 134,000 for the liver insulin receptor. In addition, insulin stimulated the dose-dependent phosphorylation of exogenous tyrosine containing substrate and a 95,000 MW plasma membrane associated protein, in a lectin-purified insulin receptor preparation. The membrane associated protein was determined to be the β subunit of the insulin receptor. Incubation of synaptosomes with insulin caused a dose-dependent inhibition of specific sodium-sensitive [3H]norepinephrine uptake. Insulin inhibition of [3H]norepinephrine uptake was mediated by a decrease in active uptake sites without any effects in theKm, and was specific for insulin since related and unrelated peptides influenced the uptake in proportion to their structural similarity with insulin. These observations indicate that synaptosomes prepared from the adult rat brain possess specific insulin receptors and insulin has inhibitory effects on norepinephrine uptake in the preparation.

Insulin receptors and insulin action in dissociated brain cells

The present study was conducted to characterize insulin receptors and insulin action in rat brain cells. Binding of [125I]insulin to cells obtained by mechanically dissociating rat brains was 86% specific, time-dependent and reached equilibrium within 90 min. The t1/2 of association was 14 min and t1/2 of dissociation was 8 min. Scatchard analysis demonstrated the typical curvilinear plot providing high affinity (0.03 nM) and low affinity (6.6 nM) binding sites. The total number of binding sites were 0.15 pmol/mg protein. Crosslinking of [125I]insulin to its receptors on dissociated brain cells followed by SDS-PAGE and autoradiography showed that the alpha-subunit of the receptor had a molecular weight of 122,000. This was in contrast with a molecular weight of 134,000 for the liver alpha-subunit. Incubation of dissociated brain cells with insulin resulted in a concentration-dependent inhibition of total [3H]norepinephrine (NE) uptake. This inhibitory effect of insulin on [3H]NE uptake was sodium ion-dependent suggesting that 80-90% of the sodium ion-dependent uptake was insulin-sensitive. Incubation of lectin-purified insulin receptors with insulin resulted in a time- and concentration-dependent stimulation of phosphorylation of the tyrosine residue of an exogenous substrate poly (Glu, Tyr) (4:1). In addition, insulin also stimulated the autophosphorylation of the beta-subunit of the insulin receptors. These observations corroborate our contention that insulin exerts neuromodulatory effects mediated by the specific insulin receptors in the brain.

Insulin inhibits specific norepinephrine uptake in neuronal cultures from rat brain

Neuronal cells in primary culture have been demonstrated to possess specific insulin receptors (Boyd et al., J. Biol. Chem., 260 (1985) 15880-15884). Incubation of these cultures with insulin causes a dose-dependent inhibition of maprotiline-sensitive [3H]norepinephrine uptake. Maximum inhibition of 95% of maprotiline-sensitive norepinephrine uptake was observed at an insulin concentration of 167 nM with an ED50 of 30 nM. Competition-inhibition and Scatchard analysis of the insulin binding data suggested that maprotiline competed for high-affinity insulin receptors. These observations suggest that both insulin and maprotiline specifically inhibit neuronal norepinephrine uptake possibly involving insulin receptors.

Use of serum C-reactive protein in differentiating septicfrom aseptic meningitis in children

LABORATORY STUDIES used with variable success ~ in the differentiation of aseptic from septic meningitis include serum and CSF white blood cell counts and differentials; CSF determinations of glucose, protein, and lactic acid 2.3; pH2; anion gap4; enzymesS; counterimmunoelectrophoresis for identification of common pathogenic bacterial antigens6; nitroblue tetrazolium dye testT; and the limulus lysate test. 8 McCarthy et al. 9 showed serum C-reactive protein to be useful in separating bacterial from viral pneumonia. Since publication of their study, rate immunonephelometry has provided a more sensitive and rapid method of quantifying CRP than the qualitative latex agglutination slide test used in their study. We studied this quantitative CRP method in patients with meningitis. PATIENTS From August to November 1980, 35 consecutive children admitted to Childrens Hospital, Louisville, Ky., with more than 10 WBC/mm 3 in their CSF, were defined as having meningitis, There were 19 girls and 16 boys, ranging in age from 8 days to 12 years (mean 1.9 years).

Phorbol esters stimulate 2-deoxyglucose uptake in glia, but not neurons

Activation of protein kinase C by phorbol esters caused a time- and dose-dependent stimulation (270% of control) of glucose uptake in cultured glia, but not in neurons from rat brain. The phorbol ester stimulation of Vmax of glial glucose uptake occurred only in glia despite nearly 2.5-fold greater phorbol ester binding in neurons. These differences in cellular responses to protein kinase C activation may be the key to understanding brain glucose regulation.

Physiologically Unique Insulin Receptors on Neuronal Cells

Neuronal cells in primary culture from the brain have been utilized in the present study to determine the physiological and biochemical roles of neuronal insulin receptors. Kinetic characteristics of 125I-insulin binding to neuronal cells were similar to those for glial and peripheral cells although the a and β subunits of the neuronal receptor were significantly smaller in size. Insulin inhibited [3H]norepinephrine uptake and stimulated [3H]-serotonin uptake in neuronal cells, the effect most likely mediated by insulin receptors. In addition, insulin upregulated, while tunicamycin failed to exert any effects on, neuronal insulin receptors. These observations indicate that the neuronal insulin receptor is physiologically distinct from the glial and peripheral receptors and suggest that insulin may have a neuromodulatory role in the brain function.

Evidence for Central Nervous System Insulin Synthesis

Since Havrankonva and co-workers described the widespread distribution of both insulinl and insulin receptors2 in the rat central nervous system (CNS), two basic questions have remained unanswered. The first is the source of this insulin within the CNS, whether locally produced or of pancreatic origin. Second is the function of insulin within the CNS. Does insulin have similar effects. centrally to those outside the CNS or does insulin have unique functions in the CNS?

Developing rat brain binds monoiodinated insulin isomers similarly to other extrahepatic target tissues

We recently reported a series of binding and metabolic studies which led to the conclusion that the developing rat brain is a target tissue for insulin. Since insulin target tissues (extrahepatic) are capable of differentiating between various monoiodoinsulin isomers, we measured the binding of the B26 monoiodoinsulin isomer compared to the A14 in newborn rat brain preparations to determine if the developing rat brain shared the same relative binding of these isomers (viz. B26 greater than A14) with other extrahepatic tissues. The B26 isomer bound 1.57, 1.50 and 1.34 times as much as did the A14 to brain membranes, glia and neurons, respectively, whereas both isomers were bound equally by liver plasma membranes. Competition-inhibition curves were generated using homologous unlabeled (127I) insulin isomers. Binding of the B26 isomer was greater than the A14 at all concentrations. Scatchard plots showed that the receptor concentrations for the two isomers were similar, and affinity profiles showed that the differences in binding could be accounted for by the greater affinity of the receptors for the B26 isomer. The results indicate that the developing rat brain shares with other extrahepatic insulin target tissues a greater affinity for B26 monoiodoinsulin isomer compared to A14. Future studies of insulin binding should avoid using mixtures of iodinated insulins so that a uniform interpretation of data is made possible.