Biomedical subjects

B A Cunningham

Publications and source records attributed to B A Cunningham.

At least 19 recordsLinked to original sources

PubMed1998

Neural cell adhesion molecule (N-CAM) domains and intracellular signaling pathways involved in the inhibition of astrocyte proliferation.

The neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferation in vitro and in vivo, and this effect is partially reversed by the glucocorticoid antagonist RU-486. The present studies have tested the hypothesis that N-CAM-mediated inhibition of astrocyte proliferation is caused by homophilic binding and involves the activation of glucocorticoid receptors. It was observed that all N-CAM Ig domains inhibited astrocyte proliferation in parallel with their ability to influence N-CAM binding. The proliferation of other N-CAM-expressing cells also was inhibited by the addition of N-CAM. In contrast, the proliferation of astrocytes from knockout mice lacking N-CAM was not inhibited by added N-CAM. These findings support the hypothesis that it is binding of soluble N-CAM to N-CAM on the astrocyte surface that leads to decreased proliferation. Signaling pathways stimulated by growth factors include activation of mitogen-activated protein (MAP) kinase. Addition of N-CAM inhibited MAP kinase activity induced by basic fibroblast growth factor in astrocytes. In accord with previous findings that RU-486 could partially prevent the proliferative effects of N-CAM, inhibition of MAP kinase activity by N-CAM was reversed by RU-486. The ability of N-CAM to inhibit astrocyte proliferation was unaffected, however, by agents that block the ability of N-CAM to promote neurite outgrowth. Together, these findings indicate that homophilic N-CAM binding leads to inhibition of astrocyte proliferation via a pathway involving the glucocorticoid receptor and that the ability of N-CAM to influence astrocyte proliferation and neurite outgrowth involves different signal pathways.

Animals↗

PubMed1997

Beta3-adrenergic receptors on white and brown adipocytes mediate beta3-selective agonist-induced effects on energy expenditure, insulin secretion, and food intake. A study using transgenic and gene knockout mice.

beta3-Adrenergic receptors (beta3-ARs) are expressed predominantly on white and brown adipocytes, and acute treatment of mice with CL 316,243, a potent and highly selective beta3-AR agonist, produces a 2-fold increase in energy expenditure, a 50-100-fold increase in insulin levels, and a 40-50% reduction in food intake. Recently, we generated gene knockout mice lacking functional beta3-ARs and demonstrated that each of these responses were mediated exclusively by beta3-ARs. However, the tissue site responsible for producing these actions is unknown. In the present study, genetically engineered mice were created in which beta3-ARs are expressed exclusively in white and brown adipocytes (WAT+BAT-mice), or in brown adipocytes only (BAT-mice). This was accomplished by injecting tissue-specific beta3-AR transgenic constructs into mouse zygotes homozygous for the beta3-AR knockout allele. Control, knockout, WAT+BAT, and BAT-mice were then treated acutely with CL, and the effects on various parameters were assessed. As previously observed, all effects of CL were completely absent in gene knockout mice lacking beta3-ARs. The effects on O2 consumption, insulin secretion, and food intake were completely rescued with transgenic re-expression of beta3-ARs in white and brown adipocytes (WAT+BAT-mice), demonstrating that each of these responses is mediated exclusively by beta3-ARs in white and/or brown adipocytes, and that beta3-ARs in other tissue sites were not required. Importantly, transgenic re-expression of beta3-ARs in brown adipocytes only (BAT-mice) failed to rescue, in any way, CL-mediated effects on insulin levels and food intake and only minimally restored effects on oxygen consumption, indicating that any effect on insulin secretion and food intake, and a full stimulation of oxygen consumption required the presence of beta3-ARs in white adipocytes. The mechanisms by which beta3-AR agonist stimulation of white adipocytes produces these responses are unknown but may involve novel mediators not previously known to effect these processes.

Adipocytes↗

PubMed1997

X-ray diffraction study of bilayer to non-bilayer phase transitions in aqueous dispersions of di-polyenoic phosphatidylethanolamines.

The low temperature phase properties of aqueous dispersions of di-18:2 and di-18:3 phosphatidylethanolamine are strongly influenced by the presence of ice. In the presence of cryoprotectants to inhibit ice formation, these lipids persist in the H(II) phase down to at least -50 degrees C. Ice formation, however, leads to a drastic reduction in the amount of available free water and a rapid reduction in the diameter of the inverted cylindrical micelles of the H(II) phase. The resulting increase in surface curvature of the micelles induces an imbalance in the forces acting in the lipid surface and the hydrophobic core which is relieved by formation of the L(alpha) phase. On reheating the lipid samples undergo an abrupt L(alpha) --> H(II) phase transition at about -20 degrees C. The radius of the water core of the inverted micelles at their point of formation is estimated to be 0.9 nm. This increases with temperature as more unfrozen water becomes available until the normal equilibrium radius of about 2.3 nm is reached at 0 degrees C when the bulk water in the sample finally melts. A small proportion of the H(II) phase lipid enters an as yet unidentified cubic phase on freezing. The spacings of the (10) planes of the H(II) phase, the (111) planes of the cubic phase and the d-spacing of the L(alpha) phase were found to be almost identical at the phase transition temperature. The cubic phase appears to disappear at low temperature but to reform on heating. Freeze-fracture studies revealed no unequivocal evidence for cubic phase lipid but the presence of residual non-bilayer lipid structures was observed even at temperatures as low as -80 degrees C. The presence of intersecting stacks of lamellar sheets in the replicas strongly suggest the existence of an epitaxial relationship between the L(alpha) and H(II) phases in these systems.

Freeze Fracturing↗

PubMed1997

Spot 14 protein-protein interactions: evidence for both homo- and heterodimer formation in vivo.

Spot 14 (S14) is a nuclear protein that is abundant only in lipogenic tissues (liver, adipose, lactating mammary), where its expression is rapidly regulated by hormones and dietary constituents. We recently showed that S14 acts at the transcriptional level in the transduction of signals for increased expression of genes encoding lipogenic enzymes. To better understand the mechanism of the regulation of gene transcription by S14, we employed a yeast two-hybrid system to identify hepatic proteins that physically interact with S14. We found that S14 has a strong propensity for homodimerization, as is the case for many transcription factors. Relevance of this finding to mammalian cells was established by transient cotransfection of S14 constructs bearing two different epitope tags. Glutathione-S-transferase-S14 and hemagglutinin-S14 fusions copurified from the transfected cells by glutathione-affinity chromatography, indicating their association in vivo. Analysis of S14 deletion mutants in the yeast system showed that an evolutionarily conserved hydrophobic heptad repeat (zipper) near the carboxyl terminus was necessary for homodimerization. In parallel studies, we observed a 36-kDa protein that specifically coimmunoprecipitated with S14 from extracts of radiolabeled rat hepatocytes. We propose that S14 is an acidic transcriptional activator that acts as a homodimer to modulate gene expression as a component of a tripartite complex with a 36-kDa hepatic protein.

Animals↗

PubMed1996

A combined SAXS/WAXS investigation of the phase behaviour of di-polyenoic membrane lipids.

Real-time measurements of the SAXS/WAXS diffraction patterns of aqueous dispersions (1:1 wt/wt) of the di-polyenoic lipids di-18:2 PC, di-18:3 PC, di-18:2 PE and di-18:3 PE were made over the temperature range 10 degrees to about -80 degrees C. The results of these measurements were compared to similar measurements performed on the corresponding di-18:0 and di-18:1 derivatives. SAXS measurements of the temperature dependence of lamellar repeat distances show that the di-polyenoic lipids undergo broad second-order transitions between their gel and liquid-crystal lamellar phases spanning 30-40 degrees C. The di-18:1 and di-18:0 derivatives, in contrast, undergo abrupt first-order transitions. The gel phases of the di-18:0 derivatives are characterised by two-component WAXS patterns with a sharp component close to 0.42 nm and a broader component at narrower spacings. On cooling, these lipids appear to undergo an initial transition to an L beta, phase followed by a conversion to an Lc phase. The gel phases of the di-18:1 derivatives also show two-component patterns but with the sharp component centred closer to 0.44 nm. The di-polyenoic lipids, in contrast, are characterised by a single broad peak centred at a spacing of about 0.42 nm, close to that of conventional L beta phases. The changes in lamellar repeat distance accompanying the transitions in the di-monoenoic and di-polyenoic lipids, all of which occur in the frozen state, are very similar, indicating that the acyl chains of the polyenoic lipids are close to their maximum extension in the gel state. The WAXS patterns of the polyenoic lipids suggest that the saturated upper parts of the acyl chains are packed on a regular hexagonal lattice while their polyunsaturated termini remain relatively disordered.

Membrane Lipids↗

PubMed1996

Reversible Ca2+-dependent translocation of protein kinase C and glucose-induced insulin release.

It has been reported that protein kinase C (PKC) interacts at multiple sites in beta-cell stimulus-secretion coupling. Nevertheless, there is still controversy concerning the importance of this enzyme in glucose-induced insulin release. The present study was undertaken to clarify whether glucose, directly, or through changes in cytoplasmic free Ca2+ concentration, [Ca2+]i, could promote translocation of PKC from the soluble to the membrane compartment. Whereas glucose, which increases [Ca2+]i, did not affect long-term distribution of PKC activity between soluble and membrane fractions, this distribution was reversibly affected acutely by the Ca2+ concentration in the extraction media. Translocation of PKC to the membrane by incubation of HIT cells for 10 min in the presence of 20 nM phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a 5-fold increase in glucose-induced insulin release. This was prevented by 50 nM concentration of the PKC inhibitor staurosporine, provided that the cells were exposed to the inhibitor before the phorbol ester. Cells pretreated with TPA demonstrated increased insulin secretion in response to glucose for several hours. This time course extended beyond the disappearance of [3H]TPA from the cells, which was complete after 1 h. Activation of PKC increased both average insulin release and the amplitude of oscillations 2-fold, but did not affect oscillation frequency. The stimulatory effect of increased PKC activity on insulin release was not matched by changes in [Ca2+]i. We suggest that stimulation of the pancreatic beta-cell with glucose promotes transient translocation of certain PKC isoforms from the cytoplasm to the plasma membrane as a direct consequence of the increase in [Ca2+]i. Such a translocation may promote phosphorylation of one or several proteins involved in the regulation of the beta-cell stimulus-secretion coupling. This results in potentiation of glucose-induced activation of insulin exocytosis, an effect then not mediated by an increase in [Ca2+]i per se. Hence, pulsatile insulin release can be obtained under conditions where overall [Ca2+]i does not change, challenging the view that oscillations in [Ca2+ ]i are indeed driving the oscillations in hormone release.

Animals↗

PubMed1996

Homophilic adhesion mediated by the neural cell adhesion molecule involves multiple immunoglobulin domains.

The neural cell adhesion molecule (N-CAM) mediates homophilic binding between a variety of cell types including neurons, neurons and glia, and neurons and muscle cells. The mechanism by which N-CAM on one cell interacts with N-CAM on another, however, is unknown. Attempts to identify which of the five immunoglobulin-like domains (Ig I-V) and the two fibronectin type III repeats (FnIII 1-2) in the extracellular region of N-CAM are involved in this process have led to ambiguous results. We have generated soluble recombinant proteins corresponding to each of the individual immunoglobulin domains and the combined FnIII 1-2 and prepared polyclonal antibodies specific for each. The purified proteins and antibodies were used in aggregation experiments with fluorescent microspheres and chicken embryo brain cells to determine possible contributions of each domain to homophilic adhesion. The recombinant domains were tested for their ability to bind to purified native N-CAM, to bind to each other, and to inhibit the aggregation of N-CAM on microspheres and the aggregation of neuronal cells. Each of the immunoglobulin domains bound to N-CAM, and in solution all of the immunoglobulin domains inhibited the aggregation of N-CAM-coated microspheres. Soluble Ig II, Ig III, and Ig IV inhibited neuronal aggregation; antibodies against whole N-CAM, the Ig III domain, and the Ig I domain all strongly inhibited neuronal aggregation, as well as the aggregation of N-CAM-coated microspheres. Of all the domains, the third immunoglobulin domain alone demonstrated the ability to self-aggregate, whereas Ig I bound to Ig V and Ig II bound to Ig IV. The combined FnIII 1-2 exhibited a slight ability to self-aggregate but did not bind to any of the immunoglobulin-like domains. These results suggest that N-CAM-N-CAM binding involves all five immunoglobulin domains and prompt the hypothesis that in homophilic cell-cell binding mediated by N-CAM these domains may interact pairwise in an antiparallel orientation.

Animals↗

PubMed1996

Malonyl coenzyme A and adiposity in the Dahl salt-sensitive rat: effects of pioglitazone.

These studies were designed to assess the effects of pioglitazone, a new oral antidiabetic agent that acts by improving insulin sensitivity, on blood pressure, plasma and tissue lipids, and insulin resistance in the Dahl salt-sensitive (Dahl-S) rat. Reaven et al had reported that male Dahl-S rats are moderately hyperinsulinemic and insulin-resistant. This was of particular interest since these rats are not obese but are hypertriglyceridemic, and on a high-salt diet they become hypertensive. In the current study, male Sprague-Dawley control and Dahl-S rats were compared when fed standard chow of high-fat, high-sucrose (HFHS) diets with or without pioglitazone (20 mg/kg body weight/d) for 3 weeks. On the standard chow diet, Dahl-S rats were hypertriglyceridemic and had high tissue levels of malonyl coenzyme A ([CoA] Dahl-S 5.0 v control 3.3 nmol/g in muscle, and Dahl-S 15.6 v control 10.7 nmol/g in liver); however, they were not hyperinsulinemic. Pioglitazone therapy decreased both malonyl CoA and plasma triglycerides toward control values, but had no effect on plasma insulin levels. On the HFHS diet, both groups became glucose-intolerant and hyperinsulinemic; however, the hyperinsulinemia was greater and more sustained in Dahl-S rats. In addition, the HFHS diet appeared to increase the mass of retroperitoneal fat in the Dahl-S but not in the control group. Treatment with pioglitazone decreased retroperitoneal fat, but as reported previously, it increased the mass of the epididymal fat pad. The results suggest that the hypertriglyceridemia of the Dahl-S rat is associated with an increase in the concentration of malonyl CoA in both liver and muscle. They also show that pioglitazone reverses both of these abnormalities independently of its effect on plasma insulin. Whether these high levels of malonyl CoA predispose the Dahl-S rat to hyperinsulinemia and possibly obesity when placed on a HFHS diet remains to be determined.

Adipose Tissue↗

PubMed1996

Glucose-induced oscillatory insulin secretion in perifused rat pancreatic islets and clonal beta-cells (HIT).

Normal insulin secretion is oscillatory in vivo and from groups of perifused islets. Stimulation of rat islets with different glucose concentrations gave insulin oscillations of similar period (5-8 min) but increasing amplitude. It has been assumed that oscillatory secretion is due to oscillations in intracellular free Ca2+, as seen in single islets and single pancreatic beta-cells. However, when islets were perifused with diazoxide and high KCl to maintain high intracellular free Ca2+, insulin oscillations of similar amplitude and period still occurred on glucose stimulation, although superimposed on elevated basal secretion. Several likely possibilities for a diffusible synchronizing factor were tested, including pyruvate, lactate, ATP, and insulin itself; nevertheless, perifusion with high concentrations of these did not prevent insulin oscillations. Clonal pancreatic beta-cells (HIT) and dissociated islets also exhibited oscillatory insulin secretion, but with the 5- to 8-min period oscillations superimposed on 15- to 20-min period oscillations. These results indicate that the mechanisms for generating and synchronizing insulin oscillations reside in the beta-cell, although the structure of the islet may modulate the oscillation pattern.

Animals↗

PubMed1995

Neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferation after injury to different regions of the adult rat brain.

After a penetrating lesion in the central nervous system, astrocytes enlarge, divide, and participate in creating an environment that adversely affects neuronal regeneration. We have recently shown that the neural cell adhesion molecule (N-CAM) partially inhibits the division of early postnatal rat astrocytes in vitro. In the present study, we demonstrate that addition of N-CAM, the third immunoglobulin-like domain of N-CAM, or a synthetic decapeptide corresponding to a putative homophilic binding site in N-CAM partially inhibits astrocyte proliferation after a stab lesion in the adult rat brain. Animals were lesioned in the cerebral cortex, hippocampus, or striatum with a Hamilton syringe and needle at defined stereotaxic positions. On one side, the lesions were concomitantly infused with N-CAM or with one of the N-CAM-related molecules. As a control, a peptide of the same composition as the N-CAM decapeptide but of random sequence was infused on the contralateral side of the brain. We consistently found that the population of dividing astrocytes was significantly smaller on the side in which N-CAM or one of the N-CAM-related molecules was infused than on the opposite side. The inhibition was greatest in the cortical lesion sites (approximately 50%) and was less pronounced in the hippocampus (approximately 25%) and striatum (approximately 20%). Two weeks after the lesion, the cerebral cortical sites infused with N-CAM continued to exhibit a significantly smaller population of dividing astrocytes than the sites on the opposite side. When N-CAM and basic fibroblast growth factor, which is known to stimulate astrocyte division in vitro, were coinfused into cortical lesion sites, astrocyte proliferation was still inhibited. These results suggest the hypothesis that, by reducing glial proliferation, N-CAM or its peptides may help create an environment that is more suitable for neuronal regeneration.

Amino Acid Sequence↗

PubMed1995

Real-time X-ray diffraction study at different scan rates of phase transitions for dipalmitoylphosphatidylcholine in KSCN.

Multibilayer arrays of dipalmitoylphosphatidylcholine (DPPC) in 1 M KSCN were characterized using real-time X-ray diffraction and differential scanning calorimetry. A phase transition sequence was observed as a function of increasing temperature which involved changes from the interdigitated subgel (Lc(inter)) to interdigitated gel (L beta(inter)) to disordered (L alpha) bilayer states. The phase transition mechanisms were unambiguously determined by comparison of results from fast and slow scans. The Lc(inter)-->L beta(inter) phase transition was shown to involve a continuous change in acyl chain spacing between the rectangular subgel acyl chain unit cell into an hexagonal gel acyl chain unit cell. The mechanism is similar to that for subgel to gel state transitions involving non-interdigitated DPPC bilayers.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

Sterols stabilize the ripple phase structure in dihexadecylphosphatidylcholine.

The presence of various sterols in mixtures with dihexadecylphosphatidylcholine (DHPC) was studied using static X-ray diffraction of temperature equilibrated samples, and real-time X-ray diffraction of samples undergoing temperature scans. It was found that these sterols eliminate the interdigitation of the alkyl chains in the DHPC sub-gel and gel-state bilayers while stabilizing the ripple gel-state at the expense of the gel-state bilayer phase. The ripple-ripple phase transition previously observed for dipalmitoylphosphatidylcholine in the presence of low molar concentrations of sterols (Wolfe et al. (1992) Phys. Rev. Lett. 68, 1085-1088) was also observed for similar DHPC-sterol mixtures. In addition, we show the first evidence that the presence of 5 alpha-cholestane-3 beta,5,6 beta-triol will cause the lipid mixtures to continue to adopt a ripple mesophase structure even after the DHPC alkyl chain becomes disordered.

Cholestanols↗

PubMed1995

Cell adhesion molecules as morphoregulators.

Many significant advances have been made recently in our understanding of the structure and function of cell adhesion molecules (CAMs). The most provocative, however, are those that indicate that CAM-mediated adhesion may lead to changes in gene expression and those that suggest that the expression of CAM genes may be regulated by the products of Hox and related genes.

Animals↗

PubMed1995

Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM.

Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.

Amino Acid Sequence↗

PubMed1995

Phosphofructokinase isozymes in pancreatic islets and clonal beta-cells (INS-1).

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal beta-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the beta-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the beta-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1994

Effects of fibrinogens on phase transitions in lipid model membrane systems.

An abnormal fibrinogen that caused aggregation of red blood cells (RBC) in a patient with gangrene was examined by real-time X-ray diffraction to determine its effects on dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) phase transitions. Similar studies were done with normal fibrinogen and results were compared. Both types of fibrinogen slightly increased the L alpha-->HII phase transition temperature and the HII phase parameters for POPE, while neither fibrinogen significantly affected the order-disordered acyl chain transitions in the lipid bilayer phase. However, fibrinogen differentially influenced the bilayer unit cell parameter of the gel and disordered bilayer and the gel state ripple phase. These results can be interpreted as indicating that fibrinogen has little effect on the balance of gel and disordered acyl chains in the lipid bilayer, but may influence membrane functions dependent on non-bilayer phases.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

Synchrotron X-ray studies of lipids and membranes: a critique.

This review gives a description of techniques, suitable for the study of lipid dispersions and unorientated membranes, that are available at synchrotron facilities to determine either the kinetics of transitional phenomena in the time after a temperature or pressure jump is initiated, or the phases present while a sample undergoes a phase transition. Included in this description is information about synchrotron X-ray sources, sample holders and temperature controllers, detection systems, as well as data reduction. Examples involving lipid dispersions are provided to illustrate the application of these methods using synchrotron radiation.

Kinetics↗

PubMed1994

Receptor dimerization determines the effects of growth hormone in primary rat adipocytes and cultured human IM-9 lymphocytes.

Binding of GH to its cell surface receptors is thought to result in the formation of a complex comprised of one molecule of hormone per two molecules of receptor. It has been proposed that this hormone-induced receptor dimerization is important for the mechanism of signal transduction. We have developed a mathematical model for quantitative evaluation of the biological responses associated with sequential receptor dimerization. Based on these predictions, we have investigated whether GH-induced receptor dimerization plays a role in two classical effects of GH, i.e. stimulation of lipogenesis in primary rat adipocytes and GH receptor down-regulation in cultured human IM-9 lymphocytes. Model predictions of biological responses linked to dimer formation yielded a bell-shaped pattern, with self-antagonism at high GH concentrations when monomeric GH-receptor complexes become predominant. The GH lipogenic bioactivity curve was indeed biphasic and first increased in a concentration-dependent manner between 10(-10)-10(-8) M GH (ED50, 0.5 nM), up to a maximum of 1.7-fold stimulation above basal. Then, the response decreased continuously above 5 x 10(-8) M GH, returning to basal levels around 10(-5) M GH. Incubation of IM-9 cells with wild-type human GH resulted in a dose-dependent loss of their surface receptors. In contrast, a human GH analog (G120R), mutated in the second binding surface of the hormone and, therefore, unable to induce GH receptor dimerization, failed to induce receptor down-regulation in the IM-9 cells. Furthermore, when added together with wild-type human GH, human GH(G120R) inhibited, in a concentration-dependent manner, the down-regulation induced by wild-type human GH. Taken together, these data support the hypothesis that receptor dimerization is critical for the stimulation by GH of both lipogenesis in primary rat adipocytes and receptor down-regulation in cultured human IM-9 lymphocytes.

Adipocytes↗