Low density lipoprotein binding and uptake by human and rat islet beta cells.

Abnormalities in lipoprotein metabolism are common in diabetes. It is unknown whether variations in form or concentration of lipoproteins influence the function of pancreatic beta cells. This study investigates whether low density lipoproteins (LDL) exhibit specific interactions with islet beta cells. Radioactively labeled LDL (125I-LDL) and fluorescently labeled LDL (DiI-LDL) were used as tracers. Rat islet cells express high affinity LDL binding sites (K(d) = 9 nM), which are also recognized by very low density lipoproteins and which are down-regulated by LDL. Binding of LDL appears restricted to the beta cells, as it was not detected on islet endocrine non-beta cells. At 37 C, LDL is taken up and lysosomally degraded by islet beta cells but not by islet non-beta cells. Human islet cells were also found to present LDL binding, uptake, and degradation. Compared with rat islet cells, human islet cells exhibit 10-fold less binding sites (2.10(7) vs. 2.10(8) per 10(3) cells) with a 2-fold lower K(d) value (5 nM) and an equal sensitivity to LDL-induced down-regulation. In conclusion, human and rat islet beta cells express LDL receptors that can internalize the lipoprotein. This pathway should be examined for its potential role in (dys)regulating pancreatic beta cell functions.

Abnormal circulating pancreatic enzyme activities in more than twenty-five percent of recent-onset insulin-dependent diabetic patients: association of hyperlipasemia with high-titer islet cell antibodies. Belgian Diabetes Registry.

Pancreatic amylase and lipase activities were measured in sera of 307 Caucasian insulin-dependent diabetes mellitus patients (IDDM) at clinical onset, 303 nondiabetic siblings of registered patients, and 207 control subjects under age 40 years. In all subject groups lipasemia and pancreatic (but not salivary) amylasemia increased with age and were significantly correlated. Using age-dependent reference ranges, reduced pancreatic enzyme levels were measured in 18% of patients, 6% of siblings, and only 2% of control subjects (p < 0.001). Increased lipase levels were noted in 10% of patients and in only 3% of siblings and 2% of control subjects (p < 0.001). Using both univariate and multivariate statistical analysis, elevated lipase activities at clinical onset were associated with higher titers of autoantibodies against islet cell cytoplasmic antigens and glucagon, but not against insulin or the 65-kDa isoform of glutamic acid decarboxylase (GAD65-Ab), or with markers of genetic predisposition or metabolic dysregulation. These findings indicate the presence of modest, but statistically significant, variations in circulating pancreatic enzyme levels in 28% of IDDM patients at clinical onset (p < 0.001 vs. 5% in control subjects). Increased lipase levels may express a form or a stage of the disease with exocrine cell damage; their association with higher titers of islet cell and glucagon autoantibodies is not yet explained. Lower lipase and isoamylase levels are thought to result from the reduced acinar cell function in the vicinity of insulin-depleted islets. It must be tested whether pancreatic enzyme activities in serum can also be altered during the preclinical stage and can thus be considered as an additional marker for the disease process in the pancreas.

Insulinlike growth factor-II/mannose 6-phosphate receptor is expressed on CCl4-exposed rat fat-storing cells and facilitates activation of latent transforming growth factor-beta in cocultures with sinusoidal endothelial cells.

Transforming growth factor beta (TGF-beta), a potent fibrogenic cytokine, is secreted in latent form. We examined which cell type in both normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver bears surface type II IGF/mannose 6-phosphate (IGF-II/M6P) receptor, known to facilitate activation of TGF-beta. In addition, the role of the IGF-II/M6P receptor in activation of latent TGF-beta was investigated in a coculture system with sinusoidal endothelial cells. Northern hybridization analysis for IGF-II/M6P receptor messenger RNA (mRNA) was performed on total RNA of different isolated and purified liver cell types. In normal liver, cells expressed little IGF-II/M6P receptor mRNA. In fibrotic liver, we found significant expression only in fat-storing cells. The presence of IGF-II/M6P receptors was established by [125I]IGF-II binding assays on freshly isolated fat-storing cells from normal and CCl4-exposed rat livers. We found specific binding of [125I]IGF-II only on CCl4 exposed fat-storing cells. As determined by polyacrylamide gel electrophoresis after affinity labeling, the specific binding involved 220 kD type II IGF receptors. Scatchard analysis revealed the presence of 3,043 +/- 1,378 IGF-II/M6P high-affinity receptors/fat-storing cell, with a Kd of 387 = 165 pmol/L. With a mink lung epithelial cell (Mv1Lu) proliferation inhibition assay, inhibition of proliferation (a measure of active TGF-beta function) was determined using conditioned media of activated fat-storing cells, cocultures of fat-storing cells, and endothelial cells and pure endothelial cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino-acid responsiveness in beta cell subpopulations with different sensitivity to glucose.

Isolated rat beta-cells differ in their individual responsiveness to glucose. The present study examines whether two beta-cell subpopulations with different thresholds for glucose stimulation also differ in their responsiveness to amino acids that are known to stimulate insulin release. The subpopulations were separated by autofluorescence-activated cell sorting using their metabolic responsiveness to 7.5 mM glucose as discriminating parameter. The 7.5 mM glucose responsive and unresponsive subpopulations were perifused in parallel in order to compare their secretory responses to leucine (10 mM) or to arginine (5 mM); responses to glucose were taken as control. Under maximal glucose stimulation (20 mM), the responsive subpopulation released two-fold more insulin than the unresponsive one whereas maximal stimulation with leucine (10 mM) elicited similar first and second phase responses in the two subpopulations. On the other hand, a maximal arginine stimulus (5 mM) amplified release only from glucose-activated beta-cells; neither did it correct the differences in glucose-induced insulin release between both subpopulations. These results indicate that rat beta-cells exhibit a heterogeneity in secretory responsiveness to glucose but not to leucine, a metabolized secretagogue which can induce release in the absence of glucose. A heterogeneity is also observed in the cellular responsiveness to arginine, and its secretory effect consists of an amplification of glucose-activated cells.

Detection of autoantibodies against islet amyloid polypeptide in human serum. Lack of association with type 1 (insulin-dependent) diabetes mellitus, or with conditions favouring amyloid deposition in islets. The Belgian Diabetes Registry.

A radiobinding assay for the detection of autoantibodies against islet amyloid polypeptide was developed, analytically validated, and--in parallel with a similar assay for the detection of autoantibodies against insulin--applied to sera from recent-onset Type 1 (insulin-dependent) diabetic patients and from age- and sex-matched control subjects. There was no difference in islet amyloid polypeptide autoantibody titres between patient groups and matched control subjects, nor within subject groups according to age. At onset of Type 1 diabetes, elevated islet amyloid polypeptide-autoantibody levels (> 97th percentile of control subjects) were only detected in 1 of 30 patients aged 0-19 years and in 2 of 35 patients aged 20-39 years. By contrast, insulin autoantibodies were frequently demonstrated, in particular at onset of diabetes under age 20 (0-19 years: 18 of 30 patients; 20-39 years: 10 of 35 patients; p < 0.01 vs matched control subjects). Islet amyloid polypeptide autoantibodies were not detectable in 3 insulinoma patients nor in 37 patients (aged 33-70 years) with Type 2 diabetes (vs 1 of 40 in matched control subjects). In positive serum, adsorption onto protein A-Sepharose removed islet amyloid polypeptide binding activity, hereby confirming its antibody nature. In conclusion, Type 1 diabetes is associated with an age-dependent autoantibody reaction against insulin but not against islet amyloid polypeptide. Conditions associated with amyloid deposition in islets (Type 2 diabetes, insulinoma and ageing) do not favour the formation of autoantibodies against islet amyloid polypeptide.

Pancreatic beta cell heterogeneity in glucose-induced insulin secretion.

Rat pancreatic beta cells differ in their individual sensitivity to glucose-inducible metabolic changes. The present study examines whether beta cells with a higher metabolic threshold require higher glucose levels for stimulation of their secretory activity. Purified beta cells were distributed according to their metabolic redox state at 7.5 mM glucose; the metabolically responsive (high responsive) and unresponsive (low responsive) subpopulations of comparable size and viability were reaggregated in the presence of [3H]tyrosine and then perfused at 2.8 mM glucose with 10-min pulses of increasing glucose concentration. Glucose elicited first-phase insulin release in both high and low responsive subpopulations from, respectively, 4.2 and 8.3 mM on. The amplitude of both secretory responses increased dose dependently, the rates in the high responsive subpopulation being 2-fold higher than in the low responsive one. At all stimulating glucose levels, fractional release of 3H-labeled insulin was 3- to 4-fold higher than that of immunoreactive insulin. Preferential release of newly formed insulin was already maximally stimulated at 4.2 mM glucose in the high responsive subpopulation, whereas it increased dose-dependently in the low responsive one. These results indicate the existence of intercellular differences in the secretory activity of glucose-exposed beta cells, both in terms of glucose sensitivity and of amplitude. This heterogeneity in beta cell secretory responsiveness parallels that which has been previously described for the cellular metabolic and biosynthetic functions. It is concluded that glucose dose-dependently recruits beta cells into both biosynthetic and secretory activities. Co-existence of inactive and activated cells can explain preferential release of newly synthesized over preformed hormone during glucose stimulation.

Direct effect of insulin and insulin-like growth factor-I on the secretory activity of rat pancreatic beta cells.

Purified pancreatic Beta cells were labelled with 3H-tyrosine before studying their secretory activity in perifusion. At 1.4 mmol/l glucose, the cells released similar fractions (0.01% per min) of their contents in preformed and in newly formed insulin. At 20 mmol/l glucose plus 10(-8) mol/l glucagon, these fractional release rates increased by 16 and 40-fold respectively. The preferential release of newly synthesized as compared to stored insulin is attributable to a heterogeneity in individual cell responses. The secretory responsiveness to glucose plus glucagon was completely suppressed by 10(-7) mol/l clonidine. Insulin induced a 20% reduction at 10(-6) mol/l, but remained without effect at 10(-7) mol/l. Insulin-like growth factor-I provoked a 30% decrease at 5.10(-9) mol/l. It is concluded that the type-I insulin-like growth factor receptors on pancreatic Beta cells mediate a suppressive action on the insulin release process. Their high affinity for insulin-like growth factor-I allows physiologic levels of this peptide to participate in the regulation of insulin release. Their low affinity for insulin provides the basis for a minor feedback action by this hormone at concentrations exceeding the normal circulating levels.

Evidence for the presence of type I insulin-like growth factor receptors on rat pancreatic A and B cells.

Purified rat pancreatic islet cells express somatomedin receptors which are identified by their affinity for insulin-like growth factor (IGF)-I, IGF-II, and insulin. Binding of [125I]IGF-I to islet A cells was half-maximally inhibited by 7.10(-10) M IGF-I, while IGF-II, insulin, and proinsulin were respectively 10-, 500-, and 10,000-fold less potent displacers of IGF-I binding. Unrelated hormones such as glucagon or GH did not compete with [125I]IGF-I binding to A cells. The concentration of IGF-I receptors on A cells was estimated at 5000 IGF-I binding sites per cell with affinity constant (Ka) of 2 X 10(9) M-1. Islet B cells were found to exhibit a reversible time- and temperature-dependent binding with [125I]IGF-I. Specificity and affinity of IGF-I binding sites were identical for islet A and B cells. Linear Scatchard plots of competitive binding data on B cells suggest 1 single class of IGF-I receptors in a concentration of 12,000 sites per cell. The presence of high affinity receptors for IGF-I on adult islet A and B cells provides a molecular basis for this growth factor to influence growth, survival, and/or function of these endocrine cell types. Their low affinity for insulin should be considered as a potential mechanism for this hormone to influence, at high concentration, the function of islet A and B cells.

Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells.

Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF-I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin.

Interplay of nutrients and hormones in the regulation of glucagon release.

The role of nutrients and hormones in the regulation of glucagon release is investigated in pancreatic A cells purified by autofluorescence-activated cell sorting. Purified A cells lack secretory activity in 1-h incubation at 1.4 mM glucose. Their release mechanism can be activated by arginine, alanine, and glutamine, alone or in combination. Glucose inhibits amino acid-induced glucagon release through a direct insulin-independent action upon pancreatic A cells. Nutrient-induced glucagon release is suppressed by somatostatin and amplified by (Bu)2cAMP or epinephrine. The epinephrine stimulus is inhibited by 10(-11) M somatostatin and abolished by 10(-10) M of this peptide. The effects of somatostatin and epinephrine are associated with parallel changes in cellular cAMP levels, which is not the case for the variations induced by amino acids or glucose. It is confirmed that calcium is an essential requirement for glucagon release. In contrast to its exquisite sensitivity for somatostatin, the glucagon release process is relatively insensitive to insulin during a 1-h exposure. The hormone affects solely epinephrine-induced glucagon release and its inhibitory action is partial and only observed at 10(-7) M. This suppressive effect of insulin is not attributable to variations in glucose handling but appears associated with the stimulatory effect of epinephrine. It is concluded that a nutrient-induced signal interacts with a hormone-inducible cAMP signal to activate the secretory process in pancreatic A cells.

Pancreatic hormone receptors on islet cells.

To assess whether islet cells are equipped with recognition units which allow an intra-islet regulation via released hormones, the presence of insulin and glucagon receptors is investigated on purified pancreatic A and B cells. Mono-[125I]glucagon is shown to bind specifically to islet B cells, with similar binding characteristics as in isolated hepatocytes but involving less receptors per cell (2.10(4) per B cell vs. 8.10(5) per liver cell). Binding is half-maximally displaced by 5.10(-9) M glucagon, a concentration known to induce half-maximal biological effects in isolated B cells. These results are compatible with a regulatory role of glucagon in the insulin release process. No specific binding of [125I]tyr-A14-insulin is detected on pancreatic A cells. In order to increase receptor assay sensitivity, [123I]tyr-A14-insulin is prepared with at least 5-fold higher specific activity. Its validity for in vitro receptor analysis is demonstrated in IM-9 lymphocytes, where insulin binding is detectable down to 10(4) cells/ml. However, no insulin-binding sites are identified on pancreatic A cells, even at 10(6) cells/ml. If isolated A cells contain high affinity insulin receptors, their number should be inferior to 400 per cell, which is 50- to 500-fold lower than in classical insulin target cells. These findings explain the insensitivity of the glucagon release process to acute exposure to insulin.

Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells.

The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insulin in inhibiting 125I-insulin binding to brain-cortical cells, which corresponds to their relative biological potencies in classical insulin-target cells; no competition was observed with glucagon and nerve growth factor, even at high concentrations. Scatchard analysis of competitive-binding data resulted in curvilinear plots with a high-affinity binding of Ka = 3.6 X 10(8) M-1. Insulin binding to foetal brain-cortical cells differed, however, in two distinct aspects from that to classical insulin-binding cell types. Firstly, dilution of 125I-insulin-bound cells in the presence of unlabelled insulin did not accelerate dissociation of the labelled hormone. Secondly, exposure of brain-cortical cells to insulin before the binding assay enhanced insulin binding, suggesting up-regulation of insulin receptors in response to insulin. In conclusion, foetal-mouse brain-cortical cells bear specific binding sites for insulin. Their insulin receptor shows a marked specificity and affinity for insulin, but differs in at least two properties from most classical insulin receptors. These differences in hormone-receptor interaction could reflect structural differences between insulin receptors on embryonic and differentiated cells.