Insulin-stimulated glucose transport in cultured fibroblasts from normal and noninsulin-dependent (type II) diabetic human subjects

We have studied the effects of insulin on glucose transport in cultured fibroblasts obtained from six normal subjects and six patients with type II or noninsulin-dependent diabetes mellitus. Initial rates of glucose transport were determined by measurement of 2-deoxy-D-glucose uptake. In both normal and diabetic fibroblasts, insulin stimulated the uptake of 2-deoxy-D-glucose in a dose-dependent manner. The maximal level of insulin stimulation of 2-deoxy-D-glucose uptake (approximately 35% over basal uptake values) and the half-maximally effective insulin concentration (approximately 1 nM) were essentially the same for both the normal and diabetic fibroblasts. Furthermore, the absolute basal and maximally insulin-stimulated 2-deoxy-D-glucose uptake values were also the same. These results demonstrate that cultured fibroblasts from patients with noninsulin-dependent diabetes mellitus have normal basal and insulin-stimulated glucose transport capacities. Thus, the postreceptor defects in the glucose transport system previously demonstrated in vivo and in freshly isolated adipocytes from type II diabetic patients are most likely due to in vivo environmental factors rather than to an intrinsic (genetic) cellular defect(s).     

Effects of metformin treatment on erythrocyte insulin binding in normal weight subjects, in obese non diabetic subjects, in type 1 and type 2 diabetic patients

We have evaluated the effects of metformin administration on erythrocyte insulin receptors in 21 subjects: 5 normal weight subjects, 5 obese non diabetics, 5 insulin-dependent diabetics (Type I) and 6 obese non insulin-dependent (Type II) diabetics. Plasma glucose, plasma insulin and erythrocyte insulin receptors were studied after 15 days of metformin (850 mg, t.d.) or placebo administered in a double blind random order. Maximum specific insulin binding to erythrocytes increased after metformin in the normals (p less than 0.01), in the obese non diabetics (p less than 0.01) and in the obese Type 2 diabetics (p less than 0.005), but not in Type I diabetics. Scatchard analysis showed that the receptor number per cell increased by 37% in the normals, by 17% in the obese non diabetics and by 182% in Type 2 diabetics. Receptor affinity increased in obese subjects but did not increase in normals and in diabetics. Only in Type II diabetics was there a significant decrease in plasma glucose. Metformin, thus, increased binding in normals by moderately increasing the capacity of cell receptors, in obese non diabetics by increasing the affinity, whereas in obese Type II diabetics it dramatically increases receptor capacity. This is consistent with the fact that metformin has a hypoglycaemic effect mainly in Type II diabetics, but not in non diabetics (whether obese or not), and could be due to a direct effect on the cell membrane.     

Insulin internalization and degradation in adipocytes from normal and type II diabetic subjects

We studied the ability of isolated adipocytes from normal and type II diabetic subjects to internalize and process [125I]insulin. Adipocytes were incubated with [125I]insulin at 16 or 37 C, and at various times total cell-associated, surface-bound, and intracellular insulin were quantitated using an acid-barbital extraction technique which quickly removes cell surface insulin, leaving behind the intracellular insulin. Insulin internalization was slow in normal adipocytes at 16 C, such that only 13% of total cell-associated insulin was intracellular after 2 h of incubation. In contrast, internalization was rapid at 37 C, such that the intracellular pool of insulin was near maximal by 30 min and accounted for approximately 40% of the total cell-associated insulin. Sephadex G-50 column chromatography of the intracellular insulin demonstrated that more than 95% of this pool coeluted with native insulin. In adipocytes from the diabetic subjects, approximately 45% of total cell-associated insulin was intracellular after 30 min of incubation at 37 C. After 60 min of incubation at 37 C, the percentages of total cell-associated and surface-bound insulin were significantly lower in adipocytes from diabetic compared to normal subjects [1.81 +/- 0.31% (+/- SEM) vs. 2.92 +/- 0.24% (P less than 0.05) and 0.97 +/- 0.14% vs. 1.72 +/- 0.15% (P less than 0.01), respectively]. The percentage of insulin in the intracellular compartment was also slightly lower in adipocytes from diabetic compared to normal subjects (0.84 +/- 0.19% vs. 1.20 +/- 0.16%; P greater than 0.05). The lysosomotropic agent chloroquine increased total cell-associated insulin, and this was due entirely to an increase in intracellular insulin. In adipocytes from normal subjects, chloroquine increased intracellular insulin by 32% at 30 min, by 89% at 60 min, by 140% at 90 min, and by 178% at 120 min. In comparison to the normal adipocytes, the chloroquine-mediated increase in intracellular insulin was lower in adipocytes from the diabetic subjects (-8.1% at 30 min, 37% at 60 min, 58% at 90 min, and 63% at 120 min; P less than 0.05 at all time points). These results indicate that insulin is rapidly internalized in human adipocytes at 37 C such that approximately half of total cell-associated insulin is intracellular the intracellular insulin is largely intact; and intracellular processing of insulin by a chloroquine-sensitive pathway(s) is impaired in adipocytes from type II diabetic subjects.     

Circulating growth hormone forms in type 1 diabetic subjects: comparison with normal subjects and acromegalics

The molecular forms of growth hormone (GH) in serum from 18 Type 1 diabetic patients with poor metabolic control were analysed using sephadex G-100 chromatography. The profiles obtained were compared with those from normal subjects whose GH secretion was stimulated by exercise and hypoglycaemia and eight acromegalic patients. In the three groups three distinct GH forms were found: little (monomeric), big and big-big-GH. Samples from normal subjects contained 45% little-GH which was less than samples from the diabetics and acromegalics (53% and 65%, respectively, P less than 0.01). Further samples from normal subjects after the onset of hypoglycaemia showed an increase in little-GH. In the three groups, the higher the proportion of little-GH, the lower the proportion of big-big-GH, while the proportion of big-GH remained similar. In the acromegalics the proportion of little-GH was strongly correlated with the log concentration of serum GH. As little-GH is cleared from the circulation quicker than the larger forms these data indicate that the main component of the frequent surges of GH secretion in poorly controlled Type 1 diabetic subjects is little-GH (monomeric forms). The sustained release of GH found in acromegaly is composed largely of monomeric forms.     

Relationship between plasma leucine concentration and clearance in normal and type 1 diabetic subjects

In a series of studies in normal and type 1 diabetic subjects, we analysed the relationship between isotope-calculated leucine clearance and plasma leucine concentration. All studies were performed under euglycaemic conditions. Plasma leucine concentrations were either experimentally decreased by means of insulin infusion, or increased by means of exogenous amino acid infusion in the presence of hyperinsulinaemia. Leucine clearance rates were compared in normal and diabetic subjects at similar plasma insulin levels. The effect of hyperinsulinaemia was examined by measuring clearance rates in normal subjects at comparable leucine levels but different insulin concentrations. Our data show that leucine clearance is inversely related to leucine concentration, and that it is not independently stimulated by hyperinsulinaemia. Type 1 diabetes is not associated with decreased leucine clearance. A general equation relating leucine concentration and clearance is proposed. These data support the view that peripheral leucine utilization is not decreased in type 1 diabetes mellitus.     

Ambulatory and exercise-induced blood pressure responses in type I diabetic patients and normal subjects

We evaluated the ambulatory blood pressure (BP) readings during daily activities and compared the values with the casual office BPs in 12 type I diabetic patients and 12 age-matched, non-diabetic normal controls. The BP responses to 750 kpm/min exercise work load on bicycle ergometer were also evaluated in all the subjects. The mean ambulatory BPs were significantly higher in the diabetic patients compared with non-diabetic subjects. However, the ambulatory and casual office BPs were remarkably similar in each group. Mean peak exercise BPs were higher in the diabetics, but significantly so for diastolic BP (P less than 0.05) and mean arterial pressure (MAP) (P less than 0.01). The mean ambulatory MAP significantly correlated with the casual office MAP in both the diabetics (r = 0.75, P less than 0.005) and non-diabetics (r = 0.58, P less than 0.02). A significantly positive relationship (r = 0.87, P less than 0.001) existed between the ambulatory and peak exercise MAP in the diabetics, but not in the non-diabetic subjects. The mean peak exercise BP responses were significantly (P less than 0.01) higher in the diabetic patients with proliferative retinopathy vs. those with non-proliferative retinopathy. We conclude that casual office BPs reflect the ambulatory values in both diabetics and non-diabetics. Exaggerated BP responses to exercise are found in diabetic patients, especially in the presence of proliferative retinopathy. These findings are suggestive of possible defects in the cardiovascular, autonomic and autoregulatory mechanisms that maintain a normal MAP during daily stresses and exercise in the diabetic patients.     

Insulin binding and activation of glycogen synthase in fibroblasts from type 1 (insulin-dependent) diabetic patients

Summary ¹²⁵I-Insulin binding and insulin stimulation of glycogen synthase were examined in fibroblasts cultured from nine Type 1 (insulin-dependent) diabetic patients with age of onset of <42 years. In all cases specific insulin binding was qualitatively and quantitatively normal. Total ¹²⁵I-insulin binding was elevated in cells from three patients with early onset diabetes (two with onset before age 1 year) due to an increase in non-specific binding. When the ability of insulin to stimulate the conversion of the glucose-6-phosphate dependent to the glucose-6-phosphate independent form of glycogen synthase was measured, all cell lines responded, albeit to differing degrees. In general, the response of cells from diabetic donors was more variable than that of control fibroblasts. A slightly lower level of cellular glycogen was evident in the cells of the diabetic patients, and this was mirrored in slightly higher levels of the independent form of the enzyme. The average maximal level of the independent form of the enzyme also was higher in the diabetic patients' cells. Fibroblasts from one of the patients with very early onset diabetes had glycogen synthase levels that were markedly lower than in any other cell line examined. In summary, fibroblasts cultured from Type 1 diabetic patients do not show major defects in either insulin binding or action. A suggestion of subtle differences in the cells from the diabetic patients, particularly those with very early onset, is evident, however. Whether these are secondary to some primary genetic defect or represent some selection during culture remains to be determined.     

Islet amyloid polypeptide in pancreatic islets from type 1 diabetic subjects

Aims/hypothesis: Islet amyloid polypeptide is originally isolated as the chief constituent of amyloid deposits in type 2 diabetic islets. Islet amyloid polypeptide hyposecretion was known in type 1 diabetics and this study aimed to detect possibly reduced islet amyloid polypeptide-positive cells in type 1 diabetic islets. Results: Non-diabetic control islets showed about 60% of islet cells were insulin cells, and 60% of insulin cells were positive for IAPP. In type 1 diabetic islets, islets were generally smaller than control islets, consisting of weaker positive cells for insulin and islet amyloid polypeptide. Medium-sized islets still retained some insulin positive cells, whereas islet amyloid polypeptide positive cells were much less or even absent, but some insulin-negative cells were weakly islet amyloid polypeptide positive. An occasional extra-large islet, representing regenerating islets, consisting of more than 100 islet cells revealed less than 35% insulin and 20% islet amyloid polypeptide positive cells with relatively increased glucagon and somatostatin cells. Both normal and type 1 diabetic islets revealed scattered, densely insulin and islet amyloid polypeptide positive sickle-shaped cytoplasm without granular appearance, consistent with degenerating insulin cells. Methods: Using commercially available rabbit anti-islet amyloid polypeptide antibody, immunostaning was performed on ten cases of type 1 diabetic pancreata and eight non-diabetic controls. Both control and type 1 diabetic pancreata were systematically immunostained for insulin, glucagon, somatostatin and islet amyloid polypeptide.

Conclusion/Interpretation: Control islets consisted of about 60% insulin cells, and about 34% of islet cells were amyloid polypeptide positive with scattered and densely positive for insulin and islet amyloid polypeptide without granular appearance, consistent with degenerating β cells. All islets, including occasional extra-large islets from type 1 diabetics, showed less insulin cells and less islet amyloid polypeptide positive cells with twice increased glucagon and somatostatin cells of the control islets, but some insulin-negative cells were positive for islet amyloid polypeptide, suggesting the presence of islet amyloid polypeptide in degenerating and extra-large regenerating islets. Thus, this immunocytochemical staining revealed generally less islet amyloid positive cells in type 1 diabetic islets, corresponding to severe hyposecretion of islet amyloid polypeptide in type 1 diabetics.     

Normal glycine transport in cultured diploid fibroblasts from hyperglycinaemic subjects

The glycine uptake in cultured fibroblasts was studied in three children with non-ketotic hyperglycinaemia, one child with D-glyceric acidaemia and hyperglycinaemia and in six normal children, using an improved assay system giving individual protein correction. It was shown that all hyperglycinaemic children had normal uptake rates and kinetic characteristics similar to the controls.     

Islet amyloid polypeptide in pancreatic islets from type 1 diabetic subjects

Aims/hypothesis:

Islet amyloid polypeptide is originally isolated as the chief constituent of amyloid deposits in type 2 diabetic islets. Islet amyloid polypeptide hyposecretion was known in type 1 diabetics and this study aimed to detect possibly reduced islet amyloid polypeptide-positive cells in type 1 diabetic islets.

Results:

Non-diabetic control islets showed about 60% of islet cells were insulin cells, and 60% of insulin cells were positive for IAPP. In type 1 diabetic islets, islets were generally smaller than control islets, consisting of weaker positive cells for insulin and islet amyloid polypeptide. Medium-sized islets still retained some insulin positive cells, whereas islet amyloid polypeptide positive cells were much less or even absent, but some insulin-negative cells were weakly islet amyloid polypeptide positive. An occasional extra-large islet, representing regenerating islets, consisting of more than 100 islet cells revealed less than 35% insulin and 20% islet amyloid polypeptide positive cells with relatively increased glucagon and somatostatin cells. Both normal and type 1 diabetic islets revealed scattered, densely insulin and islet amyloid polypeptide positive sickle-shaped cytoplasm without granular appearance, consistent with degenerating insulin cells.

Methods:

Using commercially available rabbit anti-islet amyloid polypeptide antibody, immunostaning was performed on ten cases of type 1 diabetic pancreata and eight non-diabetic controls. Both control and type 1 diabetic pancreata were systematically immunostained for insulin, glucagon, somatostatin and islet amyloid polypeptide.

Conclusion/Interpretation:

Control islets consisted of about 60% insulin cells, and about 34% of islet cells were amyloid polypeptide positive with scattered and densely positive for insulin and islet amyloid polypeptide without granular appearance, consistent with degenerating β-cells. All islets, including occasional extra-large islets from type 1 diabetics, showed less insulin cells and less islet amyloid polypeptide positive cells with twice increased glucagon and somatostatin cells of the control islets, but some insulin-negative cells were positive for islet amyloid polypeptide, suggesting the presence of islet amyloid polypeptide in degenerating and extra large regenerating islets. Thus, this immunocytochemical staining revealed generally less islet amyloid positive cells in type 1 diabetic islets, corresponding to severe hyposecretion of islet amyloid polypeptide in type 1 diabetics.Key words: immunocytochemistry, islet amyloid polypeptide, pancreatic islets, type 1 diabetes     

In situ protein Kinase C activity is increased in cultured fibroblasts from Type 1 diabetic patients with nephropathy

AIMS/HYPOTHESIS: To verify whether individual susceptibility to diabetic nephropathy resides in an intrinsic difference in Protein Kinase C (PKC) activity. METHODS: We compared the effect of different glucose concentrations on PKC activity, PKC isoform expression and diacylglycerol (DAG) content in cultured fibroblasts from 14 Type 1 diabetic patients who developed nephropathy with those in cells from 14 patients without nephropathy. We recruited 14 normal subjects as control patients. Forearm skin fibroblasts were cultured in either normal (5 mmol/l) or high (20 mmol/l) glucose concentrations. RESULTS: In normal glucose, in situ PKC activity was higher in Type 1 patients with nephropathy (10.1+/-1.4 pmol/min/mg protein; p<0.01) than in those without (6.8+/-0.8) and the normal control subjects (6.3+/-0.5). This difference was due to increased concentrations of PKCalpha isoform in the membrane fraction of fibroblasts from patients with nephropathy. DAG content was also higher in cells from Type 1 patients with nephropathy. Incubation in high glucose concentration caused a further increase in PKC activity and DAG content in quiescent fibroblasts from patients with diabetic nephropathy, with no significant changes in cells from diabetic patients without nephropathy and normal control subjects. CONCLUSION/INTERPRETATION: Differences in PKC activation could contribute to the individual susceptibility to renal damage in Type 1 diabetic patients.     

Differential proliferation of fibroblasts cultured from normal and fibrotic human lungs

We studied the behavior of the cell cycle of cultured, early-passage human diploid pulmonary fibroblastlike cells (HDPFC) when grown in the presence of several growth factors. The rates of growth and growth fractions were studied in cultures from different origins. As much as 85 +/- 5% (SE) of HDPFC cultured from specimens with early fibrosis were cycling when the cells were grown in basal media supplemented with 10% serum or growth factors. In contrast, 55% +/- 8% (SE) of HDPFC from normal lung and only 31% +/- 5% (SE) of HDPFC cultured from specimens with dense fibrosis were cycling in those culture conditions. Results obtained by removal of any of the five growth factors from the defined medium indicated that the altered growth patterns were not related to any single growth factor tested. These studies are consistent with the hypothesis that HDPFC cultured from specimens with early fibrosis have a greater proliferative potential than HDPFC from late fibrosis.     

Glucocerebrosidase processing in normal fibroblasts and in fibroblasts from patients with type I, type II, and type III Gaucher disease.

Fibroblasts from normal subjects and patients with the three types of Gaucher disease were labeled with [3H]leucine. Glucocerebrosidase antigen was immunoprecipitated using affinity-purified Sepharose-bound antibody. Normal cells initially formed a 60-kDa polypeptide antigen that was gradually replaced by a broad band of antigen averaging 63 kDa. This position corresponds with that of mature fibroblast and placental enzyme. Processing of glucocerebrosidase in six unrelated patients with type I Gaucher disease and one patient with type III Gaucher disease was exactly the same as normal. In contrast, three patients with the severe infantile (type II) form of the disease manifested a very unstable enzyme; the 60-kDa band appeared transiently and the mature 63-kDa band was never seen. These results indicate that type II Gaucher disease may well be distinguishable from type I disease by virtue of the very unstable enzyme precursor. Contrary to some earlier reports, processing of glucocerebrosidase in type I disease appears to be entirely normal.     

Oxygen free radicals in interleukin-1beta-induced glycosaminoglycan production by retro-ocular fibroblasts from normal subjects and Graves' ophthalmopathy patients.

Graves' ophthalmopathy (GO) is attributed to an autoimmune process that results in the accumulation in retro-ocular tissue of glycosaminoglycans (GAG) that are in turn responsible for the development of clinical signs and symptoms. Retro-ocular fibroblasts are thought to be the source of GAG production and deposition in GO. In the present study, we investigated interleukin (IL)-1beta-induced oxygen free radical production and the role of oxygen free radicals in IL-1beta-induced GAG production in retro-ocular fibroblasts from both normal subjects and patients with GO. Normal retro-ocular fibroblasts demonstrated no measurable oxygen free radicals whereas GO retro-ocular fibroblasts showed detectable signals by electron paramagnetic resonance (EPR) spectroscopy. IL-1beta increased the free radical production in both cells. Superoxide dismutase (SOD) activity in GO retroocular fibroblasts was higher than that in normal cells. IL-1beta dose- and time-dependently stimulated the SOD activity in both cells, with GO retro-ocular fibroblasts showing less responsiveness. IL-1beta dose-dependently increased [3H]glucosamine incorporation into GAG by both cells. An exogenous oxygen free radical-generating system failed to increase GAG. Scavenging oxygen free radicals by the use of SOD (100 U/mL) and catalase (300 U/mL) partially blocked the IL-1beta-induced GAG production in both cells. These results suggest that stress related oxygen free radicals are present in the retro-ocular tissue in GO and that oxygen free radicals are involved in GAG accumulation induced by cytokine IL-1beta.     

Sugars and fat have different effects on postprandial glucose responses in normal and type 1 diabetic subjects

Background and aims

We aimed to determine the effects on glycemic responses and potential risk of hypoglycaemia in type 1 diabetic subjects of replacing half the starch in a meal with sugars, and of adding fat to the low-sugar and high-sugar meals.

Methods and results

We studied overnight fasted subjects with type 1 diabetes (n = 11) and age-, BMI- and ethnicity-matched controls (n = 11) using a 2 × 2 factorial design. The low-sugar/low-fat meal was 110 g white-bread. In the high-sugar/low-fat meal half the white-bread starch replaced by sugars (jam and orange juice). The high-fat meals consisted of the low-fat meals plus 20 g fat (margarine). The significance of the main effects of sugars and fat and the sugar × fat, group × sugar and group × fat interactions were determined by ANOVA. In control and diabetic subjects, respectively, high-sugar significantly reduced time to peak rise by 13% (P = 0.004) and 32% (P = 0.004; group × sugar: P = 0.01) and increased peak rise by 14% and 10% (ns). Adding fat increased time to peak rise by 17-19% in both groups (P = 0.003), reduced peak rise by 31% in normal (P < 0.001) but increased peak rise in diabetic subjects by 3% (ns) (group × fat: P = 0.022). Blood glucose nadir and occurrence of hypoglycaemia were similar among the 4 meals.

Conclusions

In type 1 diabetes, insulin adjustment to avoid hypoglycemia may be useful for meals in which the proportion of carbohydrate absorbed as glucose is <0.75, however the precise level which increases hypoglycaemic risk requires further research. The results suggest that people with type 1 diabetes should not be advised to add fat to meals to reduce glycemic responses.     

Alterations in cholesterol metabolism in cultured fibroblasts from patients with Niemann-Pick disease type C

Summary Cholesterol synthesis, esterification and efflux were investigated in cultured fibroblasts from patients with Niemann-Pick disease type C. Sterol synthesis from sodium acetate was markedly increased in the two Niemann-Pick disease type C strains as compared to controls, either in the presence or absence of exogenous cholesterol supply by low-density lipoproteins. By contrast, cholesterol esterification was about 2–3-fold reduced when measured by oleic acid incorporation into cholesteryl esters and 10–15-fold reduced when measured with labelled free cholesterol as precursor, although acylcoenzyme-A: cholesterol acyltransferase activity was normal when studiedin vitro on cell homogenates. Chase experiments with¹⁴C-cholesterol demonstrated that the rate of cholesterol efflux was decreased by about 3–4-fold in fibroblasts from patients with Niemann-Pick disease type C. These results provide further evidence for alterations of sterol metabolism in Niemann-Pick disease type C and support the hypothesis of a trapping of exogenous cholesterol, which cannot enter the regulatory intracellular pools.     

Disappearance of bovine insulin from plasma in diabetic and normal subjects

Disappearance curves for radioiodinated and immunoreactive bovine insulins were determined and analyzed in 30 subjects (5 normal volunteers and 25 diabetic patients). Simultaneous determinations were made of blood glucose and of insulin binding by insulin antibodies. There was significant negative association between the disappearance of bovine insulin from plasma and the insulin-binding level of circulating insulin antibodies. The presence of diabetes, the clinical character of the diabetes (whether stable or unstable), and the presence of chronic diabetic complications, of themselves, had no apparent significant influence that could be separated from the effect of circulating levels of insulin-binding antibodies on the disappearance of bovine insulin from plasma.