Monocytes of patients with amyotrophic lateral sclerosis linked to gene mutations display altered TDP-43 subcellular distribution.

AIMS: Cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 (TDP-43) is an early determinant of motor neuron degeneration in most amyotrophic lateral sclerosis (ALS) cases. We previously disclosed this accumulation in circulating lymphomonocytes (CLM) of ALS patients with mutant TARDBP, the TDP-43-coding gene, as well as of a healthy individual carrying the parental TARDBP mutation. Here, we investigate TDP-43 subcellular localization in CLM and in the constituent cells, lymphocytes and monocytes, of patients with various ALS-linked mutant genes. METHODS: TDP-43 subcellular localization was analysed with western immunoblotting and immunocytofluorescence in CLM of healthy controls (n = 10), patients with mutant TARDBP (n = 4, 1 homozygous), valosin-containing protein (VCP; n = 2), fused in sarcoma/translocated in liposarcoma (FUS; n = 2), Cu/Zn superoxide dismutase 1 (SOD1; n = 6), chromosome 9 open reading frame 72 (C9ORF72; n = 4), without mutations (n = 5) and neurologically unaffected subjects with mutant TARDBP (n = 2). RESULTS: TDP-43 cytoplasmic accumulation was found (P < 0.05 vs. controls) in CLM of patients with mutant TARDBP or VCP, but not FUS, in line with TDP-43 subcellular localization described for motor neurons of corresponding groups. Accumulation also characterized CLM of the healthy individuals with mutant TARDBP and of some patients with mutant SOD1 or C9ORF72. In 5 patients, belonging to categories described to carry TDP-43 mislocalization in motor neurons (3 C9ORF72, 1 TARDBP and 1 without mutations), TDP-43 cytoplasmic accumulation was not detected in CLM or in lymphocytes but was in monocytes. CONCLUSIONS: In ALS forms characterized by TDP-43 mislocalization in motor neurons, monocytes display this alteration, even when not manifest in CLM. Monocytes may be used to support diagnosis, as well as to identify subjects at risk, of ALS and to develop/monitor targeted treatments.

Pregnancy induces molecular alterations reflecting impaired insulin control over glucose oxidative pathways that only in women with a family history of Type 2 diabetes last beyond pregnancy.

In circulating lymphomonocytes (CLM) of patients with Type 2 diabetes (DM2) pyruvate dehydrogenase (PDH), the major determinant of glucose oxidative breakdown, is affected by a cohort of alterations reflecting impaired insulin stimulated glucose utilization. The cohort is also expressed, although incompletely, in 40% of healthy young subjects with a DM2-family history (FH). Pregnancy restrains glucose utilization in maternal peripheral tissues to satisfy fetal requirements. Here we explore whether pregnant women develop the PDH alterations and, if so, whether there are differences between women with and without FH (FH+, FH-). Ten FH+ and 10 FH- were evaluated during pregnancy (12-14, 24-26, and 37-39 weeks) and 1 yr after (follow-up) for fasting plasma glucose and insulin as well as body mass index (BMI), and for the PDH alterations. Twenty FH- and 20 FH+ non-pregnant women served as controls. All FH+ and FH- controls exhibited normal clinical parameters and 8 FH+ had an incomplete cohort of PDH alterations. In FH- and FH+ pregnant women at 12-14 weeks clinical parameters were normal; from 24-26 weeks, with unvaried glucose, insulin and BMI rose more in FH- and only in the latter recovered the 12-14 weeks values at follow-up. In all FH-, the cohort of PDH alterations was incomplete at 24-26 weeks, complete at 37-39 weeks, and absent at follow-up but complete from 12-14 weeks including follow-up in all FH+. In FH-, the cohort is an acquired trait restricted to pregnancy signaling transiently reduced insulin-stimulated glucose utilization; in FH+, instead, it unveils the existence of an inherited DM2-related background these women all have, that is awakened by pregnancy and as such lastingly impairs insulin-stimulated glucose utilization.

Enhanced blood insulin overcomes pyruvate dehydrogenase derangements that reflect systemic insulin resistance in obese adolescents.

Pyruvate dehydrogenase (PDH) has low activity in the circulating lymphocytes (CL) of obese adolescents and adults. In vitro, it is unresponsive to insulin at 5 micro-units/ml and is activated at 50 micro-units/ml, in contrast with activation and inhibition respectively at these concentrations in CL from controls. These changes are seen as being indicative of a molecular disorder underlying insulin resistance. The aims of the present study were to determine whether a substantial enhancement of blood insulin levels restores the PDH activity in CL from obese adolescents and abolishes the in vitro alterations, and whether PDH activity and indices of insulin resistance are correlated. Six obese adolescents and six normal-weight controls underwent a 4 h frequently sampled intravenous glucose test with minimal model analysis, to bring about a sharp rise in blood insulin and provide a reliable index of insulin sensitivity (S(I)). PDH activity was evaluated in CL obtained from blood samples at set times before and after their exposure to insulin in vitro. Insulin levels rose in all subjects in the first 10 min, although to a much greater extent in the obese group, and then decreased until the end of the test (240 min; t(240)). PDH activity in CL paralleled the insulin pattern in the control subjects, whereas in the obese subjects it was below normal 3 min before the start of the test (t(-3)), but rose significantly throughout the test. PDH responses in vitro to insulin in CL taken from the control subjects at t(-3) and t(240) and in CL taken from the obese subjects at t(-3) were as reported above, but were normal (i.e. the same as in control CL) in CL taken from the obese subjects at t(240). Baseline PDH activity was inversely correlated with body mass index and with fasting insulin, and directly correlated with S(I). These results show that a brief and sharp enhancement of blood insulin overcomes derangements in PDH that reflect systemic insulin resistance in obese adolescents.

Proteasomes are a target of the anti-tumour drug vinblastine.

Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathway, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastine, whose cytostatic action relies mainly on the disruption of mitotic spindle dynamics. The effects of vinblastine on the peptidase activities of human 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteasome were assessed in the presence of specific fluorogenic peptides and (125)I-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-protein conjugates and of inhibitory kappa B alpha (I kappa B alpha), which are characteristic intracellular proteasome substrates, by Western blotting on lysates from HL60 cells incubated with or without vinblastine, illustrated the effects of vinblastine on proteasomes in vivo. We also evaluated the effects of vinblastine on the signal-induced degradation of I kappa B alpha. Vinblastine at 3--110 microM reversibly inhibited the chymotrypsin-like activity of the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of both proteasomes, but only at 110 microM vinblastine was the chymotrypsin-like activity of the 26 S proteasome inhibited; furthermore, at 25--200 microM the drug inhibited the degradation of ubiquitinated lysozyme. In HL60 cells exposed for 6 h to 0.5--10 microM vinblastine, the drug-dose-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of I kappa B alpha, occurred. Moreover, vinblastine impaired the signal-induced degradation of I kappa B alpha. Cell viability throughout the test was approx. 95%. Proteasomes can be considered to be a new and additional vinblastine target.

In obese individuals dexfenfluramine corrects molecular derangements reflecting insulin resistance.

BACKGROUND: Circulating lymphocytes of obese individuals with and without type 2 diabetes have derangements of pyruvate dehydrogenase (PDH) that are described as reflecting a disorder underlying systemic insulin resistance, namely basal activity below normal and, in vitro, unresponsiveness to insulin at 33 pmol/l and activation at 330 pmol/l instead of activation and inhibition as in controls. OBJECTIVE: To explore whether the above enzyme derangements are overcome in obese individuals on dexfenfluramine treatment, known to improve poor peripheral insulin sensitivity. METHODS: Fifteen obese diabetic patients and 15 age-matched euglycaemic obese subjects with normal glucose tolerance were enrolled for a trial composed of two 21-day periods; in the first (D-21-D0), participants received a placebo, and in the second (D0-D21), dexfenfluramine (30 mg/day). At D-21, D0 and D21 participants were evaluated for weight, BMI, fasting glycaemia (FG), fasting insulinaemia (FI), fasting insulin resistance index (FIRI), area under the glycaemic (G-AUC) and insulinaemic (I-AUC) curves from an OGT test, and for PDH activity assayed in their circulating lymphocytes before (basal activity) and after incubation with 33 or 330 pmol/l insulin. At D2, basal PDH activity and clinical parameters were assayed. RESULTS: In both groups of participants at D0 all parameters tested were constant with respect to D-21; at D2, only basal PDH activity rose significantly; at D21, basal and insulin stimulated PDH activities were normalized and weight decreased significantly, as did FG, FI, FIRI and G-AUC in the diabetic, and FI, FIRI, G-AUC and I-AUC in the non-diabetic participants. CONCLUSION: In obese, non-diabetic and diabetic individuals on dexfenfluramine treatment, amelioration of clinical parameters and indexes of poor insulin sensitivity of blood glucose homeostasis are preceded by correction, in their circulating lymphocytes, of PDH derangements described as reflecting a disorder underlying insulin resistance.

Structural and functional characterization of 20S and 26S proteasomes from bovine brain.

Two proteins were isolated, in a stable form, from bovine brain by ion exchange chromatography, gel filtration and ultracentrifugation on glycerol gradient. They were identified as 20S and 26S proteasomes on the basis of molecular mass, migration velocity on non-denaturing gels, immunoreactivity, multipeptidase activity and the 26S proteasome also for dependence on ATP for the degradation of short peptides and ubiquitinylated proteins. However, the 26S proteasome has some properties not yet described for its counterpart of other tissues and from brain of this and other species. In particular, the ATP concentration required by the 26S proteasome to reach maximal peptidase activity was approximately 40-fold lower than the one required for maximal proteolytic activity on polyubiquitinylated substrates. Moreover, plots of substrate concentration vs. velocity gave a saturation curve for the 26S proteasome only, which, for the trypsin-like and post-glutamyl peptide hydrolase activities fitted the Michaelis-Menten equation, whereas for the chymotrypsin-like activity indicated multibinding site kinetics with positive cooperativity (n = 2.32+/-0.38). As concerns the 20S proteasome, its electrophoretic pattern on native gel revealed a single protein band, a feature, to our knowledge, not yet described for the brain particle of any species.

Acute methylprednisolone administration induces a transient alteration of glucose tolerance and pyruvate dehydrogenase in humans.

BACKGROUND: Glucocorticoid administration induces alteration of glucose tolerance, and impairment of glucose oxidation may contribute to glucocorticoid-induced derangement of glucose metabolism. We investigated glucose tolerance following methylprednisolone administration in humans. In the same model, we evaluated pyruvate dehydrogenase (PDH), the rate limiting enzyme of glucose oxidation, in peripheral blood mononuclear cells. MATERIALS AND METHODS: Methylprednisolone (2 x 40 mg, iv, one dose every 12 h) was administered to six healthy volunteers. Glucose tolerance was evaluated through an oral glucose tolerance test (oGTT, 75 g glucose) at least a week before and after drug administration (2 and 24 h post-drug). To assess modifications of lipid metabolism circulating free fatty acids (FFA) and glycerol were measured, during fasting and oGTT. The active form of PDH (PDHa) was evaluated in peripheral blood mononuclear cells, both as ex vivo activity and as in vitro response to insulin (30 pmol l-1). RESULTS: Methylprednisolone induced an alteration of glucose tolerance 2 h after its administration. Such alteration was completely reversed at 24 h. Alteration of glucose tolerance was accompanied by decreased ex vivo PDHa activity. PDH responsiveness to insulin in vitro was also impaired. Circulating FFA were unmodified, but decreased glycerol levels suggested a slight inhibition of lipolysis. CONCLUSIONS: Acute methylprednisolone administration in humans induced a transient decrease of glucose tolerance 2 h after drug administration, accompanied by hyperinsulinaemia, inhibition of ex vivo PDH activity and its response to insulin in vitro. These alterations were completely abolished at 24 h, suggesting that methylprednisolone can be safely administered acutely. Furthermore, methylprednisolone induced only minor modifications of circulating FFA and glycerol, indicating minimal impact on lipid metabolism.

Derangements of pyruvate dehydrogenase in circulating lymphocytes of NIDDM patients and their healthy offspring.

Pyruvate dehydrogenase (PDH) is poorly active in circulating lymphocytes of NIDDM patients; in vitro, it is unresponsive to insulin at 5 microU/ml and activated at 50 microU/ml, instead of activated and inhibited as in healthy controls. This study examines whether healthy offspring of NIDDM patients with a family history for this disease have these alterations. Twenty seven healthy offspring (23+/-10 yr, median 18 yr) and their parents (13 diabetic with a family history for NIDDM and 11 healthy without this history) were enrolled. Twenty healthy individuals without the history and matched for age and gender with the offspring served as controls. Minimum levels for enzyme activity before and after cell stimulation with insulin at 5 microU/ml were computed for a 95% CI with no more than 5% of the controls excluded. Increased or unvaried enzyme activity in response to insulin at 50 microU/ml was defined as abnormal. All NIDDM parents and 11/27 offspring had below normal enzyme activity and defective and reversed enzyme response to insulin at 5 and 50 microU/ml; three offspring had altered enzyme response to insulin at both concentrations, four to insulin at 5 microU/ml, three to insulin at 50 microU/ml and six, together with the healthy parents, had no alterations. We conclude that in healthy individuals a family history for NIDDM is frequently signaled, irrespective of age, by molecular derangements, with an apparent genetic background, in their circulating lymphocytes.

Molecular effects of sulphonylurea agents in circulating lymphocytes of patients with non-insulin-dependent diabetes mellitus.

AIMS: In circulating lymphocytes of NIDDM patients pyruvate dehydrogenase (PDH), the major determinant in glucose consumption through oxidative pathways, is poorly active. The aim of this study is to examine whether sulphonylurea drug treatment revives PDH activity in circulating lymphocytes from NIDDM patients. METHODS: Twenty normal-weight individuals with NIDDM were enrolled in this study. They had maintained their glycaemic levels close to normal by means of a restricted diet that had no longer been successful in the proceeding 2 months. The treatment protocol consisted in 160 mg gliclazide daily for 5 weeks. Twenty healthy subjects, matched for age, body mass index and gender, were enrolled as a control group. Patients, before and after treatment, as well as controls were tested for PDH activity in their circulating lymphocytes. Nine other untreated patients and nine healthy subjects, with the above mentioned characteristics, were recruited for the assay of PDH activity in their circulating lymphocytes before and after exposure, in vitro, to gliclazide, to insulin, and to gliclazide and insulin in combination. RESULTS: In gliclazide-treated NIDDM patients, PDH activity in circulating lymphocytes recovered. In vitro, in circulating lymphocytes of untreated patients and controls insulin at 5 microU ml(-1) was ineffective and highly effective, respectively, in raising enzyme activity; gliclazide at 10 ng ml(-1) was ineffective on PDH in both groups, but in combination with insulin at 5 microU ml(-1) in both groups PDH was as active as in cells of controls exposed to insulin only. In cells of controls, gliclazide alone at 25-50 ng ml(-1) caused enzyme activation, whereas above 50 ng ml(-1) it caused inhibition; in cells of patients below 50 ng ml(-1) it had no effects, but at 50 ng ml(-1) and above raised enzyme activity to the basal level of controls. CONCLUSIONS: This study suggests that free gliclazide concentrations determine recovery of PDH activity in circulating lymphocytes of treated patients through drug-mediated enhanced insulin control over PDH or through the drug alone.

Insulin resistance in obese subjects and newly diagnosed NIDDM patients and derangements of pyruvate dehydrogenase in their circulating lymphocytes.

BACKGROUND: In circulating lymphocytes of individuals with insulin resistance and overt hyperglycaemia (NIDDM patients), alterations, affecting pyruvate dehydrogenase (PDH), the key enzyme in glucose oxidative breakdown, have been observed. They include below normal enzyme activity and, in vitro, no enzyme response to insulin at low physiological levels (5 microU/ml) as well as activation up to the basal values of controls with insulin at high physiological levels (50 microU/ml), instead of activation and inhibition respectively, as in controls. OBJECTIVE: To investigate whether these alterations characterize circulating lymphocytes of individuals with insulin resistance in whom derangements of glucose homeostasis are absent (obese subjects with normal glucose tolerance), or present but still controllable (nonobese and obese newly diagnosed NIDDM patients on an appropriate diet). SUBJECTS: Thirty obese subjects (BMI 36 +/- 3) responding normally to an oral glucose tolerance (OGT) test; 60 newly diagnosed NIDDM patients (30 nonobese, BMI 22 +/- 4 and 30 obese, BMI 38 +/- 2); 30 nonobese (BMI 21 +/- 5) and nondiabetic subjects, with no family history for NIDDM, served as controls. METHODS: Evaluation of PDH activity in circulating lymphocytes before and after exposure to insulin at 5 and 50 microU/ml, and of clinical parameters before and during an OGT test. RESULTS: 1) in circulating lymphocytes of obese nondiabetic subjects as well as obese and nonobese newly diagnosed NIDDM patients, PDH activity was significantly below normal. In vitro, enzyme response to insulin at 5 microU/ml was reduced in nonobese NIDDM patients with respect to controls, and absent in obese nondiabetic subjects and obese NIDDM patients. Enzyme response to insulin at 50 microU/ml was reversed in all individuals, which allowed enzyme activity to recover up to the basal level of controls. 2) In NIDDM patients and obese nondiabetic subjects, undergoing an OGT test, the area under the glycaemic curve (g-AUC) was as expected; the area under the insulinaemic curve (i-AUC) was increased in both groups with respect to controls, but significantly only in the latter. CONCLUSION: In individuals with insulin resistance PDH activity in their circulating lymphocytes rises up to basal levels of controls, only if these cells are exposed to insulin at high physiological concentrations, and g-AUC is normal only in those subjects who have significantly increased i-AUC. This suggests that with insulin at sufficiently high concentrations both parameters can be corrected. We conclude that the derangements responsible for the alterations of the two parameters share common features and thus the described PDH alterations in circulating lymphocytes reflect systemic insulin resistance whether accompanied by hyperglycaemia or not.

G proteins and regulation of pyruvate dehydrogenase activity by insulin in human circulating lymphocytes.

Pertussis toxin (PT) catalyzes ADP-ribosylation of G protein alpha subunits, thus preventing their role as transducers of external signals targeting metabolic pathways. In vitro, in human circulating lymphocytes insulin at physiological concentrations (5 microU/ml) determines sharp activation of pyruvate dehydrogenase (PDH), the rate limiting enzyme in glucose oxidative breakdown. This study evaluates whether the above-described effects of insulin over PDH are mediated through G proteins. Human circulating lymphocytes (six samples from different donors) were exposed to insulin (5 microU/ml), PT (1-2 micrograms/ml) or PT-9K, a mutated PT void of catalytic activity (1-10 micrograms/ml), and to insulin in combination with the two toxins, and then assessed for PDH activity. Plasma membranes from cells incubated with and without PT or PT-9K were subjected to ADP-ribosylation in the presence of [32P] NAD+ and activated PT. In circulating lymphocytes exposed to PT alone, or in combination with insulin, PDH activity falls significantly below basal values (P < 0.001); PT-9K instead has no effect on basal or on insulin-stimulated PDH activity. ADP-ribosylation of a plasma membrane component with apparent molecular mass (42 kDa) comparable to that of the Gi (inhibitory) protein alpha subunit takes place in cells exposed to PT but not in those exposed to PT-9K. In human circulating lymphocytes Gi proteins or Gi protein-like components appear to be involved in preserving basal PDH activity as well as in the mechanism by which insulin exerts its control over PDH.

Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina.

Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin-Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity.

Effect of sulfonylurea agents on pyruvate dehydrogenase activity in circulating lymphocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM).

In circulating lymphocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM) subnormal pyruvate dehydrogenase (PDH) activity returns to normal following patient treatment with sulfonylurea (gliclazide, 80 mg twice daily/5 weeks). Moreover, in vitro in cells from diabetic patients exposed to insulin at 50 microU/mL PDH activation also occurs; in cells of controls the same happens for insulin at 5 microU/mL, whereas at 50 microU/mL inhibition takes place. Therefore, the low PDH activity in cells of NIDDM patients might be caused by defective insulin control on the enzyme and its recovery in gliclazide-treated patients by drug-mediated removal of the defect. The validity of the hypothesis was verified in this study where cells of NIDDM patients before and after gliclazide treatment were exposed, in vitro, to insulin at 5 and 50 microU/mL and then tested for PDH activity. In such conditions, the profile of PDH behavior in treated patients was no longer comparable to that in untreated patients but closer to that in euglycemic controls, thus supporting the view that the recovery of PDH activity in NIDDM patients following gliclazide treatment might be the expression of an additional effect that the drug would have in these patients, aimed to renew cell responsiveness to insulin.

Levels of carnitine and glycogen in rabbit tissues during development.

This study attempts to add further light on the development of metabolic pathways in mammalians from fetal to post-natal life, by examining ongoing modifications of carnitine (in terms of total acid soluble, short chain esterified and free carnitine) and glycogen levels in the liver, heart, muscle and brain of rabbit during development.

The insulin signal and its effects on the pyruvate dehydrogenase complex in circulating lymphocytes of obese children.

1. Studies have shown that in circulating lymphocytes pyruvate dehydrogenase (PDH) is responsive to insulin. 2. To improve existing knowledge on how insulin influences PDH behaviour, situations in which cell responsiveness to insulin is impaired could be of interest. 3. PDH behaviour in circulating lymphocytes from obese children, with high plasma insulin levels and normal glucose tolerance, was examined. 4. Masking and unmasking processes of insulin receptors on the plasma membrane appear to modulate the enzyme response to insulin.

Acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of pre and post hatched chicks.

1. The behaviour of total acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of chick embryos between 11th and 21st day of development and of 8 and 180-day-old chicks is described. 2. Total acid soluble carnitine fluctuates around the same levels in the brain, liver and muscle until 18th day of development, whereas it attains a peak on that day in the heart. At hatching compared to 18th day, it suddenly increases three times in the muscle, drops not significantly in the heart and brain, but sharply in the liver (-40%). However the levels are always higher than those of the grown chick in the brain but lower in the other tissues. 3. Free carnitine levels are almost constant in all tissues during the embryonic life; if compared to adult ones, they are very much lower in the liver, heart and muscle, but higher in the brain, even in 8 day-old chick. 4. Short chain esterified, carnitine reaches a maximum on 18th day of egg incubation in the liver, brain and heart; in the muscle it stays on constant levels until this day and then rapidly increases so that at hatching it doubles the values. 5. The short chain esterified to free carnitine percentage ratio peaks in all tissues on 18th day of development, attaining figures which are well above those determined in the grown chick.

Pyruvate dehydrogenase activation by insulin in human circulating lymphocytes and the possible pathway involved.

1. The incubation of human fresh circulating lymphocytes with insulin leads to modifications in the behaviour of the pyruvate dehydrogenase complex (PDH) when the contact medium is supplemented with 50 microM Ca2+ and Mg2+. 2. To investigate the mechanism involved in the PDH responsiveness to insulin in circulating lymphocytes and the role of Ca2+ and Mg2+ in this process, the PDH activity was assayed in lymphocytes combined with insulin and/or a number of substances whose mechanism of action is partially known. 3. Of these some have been seen to mimick insulin effects on PDH, whereas other were tested for the first time in this study.

The behaviour of pyruvate dehydrogenase in circulating lymphocytes from diabetic children.

The basal and total pyruvate dehydrogenase activities were assayed in circulating lymphocytes from children with juvenile diabetes at diagnosis and after five days of insulin therapy and from control subjects. In untreated diabetic children, basal and total pyruvate dehydrogenase activities were deeply decreased and both showed very similar values; whereas, in control subjects basal activity was about 30% lower than total activity. In diabetic patients treated with insulin (in vivo situation), both basal and total activity levels were equal or even higher than those of the control subjects. The incubation of lymphocytes from diabetic patients with insulin (5 microU/ml) (in vitro situation) stimulates, but less than in vivo, the basal and total pyruvate dehydrogenase activities.

Insulin modulation of pyruvate dehydrogenase in human circulating lymphocytes.

1. In human circulating lymphocytes pyruvate dehydrogenase (PDH) complex is present in the active (PDHa) and inactive (PDHi) forms. 2. PDHi conversion into PDHa is stimulated when intact lymphocytes are incubated with 5 microU/ml insulin at pH 7.4, for 15 min at 37 degrees C in a medium supplemented with 50 microM Ca2+-Mg2+. 3. The generation of a mediator is strongly suggestive since a cell free preparation from circulating lymphocytes, treated as above described, still stimulates PDHi----PDHa conversion, when combined with either disrupted or intact lymphocytes.