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 m mUaml) as well as activation up to the basal values of controls with insulin at high physiological levels (50 m mUaml), 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 AE 3) responding normally to an oral glucose tolerance (OGT) test; 60 newly diagnosed NIDDM patients (30 nonobese, BMI 22 AE 4 and 30 obese, BMI 38 AE 2); 30 nonobese (BMI 21 AE 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 m mUaml, 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 signi®cantly below normal. In vitro, enzyme response to insulin at 5 m mUaml 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 m mUaml 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 signi®cantly 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 signi®cantly increased i-AUC. This suggests that with insulin at suf®ciently 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 re¯ect systemic insulin resistance whether accompanied by hyperglycaemia or not.

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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.

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