We compared the actions of human proinsulin and insulin on glucose turnover and on intermediary carbohydrate and lipid metabolism in non-insulin-dependent diabetes mellitus (NIDDM). Six diet-controlled weight-matched (25.4 +/- 1.0 kg/m2) NIDDM subjects underwent six separate isoglycemic clamps. Glucose turnover was measured using a primed continuous infusion of [6',6'-2H2]glucose. Each subject received three low-dose intravenous infusions of both insulin and proinsulin. Blood glucose was maintained at 6.7 +/- 0.3 mM during proinsulin and insulin infusion. Insulin (I) infusions gave steady-state levels of 0.12 +/- 0.001 (I1), 0.18 +/- 0.01 (I2), and 0.33 +/- 0.01 nM (I3). Steady-state proinsulin (P) levels were 2.5 +/- 0.1 (P1), 4.3 +/- 0.2 (P2), and 8.8 +/- 0.9 nM (P3). Hepatic glucose production was suppressed equally by proinsulin and insulin at all doses. The metabolic clearance rate of glucose was significantly increased during the insulin infusion compared with proinsulin. The use of [6',6'-2H2]glucose resulted in a mean underestimation of the glucose infusion rate of 10.0 +/- 4.0 and 6.0 +/- 2.5% during the two highest insulin and proinsulin doses, respectively. Proinsulin had a significantly weaker effect than insulin, at the lowest infusion dose, in percent suppression of plasma nonesterified fatty acids, blood glycerol, and beta-hydroxybutyrate levels (all P less than 0.05). Blood lactate levels were lower during the P1 (628 +/- 43 microM) and P2 (657 +/- 93 microM) infusions compared with I1 (776 +/- 60 microM) and I2 (878 +/- 44 microM; P less than 0.05, P less than 0.02), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)