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One approach to the location of the first coupling site of oxidative phosphorylation (Site I) and the site at which the inhibitors, rotenone and amytal, interrupt electron transport in the respiratory chain of avian and mammalian mitochondria has been based upon a comparison of the steady-state responses of the respiratory carriers nicotinamide adenine dinucleotide (NAD) and flavoprotein (FPD) to uncoupling agents and to amytal or rotenone.' On this basis, Site I and the rotenone site A B have been assigned to point A in the sequence: NAD-FPD-* (UQ,b). Discrepancies in this assignment have been noted in a number of laboratories: Several workers (cf. ref. 2) assign Site I to A but the rotenone site to B; Klingenberg3 proposes the opposite; Schatz and Racker4 assign both Site I and the rotenone site to B. M\ore recent studies of reversed electron transport have indicated an additional component, identical with neither the nonheme iron nor the sulfhydryl component of FPD, at B.5 A reconciliation of these viewpoints is now based on the finding that the NAD dehydrogenase portion of the respiratory chain contains two flavoproteins operating in sequence, with both the rotenone site and the coupling Site I between them, as in Scheme I below: NAD-FPD FPD2-F (cyt. b, UQ) cyt. cic cyt. aa3-°02 Site I Site II Site III Rotenone Antimycin A Amytal (FpSFPLFPETF) Materials and Methods.-Changes of flavoprotein absorbance and fluorescence6 in mitochondria and submitochondrial particles were measured simultaneously with a double-beam spectrophotometer7 and an attached fluorometer. Suitable filters guarded each photomultiplier against light from the opposite system. Similar procedures were used for simultaneous recording of flavoprotein fluorescence and pyridine nucleotide absorbance. Mitochondria were prepared from rat liver and

1. Mitochondria prepared from Torulopsis utilis grown in a chemostat with iron-limited growth were found to lack energy conservation but not electron flow in that segment of the respiratory chain leading from intramitochondrial NADH to the cytochromes [i.e. the site 1 segment (Lehninger, 1964)]. 2. Site 1 energy conservation was present in mitochondria prepared from cells grown under conditions of limitation by glycerol, ammonium and magnesium. Phosphate-limited growth resulted in mitochondrial preparations without respiratory control. 3. Mitochondria from cells grown under conditions of iron limitation were insensitive to the respiratory inhibitor piericidin A, whereas sensitivity was present in mitochondria prepared from glycerol-, ammonium-, magnesium- or phosphate-limited cells. 4. These observations are considered to provide indirect evidence for a role of non-haem iron in the mechanism of energy conservation and also piericidin A sensitivity in T. utilis mitochondria. 5. A readily constructed and inexpensive pH-measuring and -controlling circuit is described for use with continuous-culture apparatus.

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Leptin suppresses insulin secretion by opening ATP-sensitive K + (K ATP ) channels and hyperpolarizing -cells. We measured the intracellular concentration of ATP ([ATP] i ) in tumor-derived -cells, INS-1, and found that leptin reduced [ATP] i by ∼30%, suggesting that the opening of K ATP channels by leptin is mediated by decreased [ATP] i . A reduction in glucose availability for metabolism may explain the decreased [ATP] i , since leptin (30 min) reduced glucose transport into INS-1 cells by ∼35%, compared to vehicle-treated cells. The twofold induction of GLUT2 phosphorylation by GLP-1, an insulin secretagogue, was abolished by leptin. Therefore, the acute effect of leptin could involve covalent modification of GLUT2. These findings suggest that leptin may inhibit insulin secretion by reducing [ATP] i as a result of reduced glucose availability for the metabolic pathway. In addition, leptin reduced glucose transport by 35% in isolated rat hepatocytes that also express GLUT2, suggesting that glucose transport may also be altered by leptin in other glucose-responsive tissues such as the liver.