Thiazolidinediones (TZD) improve insulin sensitivity in human as well as in different animal models of insulin resistance and Type 2 diabetes. However, no clear link to the insulin signaling events has been identified. Using differentiated 3T3-L1 adipocytes, we found that TZD rapidly and markedly increased IRS-2 gene expression. This effect was specific for PPARgamma agonists and was not seen with PPARalpha agonists. It was rapidly induced (within 4 h) and maintained throughout the observation period of 48 h. It was also concentration dependent (EC50 approximately 50 nM) and not inhibited by cycloheximide, suggesting a direct effect on the IRS-2 promoter. There was no evidence that TZD altered IRS-2 mRNA stability, supporting that the increased mRNA levels were due to an increased gene transcription. IRS-2 protein expression was increased approximately 30% after 48 h and approximately 50% after 96 h. No effects of TZD were seen on IRS-1, PKB/Akt, or GLUT4 gene expression. TZD also increased IRS-2 mRNA levels in cultured human adipose tissue. These data show the first direct link between TZD and a critical molecule in insulin's signaling cascade in both 3T3-L1 and human adipocytes, and indicate a novel mode of action of these compounds.

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Nicotinamide nucleotide transhydrogenase from bovine heart consists of a single polypeptide of 109 kD. The complete gene for this transhydrogenase was constructed, and the protein primary structure was determined from the cDNA. As compared to the previously published sequences of partially overlapping clones, three residues differed: Ala591 (previously Phe), Val777 (previously Glu), and Ala782 (previously Arg). The Escherichia coli transhydrogenase consists of an alpha subunit of 52 kD and a beta subunit of 48 kD. Alignment of the protein primary structure of the bovine trashydrogenase with that of the transhydrogenase from E. coli showed an identity of 52%, indicating similarly folded structures. Prediction of transmembrane-spanning alpha-helices, obtained by applying several prediction algorithms to the primary structures of the revised bovine heart and E. coli transhydrogenases, yielded a model containing 10 transmembrane alpha-helices in both transhydrogenases. In E. coli transhydrogenase, four predicted alpha-helices were located in the alpha subunit and six alpha-helices were located in the beta subunit. Various conserved amino acid residues of the E. coli transhydrogenase located in or close to predicted transmembrane alpha-helixes were replaced by site-specific mutagenesis. Conserved negatively charged residues in predicted transmembrane alpha-helices possibly participating in proton translocation were identified as beta Glu82 (Asp655 in the bovine enzyme) and beta Asp213 (asp787 in the bovine enzyme) located close to the predicted alpha-helices 7 and 9 of the beta subunit. beta Glu82 was replaced by Lys or Gln and beta Asp213 by Asn or His. However, the catalytic as well as the proton pumping activity was retained. In contrast, mutagenesis of the conserved beta His91 residue (His664 in the bovine enzyme) to Ser, Thr, and Cys gave an essentially inactive enzyme. Mutation of alpha His450 (corresponding to His481 in the bovine enzyme) to Thr greatly lowered catalytic activity without abolishing proton pumping. Since no other conserved acidic or basic residues were predicted in transmembrane alpha-helices regardless of the prediction algorithm used, proton translocation by transhydrogenase was concluded to involve a basic rather than an acidic residue. The only conserved cysteine residue, beta Cys260 (Cys834 in the bovine enzyme), located in the predicted alpha-helix 10 of the E. coli transhydrogenase, previously suggested to function as a redox-active dithiol, proved not to be essential, suggesting that redox-active dithiols do not play a role in the mechanism of transhydrogenase.

Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit. The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, betaE371, which is not conserved but located in the same domain, was also mutated. Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities. However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H). It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase.

SARS‐COV‐2 frequently cause neurological disorders and is sometimes associated with onset of autoimmune diseases affecting the nervous system. Over recent years, a rare but distinct diagnosis designated myelin oligodendrocyte glycoprotein‐associated disorder (MOGAD) has been recognized in patients with attacks of optic neuritis, myelitis, or encephalomyelitis and increased levels of anti‐MOG antibodies. The cause of MOGAD is unknown. However, there have been reports of single cases of MOGAD in patients with Covid‐19 infection. We report a series of SARS‐CoV‐2 positive patients that developed MOGAD, but a homology search did not support a cross‐reactive immune response to SARS‐CoV‐2 spike‐protein and MOG.