OBJECTIVEZinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele.RESEARCH DESIGN AND METHODSSlc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols.RESULTSZnT8−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay.CONCLUSIONSZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

Type 1B diabetes (typically early onset; without islet autoantibodies) has been described in patients bearing small coding sequence mutations in the INS gene. Not all mutations in the INS gene cause the autosomal dominant Mutant INS-gene-induced Diabetes of Youth (MIDY) syndrome, but most missense mutations affecting proinsulin folding produce MIDY. MIDY patients are heterozygotes, with the expressed proinsulin mutants exerting dominant-negative (gain of toxic function) behavior in pancreatic beta cells. Herein, we focus primarily on proinsulin folding in the endoplasmic reticulum, providing insight into perturbations of this folding pathway in MIDY. Accumulated evidence indicates that in the molecular pathogenesis of the disease, misfolded proinsulin exerts dominant effects that initially inhibit insulin production, progressing to beta cell demise with diabetes.

Recently, missense mutations upstream of preproinsulin’s signal peptide (SP) cleavage site were reported to cause mutant INS gene-induced diabetes of youth (MIDY). Our objective was to understand the molecular pathogenesis using metabolic labeling and assays of proinsulin export and insulin and C-peptide production to examine the earliest events of insulin biosynthesis, highlighting molecular mechanisms underlying β-cell failure plus a novel strategy that might ameliorate the MIDY syndrome. We find that whereas preproinsulin-A(SP23)S is efficiently cleaved, producing authentic proinsulin and insulin, preproinsulin-A(SP24)D is inefficiently cleaved at an improper site, producing two subpopulations of molecules. Both show impaired oxidative folding and are retained in the endoplasmic reticulum (ER). Preproinsulin-A(SP24)D also blocks ER exit of coexpressed wild-type proinsulin, accounting for its dominant-negative behavior. Upon increased expression of ER–oxidoreductin-1, preproinsulin-A(SP24)D remains blocked but oxidative folding of wild-type proinsulin improves, accelerating its ER export and increasing wild-type insulin production. We conclude that the efficiency of SP cleavage is linked to the oxidation of (pre)proinsulin. In turn, impaired (pre)proinsulin oxidation affects ER export of the mutant as well as that of coexpressed wild-type proinsulin. Improving oxidative folding of wild-type proinsulin may provide a feasible way to rescue insulin production in patients with MIDY.

Background: Preproinsulin signal peptide mutations have been linked to human diabetes. Results: Preproinsulin mutants fail to be fully translocated across the endoplasmic reticulum membrane, accumulate in the cytosol, and lead to ␤ cell death. Conclusion: Cytosolic preproinsulin accumulation contributes to ␤ cell failure. Significance: This reveals a novel pathogenesis of diabetes associated with inefficient preproinsulin translocation and cytosolic accumulation.

Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two ␤-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single ␤-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 ␤-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5␣. Sequencing of the bla TLA-1 gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A ␤-lactamases:70 SXXK, 130 SDN, and 234 KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A ␤-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A ␤-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 ␤-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum ␤-lactamase of Ambler class A.The main mechanism of resistance to ␤-lactam antibiotics in members of the family Enterobacteriaceae is the production of ␤-lactamases (21, 35). Expanded-spectrum cephalosporins (cefotaxime, ceftazidime) have been specifically designed to resist degradation by the older broad-spectrum ␤-lactamases such as TEM-1, TEM-2, and SHV-1. With the use of these antibiotics in vivo, extended-spectrum ␤-lactamases (ESBLs) have been selected; these ESBLs most often are mutants of these older enzymes and carry a limited number of amino acid substitutions (G. Jacoby and K. Bush, http://www.lahey.org/studies /webt.htm). There is also a small but growing family of plasmid-mediated ESBLs that are not related to TEM or SHV ␤-lactamases, such as 10,14,15) and Toho (17,23), that preferentially hydrolyze cefotaxime and that belong to Ambler class A. In addition, there has been a worldwide emergence of novel ␤-lactamases, mainly among members of the family Enterobacteriaceae, that hydrolyze expanded-spectrum ␤-lactams. While they maintain the main properties of the class A ␤-lactamases, they are not closely related to the TEM, SHV, or CTX-M families of ␤-lactamases. Most of these ESBLs are plasmid mediated and include the PER-1, PER-2, VEB-1, CblA, and CepA enzymes. These ␤-lactamases are not species specific, since they have also been isolated from clinically significant gram-negative species that are not mem...