Glucose-6-phosphatase overexpression lowers glucose 6-phosphate and inhibits glycogen synthesis and glycolysis in hepatocytes without affecting glucokinase translocation. Evidence against feedback inhibition of glucokinase.

In hepatocytes glucokinase (GK) and glucose-6-phosphatase (Glc-6-Pase)(1) have converse effects on glucose 6-phosphate (and fructose 6-phosphate) levels. To establish whether hexose 6-phosphate regulates GK binding to its regulatory protein, we determined the effects of Glc-6-Pase overexpression on glucose metabolism and GK compartmentation. Glc-6-Pase overexpression (4-fold) decreased glucose 6-phosphate levels by 50% and inhibited glycogen synthesis and glycolysis with a greater negative control coefficient on glycogen synthesis than on glycolysis, but it did not affect the response coefficients of glycogen synthesis or glycolysis to glucose, and it did not increase the control coefficient of GK or cause dissociation of GK from its regulatory protein, indicating that in hepatocytes fructose 6-phosphate does not regulate GK translocation by feedback inhibition. GK overexpression increases glycolysis and glycogen synthesis with a greater control coefficient on glycogen synthesis than on glycolysis. On the basis of the similar relative control coefficients of GK and Glc-6-Pase on glycogen synthesis compared with glycolysis, and the lack of effect of Glc-6-Pase overexpression on GK translocation or the control coefficient of GK, it is concluded that the main regulatory function of Glc-6-Pase is to buffer the glucose 6-phosphate concentration. This is consistent with recent findings that hyperglycemia stimulates Glc-6-Pase gene transcription.

Snare protein expression and adenoviral transfection of amphicrine AR42J.

The amphicrine AR42J acinar cell line is an excellent model to study both exocrine and neuroendocrine exocytotic mechanisms. As a first step toward this goal, we determined the specific isoforms of the v- and t-SNARE and Munc18 families expressed in these cells. In addition, we show that dexamethasone-induced differentiation toward the exocrine phenotype causes an upregulation of several of these proteins. AR42J is notoriously difficult to transfect, limiting its usefulness as a model. However, we have now overcome this obstacle by acheiving high efficiency expression of a beta-galactosidase reporter gene and truncated SNAP-25 gene using adenoviral infection techniques. The AR42J cells can now be used to pursue and elucidate the distinct functions of individual SNARE isoforms used in endocrine and exocrine secretion within a single cell line.

Adenovirus-mediated overexpression of uncoupling protein-2 in pancreatic islets of Zucker diabetic rats increases oxidative activity and improves beta-cell function.

The discovery of uncoupling protein (UCP)-2, a ubiquitously expressed protein homologous to UCP-1, has raised the possibility that energy balance of cells might be regulated in tissues other than brown adipocytes. In normal pancreatic islets, UCP-2 is upregulated by leptin and is low in leptin-resistant islets of ZDF rats. To determine whether UCP-2 does, in fact, have uncoupling activity and, if so, whether such activity would favorably influence the abnormalities in leptin-unresponsive UCP-2-underexpressing islets of diabetic ZDF rats, we transferred the UCP-2 gene to the islets of diabetic ZDF rats and lean (+/+) ZDF control rats. Although ATP was reduced by 23% in both groups of islets, the ATP:ADP ratio increased by 42 and 141%, respectively. [3H]palmitate oxidation was increased by 50%, and [3H]glucose oxidation was 42-63% higher. Preproinsulin mRNA was 2.9-fold above control levels, and glucose-stimulated insulin secretion, which was negligible in control ZDF rat islets, was improved in UCP-2-overexpressing islets. The high fat content of the islets was not reduced, however. We conclude that UCP-2 has uncoupling function when overexpressed in leptin-insensitive islets and that its overexpression corrects the underexpression of the insulin gene and ameliorates glucose-stimulated insulin secretion, possibly by increasing the ATP:ADP ratio.

Overexpression of G11alpha and isoforms of phospholipase C in islet beta-cells reveals a lack of correlation between inositol phosphate accumulation and insulin secretion.

It has been suggested that insulin secretion from pancreatic islets may be mediated in part by activation of phospholipases C (PLCs) and phosphoinositide hydrolysis. The purpose of this study was to determine whether the relatively modest fuel-stimulated insulin secretion responses of rodent beta-cell lines might be explained by inadequate expression or activation of PLC isoforms. We have found that two insulinoma cell lines, INS-1 and betaG 40/110, completely lack PLC-delta1 expression but have levels of expression of PLC-beta1, -beta2, -beta3, -delta2, and -gamma1 that are similar to or slightly reduced from those found in fresh rat islets. Adenovirus-mediated overexpression of PLC-delta1, -beta1, or -beta3 in INS-1 or betaG 40/110 cells results in little or no enhancement in inositol phosphate (IP) accumulation and no improvement in insulin secretion when the cells are stimulated with glucose or carbachol, despite the fact that the overexpressed proteins are fully active in cell extracts. Overexpression of PLC-beta1 or -beta3 in normal rat islets elicits a larger increase in IP accumulation but, again, has no effect on insulin secretion. Because the effect of carbachol on insulin secretion is thought to be mediated through muscarinic receptors that link to the Gq/11 class of heterotrimeric G proteins, we also overexpressed G11alpha in INS-1 cells, either alone or in concert with overexpression of PLC-beta1 or -beta3. Overexpression of G11alpha enhances IP accumulation, an effect slightly potentiated by co-overexpression of PLC-beta1 or -beta3, but these maneuvers do not affect glucose or carbachol-stimulated insulin secretion. In sum, our studies show a lack of correlation between IP accumulation and insulin secretion in INS-1 cells, betaG 40/110 cells, or cultured rat islets. We conclude that overexpression of PLC isoforms and/or G11alpha is not an effective means of enhancing fuel responsiveness in the insulinoma cell lines studied.

Identification of domains mediating transcriptional activation and cytoplasmic export in the caudal homeobox protein Cdx-3.

The caudal genes have important functions in embryonic development and cell differentiation. The caudal-related protein Cdx-2/3 (the protein designated Cdx-2 in the mouse and Cdx-3 in the hamster) is expressed in the gastrointestinal epithelium and in islet and enteroendocrine cells, where it activates proglucagon gene transcription. We show here that Cdx-3 sequences amino-terminal to the homeodomain (amino acids 1-180) function as a heterologous transcriptional activation domain when fused to the LexA DNA binding domain. A Cdx-3-Pit-1 fusion protein containing only the first 83 amino acids of Cdx-3 linked to the POU domain of Pit-1 markedly stimulated the transcriptional activity of a Pit-1-responsive promoter. Analysis of the transcriptional properties of Cdx-3 mutants in fibroblasts and islet cells revealed distinct amino-terminal subdomains that function in a cell-specific manner. Point mutations within the amino-terminal A domain were associated with reduced transcriptional activity. Furthermore, internal deletions and selected point mutations within domain A, but not the B or C domains, resulted in accumulation of mutant Cdx-3 in the cytoplasm. Unexpectedly, mutation of an Asp-Lys-Asp motif within domain A identified a putative cytoplasmic membrane-associated export signal that mediates Cdx-3 compartmentalization. These experiments delineate unique activities for specific amino-terminal sequences that are functionally important for Cdx-3 biological activity.

Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats.

The terminal step in hepatic gluconeogenesis is catalyzed by glucose-6-phosphatase, an enzyme activity residing in the endoplasmic reticulum and consisting of a catalytic subunit (glucose-6-phosphatase (G6Pase)) and putative accessory transport proteins. We show that Zucker diabetic fatty rats (fa/fa), which are known to exhibit impaired suppression of hepatic glucose output, have 2.4-fold more glucose-6-phosphatase activity in liver than lean controls. To define the potential contribution of increased hepatic G6Pase to development of diabetes, we infused recombinant adenoviruses containing the G6Pase cDNA (AdCMV-G6Pase) or the beta-galactosidase gene into normal rats. Animals were studied by one of three protocols as follows: protocol 1, fed ad libitum for 7 days; protocol 2, fed ad libitum for 5 days, fasted overnight, and subjected to an oral glucose tolerance test; protocol 3, fed ad libitum for 4 days, fasted for 48 h, subjected to oral glucose tolerance test, and then allowed to refeed overnight. Hepatic glucose-6-phosphatase enzymatic activity was increased by 1.6-3-fold in microsomes isolated from AdCMV-G6Pase-treated animals in all three protocols, and the resultant metabolic profile was similar in each case. AdCMV-G6Pase-treated animals exhibited several of the abnormalities associated with early stage non-insulin-dependent diabetes mellitus, including glucose intolerance, hyperinsulinemia, decreased hepatic glycogen content, and increased peripheral (muscle) triglyceride stores. These animals also exhibited significant decreases in circulating free fatty acids and triglycerides, changes not normally associated with the disease. Our studies show that overexpression of G6Pase in liver is sufficient to perturb whole animal glucose and lipid homeostasis, possibly contributing to the development of metabolic abnormalities associated with diabetes.

Expression and characterization of an active and thermally more stable recombinant antifreeze polypeptide from ocean pout, Macrozoarces americanus, in Escherichia coli: improved expression by the modification of the secondary structure of the mRNA.

The cDNA clone coding for the ocean pout antifreeze polypeptide (AFP) was modified to improve translation of its mRNA in Escherichia coli. A recombinant AFP (rAFP), MetLys-AFP-Lys, was expressed successfully using the lambda PL promoter, and constituted 1-2% of total bacterial proteins. The rAFP was purified to homogeneity from the soluble fractions of bacterial extracts. Its identity was confirmed by amino acid analysis, automated Edman degradation, immuno-blot and activity measurements. Although the rAFP is indistinguishable from the authentic AFP in its secondary structure, thermal hysteretic activity and the alteration of ice crystal structure, it is, however, thermally more stable (approximately 4.5 degrees C increase in Tm) and is more effective in inhibiting ice growth along the a-axis. These investigations indicate that the extra amino acids in rAFP significantly improve the thermal stability and ice-binding activity of the polypeptide.

[Cloning and sequence analysis of salmon prolactin cDNA].

A cDNA library was prepared from pacific Chinook Salmon pituitaries. Salmon prolactin gene was screened using synthetic oligonucleotide probes based on partial protein sequence. A positive clone (PRL-10) was identified and sequenced. It is a full size clone containing 1.1 and coding for a preprolactin of 211 amino acids.

Molecular cloning and expression of salmon pituitary hormones.

A cDNA library was prepared from chinook salmon pituitaries. Growth hormone (GH), prolactin (PRL) and the ß subunit of gonadotropin (GTH) genes were screened using synthetic oligonucleotides as probes. Full size cDNA clones coding for these polypeptide hormones were isolated and characterized. The cDNA sequences for PRL and ßGTH have been reported earlier from our laboratories. The cDNA clone for GH contains 1148 bp and codes for a preGH of 210 amino acids. The chinook salmon GH, reported in the present investigation, differs from chum salmon GH in only 1 amino acid, and from coho salmon GH in 5 amino acids. Plasmids containing modified nucleotide sequences coding for GH, PRL and ßGTH were constructed individually into an expression vector using the heat-inducible λ pL promotor. Mature PRL, GH and unglycosylated ßGTH were expressed in the bacteria at elevated temperature.

Molecular cloning and expression of salmon prolactin cDNA.

Prolactin was purified from chum salmon pituitaries. It was resolved into two variants by reverse-phase high-performance liquid chromatography. A cDNA library was prepared from Pacific chinook salmon pituitaries. Salmon prolactin gene was screened using a synthetic oligonucleotide based on partial protein sequence. A positive clone (PRL-10) was identified and sequenced. It is a full-size clone containing 1.1 kb and coding for a preprolactin of 211 amino acids. A modified prolactin plasmid (PRL-10A), in which the 5' untranslated sequence and the nucleotide sequence coding for the signal peptide of prolactin were deleted, was reconstructed into an expression vector using the heat-inducible lambda pL promotor. Mature prolactin, a single polypeptide of 22 kDa, was efficiently expressed in the bacteria at an elevated temperature.

Structure of an antifreeze polypeptide precursor from the sea raven, Hemitripterus americanus.

The cystine-rich antifreeze polypeptides (AFP) from sea raven were fractionated by reverse-phase high performance liquid chromatography into several components, with SR2 (Mr 17,000) as the major AFP. Sea raven AFP cDNA clones were isolated from a liver cDNA library using a synthetic oligonucleotide, and the identity of one of the clones, C2-1, was confirmed by hybridization selection and cell-free translation. C2-1 encodes a pre-AFP of 195 amino acids with no evidence of any profragments. Comparison of the deduced amino acid sequence with partial peptide sequences from SR2 showed substitutions in at least four amino acid positions, suggesting that C2-1 cDNA codes for a minor component. Both the primary and the predicted secondary structures of sea raven AFP are completely different from those of other fish AFP. This further confirms that sea raven AFP belongs to a different class of antifreezes. The high frequency of reverse turns and the presence of paired hydrophilic amino acids in these structures are striking features of the protein and may contribute to their antifreeze action.

Molecular cloning and sequencing of salmon gonadotropin beta subunit.

Gonadotropin (GTH) was purified from the pituitaries of the Pacific chinook salmon using a combination of stepwise ethanol precipitation and concanavalin-A affinity chromatography. The alpha and beta subunits were dissociated and fractionated by C-18 reverse-phase high-performance liquid chromatography with a 0.1% trifluoroacetic acid/acetonitrile gradient. An enriched cDNA library was screened for the beta-GTH gene(s) using two synthetic oligonucleotides based on partial protein data. A positive, full-size clone (E3) was identified and sequenced. It contains 657 base pairs and codes for a 142-amino-acid precursor protein. The mature salmon beta-GTH (119 amino acids) is structurally homologous to human luteinizing hormone and chorionic gonadotropin. The effect of testosterone implantation on pituitary GTH and beta-GTH mRNA was examined with radioimmunoassay and Northern blot analysis. There was a corresponding increase in both the pituitary GTH and mRNA levels.

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus.

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.