WNT-4 mRNA expression in human adrenocortical tumors and cultured adrenal cells.

The members of the Wnt glycoprotein family are important in embryogenesis and adult tissue homeostasis, and deletion of WNT-4 gene in mice leads to improper development of many organs including the adrenals. The objective of this study was to investigate the expression of WNT-4 gene in human adrenals and adrenocortical tumors. The WNT-4 mRNA expression (analyzed by quantitative real-time RT-PCR) was significantly higher in Conn's adenomas (p<0.01) and lower in Cushing's adenomas, virilizing carcinomas and fetal adrenals (p<0.05) compared with normal adult adrenals. WNT-4 mRNA expression was clearly upregulated by ACTH and 8-bromo-cAMP (8-BrcAMP) in primary cultures of normal adult adrenocortical cells, but downregulated by 8-BrcAMP and 12- O-tetradecanoylphorbol-13-acetate (TPA) in human NCI-H295R adrenocortical carcinoma cells. Angiotensin II tended to increase WNT-4 mRNA expression at 24 hours and decreased it at 48 hours time point in both cell culture types. The abundant WNT-4 mRNA expression in Conn's adenomas and its hormonal regulation in adrenocortical cells suggest a role for WNT-4 in human adrenocortical function.

Concurrent overexpression of ornithine decarboxylase and spermidine/spermine N(1)-acetyltransferase further accelerates the catabolism of hepatic polyamines in transgenic mice.

We have generated a hybrid transgenic mouse line overexpressing both ornithine decarboxylase (ODC) and spermidine/spermine N(1)-acetyltransferase (SSAT) under the control of the mouse metallothionein (MT) I promoter. In comparison with singly transgenic animals overexpressing SSAT, the doubly transgenic mice unexpectedly displayed much more striking signs of activated polyamine catabolism, as exemplified by a massive putrescine accumulation and an extreme reduction of hepatic spermidine and spermine pools. Interestingly, the profound depletion of the higher polyamines in the hybrid animals occurred in the presence of strikingly high ODC activity and tremendous putrescine accumulation. Polyamine catabolism in the doubly transgenic mice could be enhanced further by administration of zinc or the polyamine analogue N(1),N(11)-diethylnorspermine. In tracer experiments with [(14)C]spermidine we found that, in comparison with syngenic animals, both MT-ODC and MT-SSAT mice possessed an enhanced efflux mechanism for hepatic spermidine. In the MT-ODC animals this mechanism apparently operated in the absence of measurable SSAT activity. In the hybrid animals, spermidine efflux was stimulated further in comparison with the singly transgenic animals. In spite of a dramatic accumulation of putrescine and a profound reduction of the spermidine and spermine pools, only marginal changes were seen in the level of ODC antizyme. Even though the hybrid animals showed no liver or other organ-specific overt toxicity, except an early and permanent loss of hair, their life span was greatly reduced. These results can be understood from the perspective that catabolism is the overriding regulatory mechanism in the metabolism of the polyamines and that, even under conditions of severe depletion of spermidine and spermine, extremely high tissue pools of putrescine are not driven further to replenish the pools of the higher polyamines.

Mouse hexokinase II gene: structure, cDNA, promoter analysis, and expression pattern.

In mammalian tissues, the phosphorylation of intracellular glucose to glucose-6-phosphate (Glu-6-P) is facilitated by four distinct hexokinase (HK) isoenzymes, designated as HKI-IV. Because of the role of HKII as a leading glycolytic enzyme in insulin-sensitive tissues such as skeletal muscle, heart, and adipose tissue, defects in HKII function could contribute to the development of insulin resistance and perhaps Type 2 diabetes. As a first step towards elucidation of the physiological role of HKII in insulin resistance and type 2 diabetes using mouse knock-out models, we determined the genomic structure, sequence of the cDNA and of 4.8 kb of the 5' regulatory region, and tissue-specific expression of the mouse HKII gene. The gene comprises 18 exons that span approximately 50 kb of DNA. Nucleotide sequence of the proximal promoter revealed a number of conserved putative transcription factor binding motifs. We also found numerous repeat elements throughout the mouse HKII gene. The mouse HKII cDNA is approximately 5.5 kb in length and contains an open reading frome of 2751 bp encoding a protein of 917 amino acids. The mouse HKII gene is predominantly expressed in skeletal muscle, heart, and adipose tissue. The transcription initiation and polyadenylation sites for the mouse HKII mRNA were similar to those of the rat and human genes.

Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes.

Type 2 diabetes is characterized by decreased rates of insulin-stimulated glucose uptake and utilization, reduced hexokinase II mRNA and enzyme production, and low basal levels of glucose 6-phosphate in insulin-sensitive skeletal muscle and adipose tissues. Hexokinase II is primarily expressed in muscle and adipose tissues where it catalyzes the phosphorylation of glucose to glucose 6-phosphate, a possible rate-limiting step for glucose disposal. To investigate the role of hexokinase II in insulin action and in glucose homeostasis as well as in mouse development, we generated a hexokinase II knock-out mouse. Mice homozygous for hexokinase II deficiency (HKII(-/-)) died at approximately 7.5 days post-fertilization, indicating that hexokinase II is vital for mouse embryogenesis after implantation and before organogenesis. HKII(+/-) mice were viable, fertile, and grew normally. Surprisingly, even though HKII(+/-) mice had significantly reduced (by 50%) hexokinase II mRNA and activity levels in skeletal muscle, heart, and adipose tissue, they did not exhibit impaired insulin action or glucose tolerance even when challenged with a high-fat diet.

Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue.

We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT-SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and >90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1-2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues.