Growth hormone improves body composition, fasting blood glucose, glucose tolerance and liver triacylglycerol in a mouse model of diet-induced obesity and type 2 diabetes.

AIMS/HYPOTHESIS: Growth hormone has been used experimentally in two studies to treat individuals with type 2 diabetes, with both reporting beneficial effects on glucose metabolism. However, concerns over potential diabetogenic actions of growth hormone complicate its anticipated use to treat type 2 diabetes. Thus, an animal model of type 2 diabetes could help evaluate the effects of growth hormone for treating this condition. METHODS: Male C57BL/6J mice were placed on a high-fat diet to induce obesity and type 2 diabetes. Starting at 16 weeks of age, mice were treated once daily for 6 weeks with one of four different doses of growth hormone. Body weight, body composition, fasting blood glucose, insulin, glucose tolerance, liver triacylglycerol, tissue weights and blood chemistries were determined. RESULTS: Body composition measurements revealed a dose-dependent decrease in fat and an increase in lean mass. Analysis of fat loss by depot revealed that subcutaneous and mesenteric fat was the most sensitive to growth hormone treatment. In addition, growth hormone treatment resulted in improvement in glucose metabolism, with the highest dose normalising glucose, glucose tolerance and liver triacylglycerol. In contrast, insulin levels were not altered by the treatment, nor did organ weights change. However, fasting plasma leptin and resistin were significantly decreased after growth hormone treatment. CONCLUSIONS/INTERPRETATION: Growth hormone therapy improves glucose metabolism in this mouse model of obesity and type 2 diabetes, providing a means to explore the molecular mechanism(s) of this treatment.

Enzymological properties and immunological characterization of alpha-galactosidase isoenzymes from normal and Fabry human liver.

1. A method is described for the rapid isolation of alpha-galactosidases A and B (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from normal human liver. 2. When the same method is applied to Fabry liver, most of the alpha-galactosidase activity is recovered in the fraction corresponding to normal alpha-galactosidase B. In agreement with Romeo, G., D'Urso, M., Pisacane, A., Blum, E., De Falco, A. and Ruffilli, A. (1975) Biochem. Genet. 13, 615-628) [18], a small amount of alpha-galactosidase activity is found in the fraction corresponding to normal alpha-galactosidase A. 3. The kinetic properties of the B-like activity from Fabry liver are similar to those of normal alpha-galactosidase B. In agreement with Romeo et al. [18], it was found that the kinetic properties of the A-like activity from Fabry liver are similar to those of normal alpha-galactosidase A. 4. Using antisera raised against normal alpha-galactosidase A and normal alpha-galactosidase B, it is shown that the normal alpha-galactosidase isoenzymes are immunologically distinct and that the B-like activity from Fabry liver is immunologically related to normal alpha-galactosidase B. Furthermore, the A-like activity from Fabry liver is immunologically related to normal alpha-galactosidase B and not to normal alpha-galactosidase A. 5. Normal alpha-galactosidase B is converted into an A-like form during storage. 6. It is concluded that the B-like alpha-galactosidase in Fabry tissues is identical to normal alpha-galactosidase B, and that the small amount of A-like activity found in Fabry material is due to a modified form of alpha-galactosidase B.

Use of immobilized antibodies in investigating acid alpha-glucosidase in urine in relation to Pompe's disease.

(1) A simple method is described for the isolation of the lysosomal enzyme, acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from normal human liver. Antibodies raised against the purified enzyme were immobilized by covalent coupling to Sepharose 4B. (2) Acid alpha-glucosidase can be quantitatively removed from normal urine by incubating with an excess of immobilized antibody. With p-nitrophenyl-alpha-glucoside as substrate, acid alpha-glucosidase accounts for 91 +/- 3% of the total alpha-glucosidase activity at pH 4.0 IN Normal urine. (3) In urine from a patient with the infantile form of Pompe's disease ('acid maltase deficiency'), no alpha-glucosidase activity could be removed by the immobilized antibody, in agreement with the fact that acid alpha-glucosidase is absent in these patients. (4) In urine from patients with the late-onset form of Pompe's disease, 46 +/- 11% of the alpha-glucosidase activity at pH 4.0 can be removed by incubation with immobilized antibodies, indicating that residual acid alpha-glucosidase activity is present in urine of these patients. The residual acid alpha-glucosidase activity amounts to about 5% of that in the urine of control persons. (5) If acid alpha-glucosidase is adsorbed to immobilized antibodies, the activity can still be measured with p-nitrophenyl-alpha-glucoside as substrate. The Km for p-nitrophenyl-alpha-glucoside is not significantly changed by adsorbing purified acid alpha-glucosidase to immobilized antibodies. (6) The properties of acid alpha-glucosidase from urine of patients with late-onset Pompe's disease were compared with those of acid alpha-glucosidase from normal urine, both adsorbed to immobilized antiserum. The pH-activity profile of the enzyme from urine of patients with late-onset Pompe's disease can not be distinguished from that of the normal urinary enzyme. The Km for p-nitro-phenyl-alpha-glucoside of the two enzymes is identical, both at pH 4 and 3. The titration curves of the two enzymes with immobilized antibodies are identical.

Monoclonal antibodies against human acid alpha-glucosidase.

Acid alpha-glucosidase purified from human placenta was used to immunize a mouse (strain Balb/cHeA) according to a procedure described earlier (Stähli, C., Staehlin, T., Miggiano, V., Schmidt, J. and Häring, P. (1980) J. Immunol. Methods 32, 297-304). After fusion of spleen cells with myeloma cells, about 10% of the hybrid clones obtained produced antibodies against acid alpha-glucosidase. Finally, eight stable clones producing antibodies against the enzyme were obtained. When purified acid alpha-glucosidase is analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, two major protein bands (mol. wt. 76000 and 70000,) a minor band of mol. wt. 9600 and several minor bands with a mol. wt. of 67000 or lower are seen. Since all these components react with the monoclonal antibodies, they must have at least one antigenic determinant in common.

Identification, isolation, and cloning of growth hormone (GH)-inducible interscapular brown adipose complementary deoxyribonucleic acid from GH antagonist mice.

In a dwarf mouse line that expresses a GH antagonist, we found that the interscapular brown adipose tissue (iBAT) mass is significantly greater than that in nontransgenic littermates. We proposed that gene expression in iBAT may be up- or down-regulated by GH. To identify these genes, we employed the PCR-select subtraction approach to construct subtractive libraries from iBAT total RNAs. We have generated forward and reverse subtractive libraries. Clones were screened by differential hybridization and identified by BLAST similarity to expressed sequence tags and complementary DNA sequences. Four novel expressed sequence tags were isolated from the reverse subtractive library. Of them, clone 42, was further analyzed. It encodes a 2475-bp messenger RNA with an open reading frame of 346 amino acids. Northern blot analysis demonstrated two RNA isoforms (2.5 and 1.3 kb) in various tissues. Differential expression of both isoforms was verified in GH antagonist and nontransgenic mouse iBAT. BLAST searches suggested that clone 42 is highly homologous to a gene found in a human female fetal brain and a related gene found in a human pituitary tumor.

Inability of a mouse cell line transformed to produce biologically active recombinant human insulin-like growth factor I (IGF-I) to respond to exogenously added IGF-I.

A plasmid expression vector encoding human insulin-like growth factor I (hIGF-I) in the form of a 97-amino acid precursor protein containing the first 27 amino acids of prebovine GH and the 70 amino acids of hIGF-I has been used to transform mouse L cells. A stably transformed mouse L cell clone has been isolated which expresses and secretes hIGF-I. The secreted peptide comprises 3% of the protein in conditioned medium. IGF-I can be purified to homogeneity in 2 chromatographic steps. One liter of conditioned medium yields approximately 200 micrograms purified peptide. Amino-terminal sequence analysis confirms that the signal peptide has been proteolytically hydrolyzed from the precursor protein before secretion to form [Ala0]hIGF-I. The recombinant peptide and serum-derived hIGF-I are equipotent as inhibitors of the binding of [125I]IGF-I to the type 1 receptor of human placenta and to a crude preparation of acid-stable human serum binding proteins. The peptides are equipotent in 2 in vitro assays, the stimulation of the rate of 2-[1,2-N-3H]deoxyglucose transport in BC3H1 cells and the stimulation of [methyl-3-3H]thymidine incorporation into DNA in A10 cells. In contrast to a control mouse L cell line, DNA synthesis in the [Ala0]IGF-I-secreting line is completely unresponsive to [Thr59]IGF-I, while it responds normally to calf serum (10%). Thus, the [Ala0]IGF-I-secreting line is selectively desensitized to IGF-I. The binding of [125I]IGF-I to both lines is identical, indicating that the loss of responsiveness to IGF-I is not due to a loss of cell surface receptor. The ability to render mouse L cells unresponsive to IGF-I is transferred in the conditioned medium of the [Ala0]IGF-I-secreting cell line. In addition, pretreatment of control cells with [Thr59]IGF-I (10 nM) results in attenuation of the response to a subsequent dose of IGF-I. These data indicate that prolonged exposure to high levels of IGF-I may cause a postreceptor-mediated desensitization to IGF-I. Alternatively, IGF-I may promote secretion of an inhibitor of IGF-mediated DNA synthesis.

A growth hormone (GH) analog can antagonize the ability of native GH to promote differentiation of 3T3-F442A preadipocytes and stimulate insulin-like and lipolytic activities in primary rat adipocytes.

The effect of amino acid substitutions introduced to the third alpha-helix in bovine GH (bGH) was investigated. A GH analog (bGH-M8), in which three amino acids were substituted to form an idealized amphiphilic alpha-helix, possessed the same specific binding affinity as wild-type bGH to cell membranes prepared from 3T3-F442A cells or rat adipocytes. However, bGH-M8 failed to stimulate preadipocyte differentiation, as measured by the level of glycerol-3-phosphate dehydrogenase activity. An equimolar concentration of bGH-M8 was inhibitory for this adipogenic effect caused by bGH at a concentration of 30 pM. bGH-M8 also failed to induce an insulin-like response and reduced lipolytic potency in rat primary adipocytes. A 10-fold excess of bGH-M8 abolished the effect of wild-type bGH in the insulin-like and lipolytic assays. Thus, bGH-M8 inhibited these actions of wild-type bGH and, therefore, appears to be a competitive antagonist. These results suggest that a major biologically active domain resides in the third alpha-helix of bGH, which is independent of amino acids important in the initial interaction of GH with its receptor.

Activation of the mouse metallothionein-I promoter in transiently transfected avian cells.

The induction of the mouse metallothionein-I (MT-I) transcriptional regulatory region by heavy metals in cultured avian cells was studied in a transient-expression assay system for a growth hormone. The MT-I promoter was shown to be inducible by 10 microM to 90 microM ZnCl2 in a dose-dependent manner.

Production, characterization and functional activities of v-Ski in cultured cells.

The v-ski oncogene was introduced into mammalian cells in order to study its biochemical and biological properties. v-Ski, produced at relatively high levels by mouse L cells stably transfected with this DNA, was localized to the cell nucleus, was of correct apparent molecular mass, and was capable of complexing with DNA. Transient transfection of reporter plasmids into control or Ski producing mouse L cells revealed that Ski acts as a transcriptional activator of various transcriptional regulatory elements, including CMVie, RSV LTR and SV40. These results indicate that mouse L cells contain the nuclear cofactor(s) required for the ability of v-Ski to bind to DNA and also suggest that the v-Ski present within the cells is functional.

A comparison of bovine growth-hormone gene expression in mouse L cells directed by the Moloney murine-leukemia virus long terminal repeat, simian virus-40 early promoter or cytomegalovirus immediate-early promoter.

Three transcriptional regulatory regions including a human cytomegalovirus immediate-early region (CMVIE) promoter, the simian virus-40 early region (SV40E) promoter, and Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR) were ligated to the bovine growth hormone (bGH)-coding gene. Using a bGH transient expression system, the CMVIE and SV40E gene transcriptional regulatory regions were found to be approximately two-fold more efficient than the MoMLV LTR in directing expression of bGH in mouse L cells.

A family with different clinical forms of acid maltase deficiency (glycogenosis type II): biochemical and genetic studies.

In the same family, the generalized or infantile form of acid maltase deficiency (glycogenosis type II, Pompe disease) and the muscular or adult-onset form affected different individuals. Autosomal-recessive inheritance for the two clinical forms was demonstrated in this family by assay of acid alpha-glucosidase in muscle, lymphocytes, cultured fibroblasts, and urine of asymptomatic relatives. Current biochemical techniques do not discriminate between persons heterozygous for the generalized form and those heterozygous for the muscular form. To explain the coexistence of both forms in the same family, the infant with the generalized form or her grandfather with the muscular form must have been a genetic compound of different mutant alleles for acid alpha-glucosidase.

Elevation of internal 6-methyladenine mRNA methyltransferase activity after cellular transformation.

A comparison of internal 6-methyladenine mRNA methyltransferase activity in a variety of cell types demonstrated an 8-15-fold increase as a result of cellular transformation. Utilizing adenovirus transformed rat embryo cells, it was found that the increase in methyltransferase activity was concomitant with or occurred rapidly after transformation. An 80-fold increase in activity was observed in the cells isolated from the transformed foci and remained elevated through subsequent passages. The relationship between methyltransferase activity and tumor formation was also investigated. High level expression of the avian ski oncogene in mouse L cells causes a reversion of the transformed phenotype to a non-transformed state, and resulted in a 47% reduction in the specific activity of the methyltransferase as compared with mock transfected cells.

In vitro mutagenesis of growth hormone receptor Asn-linked glycosylation sites.

Site-directed mutagenesis was used to replace asparagine (Asn) residues with glutamine (Gln) at the five potential N-linked glycosylation sites located at positions 28, 97, 138, 143, and 182 in the extracellular domain of the porcine growth hormone receptor (pGHR). These mutated pGHR cDNAs were stably expressed in mouse L cells. Single substitution of the Asn residues did not alter growth hormone binding when compared to cells which express native pGHR (KD approximately 1 nM). However, substitution of the five potential Asn-linked sites together (pGHR delta 5) resulted in a 20-fold reduced GH binding affinity (KD = 20 nM). Residues Asn97, Asn138, and Asn182 were apparently glycosylated and upon cross-linking with 125I-labeled pGH migrated as a molecular complex of approximately 130 kDa. Native pGHR and pGHR analogs with substitutions of N28Q and N143Q when cross-linked to 125I-labeled pGH, migrated with a Mr of 138 kDa. The fully deglycosylated cross-linked receptor, pGHR delta 5, migrated as a complex of 108 kDa. Therefore, each carbohydrate moiety contributed approximately 10 kDa to the total molecular mass of the pGHR, in sum contributing 30 kDa to the total Mr of the glycosylated pGHR. pGHR delta 5 was able to internalize nearly all the bound 125I-labeled pGH within 10 min, whereas native pGHR and individual Asn substituted pGHR analogs internalized 25% of bound 125I-labeled pGH at 10 min. Also, mutagenesis of the pGHR five potential Asn-linked glycosylation sites, either singly or together, did not alter the ability of GH to induce tyrosine phosphorylation of a 95-kDa protein. Together, the results indicate that three of the five pGHR Asn residues are apparently glycosylated and are necessary for maintenance of a high affinity GH binding site and for GH internalization. However, glycosylation of the pGHR is not critical for eliciting tyrosine phosphorylated proteins following the GH/GHR interaction.

Expression of a functional porcine growth hormone receptor cDNA in mouse L cells.

Porcine (p) growth hormone receptor (GHR) complementary DNA (cDNA) has been cloned and the primary amino acid structure was deduced from the nucleotide sequence. A comparison of pGHR to other GHRs revealed an approximately 70% similarity in amino acid sequence (Cioffi et al., 1990). Hybridization of this receptor cDNA to RNA samples isolated from various porcine tissues revealed a single RNA band of 4.2 kb. The full-length pGHR cDNA was subcloned into an eukaryotic expression vector, transcription of which was directed by the mouse metallothionein-I transcriptional regulatory sequence. Stable mouse L cell lines which express the pGHR cDNA were established. Approximately 80% of the cell lines were found to possess pGHR mRNA (approximately 2 kb) which corresponds to the length of the cloned pGHR cDNA. Binding studies showed that the stable cell lines were capable of specifically binding 125I-labeled pGH with a dissociation constant (Kd) of approximately 1.0 nM. The apparent molecular mass of the receptor, as determined by cross-linking studies, was found to be 118 kDa. Also, the receptor-ligand complex could be internalized. These results suggest that an active form of pGHR had been cloned and stably expressed in mouse L cells.

Transgenic animals and nutrition research.

Transgenic animals are useful tools for the study of biological functions of proteins and secondary gene products synthesized by the action of protein catalysts. Research in nutrition and allied fields is benefiting from their use as models to contrast normal and altered metabolism. Although food, nutritional products, and ingredients from transgenic animals have not yet reached consumers, the technologies for their production are maturing and yielding exciting results in experimental and farm animals. Regulatory governmental bodies are already issuing guidelines and legislation in anticipation of the advent of these products and ingredients. This review summarizes available technology for the production of transgenic animals, discusses their scientific and commercial potential, and examines ancillary issues relevant to the field of nutrition.

The identification of type 1 Gaucher disease patients, asymptomatic cases and carriers in The Netherlands using urine samples: an evaluation.

The feasibility of using urine samples for the identification of patients with Gaucher disease and carriers has been investigated. It was found that the pH of a urine sample should be pH 6.0 or lower to ensure stability of lysosomal hydrolases. Two parameters of glucocerebrosidase, which is deficient in Gaucher disease, were studied using urine samples from control subjects, obligate carriers and patients. Firstly, the relative level of glucocerebrosidase activity was measured by relating the activity of the enzyme to that of another lysosomal hydrolase. Secondly, the enzymic activity of glucocerebrosidase per unit of protein was measured using an immunological method. The first method allowed discrimination of nearly all obligate carriers of type 1 Gaucher disease from normal individuals. The second method allowed clear discrimination of the majority of carriers from normal individuals, but some obligate carriers were not distinguishable from normal subjects on the basis of this parameter. However, the combination of both methods allowed discrimination between all obligate carriers examined so far (n = 34) and controls (n = 86). There was variability between healthy individuals in the relative amount of glucocerebrosidase in urine samples. A small proportion of healthy individuals have a relatively high activity of glucocerebrosidase in urine samples, reminiscent of observations made in white blood cells by other investigators. In urine samples from two unrelated parents of Gaucher disease patients a level of glucocerebrosidase activity was present that could not be distinguished from that in samples of patients. These individuals represent cases with subclinical manifestation of Gaucher disease, illustrating once more the remarkable heterogeneity in clinical expression of this disorder.

Glycoconjugates in human and transgenic animal milk.

Human milk samples contain a particularly rich collection of oligosaccharides compared with other milk samples. The synthesis of these molecules should depend on the expression of glycosyltransferases and the presence of sugar nucleotides in lactating mammary glands. We set out to produce transgenic animals expressing glycosyltransferases during lactation with the purpose of exploring the following issues: a) Is it possible to synthesize human milk oligosaccharides in lactating mammary glands of nonhuman animals?, b) Is it possible to express during lactation homologous, tissue specific glycosyltransferases that are not normally expressed in lactating mammary tissue?, and c) What is the effect of expressing a human glycosyltransferase in different animal species? Simultaneously, we embarked on a research program to study short-chain neutral human milk oligosaccharides--no larger than hexasaccharides--to understand the natural variation of milk sugars and glycoproteins. The reagents and methods developed to study human milk oligosaccharides and glycoproteins were also applied to the study of milk from transgenic animals. Our results indicate that mice predictably express transgene-encoded glycosyltransferases and their secondary gene products, oligosaccharides and remodeled glycoproteins. This was true even when the transgene encoded a homologous galactosyltransferase. Also, it was possible to synthesize fucosylated glycoconjugates in mouse milk using two different fucosyltransferases, thus demonstrating that is feasible to emulate the synthesis occurring in the human lactating mammary gland. Experiments with transgenic rabbits yielded different phenotypes, some of them unexpected. Taken together, our results answer the questions stated above but open even more intriguing areas of inquiry.