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.

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.

Expression of fungal desaturase genes in cultured mammalian cells.

Long-chain polyunsaturated fatty acids (LC-PUFA) are important components of cellular structure and function. Most of LC-PUFA are derived from linoleic acid and alpha-linolenic acid. In plants and fungi, these two acids can be synthesized from oleic acid via the action of two enzymes, delta12 and delta15-desaturases. Due to lack of these enzymatic activities and the ability to synthesize these two essential fatty acids, animals must obtain them from the diet. In this report, we demonstrated the expression of a fungal delta12-desaturase gene in mouse L cells incubated in serum-free medium. The results showed a significant increase in the amount of linoleic acid with a concomitant decrease of oleic acid in cellular lipids. Most of the newly formed linoleic acid was incorporated into cellular phospholipids, particularly phosphatidylcholine. The increase of linoleic acid provided the substrate for the endogenous synthesis of (n-6) LC-PUFA, such as eicosadienoic acid (EDA), dihomo-gamma-linoleic acid (DGLA) and arachidonic acid (AA). Prolonged incubation further increased the levels of linoleic acid derived from oleic acid by the action of delta12-desaturase, and the levels of 20:2n-6 produced from linoleic acid by the action of the endogenous elongase. However, prolonged incubation suppressed significantly the formation of DGLA and AA. In a separate study, a fungal delta6-desaturase gene has also been expressed in the mouse L cells incubated in serum-containing medium. The result shows a significant increase in levels of 20:3n-6 and 20:4n-6. These findings demonstrate that through genetic modification, it is possible to (1) generate cell lines which no longer require dietary 'essential' fatty acids and (2) alter the endogenous fatty acid metabolism to enhance the production of LC-PUFA and their derivatives.

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.

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells.

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.

cDNA cloning and characterization of human Delta5-desaturase involved in the biosynthesis of arachidonic acid.

Two human expressed sequence tag (EST) cDNA sequences with identity with Delta(5)- and Delta(6)-desaturases from a filamentous fungus, Mortierella alpina, were identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. (Palo Alto, CA, U.S.A.). An oligonucleotide complementary to the 3' EST cDNA sequences was used to screen human liver cDNA using rapid amplification of cDNA ends (RACE)-PCR. The amplified DNA fragment had 98% identity with a putative open reading frame (ORF) predicted from a human genomic sequence, and encoded 444 amino acids. Expression of this ORF in mouse fibroblast cells demonstrated that the encoded protein was a Delta(5)-desaturase, as determined by the conversion of dihomo-gamma-linolenic acid (C(20:3,n-6)) into arachidonic acid (C(20:4,n-6)). The human Delta(5)-desaturase contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. A tissue expression profile revealed that this gene is highly expressed in fetal liver, fetal brain, adult brain and adrenal gland. A search of the existing databases led to localization of this ORF within a 14 kb interval flanked by the flap endonuclease-1 (FEN1) and vitelliform macular dystrophy (Best's disease; VMD2) loci of chromosome 11q12.

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.

DNA binding and transcriptional activation by the Ski oncoprotein mediated by interaction with NFI.

The Ski oncoprotein has been found to bind non-specifically to DNA in association with unindentified nuclear factors. In addition, Ski has been shown to activate transcription of muscle-specific and viral promoters/enhancers. The present study was undertaken to identify Ski's DNA binding and transcriptional activation partners by identifying specific DNA binding sites. We used nuclear extracts from a v-Ski-transduced mouse L-cell line and selected Ski-bound sequences from a pool of degenerate oligonucleotides with anti-Ski monoclonal antibodies. Two sequences were identified by this technique. The first (TGGC/ANNNNNT/GCCAA) is the previously identified binding site of the nuclear factor I (NFI) family of transcription factors. The second (TCCCNNGGGA) is the binding site of Olf-1/EBF. By electophoretic mobility shift assays we find that Ski is a component of one or more NFI complexes but we fail to detect Ski in Olf-1/EBF complexes. We show that Ski binds NFI proteins and activates transcription of NFI reporters, but only in the presence of NFI. We also find that homodimerization of Ski is essential for co-activation with NFI. However, the C-terminal dimerization domain of c-Ski, which is missing in v-Ski, can be substituted by the leucine zipper domain of GCN4.

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.

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.

Remodeling of mouse milk glycoconjugates by transgenic expression of a human glycosyltransferase.

The mammary gland is a unique biosynthetic tissue that produces a variety of species-specific glycoconjugates, but the factors regulating the production of specific glycoconjugates are not well understood. To explore the underlying regulation, a fusion gene containing a cDNA encoding the human alpha 1,2-fucosyltransferase (alpha 1,2FT), which generates the H-blood group antigen, flanked by the murine whey acidic protein promoter and a polyadenylation signal, was introduced into mice. Milk samples from transgenic animals contained soluble forms of the alpha 1,2FT, as revealed by Western blots of milk samples using an anti-alpha 1,2FT antiserum and by the demonstration of alpha 1,2FT enzyme activity. Milk from transgenic animals also contained large quantities of 2'-fucosyllactose (Fuc alpha 1-2Gal beta 1-4Glc) and modified glycoproteins containing the H-antigen, whereas milk from control animals lacked these glycoconjugates. Expression levels of 2'-fucosyllactose were high in most animals and represented 1/3 to nearly 1/2 of the total milk oligosaccharides. These results demonstrate that heterologous transgenic expression of a glycosyltransferase can result in the expression of both the transgene and its secondary gene products and that the structures of milk oligosaccharides can be remodeled depending on expression of the appropriate enzyme. Furthermore, these results suggest that the lactating mammary gland may be a unique biosynthetic reactor for the production of biologically active oligosaccharides and glycoconjugates.

A 40-amino acid segment of the growth hormone receptor cytoplasmic domain is essential for GH-induced tyrosine-phosphorylated cytosolic proteins.

It has become evident that intracellular protein phosphorylation plays an important role in mediating signal transduction of hormones and growth factors, including growth hormone (GH). We have previously demonstrated that GH can stimulate tyrosine phosphorylation of cellular proteins with approximate molecular masses of 95,000 daltons (pp95) in GH-treated 3T3-F442A preadipocytes and in mouse L cells that express recombinant porcine or bovine GH receptors. In the present study, a series of GH receptor (GHR) truncation analogs were constructed and examined for their abilities to induce pp95. The results revealed that a region of approximately 40 amino acids in the porcine GHR cytoplasmic domain is essential for induction of pp95. The results also established that the 115 amino acids (517-638) near the C terminus of porcine GHR are not required for pp95 induction. Moreover, the basal levels of GH-induced pp95 in parental mouse L cells was suppressed by expression of these GHR truncation analogs. This suggests that pp95 induced by GH may be mediated by GHR dimerization and can be inhibited by overexpression of truncated porcine GHRs.

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.

A growth hormone (GH) analog that antagonizes the lipolytic effect but retains full insulin-like (antilipolytic) activity of GH.

An analog of bovine growth hormone (bGH-M8: [Leu117, Arg119, Asp122]-bGH) with an idealized amphiphilic third alpha-helix has been proposed to be a functional antagonist of GH. In accordance with this proposition, bGH-M8 profoundly inhibited bGH-stimulated lipolysis by chicken adipose tissue in vitro. bGH-M8 alone was a weak agonist in the lipolytic assay (1.9% the potency of bGH). The present evidence indicates that bGH-M8 is a competitive antagonist of the lipolytic action of GH based upon the following results: (i) increasing concentrations of bGH-M8 (antagonist) produce progressively greater inhibition of GH-stimulated lipolysis; (ii) increasing concentrations of bGH (agonist) are capable of overcoming this antagonism; and (iii) Schild plot analysis (slope = -0.94) suggests a receptor antagonist with an equilibrium dissociation constant (KB) of 4.54 nM. In contrast to the antagonistic effects of bGH-M8 on bGH-stimulated lipolysis, bGH-M8 retained full insulin-like ("antilipolytic") activity (i.e., inhibition of glucagon-induced lipolysis). bGH-M8 and bGH were similarly potent in eliciting antilipolytic effects in vitro. Moreover, the antilipolytic effects of bGH-M8 and bGH were additive. Therefore, the third alpha-helix (particularly residues 117, 119, and 122) of bGH contains major structural determinants for the lipolytic effects of GH. The ability of bGH-M8 to act as an antagonist for at least one action of GH (lipolysis) while being a full agonist for another (antilipolysis) suggests that different domains of GH are responsible for its various biologic activities, possibly involving different binding sites and/or signal transduction mechanisms.

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.

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.

Effects of a leucine analog on growth hormone processing and secretion by cultured cells.

Bovine and rat growth hormones (bGH and rGH, respectively) possess signal peptides that direct the hormone to the secretory pathway and are proteolytically cleaved prior to secretion. Previous in vitro translation studies indicated that incorporation of the polar leucine analog beta-hydroxyleucine into de novo synthesized polypeptides inhibits signal peptide function. To test the effects of this analog on GH secretion by cultured animal cells, transfections of mouse L-cells with a bGH expression plasmid or metabolic labeling of endogenous rGH in anterior pituitary cells was performed in the absence or presence of beta-hydroxyleucine. Transient expression of bGH in mouse L-cells or endogenous expression of rGH in anterior pituitary cells resulted in an accumulation of GH in the culture medium. Treatment with beta-hydroxyleucine resulted in a block in secretion as evidenced by an accumulation of GHs within these cells. Amino-terminal sequencing of the intracellular form of the analog-substituted GHs demonstrated accurate signal peptide cleavage. In contrast, in vitro translations of bGH RNA performed in the presence of beta-hydroxyleucine and microsomal membranes resulted in the inhibition of signal peptide cleavage. The results suggest that beta-hydroxyleucine can uncouple signal peptide processing and protein secretion in cultured cells.

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.

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.