Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes melitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2–5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity.

Disappearance of body fat in normal rats induced by adenovirus-mediated leptin gene therapy

Sustained hyperleptinemia of 8 ng/ml was induced for 28 days in normal Wistar rats by infusing a recombinant adenovirus containing the rat leptin cDNA (AdCMV-leptin). Hyperleptinemic rats exhibited a 30–50% reduction in food intake and gained only 22 g over the experimental period versus 115–132 g in control animals that received saline infusions or a recombinant virus containing the β-galactosidase gene (AdCMV-βGal). Body fat was absent in hyperleptinemic rats, whereas control rats pair-fed to the hyperleptinemic rats retained ≈50% body fat. Further, plasma triglycerides and insulin levels were significantly lower in hyperleptinemic versus pair-fed controls, while fatty acid and glucose levels were similar in the two groups, suggestive of enhanced insulin sensitivity in the hyperleptinemic animals. Thus, despite equivalent reductions in food intake and weight gain in hyperleptinemic and pair-fed animals, identifiable fat tissue was completely ablated only in the former group, raising the possibility of a specific lipoatrophic activity for leptin.

Correction of obesity and diabetes in genetically obese mice by leptin gene therapy

The ob/ob mouse is genetically deficient in leptin and exhibits both an obese and a mild non-insulin-dependent diabetic phenotype. To test the hypothesis that correction of the obese phenotype by leptin gene therapy will lead to the spontaneous correction of the diabetic phenotype, the ob/ob mouse was treated with a recombinant adenovirus expressing the mouse leptin cDNA. Treatment resulted in dramatic reductions in both food intake and body weight, as well as the normalization of serum insulin levels and glucose tolerance. The subsequent diminishment in serum leptin levels resulted in the rapid resumption of food intake and a gradual gain of body weight, which correlated with the gradual return of hyperinsulinemia and insulin resistance. These results not only demonstrated that the obese and diabetic phenotypes in the adult ob/ob mice are corrected by leptin gene treatment but also provide confirming evidence that body weight control may be critical in the long-term management of non-insulin-dependent diabetes mellitus in obese patients.

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin’s biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin’s effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.

Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressing Plasmodium yoelii (Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-γ-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P < 0.0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-γ production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-γ production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.

Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them

The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death. Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes. We expressed human granzyme A in bacteria as a proenzyme capable of in vitro activation by enterokinase. The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin. An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates. Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment. PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack. PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.

Physiological response to long-term peripheral and central leptin infusion in lean and obese mice

Recent data have identified leptin as an afferent signal in a negative-feedback loop regulating the mass of the adipose tissue. High leptin levels are observed in obese humans and rodents, suggesting that, in some cases, obesity is the result of leptin insensitivity. This hypothesis was tested by comparing the response to peripherally and centrally administered leptin among lean and three obese strains of mice: diet-induced obese AKR/J, New Zealand Obese (NZO), and Ay. Subcutaneous leptin infusion to lean mice resulted in a dose-dependent loss of body weight at physiologic plasma levels. Chronic infusions of leptin intracerebroventricularly (i.c.v.) at doses of 3 ng/hr or greater resulted in complete depletion of visible adipose tissue, which was maintained throughout 30 days of continuous i.c.v. infusion. Direct measurement of energy balance indicated that leptin treatment did not increase total energy expenditure but prevented the decrease that follows reduced food intake. Diet-induced obese mice lost weight in response to peripheral leptin but were less sensitive than lean mice. NZO mice were unresponsive to peripheral leptin but were responsive to i.c.v. leptin. Ay mice did not respond to subcutaneous leptin and were 1/100 as sensitive to i.c.v. leptin. The decreased response to leptin in diet-induced obese, NZO, and Ay mice suggests that obesity in these strains is the result of leptin resistance. In NZO mice, leptin resistance may be the result of decreased transport of leptin into the cerebrospinal fluid, whereas in Ay mice, leptin resistance probably results from defects downstream of the leptin receptor in the hypothalamus.

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of ≈142 residues designated PrP 27–30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27–30 was expressed in Escherichia coli and purified. After refolding rPrP into an α-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113–125 characterize a hydrophobic cluster, which packs against an irregular β-sheet, whereas residues 90–112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200–227) and a structured loop (residues 165–171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice

Leptin is a circulating protein involved in the long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety signals. We investigated the interaction between leptin and CCK-8 in the short-term regulation of food intake induced by 24-hr fasting in lean mice. Leptin, injected intraperitoneally (i.p.) at low doses (4–120 μg/kg), which did not influence feeding behavior for the first 3 hr postinjection, decreased food intake dose dependently by 47–83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was not observed between leptin and bombesin. The food-reducing effect of leptin injected with CCK was not associated with alterations in gastric emptying or locomotor behavior. Leptin–CCK action was blocked by systemic capsaicin at a dose inducing functional ablation of sensory afferent fibers and by devazepide, a CCK-A receptor antagonist but not by the CCK-B receptor antagonist, L-365,260. The decrease in food intake which occurs 5 hr after i.p. injection of leptin alone was also blunted by devazepide. Coinjection of leptin and CCK enhanced the number of Fos-positive cells in the hypothalamic paraventricular nucleus by 60%, whereas leptin or CCK alone did not modify Fos expression. These results indicate the existence of a functional synergistic interaction between leptin and CCK leading to early suppression of food intake which involves CCK-A receptors and capsaicin-sensitive afferent fibers.

Constitutive and impaired signaling of leptin receptors containing the Gln → Pro extracellular domain fatty mutation

Leptin (OB), an adipocyte-secreted circulating hormone, and its receptor (OB-R) are key components of an endocrine loop that regulates mammalian body weight. In this report we have analyzed signal transduction activities of OB-R containing the fatty mutation [OB-R(fa)], a single amino acid substitution at position 269 (Gln → Pro) in the OB-R extracellular domain that results in the obese phenotype of the fatty rat. We find that this mutant receptor exhibits both ligand-independent transcriptional activation via interleukin 6 and hematopoietin receptor response elements and ligand-independent activation of signal transducer and activator of transcription (STAT) proteins 1 and 3. However, OB-R(fa) is unable to constitutively activate STAT5B and is highly impaired for ligand induced activation of STAT5B compared with OB-R(wt). Introduction of the fatty mutation into a OB-R/G-CSF-R chimera generates a receptor with constitutive character that is similar but distinct from that of OB-R(fa). Constitutive mutant OB-R(fa) receptor signaling is repressed by coexpression of OB-R(wt). The implications of an extracellular domain amino acid substitution generating a cytokine receptor with a partially constitutive phenotype are discussed both in terms of the mechanism of OB-R triggering and the biology of the fatty rat.

Functional consequences of NR2 subunit composition in single recombinant N-methyl-d-aspartate receptors

Single-channel recordings were obtained from Chinese hamster ovary cells transfected with the N-methyl-d-aspartate (NMDA) receptor subunit NR1 in combination with NR2A, NR2B, NR2C, or NR2A/NR2B. NMDA-activated currents were recorded under control conditions and in the presence of a thiol reductant (DTT), an oxidant (5,5′-dithio-bis[2-nitrobenzoic acid], DTNB), or the noncompetitive antagonist CP101,606 (CP). For all subunit combinations, DTT increased the frequency of channel opening when compared with DTNB. In addition, channels obtained from NR1/NR2A-transfected cells also exhibited a pronounced difference in mean open dwell-time between redox conditions. CP dramatically reduced both the open dwell-time and frequency of channel opening of NR1/NR2B-containing receptors, but only modestly inhibited NR1/NR2A and NR1/NR2C channel activity. A small number of patches obtained from cells transfected with NR1/NR2A/NR2B had channels with properties intermediate to NR1/NR2A and NR1/NR2B receptors, including insensitivity to CP block but redox properties similar to NR1/NR2B, consistent with the coassembly of NR2A with NR2B. Hence, NMDA receptors containing multiple types of NR2 subunits can have functionally distinguishable attributes.

Characterization of recombinant phytochrome from the cyanobacterium Synechocystis

The complete sequence of the Synechocystis chromosome has revealed a phytochrome-like sequence that yielded an authentic phytochrome when overexpressed in Escherichia coli. In this paper we describe this recombinant Synechocystis phytochrome in more detail. Islands of strong similarity to plant phytochromes were found throughout the cyanobacterial sequence whereas C-terminal homologies identify it as a likely sensory histidine kinase, a family to which plant phytochromes are related. An ≈300 residue portion that is important for plant phytochrome function is missing from the Synechocystis sequence, immediately in front of the putative kinase region. The recombinant apoprotein is soluble and can easily be purified to homogeneity by affinity chromatography. Phycocyanobilin and similar tetrapyrroles are covalently attached within seconds, an autocatalytic process followed by slow conformational changes culminating in red-absorbing phytochrome formation. Spectral absorbance characteristics are remarkably similar to those of plant phytochromes, although the conformation of the chromophore is likely to be more helical in the Synechocystis phytochrome. According to size-exclusion chromatography the native recombinant apoproteins and holoproteins elute predominantly as 115- and 170-kDa species, respectively. Both tend to form dimers in vitro and aggregate under low salt conditions. Nevertheless, the purity and solubility of the recombinant gene product make it a most attractive model for molecular studies of phytochrome, including x-ray crystallography.

Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta

Leptin is a 167-aa protein that is secreted from adipose tissue and is important in the regulation of energy balance. It also functions in hematopoiesis and reproduction. To assess whether leptin is involved in fetal growth and development we have examined the distribution of mRNAs encoding leptin and the leptin receptor (which has at least six splice variants) in the 14.5-day postcoitus mouse fetus and in the placenta using reverse transcription–PCR and in situ hybridization. High levels of gene expression for leptin, the leptin receptor, and the long splice variant of the leptin receptor with an intracellular signaling domain were observed in the placenta, fetal cartilage/bone, and hair follicles. Receptor expression also was detected in the lung, as well as the leptomeninges and choroid plexus of the fetal brain. Western blotting and immunocytochemistry, using specific antibodies, demonstrated the presence of leptin and leptin receptor protein in these tissues. These results suggest that leptin may play a role in the growth and development of the fetus, both through placental and fetal expression of the leptin and leptin receptor genes. In the fetus, leptin may be multifunctional and have both paracrine and endocrine effects.

Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: Role in restricted conformational mobility and compaction of native state

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193–7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein’s surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen–deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein’s interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.

Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells.