Induction of insulin secretion in engineered liver cells by nitric oxide

Background  

Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway.     

Genetically engineered K cells provide sufficient insulin to correct hyperglycemia in a nude murine model

A gene therapy-based treatment of type 1 diabetes mellitus requires the development of a surrogate β cell that can synthesize and secrete functionally active insulin in response to physiologically relevant changes in ambient glucose levels. In this study, the murine enteroendocrine cell line STC-1 was genetically modified by stable transfection. Two clone cells were selected (STC-1-2 and STC-1-14) that secreted the highest levels of insulin among the 22 clones expressing insulin from 0 to 157.2 μIU/ml/10⁶ cells/d. After glucose concentration in the culture medium was increased from 1 mM to 10 mM, secreted insulin rose from 40.3±0.8 to 56.3±3.2 μIU/ml (STC-1-2), and from 10.8±0.8 to 23.6±2.3 μIU/ml (STC-1-14). After STC-1-14 cells were implanted into diabetic nude mice, their blood glucose levels were reduced to normal. Body weight loss was also ameliorated. Our data suggested that genetically engineered K cells secrete active insulin in a glucose-regulated manner, and in vivo study showed that hyperglycemia could be reversed by implantation of the cells, suggesting that the use of genetically engineered K cells to express human insulin might provide a glucose-regulated approach to treat diabetic hyperglycemia.     

Insulin is secreted upon glucose stimulation by both gastrointestinal enteroendocrine K-cells and L-cells engineered with the preproinsulin gene

Transgenic mice carrying the human insulin gene driven by the K-cell glucose-dependent insulinotropic peptide (GIP) promoter secrete insulin and display normal glucose tolerance tests after their pancreatic p-cells have been destroyed. Establishing the existence of other types of cells that can process and secrete transgenic insulin would help the development of new gene therapy strategies to treat patients with diabetes mellitus. It is noted that in addition to GIP secreting K-cells, the glucagon-like peptide 1 (GLP-1) generating L-cells share/ many similarities to pancreatic p-cells, including the peptidases required for proinsulin processing, hormone storage and a glucose-stimulated hormone secretion mechanism. In the present study, we demonstrate that not only K-cells, but also L-cells engineered with the human preproinsulin gene are able to synthesize, store and, upon glucose stimulation, release mature insulin. When the mouse enteroendocrine STC-1 cell line was transfected with the human preproinsulin gene, driven either by the K-cell specific GIP promoter or by the constitutive cytomegalovirus (CMV) promoter, human insulin co-localizes in vesicles that contain GIP (GIP or CMV promoter) or GLP-1 (CMV promoter). Exposure to glucose of engineered STC-1 cells led to a marked insulin secretion, which was 7-fold greater when the insulin gene was driven by the CMV promoter (expressed both in K-cells and L-cells) than when it was driven by the GIP promoter (expressed only in K-cells). Thus, besides pancreatic p-cells, both gastrointestinal enteroendocrine K-cells and L-cells can be selected as the target cell in a gene therapy strategy to treat patients with type 1 diabetes mellitus.     

Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer

Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD.     

Enhanced plasmid DNA production by enzyme-controlled glucose release and an engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To evaluate the combination of a culture medium employing glucoamylase-mediated glucose reléase from a gluco-polysaccharide and an E. coli strain engineered in its glucose transport system for improving plasmid DNA (pDNA) production.

Results

The production of pDNA was tested using E. coli DH5α grown in shake-flasks and the recently developed VH33 Δ(recA deoR)-engineered strain, which utilizes glucose more efficiently than wild type strains. Three glucoamylase concentrations for releasing glucose from the polysaccharide carbon source were used: 1, 2 and 3 U l^?1. Both strains reached similar cell densities ranging from 5 to 8.8 g l^?1 under the different conditions. The highest pDNA yields on biomass (Y_pDNA/X) for both strains were obtained when 3 U enzyme l^?1were used. Under these conditions, 35 ± 3 mgof pDNA l^?1 were produced by DH5α after 24 h of culture. Under the same conditions, the engineered strain produced 66 ± 1 mgpDNAl^?1 after 20 h. pDNA supercoiled fractionswere close to 80 % for both strains.

Conclusions

The pDNA concentration achieved by the engineered E. coli was 89 % higher than that of DH5α. The combination of the engineered strain and enzyme-controlled glucose release is an attractive alternative for pDNA production in shake-flasks.

     

13C Metabolic Flux Analysis of Escherichia coli Engineered for Gamma‐Aminobutyrate Production

Escherichia coli is engineered for γ‐aminobutyrate (GABA) production in glucose minimal medium. For this, overexpression of mutant glutamate decarboxylase (GadB) and mutant glutamate/GABA antiporter (GadC), as well as deletion of GABA transaminase (GabT), are accomplished. In addition, the carbon flux to the tricarboxylic acid cycle is engineered by the overexpression of gltA, ppc, or both. The overexpression of citrate synthase (CS), encoded by gltA, increases GABA productivity, as expected. Meanwhile, the overexpression of phosphoenolpyruvate carboxylase (PPC) causes a decrease in the rate of glucose uptake, resulting in a decrease in GABA production. The phenotypes of the strains are characterized by ¹³C metabolic flux analysis (¹³C MFA). The results reveal that CS overexpression increases glycolysis and anaplerotic reaction rates, as well as the citrate synthesis rate, while PPC overexpression causes little changes in metabolic fluxes, but reduces glucose uptake rate. The engineered strain produces 1.2 g L^?1 of GABA from glucose. Thus, by using ¹³C MFA, important information is obtained for designing metabolically engineered strains for efficient GABA production.     

Engineering industrial <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for xylose fermentation and comparison for switchgrass conversion

Saccharomyces’ physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04–0.17 h⁻¹. Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17–0.31 g/l/h, with ethanol yields 0.18–0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13–17% more ethanol than the parental control strains because of their ability to ferment xylose.     

Production of insulinomimetic antibodies against rat adipocyte membranes by hybridoma cells

SJL mice were injected intraperitoneally with adipocyte plasma membranes or with intrinsic membrane proteins obtained by extraction of plasma membranes with dimethylmaleic anhydride. Three days after the boost injection, the spleens were removed and fused with NS-1, a thioguanine-resistant myeloma cell line derived from P₃X63 Ag⁸ (Balb/c). Following selection for hybrids with hypoxanthine, aminopterin, and thymidine, medium of the hybrid cells was tested for its ability to bind to the plasma membrane of the adipocyte and to stimulate the oxidation of D-(1-¹⁴C) glucose to ¹⁴CO₂. Approximately 40% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with plasma membranes produced immunoglobulin that bound to adipocyte plasma membranes. About 30% of these mimicked the ability of insulin to stimulate the oxidation of D-(1-¹⁴C) glucose to ¹⁴CO₂ in adipocytes. Media from 51% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with intrinsic membrane proteins produced immunoglobulin that bound to the plasma membrane and 48% of those stimulated glucose oxidation. The bioactivity of the hybrid cell media could be blocked by adsorption with intrinsic membrane proteins or by the removal of immunoglobulins using formalin-fixed Staphylococcus aureus. The hybrids generated in this study can be divided into three categories: (1) hybrids that secrete antibodies that can bind to plasma membranes and mimic insulin action of glucose transport; (2) hybrids that secrete antibodies that bind to plasma membranes but do not stimulate the oxidation of D-(1-¹⁴C) glucose to ¹⁴CO₂; and (3) hybrids that produce no antimembrane antibodies. The data suggest that interaction of immunoglobulins with specific membrane proteins is essential in mimicking the action of insulin on glucose transport and oxidation in the rat adipocyte.     

Producing Biochemicals in Yarrowia lipolytica from Xylose through a Strain Mating Approach

Enabling xylose catabolism is challenging, especially for unconventional yeasts and previously engineered background strains. In this study, the efficacy of a yeast mating approach with Yarrowia lipolytica that can combine a previously engineering and evolved xylose phenotype with a metabolite overproduction phenotype is demonstrated. Specifically, several engineered Y. lipolytica strains that produce α‐linolenic acid (ALA), riboflavin, and triacetic acid lactone (TAL) with an engineered and adapted xylose‐utilizing strain to obtain three diploid strains that rapidly produce these molecules directly from xylose are mated. Titers of 0.52 g L^?1 ALA, 96.6 mg L^?1 riboflavin, and 2.9 g L^?1 TAL, are obtained from xylose in flask cultures and 1.42 g L^?1 production of ALA is obtained using bioreactor condition. This total production level is generally on par or higher than the parental strain cultivated on glucose, although specific productivities decreased as a result of improved overall cell growth by the diploid strains. In the case of ALA, this lipid content reached similar levels to that of flaxseed oil. This result showcases the first study using strain mating in Y. lipolytica for producing biomolecules from xylose, and thus demonstrates the utility of this approach as a routine tool for metabolic engineering.     

High passage MIN6 cells have impaired insulin secretion with impaired glucose and lipid oxidation

Type 2 diabetes is a metabolic disorder characterized by the inability of beta-cells to secrete enough insulin to maintain glucose homeostasis. MIN6 cells secrete insulin in response to glucose and other secretagogues, but high passage (HP) MIN6 cells lose their ability to secrete insulin in response to glucose. We hypothesized that metabolism of glucose and lipids were defective in HP MIN6 cells causing impaired glucose stimulated insulin secretion (GSIS). HP MIN6 cells had no first phase and impaired second phase GSIS indicative of global functional impairment. This was coupled with a markedly reduced ATP content at basal and glucose stimulated states. Glucose uptake and oxidation were higher at basal glucose but ATP content failed to increase with glucose. HP MIN6 cells had decreased basal lipid oxidation. This was accompanied by reduced expressions of Glut1, Gck, Pfk, Srebp1c, Ucp2, Sirt3, Nampt. MIN6 cells represent an important model of beta cells which, as passage numbers increased lost first phase but retained partial second phase GSIS, similar to patients early in type 2 diabetes onset. We believe a number of gene expression changes occurred to produce this defect, with emphasis on Sirt3 and Nampt, two genes that have been implicated in maintenance of glucose homeostasis.     

Combinatorial insulin secretion dynamics of recombinant hepatic and enteroendocrine cells

One of the most promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-β cells that secrete insulin in response to physiologic stimuli. A normal pancreatic β cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal β cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with 1 of 3 adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards preclinical studies.     

Obesity‐Induced Diabetes in Mouse Strains Treated With Gold Thioglucose: A Novel Animal Model for Studying β‐Cell Dysfunction

An obesity‐induced diabetes model using genetically normal mouse strains would be invaluable but remains to be established. One reason is that several normal mouse strains are resistant to high‐fat diet‐induced obesity. In the present study, we show the effectiveness of gold thioglucose (GTG) in inducing hyperphagia and severe obesity in mice, and demonstrate the development of obesity‐induced diabetes in genetically normal mouse strains. GTG treated DBA/2, C57BLKs, and BDF1 mice gained weight rapidly and exhibited significant increases in nonfasting plasma glucose levels 8–12 weeks after GTG treatment. These mice showed significantly impaired insulin secretion, particularly in the early phase after glucose load, and reduced insulin content in pancreatic islets. Interestingly, GTG treated C57BL/6 mice did not become diabetic and retained normal early insulin secretion and islet insulin content despite being as severely obese and insulin resistant as the other mice. These results suggest that the pathogenesis of obesity‐induced diabetes in GTG‐treated mice is attributable to the inability of their pancreatic β‐cells to secrete enough insulin to compensate for insulin resistance. Mice developing obesity‐induced diabetes after GTG treatment might be a valuable tool for investigating obesity‐induced diabetes. Furthermore, comparing the genetic backgrounds of mice with different susceptibilities to diabetes may lead to the identification of novel genetic factors influencing the ability of pancreatic β‐cells to secrete insulin.     

High yield production of four-carbon dicarboxylic acids by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Several metabolic engineered Escherichia coli strains were constructed and evaluated for four-carbon dicarboxylic acid production. Fumarase A, fumarase B and fumarase C single, double and triple mutants were constructed in a ldhA adhE mutant background overexpressing the pyruvate carboxylase from Lactococcus lactis. All the mutants produced succinate as the main four-carbon (C4) dicarboxylic acid product when glucose was used as carbon source with the exception of the fumAC and the triple fumB fumAC deletion strains, where malate was the main C4-product with a yield of 0.61–0.67 mol (mole glucose)^?1. Additionally, a mdh mutant strain and a previously engineered high-succinate-producing strain (SBS550MG-Cms pHL413-Km) were investigated for aerobic malate production from succinate. These strains produced 40.38 mM (5.41 g/L) and 50.34 mM (6.75 g/L) malate with a molar yield of 0.53 and 0.55 mol (mole succinate)^?1, respectively. Finally, by exploiting the high-succinate production capability, the strain SBS550MG-Cms243 pHL413-Km showed significant malate production in a two-stage process from glucose. This strain produced 133 mM (17.83 g/L) malate in 47 h, with a high yield of 1.3 mol (mole glucose)^?1 and productivity of 0.38 g L^?1 h^?1.     

Construction and Analysis of High-Ethanol-Producing Fusants with Co-Fermentation Ability through Protoplast Fusion and Double Labeling Technology

Double labeling of resistance markers and report genes can be used to breed engineered Saccharomyces cerevisiae strains that can assimilate xylose and glucose as a mixed carbon source for ethanol fermentation and increased ethanol production. In this study Saccharomyces cerevisiae W5 and Candida shehatae 20335 were used as parent strains to conduct protoplast fusion and the resulting fusants were screened by double labeling. High performance liquid chromatography (HPLC) was used to assess the ethanol yield following the fermentation of xylose and glucose, as both single and mixed carbon sources, by the fusants. Interestingly, one fusant (ZLYRHZ7) was demonstrated to have an excellent fermentation performance, with an ethanol yield using the mixed carbon source of 0.424 g g⁻¹, which compares with 0.240 g g⁻¹ (W5) and 0.353 g g⁻¹ (20335) for the parent strains. This indicates an improvement in the ethanol yield of 43.4% and 16.7%, respectively.     

Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters

Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular β-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (ΔG°=+3.6 kJ mol⁻¹). A thermodynamic “push” from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae.     

Genetic engineering of soft-rot bacteria for ethanol production from lignocellulose

Summary The soft-rot bacteriaErwinia carotovora SR38 andErwinia chrysanthemi EC16 have been genetically engineered to efficiently produce ethanol and carbon dioxide as primary fermentation products from cellobiose, glucose and xylose. These organisms have the native ability to secrete a battery of hydrolases and lyases to aid in the solubilization of lignocellulose. Both strains of ethanologenicErwinia fermented cellobiose at twice the rate of the cellobioseutilizing yeasts (Spindler et al., 1992. Biotechnology Letters 14: 403–407) and may be useful in simultaneous saccharification and fermentation processes.     

Variation in Type 2 Diabetes-Related Phenotypes among Apolipoprotein E-Deficient Mouse Strains

We recently have found that apolipoprotein E-deficient (Apoe^-/-) mice with the C57BL/6 background develop type 2 diabetes when fed a Western diet for 12 weeks. In the present study we constructed multiple Apoe^-/- mouse strains to find diabetes-related phenotyptic variations that might be linked to atherosclerosis development. Evaluation of both early and advanced lesion formation in aortic root revealed that C57BL/6, SWR/J, and SM/J Apoe^-/- mice were susceptible to atherosclerosis and that C3H/HeJ and BALB/cJ Apoe^-/- mice were relatively resistant. On a chow diet, fasting plasma glucose varied among strains with C3H/HeJ having the highest (171.1 ± 9.7 mg/dl) and BALB/cJ the lowest level (104.0 ± 6.6 mg/dl). On a Western diet, fasting plasma glucose rose significantly in all strains, with C57BL/6, C3H/HeJ and SWR/J exceeding 250 mg/dl. BALB/cJ and C3H/HeJ were more tolerant to glucose loading than the other 3 strains. C57BL/6 was sensitive to insulin while other strains were not. Non-fasting blood glucose was significantly lower in C3H/HeJ and BALB/cJ than C57BL/6, SM/J, and SWR/J. Glucose loading induced the 1st and the 2nd phase of insulin secretion in BALB/cJ, but the 2nd phase was not observed in other strains. Morphological analysis showed that BALB/cJ had the largest islet area (1,421,493 ± 61,244 μm²) and C57BL/6 had the smallest one (747,635 ± 41,798 μm²). This study has demonstrated strain-specific variations in the metabolic and atherosclerotic phenotypes, thus laying the basis for future genetic characterization.