Kathy D. Shelton

Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase.

Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxPtechnology in combination with gene targeting to perform global, β cell-, and hepatocyte-specific gene knock-outs of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively. β cell- and hepatocyte-specific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a β cell or hepatocyte-specific knock-out of GK. Animals either globally deficient in GK, or lacking GK just in β cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the β cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knock-out mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both β cell and hepatic GK contribute to the hyperglycemia of MODY-2.

Sulfonylurea Receptor Type 1 Knock-out Mice Have Intact Feeding-stimulated Insulin Secretion despite Marked Impairment in Their Response to Glucose

The ATP-sensitive potassium channel is a key molecular complex for glucose-stimulated insulin secretion in pancreatic β cells. In humans, mutations in either of the two subunits for this channel, the sulfonylurea type 1 receptor (Sur1) or Kir6.2, cause persistent hyperinsulinemic hypoglycemia of infancy. We have generated and characterized Sur1 null mice. Interestingly, these animals remain euglycemic for a large portion of their life despite constant depolarization of membrane, elevated cytoplasmic free Ca2+ concentrations, and intact sensitivity of the exocytotic machinery to Ca2+. A comparison of glucose- and meal-stimulated insulin secretion showed that, although Sur1 null mice do not secrete insulin in response to glucose, they secrete nearly normal amounts of insulin in response to feeding. Because Sur1 null mice lack an insulin secretory response to GLP-1, even though their islets exhibit a normal rise in cAMP by GLP-1, we tested their response to cholinergic stimulation. We found that perfused Sur1 null pancreata secreted insulin in response to the cholinergic agonist carbachol in a glucose-dependent manner. Together, these findings suggest that cholinergic stimulation is one of the mechanisms that compensate for the severely impaired response to glucose and GLP-1 brought on by the absence of Sur1, thereby allowing euglycemia to be maintained.

Phosphoenolpyruvate Carboxykinase Is Necessary for the Integration of Hepatic Energy Metabolism

ABSTRACT We used an allelogenic Cre/loxP gene targeting strategy in mice to determine the role of cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in hepatic energy metabolism. Mice that lack this enzyme die within 3 days of birth, while mice with at least a 90% global reduction of PEPCK, or a liver-specific knockout of PEPCK, are viable. Surprisingly, in both cases these animals remain euglycemic after a 24-h fast. However, mice without hepatic PEPCK develop hepatic steatosis after fasting despite up-regulation of a variety of genes encoding free fatty acid-oxidizing enzymes. Also, marked alterations in the expression of hepatic genes involved in energy metabolism occur in the absence of any changes in plasma hormone concentrations. Given that a ninefold elevation of the hepatic malate concentration occurs in the liver-specific PEPCK knockout mice, we suggest that one or more intermediary metabolites may directly regulate expression of the affected genes. Thus, hepatic PEPCK may function more as an integrator of hepatic energy metabolism than as a determinant of gluconeogenesis.

Adenovirus-mediated Knockout of a Conditional Glucokinase Gene in Isolated Pancreatic Islets Reveals an Essential Role for Proximal Metabolic Coupling Events in Glucose-stimulated Insulin Secretion

The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between β cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gk lox). Analysis of AdenoCre-treated islets indicated that the gk lox allele in ∼30% of islet cells was converted to a nonexpressing variant (gk del). This resulted in a heterogeneous population of β cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet β cells fromgk lox/lox mice. In AdenoCre-infected islets, approximately one-third of the β cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low-responding cells were not sigmoidal and reached a maximum at ∼5 mmglucose. In contrast, the normal response cells showed a sigmoidal response with an K catS0.5 of ∼8 mm. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in β cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.

BZP, a novel serum-responsive zinc finger protein that inhibits gene transcription.

We report the fortuitous isolation of cDNA clones encoding a novel zinc finger DNA-binding protein termed BZP. The protein encoded is 114 kDa and contains eight zinc finger motifs, seven of which are present in two clusters at opposite ends of the molecule. Both finger clusters bound to the 9-bp sequence AAAGGTGCA with apparent Kds of approximately 2.5 nM. Two of the finger motifs within the amino- and carboxy-terminal finger clusters share 63% amino acid identity. BZP inhibited transcription of the herpes simplex virus thymidine kinase promoter when copies of the 9-bp target motif were linked in cis, suggesting that it functions as a transcriptional repressor. BZP mRNA and immunoreactivity were detected in several established cell lines but were most abundant in hamster insulinoma (HIT) cells, the parental source of the cDNAs. In mouse tissues, BZP mRNA and immunoreactivity were identified in cells of the endocrine pancreas, anterior pituitary, and central nervous system. Interestingly, in HIT cells proliferating in culture, BZP immunoreactivity was predominately nuclear in location, whereas it was usually located in the cytoplasm in most neural and neuroendocrine tissues. Serum deprivation of HIT cells caused BZP immunoreactivity to become predominantly cytoplasmic in location and attenuated its inhibitory effect on transcription, thereby suggesting that the both the subcellular location and the function of this protein are modulated by factors in serum.

Glucokinase Thermolability and Hepatic Regulatory Protein Binding Are Essential Factors for Predicting the Blood Glucose Phenotype of Missense Mutations

To better understand how glucokinase (GK) missense mutations associated with human glycemic diseases perturb glucose homeostasis, we generated and characterized mice with either an activating (A456V) or inactivating (K414E) mutation in the gk gene. Animals with these mutations exhibited alterations in their blood glucose concentration that were inversely related to the relative activity index of GK. Moreover, the threshold for glucose-stimulated insulin secretion from islets with either the activating or inactivating mutation were left- or right-shifted, respectively. However, we were surprised to find that mice with the activating mutation had markedly reduced amounts of hepatic GK activity. Further studies of bacterially expressed mutant enzymes revealed that GKA456V is as stable as the wild type enzyme, whereas GKK414E is thermolabile. However, the ability of GK regulatory protein to inhibit GKA456V was found to be less than that of the wild type enzyme, a finding consistent with impaired hepatic nuclear localization. Taken together, this study indicates that it is necessary to have knowledge of both thermolability and the interactions of mutant GK enzymes with GK regulatory protein when attempting to predict in vivo glycemic phenotypes based on the measurement of enzyme kinetics.

Strategies for the Use of Site-Specific Recombinases in Genome Engineering

Conventional gene targeting has been very useful in the study of gene function and regulation in mice. However, the methodologies involved have several limitations. First, mutations that cause embryonic lethality largely preclude studies of gene function at a later stage in development. Second, conditional and/or tissue-specific alterations of gene expression cannot be achieved using these methods. In addition, classical gene targeting can be difficult and time consuming. Strategies that make use of site-specific recombinases such as Cre and/or Flp have been developed in recent years to overcome these limitations. These new techniques include global and conditional knockouts, recombinase-mediated DNA insertion (RMDI), and recombinase-mediated cassette exchange (RMCE). Together, they have tremendously increased the number and variety of genetic manipulations that can be achieved.

Isolation and characterization of the mouse cytosolic phosphoenolpyruvate carboxykinase (GTP) gene: evidence for tissue-specific hypersensitive sites

A 72 kilobase pair DNA fragment that contains the mouse phosphoenolpyruvate carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacterial artificial chromosome library. The region from approximately -5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Additionally, the chromatin structure of the PEPCK gene in mouse liver resembles that seen in rat. Backcross panel analysis of a microsatellite sequence confirms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. The same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferentiated state but that something else is required for transcription.