Increased glucose production in mice overexpressing human fructose-1,6-bisphosphatase in the liver

Increased endogenous glucose production (EGP) predominantly from the liver is a characteristic feature of type 2 diabetes, which positively correlates with fasting hyperglycemia. Gluconeogenesis is the biochemical pathway shown to significantly contribute to increased EGP in diabetes. Fructose-1,6-bisphosphatase (FBPase) is a regulated enzyme in gluconeogenesis that is increased in animal models of obesity and insulin resistance. However, whether a specific increase in liver FBPase can result in increased EGP has not been shown. The objective of this study was to determine the role of upregulated liver FBPase in glucose homeostasis. To achieve this goal, we generated human liver FBPase transgenic mice under the control of the transthyretin promoter, using insulator sequences to flank the transgene and protect it from site-of-integration effects. This resulted in a liver-specific model, as transgene expression was not detected in other tissues. Mice were studied under the following conditions: 1) at two ages (24 wk and 1 yr old), 2) after a 60% high-fat diet, and 3) when bred to homozygosity. Hemizygous transgenic mice had an approximately threefold increase in total liver FBPase mRNA with concomitant increases in FBPase protein and enzyme activity levels. After high-fat feeding, hemizygous transgenics were glucose intolerant compared with negative littermates (P < 0.02). Furthermore, when bred to homozygosity, chow-fed transgenic mice showed a 5.5-fold increase in liver FBPase levels and were glucose intolerant compared with negative littermates, with a significantly higher rate of EGP (P < 0.006). This is the first study to show that FBPase regulates EGP and whole body glucose homeostasis in a liver-specific transgenic model. Our homozygous transgenic model may be useful for testing human FBPase inhibitor compounds with the potential to treat patients with type 2 diabetes.     

Interleukin-6 gene expression is increased in insulin-resistant rat skeletal muscle following insulin stimulation

IL-6 expression in skeletal muscle is stimulated by contractions. We sought to examine whether hyperinsulinaemia increases IL-6 mRNA in skeletal muscle and whether any increase is modified in insulin resistant muscle. We hypothesized that intramuscular IL-6 mRNA would be increased in response to insulin, but such an affect would be unaffected by insulin resistance because the primary insulin sensitive signalling protein responsible for activating IL-6 functions normally in insulin resistant muscle. Transgenic rats over-expressing the gluconeogenic regulatory enzyme phosphoenolpyruvate carboxykinase (PEPCK) were studied. White gastrocnemius muscle samples were obtained under hyperinsulinaemic, euglycaemic clamp (4 mU kg(-1)min(-1) insulin, plasma glucose concentration 4-6 mmol L(-1)) and basal conditions in both PEPCK (basal n=4; insulin n=5) and wild-type (CON) (basal n=5; insulin n=4) rats, which were previously injected with a bolus of 2-[1-14C]deoxyglucose (2-DG) into the carotid artery. Muscle samples were assayed for 2-DG uptake and IL-6 mRNA. No differences in 2-DG uptake or IL-6 mRNA were observed when comparing groups under basal conditions. Under clamp conditions, 2-DG uptake was lower (P<0.05) in PEPCK compared with CON. Insulin stimulation in CON did not change IL-6 mRNA compared with basal levels. In contrast, there was an approximately 8-fold increase (P<0.05) in IL-6 mRNA in insulin-stimulated PEPCK compared with CON basal levels. Insulin stimulation increases IL-6 gene expression in insulin resistant, but not healthy, skeletal muscle, suggesting that IL-6 expression in skeletal muscle is sensitive to changes in insulin in circumstances of insulin resistance. It is likely that the differences observed when comparing healthy with insulin resistant muscle are due to the differential activation of insulin sensitive signalling proteins responsible for activating IL-6.     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

Polymorphisms in genes of the steroid hormone biosynthesis and metabolism pathways and endometrial cancer risk

Objectives: The incidence of endometrial cancer has recently increased substantially and studies have shown that altered levels of exogenous and endogenous hormones are associated with individual variation in endometrial cancer risk. The environmental and reproductive risk factors that influence these hormones are well known, however, genetic variants involved in hormone biosynthesis and estrogen metabolism have not been well established in endometrial cancer. Methods: To determine whether polymorphisms in genes of the steroid hormone biosynthesis and metabolism pathways are associated with endometrial cancer risk, 28 polymorphisms in 18 genes were genotyped in 191 endometrial cancer cases and 291 healthy controls. Results: The GSTM1 deletion and the variant (GG) genotype of the CYP1B1 rs1800440 polymorphism were associated with a decreased risk of developing endometrial cancer. Furthermore, combinations of haplotypes in CYP1A1, CYP1B1 and GSTs were associated with a decreased risk. The analysis of the repeat polymorphisms revealed that women with the long repeat allele length of the ESR1 (GT)n repeat polymorphism were at an increased risk of developing endometrial cancer. Conversely, women with two long repeat length alleles of the (CAG)n repeat polymorphism in the AR correlated with a decrease in endometrial cancer risk compared to women with one or two alleles with the short repeat length. Conclusions: The findings are consistent with our hypothesis that variability in genes involved in steroidogenesis and estrogen metabolism may alter the risk of developing endometrial cancer, suggesting that they may be useful as biomarkers for genetic susceptibility to endometrial cancer.     

Evaluating the glucose tolerance test in mice

The objective of this study was to determine the optimal conditions under which to assess glucose tolerance in chow- and high-fat-fed C57BL/6J mice. Mice were fed either chow or high-fat diet for 8 wk. Variables tested were fasting duration (0-, 3-, 6-, and 24-h and overnight fasting), route of administration (intraperitoneal vs. oral) load of glucose given (2, 1, or 0.5 g/kg and fixed 50-mg dose), and state of consciousness. Basal glucose concentrations were increased in high-fat- compared with chow-fed mice following 6 h of fasting (9.1 +/- 0.3 vs. 7.9 +/- 0.4 mmol/l P = 0.01). Glucose tolerance was most different and therefore significant (P = 0.001) in high-fat-fed mice after 6 h of fasting (1,973 +/- 96 vs. 1,248 +/- 83 mmol.l(-1).120 min(-1)). The difference in glucose tolerance was greater following an OGTT (142%), in contrast to an IPGTT, with a 127% difference between high fat and chow. We also found that administering 2 g/kg of glucose resulted in a greater level of significance (P = 0.0008) in glucose intolerance in high-fat- compared with chow-fed mice. A fixed dose of 50 mg glucose regardless of body weight was enough to show glucose intolerance in high-fat- vs. chow-fed mice. Finally, high-fat-fed mice showed glucose intolerance compared with their chow-fed counterparts whether they were tested under conscious or anesthetized conditions. We conclude that 2 g/kg glucose administered orally following 6 h of fasting is best to assess glucose tolerance in mice under these conditions.