Thymidine Kinase 2 Deficiency-Induced mtDNA Depletion in Mouse Liver Leads to Defect beta-Oxidation

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/-) mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/-) mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial beta-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/-) mice causes impaired mitochondrial function that leads to defect beta-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies

Heat-sensitive TRP channels detected in pancreatic beta cells by microfluorometry and western blot

Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot. Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein. Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells. Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes

LXR Driven Induction of HDL-Cholesterol is Independent of Intestinal Cholesterol Absorption and ABCA1 Protein Expression

We investigated whether: (1) liver X receptor (LXR)-driven induction of high-density lipoprotein cholesterol (HDL-C) and other LXR-mediated effects on cholesterol metabolism depend on intestinal cholesterol absorption; and (2) combined treatment with the LXR agonist GW3965 and the cholesterol absorption inhibitor ezetimibe results in synergistic effects on cholesterol metabolism that could be beneficial for treatment of atherosclerosis. Mice were fed 0.2 % cholesterol and treated with GW3965+ezetimibe, GW3965 or ezetimibe. GW3965+ezetimibe treatment elevated serum HDL-C and Apolipoprotein (Apo) AI, effectively reduced the intestinal cholesterol absorption and increased the excretion of faecal neutral sterols. No changes in intestinal ATP-binding cassette (ABC) A1 or ABCG5 protein expression were observed, despite increased mRNA expression, while hepatic ABCA1 was slightly reduced. The combined treatment caused a pronounced down-regulation of intestinal Niemann-Pick C1-like 1 (NPC1L1) and reduced hepatic and intestinal cholesterol levels. GW3965 did not affect the intestinal cholesterol absorption, but increased serum HDL-C and ApoAI levels. GW3965 also increased Apoa1 mRNA levels in primary mouse hepatocytes and HEPA1-6 cells. Ezetimibe reduced the intestinal cholesterol absorption, ABCA1 and ABCG5, but did not affect the serum HDL-C or ApoAI levels. Thus, the LXR-driven induction of HDL-C and ApoAI was independent of the intestinal cholesterol absorption and increased expression of intestinal or hepatic ABCA1 was not required. Inhibited influx of cholesterol via NPC1L1 and/or low levels of intracellular cholesterol prevented post-transcriptional expression of intestinal ABCA1 and ABCG5, despite increased mRNA levels. Combined LXR activation and blocked intestinal cholesterol absorption induced effective faecal elimination of cholesterol

A liver‐humanized mouse model of carbamoyl phosphate synthetase 1‐deficiency

A liver‐humanized mouse model for CPS1‐deficiency was generated by the high‐level repopulation of the mouse liver with CPS1‐deficient human hepatocytes. When compared with mice that are highly repopulated with CPS1‐proficient human hepatocytes, mice that are repopulated with CPS1‐deficient human hepatocytes exhibited characteristic symptoms of human CPS1 deficiency including an 80% reduction in CPS1 metabolic activity, delayed clearance of an ammonium chloride infusion, elevated glutamine and glutamate levels, and impaired metabolism of [15N]ammonium chloride into urea, with no other obvious phenotypic differences. Because most metabolic liver diseases result from mutations that alter critical pathways in hepatocytes, a model that incorporates actual disease‐affected, mutant human hepatocytes is useful for the investigation of the molecular, biochemical, and phenotypic differences induced by that mutation. The model is also expected to be useful for investigations of modified RNA, gene, and cellular and small molecule therapies for CPS1‐deficiency. Liver‐humanized models for this and other monogenic liver diseases afford the ability to assess the therapy on actual disease‐affected human hepatocytes, in vivo, for long periods of time and will provide data that are highly relevant for investigations of the safety and efficacy of gene‐editing technologies directed to human hepatocytes and the translation of gene‐editing technology to the clinic

Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts.

Induced pluripotent stem cell (iPSC)-technology is an important platform in medicine and disease modeling. Physiological degeneration and disease onset are common occurrences in the aging population. iPSCs could offer regenerative medical options for age-related degeneration and disease in the elderly. However, reprogramming somatic cells from the elderly is inefficient when successful at all. Perhaps due to their low rates of replication in culture, traditional transduction and reprogramming approaches with centenarian fibroblasts met with little success. A simple and reproducible reprogramming process is reported here which enhances interactions of the cells with the viral vectors that leads to improved iPSC generation. The improved methods efficiently generates fully reprogrammed iPSC lines from 105-107 years old subjects in feeder-free conditions using an episomal, Sendai-Virus (SeV) reprogramming vector expressing four reprogramming factors. In conclusion, dermal fibroblasts from human subjects older than 100 years can be efficiently and reproducibly reprogrammed to fully pluripotent cells with minor modifications to the standard reprogramming procedures. Efficient generation of iPSCs from the elderly may provide a source of cells for the regeneration of tissues and organs with autologous cells as well as cellular models for the study of aging, longevity and age-related diseases

Genes differentially expressed in liver tissue of 14 days old TK2^+/+ and TK2^−/− mice identified by a PCR-focused array.

<p>Data are expressed as mean fold change in TK2^−/− samples relative to TK2^+/+ control samples. The data analysis, including statistical comparisons, were done using Qiagen’s software RT² Profiler PCR Array Data Analysis version 3.5. Genes of TK2^−/− mice (n = 3) that differed by >2-fold compared with TK2^+/+ controls (n = 3) are included and highlighted in bold when p≤0.05.</p

Mitochondrial ATP production rates standardized with citrate synthase activity.

<p>CS: citrate synthase; G: glutamate; M: malate; S: succinate; P: pyruvate; PC: palmitoyl-L-carnitine; R: rotenone. Mitochondrial CS activity was similar in the genotypes. For each time point data from 14 days old mice, three from each genotype, have been used (n = 3) and are presented as mean ± SEM. Statistical comparisons (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− were made but no statistical differences were detected.</p

Mitochondrial palmitate oxidation rate in liver homogenates of 14 days old TK2^+/+ and TK2^−/− mice.

<p>Three independent measurements were performed (TK2^+/+ n = 3, TK2^−/− n = 3) and comparisons between the genotypes were made within each measurement. Data presented as per cent activity (mean ± SEM) compared to TK2^+/+. The P-value for statistical comparison (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− is shown, *p≤0.05.</p

Serum levels of cholesterol and triglycerides in ApoB-containing, ApoA1-containing and total lipoprotein particles, as well as total serum nonesterified fatty acids (NEFA).

<p>For each time point data from at least four different mice have been used (n≥4) and are presented as mean ± SEM. The P-value for statistical comparison (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− of the same age group is shown, *p≤0.05, **p≤0.01.</p

Hepatic expression of adipophilin, voltage dependent anion channel (VDAC) and cytochrome oxidase subunit II (COXII).

<p>Protein expression in livers of A) 7 days old and B) 12 days old TK2^+/+ and TK2^−/− mice detected with Western blot. Protein samples are from three individuals of each genotype and age. Bio-RAD Quantity one software was used to determine the intensity of the bands and statistical comparisons (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− were done. VDAC was used to normalize the data. Only 12 days old TK2^−/− mice exhibit 150% higher expression of adipophilin compared to TK2^+/+ mice (p≤0.05), no other difference was detected.</p