Andras Franko

CREB-1α Is Recruited to and Mediates Upregulation of the Cytochrome c Promoter during Enhanced Mitochondrial Biogenesis Accompanying Skeletal Muscle Differentiation

ABSTRACT To further understand pathways coordinating the expression of nuclear genes encoding mitochondrial proteins, we studied mitochondrial biogenesis during differentiation of myoblasts to myotubes. This energy-demanding process was accompanied by a fivefold increase of ATP turnover, covered by an eightfold increase of mitochondrial activity. While no change in mitochondrial DNA copy number was observed, mRNAs as well as proteins for nucleus-encoded cytochrome c, cytochrome c oxidase subunit IV, and mitochondrial transcription factor A (TFAM) increased, together with total cellular RNA and protein levels. Detailed analysis of the cytochrome c promoter by luciferase reporter, binding affinity, and electrophoretic mobility shift assays as well as mutagenesis studies revealed a critical role for cyclic AMP responsive element binding protein 1 (CREB-1) for promoter activation. Expression of two CREB-1 isoforms was observed by using specific antibodies and quantitative reverse transcription-PCR, and a shift from phosphorylated CREB-1Δ in myoblasts to phosphorylated CREB-1α protein in myotubes was shown, while mRNA ratios remained unchanged. Chromatin immunoprecipitation assays confirmed preferential binding of CREB-1α in situ to the cytochrome c promoter in myotubes. Overexpression of constitutively active and dominant-negative forms supported the key role of CREB-1 in regulating the expression of genes encoding mitochondrial proteins during myogenesis and probably also in other situations of enhanced mitochondrial biogenesis.

α-Actinin 4 and BAT1 interaction with the Cytochrome c promoter upon skeletal muscle differentiation

To identify common regulatory features of nuclear genes encoding mitochondrial proteins we searched for regulatory elements in the Cytochrome c promoter during skeletal muscle differentiation in cell culture. A consensus element with the sequence GCTGCCGCAC-(N4-20)-GGSCGYGGG was found in both rat Cyt c and coxIV promoters. This new sequence element with yet undescribed function, but high abundance in promoters of nuclear genes encoding mitochondrial proteins available from the databases, showed a striking change in protein binding in electromobility shift assays when myoblasts were compared to myotubes. Proteins involved in the observed protein-DNA complexes were isolated from myotubes and identified by MALDI-TOF as BAT1, a DEAD-box protein of yet unknown function, heat shock protein HSP84, and α-actinin 4, a non-muscle isoform of the structural protein α-actinin. α-actinin 4 was found to be preferentially localized in the nucleus upon induction of myogenesis, suggesting a signaling function during muscle differentiation. In conclusion, the analyzed sequence motif may be a new candidate for common regulatory elements specific for nuclear encoded mitochondrial genes, and α-actinin 4 may be involved in their regulation.

Liver adapts mitochondrial function to insulin resistant and diabetic states in mice

BACKGROUND & AIMS To determine if diabetic and insulin-resistant states cause mitochondrial dysfunction in liver or if there is long term adaptation of mitochondrial function to these states, mice were (i) fed with a high-fat diet to induce obesity and T2D (HFD), (ii) had a genetic defect in insulin signaling causing whole body insulin resistance, but not full blown T2D (IR/IRS-1(+/-) mice), or (iii) were analyzed after treatment with streptozocin (STZ) to induce a T1D-like state. METHODS Hepatic lipid levels were measured by thin layer chromatography. Mitochondrial respiratory chain (RC) levels and function were determined by Western blot, spectrophotometric, oxygen consumption and proton motive force analysis. Gene expression was analyzed by real-time PCR and microarray. RESULTS HFD caused insulin resistance and hepatic lipid accumulation, but RC was largely unchanged. Livers from insulin resistant IR/IRS-1(+/-) mice had normal lipid contents and a normal RC, but mitochondria were less well coupled. Livers from severely hyperglycemic and hypoinsulinemic STZ mice had massively depleted lipid levels, but RC abundance was unchanged. However, liver mitochondria isolated from these animals showed increased abundance and activity of the RC, which was better coupled. CONCLUSIONS Insulin resistance, induced either by obesity or genetic manipulation and steatosis do not cause mitochondrial dysfunction in mouse liver. Also, mitochondrial dysfunction is not a prerequisite for liver steatosis. However, severe insulin deficiency and high blood glucose levels lead to an enhanced performance and better coupling of the RC. This may represent an adaptation to fuel overload and the high energy-requirement of an unsuppressed gluconeogenesis.

Complete failure of insulin-transmitted signaling, but not obesity-induced insulin resistance, impairs respiratory chain function in muscle

The role of mitochondrial dysfunction in the development of insulin resistance and type 2 diabetes remains controversial. In order to specifically define the relationship between insulin receptor (InsR) signaling, insulin resistance, hyperglycemia, hyperlipidemia and mitochondrial function, we analyzed mitochondrial performance of insulin-sensitive, slow-oxidative muscle in four different mouse models. In obese but normoglycemic ob/ob mice as well as in obese but diabetic mice under high-fat diet, mitochondrial performance remained unchanged even though intramyocellular diacylglycerols (DAGs), triacylglycerols (TAGs), and ceramides accumulated. In contrast, in muscle-specific InsR knockout (MIRKO) and streptozotocin (STZ)-treated hypoinsulinemic, hyperglycemic mice, levels of mitochondrial respiratory chain complexes and mitochondrial function were markedly reduced. In STZ, but not in MIRKO mice, this was caused by reduced transcription of mitochondrial genes mediated via decreased PGC-1α expression. We conclude that mitochondrial dysfunction is not causally involved in the pathogenesis of obesity-associated insulin resistance under normoglycemic conditions. However, obesity-associated type 2 diabetes and accumulation of DAGs or TAGs is not associated with impaired mitochondrial function. In contrast, chronic hypoinsulinemia and hyperglycemia as seen in STZ-treated mice as well as InsR deficiency in muscle of MIRKO mice lead to mitochondrial dysfunction. We postulate that decreased mitochondrial mass and/or performance in skeletal muscle of non-diabetic, obese or type 2 diabetic, obese patients observed in clinical studies must be explained by genetic predisposition, physical inactivity, or other still unknown factors.

Efficient Isolation of Pure and Functional Mitochondria from Mouse Tissues Using Automated Tissue Disruption and Enrichment with Anti-TOM22 Magnetic Beads

To better understand molecular mechanisms regulating changes in metabolism, as observed e.g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique.

The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.

Bezafibrate Improves Insulin Sensitivity and Metabolic Flexibility in STZ-Induced Diabetic Mice

Bezafibrate (BEZ), a pan activator of peroxisome proliferator–activated receptors (PPARs), has been generally used to treat hyperlipidemia for decades. Clinical trials with type 2 diabetes patients indicated that BEZ also has beneficial effects on glucose metabolism, although the underlying mechanisms of these effects remain elusive. Even less is known about a potential role for BEZ in treating type 1 diabetes. Here we show that BEZ markedly improves hyperglycemia and glucose and insulin tolerance in mice with streptozotocin (STZ)-induced diabetes, an insulin-deficient mouse model of type 1 diabetes. BEZ treatment of STZ mice significantly suppressed the hepatic expression of genes that are annotated in inflammatory processes, whereas the expression of PPAR and insulin target gene transcripts was increased. Furthermore, BEZ-treated mice also exhibited improved metabolic flexibility as well as an enhanced mitochondrial mass and function in the liver. Finally, we show that the number of pancreatic islets and the area of insulin-positive cells tended to be higher in BEZ-treated mice. Our data suggest that BEZ may improve impaired glucose metabolism by augmenting hepatic mitochondrial performance, suppressing hepatic inflammatory pathways, and improving insulin sensitivity and metabolic flexibility. Thus, BEZ treatment might also be useful for patients with impaired glucose tolerance or diabetes.

Bezafibrate ameliorates diabetes via reduced steatosis and improved hepatic insulin sensitivity in diabetic TallyHo mice

Objective Recently, we have shown that Bezafibrate (BEZ), the pan-PPAR (peroxisome proliferator-activated receptor) activator, ameliorated diabetes in insulin deficient streptozotocin treated diabetic mice. In order to study whether BEZ can also improve glucose metabolism in a mouse model for fatty liver and type 2 diabetes, the drug was applied to TallyHo mice. Methods TallyHo mice were divided into an early (ED) and late (LD) diabetes progression group and both groups were treated with 0.5% BEZ (BEZ group) or standard diet (SD group) for 8 weeks. We analyzed plasma parameters, pancreatic beta-cell morphology, and mass as well as glucose metabolism of the BEZ-treated and control mice. Furthermore, liver fat content and composition as well as hepatic gluconeogenesis and mitochondrial mass were determined. Results Plasma lipid and glucose levels were markedly reduced upon BEZ treatment, which was accompanied by elevated insulin sensitivity index as well as glucose tolerance, respectively. BEZ increased islet area in the pancreas. Furthermore, BEZ treatment improved energy expenditure and metabolic flexibility. In the liver, BEZ ameliorated steatosis, modified lipid composition and increased mitochondrial mass, which was accompanied by reduced hepatic gluconeogenesis. Conclusions Our data showed that BEZ ameliorates diabetes probably via reduced steatosis, enhanced hepatic mitochondrial mass, improved metabolic flexibility and elevated hepatic insulin sensitivity in TallyHo mice, suggesting that BEZ treatment could be beneficial for patients with NAFLD and impaired glucose metabolism.

Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application.

Mouse Genetics and Metabolic Mouse Phenotyping

The mouse is widely considered as a toolbox for modeling human diseases: mice are easy to handle and breed, there exist inbred strains, and the mouse genome sequence is available. Mutant mouse lines can be generated by different technologies, and standardized phenotyping of these mutant mouse lines produces a huge amount of valuable data. Useful resources for the scientific community are archives of mutant lines and strains as well as databases delivering information about the mouse lines and their availability. The phenotypic characterization of mutant mouse lines is the bottleneck within the pipeline from the generation via phenotyping to archiving of mutant mouse lines. Mouse clinics generate large data sets by the standardized, comprehensive phenotypic characterization of mutant mouse lines. There is a portfolio of phenotyping protocols available for a broad spectrum of disease areas that is considered as an international standard. For the investigation of human diseases like diabetes, obesity or the metabolic syndrome, metabolic tests to analyze mutant mouse lines become increasingly important. In this respect, challenge experiments have become the major focus to induce disease phenotypes in mutant mice that would remain undiscovered without the environmental challenges. These experimental setups reflect human conditions, where genetic predisposition and the environmental factors originating from different life style act together and enhance each other.