Mechanisms underlying mitochondrial function and biogenesis: Implications for type 2 diabetes mellitus and obesity

The ample supply of food, in conjunction with a sedentary lifestyle and certain genetic risk factors contribute to the rise in obesity, insulin resistance and type 2 diabetes mellitus. Reduced mitochondrial capacity for oxidative metabolism has been implicated as one possible cause of insulin resistance in several tissues; such as liver and skeletal muscle. The adipose-derived hormone leptin and the metabolic sensor 5’-AMP-activated protein kinase, are two key regulators that modulate intracellular fuel handling. The aim of this thesis is to investigate the effects of these metabolic signals on tissue-specific mitochondrial respiration and biogenesis. The aim of study I was to investigate the role of the AMPK γ3 subunit in determining mitochondrial function in glycolytic skeletal muscle. The AMPK signaling axis is a metabolic switch regulated by the intracellular energy charge. A single-nucleotide mutation (R225Q) in the AMPKγ3 subunit causes elevated basal enzyme activity. Transgenic expression in mice (Tg-AMPKγ3R225Q) increased expression of regulators and mediators of substrate oxidation, as well as components of mitochondrial dynamics and electron transport. In summary, this single nucleotide mutation is associated with mitochondrial biogenesis, concomitant with increased expression of transcription factors that regulate mitochondrial proteins. The focus of study II was to characterize tissue-specific mitochondrial function in permeabilized tissue from lean and leptin receptor-deficient obese db/db mice. Respiratory capacity in oxidative soleus muscle was similar between genotypes, except for decreased complex II function in db/db mice. Oxidative function in glycolytic EDL muscle was higher in db/db mice than in lean littermates; likely as a result of increased mitochondrial biogenesis. Maximal respiratory capacity in liver from db/db mice was blunted, concomitant with increased mitochondrial fission. In summary, mitochondrial respiratory performance is controlled by tissue-specific mechanisms and is not uniformly altered in obesity. The aim of study III was to determine tissue-specific mitochondrial respiration in obese leptin-deficient ob/ob mice, and lean littermates, following treatment with leptin or saline. Oxidative capacity in soleus muscle was unaffected in saline- and leptin-treated ob/ob mice, whereas maximal electron transport capacity was increased with obesity in EDL muscle. Regulation of transcription and mitochondrial fission in EDL was altered in saline-treated ob/ob mice, and only partially normalized with leptin repletion. In liver, maximal respiratory capacity and mediators of lipid oxidation were reduced with in saline- and leptin-treated ob/ob mice; while leptin treatment normalized indicators of mitochondrial stress. In conclusion, mitochondrial respiratory function is a dynamic process that is tightly regulated to meet the energy needs of the cell. Despite profound alterations in whole-body or intracellular energy sensing, mitochondrial adaptation can occur and respiratory adaptations are comparatively modest. This highlights the need to target several pathways of metabolic regulation to modulate mitochondrial function to improve systemic homeostasis

Gain-of-function R225Q mutation in AMP-activated protein kinase gamma3 subunit increases mitochondrial biogenesis in glycolytic skeletal muscle

AMP-activated protein kinase (AMPK) is a heterotrimeric complex, composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma), that works as a cellular energy sensor. The existence of multiple heterotrimeric complexes provides a molecular basis for the multiple roles of this highly conserved signaling system. The AMPK gamma3 subunit is predominantly expressed in skeletal muscle, mostly in type II glycolytic fiber types. We determined whether the AMPK gamma3 subunit has a role in signaling pathways that mediate mitochondrial biogenesis in skeletal muscle. We provide evidence that overexpression or ablation of the AMPK gamma3 subunit does not appear to play a critical role in defining mitochondrial content in resting skeletal muscle. However, overexpression of a mutant form (R225Q) of the AMPK gamma3 subunit (Tg-AMPKgamma3(225Q)) increases mitochondrial biogenesis in glycolytic skeletal muscle. These adaptations are associated with an increase in expression of the co-activator PGC-1alpha and several transcription factors that regulate mitochondrial biogenesis, including NRF-1, NRF-2, and TFAM. Succinate dehydrogenase staining, a marker of the oxidative profile of individual fibers, was also increased in transversal skeletal muscle sections of white gastrocnemius muscle from Tg-AMPKgamma3(225Q) mice, independent of changes in fiber type composition. In conclusion, a single nucleotide mutation (R225Q) in the AMPK gamma3 subunit is associated with mitochondrial biogenesis in glycolytic skeletal muscle, concomitant with increased expression of the co-activator PGC-1alpha and several transcription factors that regulate mitochondrial proteins, without altering fiber type composition

Effects of AMPK activation on insulin sensitivity and metabolism in leptin-deficient ob/ob mice

AMP-activated protein kinase (AMPK) is a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), which act as a metabolic sensor to regulate glucose and lipid metabolism. A mutation in the γ3 subunit (AMPKγ3(R225Q)) increases basal AMPK phosphorylation, while concomitantly reducing sensitivity to AMP. AMPKγ3(R225Q) (γ3(R225Q)) transgenic mice are protected against dietary-induced triglyceride accumulation and insulin resistance. We determined whether skeletal muscle-specific expression of AMPKγ3(R225Q) prevents metabolic abnormalities in leptin-deficient ob/ob (ob/ob-γ3(R225Q)) mice. Glycogen content was increased, triglyceride content was decreased, and diacylglycerol and ceramide content were unaltered in gastrocnemius muscle from ob/ob-γ3(R225Q) mice, whereas glucose tolerance was unaltered. Insulin-stimulated glucose uptake in extensor digitorum longus muscle during the euglycemic-hyperinsulinemic clamp was increased in lean γ3(R225Q) mice, but not in ob/ob-γ3(R225Q) mice. Acetyl-CoA carboxylase phosphorylation was increased in gastrocnemius muscle from γ3(R225Q) mutant mice independent of adiposity. Glycogen and triglyceride content were decreased after leptin treatment (5 days) in ob/ob mice, but not in ob/ob-γ3(R225Q) mice. In conclusion, metabolic improvements arising from muscle-specific expression of AMPKγ3(R225Q) are insufficient to ameliorate insulin resistance and obesity in leptin-deficient mice. Central defects due to leptin deficiency may override any metabolic benefit conferred by peripheral overexpression of the AMPKγ3(R225Q) mutation

Quantitative Analysis of BTF3, HINT1, NDRG1 and ODC1 Protein Over-Expression in Human Prostate Cancer Tissue

Prostate carcinoma is the most common cancer in men with few, quantifiable, biomarkers. Prostate cancer biomarker discovery has been hampered due to subjective analysis of protein expression in tissue sections. An unbiased, quantitative immunohistochemical approach provided here, for the diagnosis and stratification of prostate cancer could overcome this problem. Antibodies against four proteins BTF3, HINT1, NDRG1 and ODC1 were used in a prostate tissue array (> 500 individual tissue cores from 82 patients, 41 case pairs matched with one patient in each pair had biochemical recurrence). Protein expression, quantified in an unbiased manner using an automated analysis protocol in ImageJ software, was increased in malignant vs non-malignant prostate (by 2-2.5 fold, p<0.0001). Operating characteristics indicate sensitivity in the range of 0.68 to 0.74; combination of markers in a logistic regression model demonstrates further improvement in diagnostic power. Triple-labeled immunofluorescence (BTF3, HINT1 and NDRG1) in tissue array showed a significant (p<0.02) change in co-localization coefficients for BTF3 and NDRG1 co-expression in biochemical relapse vs non-relapse cancer epithelium. BTF3, HINT1, NDRG1 and ODC1 could be developed as epithelial specific biomarkers for tissue based diagnosis and stratification of prostate cancer

Effects of AMPK activation on insulin sensitivity and metabolism in leptin-deficient ob/ob mice

AMP-activated protein kinase (AMPK) is a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), which act as a metabolic sensor to regulate glucose and lipid metabolism. A mutation in the γ3 subunit (AMPKγ3(R225Q)) increases basal AMPK phosphorylation, while concomitantly reducing sensitivity to AMP. AMPKγ3(R225Q) (γ3(R225Q)) transgenic mice are protected against dietary-induced triglyceride accumulation and insulin resistance. We determined whether skeletal muscle-specific expression of AMPKγ3(R225Q) prevents metabolic abnormalities in leptin-deficient ob/ob (ob/ob-γ3(R225Q)) mice. Glycogen content was increased, triglyceride content was decreased, and diacylglycerol and ceramide content were unaltered in gastrocnemius muscle from ob/ob-γ3(R225Q) mice, whereas glucose tolerance was unaltered. Insulin-stimulated glucose uptake in extensor digitorum longus muscle during the euglycemic-hyperinsulinemic clamp was increased in lean γ3(R225Q) mice, but not in ob/ob-γ3(R225Q) mice. Acetyl-CoA carboxylase phosphorylation was increased in gastrocnemius muscle from γ3(R225Q) mutant mice independent of adiposity. Glycogen and triglyceride content were decreased after leptin treatment (5 days) in ob/ob mice, but not in ob/ob-γ3(R225Q) mice. In conclusion, metabolic improvements arising from muscle-specific expression of AMPKγ3(R225Q) are insufficient to ameliorate insulin resistance and obesity in leptin-deficient mice. Central defects due to leptin deficiency may override any metabolic benefit conferred by peripheral overexpression of the AMPKγ3(R225Q) mutation

Case Report: First longitudinal study of a patient with CALR positive clonal hematopoiesis of indeterminate potential developing into pre-fibrotic myelofibrosis

Initial diagnosis of overt myeloproliferative neoplasms (MPNs) represents the juncture during clonal evolution when symptoms or complications prompt an afflicted individual to seek medical attention. In 30-40% of the MPN subgroups essential thrombocythemia (ET) and myelofibrosis (MF), somatic mutations in the calreticulin gene (CALR) are drivers of the disease resulting in constitutive activation of the thrombopoietin receptor (MPL). In the current study, we describe a healthy CALR mutated individual during a 12 year follow-up from initial identification of CALR clonal hematopoiesis of indeterminate potential (CHIP) to the diagnosis of pre-MF. The pre-diagnostic exponential development dynamics of the malignant clone demonstrated close correlation with the platelet counts, neutrophil-to-lymphocyte (NLR) ratio, and inversely correlated to hemoglobin and erythrocyte counts. Backward extrapolation of the growth rate indicated the potential for discovery of the malignant clone many years prior to presentation of overt disease, opening a window of opportunity for early treatment intervention. We did not find any additional mutations associated with MPNs and the current case report provides novel information regarding the development of a driver mutation and the association with blood cell counts prior to clinical manifestation of symptoms suggesting that pre-diagnostic dynamics may supplement future diagnostic criteria for early diagnosis and intervention in MPN patients