Ian R. Lanza

Endurance Exercise as a Countermeasure for Aging

OBJECTIVE— We determined whether reduced insulin sensitivity, mitochondrial dysfunction, and other age-related dysfunctions are inevitable consequences of aging or secondary to physical inactivity. RESEARCH DESIGN AND METHODS— Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and ATP production in mitochondria isolated from vastus lateralis biopsies of 42 healthy sedentary and endurance-trained young (18–30 years old) and older (59–76 years old) subjects. Expression of proteins involved in fuel metabolism was measured by mass spectrometry. Citrate synthase activity, mitochondrial DNA (mtDNA) abundance, and expression of nuclear-encoded transcription factors for mitochondrial biogenesis were measured. SIRT3, a mitochondrial sirtuin linked to lifespan-enhancing effects of caloric restriction, was measured by immunoblot. RESULTS— Insulin-induced glucose disposal and suppression of endogenous glucose production were higher in the trained young and older subjects, but no age effect was noted. Age-related decline in mitochondrial oxidative capacity was absent in endurance-trained individuals. Although endurance-trained individuals exhibited higher expression of mitochondrial proteins, mtDNA, and mitochondrial transcription factors, there were persisting effects of age. SIRT3 expression was lower with age in sedentary but equally elevated regardless of age in endurance-trained individuals. CONCLUSIONS— The results demonstrate that reduced insulin sensitivity is likely related to changes in adiposity and to physical inactivity rather than being an inevitable consequence of aging. The results also show that regular endurance exercise partly normalizes age-related mitochondrial dysfunction, although there are persisting effects of age on mtDNA abundance and expression of nuclear transcription factors and mitochondrial protein. Furthermore, exercise may promote longevity through pathways common to effects of caloric restriction.

A PGC-1α isoform induced by resistance training regulates skeletal muscle hypertrophy

PGC-1α is a transcriptional coactivator induced by exercise that gives muscle many of the best known adaptations to endurance-type exercise but has no effects on muscle strength or hypertrophy. We have identified a form of PGC-1α (PGC-1α4) that results from alternative promoter usage and splicing of the primary transcript. PGC-1α4 is highly expressed in exercised muscle but does not regulate most known PGC-1α targets such as the mitochondrial OXPHOS genes. Rather, it specifically induces IGF1 and represses myostatin, and expression of PGC-1α4 in vitro and in vivo induces robust skeletal muscle hypertrophy. Importantly, mice with skeletal muscle-specific transgenic expression of PGC-1α4 show increased muscle mass and strength and dramatic resistance to the muscle wasting of cancer cachexia. Expression of PGC-1α4 is preferentially induced in mouse and human muscle during resistance exercise. These studies identify a PGC-1α protein that regulates and coordinates factors involved in skeletal muscle hypertrophy.

Quantitative Metabolomics by 1H-NMR and LC-MS/MS Confirms Altered Metabolic Pathways in Diabetes

Insulin is as a major postprandial hormone with profound effects on carbohydrate, fat, and protein metabolism. In the absence of exogenous insulin, patients with type 1 diabetes exhibit a variety of metabolic abnormalities including hyperglycemia, glycosurea, accelerated ketogenesis, and muscle wasting due to increased proteolysis. We analyzed plasma from type 1 diabetic (T1D) humans during insulin treatment (I+) and acute insulin deprivation (I-) and non-diabetic participants (ND) by 1H nuclear magnetic resonance spectroscopy and liquid chromatography-tandem mass spectrometry. The aim was to determine if this combination of analytical methods could provide information on metabolic pathways known to be altered by insulin deficiency. Multivariate statistics differentiated proton spectra from I- and I+ based on several derived plasma metabolites that were elevated during insulin deprivation (lactate, acetate, allantoin, ketones). Mass spectrometry revealed significant perturbations in levels of plasma amino acids and amino acid metabolites during insulin deprivation. Further analysis of metabolite levels measured by the two analytical techniques indicates several known metabolic pathways that are perturbed in T1D (I-) (protein synthesis and breakdown, gluconeogenesis, ketogenesis, amino acid oxidation, mitochondrial bioenergetics, and oxidative stress). This work demonstrates the promise of combining multiple analytical methods with advanced statistical methods in quantitative metabolomics research, which we have applied to the clinical situation of acute insulin deprivation in T1D to reflect the numerous metabolic pathways known to be affected by insulin deficiency.

Functional Assessment of Isolated Mitochondria In Vitro

Mitochondria play a pivotal role in cellular function, not only as a major site of ATP production, but also by regulating energy expenditure, apoptosis signaling, and production of reactive oxygen species. Altered mitochondrial function is reported to be a key underlying mechanism of many pathological states and in the aging process. Functional measurements of intact mitochondria isolated from fresh tissue provides distinct information regarding the function of these organelles that complements conventional mitochondrial assays using previously frozen tissue as well as in vivo assessment using techniques such as magnetic resonance and near-infrared spectroscopy. This chapter describes the process by which mitochondria are isolated from small amounts of human skeletal muscle obtained by needle biopsy and two approaches used to assess mitochondrial oxidative capacity and other key components of mitochondrial physiology. We first describe a bioluminescent approach for measuring the rates of mitochondrial ATP production. Firefly luciferase catalyzes a light-emitting reaction whereby the substrate luciferin is oxidized in an ATP-dependent manner. A luminometer is used to quantify the light signal, which is proportional to ATP concentration. We also review a method involving polarographic measurement of oxygen consumption. Measurements of oxygen consumption, which previously required large amounts of tissue, are now feasible with very small amounts of sample obtained by needle biopsy due to recent advances in the field of high-resolution respirometry. We illustrate how careful attention to substrate combinations and inhibitors allows an abundance of unique functional information to be obtained from isolated mitochondria, including function at various energetic states, oxidative capacity with electron flow through distinct complexes, coupling of oxygen consumption to ATP production, and membrane integrity. These measurements, together with studies of mitochondrial DNA abundance, mRNA levels, protein expression, and synthesis rates of mitochondrial proteins provide insightful mechanistic information about mitochondria in a variety of tissue types.

Mitochondrial function as a determinant of life span

Average human life expectancy has progressively increased over many decades largely due to improvements in nutrition, vaccination, antimicrobial agents, and effective treatment/prevention of cardiovascular disease, cancer, etc. Maximal life span, in contrast, has changed very little. Caloric restriction (CR) increases maximal life span in many species, in concert with improvements in mitochondrial function. These effects have yet to be demonstrated in humans, and the duration and level of CR required to extend life span in animals is not realistic in humans. Physical activity (voluntary exercise) continues to hold much promise for increasing healthy life expectancy in humans, but remains to show any impact to increase maximal life span. However, longevity in Caenorhabditis elegans is related to activity levels, possibly through maintenance of mitochondrial function throughout the life span. In humans, we reported a progressive decline in muscle mitochondrial DNA abundance and protein synthesis with age. Other investigators also noted age-related declines in muscle mitochondrial function, which are related to peak oxygen uptake. Long-term aerobic exercise largely prevented age-related declines in mitochondrial DNA abundance and function in humans and may increase spontaneous activity levels in mice. Notwithstanding, the impact of aerobic exercise and activity levels on maximal life span is uncertain. It is proposed that age-related declines in mitochondrial content and function not only affect physical function, but also play a major role in regulation of life span. Regular aerobic exercise and prevention of adiposity by healthy diet may increase healthy life expectancy and prolong life span through beneficial effects at the level of the mitochondrion.

Muscle mitochondrial changes with aging and exercise

Aging has been reported to be accompanied by reduced mitochondrial function and insulin sensitivity. Whether these deleterious effects result from chronological age or lifestyle-related factors such as adiposity and physical inactivity remains debatable. The beneficial effects of exercise on mitochondrial function and insulin sensitivity are well documented; however, it is unclear whether exercise can effectively prevent, reverse, or delay the onset of these age-related dysfunctions. Other investigators and we have found that endurance exercise enhances mitochondrial function across the life span, highlighting the beneficial role of exercise in combating age-related mitochondrial dysfunction. The literature is mixed concerning the ability of endurance exercise to normalize age-related insulin resistance; however, emerging evidence points to adiposity rather than age per se as a primary determinant of age-related declines in insulin sensitivity. Recent data from our laboratory also shed some light on the controversial relation between mitochondrial function and insulin sensitivity. Although some investigators purport a causal role of mitochondrial dysfunction in the etiology of insulin resistance, we provide evidence that the reverse may be true based on the fact that insulin stimulates mitochondrial function in healthy control subjects but not in patients with type 2 diabetes. Furthermore, we find that these 2 variables are completely dissociated in some populations, such as Asian Indians, who exhibit elevated mitochondrial capacity despite marked insulin resistance compared with European Americans. Our data not only point to regular endurance exercise as a viable strategy to delay the onset of age-related dysfunctions but they suggest that mitochondrial function and insulin resistance may be linked by additional factors such as physical activity.

Effects of old age on human skeletal muscle energetics during fatiguing contractions with and without blood flow

We recently reported lower glycolytic flux (ATPGLY) and increased reliance on oxidative ATP synthesis (ATPOX) in contracting muscle of older compared to young humans. To further investigate this age‐related difference in the pathways of ATP synthesis, we used magnetic resonance spectroscopy to determine the rates of ATPOX, ATPGLY and net phosphocreatine hydrolysis in vivo during maximal muscle contractions under free‐flow (FF) and ischaemic (ISC) conditions in the ankle dorsiflexors of 20 young (27 ± 3 years; 10 male, 10 female) and 18 older (70 ± 5 years; 10 male, 8 female) adults. We hypothesized that ATPGLY would be higher in young compared to old during FF contractions, but that old would be unable to increase ATPGLY during ISC to match that of the young, which would suggest impaired glycolytic ATP synthesis with old age. Peak glycolytic flux during FF was lower in older (0.8 ± 0.1 mm ATP s−1) compared to young (1.4 ± 0.1 mm ATP s−1, P < 0.001) subjects. During ISC, peak ATPGLY increased in old to a level similar to that of young (1.4 ± 0.2 mm ATP s−1, 1.3 ± 0.2 mm ATP s−1, respectively; P= 0.86), suggesting that glycolytic function remains intact in aged muscle in vivo. Notably, older adults fatigued less than young during both FF and ISC (P≤ 0.004). These results provide novel evidence of unimpaired in vivo glycolytic function in the skeletal muscle of older adults during maximal isometric dorsiflexion, and suggest a potential role for differences in metabolic economy and as a result, metabolite accumulation, in the fatigue resistance of the old.

Methods for Assessing Mitochondrial Function in Diabetes

A growing body of research is investigating the potential contribution of mitochondrial function to the etiology of type 2 diabetes. Numerous in vitro, in situ, and in vivo methodologies are available to examine various aspects of mitochondrial function, each requiring an understanding of their principles, advantages, and limitations. This review provides investigators with a critical overview of the strengths, limitations and critical experimental parameters to consider when selecting and conducting studies on mitochondrial function. In vitro (isolated mitochondria) and in situ (permeabilized cells/tissue) approaches provide direct access to the mitochondria, allowing for study of mitochondrial bioenergetics and redox function under defined substrate conditions. Several experimental parameters must be tightly controlled, including assay media, temperature, oxygen concentration, and in the case of permeabilized skeletal muscle, the contractile state of the fibers. Recently developed technology now offers the opportunity to measure oxygen consumption in intact cultured cells. Magnetic resonance spectroscopy provides the most direct way of assessing mitochondrial function in vivo with interpretations based on specific modeling approaches. The continuing rapid evolution of these technologies offers new and exciting opportunities for deciphering the potential role of mitochondrial function in the etiology and treatment of diabetes.

Cystathionine Beta-Synthase (CBS) Contributes to Advanced Ovarian Cancer Progression and Drug Resistance

Background Epithelial ovarian cancer is the leading cause of gynecologic cancer deaths. Most patients respond initially to platinum-based chemotherapy after surgical debulking, however relapse is very common and ultimately platinum resistance emerges. Understanding the mechanism of tumor growth, metastasis and drug resistant relapse will profoundly impact the therapeutic management of ovarian cancer. Methods/Principal Findings Using patient tissue microarray (TMA), in vitro and in vivo studies we report a role of of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme in ovarian carcinoma. We report here that the expression of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme, is common in primary serous ovarian carcinoma. The in vitro effects of CBS silencing can be reversed by exogenous supplementation with the GSH and H2S producing chemical Na2S. Silencing CBS in a cisplatin resistant orthotopic model in vivo by nanoliposomal delivery of CBS siRNA inhibits tumor growth, reduces nodule formation and sensitizes ovarian cancer cells to cisplatin. The effects were further corroborated by immunohistochemistry that demonstrates a reduction of H&E, Ki-67 and CD31 positive cells in si-RNA treated as compared to scrambled-RNA treated animals. Furthermore, CBS also regulates bioenergetics of ovarian cancer cells by regulating mitochondrial ROS production, oxygen consumption and ATP generation. This study reports an important role of CBS in promoting ovarian tumor growth and maintaining drug resistant phenotype by controlling cellular redox behavior and regulating mitochondrial bioenergetics. Conclusion The present investigation highlights CBS as a potential therapeutic target in relapsed and platinum resistant ovarian cancer.

Regulation of skeletal muscle mitochondrial function: genes to proteins

The impact of ageing on mitochondrial function and the deterministic role of mitochondria on senescence continue to be topics of vigorous debate. Many studies report that skeletal muscle mitochondrial content and function are reduced with ageing and metabolic diseases associated with insulin resistance. However, an accumulating body of literature suggests that physical inactivity typical of ageing may be a more important determinant of mitochondrial function than chronological age, per se. Reports of age‐related declines in mitochondrial function have spawned a vast body of literature devoted to understanding the underlying mechanisms. These mechanisms include decreased abundance of mtDNA, reduced mRNA levels, as well as decreased synthesis and expression of mitochondrial proteins, ultimately resulting in decreased function of the whole organelle. Effective therapies to prevent, reverse or delay the onset of the aforementioned mitochondrial changes, regardless of their inevitability or precise underlying causes, require an intimate understanding of the processes that regulate mitochondrial biogenesis, which necessitates the coordinated regulation of nuclear and mitochondrial genomes. Herein we review the current thinking on regulation of mitochondrial biogenesis by transcription factors and transcriptional co‐activators and the role of hormones and exercise in initiating this process. We review how exercise may help preserve mitochondrial content and functionality across the lifespan, and how physical inactivity is emerging as a major determinant of many age‐associated changes at the level of the mitochondrion. We also review evidence that some mitochondrial changes with ageing are independent of exercise or physical activity and appear to be inevitable consequences of old age.