Chad A. Galloway

Mitochondrial Dynamics in Diabetes

Mitochondria are at the center of cellular energy metabolism and regulate cell life and death. The cell biological aspect of mitochondria, especially mitochondrial dynamics, has drawn much attention through implications in human pathology, including neurological disorders and metabolic diseases. Mitochondrial fission and fusion are the main processes governing the morphological plasticity and are controlled by multiple factors, including mechanochemical enzymes and accessory proteins. Emerging evidence suggests that mitochondrial dynamics plays an important role in metabolism-secretion coupling in pancreatic β-cells as well as complications of diabetes. This review describes an overview of mechanistic and functional aspects of mitochondrial fission and fusion, and comments on the recent advances connecting mitochondrial dynamics with diabetes and diabetic complications.

Control of Mitochondrial Morphology Through Differential Interactions of Mitochondrial Fusion and Fission Proteins

Mitochondria in mammals are organized into tubular networks that undergo frequent shape change. Mitochondrial fission and fusion are the main components mediating the mitochondrial shape change. Perturbation of the fission/fusion balance is associated with many disease conditions. However, underlying mechanisms of the fission/fusion balance are not well understood. Mitochondrial fission in mammals requires the dynamin-like protein DLP1/Drp1 that is recruited to the mitochondrial surface, possibly through the membrane-anchored protein Fis1 or Mff. Additional dynamin-related GTPases, mitofusin (Mfn) and OPA1, are associated with the outer and inner mitochondrial membranes, respectively, and mediate fusion of the respective membranes. In this study, we found that two heptad-repeat regions (HR1 and HR2) of Mfn2 interact with each other, and that Mfn2 also interacts with the fission protein DLP1. The association of the two heptad-repeats of Mfn2 is fusion inhibitory whereas a positive role of the Mfn2/DLP1 interaction in mitochondrial fusion is suggested. Our results imply that the differential binding of Mfn2-HR1 to HR2 and DLP1 regulates mitochondrial fusion and that DLP1 may act as a regulatory factor for efficient execution of both fusion and fission of mitochondria.

Mitochondrial Morphology in Metabolic Diseases

SIGNIFICANCE Mitochondria are the cellular energy-producing organelles and are at the crossroad of determining cell life and death. As such, the function of mitochondria has been intensely studied in metabolic disorders, including diabetes and associated maladies commonly grouped under all-inclusive pathological condition of metabolic syndrome. More recently, the altered metabolic profiles and function of mitochondria in these ailments have been correlated with their aberrant morphologies. This review describes an overview of mitochondrial fission and fusion machineries, and discusses implications of mitochondrial morphology and function in these metabolic maladies. RECENT ADVANCES Mitochondria undergo frequent morphological changes, altering the mitochondrial network organization in response to environmental cues, termed mitochondrial dynamics. Mitochondrial fission and fusion mediate morphological plasticity of mitochondria and are controlled by membrane-remodeling mechanochemical enzymes and accessory proteins. Growing evidence suggests that mitochondrial dynamics play an important role in diabetes establishment and progression as well as associated ailments, including, but not limited to, metabolism-secretion coupling in the pancreas, nonalcoholic fatty liver disease progression, and diabetic cardiomyopathy. CRITICAL ISSUES While mitochondrial dynamics are intimately associated with mitochondrial bioenergetics, their cause-and-effect correlation remains undefined in metabolic diseases. FUTURE DIRECTIONS The involvement of mitochondrial dynamics in metabolic diseases is in its relatively early stages. Elucidating the role of mitochondrial dynamics in pathological metabolic conditions will aid in defining the intricate form-function correlation of mitochondria in metabolic pathologies and should provide not only important clues to metabolic disease progression, but also new therapeutic targets.

Hepatic very-low-density lipoprotein and apolipoprotein B production are increased following in vivo induction of betaine–homocysteine S-methyltransferase

We have previously reported a positive correlation between the expression of BHMT (betaine-homocysteine S-methyltransferase) and ApoB (apolipoprotein B) in rat hepatoma McA (McArdle RH-7777) cells [Sowden, Collins, Smith, Garrow, Sparks and Sparks (1999) Biochem. J. 341, 639-645]. To examine whether a similar relationship occurs in vivo, hepatic BHMT expression was induced by feeding rats a Met (L-methionine)-restricted betaine-containing diet, and parameters of ApoB metabolism were evaluated. There were no generalized metabolic abnormalities associated with Met restriction for 7 days, as evidenced by control levels of serum glucose, ketones, alanine aminotransferase and L-homocysteine levels. Betaine plus the Met restriction resulted in lower serum insulin and non-esterified fatty acid levels. Betaine plus Met restriction induced hepatic BHMT 4-fold and ApoB mRNA 3-fold compared with Met restriction alone. No changes in percentage of edited ApoB mRNA were observed on the test diets. An increase in liver ApoB mRNA correlated with an 82% and 46% increase in ApoB and triacylglycerol production respectively using in vivo Triton WR 1339. Increased secretion of VLDL (very-low-density lipoprotein) with Met restriction plus betaine was associated with a 45% reduction in liver triacylglycerol compared with control. Nuclear run-off assays established that transcription of both bhmt and apob genes was also increased in Met-restricted plus betaine diets. No change in ApoB mRNA stability was detected in BHMT-transfected McA cells. Hepatic ApoB and BHMT mRNA levels were also increased by 1.8- and 3-fold respectively by betaine supplementation of Met-replete diets. Since dietary betaine increased ApoB mRNA, VLDL ApoB and triacylglycerol production and decreased hepatic triacylglycerol, results suggest that induction of apob transcription may provide a potential mechanism for mobilizing hepatic triacylglycerol by increasing ApoB available for VLDL assembly and secretion.

Mitochondrial morphology-emerging role in bioenergetics.

Dynamic change in mitochondrial shape is a cellular process mediated mainly by fission and fusion of mitochondria. Studies have shown that mitochondrial fission and fusion are directly and indirectly associated with mitochondrial maintenance, bioenergetic demand, and cell death. Changes in mitochondrial morphology are frequently observed in response to changes in the surrounding cellular milieu, such as metabolic flux, that influence cellular bioenergetics. Connections between morphological regulation and the bioenergetic status of mitochondria are emerging as reciprocally responsive processes, though the nature of the signaling remains to be defined. Given the pivotal role mitochondria play in cellular fate, tight regulation of fission and fusion is therefore critical to preserving normal cellular physiology. Here we describe recent advancements in the understanding of the mechanisms governing mitochondrial morphology and their emerging role in mitochondrial bioenergetics.

Transgenic Control of Mitochondrial Fission Induces Mitochondrial Uncoupling and Relieves Diabetic Oxidative Stress

Mitochondria are the essential eukaryotic organelles that produce most cellular energy. The energy production and supply by mitochondria appear closely associated with the continuous shape change of mitochondria mediated by fission and fusion, as evidenced not only by the hereditary diseases caused by mutations in fission/fusion genes but also by aberrant mitochondrial morphologies associated with numerous pathologic insults. However, how morphological change of mitochondria is linked to their energy-producing activity is poorly understood. In this study, we found that perturbation of mitochondrial fission induces a unique mitochondrial uncoupling phenomenon through a large-scale fluctuation of a mitochondrial inner membrane potential. Furthermore, by genetically controlling mitochondrial fission and thereby inducing mild proton leak in mice, we were able to relieve these mice from oxidative stress in a hyperglycemic model. These findings provide mechanistic insight into how mitochondrial fission participates in regulating mitochondrial activity. In addition, these results suggest a potential application of mitochondrial fission to control mitochondrial reactive oxygen species production and oxidative stress in many human diseases.

Perspectives on: SGP symposium on mitochondrial physiology and medicine: what comes first, misshape or dysfunction? The view from metabolic excess.

In typical cultured animal cells, mitochondria exist in shapes ranging from long interconnected tubules, often clustered in the perinuclear region of the cell, to small spherical forms in the cell periphery. However, mitochondrial appearance at any given moment in time is only a snapshot of dynamic

Decreasing mitochondrial fission alleviates hepatic steatosis in a murine model of nonalcoholic fatty liver disease

Mitochondria produce the majority of cellular ATP through oxidative phosphorylation, and their capacity to do so is influenced by many factors. Mitochondrial morphology is recently suggested as an important contributor in controlling mitochondrial bioenergetics. Mitochondria divide and fuse continuously, which is affected by environmental factors, including metabolic alterations. Underscoring its bioenergetic influence, altered mitochondrial morphology is reported in tissues of patients and in animal models of metabolic dysfunction. In this study, we found that mitochondrial fission plays a vital role in the progression of nonalcoholic fatty liver disease (NAFLD). The development of hepatic steatosis, oxidative/nitrative stress, and hepatic tissue damage, induced by a high-fat diet, were alleviated in genetically manipulated mice suppressing mitochondrial fission. The alleviation of steatosis was recapitulated in primary hepatocytes with the inhibition of mitochondrial fission. Mechanistically, our study indicates that fission inhibition enhances proton leak under conditions of free fatty acid incubation, implicating bioenergetic change through manipulating mitochondrial fission. Taken together, our results suggest a mechanistic role for mitochondrial fission in the etiology of NAFLD. The efficacy of decreasing mitochondrial fission in the suppression of NAFLD suggests that mitochondrial fission represents a novel target for therapeutic treatment of NAFLD.

Metabolic regulation of ApoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor

Apolipoprotein B (apoB) mRNA editing is a nuclear event that minimally requires the RNA substrate, APOBEC-1 and APOBEC-1 Complementation Factor (ACF). The co-localization of these macro-molecules within the nucleus and the modulation of hepatic apoB mRNA editing activity have been described following a variety of metabolic perturbations, but the mechanism that regulates editosome assembly is unknown. APOBEC-1 was effectively co-immunoprecipitated with ACF from nuclear, but not cytoplasmic extracts. Moreover, alkaline phosphatase treatment of nuclear extracts reduced the amount of APOBEC-1 co-immunoprecipitated with ACF and inhibited in vitro editing activity. Ethanol stimulated apoB mRNA editing was associated with a 2- to 3-fold increase in ACF phosphorylation relative to that in control primary hepatocytes. Significantly, phosphorylated ACF was restricted to nuclear extracts where it co-sedimented with 27S editing competent complexes. Two-dimensional phosphoamino acid analysis of ACF immunopurified from hepatocyte nuclear extracts demonstrated phosphorylation of serine residues that was increased by ethanol treatment. Inhibition of protein phosphatase I, but not PPIIA or IIB, stimulated apoB mRNA editing activity coincident with enhanced ACF phosphorylation in vivo. These data demonstrate that ACF is a metabolically regulated phosphoprotein and suggest that this post-translational modification increases hepatic apoB mRNA editing activity by enhancing ACF nuclear localization/retention, facilitating the interaction of ACF with APOBEC-1 and thereby increasing the probability of editosome assembly and activity.

Mitochondrial dynamics in diabetic cardiomyopathy.

SIGNIFICANCE Cardiac function is energetically demanding, reliant on efficient well-coupled mitochondria to generate adenosine triphosphate and fulfill the cardiac demand. Predictably then, mitochondrial dysfunction is associated with cardiac pathologies, often related to metabolic disease, most commonly diabetes. Diabetic cardiomyopathy (DCM), characterized by decreased left ventricular function, arises independently of coronary artery disease and atherosclerosis. Dysregulation of Ca(2+) handling, metabolic changes, and oxidative stress are observed in DCM, abnormalities reflected in alterations in mitochondrial energetics. Cardiac tissue from DCM patients also presents with altered mitochondrial morphology, suggesting a possible role of mitochondrial dynamics in its pathological progression. RECENT ADVANCES Abnormal mitochondrial morphology is associated with pathologies across diverse tissues, suggesting that this highly regulated process is essential for proper cell maintenance and physiological homeostasis. Highly structured cardiac myofibers were hypothesized to limit alterations in mitochondrial morphology; however, recent work has identified morphological changes in cardiac tissue, specifically in DCM. CRITICAL ISSUES Mitochondrial dysfunction has been reported independently from observations of altered mitochondrial morphology in DCM. The temporal relationship and causative nature between functional and morphological changes of mitochondria in the establishment/progression of DCM is unclear. FUTURE DIRECTIONS Altered mitochondrial energetics and morphology are not only causal for but also consequential to reactive oxygen species production, hence exacerbating oxidative damage through reciprocal amplification, which is integral to the progression of DCM. Therefore, targeting mitochondria for DCM will require better mechanistic characterization of morphological distortion and bioenergetic dysfunction.