Daniel A. Kane

Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H(2)O(2)-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H(2)O(2) emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H(2)O(2) emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity.

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.

Mitochondrial creatine kinase activity and phosphate shuttling are acutely regulated by exercise in human skeletal muscle.

•  ATP transfer from mitochondria to the cytoplasm occurs mainly through phosphate transfer to creatine by mitochondrial creatine kinase (miCK) but also by transport and/or diffusion of ADP and ATP through specific mitochondrial transport protein complexes. •  Determining the effect of exercise on phosphate shuttling may require contractile signals in situ and varying creatine concentrations to alter miCK activity. •  Mitochondrial respiratory sensitivity to ADP was assessed in permeabilized muscle fibre bundles (PmFBs) before and after 2 h cycling exercise in human skeletal muscle. •  In relaxed PmFBs, ADP sensitivity decreased post‐exercise when miCK phosphate shuttling was low (no creatine) with no change in net ADP sensitivity in the presence of creatine, whereas in contracting fibres post‐exercise ADP sensitivity was higher with creatine. •  This shows miCK activity is increased post‐exercise, especially during contraction in PmFBs, and suggests exercise regulates phosphate shuttling, which would improve maintenance of energy homeostasis during contraction.

Mitochondrial fission mediates ceramide-induced metabolic disruption in skeletal muscle.

Ceramide is a sphingolipid that serves as an important second messenger in an increasing number of stress-induced pathways. Ceramide has long been known to affect the mitochondria, altering both morphology and physiology. We sought to assess the impact of ceramide on skeletal muscle mitochondrial structure and function. A primary observation was the rapid and dramatic division of mitochondria in ceramide-treated cells. This effect is likely to be a result of increased Drp1 (dynamin-related protein 1) action, as ceramide increased Drp1 expression and Drp1 inhibition prevented ceramide-induced mitochondrial fission. Further, we found that ceramide treatment reduced mitochondrial O2 consumption (i.e. respiration) in cultured myotubes and permeabilized red gastrocnemius muscle fibre bundles. Ceramide treatment also increased H2O2 levels and reduced Akt/PKB (protein kinase B) phosphorylation in myotubes. However, inhibition of mitochondrial fission via Drp1 knockdown completely protected the myotubes and fibre bundles from ceramide-induced metabolic disruption, including maintained mitochondrial respiration, reduced H2O2 levels and unaffected insulin signalling. These data suggest that the forced and sustained mitochondrial fission that results from ceramide accrual may alter metabolic function in skeletal muscle, which is a prominent site not only of energy demand (via the mitochondria), but also of ceramide accrual with weight gain.

Lactate oxidation at the mitochondria: a lactate-malate-aspartate shuttle at work.

Lactate, the conjugate base of lactic acid occurring in aqueous biological fluids, has been derided as a “dead-end” waste product of anaerobic metabolism. Catalyzed by the near-equilibrium enzyme lactate dehydrogenase (LDH), the reduction of pyruvate to lactate is thought to serve to regenerate the NAD+ necessary for continued glycolytic flux. Reaction kinetics for LDH imply that lactate oxidation is rarely favored in the tissues of its own production. However, a substantial body of research directly contradicts any notion that LDH invariably operates unidirectionally in vivo. In the current Perspective, a model is forwarded in which the continuous formation and oxidation of lactate serves as a mitochondrial electron shuttle, whereby lactate generated in the cytosol of the cell is oxidized at the mitochondria of the same cell. From this perspective, an intracellular lactate shuttle operates much like the malate-aspartate shuttle (MAS); it is also proposed that the two shuttles are necessarily interconnected in a lactate-MAS. Among the requisite features of such a model, significant compartmentalization of LDH, much like the creatine kinase of the phosphocreatine shuttle, would facilitate net cellular lactate oxidation in a variety of cell types.

Physical and functional association of lactate dehydrogenase (LDH) with skeletal muscle mitochondria

Background: The notion of mitochondrial lactate oxidation in skeletal muscle is controversial. Results: Mitochondrial respiration increased in the presence of substrates and cofactors for the lactate dehydrogenase (LDH) reaction. Respiration was inhibited when mitochondrial pyruvate transport was blocked. Conclusion: Extra-matrix LDH is associated with muscle mitochondria. Significance: LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria. The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD+ significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD+-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

Progesterone increases skeletal muscle mitochondrial H2O2 emission in nonmenopausal women.

The luteal phase of the female menstrual cycle is associated with both 1) elevated serum progesterone (P4) and estradiol (E2), and 2) reduced insulin sensitivity. Recently, we demonstrated a link between skeletal muscle mitochondrial H(2)O(2) emission (mE(H2O2)) and insulin resistance. To determine whether serum levels of P4 and/or E(2) are related to mitochondrial function, mE(H2O2) and respiratory O(2) flux (Jo(2)) were measured in permeabilized myofibers from insulin-sensitive (IS, n = 24) and -resistant (IR, n = 8) nonmenopausal women (IR = HOMA-IR > 3.6). Succinate-supported mE(H2O2) was more than 50% greater in the IR vs. IS women (P < 0.05). Interestingly, serum P4 correlated positively with succinate-supported mE(H2O2) (r = 0. 53, P < 0.01). To determine whether P4 or E2 directly affect mitochondrial function, saponin-permeabilized vastus lateralis myofibers biopsied from five nonmenopausal women in the early follicular phase were incubated in P4 (60 nM), E2 (1.4 nM), or both. P4 alone inhibited state 3 Jo(2), supported by multisubstrate combination (P < 0.01). However, E2 alone or in combination with P4 had no effect on Jo(2). In contrast, during state 4 respiration, supported by succinate and glycerophosphate, mE(H2O2) was increased with P4 alone or in combination with E2 (P < 0.01). The results suggest that 1) P4 increases mE(H2O2) with or without E2; 2) P4 alone inhibits Jo(2) but not when E2 is present; and 3) P4 is related to the mE(H2O2) previously linked to skeletal muscle insulin resistance.

Metformin Improves Insulin Signaling in Obese Rats via Reduced IKK Action in a Fiber-Type Specific Manner

Metformin is a widely used insulin-sensitizing drug, though its mechanisms are not fully understood. Metformin has been shown to activate AMPK in skeletal muscle; however, its effects on the inhibitor of κB kinaseβ (IKKβ) in this same tissue are unknown. The aim of this study was to (1) determine the ability of metformin to attenuate IKKβ action, (2) determine whether changes in AMPK activity are associated with changes in IKKβ action in skeletal muscle, and (3) examine whether changes in AMPK and IKKβ function are consistent with improved insulin signaling. Lean and obese male Zuckers received either vehicle or metformin by oral gavage daily for four weeks (four groups of eight). Proteins were measured in white gastrocnemius (WG), red gastrocnemius (RG), and soleus. AMPK phosphorylation increased (P < .05) in WG in both lean (57%) and obese (106%), and this was supported by an increase in phospho-ACC in WG. Further, metformin increased IκBα levels in both WG (150%) and RG (67%) of obese rats, indicative of reduced IKKβ activity (P < .05), and was associated with reduced IRS1-pSer307 (30%) in the WG of obese rats (P < .02). From these data we conclude that metformin treatment appears to exert an inhibitory influence on skeletal muscle IKKβ activity, as evidenced by elevated IκBα levels and reduced IRS1-Ser307 phosphorylation in a fiber-type specific manner.

Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore

The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) -sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix.

Lactate metabolism: historical context, prior misinterpretations, and current understanding

Lactate (La−) has long been at the center of controversy in research, clinical, and athletic settings. Since its discovery in 1780, La− has often been erroneously viewed as simply a hypoxic waste product with multiple deleterious effects. Not until the 1980s, with the introduction of the cell-to-cell lactate shuttle did a paradigm shift in our understanding of the role of La− in metabolism begin. The evidence for La− as a major player in the coordination of whole-body metabolism has since grown rapidly. La− is a readily combusted fuel that is shuttled throughout the body, and it is a potent signal for angiogenesis irrespective of oxygen tension. Despite this, many fundamental discoveries about La− are still working their way into mainstream research, clinical care, and practice. The purpose of this review is to synthesize current understanding of La− metabolism via an appraisal of its robust experimental history, particularly in exercise physiology. That La− production increases during dysoxia is beyond debate, but this condition is the exception rather than the rule. Fluctuations in blood [La−] in health and disease are not typically due to low oxygen tension, a principle first demonstrated with exercise and now understood to varying degrees across disciplines. From its role in coordinating whole-body metabolism as a fuel to its role as a signaling molecule in tumors, the study of La− metabolism continues to expand and holds potential for multiple clinical applications. This review highlights La−’s central role in metabolism and amplifies our understanding of past research.