The Mighty Mitochondria Are Unifying Organelles and Metabolic Hubs in Multiple Organs of Obesity, Insulin Resistance, Metabolic Syndrome, and Type 2 Diabetes: An Observational Ultrastructure Study

Abstract

Mitochondria (Mt) are essential cellular organelles for the production of energy and thermogenesis. Mt also serve a host of functions in addition to energy production, which include cell signaling, metabolism, cell death, and aging. Due to the central role of Mt in metabolism as metabolic hubs, there has been renewed interest in how Mt impact metabolic pathways and multiple pathologies. This review shares multiple observational ultrastructural findings in multiple cells and organs to depict aberrant mitochondrial (aMt) remodeling in pre-clinical rodent models. Further, it is intended to show how remodeling of Mt are associated with obesity, insulin resistance, metabolic syndrome (MetS), and type 2 diabetes mellitus (T2DM). Specifically, Mt remodeling in hypertensive and insulin-resistant lean models (Ren2 rat models), lean mice with streptozotocin-induced diabetes, obesity models including diet-induced obesity, genetic leptin-deficient ob/ob, and leptin receptor-deficient db/db diabetic mice are examined. Indeed, aMt dysfunction and damage have been implicated in multiple pathogenic diseases. Manipulation of Mt such as the induction of Mt biogenesis coupled with improvement of mitophagy machinery may be helpful to remove leaky damaged aMt in order to prevent the complications associated with the generation of superoxide-derived reactive oxygen species and the subsequent reactive species interactome. A better understanding of Mt remodeling may help to unlock many of the mysteries in obesity, insulin resistance, MetS, T2DM, and the associated complications of diabetic end-organ disease.

1. Introduction

Mitochondria (Mt) are highly dynamic and mobile cellular organelles that contribute to homeostasis by maintaining energy for cells to function in the form of adenosine-5′-triphosphate (ATP) [1,2,3,4,5,6,7,8]. They are unifying organelles and metabolic hubs, which ultimately determine cellular homeostasis for survival, and cellular death when they are remodeled and damaged to an aberrant phenotype. Further, a decrease or loss of ATP from Mt places cells in great danger of survival. Specifically, Mt serve to produce energy from the tricarboxylic acid (TCA) or Krebs cycle and fatty acid beta-oxidation in the electron transport chain (ETC) in the form of ATP, which are essential for proper cellular functioning and life. Additionally, Mt generate low levels of reactive oxygen species (ROS) in the form of superoxide via the electron transport chain (ETC) that are important for physiologic cell signaling and maintaining cellular homeostasis (Figure 1) [1,2,3,4,5,6,7,8].

These dynamic organelles also have a highly adaptive capacity to respond to physiologic stressors, especially metabolic stressors, in the form of Mt fusion, fission, biogenesis, and mitophagy. However, these adaptive capacities of the Mt can be exceeded due to chronic stressors such as nutrient excess in obesity and T2DM and develop aberrant Mt (aMt) with defective changes in morphology and phenotypes that are associated with multiple disease states and especially in obesity, IR, MetS, and T2DM (Figure 2).

Importantly, dysfunction and damage to Mt result in decreased ATP generation and the excessive generation of Mt superoxide—Mt ROS (mtROS), which are capable of propagating cellular dysfunction or loss via apoptosis that can result in multiple organs, which can result in multiple human pathologies (Figure 3).

Mt have a variety of sizes, shapes, and structures, and they are functionally complex and intimately integrated with many aspects of cellular metabolism [1,2,3,4,5,6,7,8]. Mt are often depicted as discrete organelles; however, they form a reticular-tubular type of network within cells depending on cell type and environment and interact intimately with the endoplasmic reticulum and nuclear membranes. The interconnected reticular network or threaded granules of Mt are not stationary but capable of moving about the cell to where energy needs may be the greatest and where cellular signaling by Mt are necessary [9,10,11]. This movement of Mt is supported by the complex Mt interaction with molecular motors along the actin cytoskeleton and microtubule networks that are also dependent on the energy production by the Mt [9,10,11]. Mt contain two ribosomal and 22 transfer RNA genes that are required for mitochondrial protein synthesis and 13 essential protein-encoding genes from the circular mitochondria deoxyribonucleic acid (mtDNA) that are necessary for the formation of ETC complexes (I, III, IV, V) [7,8,9]. It is important to note here that complex II proteins are encoded by nuclear DNA [12].

The ETC within the Mt is a complex interaction and may be described as follows: Mitochondria generate energy from electrons that are passed from their donors at lower redox potential to acceptors at higher redox potential through the ETC of its complexes (I–IV). Along with this electron movement, H+ protons are pumped from the matrix to the inner membraneous space that generates an energy potential difference across the inner membrane. This energy potential (preserved mitochondrial membrane potential (ΔΨm)) is then transferred to ATP from adenosine diphosphate (ADP) or dissipated as heat (non-shivering thermogenesis via UCP) as H+ protons leak back toward the matrix of the ETC complex (V) [8]. In this process, most of the electrons are eventually passed to molecular oxygen and a small portion is leaked during this transport. This mechanism results in the one-electron reduction of oxygen to superoxide. The superoxide (^•O₂⁻) generating ROS (mt-ROS) may become destructive to the cell due to free radical damage in response to nutrient excess; however, at low levels of production, superoxide serves as a metabolic cell signaling mechanism. The ETC becomes overwhelmed when there is nutrient excess such as hyperglycemia and hyperlipidemia associated with T2DM. Mt-derived superoxide is capable of interacting with the reactive species interactome [13,14], which includes not only mt-ROS but also reactive nitrogen species and reactive sulfur species such as occurs in the presence of iron-sulfur clusters in complex I or the reactions when there is impaired folate one-carbon metabolism and hyperhomocysteinemia (HHcy) that form reactive species due to homocysteine (Hcy) autoxidation, mixed disulfide formation, the interaction of reactive Hcy thiolactones, and homocysteinylation [15]. Thus, these affected cells will experience damage associated with increased oxidative-redox stress of the reactive species interactome (RSI) [13]. This RSI is an interactome consisting of functional interactions between molecules within a cell due to a combination or sum of reactive oxygen, nitrogen, and sulfur species (RONSS) [11,12,13].

The intersection of the MetS and aberrant mitochondria (aMt) may not only have immediate but also long-term consequences in regard to the constellation of metabolic abnormalities and clinical disease states with multiple organ vulnerabilities associated with age-related diseases (Figure 2, Figure 3 and Figure 4) [14,15,16,17,18,19].

The dynamic, shape-shifting morphology and functional complexity of Mt are not to be underestimated. This review demonstrates that multiple different organs and cells have the capability for the Mt to undergo a rather remarkable remodeling of their structure not only in their phenotypic morphology but also in their function. The primary functional mechanistic studies of Mt are extremely necessary and vitally important to the contribution of the abnormally remodeled aMt detected by transmission electron microscopy (TEM). However, TEM still remains the ultimate gold standard for the study of Mt and their precise morphology and their varying ultrastructural phenotypes in different disease states in multiple cells and organs. Once the Mt become aberrantly remodeled to an aMt phenotype and impaired mitophagy is present, they contribute mechanistically via mtROS and the reactive species interactome to the ongoing cellular dysfunction and damage.

Importantly, Mt are known to be susceptible to different genetic and environmental injuries, the accumulation of mtDNA mutations, and mtDNA copy number depletion, and the epigenetic modification of the Mt genome may help to explain the prevalence of Mt-related diseases including (T2DM) [7,9,20].

2. Brief Overview of Mitochondrial Dynamics (Fusion and Fission), Biogenesis, and Mitophagy

Before examining the Mt in each of the individual organs, it is necessary to briefly identify each of the Mt remodeling mechanisms as it pertains to fusion, fission, biogenesis, and mitophagy (Figure 5).

The previous figure illustrates the possible various morphological mechanisms and remodeling of Mt in health and disease. The functional mechanisms are extremely complex and much has been learned about fusion, fission, biogenesis, and mitophagy with the use of genetic knockout/knockin models. For a more complete discussion of the complicated interactions regarding the functional molecular mechanisms, the following reference is highly recommended [20].

It is also important to note that extramitochondrial oxygen consumption also occurs via nonenzymatic and other enzymatic reactions, which are derived from NADPH oxidase, xanthine oxidase, uncoupled nitric oxide synthase, D-amino oxidase, p450 cytochromes, and proline hydroxylases [20]. In the following sections, multiple cells from different organs are examined to demonstrate how normal Mt are remodeled to an aMt phenotype in obesity, insulin resistance, MetS, and T2DM.

3. Myocardial Remodeling: Increased Mitochondrial Biogenesis, Impaired Mitophagy, and Accumulation of Aberrant Mt (aMt) in Obesity, IR, MetS, and T2DM

The myocardium is very unique because it requires a constant source of ATP in order to provide adequate continuous myocardial contraction and this specialized muscle must be able to increase its ATP production for any increase in heart rate or respond to an increased end-diastolic pressure to promote homeostasis [16,21,22,23]. Importantly, diabetic cardiomyopathy (DC) that occurs in T2DM is associated with dysfunctional Mt (aMt). DC may be considered a condition of myocardial dysfunction in the absence of overt clinical coronary artery disease, valvular disease, and other conventional cardiovascular risk factors, such as hypertension and dyslipidemia that occur with diabetes. DC may be characterized by multiple mechanisms, which include: mitochondrial dysfunction (aMt), disruption in cardiac insulin signaling, increased oxidative stress, inflammation, decreased nitric oxide bioavailability, elevated advanced glycation end products, accumulation of extracellular matrix (ECM) (fibrosis), ECM and cardiomyocyte stiffening, impaired calcium handling of Mt and cardiomyocytes, endoplasmic reticulum stress, cardiac autonomic neuropathy, microvascular dysfunction, renin–angiotensin–aldosterone system (RAAS) activation, and importantly, the emerging and novel exosome pathways that are now being considered [21,22]. Indeed, healthy Mt are critical for the mechanical function of cardiomyocytes [23].

The myocardium must also be prepared to rapidly adapt to stressors such as hypertension and increased demands to work against an increased stiffened vascular system that occurs when there is systemic vascular stiffening and increased myocardial end-diastolic load [16,21,22,23]. The myocardium does this, due to its unique compensatory mechanism of adaptive Mt biogenesis. This can be appreciated by observing the marked increased expansion in the number of intermyofibrillar (IMF) Mt. However, if these increased stressors are sustained such as occurs in nutrient excess, the Mt will also develop impaired mitophagy as observed in obesity, IR, MetS, impaired glucose tolerance, and T2DM. Uncoupling of mitophagy from biogenesis due to impaired mitophagy and accumulation of aMt that occurs in obesity, IR, and T2DM is intriguing and not totally understood. However, there is ongoing research to figure out why impaired mitophagy is associated with Mt biogenesis. Further, this disequilibrium (Mt uncoupling) between impaired mitophagy and Mt biogenesis results in cardiomyocyte dysfunction, abnormal remodeling with fibrosis and stiffening of the heart, and even myocyte death due to apoptosis [24]. The following TEM images allow one to view myocardial Mt biogenesis and aMt accumulation due to impaired mitophagy in the female DIO-Western (Figure 6) [25], obese, female diabetic db/db (Figure 7) [26], and the IR, impaired glucose tolerance, hypertensive, lean, male Ren2 models (Figure 8) [27].

While the images in Figure 6 were of the 20-week-old female DIO Western models [25], similar Mt remodeling in the younger 16-week-old female diabetic db/db mice were noted to display increased biogenesis and impaired mitophagy with the accumulation of aMt (Figure 7) [26].

Mt fusion and fission (mitochondrial dynamics), biogenesis, and mitophagy are all important components of the Mt quality control system in the myocardium. However, the co-occurrence of both increased Mt biogenesis and importantly impaired mitophagy with aMt accumulation will result in cardiac contractile dysfunction and decreased cardiac output. The co-occurrence of impaired mitophagy will allow the leaky aMt to continuously leak an excess of superoxide—mtROS from the dysfunctional ETC into the cytosol of the cardiomyocyte. Mitophagy plays a protective role in diabetic cardiomyopathy, principally through the clearance of abnormal—dysfunctional aMt. Importantly, impaired mitophagy will be detrimental to myocardial contractility. It is important to note here that treatment with empagliflozin (a sodium glucose transporter-2 inhibitor) improved the Mt expansion and aMt in the diabetic db/db models with 5 weeks of treatment that were associated with improvement of diastolic dysfunction (Figure 7D, insert d) [26].

A close relationship exists between both the structural and functional relationship between Mt and the endoplasmic reticulum. This ultrastructural relationship to demonstrate the tight structural arrangement between the myocardial ER and aMt ultrastructure was identified in male Ren2 hypertensive, insulin-resistant models with impaired glucose tolerance (Figure 8) [27].

The ER and Mt are known to interact dynamically, structurally, physiologically, and functionally [28,29,30,31]. Further, this arrangement of close proximity and touching of the Mt and ER is known as the mitochondria-associated ER membrane(s) or (MAMs) [30,31]. Importantly, one of the most critical aspects of this interaction is calcium signaling and ROS exchange between these two organelles in addition to the supply of ATP from the Mt to the ER. This crosstalk between the membranes of the aMt and the ER could result in an increased transfer of both calcium and ROS. The interaction and crosstalk between these two organelles could result in ER stress that could be associated with the misfolding of proteins by the ER and further in combination with ROS leakage from the aMt could lead not only to cellular dysfunction with loss of function but also cell death via apoptosis [30,31]. The Mt-ER close interaction allows for 3 major functions, which include (1) calcium buffering; (2) Mt fission through Drp-1; (3) phospholipid transfer [31]. Importantly, it is also known that low levels of mitofusin 2 have been shown to have a positive correlation with obesity and T2DM [31]. However, if the Mt have undergone remodeling to an aberrant phenotype (depicted in Figure 8) there will develop an excess of extramitochondrial calcium accumulation. Normal Mt are known to act as a sink for calcium and acts as a calcium buffering mechanism. The release of Mt calcium content from aMt may induce cardiomyocyte apoptosis.

4. Mitochondria Remodeling in the Brain

Brain mitochondria are essential to regulate neuronal synaptic transmission with high energetic requirements. Therefore, the remodeling of the normal Mt to an aMt phenotype could result in a loss of these normal functions and result in neuronal dysfunction in T2DM due to impaired mitochondrial bioenergetics and brain hypometabolism [32,33,34,35,36,37]. Mitochondrial dysfunction and damage are implicated in the pathogenesis of multiple neurological diseases. Virtually, all of the cells within the brain frontal cortex in layer III of the grey matter in the obese diabetic, female 20-week-old db/db models demonstrate aMt (Figure 9) [32,33,34,35,36].

The mural cells of the neurovascular unit including the pericyte and endothelial cells (ECs) undergo remodeling of Mt to become aMt in the diabetic db/db models (Figure 10).

Additionally, reactive-activated microglia cell(s) (aMGCs) were also found to undergo marked Mt remodeling to form aMt in these same models discussed previously (Figure 11) [33,36].

These aMGCs depicted not only Mt remodeling to develop aMt but also these same MGCs underwent concurrent remodeling from a ramified state to a reactive-activated state, which encircled and damaged the neurovascular unit. This resulted in blood-brain barrier disruption in brain endothelial cells (BEC) and remodeling of the nucleus to form nuclear chromatin condensation. The neuroglia cells including the aMGCs, astrocytes (ACs), and oligodendrocytes demonstrated aMt remodeling in the above models, for those who are interested in viewing both the ramified and activated MGCs and their aMt a video has been created to demonstrate these remodeling changes in an en bloc focused ion beam scanning electron microscopy FIB/SEM video in the supplement [33].

Importantly, there is a specific Mt carbonic anhydrase inhibitor that has been observed to have an effect on protecting healthy Mt from undergoing aMt changes named topiramate. Topiramate (approved for the treatment of seizures) has been shown to decrease ROS and oxidative stress in the brain by the specific inhibition of the Mt carbonic anhydrase in streptozotocin-induced diabetes [38,39,40,41,42]. In the male CD-1 streptozotocin-induced diabetic model, the brain had increased permeability to ¹⁴C-sucrose due to the attenuation and/or loss of the BBB via the attenuation or loss of EC tight and adherens junctions. Additionally, this model had an attenuation and/or loss of the supportive pericytes of the neurovascular unit due to increased oxidative stress at 16-weeks post-induction of diabetes. This model is known to have increased oxidative stress and increased permeability in the midbrain. In this same experiment, observations of aMt were present in the EC cytoplasm that were prevented with topiramate (50 mg/kg body weight) (Figure 12) [38].

These brain observational images strongly support the central finding of aMt in multiple brain cells in obesity, IR, and T2DM in the diabetic db/db models and streptozotocin-induced diabetes in the brain. Impaired brain insulin signaling due to IR is related to an increased risk of late-onset Alzheimer’s disease (LOAD) [36]. There are at least five major intersecting links to consider when discussing the relationship of T2DM and the increased risk of LOAD: (1) Aging; (2) Metabolic (hyperglycemia and advanced glycation end products (AGE) and its receptor RAGE resulting in AGE/RAGE interactions and hyperinsulinemia—IR (a linking linchpin); (3) Oxidative-redox stress (ROS, RONS, RSS) resulting in a RONSS interactome as discussed in this review of the leaky aMt; (4) Inflammation (peripheral macrophages and importantly the central brain microglia); (5) Vascular (macrovascular with accelerated atherosclerosis-vascular stiffening and microvascular NVU/neuroglial remodeling) with subsequent impaired cerebral blood flow. Peripheral IR and brain IR contribute to impaired mitophagy and disrupts the lysosomal degradation pathway [43].

Since T2DM and LOAD are both age-related and chronic diseases, it is not unusual for them to co-occur along with cerebrocardiovascular disease in aging global societies. Both have multifactorial causations and risks with IR as a linking linchpin between these two disparate diseases (Figure 4 and Figure 13).

A great deal still remains unknown; however, a wonderful discussion regarding what is and what is not known regarding brain insulin resistance (BIR) can be found and is strongly suggested [43,44,45]. Indeed, peripheral IR is a definite core feature of T2DM that is rapidly emerging as a core feature in LOAD as in BIR, which may be defined as the failure of brain cells to respond to insulin [45,46]. Additionally, the combination of T2DM and LOAD suggest that the two disparate diseases may be synergistic when they co-occur and demonstrate the greatest decrease in insulin signaling proteins [47].

Glutamate (an excitatory neurotransmitter), plays a major role in determining certain neurological disorders including LOAD and Parkinson’s disease (PD). Glutamate neurotoxicity (GNT) is characterized by increasing damage of cellular components and organelles, including mitochondria. Since the aMt phenotype is present in BIR and T2DM, the Mt will be become increasingly dysfunctional and damaged due to GNT [48].

Indeed, aMt dysfunction, neuronal dysfunction, and damage are key features in T2DM, LOAD, and PD that can lead to synergistic neuronal toxicity and death [49]. Importantly, bacterial and viral infections are of current interest since the SARS-CoV-2 virus can lead to neuroglial activation and eventually lead to neurodegenerative progressive syndromes that have been proposed to be an additional trigger for LOAD and PD [50]. This may be very pertinent since we are all still in the midst of the coronavirus disease-19 (COVID-19) pandemic that may place some survivors at an increased risk for developing neurodegenerative diseases such as LOAD and PD over the future coming years. This may be especially true in vulnerable individuals with obesity, MetS, IR, and T2DM with dysfunctional and damaged BECs, aMt, and neuroglia activation with the aberrant remodeling of the NVU with dysfunctional and damaged BECs and endothelial glycocalyx [50,51].

Importantly, cytoadherence of red blood cells to BECs of the NVU was found to be present in the subcortical white matter (SCWM) regions of the diabetic db/db models (Figure 14) [32,36].

4.1. Brain Endothelial Cell Glycocalyx (ecGCx) of the Neurovascular Unit (NVU)

The ecGCx is vasculoprotective and acts as the first barrier of the tripartite BBB of the NVU, which consists of: (1) ecGCx; (2) BEC; (3) abluminal basement membrane (BM), pericytes and pericyte foot processes within the BM, and astrocyte foot processes [52]. The ecGCx also covers the stromal fenestrated capillaries and the epithelial ependymal glycocalyx of the blood-cerebrospinal fluid barrier [52]. An intact ecGCx is important for the vascular integrity of arteries, arterioles, capillaries, and post-capillary venules. It is a gel-like, sugar-protein protective surface layer coating of the luminal BEC of the NVU. This protective surface coating delimits the blood and its constituents (erythrocytes, leukocytes, and thrombocytes-platelets). The ecGCx is primarily synthesized by the BECs with contributions by plasma albumin, orosomucoids, fibrinogen, glycoproteins, and glycolipids [50,51,52,53,54,55,56]. The ecGCx is anchored to the BEC luminal plasma membranes by highly sulfated proteoglycans (syndecans and glycipans), glycoproteins (including selectins such as various cellular adhesion molecules and integrins), and non-sulfated hyaluronan (a glycosaminoglycan) via BEC cluster of differentiation 44 (CD44). Hyaluronan (HA) may also be free-floating (unbound), attached to the assembly proteins such as the BEC hyaluronan synthases, or Form HA-HA stable complexes. The ecGCx is also anchored via the proteoglycan (glypican) to the caveolae and this plays a key role in mechanotransduction of BEC luminal fluid shear stress-induced synthesis of essential nitric oxide (NO) via glypican caveolae interactions located within the BEC lipid rafts (Figure 15, Figure 16 and Figure 17) [52,53]. The ecGCx has a net negative charge largely due to the sulfation of glycosaminoglycan side chains which allow for strong electrostatic binding to the polyvalent cation lanthanum nitrate (La(3+) nitrate) (LAN). This allows the ecGCx to be identified with TEM staining in perfusion fixed animal models (Figure 15) [52,57].

The diffuse electron density of the ecGCx does not allow one to identify any substructures within this surface layer of the EC, and therefore, an illustration has been generated to demonstrate its contents (Figure 16).

The ecGCx serves as the first luminal barrier of the NVU and lanthanum nitrate (LAN) staining has been utilized to define the ecGCx by our laboratory and others [52,57,58]. LAN staining allows the ecGCx to be visualized with TEM studies due to the strong electrostatic attraction between the highly positive charge of LAN to the negatively charged ecGCx. We have previously utilized LAN staining in the diabetic BTBR ob/ob models and found it to be both reliable and reproducible [52], while others have utilized LAN staining in the diabetic db/db models in the pulmonary system, which demonstrated an attenuation of the ecGCx in the diabetic db/db preclinical models of the lung [58]. LAN staining revealed that the ecGCx was markedly attenuated and even lost in the obese, insulin-resistant, diabetic BTBR ob/ob models in the frontal cortical layer III and CA-1 regions of the hippocampus (Figure 17) [52].

Interestingly, when the BTBR ob/ob models were treated for 16-weeks prior to sacrifice with leptin (15 μg/day via implanted intraperitoneal pump), it was observed that restoring leptin protected the ecGCx from being attenuated and/or lost (Figure 18) [52].

If the NVU ecGCx becomes dysfunctional, attenuated, and/or lost via shedding as in the obese, insulin-resistant diabetic BTBR ob/ob model [52], then there would be a decrease in bioavailable NO to signal pericytes in the capillary NVU and NVU uncoupling would develop due to the loss of regional mechanotransduction at these BEC regions. This would result in regional decreased cerebral blood flow and regional ischemia. If ecGCx loss is an early event, as suspected in the BTBR ob/ob models, this would add to the oxidative stress of the BECs and possibly contribute to an even greater increase in mtROS as well as NADPH oxidase-derived ROS. An intact glycocalyx and Mt of BECs are of utmost importance in maintaining the NVU BBB homeostasis. Damage to the glycocalyx in T2DM can have multiple pathophysiological consequences, which include: (1) increased vascular permeability; (2) edema formation; (3) increased adhesion of circulating inflammatory cells to the endothelium; (4) accelerated inflammatory processes; (5) activation of the coagulation cascade; (6) platelet aggregation [50,51,52,53,54,55,56]. Excess nutrients (increased glucose and fatty acids) in obesity, MetS, IR, T2DM, and the associated glucotoxicity will result in leaky aMt phenotypes and an increase in mtROS. In turn, this will lead to dysfunction, attenuation, and/or shedding of the ecGCx and a further increase of aMt and mtROS. Additionally, this increase in EC mtROS could result in contributing to even further ecGCx shedding or impairment in ecGCx regeneration. This sequence of events between aMt, mtROS, and ecGCx shedding could be bidirectional, such that a vicious cycle may ensue wherein one abnormality may lead to another. Decreasing mtROS from leaky aMt results in improved homeostasis [35,38,57,58,59,60] and likewise restoring the ecGCx also improves homeostasis and function [60,61,62,63]. ecGCx with attenuation and/or shedding in BECs may be playing a bidirectional role in the development and progression of impaired cognition and neurodegeneration. Importantly, there may exist a bidirectional role between the accumulation of aMt (due to impaired mitophagy) and the dysfunction or loss of the ecGCx in BECs (Figure 19) [32,33,34,35,36,59,60,61,62,63,64,65].

While more research is necessary to confirm this bidirectional relationship between aMt and ecGCx attenuation or loss via shedding, it is nevertheless a very intriguing association and presents an emerging opportunity to further unlock some of the mysteries associated with Mt in obesity, IR, MetS, and T2DM and possibly the associated complications of diabetic end-organ disease including the brain. Additionally, this concept may allow for future interventions to interrupt this bidirectional vicious cycle by utilizing Mt carbonic anhydrase inhibition with topiramate [38], uncoupling proteins such as UCP2 [61], and the emerging role of Mt transfer [62].

5. Descending Aorta Mitochondria Remodeling

It is important to discuss Mt remodeling in the descending thoracic aorta to better understand its relationship to vascular stiffening and the heart-brain-kidney (HBK) axis as discussed in Figure 3 and how this links aortic stiffening and its relationship for the progression to end-organ damage in HBK axis in obesity, IR, impaired glucose tolerance, MetS, and T2DM.

The vascular tunica media, which contains vascular smooth muscle cell(s) (VSMCs) depict aMt remodeling when there are cellular stressors such as hypertension, insulin resistance, and impaired glucose tolerance as occurs in the Ren2 descending aorta tunica media VSMCs (Figure 20) [66].

Cell-cell communication is a process necessary for physiological tissue homeostasis and is frequently impaired or altered in many disease states such as obesity, IR, impaired glucose tolerance, T2DM, and hypertension [66,67]. Importantly, intercellular transfer of mitochondria is rarely observed but is known to occur in certain stress states such as hypertension in the aorta [66,67]. In the stressed cells of the descending aortic tunica media in the Ren2 rat model, the author was able to identify this rare process between two VSMCs from an ex vivo model and indeed, Mt know no boundaries (Figure 21) [68].

Additionally, the obese, insulin-resistant, and diabetic db/db models revealed aMt in the proliferative VSMCs of the tunic media similar to the hypertensive Ren2 model [66] (Figure 22) [69].

aMt are a prominent feature in the VSMC in the hypertensive Ren2models [66]. Additionally, they are a characteristic finding in the obese, diabetic, female db/db models [69] and strongly suggest an important role in the remodeling of the descending thoracic aorta that results in stiffening of the thoracic aorta.

5.1. Perivascular Adipose Tissue (PVAT) of the Descending Thoracic Aorta in Obese, Insulin Resistant, and Diabetic db/db Model: The Tunica Adiposa of the Adventitia

The PVAT of the descending thoracic aorta allows one to examine mitochondria remodeling in the adipose tissue of both brown adipose tissue (BAT) and white adipose tissue (WAT) depots in the tunica adiposa of the aortic adventitia. In healthy human individuals and preclinical rodent models, the descending thoracic aorta PVAT consists of BAT in the tunica adiposa in contrast to WAT in the abdominal aorta [70]. However, in obesity, insulin resistance, and T2DM as in the db/db model, the PVAT BAT undergoes a near complete transdifferentiation to WAT, which is dysfunctional and is associated with marked Mt remodeling to aMt and adipocytes that rupture and incite inflammation to develop crown-like structures [69]. This transdifferentiation from BAT to WAT results in the loss of the protective functions of the PVAT to the aortic vascular wall and it remodels to a stiffened vessel that is damaging to the end organs of the HBK axis capillaries with high flow and low resistance to result in end-organ dysfunction, damage, and disease. (Figure 23).

The transdifferentiation of PVAT from BAT to WAT (Figure 24) contributes to aortic vascular stiffening as well as remodeling damage not only in the heart but also the brain and the kidney as in the HBK axis [26,35,71]. While BAT is the predominant adipose tissue in the PVAT in control models, the db/db models of obesity, insulin resistance, and T2DM undergoes a remodeling transdifferentiation to WAT and aMt remodeling (Figure 24) [69].

Importantly, the remodeling of the tunica adiposa results in the loss of the anticontractile and protective mechanisms of the normal healthy BAT in the descending thoracic aorta [69]. The transdifferentiated and remodeled WAT of the PVAT in the upper 1/3 of the descending thoracic aorta of db/db models demonstrates aMt and inflammatory crown-like structures (CLS) (Figure 25).

aMt in the transdifferentiated WAT adipocytes may be characterized by hyperlucency with a loss of electron-dense Mt matrix and loss of crista (Figure 26) [69].

Section 5 and Section 5.1 have shared multiple TEM images depicting multiple Mt remodeling changes in the descending thoracic aorta and these changes are important to better understand the overall remodeling of the descending thoracic aorta and aortic stiffening. Aortic vascular stiffening may be considered as the nexus between the HBK axis with subsequent cellular and Mt remodeling in these end-organs that are being affected by an abnormally increased aortic pulse pressure and the damage to the tissues and capillaries that they serve [72].

6. Thoracic Aorta Vascular Stiffening as the Nexus between the Heart-Brain-Kidney (HBK) Axis

Vascular stiffening of the descending thoracic aorta may be considered as the nexus between the HBK axis [72]. Importantly, the combined aMt in the VSMCs in the Ren 2 of the tunica media [66] and the WAT of the tunica adiposa of the thoracic aorta adventitia [69] are very important to the development of the descending thoracic aorta stiffening (Figure 27).

A healthy elastic aorta has a phenomenal cushioning function to the high pressure exerted by systolic flow from the left ventricle of the heart, and this cushioning effect limits the arterial pulsatility and serves to protect the microvasculature in the high flow and low resistance microvessels such as occurs in the HBK axis. However, when thoracic aorta stiffening develops as in the hypertensive Ren2 and diabetic db/db preclinical models this protective elastic cushioning is diminished and/or lost, and these microvessels will respond to this injury in a response to injury wound healing response, which results in dysfunction and damage to the heart, brain, and kidney [26,35,71]. It is important to note that even though aortic stiffening precedes isolated systolic hypertension and is related to target organ damage in the heart, brain, and kidney that the descending thoracic aorta is also a target organ that is significantly affected by aging and other various states such as obesity, MetS, diabetes (T1 and T2DM), smoking, elevated lipids (triglycerides and low-density lipoprotein (LDL) cholesterol, and chronic kidney disease [38,73,74]. Plus, aortic stiffening has a central and important role in providing a vicious cycle of hemodynamic dysfunction that may be characterized by excessive pulsatility (increased PWV) that contributes to a HBK axis [26,35,71,72].

Indeed, aortic stiffness is a complex phenomenon that arises from multiple structural alterations in the aortic wall with impaired endothelial function, increased VSMC tone, phenotypic modulation to a proliferative phenotype of adventitial fibroblast to myofibroblasts, chronic low-grade inflammation, and the loss of the PVATs tunica adiposa anticontractile effect [72,73,74]. Increased aMt in VSMCs in hypertension, obesity, MetS, and T2DM suggests that increased superoxide (mtROS) along with other RONSS interactome would over time deplete the vascular wall antioxidants such as superoxide dismutases (MnSOD, CuZnSOD, EcSOD), catalase, glutathione peroxidase, paraoxonase, thioredoxin peroxidase, and heme oxygenases [73] and contribute to aortic vascular stiffening.

Superoxide released from the aMt would also instigate the RONSS interactome in the VSMCs within the tunica media and result in the activation of latent matrix metalloproteinases (MMPs) with subsequent collagen type I deposition as well as elastin fragmentation and remodeling. This vascular extracellular matrix remodeling would also result in aortic stiffening with increased PWV and impaired pulsatile reflection to the heart resulting in increased afterload and remodeling of the myocardium in a stress injury response and response to injury remodeling with interstitial fibrosis [74]. Concurrently, this increased pulsatile force, which also occurs in the carotid arteries would be transferred to the brain microcirculation (neurovascular units) and similarly to the renal arteries with damaging effects to the renal glomeruli and PTCs to result in both a glomerular and tubular remodeling nephropathy with early microalbuminuria and later macroalbuminuria.

Thus, the aMt that are found within the Ren2 and the diabetic db/db VSMCs could result in both hypertension and, importantly, aortic vascular stiffening with damage to the target organs with high flow and low resistance such as the heart, brain, and kidney [26,35,71,72,73,74,75,76].

Elevated homocysteine levels referred to as hyperhomocysteinemia (HHcy) are a biomarker of impaired FOCM [15] and are strongly and independently correlated to arterial stiffness measured by aortic pulse wave velocity [77]. Plus, it is known that T2DM is associated with impaired FOCM and HHcy including the preclinical diabetic db/db models [15,77,78,79,80,81,82,83]. HHcy results in increased ROS and the RONSS interactome, which would also accelerate vascular stiffening. HHcy promotes oxidant injury to vascular cells including the endothelial cells and VSMCs via ROS due to the process of Hcy autoxidation, formation of Hcy disulfides, the interaction of Hcy thiolactones, and protein homocysteinylation [15,79,80,81,84,85]. The ROS produced by HHcy then would be capable of acting synergistically with the superoxide generated from aMt to interact with nitrogen and sulfur species to form RONSS and eventually implicate the RONSS interactome [15]. The RONSS interactome within the tunica media VSMCs and the tunica adiposa of the adventitial layer would then be capable of contributing to the overall oxidative-redox stress of the aortic vascular wall to result in remodeling and provide a compelling contribution to the thoracic descending aorta stiffening that negatively affects the HBK axis [78,83,85].

7. aMt Remodeling in Obesity, IR, MetS, T2DM and Other Important Organs: Skeletal Muscle, Visceral White Adipose Tissue, Liver, Pancreatic Islet β-Cells, and Kidney

Mitochondria remodeling to an aMt phenotype also occurred in soleus slow-twitch skeletal muscle [86,87]. Visceral, omental WAT had similar remodeling with aMt and the development of crown-like structures as discussed in Section 5.1 of the PVAT [86]. Liver hepatocytes developed aMt [16]. Pancreatic islet beta-cells with a concurrent dilated Golgi apparatus to suggest endoplasmic reticulum stress [86,88]. Basilar kidney proximal tubule cells had a marked remodeling change to aberrant fragmented Mt with loss of elongation and formation of spherical Mt suggesting fission with concurrent disordered and chaotic basilar canalicular system [86,89,90].

8. Conclusions

Mt remodeling to an aMt phenotype and Mt dysfunction is a common thread that weaves across the multiple organ systems in obesity, IR, MetS, and the T2DM mosaic fabric of disease. The images presented in this narrative and ultrastructure Mt review still brings the author back to the story and the presentation shared by Michael Brownlee when he delivered his classic presentation and later published his findings entitled: “The Pathobiology of Diabetic Complications: A Unifying Mechanism” as a title for the Banting lecture in 2004 [91]. Specifically, Brownlee had a section in this manuscript regarding putting the pieces of the puzzle together. He spoke of four puzzle pieces, including (1) increased flux across the polyol pathway; (2) advanced glycation end-products (AGE); (3) hyperglycemia-induced activation of protein kinase C; (4) increased flux across the hexosamine pathway (all due to hyperglycemia).

This review has demonstrated remodeling of the healthy Mt in control models to an aberrant phenotype (aMt) in obesity, IR, and T2DM that certainly would be an important and additional piece of this puzzle. The additional ‘fifth piece’ of the puzzle, (aMt), would increase mtROS and the RONSS interactome, which could induce DNA strand breaks and activate nuclear poly (ADP-ribose) polymerase (PARP) and result in decreased glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and subsequently increase each of the four previous Brownlee puzzle pieces [92].

Mt may be considered to be unifying organelles and metabolic hubs in multiple organs of obesity, IR, MetS, and T2DM. This review has shared multiple ultrastructural TEM images depicting that Mt undergo remarkable remodeling in multiple organs and cells with a common finding of aMt depicting hyperlucency, attenuation and/or loss of electron-dense Mt matrix, and attenuation and/or loss of cristae. These aMt were a constant unifying theme in multiple different organs and cells studied in this observational TEM study.

The presence of multiple complex, interacting cycles within the Mt matrix include the folate and methionine cycles of FOCM [15]. The Mt matrix FOCM transfers one-carbon units for many biochemical processes, which include (1) purine and deoxythymidine monophosphate (dTMP) biosynthesis; (2) Mt protein translation; (3) cell proliferation, protein synthesis, and Mt respiration. Therefore, impaired FOCM in T2DM [15] may contribute to impaired cell proliferation, protein synthesis, and Mt respiration in addition to an accumulation of mtDNA deletions [15,93].

In myocardial cells (Section 3) it was quite evident that there was compensatory Mt biogenesis in the intermyofibrillar regions; however, this increase in Mt due to biogenesis was also plagued with findings of multiple aMt. This accumulation of aMt strongly suggested that there was also concurrent impaired mitophagy that allowed these aMt to accumulate and leak damaging superoxide mtROS. These superoxide free radicals would then interact with nitrogen and sulfur species to generate not only ROS but also the generation of RNS and reactive sulfur species RSS to generate the RONSS and act as a RONSS interactome to enhance the overall oxidative-redox stress within myocardial cells and the multiple cells and organs that were also shared in this review. In the brain (Section 4) these aMt would not only increase mtROS and activation of the RONSS interactome but also contribute to hypometabolism that is present in both T2DM and late-onset Alzheimer’s disease (LOAD) due to impaired oxidative phosphorylation and generation of ATP [37,39,64,94,95]. Importantly, the aMt may be related to attenuation and/or loss of the ecGCx and this relation between aMt and loss of the ecGCx may be bidirectional (Section 4.1). Additionally, there was an attenuation and/or loss of subsarcolemmal, intermyofibrillar, and pericapillary Mt in skeletal muscle (Section 7); a marked remodeling of PTCs from elongated basilar Mt to spherical Mt suggesting increased fission (Section 7); aMt in descending aorta VSMC (Section 7); PVAT tunica adiposa adipocytes (Section 5.1); liver hepatocytes (Section 7) and pancreatic islet beta cells (Section 7). Further, there was also an impact of aMt on the cytoadhesion of RBCs to activated ECs in the capillaries of the diabetic db/db models in the brain (Section 4—Figure 14).

Novel emerging information has identified the presence of specific mitochondrial matrix metalloproteinases (MMP-2 and MMP-9). These proteinases-gelatinases (referred to as moonlighting proteins) would be capable of creating an increase in MOMP and the release of mtROS and cytochrome c due to nutrient excess such as hyperglycemia and increased superoxide, which adds another dimension to the formation of leaky aMt [96].

The persistent findings of aMt hyperlucency, vesiculation/vacuolization, loss of Mt matrix electron density, and cristae strongly suggest impaired mitophagy. Importantly, healthy Mt cardiolipin (a phospholipid of the inner membrane) not only plays an important role in the process of energy generation of ATP via the ETC but also in the process of preserving mitophagy. Therefore, if cardiolipin is impaired or decreased as occurs in obesity, IR, MetS, and T2DM, its functional impairment due to increased mtROS within the Mt inner membranes could impair not only Mt ETC function in producing ATP but also mitophagy [97,98,99].

Importantly, in Section 3 the ultrastructural findings of increased Mt biogenesis and impaired mitophagy were demonstrated (Figure 6, Figure 7 and Figure 8); however, the possible causes for this remodeling phenomenon were not discussed. In this regard, Palikaras et al. [24,100] have shared that there is a delicate balance wherein mitochondria biogenesis and mitophagy are coupled in order to maintain cellular homeostasis. However, uncoupling of Mt biogenesis and mitophagy may result in dyshomeostasis with impaired mitophagy, which results in the accumulation of aMt as previously discussed and depicted in different models, cells, and organs in previous Section 3. This group has been actively working with Caenorhabditis elegans in order to sort out these complicated interacting mechanisms and what may be done in the future to prevent Mt biogenesis and mitophagy uncoupling in order to translate their findings into mammals including humans. Further, the associated glucotoxicity, lipotoxicity, inflammation, oxidative stress, and hyperinsulinemia in T2DM combine to activate 5′ AMP-activated protein kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1), which impairs mitophagy via mTORC1 to cause phosphorylation of Unc51-like kinase, hATG1 (ULK1) [101]. Additionally, there may also be impairment of autophagy-related proteins ATG/microtubule-associated proteins 1A/1B light chain LC3 (ATG/LC3) machinery in T2DM [102]. This allows the accumulation of aMt to leak mtROS and mtDNA to promote cellular apoptosis as well as instigating inflammation. As noted in Figure 5 in the introduction Section 1, selective mitophagy is governed by Pink1/Parkin and impairment of this pathway is associated with diabetes and neurodegeneration [103].

The problem with T2DM is global, and the International Diabetes Federation has reported that the number of adults aged 18–99 years old with T2DM is 451 million strong and also predicts that T2DM may rise to 693 million in 2 more years by 2024 [104]. This increased risk for T2DM in both obese and aging populations that are coupled with the global demographics requires a better understanding of the molecular changes. This review has been an attempt to better understand the multiple pathways with a focus on ultrastructural Mt remodeling changes, which includes aMt as a nexus for the convergence of obesity, IR, and T2DM and their multiple end-organ complications [12,14,20,105,106]. While the focus in this narrative review and ultrastructural observational findings has been on aMt and impaired mitophagy allowing the accumulation of aMt, it is also important to note that there is also impaired Mt dynamics (fission and fusion) and impaired Mt biogenesis in conditions of insulin resistance in general and particularly in T2DM in addition to impaired mitophagy and the accumulation of aMt [105].

The observational TEM findings of aMt in multiple cells and organs including the brain strongly place the Mt at the very core of associating IR and T2DM and LOAD (Section 4 and Section 4.1). These findings are in strong support of a mitochondrial cascade hypothesis for LOAD as put forward by Swerdlow et al. [107]. Additionally, there are at least nine well-accepted hypotheses and three emerging hypotheses for the development of LOAD [36]. In most cases, the Mt cascade hypothesis does not exclude any of these other nine accepted hypotheses; instead, the Mt cascade hypothesis is supportive [107]. Therefore, the author remains very excited regarding the future potential of mitochondrial transfer [108]. Importantly, the intercellular transfer of Mt is a universal biological event (Section 5 and Figure 21) and indeed Mt can cross cell boundaries [68,109]. This evolving field of Mt transfer research is very exciting, and while there is a paucity of specific literature in regard to T2DM, there are papers that discuss the importance of mesenchymal stem cell therapy and artificial Mt transfer with the following references being highly recommended [108,109,110,111,112,113].

There are certain limitations to this review, in that this study only utilizes ultrastructural TEM images to demonstrate mitochondrial remodeling at a single point in time; however, one can note the marked remodeling of the aMt as compared to the mitochondria in normal control models in the images that were presented.

It has now been 70 years since George Emil Palade published the first TEM images of the mitochondria in 1952 [114] and we are still vigorously studying the mitochondrial dynamics of fusion and fission, biogenesis, and mitophagy, which are Mt processes that are impaired in obesity, IR, MetS, and T2DM. Indeed, this review has demonstrated the normal Mt phenotype in healthy control models and depicted how the remodeled aMt phenotypes in obesity, IR, MetS, and T2DM accumulate and are associated with and play a responsible central role for cellular dysfunction, damage, and apoptosis in multiple cells and organs (Figure 3 and Figure 27). It is hoped that the crossover of knowledge between the ultrastructure images presented in this review and the discipline of functional molecular biology may lead to advances in the study of mitochondria and the remodeling phenotype of aberrant Mt due to impaired mitophagy found in preclinical models of obesity, IR, MetS, and T2DM.