New perspectives in diabetic neuropathy.

Diabetes prevalence continues to climb with the aging population. Type 2 diabetes (T2D), which constitutes most cases, is metabolically acquired. Diabetic peripheral neuropathy (DPN), the most common microvascular complication, is length-dependent damage to peripheral nerves. DPN pathogenesis is complex, but, at its core, it can be viewed as a state of impaired metabolism and bioenergetics failure operating against the backdrop of long peripheral nerve axons supported by glia. This unique peripheral nerve anatomy and the injury consequent to T2D underpins the distal-to-proximal symptomatology of DPN. Earlier work focused on the impact of hyperglycemia on nerve damage and bioenergetics failure, but recent evidence additionally implicates contributions from obesity and dyslipidemia. This review will cover peripheral nerve anatomy, bioenergetics, and glia-axon interactions, building the framework for understanding how hyperglycemia and dyslipidemia induce bioenergetics failure in DPN. DPN and painful DPN still lack disease-modifying therapies, and research on novel mechanism-based approaches is also covered.

Gut microbiota in a mouse model of obesity and peripheral neuropathy associated with plasma and nerve lipidomics and nerve transcriptomics.

BACKGROUND: Peripheral neuropathy (PN) is a common complication in obesity, prediabetes, and type 2 diabetes, though its pathogenesis remains incompletely understood. In a murine high-fat diet (HFD) obesity model of PN, dietary reversal (HFD-R) to a low-fat standard diet (SD) restores nerve function and the nerve lipidome to normal. As the gut microbiome represents a potential link between dietary fat intake and nerve health, the current study assessed shifts in microbiome community structure by 16S rRNA profiling during the paradigm of dietary reversal (HFD-R) in various gut niches. Dietary fat content (HFD versus SD) was also correlated to gut flora and metabolic and PN phenotypes. Finally, PN-associated microbial taxa that correlated with the plasma and sciatic nerve lipidome and nerve transcriptome were used to identify lipid species and genes intimately related to PN phenotypes. RESULTS: Microbiome structure was altered in HFD relative to SD but rapidly reversed with HFD-R. Specific taxa variants correlating positively with metabolic health associated inversely with PN, while specific taxa negatively linked to metabolic health positively associated with PN. In HFD, PN-associated taxa variants, including Lactobacillus, Lachnoclostridium, and Anaerotruncus, also positively correlated with several lipid species, especially elevated plasma sphingomyelins and sciatic nerve triglycerides. Negative correlations were additionally present with other taxa variants. Moreover, relationships that emerged between specific PN-associated taxa variants and the sciatic nerve transcriptome were related to inflammation, lipid metabolism, and antioxidant defense pathways, which are all established in PN pathogenesis. CONCLUSIONS: The current results indicate that microbiome structure is altered with HFD, and that certain taxa variants correlate with metabolic health and PN. Apparent links between PN-associated taxa and certain lipid species and nerve transcriptome-related pathways additionally provide insight into new targets for microbiota and the associated underlying mechanisms of action in PN. Thus, these findings strengthen the possibility of a gut-microbiome-peripheral nervous system signature in PN and support continuing studies focused on defining the connection between the gut microbiome and nerve health to inform mechanistic insight and therapeutic opportunities. Video Abstract.

A High-Fat Diet Disrupts Nerve Lipids and Mitochondrial Function in Murine Models of Neuropathy.

As the prevalence of prediabetes and type 2 diabetes (T2D) continues to increase worldwide, accompanying complications are also on the rise. The most prevalent complication, peripheral neuropathy (PN), is a complex process which remains incompletely understood. Dyslipidemia is an emerging risk factor for PN in both prediabetes and T2D, suggesting that excess lipids damage peripheral nerves; however, the precise lipid changes that contribute to PN are unknown. To identify specific lipid changes associated with PN, we conducted an untargeted lipidomics analysis comparing the effect of high-fat diet (HFD) feeding on lipids in the plasma, liver, and peripheral nerve from three strains of mice (BL6, BTBR, and BKS). HFD feeding triggered distinct strain- and tissue-specific lipid changes, which correlated with PN in BL6 mice versus less robust murine models of metabolic dysfunction and PN (BTBR and BKS mice). The BL6 mice showed significant changes in neutral lipids, phospholipids, lysophospholipids, and plasmalogens within the nerve. Sphingomyelin (SM) and lysophosphatidylethanolamine (LPE) were two lipid species that were unique to HFD BL6 sciatic nerve compared to other strains (BTBR and BKS). Plasma and liver lipids were significantly altered in all murine strains fed a HFD independent of PN status, suggesting that nerve-specific lipid changes contribute to PN pathogenesis. Many of the identified lipids affect mitochondrial function and mitochondrial bioenergetics, which were significantly impaired in ex vivo sural nerve and dorsal root ganglion sensory neurons. Collectively, our data show that consuming a HFD dysregulates the nerve lipidome and mitochondrial function, which may contribute to PN in prediabetes.

A Role for Fatty Acids in Peripheral Neuropathy Associated with Type 2 Diabetes and Prediabetes.

Significance: As the global prevalence of diabetes rises, diabetic complications are also increasing at an alarming rate. Peripheral neuropathy (PN) is the most prevalent complication of diabetes and prediabetes, and is characterized by progressive sensory loss resulting from nerve damage. While hyperglycemia is the major risk factor for PN in type 1 diabetes (T1D), the metabolic syndrome (MetS) underlies the onset and progression of PN in type 2 diabetes (T2D) and prediabetes. Recent Advances: Recent reports show that dyslipidemia, a MetS component, is strongly associated with PN in T2D and prediabetes. Dyslipidemia is characterized by an abnormal plasma lipid profile with uncontrolled lipid levels, and both clinical and preclinical studies implicate a role for dietary fatty acids (FAs) in PN pathogenesis. Molecular studies further show that saturated and unsaturated FAs differentially regulate the nerve lipid profile and nerve function. Critical Issues: We first review the properties of FAs and the neuroanatomy of the peripheral nervous system (PNS). Second, we discuss clinical and preclinical studies that implicate the involvement of FAs in PN. Third, we summarize the potential effects of FAs on nerve function and lipid metabolism within the peripheral nerves, sensory neurons, and Schwann cells. Future Directions: Future directions will focus on identifying molecular pathways in T2D and prediabetes that are modulated by FAs in PN. Determining pathophysiological mechanisms that underlie the injurious effects of saturated FAs and beneficial properties of unsaturated FAs will provide mechanistic targets for developing new targeted therapies to treat PN associated with T2D and prediabetes. Antioxid. Redox Signal. 37, 560-577.

Plasma Metabolomics and Lipidomics Differentiate Obese Individuals by Peripheral Neuropathy Status.

CONTEXT: Peripheral neuropathy (PN) is a frequent prediabetes and type 2 diabetes (T2D) complication. Multiple clinical studies reveal that obesity and dyslipidemia can also drive PN progression, independent of glycemia, suggesting a complex interplay of specific metabolite and/or lipid species may underlie PN. OBJECTIVE: This work aimed to identify the plasma metabolomics and lipidomics signature that underlies PN in an observational study of a sample of individuals with average class 3 obesity. METHODS: We performed plasma global metabolomics and targeted lipidomics on obese participants with (n = 44) and without PN (n = 44), matched for glycemic status, vs lean nonneuropathic controls (n = 43). We analyzed data by Wilcoxon, logistic regression, partial least squares-discriminant analysis, and group-lasso to identify differential metabolites and lipids by obesity and PN status. We also conducted subanalysis by prediabetes and T2D status. RESULTS: Lean vs obese comparisons, regardless of PN status, identified the most significant differences in gamma-glutamyl and branched-chain amino acid metabolism from metabolomics analysis and triacylglycerols from lipidomics. Stratification by PN status within obese individuals identified differences in polyamine, purine biosynthesis, and benzoate metabolism. Lipidomics found diacylglycerols as the most significant subpathway distinguishing obese individuals by PN status, with additional contributions from phosphatidylcholines, sphingomyelins, ceramides, and dihydroceramides. Stratifying the obese group by glycemic status did not affect discrimination by PN status. CONCLUSION: Obesity may be as strong a PN driver as prediabetes or T2D in a sample of individuals with average class 3 obesity, at least by plasma metabolomics and lipidomics profile. Metabolic and complex lipid pathways can differentiate obese individuals with and without PN, independent of glycemic status.

Plasma lipid metabolites associate with diabetic polyneuropathy in a cohort with type 2 diabetes.

OBJECTIVE: The global rise in type 2 diabetes is associated with a concomitant increase in diabetic complications. Diabetic polyneuropathy is the most frequent type 2 diabetes complication and is associated with poor outcomes. The metabolic syndrome has emerged as a major risk factor for diabetic polyneuropathy; however, the metabolites associated with the metabolic syndrome that correlate with diabetic polyneuropathy are unknown. METHODS: We conducted a global metabolomics analysis on plasma samples from a subcohort of participants from the Danish arm of Anglo-Danish-Dutch study of Intensive Treatment of Diabetes in Primary Care (ADDITION-Denmark) with and without diabetic polyneuropathy versus lean control participants. RESULTS: Compared to lean controls, type 2 diabetes participants had significantly higher HbA1c (p = 0.0028), BMI (p = 0.0004), and waist circumference (p = 0.0001), but lower total cholesterol (p = 0.0001). Out of 991 total metabolites, we identified 15 plasma metabolites that differed in type 2 diabetes participants by diabetic polyneuropathy status, including metabolites belonging to energy, lipid, and xenobiotic pathways, among others. Additionally, these metabolites correlated with alterations in plasma lipid metabolites in type 2 diabetes participants based on neuropathy status. Further evaluating all plasma lipid metabolites identified a shift in abundance, chain length, and saturation of free fatty acids in type 2 diabetes participants. Importantly, the presence of diabetic polyneuropathy impacted the abundance of plasma complex lipids, including acylcarnitines and sphingolipids. INTERPRETATION: Our explorative study suggests that diabetic polyneuropathy in type 2 diabetes is associated with novel alterations in plasma metabolites related to lipid metabolism.

High Dietary Fat Consumption Impairs Axonal Mitochondrial Function In Vivo.

Peripheral neuropathy (PN) is the most common complication of prediabetes and diabetes. PN causes severe morbidity for Type 2 diabetes (T2D) and prediabetes patients, including limb pain followed by numbness resulting from peripheral nerve damage. PN in T2D and prediabetes is associated with dyslipidemia and elevated circulating lipids; however, the molecular mechanisms underlying PN development in prediabetes and T2D are unknown. Peripheral nerve sensory neurons rely on axonal mitochondria to provide energy for nerve impulse conduction under homeostatic conditions. Models of dyslipidemia in vitro demonstrate mitochondrial dysfunction in sensory neurons exposed to elevated levels of exogenous fatty acids. Herein, we evaluated the effect of dyslipidemia on mitochondrial function and dynamics in sensory axons of the saphenous nerve of a male high-fat diet (HFD)-fed murine model of prediabetes to identify mitochondrial alterations that correlate with PN pathogenesis in vivo We found that the HFD decreased mitochondrial membrane potential (MMP) in axonal mitochondria and reduced the ability of sensory neurons to conduct at physiological frequencies. Unlike mitochondria in control axons, which dissipated their MMP in response to increased impulse frequency (from 1 to 50 Hz), HFD mitochondria dissipated less MMP in response to axonal energy demand, suggesting a lack of reserve capacity. The HFD also decreased sensory axonal Ca2+ levels and increased mitochondrial lengthening and expression of PGC1α, a master regulator of mitochondrial biogenesis. Together, these results suggest that mitochondrial dysfunction underlies an imbalance of axonal energy and Ca2+ levels and impairs impulse conduction within the saphenous nerve in prediabetic PN.SIGNIFICANCE STATEMENT Diabetes and prediabetes are leading causes of peripheral neuropathy (PN) worldwide. PN has no cure, but development in diabetes and prediabetes is associated with dyslipidemia, including elevated levels of saturated fatty acids. Saturated fatty acids impair mitochondrial dynamics and function in cultured neurons, indicating a role for mitochondrial dysfunction in PN progression; however, the effect of elevated circulating fatty acids on the peripheral nervous system in vivo is unknown. In this study, we identify early pathogenic events in sensory nerve axons of mice with high-fat diet-induced PN, including alterations in mitochondrial function, axonal conduction, and intra-axonal calcium, that provide important insight into potential PN mechanisms associated with prediabetes and dyslipidemia in vivo.

Cytoplasmic TDP43 Binds microRNAs: New Disease Targets in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, and incurable neurodegenerative disease. Recent studies suggest that dysregulation of gene expression by microRNAs (miRNAs) may play an important role in ALS pathogenesis. The reversible nature of this dysregulation makes miRNAs attractive pharmacological targets and a potential therapeutic avenue. Under physiological conditions, miRNA biogenesis, which begins in the nucleus and includes further maturation in the cytoplasm, involves trans-activation response element DNA/RNA-binding protein of 43 kDa (TDP43). However, TDP43 mutations or stress trigger TDP43 mislocalization and inclusion formation, a hallmark of most ALS cases, that may lead to aberrant protein/miRNA interactions in the cytoplasm. Herein, we demonstrated that TDP43 exhibits differential binding affinity for select miRNAs, which prompted us to profile miRNAs that preferentially bind cytoplasmic TDP43. Using cellular models expressing TDP43 variants and miRNA profiling analyses, we identified differential levels of 65 cytoplasmic TDP43-associated miRNAs. Of these, approximately 30% exhibited levels that differed by more than 3-fold in the cytoplasmic TDP43 models relative to our control model. The hits included both novel miRNAs and miRNAs previously associated with ALS that potentially regulate several predicted genes and pathways that may be important for pathogenesis. Accordingly, these findings highlight specific miRNAs that may shed light on relevant disease pathways and could represent potential biomarkers and reversible treatment targets for ALS.

Evolving concepts on the role of dyslipidemia, bioenergetics, and inflammation in the pathogenesis and treatment of diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is one of the most widespread and disabling neurological conditions, accounting for half of all neuropathy cases worldwide. Despite its high prevalence, no approved disease modifying therapies exist. There is now a growing body of evidence that DPN secondary to type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) represents different disease processes, with T2DM DPN best understood within the context of metabolic syndrome rather than hyperglycemia. In this review, we highlight currently understood mechanisms of DPN, along with their corresponding potential therapeutic targets. We frame this discussion within a practical overview of how the field evolved from initial human observations to murine pathomechanistic and therapeutic models into ongoing and human clinical trials, with particular emphasis on T2DM DPN and metabolic syndrome.

Integrated lipidomic and transcriptomic analyses identify altered nerve triglycerides in mouse models of prediabetes and type 2 diabetes.

Peripheral neuropathy (PN) is a complication of prediabetes and type 2 diabetes (T2D). Increasing evidence suggests that factors besides hyperglycaemia contribute to PN development, including dyslipidaemia. The objective of this study was to determine differential lipid classes and altered gene expression profiles in prediabetes and T2D mouse models in order to identify the dysregulated pathways in PN. Here, we used high-fat diet (HFD)-induced prediabetes and HFD/streptozotocin (STZ)-induced T2D mouse models that develop PN. These models were compared to HFD and HFD-STZ mice that were subjected to dietary reversal. Both untargeted and targeted lipidomic profiling, and gene expression profiling were performed on sciatic nerves. Lipidomic and transcriptomic profiles were then integrated using complex correlation analyses, and biological meaning was inferred from known lipid-gene interactions in the literature. We found an increase in triglycerides (TGs) containing saturated fatty acids. In parallel, transcriptomic analysis confirmed the dysregulation of lipid pathways. Integration of lipidomic and transcriptomic analyses identified an increase in diacylglycerol acyltransferase 2 (DGAT2), the enzyme required for the last and committed step in TG synthesis. Increased DGAT2 expression was present not only in the murine models but also in sural nerve biopsies from hyperlipidaemic diabetic patients with PN. Collectively, these findings support the hypothesis that abnormal nerve-lipid signalling is an important factor in peripheral nerve dysfunction in both prediabetes and T2D.This article has an associated First Person interview with the joint first authors of the paper.

Disorders of mitochondrial dynamics in peripheral neuropathy: Clues from hereditary neuropathy and diabetes.

Peripheral neuropathy is a common and debilitating complication of diabetes and prediabetes. Recent clinical studies have identified an association between the development of neuropathy and dyslipidemia in prediabetic and diabetic patients. Despite the prevalence of this complication, studies identifying molecular mechanisms that underlie neuropathy progression in prediabetes or diabetes are limited. However, dysfunctional mitochondrial pathways in hereditary neuropathy provide feasible molecular targets for assessing mitochondrial dysfunction in neuropathy associated with prediabetes or diabetes. Recent studies suggest that elevated levels of dietary saturated fatty acids (SFAs) associated with dyslipidemia impair mitochondrial dynamics in sensory neurons by inducing mitochondrial depolarization, compromising mitochondrial bioenergetics, and impairing axonal mitochondrial transport. This causes lower neuronal ATP and apoptosis. Conversely, monounsaturated fatty acids (MUFAs) restore nerve and sensory mitochondrial function. Understanding the mitochondrial pathways that contribute to neuropathy progression in prediabetes and diabetes may provide therapeutic targets for the treatment of this debilitating complication.

The Divergent Roles of Dietary Saturated and Monounsaturated Fatty Acids on Nerve Function in Murine Models of Obesity.

Neuropathy is the most common complication of prediabetes and diabetes and presents as distal-to-proximal loss of peripheral nerve function in the lower extremities. Neuropathy progression and disease severity in prediabetes and diabetes correlates with dyslipidemia in man and murine models of disease. Dyslipidemia is characterized by elevated levels of circulating saturated fatty acids (SFAs) that associate with the progression of neuropathy. Increased intake of monounsaturated fatty acid (MUFA)-rich diets confers metabolic health benefits; however, the impact of fatty acid saturation in neuropathy is unknown. This study examines the differential effect of SFAs and MUFAs on the development of neuropathy and the molecular mechanisms underlying the progression of the complication. Male mice Mus musculus fed a high-fat diet rich in SFAs developed robust peripheral neuropathy. This neuropathy was completely reversed by switching the mice from the SFA-rich high-fat diet to a MUFA-rich high-fat diet; nerve conduction velocities and intraepidermal nerve fiber density were restored. A MUFA oleate also prevented the impairment of mitochondrial transport and protected mitochondrial membrane potential in cultured sensory neurons treated with mixtures of oleate and the SFA palmitate. Moreover, oleate also preserved intracellular ATP levels, prevented apoptosis induced by palmitate treatment, and promoted lipid droplet formation in sensory neurons, suggesting that lipid droplets protect sensory neurons from lipotoxicity. Together, these results suggest that MUFAs reverse the progression of neuropathy by protecting mitochondrial function and transport through the formation of intracellular lipid droplets in sensory neurons.SIGNIFICANCE STATEMENT There is a global epidemic of prediabetes and diabetes, disorders that represent a continuum of metabolic disturbances in lipid and glucose metabolism. In the United States, 80 million individuals have prediabetes and 30 million have diabetes. Neuropathy is the most common complication of both disorders, carries a high morbidity, and, despite its prevalence, has no treatments. We report that dietary intervention with monounsaturated fatty acids reverses the progression of neuropathy and restores nerve function in high-fat diet-fed murine models of peripheral neuropathy. Furthermore, the addition of the monounsaturated fatty acid oleate to sensory neurons cultured under diabetic conditions shows that oleate prevents impairment of mitochondrial transport and mitochondrial dysfunction through a mechanism involving formation of axonal lipid droplets.

Mitochondrial uncoupling has no effect on microvascular complications in type 2 diabetes.

Diabetic peripheral neuropathy (DPN), diabetic kidney disease (DKD), and diabetic retinopathy (DR) contribute to significant morbidity and mortality in diabetes patients. The incidence of these complications is increasing with the diabetes epidemic, and current therapies minimally impact their pathogenesis in type 2 diabetes (T2D). Improved mechanistic understanding of each of the diabetic complications is needed in order to develop disease-modifying treatments for patients. We recently identified fundamental differences in mitochondrial responses of peripheral nerve, kidney, and retinal tissues to T2D in BKS-db/db mice. However, whether these mitochondrial adaptations are the cause or consequence of tissue dysfunction remains unclear. In the current study BKS-db/db mice were treated with the mitochondrial uncoupler, niclosamide ethanolamine (NEN), to determine the effects of mitochondrial uncoupling therapy on T2D, and the pathogenesis of DPN, DKD and DR. Here we report that NEN treatment from 6-24 wk of age had little effect on the development of T2D and diabetic complications. Our data suggest that globally targeting mitochondria with an uncoupling agent is unlikely to provide therapeutic benefit for DPN, DKD, or DR in T2D. These data also highlight the need for further insights into the role of tissue-specific metabolic reprogramming in the pathogenesis of diabetic complications.

Chain length of saturated fatty acids regulates mitochondrial trafficking and function in sensory neurons.

Dyslipidemia associated with T2D leads to diabetic neuropathy, a complication characterized by sensory neuronal dysfunction and peripheral nerve damage. Sensory dorsal root ganglion (DRG) neurons are dependent on axonal mitochondrial energy production facilitated by mitochondrial transport mechanisms that distribute mitochondria throughout the axon. Because long-chain saturated FAs (SFAs) damage DRG neurons and medium-chain SFAs are reported to improve neuronal function, we evaluated the impact of SFA chain length on mitochondrial trafficking, mitochondrial function, and apoptosis. DRG neurons were exposed to SFAs with C12:0-C18:0 chain lengths and evaluated for changes in mitochondrial trafficking, mitochondrial polarization, and apoptosis. DRG neurons treated with C16:0 and C18:0 SFAs showed a significant decrease in the percentage of motile mitochondria and velocity of mitochondrial trafficking, whereas C12:0 and C14:0 SFAs had no impact on motility. Treatment with C16:0 and C18:0 SFAs exhibited mitochondrial depolarization correlating with impaired mitochondrial motility; the C12:0- and C14:0-treated neurons retained mitochondrial polarization. The reduction in mitochondrial trafficking and function in C16:0- and C18:0-treated DRG neurons correlated with apoptosis that was blocked in C12:0 and C14:0 SFA treatments. These results suggest that SFA chain length plays an important role in regulating axonal mitochondrial trafficking and function in DRG neurons.

Conserved Transcriptional Signatures in Human and Murine Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes. In this study, we employed a systems biology approach to identify DPN-related transcriptional pathways conserved across human and various murine models. Eight microarray datasets on peripheral nerve samples from murine models of type 1 (streptozotocin-treated) and type 2 (db/db and ob/ob) diabetes of various ages and human subjects with non-progressive and progressive DPN were collected. Differentially expressed genes (DEGs) were identified between non-diabetic and diabetic samples in murine models, and non-progressive and progressive human samples using a unified analysis pipeline. A transcriptional network for each DEG set was constructed based on literature-derived gene-gene interaction information. Seven pairwise human-vs-murine comparisons using a network-comparison program resulted in shared sub-networks including 46 to 396 genes, which were further merged into a single network of 688 genes. Pathway and centrality analyses revealed highly connected genes and pathways including LXR/RXR activation, adipogenesis, glucocorticoid receptor signalling, and multiple cytokine and chemokine pathways. Our systems biology approach identified highly conserved pathways across human and murine models that are likely to play a role in DPN pathogenesis and provide new possible mechanism-based targets for DPN therapy.

Juvenile murine models of prediabetes and type 2 diabetes develop neuropathy.

Peripheral neuropathy (neuropathy) is a common complication of obesity and type 2 diabetes in children and adolescents. To model this complication in mice, 5-week-old male C57BL/6J mice were fed a high-fat diet to induce diet-induced obesity (DIO), a model of prediabetes, and a cohort of these animals was injected with low-dose streptozotocin (STZ) at 12 weeks of age to induce hyperglycemia and type 2 diabetes. Neuropathy assessments at 16, 24 and 36 weeks demonstrated that DIO and DIO-STZ mice displayed decreased motor and sensory nerve conduction velocities as early as 16 weeks, hypoalgesia by 24 weeks and cutaneous nerve fiber loss by 36 weeks, relative to control mice fed a standard diet. Interestingly, neuropathy severity was similar in DIO and DIO-STZ mice at all time points despite significantly higher fasting glucose levels in the DIO-STZ mice. These mouse models provide critical tools to better understand the underlying pathogenesis of prediabetic and diabetic neuropathy from youth to adulthood, and support the idea that hyperglycemia alone does not drive early neuropathy.This article has an associated First Person interview with the first author of the paper.

Transcriptional networks of progressive diabetic peripheral neuropathy in the db/db mouse model of type 2 diabetes: An inflammatory story.

Diabetic peripheral neuropathy is the most common complication of diabetes and a source of considerable morbidity. Numerous molecular pathways are linked to neuropathic progression, but it is unclear whether these pathways are altered throughout the course of disease. Moreover, the methods by which these molecular pathways are analyzed can produce significantly different results; as such it is often unclear whether previously published pathways are viable targets for novel therapeutic approaches. In the current study we examine changes in gene expression patterns in the sciatic nerve (SCN) and dorsal root ganglia (DRG) of db/db diabetic mice at 8, 16, and 24 weeks of age using microarray analysis. Following the collection and verification of gene expression data, we utilized both self-organizing map (SOM) analysis and differentially expressed gene (DEG) analysis to detect pathways that were altered at all time points. Though there was some variability between SOM and DEG analyses, we consistently detected altered immune pathways in both the SCN and DRG over the course of disease. To support these results, we further used multiplex analysis to assess protein changes in the SCN of diabetic mice; we found that multiple immune molecules were upregulated at both early and later stages of disease. In particular, we found that matrix metalloproteinase-12 was highly upregulated in microarray and multiplex data sets suggesting it may play a role in disease progression.

Dyslipidemia impairs mitochondrial trafficking and function in sensory neurons.

Mitochondrial trafficking plays a central role in dorsal root ganglion (DRG) neuronal cell survival and neurotransmission by transporting mitochondria from the neuronal cell body throughout the bundles of DRG axons. In type 2 diabetes (T2DM), dyslipidemia and hyperglycemia damage DRG neurons and induce mitochondrial dysfunction; however, the impact of free fatty acids and glucose on mitochondrial trafficking in DRG neurons remains unknown. To evaluate the impact of free fatty acids compared to hyperglycemia on mitochondrial transport, primary adult mouse DRG neuron cultures were treated with physiologic concentrations of palmitate and glucose and assessed for alterations in mitochondrial trafficking, mitochondrial membrane potential, and mitochondrial bioenergetics. Palmitate treatment significantly reduced the number of motile mitochondria in DRG axons, but physiologic concentrations of glucose did not impair mitochondrial trafficking dynamics. Palmitate-treated DRG neurons also exhibited a reduction in mitochondrial velocity, and impaired mitochondrial trafficking correlated with mitochondrial depolarization in palmitate-treated DRG neurons. Finally, we found differential bioenergetic effects of palmitate and glucose on resting and energetically challenged mitochondria in DRG neurons. Together, these results suggest that palmitate induces DRG neuron mitochondrial depolarization, inhibiting axonal mitochondrial trafficking and altering mitochondrial bioenergetic capacity.-Rumora, A. E., Lentz, S. I., Hinder, L. M., Jackson, S. W., Valesano, A., Levinson, G. E., Feldman, E. L. Dyslipidemia impairs mitochondrial trafficking and function in sensory neurons.

Comparative RNA-Seq transcriptome analyses reveal distinct metabolic pathways in diabetic nerve and kidney disease.

Treating insulin resistance with pioglitazone normalizes renal function and improves small nerve fibre function and architecture; however, it does not affect large myelinated nerve fibre function in mouse models of type 2 diabetes (T2DM), indicating that pioglitazone affects the body in a tissue-specific manner. To identify distinct molecular pathways regulating diabetic peripheral neuropathy (DPN) and nephropathy (DN), as well those affected by pioglitazone, we assessed DPN and DN gene transcript expression in control and diabetic mice with or without pioglitazone treatment. Differential expression analysis and self-organizing maps were then used in parallel to analyse transcriptome data. Differential expression analysis showed that gene expression promoting cell death and the inflammatory response was reversed in the kidney glomeruli but unchanged or exacerbated in sciatic nerve by pioglitazone. Self-organizing map analysis revealed that mitochondrial dysfunction was normalized in kidney and nerve by treatment; however, conserved pathways were opposite in their directionality of regulation. Collectively, our data suggest inflammation may drive large fibre dysfunction, while mitochondrial dysfunction may drive small fibre dysfunction in T2DM. Moreover, targeting both of these pathways is likely to improve DN. This study supports growing evidence that systemic metabolic changes in T2DM are associated with distinct tissue-specific metabolic reprogramming in kidney and nerve and that these changes play a critical role in DN and small fibre DPN pathogenesis. These data also highlight the potential dangers of a 'one size fits all' approach to T2DM therapeutics, as the same drug may simultaneously alleviate one complication while exacerbating another.

Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element-Binding Protein B.

Myofibroblast differentiation is characterized by an increased level of expression of cytoskeletal smooth muscle α-actin. In human and murine fibroblasts, the gene encoding smooth muscle α-actin (Acta2) is tightly regulated by a network of transcription factors that either activate or repress the 5' promoter-enhancer in response to environmental cues signaling tissue repair and remodeling. Purine-rich element-binding protein B (Purß) suppresses the expression of Acta2 by cooperatively interacting with the sense strand of a 5' polypurine sequence containing an inverted MCAT cis element required for gene activation. In this study, we evaluated the chemical basis of nucleoprotein complex formation between the Purß repressor and the purine-rich strand of the MCAT element in the mouse Acta2 promoter. Quantitative single-stranded DNA (ssDNA) binding assays conducted in the presence of increasing concentrations of monovalent salt or anionic detergent suggested that the assembly of a high-affinity nucleoprotein complex is driven by a combination of electrostatic and hydrophobic interactions. Consistent with the results of pH titration analysis, site-directed mutagenesis revealed several basic amino acid residues in the intermolecular (R267) and intramolecular (K82 and R159) subdomains that are essential for Purß transcriptional repressor function in Acta2 promoter-reporter assays. In keeping with their diminished Acta2 repressor activity in fibroblasts, purified Purß variants containing an R267A mutation exhibited reduced binding affinity for purine-rich ssDNA. Moreover, certain double and triple-point mutants were also defective in binding to the Acta2 corepressor protein, Y-box-binding protein 1. Collectively, these findings establish the repertoire of noncovalent interactions that account for the unique structural and functional properties of Purß.