Small cohort studies raise the hypothesis that corneal nerve abnormalities (including corneal nerve fibre length [CNFL]) are valid non-invasive imaging endpoints for diabetic sensorimotor polyneuropathy (DSP). We aimed to establish concurrent validity and diagnostic thresholds in a large cohort of participants with and without DSP.

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.

Neurochemical monitoring via sampling probes is valuable for deciphering neurotransmission in vivo. Microdialysis is commonly used; however, the spatial resolution is poor.

This study focused on altered mitochondrial dynamics as a potential mechanism for diabetic peripheral neuropathy (DPN). We employed both an in vitro sensory neuron model and an in situ analysis of human intraepidermal nerve fibers (IENFs) from cutaneous biopsies to measure alterations in the size distribution of mitochondria as a result of hyperglycemia and diabetes, respectively.

Obesity, prediabetes, and diabetes are growing in prevalence worldwide. These metabolic disorders are associated with neurodegenerative diseases, particularly Alzheimer's disease and Alzheimer's disease related dementias. Innate inflammatory signaling plays a critical role in this association, potentially via the early activation of the cGAS/STING pathway. To determine acute systemic metabolic and inflammatory responses and corresponding changes in the brain, we used a high fat diet fed obese mouse model of prediabetes and cognitive impairment. We observed acute systemic changes in metabolic and inflammatory responses, with impaired glucose tolerance, insulin resistance, and alterations in peripheral immune cell populations. Central inflammatory changes included microglial activation in a pro-inflammatory environment with cGAS/STING activation. Blocking gap junctions in neuron-microglial co-cultures significantly decreased cGAS/STING activation. Collectively these studies suggest a role for early activation of the innate immune system both peripherally and centrally with potential inflammatory crosstalk between neurons and glia.

The free calcium (Ca2+) levels in pancreatic beta cell organelles have been the subject of many recent investigations. Under pathophysiological conditions, disturbances in these pools have been linked to altered intracellular communication and cellular dysfunction. To facilitate studies of subcellular Ca2+ signaling in beta cells and, particularly, signaling between the endoplasmic reticulum (ER) and mitochondria, we designed a novel dual Ca2+ sensor which we termed DS-1. DS-1 encodes two stoichiometrically fluorescent proteins within a single plasmid, G-CEPIA-er, targeted to the ER and R-CEPIA3-mt, targeted to mitochondria. Our goal was to simultaneously measure the ER and mitochondrial Ca2+ in cells in real time. The Kds of G-CEPIA-er and R-CEPIA3-mt for Ca2+ are 672 and 3.7 μM, respectively. Confocal imaging of insulin-secreting INS-1 832/13 expressing DS-1 confirmed that the green and red fluorophores correctly colocalized with organelle-specific fluorescent markers as predicted. Further, we tested whether DS-1 exhibited the functional properties expected by challenging an INS-1 cell to glucose concentrations or drugs having well-documented effects on the ER and mitochondrial Ca2+ handling. The data obtained were consistent with those seen using other single organelle targeted probes. These results taken together suggest that DS-1 is a promising new approach for investigating Ca2+ signaling within multiple organelles of the cell.

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.

Members of the Rab3 gene family are considered central to membrane trafficking of synaptic vesicles at mammalian central excitatory synapses. Recent evidence, however, indicates that the Rab27B-GTPase, which is highly homologous to the Rab3 family, is also enriched on SV membranes and co-localize with Rab3A and Synaptotagmin at presynaptic terminals. While functional roles of Rab3A have been well-established, little functional information exists on the role of Rab27B in synaptic transmission. Here we report on functional effects of Rab27B at SC-CA1 and DG-MF hippocampal synapses. The data establish distinct functional actions of Rab27B and demonstrate functions of Rab27B that differ between SC-CA1 and DG-MF synapses. Rab27B knockout reduced frequency facilitation compared to wild-type (WT) controls at the DG/MF-CA3 synaptic region, while increasing facilitation at the SC-CA1 synaptic region. Remarkably, Rab27B KO resulted in a complete elimination of LTP at the MF-CA3 synapse with no effect at the SC-CA1 synapse. These actions are similar to those previously reported for Rab3A KO. Specificity of action on LTP to Rab27B was confirmed as LTP was rescued in response to lentiviral infection and expression of human Rab27B, but not to GFP, in the DG in the Rab27B KO mice. Notably, the effect of Rab27B KO on MF-CA3 LTP occurred in spite of continued expression of Rab3A in the Rab27B KO. Overall, the results provide a novel perspective in suggesting that Rab27B and Rab3A act synergistically, perhaps via sequential effector recruitment or signaling for presynaptic LTP expression in this hippocampal synaptic region.

Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

A relationship between the actions of TSH and IGF-1 was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR and IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the noncanonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunofluorescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without and with intact TSHR expression. IGF-1 treatment of TSHR(+/+) fibroblasts resulted in reduced nuclear and cytoplasmic staining for IGF-1Rα, whereas it enhanced the nuclear signal in TSHR(-/-) cells. In contrast, IGF-1 enhanced cytoplasmic IGF-1Rβ in TSHR(-/-) fibroblasts while increasing the nuclear signal in TSHR(+/+) cells. These findings indicate the intimate relationship between TSHR and IGF-1R found earlier in human orbital fibroblasts also exists in mouse lung fibroblasts. Furthermore, the presence of TSHR in these fibroblasts influenced not only the levels of IGF-1R protein but also its subcellular distribution and response to IGF-1. They suggest that the mouse might serve as a suitable model for delineating the molecular mechanisms overarching these two receptors.

Diabetic neuropathy (DN) is a debilitating complication of type 1 and type 2 diabetes. Rodent models of DN do not fully replicate the pathology observed in human patients. We examined DN in streptozotocin (STZ)-induced [B6] and spontaneous type 1 diabetes [B6Ins2(Akita)] and spontaneous type 2 diabetes [B6-db/db, BKS-db/db]. Despite persistent hyperglycemia, the STZ-treated B6 and B6Ins2(Akita) mice were resistant to the development of DN. In contrast, DN developed in both type 2 diabetes models: the B6-db/db and BKS-db/db mice. The persistence of hyperglycemia and development of DN in the B6-db/db mice required an increased fat diet while the BKS-db/db mice developed severe DN and remained hyperglycemic on standard mouse chow. Our data support the hypothesis that genetic background and diet influence the development of DN and should be considered when developing new models of DN.

The conserved protein kinase mTOR (mechanistic target of rapamycin) responds to diverse environmental cues to control cell metabolism and promote cell growth, proliferation, and survival as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. Our prior work demonstrated that an alkaline intracellular pH (pHi) increases mTORC2 activity and cell survival in complete media in part by activating AMP-activated protein kinase, a kinase best known to sense energetic stress. It is important to note that an alkaline pHi represents an underappreciated hallmark of cancer cells that promotes their oncogenic behaviors. In addition, mechanisms that control mTORC1 and mTORC2 signaling and function remain incompletely defined, particularly in response to stress conditions. Here, we demonstrate that an alkaline pHi increases phosphatidylinositide 3-kinase (PI3K) activity to promote mTORC1 and mTORC2 signaling in the absence of serum growth factors. Alkaline pHi increases mTORC1 activity through PI3K-Akt signaling, which mediates inhibitory phosphorylation of the upstream proteins tuberous sclerosis complex 2 and proline-rich Akt substrate of 40 kDa and dissociates tuberous sclerosis complex from lysosomal membranes, thus enabling Rheb-mediated activation of mTORC1. Thus, alkaline pHi mimics growth factor-PI3K signaling. Functionally, we also demonstrate that an alkaline pHi increases cap-dependent protein synthesis through inhibitory phosphorylation of eIF4E binding protein 1 and suppresses apoptosis in a PI3K- and mTOR-dependent manner. We speculate that an alkaline pHi promotes a low basal level of cell metabolism (e.g., protein synthesis) that enables cancer cells within growing tumors to proliferate and survive despite limiting growth factors and nutrients, in part through elevated PI3K-mTORC1 and/or PI3K-mTORC2 signaling.

The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.

To quantify peripheral nerve lesions in symptomatic and asymptomatic hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PNP) by analyzing the magnetization transfer ratio (MTR) of the sciatic nerve, and to test its potential as a novel biomarker for macromolecular changes.

Congenital erythropoietic porphyria (CEP) is a rare genetic disorder leading to accumulation of uro/coproporphyrin-I in tissues due to inhibition of uroporphyrinogen-III synthase. Clinical manifestations of CEP include bone fragility, severe photosensitivity and photomutilation. Currently there is no specific treatment for CEP, except bone marrow transplantation, and there is an unmet need for treating this orphan disease. Fluorescent porphyrins cause protein aggregation, which led us to hypothesize that uroporphyrin-I accumulation leads to protein aggregation and CEP-related bone phenotype. We developed a zebrafish model that phenocopies features of CEP. As in human patients, uroporphyrin-I accumulated in the bones of zebrafish, leading to impaired bone development. Furthermore, in an osteoblast-like cell line, uroporphyrin-I decreased mineralization, aggregated bone matrix proteins, activated endoplasmic reticulum stress and disrupted autophagy. Using high-throughput drug screening, we identified acitretin, a second-generation retinoid, and showed that it reduced uroporphyrin-I accumulation and its deleterious effects on bones. Our findings provide a new CEP experimental model and a potential repurposed therapeutic.