Accelerated onset of diabetes in non-obese diabetic mice fed a refined high-fat diet.

AIM: Type 1 diabetes results from autoimmune events influenced by environmental variables, including changes in diet. This study investigated how feeding refined versus unrefined (aka 'chow') diets affects the onset and progression of hyperglycaemia in non-obese diabetic (NOD) mice. METHODS: Female NOD mice were fed either unrefined diets or matched refined low- and high-fat diets. The onset of hyperglycaemia, glucose tolerance, food intake, energy expenditure, circulating insulin, liver gene expression and microbiome changes were measured for each dietary group. RESULTS: NOD mice consuming unrefined (chow) diets developed hyperglycaemia at similar frequencies. By contrast, mice consuming the defined high-fat diet had an accelerated onset of hyperglycaemia compared to the matched low-fat diet. There was no change in food intake, energy expenditure, or physical activity within each respective dietary group. Microbiome changes were driven by diet type, with chow diets clustering similarly, while refined low- and high-fat bacterial diversity also grouped closely. In the defined dietary cohort, liver gene expression changes in high-fat-fed mice were consistent with a greater frequency of hyperglycaemia and impaired glucose tolerance. CONCLUSION: Glucose intolerance is associated with an enhanced frequency of hyperglycaemia in female NOD mice fed a defined high-fat diet. Using an appropriate matched control diet is an essential experimental variable when studying changes in microbiome composition and diet as a modifier of disease risk.

ICAM-1 Abundance Is Increased in Pancreatic Islets of Hyperglycemic Female NOD Mice and Is Rapidly Upregulated by NF-κB in Pancreatic ß-Cells.

Type 1 diabetes (T1D) is classified as an autoimmune disease where pancreatic ß-cells are specifically targeted by cells of the immune system. The molecular mechanisms underlying this process are not completely understood. Herein, we identified that the Icam1 gene and ICAM-1 protein were selectively elevated in female NOD mice relative to male mice, fitting with the sexual dimorphism of diabetes onset in this key mouse model of T1D. In addition, ICAM-1 abundance was greater in hyperglycemic female NOD mice than in age-matched normoglycemic female NOD mice. Moreover, we discovered that the Icam1 gene was rapidly upregulated in response to IL-1ß in mouse, rat, and human islets and in 832/13 rat insulinoma cells. This early temporal genetic regulation requires key components of the NF-κB pathway and was associated with rapid recruitment of the p65 transcriptional subunit of NF-κB to corresponding κB elements within the Icam1 gene promoter. In addition, RNA polymerase II recruitment to the Icam1 gene promoter in response to IL-1ß was consistent with p65 occupancy at κB elements, histone chemical modifications, and increased mRNA abundance. Thus, we conclude that ß-cells undergo rapid genetic reprogramming by IL-1ß to enhance expression of the Icam1 gene and that elevations in ICAM-1 are associated with hyperglycemia in NOD mice. These findings are highly relevant to, and highlight the importance of, pancreatic ß-cell communication with the immune system. Collectively, these observations reveal a portion of the complex molecular events associated with onset and progression of T1D.

Channel Expansion in the Ligand-Binding Domain of the Glucocorticoid Receptor Contributes to the Activity of Highly Potent Glucocorticoid Analogues.

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR) and are commonly used as anti-inflammatory and immunosuppressant medications. Chronic GC use has been linked with unwanted complications such as steroid-induced diabetes mellitus (SIDM), although the mechanisms for these effects are not completely understood. Modification of six GC parent molecules with 2-mercaptobenzothiazole resulted in consistently less promoter activity in transcriptional activation assays using a 3xGRE reporter construct while constantly reducing inflammatory pathway activity. The most selective candidate, DX1, demonstrated a significant reduction (87%) in transactivation compared to commercially available dexamethasone. DX1 also maintained 90% of the anti-inflammatory potential of dexamethasone while simultaneously displaying a reduced toxicity profile. Additionally, two novel and highly potent compounds, DX4 and PN4, were developed and shown to elicit similar mRNA expression at attomolar concentrations that dexamethasone exhibits at nanomolar dosages. To further explain these results, Molecular Dynamic (MD) simulations were performed to examine structural changes in the ligand-binding domain of the glucocorticoid receptor in response to docking with the top ligands. Differing interactions with the transcriptional activation function 2 (AF-2) region of the GR may be responsible for lower transactivation capacity in DX1. DX4 and PN4 lose contact with Arg611 due to a key interaction changing from a stronger hydrophilic to a weaker hydrophobic one, which leads to the formation of an unoccupied channel at the location of the deacylcortivazol (DAC)-expanded binding pocket. These findings provide insights into the structure-function relationships important for regulating anti-inflammatory activity, which has implications for clinical utility.

NOD mice have distinct metabolic and immunologic profiles when compared with genetically similar MHC-matched ICR mice.

Nonobese diabetic (NOD) mice are the most commonly used rodent model to study mechanisms relevant to the autoimmunity and immunology of type 1 diabetes. Although many different strains of mice have been used as controls for studies comparing nondiabetic lines to the NOD strain, we hypothesized that the parental strain that gave rise to the NOD line might be one of the best options. Therefore, we compared female ICR and NOD mice, which are matched at key major histocompatibility complex (MHC) loci, to understand their metabolic and immunologic similarities and differences. Several novel observations emerged: 1) NOD mice have greater circulating proinsulin when compared with ICR mice. 2) NOD mice display CD3+ and IBA1+ cell infiltration into and near pancreatic islets before hyperglycemia. 3) NOD mice show increased expression of the Il1b and Cxcl11 genes in islets when compared with islets from age-matched ICR mice. 4) NOD mice have a greater abundance of STAT1 and ICAM-1 protein in islets when compared with ICR mice. These data show that ICR mice, which are genetically similar to NOD mice, do not retain the same immunologic outcomes. Thus, ICR mice are an excellent choice as a genetically similar and MHC-matched control for NOD mice in studies designed to understand mechanisms relevant to autoimmune-mediated diabetes onset as well as novel therapeutic interventions.NEW & NOTEWORTHY Nonobese diabetic (NOD) mice have more proinsulin in circulation and STAT1 protein in islets compared with the major histocompatibility complex (MHC)-matched ICR line. NOD mice also display greater expression of cytokines and chemokines in pancreatic islets consistent with immune cell infiltration before hyperglycemia when compared with age-matched ICR mice. Thus, ICR mice represent an excellent control for autoimmunity and inflammation studies using the NOD line of mice.

Hepatic IKKε expression is dispensable for high-fat feeding-induced increases in liver lipid content and alterations in glucose tolerance.

There are endocrine and immunological changes that occur during onset and progression of the overweight and obese states. The inhibitor of nuclear factor-κB kinase-ε (IKKε) was originally described as an inducible protein kinase; whole body gene deletion or systemic pharmaceutical targeting of this kinase improved insulin sensitivity and glucose tolerance in mice. To investigate the primary sites of action associated with IKKε during weight gain, we describe the first mouse line with conditional elimination of IKKε in the liver (IKKεAlb-/-). IKKεAlb-/- mice and littermate controls gain weight, show similar changes in body composition, and do not display any improvements in insulin sensitivity or whole body glucose tolerance. These studies were conducted using breeder chow diets and matched low- vs. high-fat diets. While glycogen accumulation in the liver is reduced in IKKεAlb-/- mice, lipid storage in liver is similar in IKKεAlb-/- mice and littermate controls. Our results using IKKεAlb-/- mice suggest that the primary action of this kinase to impact insulin sensitivity during weight gain lies predominantly within extrahepatic tissues.

Evidence that hindbrain astrocytes in the rat detect low glucose with a glucose transporter 2-phospholipase C-calcium release mechanism.

Astrocytes generate robust cytoplasmic calcium signals in response to reductions in extracellular glucose. This calcium signal, in turn, drives purinergic gliotransmission, which controls the activity of catecholaminergic (CA) neurons in the hindbrain. These CA neurons are critical to triggering glucose counter-regulatory responses (CRRs) that, ultimately, restore glucose homeostasis via endocrine and behavioral means. Although the astrocyte low-glucose sensor involvement in CRR has been accepted, it is not clear how astrocytes produce an increase in intracellular calcium in response to a decrease in glucose. Our ex vivo calcium imaging studies of hindbrain astrocytes show that the glucose type 2 transporter (GLUT2) is an essential feature of the astrocyte glucosensor mechanism. Coimmunoprecipitation assays reveal that the recombinant GLUT2 binds directly with the recombinant Gq protein subunit that activates phospholipase C (PLC). Additional calcium imaging studies suggest that GLUT2 may be connected to a PLC-endoplasmic reticular-calcium release mechanism, which is amplified by calcium-induced calcium release (CICR). Collectively, these data help outline a potential mechanism used by astrocytes to convert information regarding low-glucose levels into intracellular changes that ultimately regulate the CRR.

Oral Corticosterone Administration Reduces Insulitis but Promotes Insulin Resistance and Hyperglycemia in Male Nonobese Diabetic Mice.

Steroid-induced diabetes is the most common form of drug-induced hyperglycemia. Therefore, metabolic and immunological alterations associated with chronic oral corticosterone were investigated using male nonobese diabetic mice. Three weeks after corticosterone delivery, there was reduced sensitivity to insulin action measured by insulin tolerance test. Body composition measurements revealed increased fat mass and decreased lean mass. Overt hyperglycemia (>250 mg/dL) manifested 6 weeks after the start of glucocorticoid administration, whereas 100% of the mice receiving the vehicle control remained normoglycemic. This phenotype was fully reversed during the washout phase and readily reproducible across institutions. Relative to the vehicle control group, mice receiving corticosterone had a significant enhancement in pancreatic insulin-positive area, but a marked decrease in CD3+ cell infiltration. In addition, there were striking increases in both citrate synthase gene expression and enzymatic activity in skeletal muscle of mice in the corticosterone group relative to vehicle control. Moreover, glycogen synthase expression was greatly enhanced, consistent with elevations in muscle glycogen storage in mice receiving corticosterone. Corticosterone-induced hyperglycemia, insulin resistance, and changes in muscle gene expression were all reversed by the end of the washout phase, indicating that the metabolic alterations were not permanent. Thus, male nonobese diabetic mice allow for translational studies on the metabolic and immunological consequences of glucocorticoid-associated interventions in a mouse model with genetic susceptibility to autoimmune disease.

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet ß-Cell Function.

Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet ß-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.

CCL20 is elevated during obesity and differentially regulated by NF-κB subunits in pancreatic ß-cells.

Enhanced leukocytic infiltration into pancreatic islets contributes to inflammation-based diminutions in functional ß-cell mass. Insulitis (aka islet inflammation), which can be present in both T1DM and T2DM, is one factor influencing pancreatic ß-cell death and dysfunction. IL-1ß, an inflammatory mediator in both T1DM and T2DM, acutely (within 1h) induced expression of the CCL20 gene in rat and human islets and clonal ß-cell lines. Transcriptional induction of CCL20 required the p65 subunit of NF-κB to replace the p50 subunit at two functional κB sites within the CCL20 proximal gene promoter. The NF-κB p50 subunit prevents CCL20 gene expression during unstimulated conditions and overexpression of p50 reduces CCL20, but enhances cyclooxygenase-2 (COX-2), transcript accumulation after exposure to IL-1ß. We also identified differential recruitment of specific co-activator molecules to the CCL20 gene promoter, when compared with the CCL2 and COX2 genes, revealing distinct transcriptional requirements for individual NF-κB responsive genes. Moreover, IL-1ß, TNF-α and IFN-γ individually increased the expression of CCR6, the receptor for CCL20, on the surface of human neutrophils. We further found that the chemokine CCL20 is elevated in serum from both genetically obese db/db mice and in C57BL6/J mice fed a high-fat diet. Taken together, these results are consistent with a possible activation of the CCL20-CCR6 axis in diseases with inflammatory components. Thus, interfering with this signaling pathway, either at the level of NF-κB-mediated chemokine production, or downstream receptor activation, could be a potential therapeutic target to offset inflammation-associated tissue dysfunction in obesity and diabetes.

IL-1ß reciprocally regulates chemokine and insulin secretion in pancreatic ß-cells via NF-κB.

Proinflammatory cytokines impact islet ß-cell mass and function by altering the transcriptional activity within pancreatic ß-cells, producing increases in intracellular nitric oxide abundance and the synthesis and secretion of immunomodulatory proteins such as chemokines. Herein, we report that IL-1ß, a major mediator of inflammatory responses associated with diabetes development, coordinately and reciprocally regulates chemokine and insulin secretion. We discovered that NF-κB controls the increase in chemokine transcription and secretion as well as the decrease in both insulin secretion and proliferation in response to IL-1ß. Nitric oxide production, which is markedly elevated in pancreatic ß-cells exposed to IL-1ß, is a negative regulator of both glucose-stimulated insulin secretion and glucose-induced increases in intracellular calcium levels. By contrast, the IL-1ß-mediated production of the chemokines CCL2 and CCL20 was not influenced by either nitric oxide levels or glucose concentration. Instead, the synthesis and secretion of CCL2 and CCL20 in response to IL-1ß were dependent on NF-κB transcriptional activity. We conclude that IL-1ß-induced transcriptional reprogramming via NF-κB reciprocally regulates chemokine and insulin secretion while also negatively regulating ß-cell proliferation. These findings are consistent with NF-κB as a major regulatory node controlling inflammation-associated alterations in islet ß-cell function and mass.

Synergistic expression of the CXCL10 gene in response to IL-1ß and IFN-γ involves NF-κB, phosphorylation of STAT1 at Tyr701, and acetylation of histones H3 and H4.

The CXCL10 gene encodes a peptide that chemoattracts a variety of leukocytes associated with type 1 and type 2 diabetes. The present study was undertaken to determine the molecular mechanisms required for expression of the CXCL10 gene in response to IL-1ß and IFN-γ using rat islets and ß cell lines. IL-1ß induced the expression of the CXCL10 gene and promoter activity, whereas the combination of IL-1ß plus IFN-γ was synergistic. Small interfering RNA-mediated suppression of NF-κB p65 markedly inhibited the ability of cytokines to induce the expression of the CXCL10 gene, whereas targeting STAT1 only diminished the synergy provided by IFN-γ. Furthermore, we found that a JAK1 inhibitor dose dependently reduced IFN-γ-controlled CXCL10 gene expression and promoter activity, concomitant with a decrease in STAT1 phosphorylation at Tyr(701). We further discovered that, although the Tyr(701) phosphorylation site is inducible (within 15 min of IFN-γ exposure), the Ser(727) site within STAT1 is constitutively phosphorylated. Thus, we generated single-mutant STAT1 Y701F and double-mutant STAT1 Y701F/S727A adenoviruses. Using these recombinant adenoviruses, we determined that overexpression of either the single- or double-mutant STAT1 decreased the IFN-γ-mediated potentiation of CXCL10 gene expression, promoter activity, and secretion of protein. Moreover, the Ser(727) phosphorylation was neither contingent on a functional Y701 site in ß cells nor was it required for cytokine-mediated expression of the CXCL10 gene. We conclude that the synergism of IL-1ß and IFN-γ to induce expression of the CXCL10 gene requires NF-κB, STAT1 phosphorylated at Tyr(701), recruitment of coactivators, and acetylation of histones H3 and H4.

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription.

Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing ß-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1ß markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and ß-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1ß to promote transcription. In addition, IL-1ß induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1ß induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1ß-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic ß-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.

Islet beta-cells and intercellular adhesion molecule-1 (ICAM-1): Integrating immune responses that influence autoimmunity and graft rejection.

Type 1 diabetes (T1D) develops due to autoimmune targeting of the pancreatic islet ß-cells. Clinical symptoms arise from reduced insulin in circulation. The molecular events and interactions between discrete immune cell populations, infiltration of such leukocytes into pancreatic and islet tissue, and selective targeting of the islet ß-cells during autoimmunity and graft rejection are not entirely understood. One protein central to antigen presentation, priming of immune cells, trafficking of leukocytes, and vital for leukocyte effector function is the intercellular adhesion molecule-1 (ICAM-1). The gene encoding ICAM-1 is transcriptionally regulated and rapidly responsive (i.e., within hours) to pro-inflammatory cytokines. ICAM-1 is a transmembrane protein that can be glycosylated; its presence on the cell surface provides co-stimulatory functions for immune cell activation and stabilization of cell-cell contacts. ICAM-1 interacts with the ß2-integrins, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), which are present on discrete immune cell populations. A whole-body ICAM-1 deletion protects NOD mice from diabetes onset, strongly implicating this protein in autoimmune responses. Since several different cell types express ICAM-1, its biology is fundamentally essential for various physiological and pathological outcomes. Herein, we review the role of ICAM-1 during both autoimmunity and islet graft rejection to understand the mechanism(s) leading to islet ß-cell death and dysfunction that results in insufficient circulating quantities of insulin to control glucose homeostasis.

Lysosomal glucose sensing and glycophagy in metabolism.

Lysosomes are cellular organelles that function to catabolize both extra- and intracellular cargo, act as a platform for nutrient sensing, and represent a core signaling node integrating bioenergetic cues to changes in cellular metabolism. Although lysosomal amino acid and lipid sensing in metabolism has been well characterized, lysosomal glucose sensing and the role of lysosomes in glucose metabolism is unrefined. This review will highlight the role of the lysosome in glucose metabolism with a focus on lysosomal glucose and glycogen sensing, glycophagy, and lysosomal glucose transport and how these processes impact autophagy and energy metabolism. Additionally, the role of lysosomal glucose metabolism in genetic and metabolic diseases will be briefly discussed.

Pharmacological inhibition of lipolysis prevents adverse metabolic outcomes during glucocorticoid administration.

OBJECTIVE: Glucocorticoids are one of the most commonly prescribed classes of anti-inflammatory drugs; however, chronic treatment promotes iatrogenic (drug-induced) diabetes. As part of their physiological role, glucocorticoids stimulate lipolysis to spare glucose. We hypothesized that persistent stimulation of lipolysis during glucocorticoid therapy plays a causative role in the development of iatrogenic diabetes. METHODS: Male C57BL/6J mice were given 100 µg/mL corticosterone (Cort) in the drinking water for two weeks and were fed either normal chow (TekLad 8640) or the same diet supplemented with an adipose triglyceride lipase inhibitor (Atglistatin - 2  g/kg diet) to inhibit the first step of lipolysis. RESULTS: Herein, we report for the first time that glucocorticoid administration promotes a unique state of substrate excess and energetic overload in skeletal muscle that primarily results from the rampant mobilization of endogenous fuels. Inhibiting lipolysis protected mice from Cort-induced gains in fat mass, excess ectopic lipid accrual, hyperinsulinemia, and hyperglycemia. The role lipolysis plays in Cort-mediated pathology appears to differ between tissues. Within skeletal muscle, Cort-induced lipolysis facilitated diversion of glucose-derived carbons toward the pentose phosphate and hexosamine biosynthesis pathways but contributed to <3% of the Cort-induced genomic adaptations. In contrast, Cort stimulation of lipolysis accounted for ∼35% of the genomic changes in the liver but had minimal impact on hepatic metabolites reported. CONCLUSIONS: These data support the idea that activation of lipolysis plays a causal role in the progression toward iatrogenic diabetes during glucocorticoid therapy with differential impact on skeletal muscle and liver.

Special Issue: Emerging Paradigms in Insulin Resistance.

This Biomedicines Special Issue was designed to attract articles that focused on different facets of biology relating to insulin resistance, defined as reduced cellular and organismal response to the insulin hormone, and its underlying mechanisms [...].

Artemisia dracunculus L. Ethanolic Extract and an Isolated Component, DMC2, Ameliorate Inflammatory Signaling in Pancreatic ß-Cells via Inhibition of p38 MAPK.

Non-resolving pancreatic islet inflammation is widely viewed as a contributor to decreases in ß-cell mass and function that occur in both Type 1 and Type 2 diabetes. Therefore, strategies aimed at reducing or eliminating pathological inflammation would be useful to protect islet ß-cells. Herein, we described the use of 2',4'-dihydroxy-4-methoxydihydrochalcone (DMC2), a bioactive molecule isolated from an ethanolic extract of Artemisia dracunculus L., as a novel anti-inflammatory agent. The ethanolic extract, termed PMI 5011, reduced IL-1ß-mediated NF-κB activity. DMC2 retained this ability, indicating this compound as the likely source of anti-inflammatory activity within the overall PMI 5011 extract. We further examined NF-κB activity using promoter-luciferase reporter constructs, Western blots, mRNA abundance, and protein secretion. Specifically, we found that PMI 5011 and DMC2 each reduced the ability of IL-1ß to promote increases in the expression of the Ccl2 and Ccl20 genes. These genes encode proteins that promote immune cell recruitment and are secreted by ß-cells in response to IL-1ß. Phosphorylation of IκBα and the p65 subunit of NF-κB were not reduced by either PMI 5011 or DMC2; however, phosphorylation of p38 MAPK was blunted in the presence of DMC2. Finally, we observed that while PMI 5011 impaired glucose-stimulated insulin secretion, insulin output was preserved in the presence of DMC2. In conclusion, PMI 5011 and DMC2 reduced inflammation, but only DMC2 did so with the preservation of glucose-stimulated insulin secretion.

Stereoisomers of an Aryl Pyrazole Glucocorticoid Receptor Agonist Scaffold Elicit Differing Anti-inflammatory Responses.

Glucocorticoids (GCs) are heavily prescribed to control inflammation in various human diseases; however, side effects associated with GCs are well documented and lead to serious metabolic and immunological complications with long-term use. The paradigm for GC function includes two well described modes of activity: dimer formation of the glucocorticoid receptor (GR) promotes transactivation, while monomeric interaction with co-regulators promotes transrepression. Previously, a set of aryl pyrazole-derived glucocorticoid receptor agonists (APGRAs) with potency rivaling current commercially available glucocorticoids were described. In this study, a further series of existing and novel stereopure APGRAs were thoroughly examined for biological activity and evaluated for structure-activity relationships (SARs). The si isomers with an upward OH moiety were ∼70% more active on average than the re isomers. Additionally, AP13 was found to elicit 79% transrepression of dexamethasone while eliciting less than half the transactivation response in 832/13 cells, a rat insulinoma cell line.

Adipocyte-Specific Laminin Alpha 4 Deletion Preserves Adipose Tissue Health despite Increasing Adiposity.

Laminins are heterotrimeric glycoproteins with structural and functional roles in basement membranes. The predominant laminin alpha chain found in adipocyte basement membranes is laminin α4 (LAMA4). Global LAMA4 deletion in mice leads to reduced adiposity and increased energy expenditure, but also results in vascular defects that complicate the interpretation of metabolic data. Here, we describe the generation and initial phenotypic analysis of an adipocyte-specific LAMA4 knockout mouse (Lama4AKO). We first performed an in-silico analysis to determine the degree to which laminin α4 was expressed in human and murine adipocytes. Next, male Lama4AKO and control mice were fed chow or high-fat diets and glucose tolerance was assessed along with serum insulin and leptin levels. Adipocyte area was measured in both epididymal and inguinal white adipose tissue (eWAT and iWAT, respectively), and eWAT was used for RNA-sequencing. We found that laminin α4 was highly expressed in human and murine adipocytes. Further, chow-fed Lama4AKO mice are like control mice in terms of body weight, body composition, and glucose tolerance, although they have larger eWAT adipocytes and lower insulin levels. High-fat-fed Lama4AKO mice are fatter and more glucose tolerant when compared to control mice. Transcriptionally, the eWAT of high-fat fed Lama4AKO mice resembles that of chow-fed control mice. We conclude from these findings that adipocyte-specific LAMA4 deletion is protective in an obesogenic environment, even though overall adiposity is increased.

Intact mitochondrial substrate efflux is essential for prevention of tubular injury in a sex-dependent manner.

The importance of healthy mitochondrial function is implicated in the prevention of chronic kidney disease (CKD) and diabetic kidney disease (DKD). Sex differences also play important roles in DKD. Our previous studies revealed that mitochondrial substrate overload (modeled by homozygous deletion of carnitine acetyl-transferase [CrAT]) in proximal tubules causes renal injury. Here, we demonstrate the importance of intact mitochondrial substrate efflux by titrating the amount of overload through the generation of a heterozygous CrAT-KO model (PT-CrATHET mouse). Intriguingly, these animals developed renal injury similarly to their homozygous counterparts. Mitochondria were structurally and functionally impaired in both sexes. Transcriptomic analyses, however, revealed striking sex differences. Male mice shut down fatty acid oxidation and several other metabolism-related pathways. Female mice had a significantly weaker transcriptional response in metabolism, but activation of inflammatory pathways was prominent. Proximal tubular cells from PT-CrATHET mice of both sexes exhibited a shift toward a more glycolytic phenotype, but female mice were still able to oxidize fatty acid-based substrates. Our results demonstrate that maintaining mitochondrial substrate metabolism balance is crucial to satisfying proximal tubular energy demand. Our findings have potentially broad implications, as both the glycolytic shift and the sexual dimorphisms discovered herein offer potentially new modalities for future interventions for treating kidney disease.