Post mortem analysis of hepatic volume and lipid content by magnetic resonance imaging and spectroscopy in fixed murine neonates.

Maternal obesity and hyperglycemia are linked to an elevated risk for obesity, diabetes, and steatotic liver disease in the adult offspring. To establish and validate a noninvasive workflow for perinatal metabolic phenotyping, fixed neonates of common mouse strains were analyzed postmortem via magnetic resonance imaging (MRI)/magnetic resonance spectroscopy (MRS) to assess liver volume and hepatic lipid (HL) content. The key advantage of nondestructive MRI/MRS analysis is the possibility of further tissue analyses, such as immunohistochemistry, RNA extraction, and even proteomics, maximizing the data that can be gained per individual and therefore facilitating comprehensive correlation analyses. This study employed an MRI and 1H-MRS workflow to measure liver volume and HL content in 65 paraformaldehyde-fixed murine neonates at 11.7 T. Liver volume was obtained using semiautomatic segmentation of MRI acquired by a RARE sequence with 0.5-mm slice thickness. HL content was measured by a STEAM sequence, applied with and without water suppression. T1 and T2 relaxation times of lipids and water were measured for respective correction of signal intensity. The HL content, given as CH2/(CH2 + H2O), was calculated, and the intrasession repeatability of the method was tested. The established workflow yielded robust results with a variation of ~3% in repeated measurements for HL content determination. HL content measurements were further validated by correlation analysis with biochemically assessed triglyceride contents (R2 = 0.795) that were measured in littermates. In addition, image quality also allowed quantification of subcutaneous adipose tissue and stomach diameter. The highest HL content was measured in C57Bl/6N (4.2%) and the largest liver volume and stomach diameter in CBA (53.1 mm3 and 6.73 mm) and NMRI (51.4 mm3 and 5.96 mm) neonates, which also had the most subcutaneous adipose tissue. The observed effects were independent of sex and litter size. In conclusion, we have successfully tested and validated a robust MRI/MRS workflow that allows assessment of morphology and HL content and further enables paraformaldehyde-fixed tissue-compatible subsequent analyses in murine neonates.

MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins.

Impairments of mitochondrial functions are linked to human ageing and pathologies such as cancer, cardiomyopathy, neurodegeneration and diabetes. Specifically, aberrations in ultrastructure of mitochondrial inner membrane (IM) and factors regulating them are linked to diabetes. The development of diabetes is connected to the 'Mitochondrial Contact Site and Cristae Organising System' (MICOS) complex which is a large membrane protein complex defining the IM architecture. MIC26 and MIC27 are homologous apolipoproteins of the MICOS complex. MIC26 has been reported as a 22 kDa mitochondrial and a 55 kDa glycosylated and secreted protein. The molecular and functional relationship between these MIC26 isoforms has not been investigated. In order to understand their molecular roles, we depleted MIC26 using siRNA and further generated MIC26 and MIC27 knockouts (KOs) in four different human cell lines. In these KOs, we used four anti-MIC26 antibodies and consistently detected the loss of mitochondrial MIC26 (22 kDa) and MIC27 (30 kDa) but not the loss of intracellular or secreted 55 kDa protein. Thus, the protein assigned earlier as 55 kDa MIC26 is nonspecific. We further excluded the presence of a glycosylated, high-molecular weight MIC27 protein. Next, we probed GFP- and myc-tagged variants of MIC26 with antibodies against GFP and myc respectively. Again, only the mitochondrial versions of these tagged proteins were detected but not the corresponding high-molecular weight MIC26, suggesting that MIC26 is indeed not post-translationally modified. Mutagenesis of predicted glycosylation sites in MIC26 also did not affect the detection of the 55 kDa protein band. Mass spectrometry of a band excised from an SDS gel around 55 kDa could not confirm the presence of any peptides derived from MIC26. Taken together, we conclude that both MIC26 and MIC27 are exclusively localized in mitochondria and that the observed phenotypes reported previously are exclusively due to their mitochondrial function.

Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism.

Type 2 diabetes is characterized by insulin hypersecretion followed by reduced glucose-stimulated insulin secretion (GSIS). Here we show that acute stimulation of pancreatic islets with the insulin secretagogue dextrorphan (DXO) or glibenclamide enhances GSIS, whereas chronic treatment with high concentrations of these drugs reduce GSIS but protect islets from cell death. Bulk RNA sequencing of islets shows increased expression of genes for serine-linked mitochondrial one-carbon metabolism (OCM) after chronic, but not acute, stimulation. In chronically stimulated islets, more glucose is metabolized to serine than to citrate, and the mitochondrial ATP/ADP ratio decreases, whereas the NADPH/NADP+ ratio increases. Activating transcription factor-4 (Atf4) is required and sufficient to activate serine-linked mitochondrial OCM genes in islets, with gain- and loss-of-function experiments showing that Atf4 reduces GSIS and is required, but not sufficient, for full DXO-mediated islet protection. In sum, we identify a reversible metabolic pathway that provides islet protection at the expense of secretory function.

E96V Mutation in the Kdelr3 Gene Is Associated with Type 2 Diabetes Susceptibility in Obese NZO Mice.

Type 2 diabetes (T2D) represents a multifactorial metabolic disease with a strong genetic predisposition. Despite elaborate efforts in identifying the genetic variants determining individual susceptibility towards T2D, the majority of genetic factors driving disease development remain poorly understood. With the aim to identify novel T2D risk genes we previously generated an N2 outcross population using the two inbred mouse strains New Zealand obese (NZO) and C3HeB/FeJ (C3H). A linkage study performed in this population led to the identification of the novel T2D-associated quantitative trait locus (QTL) Nbg15 (NZO blood glucose on chromosome 15, Logarithm of odds (LOD) 6.6). In this study we used a combined approach of positional cloning, gene expression analyses and in silico predictions of DNA polymorphism on gene/protein function to dissect the genetic variants linking Nbg15 to the development of T2D. Moreover, we have generated congenic strains that associated the distal sublocus of Nbg15 to mechanisms altering pancreatic beta cell function. In this sublocus, Cbx6, Fam135b and Kdelr3 were nominated as potential causative genes associated with the Nbg15 driven effects. Moreover, a putative mutation in the Kdelr3 gene from NZO was identified, negatively influencing adaptive responses associated with pancreatic beta cell death and induction of endoplasmic reticulum stress. Importantly, knockdown of Kdelr3 in cultured Min6 beta cells altered insulin granules maturation and pro-insulin levels, pointing towards a crucial role of this gene in islets function and T2D susceptibility.

Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis.

Impaired proinsulin-to-insulin processing in pancreatic ß-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. 1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. 3-8); nonetheless, the role of specific SL species in ß-cell function and demise is unclear. Here we define the lipid signature of T2D-associated ß-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. ß-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in ß-cell function and T2D-associated ß-cell failure.

Selective ablation of P53 in pancreatic beta cells fails to ameliorate glucose metabolism in genetic, dietary and pharmacological models of diabetes mellitus.

OBJECTIVE: Beta cell dysfunction and death are critical steps in the development of both type 1 and type 2 diabetes (T1D and T2D), but the underlying mechanisms are incompletely understood. Activation of the essential tumor suppressor and transcription factor P53 (also known as TP53 and Trp53 in mice) was linked to beta cell death in vitro and has been reported in several diabetes mouse models and beta cells of humans with T2D. In this article, we set out to determine the beta cell specific role of P53 in beta cell dysfunction, cell death and development of diabetes in vivo. METHODS: We generated beta cell specific P53 knockout (P53BKO) mice and used complementary genetic, dietary and pharmacological models of glucose intolerance, beta cell dysfunction and diabetes development to evaluate the functional role of P53 selectively in beta cells. We further analyzed the effect of P53 ablation on beta cell survival in isolated pancreatic islets exposed to diabetogenic stress inducers ex vivo by flow cytometry. RESULTS: Beta cell specific ablation of P53/Trp53 failed to ameliorate glucose tolerance, insulin secretion or to increase beta cell numbers in genetic, dietary and pharmacological models of diabetes. Additionally, loss of P53 in beta cells did not protect against streptozotocin (STZ) induced hyperglycemia and beta cell death, although STZ-induced activation of classical pro-apoptotic P53 target genes was significantly reduced in P53BKO mice. In contrast, Olaparib mediated PARP1 inhibition protected against acute ex vivo STZ-induced beta cell death and islet destruction. CONCLUSIONS: Our study reveals that ablation of P53 specifically in beta cells is unexpectedly unable to attenuate beta cell failure and death in vivo and ex vivo. While during development and progression of diabetes, P53 and P53-regulated pathways are activated, our study suggests that P53 signaling is not essential for loss of beta cells or beta cell dysfunction. P53 in other cell types and organs may predominantly regulate systemic glucose homeostasis.

Positive allosteric γ-aminobutyric acid type A receptor modulation prevents lipotoxicity-induced injury in hepatocytes in vitro.

AIM: To determine if a novel positive allosteric modulator of the γ-aminobutyric acid type A (GABAA ) receptor, the thioacrylamide-derivative HK4, which does not penetrate the blood-brain barrier, protects human hepatocytes against lipotoxicity-induced injury. MATERIALS AND METHODS: Allosteric modulation of the GABAA receptor by HK4 was determined by patch clamp in HEK-293 cells, calcium influx in INS-1E cells and by using the specific GABAA channel blockers picrotoxin and tert-butylbicyclophosphorothionate (TBPS) in HepG2 cells. Apoptosis was analysed using caspase 3/7, terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) and array assays in HepG2 cells and/or human primary hepatocytes. Phosphorylation of STAT3 and the NF-κB subunit p65, protein disulphide isomerase (PDI) and poly-ADP-ribose polymerase-1 (PARP-1) was detected by Western blotting. RESULTS: Patch clamping, calcium influx measurements and apoptosis assays with the non-competitive GABAA channel blockers picrotoxin and TBPS proved HK4 as a selective positive allosteric modulator of the GABAA receptor. In HepG2 cells, which expressed the main GABAA receptor subunits, HK4 prevented palmitate-induced apoptosis. This protective effect was mediated by downregulation of caspase 3/7 activity and was additionally verified by TUNEL assay. HK4 effectively prevented palmitate-induced apoptosis in human primary hepatocytes. HK4 reduced STAT3 and NF-κB phosphorylation, reduced cleaved PARP-1 expression and upregulated the endoplasmic reticulum (ER) chaperone PDI. CONCLUSIONS: HK4 reduced lipotoxic-induced apoptosis by preventing inflammation, DNA damage and ER stress. We propose that the effect of HK4 is mediated by STAT3 and NF-κB. It is suggested that thioacrylamide compounds represent an innovative pharmacological tool to treat or prevent non-alcoholic steatohepatitis as first-in-class drugs.

Interaction between magnesium and methylglyoxal in diabetic polyneuropathy and neuronal models.

OBJECTIVE: The lack of effective treatments against diabetic sensorimotor polyneuropathy demands the search for new strategies to combat or prevent the condition. Because reduced magnesium and increased methylglyoxal levels have been implicated in the development of both type 2 diabetes and neuropathic pain, we aimed to assess the putative interplay of both molecules with diabetic sensorimotor polyneuropathy. METHODS: In a cross-sectional study, serum magnesium and plasma methylglyoxal levels were measured in recently diagnosed type 2 diabetes patients with (n = 51) and without (n = 184) diabetic sensorimotor polyneuropathy from the German Diabetes Study baseline cohort. Peripheral nerve function was assessed using nerve conduction velocity and quantitative sensory testing. Human neuroblastoma cells (SH-SY5Y) and mouse dorsal root ganglia cells were used to characterize the neurotoxic effect of methylglyoxal and/or neuroprotective effect of magnesium. RESULTS: Here, we demonstrate that serum magnesium concentration was reduced in recently diagnosed type 2 diabetes patients with diabetic sensorimotor polyneuropathy and inversely associated with plasma methylglyoxal concentration. Magnesium, methylglyoxal, and, importantly, their interaction were strongly interrelated with methylglyoxal-dependent nerve dysfunction and were predictive of changes in nerve function. Magnesium supplementation prevented methylglyoxal neurotoxicity in differentiated SH-SY5Y neuron-like cells due to reduction of intracellular methylglyoxal formation, while supplementation with the divalent cations zinc and manganese had no effect on methylglyoxal neurotoxicity. Furthermore, the downregulation of mitochondrial activity in mouse dorsal root ganglia cells and consequently the enrichment of triosephosphates, the primary source of methylglyoxal, resulted in neurite degeneration, which was completely prevented through magnesium supplementation. CONCLUSIONS: These multifaceted findings reveal a novel putative pathophysiological pathway of hypomagnesemia-induced carbonyl stress leading to neuronal damage and merit further investigations not only for diabetic sensorimotor polyneuropathy but also other neurodegenerative diseases associated with magnesium deficiency and impaired energy metabolism.

ATM and P53 differentially regulate pancreatic beta cell survival in Ins1E cells.

Pancreatic beta cell death is a hallmark of type 1 and 2 diabetes (T1D/T2D), but the underlying molecular mechanisms are incompletely understood. Key proteins of the DNA damage response (DDR), including tumor protein P53 (P53, also known as TP53 or TRP53 in rodents) and Ataxia Telangiectasia Mutated (ATM), a kinase known to act upstream of P53, have been associated with T2D. Here we test and compare the effect of ATM and P53 ablation on beta cell survival in the rat beta cell line Ins1E. We demonstrate that ATM and P53 differentially regulate beta cell apoptosis induced upon fundamentally different types of diabetogenic beta cell stress, including DNA damage, inflammation, lipotoxicity and endoplasmic reticulum (ER) stress. DNA damage induced apoptosis by treatment with the commonly used diabetogenic agent streptozotocin (STZ) is regulated by both ATM and P53. We show that ATM is a key STZ induced activator of P53 and that amelioration of STZ induced cell death by inhibition of ATM mainly depends on P53. While both P53 and ATM control lipotoxic beta cell apoptosis, ATM but not P53 fails to alter inflammatory beta cell death. In contrast, tunicamycin induced (ER stress associated) apoptosis is further increased by ATM knockdown or inhibition, but not by P53 knockdown. Our results reveal differential roles for P53 and ATM in beta cell survival in vitro in the context of four key pathophysiological types of diabetogenic beta cell stress, and indicate that ATM can use P53 independent signaling pathways to modify beta cell survival, dependent on the cellular insult.

The microRNA-200 family regulates pancreatic beta cell survival in type 2 diabetes.

Pancreatic beta cell death is a hallmark of type 1 (T1D) and type 2 (T2D) diabetes, but the molecular mechanisms underlying this aspect of diabetic pathology are poorly understood. Here we report that expression of the microRNA (miR)-200 family is strongly induced in islets of diabetic mice and that beta cell-specific overexpression of miR-200 in mice is sufficient to induce beta cell apoptosis and lethal T2D. Conversely, mir-200 ablation in mice reduces beta cell apoptosis and ameliorates T2D. We show that miR-200 negatively regulates a conserved anti-apoptotic and stress-resistance network that includes the essential beta cell chaperone Dnajc3 (also known as p58IPK) and the caspase inhibitor Xiap. We also observed that mir-200 dosage positively controls activation of the tumor suppressor Trp53 and thereby creates a pro-apoptotic gene-expression signature found in islets of diabetic mice. Consequently, miR-200-induced T2D is suppressed by interfering with the signaling of Trp53 and Bax, a proapoptotic member of the B cell lymphoma 2 protein family. Our results reveal a crucial role for the miR-200 family in beta cell survival and the pathophysiology of diabetes.

SIK2 regulates insulin secretion.

Correct regulation of insulin secretion by the pancreas is crucial for organismal function and survival. The AMPK-related kinase SIK2 (salt-inducible kinase 2) is now shown to be stabilized in pancreatic ß-cells following glucose stimulation, leading to improved systemic glucose homeostasis by regulating cellular calcium flux and insulin secretion.