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Background: Metformin and sodium-glucose-cotransporter-2 inhibitors (SGLT2i) are cornerstone therapies for managing hyperglycemia in diabetes. However, their detailed impacts on metabolic processes, particularly within the citric acid (TCA) cycle and its anaplerotic pathways, remain unclear. This study investigates the tissue-specific metabolic effects of metformin, both as a monotherapy and in combination with SGLT2i, on the TCA cycle and associated anaplerotic reactions in both mice and humans., Methods: Metformin-specific metabolic changes were initially identified by comparing metformin-treated diabetic mice (MET) with vehicle-treated db/db mice (VG). These findings were then assessed in two human cohorts (KORA and QBB) and a longitudinal KORA study of metformin-naïve patients with Type 2 Diabetes (T2D). We also compared MET with db/db mice on combination therapy (SGLT2i + MET). Metabolic profiling analyzed 716 metabolites from plasma, liver, and kidney tissues post-treatment, using linear regression and Bonferroni correction for statistical analysis, complemented by pathway analyses to explore the pathophysiological implications., Results: Metformin monotherapy significantly upregulated TCA cycle intermediates such as malate, fumarate, and α-ketoglutarate (α-KG) in plasma, and anaplerotic substrates including hepatic glutamate and renal 2-hydroxyglutarate (2-HG) in diabetic mice. Downregulated hepatic taurine was also observed. The addition of SGLT2i, however, reversed these effects, such as downregulating circulating malate and α-KG, and hepatic glutamate and renal 2-HG, but upregulated hepatic taurine. In human T2D patients on metformin therapy, significant systemic alterations in metabolites were observed, including increased malate but decreased citrulline. The bidirectional modulation of TCA cycle intermediates in mice influenced key anaplerotic pathways linked to glutaminolysis, tumorigenesis, immune regulation, and antioxidative responses., Conclusion: This study elucidates the specific metabolic consequences of metformin and SGLT2i on the TCA cycle, reflecting potential impacts on the immune system. Metformin shows promise for its anti-inflammatory properties, while the addition of SGLT2i may provide liver protection in conditions like metabolic dysfunction-associated steatotic liver disease (MASLD). These observations underscore the importance of personalized treatment strategies., (© 2024. The Author(s).)

Combining a Sodium-Glucose-Cotransporter-2-inhibitor (SGLT2i) with metformin is recommended for managing hyperglycemia in patients with type 2 diabetes (T2D) who have cardio-renal complications. Our study aimed to investigate the metabolic effects of SGLT2i and metformin, both individually and synergistically. We treated leptin receptor-deficient (db/db) mice with these drugs for two weeks and conducted metabolite profiling, identifying 861 metabolites across kidney, liver, muscle, fat, and plasma. Using linear regression and mixed-effects models, we identified two SGLT2i-specific metabolites, X-12465 and 3-hydroxybutyric acid (3HBA), a ketone body, across all examined tissues. The levels of 3HBA were significantly higher under SGLT2i monotherapy compared to controls and were attenuated when combined with metformin. We observed similar modulatory effects on metabolites involved in protein catabolism (e.g., branched-chain amino acids) and gluconeogenesis. Moreover, combination therapy significantly raised pipecolate levels, which may enhance mTOR1 activity, while modulating GSK3, a common target of SGLT2i and 3HBA inhibition. The combination therapy also led to significant reductions in body weight and lactate levels, contrasted with monotherapies. Our findings advocate for the combined approach to better manage muscle loss, and the risks of DKA and lactic acidosis, presenting a more effective strategy for T2D treatment., Competing Interests: Declaration of competing interest Markus F. Scheerer was employed at Helmholtz Zentrum München during his PhD thesis and is currently employed in the CardioRenal Medical Department of Bayer AG, however, the company was not involved in work related to data and manuscript generation. Susanne Neschen was employed by the Helmholtz Zentrum München during the execution of this study. She is currently an employee of Sanofi Aventis Deutschland GmbH, however, the company was not involved in work related to data and manuscript generation., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Biological exploration of early biomarkers for chronic kidney disease (CKD) in (pre)diabetic individuals is crucial for personalized management of diabetes. Here, we evaluated two candidate biomarkers of incident CKD (sphingomyelin (SM) C18:1 and phosphatidylcholine diacyl (PC aa) C38:0) concerning kidney function in hyperglycemic participants of the Cooperative Health Research in the Region of Augsburg (KORA) cohort, and in two biofluids and six organs of leptin receptor-deficient (db/db) mice and wild type controls. Higher serum concentrations of SM C18:1 and PC aa C38:0 in hyperglycemic individuals were found to be associated with lower estimated glomerular filtration rate (eGFR) and higher odds of CKD. In db/db mice, both metabolites had a significantly lower concentration in urine and adipose tissue, but higher in the lungs. Additionally, db/db mice had significantly higher SM C18:1 levels in plasma and liver, and PC aa C38:0 in adrenal glands. This cross-sectional human study confirms that SM C18:1 and PC aa C38:0 associate with kidney dysfunction in pre(diabetic) individuals, and the animal study suggests a potential implication of liver, lungs, adrenal glands, and visceral fat in their systemic regulation. Our results support further validation of the two phospholipids as early biomarkers of renal disease in patients with (pre)diabetes.

Early and precise identification of individuals with prediabetes and type 2 diabetes (T2D) at risk for progressing to chronic kidney disease (CKD) is essential to prevent complications of diabetes. Here, we identify and evaluate prospective metabolite biomarkers and the best set of predictors of CKD in the longitudinal, population-based Cooperative Health Research in the Region of Augsburg (KORA) cohort by targeted metabolomics and machine learning approaches. Out of 125 targeted metabolites, sphingomyelin C18:1 and phosphatidylcholine diacyl C38:0 were identified as candidate metabolite biomarkers of incident CKD specifically in hyperglycemic individuals followed during 6.5 years. Sets of predictors for incident CKD developed from 125 metabolites and 14 clinical variables showed highly stable performances in all three machine learning approaches and outperformed the currently established clinical algorithm for CKD. The two metabolites in combination with five clinical variables were identified as the best set of predictors, and their predictive performance yielded a mean area value under the receiver operating characteristic curve of 0.857. The inclusion of metabolite variables in the clinical prediction of future CKD may thus improve the risk prediction in people with prediabetes and T2D. The metabolite link with hyperglycemia-related early kidney dysfunction warrants further investigation., (© 2020 by the American Diabetes Association.)

Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in β cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of t 6 A37 in tRNA Lys UUU to ms 2 t 6 A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms 2 t 6 A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2 -/- β cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans.

Aims and Methods: Glucose homeostasis and energy balance are under control by peripheral and brain processes. Especially insulin signaling in the brain seems to impact whole body glucose homeostasis and interacts with fatty acid signaling. In humans circulating saturated fatty acids are negatively associated with brain insulin action while animal studies suggest both positive and negative interactions of fatty acids and insulin brain action. This apparent discrepancy might reflect a difference between acute and chronic fatty acid signaling. To address this question we investigated the acute effect of an intracerebroventricular palmitic acid administration on peripheral glucose homeostasis. We developed and implemented a method for simultaneous monitoring of brain activity and peripheral insulin action in freely moving mice by combining radiotelemetry electrocorticography (ECoG) and euglycemic-hyperinsulinemic clamps. This method allowed gaining insight in the early kinetics of brain fatty acid signaling and its contemporaneous effect on liver function in vivo , which, to our knowledge, has not been assessed so far in mice., Results: Insulin-induced brain activity in the theta and beta band was decreased by acute intracerebroventricular application of palmitic acid. Peripherally it amplified insulin action as demonstrated by a significant inhibition of endogenous glucose production and increased glucose infusion rate. Moreover, our results further revealed that the brain effect of peripheral insulin is modulated by palmitic acid load in the brain., Conclusion: These findings suggest that insulin action is amplified in the periphery and attenuated in the brain by acute palmitic acid application. Thus, our results indicate that acute palmitic acid signaling in the brain may be different from chronic effects., Competing Interests: No conflict of interest has been declared by the authors. SN is an employee of Sanofi-Aventis GmbH, Frankfurt, Germany since 2015., (© Georg Thieme Verlag KG Stuttgart · New York.)

The processes contributing to β cell dysfunction in type 2 diabetes (T2D) are uncertain, largely because it is difficult to access β cells in their intact immediate environment. We examined the pathophysiology of β cells under T2D progression directly in pancreatic tissues. We used MALDI imaging of Langerhans islets (LHIs) within mouse tissues or from human tissues to generate in situ-omics data, which we supported with in vitro experiments. Molecular interaction networks provided information on functional pathways and molecules. We found that stearoylcarnitine accumulated in β cells, leading to arrest of insulin synthesis and energy deficiency via excessive β-oxidation and depletion of TCA cycle and oxidative phosphorylation metabolites. Acetylcarnitine and an accumulation of N-acyl taurines, a group not previously detected in β cells, provoked insulin secretion. Thus, β cell dysfunction results from enhanced insulin secretion combined with an arrest of insulin synthesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Dietary intake of saturated fat is a likely contributor to nonalcoholic fatty liver disease (NAFLD) and insulin resistance, but the mechanisms that initiate these abnormalities in humans remain unclear. We examined the effects of a single oral saturated fat load on insulin sensitivity, hepatic glucose metabolism, and lipid metabolism in humans. Similarly, initiating mechanisms were examined after an equivalent challenge in mice., Methods: Fourteen lean, healthy individuals randomly received either palm oil (PO) or vehicle (VCL). Hepatic metabolism was analyzed using in vivo 13C/31P/1H and ex vivo 2H magnetic resonance spectroscopy before and during hyperinsulinemic-euglycemic clamps with isotope dilution. Mice underwent identical clamp procedures and hepatic transcriptome analyses., Results: PO administration decreased whole-body, hepatic, and adipose tissue insulin sensitivity by 25%, 15%, and 34%, respectively. Hepatic triglyceride and ATP content rose by 35% and 16%, respectively. Hepatic gluconeogenesis increased by 70%, and net glycogenolysis declined by 20%. Mouse transcriptomics revealed that PO differentially regulates predicted upstream regulators and pathways, including LPS, members of the TLR and PPAR families, NF-κB, and TNF-related weak inducer of apoptosis (TWEAK)., Conclusion: Saturated fat ingestion rapidly increases hepatic lipid storage, energy metabolism, and insulin resistance. This is accompanied by regulation of hepatic gene expression and signaling that may contribute to development of NAFLD.REGISTRATION. ClinicalTrials.gov NCT01736202., Funding: Germany: Ministry of Innovation, Science, and Research North Rhine-Westfalia, German Federal Ministry of Health, Federal Ministry of Education and Research, German Center for Diabetes Research, German Research Foundation, and German Diabetes Association. Portugal: Portuguese Foundation for Science and Technology, FEDER - European Regional Development Fund, Portuguese Foundation for Science and Technology, and Rede Nacional de Ressonância Magnética Nuclear., Competing Interests: The authors have declared that no conflict of interest exists.

Objective: Recently, we have shown that Bezafibrate (BEZ), the pan-PPAR (peroxisome proliferator-activated receptor) activator, ameliorated diabetes in insulin deficient streptozotocin treated diabetic mice. In order to study whether BEZ can also improve glucose metabolism in a mouse model for fatty liver and type 2 diabetes, the drug was applied to TallyHo mice., Methods: TallyHo mice were divided into an early (ED) and late (LD) diabetes progression group and both groups were treated with 0.5% BEZ (BEZ group) or standard diet (SD group) for 8 weeks. We analyzed plasma parameters, pancreatic beta-cell morphology, and mass as well as glucose metabolism of the BEZ-treated and control mice. Furthermore, liver fat content and composition as well as hepatic gluconeogenesis and mitochondrial mass were determined., Results: Plasma lipid and glucose levels were markedly reduced upon BEZ treatment, which was accompanied by elevated insulin sensitivity index as well as glucose tolerance, respectively. BEZ increased islet area in the pancreas. Furthermore, BEZ treatment improved energy expenditure and metabolic flexibility. In the liver, BEZ ameliorated steatosis, modified lipid composition and increased mitochondrial mass, which was accompanied by reduced hepatic gluconeogenesis., Conclusions: Our data showed that BEZ ameliorates diabetes probably via reduced steatosis, enhanced hepatic mitochondrial mass, improved metabolic flexibility and elevated hepatic insulin sensitivity in TallyHo mice, suggesting that BEZ treatment could be beneficial for patients with NAFLD and impaired glucose metabolism.

Metformin is the first-line oral medication to increase insulin sensitivity in patients with type 2 diabetes (T2D). Our aim was to investigate the pleiotropic effect of metformin using a nontargeted metabolomics approach. We analyzed 353 metabolites in fasting serum samples of the population-based human KORA (Cooperative Health Research in the Region of Augsburg) follow-up survey 4 cohort. To compare T2D patients treated with metformin (mt-T2D, n = 74) and those without antidiabetes medication (ndt-T2D, n = 115), we used multivariable linear regression models in a cross-sectional study. We applied a generalized estimating equation to confirm the initial findings in longitudinal samples of 683 KORA participants. In a translational approach, we used murine plasma, liver, skeletal muscle, and epididymal adipose tissue samples from metformin-treated db/db mice to further corroborate our findings from the human study. We identified two metabolites significantly (P < 1.42E-04) associated with metformin treatment. Citrulline showed lower relative concentrations and an unknown metabolite X-21365 showed higher relative concentrations in human serum when comparing mt-T2D with ndt-T2D. Citrulline was confirmed to be significantly (P < 2.96E-04) decreased at 7-year follow-up in patients who started metformin treatment. In mice, we validated significantly (P < 4.52E-07) lower citrulline values in plasma, skeletal muscle, and adipose tissue of metformin-treated animals but not in their liver. The lowered values of citrulline we observed by using a nontargeted approach most likely resulted from the pleiotropic effect of metformin on the interlocked urea and nitric oxide cycle. The translational data derived from multiple murine tissues corroborated and complemented the findings from the human cohort., (© 2016 by the American Diabetes Association.)

Bezafibrate (BEZ), a pan activator of peroxisome proliferator-activated receptors (PPARs), has been generally used to treat hyperlipidemia for decades. Clinical trials with type 2 diabetes patients indicated that BEZ also has beneficial effects on glucose metabolism, although the underlying mechanisms of these effects remain elusive. Even less is known about a potential role for BEZ in treating type 1 diabetes. Here we show that BEZ markedly improves hyperglycemia and glucose and insulin tolerance in mice with streptozotocin (STZ)-induced diabetes, an insulin-deficient mouse model of type 1 diabetes. BEZ treatment of STZ mice significantly suppressed the hepatic expression of genes that are annotated in inflammatory processes, whereas the expression of PPAR and insulin target gene transcripts was increased. Furthermore, BEZ-treated mice also exhibited improved metabolic flexibility as well as an enhanced mitochondrial mass and function in the liver. Finally, we show that the number of pancreatic islets and the area of insulin-positive cells tended to be higher in BEZ-treated mice. Our data suggest that BEZ may improve impaired glucose metabolism by augmenting hepatic mitochondrial performance, suppressing hepatic inflammatory pathways, and improving insulin sensitivity and metabolic flexibility. Thus, BEZ treatment might also be useful for patients with impaired glucose tolerance or diabetes., (© 2016 by the American Diabetes Association.)

Although it is widely accepted that ectopic lipid accumulation in the liver is associated with hepatic insulin resistance, the underlying molecular mechanisms have not been well characterized.Here we employed time resolved quantitative proteomic profiling of mice fed a high fat diet to determine which pathways were affected during the transition of the liver to an insulin-resistant state. We identified several metabolic pathways underlying altered protein expression. In order to test the functional impact of a critical subset of these alterations, we focused on the epoxyeicosatrienoic acid (EET) eicosanoid pathway, whose deregulation coincided with the onset of hepatic insulin resistance. These results suggested that EETs may be positive modulators of hepatic insulin signaling. Analyzing EET activity in primary hepatocytes, we found that EETs enhance insulin signaling on the level of Akt. In contrast, EETs did not influence insulin receptor or insulin receptor substrate-1 phosphorylation. This effect was mediated through the eicosanoids, as overexpression of the deregulated enzymes in absence of arachidonic acid had no impact on insulin signaling. The stimulation of insulin signaling by EETs and depression of the pathway in insulin resistant liver suggest a likely role in hepatic insulin resistance. Our findings support therapeutic potential for inhibiting EET degradation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Insulin regulates glycaemia, lipogenesis and increases mRNA translation. Cells with reduced eukaryotic initiation factor 6 (eIF6) do not increase translation in response to insulin. The role of insulin-regulated translation is unknown. Here we show that reduction of insulin-regulated translation in mice heterozygous for eIF6 results in normal glycaemia, but less blood cholesterol and triglycerides. eIF6 controls fatty acid synthesis and glycolysis in a cell autonomous fashion. eIF6 acts by exerting translational control of adipogenic transcription factors like C/EBPβ, C/EBPδ and ATF4 that have G/C rich or uORF sequences in their 5' UTR. The outcome of the translational activation by eIF6 is a reshaping of gene expression with increased levels of lipogenic and glycolytic enzymes. Finally, eIF6 levels modulate histone acetylation and amounts of rate-limiting fatty acid synthase (Fasn) mRNA. Since obesity, type 2 diabetes, and cancer require a Fasn-driven lipogenic state, we propose that eIF6 could be a therapeutic target for these diseases.

This article presents a detailed description of intraperitoneal and oral glucose tolerance tests in mice. The former is widely used in initial high-throughput phenotyping of mutant mice to assess a diabetic phenotype and alterations in glucose homeostasis. Each protocol provides a comprehensive description of each step in the workflow, including variation of the standard protocol under particular circumstances (e.g., sensitivity to food deprivation, excessive deviations in body composition, or need for extra blood samples for additional analyses). We also describe how reduction of body mass and body temperature can be used as additional readouts to monitor metabolic function in response to food deprivation., (Copyright © 2015 John Wiley & Sons, Inc.)

Combined use of metformin and a sodium glucose cotransporter 2 inhibitor (SGLT2I) is a promising treatment strategy for type 2 diabetes. The mechanism by which combination treatment provides better glycemic control than metformin or SGLT2I monotherapy remains elusive. Therefore, we investigated the physiological mechanism by which both compounds lower blood glucose concentrations in diabetic mice. We compared the potential of metformin and the SGLT2I AVE2268 alone or in combination to mitigate hyperglycemia and modulate glucose fluxes in db/db and diabetic Tallyho/JngJ mice. SGLT2I treatment alone elicited a rapid decline in circulating blood glucose levels, which appeared to induce endogenous glucose production. Supplementation of metformin dampened this counterresponse, and therefore, combination therapy more efficiently maintained glycemic control. Finally, combination treatment blunted postprandial glucose excursions and improved HbA1c levels within 2 weeks. We conclude that coapplication of metformin enhances the glucose-lowering actions of SGLT2I by restraining endogenous glucose production, which may provide long-term improvement of glycemic control in type 2 diabetic patients., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Insulin resistance (IR) lies at the origin of type 2 diabetes. It induces initial compensatory insulin secretion until insulin exhaustion and subsequent excessive levels of glucose (hyperglycemia). A high-calorie diet is a major risk factor contributing to the development of this metabolic disease. For this study, a time-course experiment was designed that consisted of two groups of mice. The aim of this design was to reproduce the dietary conditions that parallel the progress of IR over time. The first group was fed with a high-fatty-acid diet for several weeks and followed by 1 week of a low-fatty-acid intake, while the second group was fed with a low-fatty-acid diet during the entire experiment. The metabolomic fingerprint of C3HeB/FeJ mice liver tissue extracts was determined by means of two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-ToF-MS). This article addresses the application of ANOVA-simultaneous component analysis (ASCA) to the found metabolomic profile. By performing hyphenated high-throughput analytical techniques together with multivariate chemometric methodology on metabolomic analysis, it enables us to investigate the sources of variability in the data related to each experimental factor of the study design (defined as time, diet and individual). The contribution of the diet factor in the dissimilarities between the samples appeared to be predominant over the time factor contribution. Nevertheless, there is a significant contribution of the time-diet interaction factor. Thus, evaluating the influences of the factors separately, as it is done in classical statistical methods, may lead to inaccurate interpretation of the data, preventing achievement of consistent biological conclusions.

A combinatory approach using metabolomics and gut microbiome analysis techniques was performed to unravel the nature and specificity of metabolic profiles related to gut ecology in obesity. This study focused on gut and liver metabolomics of two different mouse strains, the C57BL/6J (C57J) and the C57BL/6N (C57N) fed with high-fat diet (HFD) for 3 weeks, causing diet-induced obesity in C57N, but not in C57J mice. Furthermore, a 16S-ribosomal RNA comparative sequence analysis using 454 pyrosequencing detected significant differences between the microbiome of the two strains on phylum level for Firmicutes, Deferribacteres and Proteobacteria that propose an essential role of the microbiome in obesity susceptibility. Gut microbial and liver metabolomics were followed by a combinatory approach using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and ultra performance liquid chromatography time of tlight MS/MS with subsequent multivariate statistical analysis, revealing distinctive host and microbial metabolome patterns between the C57J and the C57N strain. Many taurine-conjugated bile acids (TBAs) were significantly elevated in the cecum and decreased in liver samples from the C57J phenotype likely displaying different energy utilization behavior by the bacterial community and the host. Furthermore, several metabolite groups could specifically be associated with the C57N phenotype involving fatty acids, eicosanoids and urobilinoids. The mass differences based metabolite network approach enabled to extend the range of known metabolites to important bile acids (BAs) and novel taurine conjugates specific for both strains. In summary, our study showed clear alterations of the metabolome in the gastrointestinal tract and liver within a HFD-induced obesity mouse model in relation to the host-microbial nutritional adaptation.

A metabolic disorder such as Type-2 Diabetes mellitus (T2DM) is a complex disease induced by genetic, environmental, and nutritional factors. The db/db mouse model, bearing a nonfunctional leptin receptor, is widely used to investigate the pathophysiology of T2DM. Fecal extracts of db/db and wild-type littermates were studied to unravel a broad spectrum of new and relevant metabolites related to T2DM as proxies of the interplay of gut microbiome and murine metabolomes. The nontargeted metabolomics approach consists of an integrated analytical concept of high-resolution mass spectrometry FT-ICR-MS, followed by UPLC-TOF-MS/MS experiments. We demonstrate that a metabolic disorder such as T2DM affects the gastrointestinal tract environment, thereby influencing different metabolic pathways and their respective metabolites in diabetic mice. Fatty acids, bile acids concerning cholic and deoxycholic acid, and steroid metabolism were highly discriminative comparing fecal meta-metabolomes of wt and db/db mice. Furthermore, sulfur-(S)-containing metabolites including N-acyl taurines were altered in diabetic mice, enabling us to focus on S-containing metabolites, especially the sulfate and taurine conjugates of bile and fatty acids. Different sulfate containing bile acids including sulfocholic acid, oxocholic acid sulfate, taurocholic acid sulfate, and cyprinol sulfate were significantly altered in diabetic mice. Moreover, we identified 12 new sulfate and taurine conjugates of hydroxylated fatty acids with significant importance in T2DM metabolism in db/db mice.

Background & Aims: To determine if diabetic and insulin-resistant states cause mitochondrial dysfunction in liver or if there is long term adaptation of mitochondrial function to these states, mice were (i) fed with a high-fat diet to induce obesity and T2D (HFD), (ii) had a genetic defect in insulin signaling causing whole body insulin resistance, but not full blown T2D (IR/IRS-1(+/-) mice), or (iii) were analyzed after treatment with streptozocin (STZ) to induce a T1D-like state., Methods: Hepatic lipid levels were measured by thin layer chromatography. Mitochondrial respiratory chain (RC) levels and function were determined by Western blot, spectrophotometric, oxygen consumption and proton motive force analysis. Gene expression was analyzed by real-time PCR and microarray., Results: HFD caused insulin resistance and hepatic lipid accumulation, but RC was largely unchanged. Livers from insulin resistant IR/IRS-1(+/-) mice had normal lipid contents and a normal RC, but mitochondria were less well coupled. Livers from severely hyperglycemic and hypoinsulinemic STZ mice had massively depleted lipid levels, but RC abundance was unchanged. However, liver mitochondria isolated from these animals showed increased abundance and activity of the RC, which was better coupled., Conclusions: Insulin resistance, induced either by obesity or genetic manipulation and steatosis do not cause mitochondrial dysfunction in mouse liver. Also, mitochondrial dysfunction is not a prerequisite for liver steatosis. However, severe insulin deficiency and high blood glucose levels lead to an enhanced performance and better coupling of the RC. This may represent an adaptation to fuel overload and the high energy-requirement of an unsuppressed gluconeogenesis., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Metabolomics has entered the well-established omic sciences as it is an indispensable information resource to achieve a global picture of biological systems. The aim of the present study was to estimate the influence of blood removal from mice liver as part of sample preparation for metabolomic and proteomic studies. For this purpose, perfused mice liver tissue (i.e. with blood removed) and unperfused mice liver tissue (i.e. containing blood) were compared by two-dimensional gas chromatography time of flight mass spectrometry (GC × GC-TOFMS) for the metabolomic part, and by liquid chromatography tandem mass spectrometry (LC-MS/MS) for the proteomic part. Our data showed significant differences between the unperfused and perfused liver tissue samples. Furthermore, we also observed an overlap of blood and tissue metabolite profiles in our data, suggesting that the perfusion of liver tissue prior to analysis is beneficial for an accurate metabolic profile of this organ., (Copyright © 2013 John Wiley & Sons, Ltd.)

Aims/hypothesis: Diabetic retinopathy is a major complication of type 2 diabetes and the leading cause of blindness in adults of working age. Neuronal defects are known to occur early in disease, but the source of this dysfunction is unknown. The aim of this study was to examine differences in the retinal membrane proteome among non-diabetic mice and mouse models of diabetes either with or without metformin treatment., Methods: Alterations in the retinal membrane proteome of 10-week-old diabetic db/db mice, diabetic db/db mice orally treated with the anti-hyperglycaemic metformin, and congenic wild-type littermates were examined using label-free mass spectrometry. Pathway enrichment analysis was completed with Genomatix and Ingenuity. Alterations in Slc17a7 mRNA and vesicular glutamate transporter 1 (VGLUT1) protein expression were evaluated using real-time quantitative PCR and IMMUNOFLUORESCENCE., Results: A total of 98 proteins were significantly differentially abundant between db/db and wild-type animals. Pathway enrichment analysis indicated decreases in levels of proteins related to synaptic transmission and cell signalling. Metformin treatment produced 63 differentially abundant proteins compared with untreated db/db mice, of which only 43 proteins were found to occur in both datasets, suggesting that treatment only partially normalises the alterations induced by diabetes. VGLUT1, which is responsible for loading glutamate into synaptic vesicles, was found to be differentially abundant in db/db mice and was not normalised by metformin. The decrease in Slc17a7/VGLUT1 was confirmed by transcriptomic and immunocytochemical analysis., Conclusions/interpretation: These findings expand the knowledge of the protein changes in diabetic retinopathy and suggest that membrane-associated signalling proteins are susceptible to changes that are partially ameliorated by treatment

Genetic predisposition and environmental factors contribute to an individual's susceptibility to develop hepatosteatosis. In a systematic, comparative survey we focused on genotype-dependent and -independent adaptations early in the pathogenesis of hepatosteatosis by characterizing C3HeB/FeJ, C57BL/6NTac, C57BL/6J, and 129P2/OlaHsd mice after 7, 14, or 21 days high-fat-diet exposure. Strain-specific metabolic responses during diet challenge and liver transcript signatures in mild hepatosteatosis outline the suitability of particular strains for investigating the relationship between hepatocellular lipid content and inflammation, glucose homeostasis, insulin action, or organelle physiology. Genetic background-independent transcriptional adaptations in liver paralleling hepatosteatosis suggest an early increase in the organ's vulnerability to oxidative stress damage what could advance hepatosteatosis to steatohepatitis. "Universal" adaptations in transcript signatures and transcription factor regulation in liver link insulin resistance, type 2 diabetes mellitus, cancer, and thyroid hormone metabolism with hepatosteatosis, hence, facilitating the search for novel molecular mechanisms potentially implicated in the pathogenesis of human non-alcoholic-fatty-liver-disease.

Male New Zealand Obese (NZO) mice progress through pathophysiological stages similar to humans developing obesity-associated type 2 diabetes (T2D). The current challenge is to establish quantitative proteomics from small plasma sample amounts. We established an analytical workflow that facilitates a reproducible depletion of high-abundance proteins, has high throughput applicability, and allows absolute quantification of proteins from mouse plasma samples by LC-SRM-MS. The ProteoMiner equalizing technology was adjusted to the small sample amount, and reproducibility of the identifications was monitored by spike proteins. Based on the label-free relative quantification of proteins in depleted plasma of a test set of NZO mice, assays for potential candidates were designed for the setup of a targeted selected reaction monitoring (SRM) approach and absolute quantification. We could demonstrate that apolipoprotein E (Apoe), mannose-binding lectin 2 (Mbl2), and parotid secretory protein (Psp) are present at significantly different quantities in depleted plasma of diabetic NZO mice compared to non-diabetic controls using AQUA peptides. Quantification was validated for Mbl2 using the ELISA technology on non-depleted plasma. We conclude that the depletion technique is applicable to restricted sample amounts and suitable for the identification of T2D signatures in plasma.

Protein expression analysis is one of the most powerful tools to further the understanding of biological systems. Progress in the field of mass spectrometry has shifted focus from gel-based approaches to the upcoming LC-selected reaction monitoring (SRM) technique which combines high technical accuracy with absolute quantification of proteins and the capability for high-throughput analyses. Due to these properties, LC-SRM has the potential to become the foundation for biomarker analysis, targeted hypothesis driven proteomic studies and contribute to the field of systems biology. While the performance of LC-SRM applied to samples from various bodily fluids, particularly plasma, and microorganisms has been extensively investigated, there is only little experience with its application to animal tissue samples. Here, we show that a conventional one-dimensional LC-SRM workflow applied to mouse liver tissue suffers from a shortcoming in terms of sensitivity for lower abundance proteins. This problem could be solved through the extension of the standard workflow by an additional dimension of separation at the peptide level prior to online LC-SRM. For this purpose, we used off-gel electrophoresis (OGE) which is also shown to outperform strong cation exchange (SCX) in terms of resolution, gain of signal intensity, and predictability of separation. The extension of the SRM workflow by a high resolving peptide separation technique is an ideal combination as it allows the addition of stable isotope standards directly after trytic digestion and will increase the dynamic range of protein abundances amenable by SRM in animal tissue.

Under the label of the German Mouse Clinic (GMC), a concept has been developed and implemented that allows the better understanding of human diseases on the pathophysiological and molecular level. This includes better understanding of the crosstalk between different organs, pleiotropy of genes, and the systemic impact of envirotypes and drugs. In the GMC, experts from various fields of mouse genetics and physiology, in close collaboration with clinicians, work side by side under one roof. The GMC is an open-access platform for the scientific community by providing phenotypic analysis in bilateral collaborations ("bottom-up projects") and as a partner and driver in international large-scale biology projects ("top-down projects"). Furthermore, technology development is a major topic in the GMC. Innovative techniques for primary and secondary screens are developed and implemented into the phenotyping pipelines (e.g., detection of volatile organic compounds, VOCs).

Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20 years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/[2])., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Mitochondrial fatty acid oxidation provides an important energy source for cellular metabolism, and decreased mitochondrial fatty acid oxidation has been implicated in the pathogenesis of type 2 diabetes. Paradoxically, mice with an inherited deficiency of the mitochondrial fatty acid oxidation enzyme, very long-chain acyl-CoA dehydrogenase (VLCAD), were protected from high-fat diet-induced obesity and liver and muscle insulin resistance. This was associated with reduced intracellular diacylglycerol content and decreased activity of liver protein kinase Cvarepsilon and muscle protein kinase Ctheta. The increased insulin sensitivity in the VLCAD(-/-) mice were protected from diet-induced obesity and insulin resistance due to chronic activation of AMPK and PPARalpha, resulting in increased fatty acid oxidation and decreased intramyocellular and hepatocellular diacylglycerol content.

Objective: Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance are poorly understood. In this study, we examine the role of serine phosphorylation of insulin receptor substrate (IRS)-1 in mediating fat-induced insulin resistance in skeletal muscle in vivo., Research Design and Methods: To directly assess the role of serine phosphorylation in mediating fat-induced insulin resistance in skeletal muscle, we generated muscle-specific IRS-1 Ser(302), Ser(307), and Ser(612) mutated to alanine (Tg IRS-1 Ser-->Ala) and IRS-1 wild-type (Tg IRS-1 WT) transgenic mice and examined insulin signaling and insulin action in skeletal muscle in vivo., Results: Tg IRS-1 Ser-->Ala mice were protected from fat-induced insulin resistance, as reflected by lower plasma glucose concentrations during a glucose tolerance test and increased insulin-stimulated muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. In contrast, Tg IRS-1 WT mice exhibited no improvement in glucose tolerance after high-fat feeding. Furthermore, Tg IRS-1 Ser-->Ala mice displayed a significant increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity and Akt phosphorylation in skeletal muscle in vivo compared with WT control littermates., Conclusions: These data demonstrate that serine phosphorylation of IRS-1 plays an important role in mediating fat-induced insulin resistance in skeletal muscle in vivo.

Uncoupling of oxidative phosphorylation represents a potential target for the treatment of hyperglycemia and insulin resistance in obesity and type 2 diabetes. The present study investigated whether the expression of uncoupling protein 1 in skeletal muscles of transgenic (mUCP1 TG) mice modulates insulin action in major insulin target tissues in vivo. Euglycemic-hyperinsulinemic clamps (17 pM x kg lean body mass(-1) x min(-1)) were performed in 9-mo-old hemizygous male mUCP1 TG mice and wild-type (WT) littermates matched for body composition. mUCP1 TG mice exhibited fasting hypoglycemia and hypoinsulinemia compared with WT mice, whereas fasting hepatic glucose production rates were comparable in both genotypes. mUCP1 TG mice were markedly more sensitive to insulin action compared with WT mice and displayed threefold higher glucose infusion rates, enhanced skeletal muscle and white adipose tissue glucose uptake, and whole body glycolysis rates. In the absence of alterations in plasma adiponectin concentrations, acceleration of insulin-stimulated glucose turnover in skeletal muscle of mUCP1 TG mice was accompanied by increased phosphorylated Akt-to-Akt and phosphorylated AMP-activated protein kinase (AMPK)-to-AMPK ratios compared with WT mice. UCP1-mediated uncoupling of oxidative phosphorylation in skeletal muscle was paralleled by AMPK activation and thereby stimulated insulin-mediated glucose uptake in skeletal muscle.

The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.

Objective: We have previously reported that a high-fat, carbohydrate-free diet prevents diabetes and beta-cell destruction in the New Zealand Obese (NZO) mouse strain. Here we investigated the effect of diets with and without carbohydrates on obesity and development of beta-cell failure in a second mouse model of type 2 diabetes, the db/db mouse., Results: When kept on a carbohydrate-containing standard (SD; with (w/w) 5.1, 58.3, and 17.6% fat, carbohydrates and protein, respectively) or high-fat diet (HFD; 14.6, 46.7 and 17.1%), db/db mice developed severe diabetes (blood glucose >20 mmol/l, weight loss, polydipsia and polyurea) associated with a selective loss of pancreatic beta-cells, reduced GLUT2 expression in the remaining beta-cells, and reduced plasma insulin levels. In contrast, db/db mice kept on a high-fat, carbohydrate-free diet (CFD; with 30.2 and 26.4% (w/w) fat or protein) did not develop diabetes and exhibited near-normal, hyperplastic islets in spite of a morbid obesity (fat content >60%) associated with hyperinsulinaemia., Conclusion: These data indicate that in genetically different mouse models of obesity-associated diabetes, obesity and dietary fat are not sufficient, and dietary carbohydrates are required, for beta-cell destruction.

Disruptions of the melanocortin signaling system have been linked to obesity. We investigated a possible role of the central nervous melanocortin system (CNS-Mcr) in the control of adiposity through effects on nutrient partitioning and cellular lipid metabolism independent of nutrient intake. We report that pharmacological inhibition of melanocortin receptors (Mcr) in rats and genetic disruption of Mc4r in mice directly and potently promoted lipid uptake, triglyceride synthesis, and fat accumulation in white adipose tissue (WAT), while increased CNS-Mcr signaling triggered lipid mobilization. These effects were independent of food intake and preceded changes in adiposity. In addition, decreased CNS-Mcr signaling promoted increased insulin sensitivity and glucose uptake in WAT while decreasing glucose utilization in muscle and brown adipose tissue. Such CNS control of peripheral nutrient partitioning depended on sympathetic nervous system function and was enhanced by synergistic effects on liver triglyceride synthesis. Our findings offer an explanation for enhanced adiposity resulting from decreased melanocortin signaling, even in the absence of hyperphagia, and are consistent with feeding-independent changes in substrate utilization as reflected by respiratory quotient, which is increased with chronic Mcr blockade in rodents and in humans with loss-of-function mutations in MC4R. We also reveal molecular underpinnings for direct control of the CNS-Mcr over lipid metabolism. These results suggest ways to design more efficient pharmacological methods for controlling adiposity.

Individuals born with a low birth weight (LBW) have an increased prevalence of type 2 diabetes, but the mechanisms responsible for this association are unknown. Given the important role of insulin resistance in the pathogenesis of type 2 diabetes, we examined insulin sensitivity in a rat model of LBW due to intrauterine fetal stress. During the last 7 days of gestation, rat dams were treated with dexamethasone and insulin sensitivity was assessed in the LBW offspring by a hyperinsulinemic euglycemic clamp. The LBW group had liver-specific insulin resistance associated with increased levels of PEPCK expression. These changes were associated with pituitary hyperplasia of the ACTH-secreting cells, increased morning plasma ACTH concentrations, elevated corticosterone secretion during restraint stress, and an approximately 70% increase in 24-h urine corticosterone excretion. These data support the hypothesis that prenatal stress can result in chronic hyperactivity of the hypothalamic-pituitary-adrenal axis, resulting in increased plasma corticosterone concentrations, upregulation of hepatic gluconeogenesis, and hepatic insulin resistance.

Nonalcoholic fatty liver disease (NAFLD) is a major contributing factor to hepatic insulin resistance in type 2 diabetes. Diacylglycerol acyltransferase (Dgat), of which there are two isoforms (Dgat1 and Dgat2), catalyzes the final step in triglyceride synthesis. We evaluated the metabolic impact of pharmacological reduction of DGAT1 and -2 expression in liver and fat using antisense oligonucleotides (ASOs) in rats with diet-induced NAFLD. Dgat1 and Dgat2 ASO treatment selectively reduced DGAT1 and DGAT2 mRNA levels in liver and fat, but only Dgat2 ASO treatment significantly reduced hepatic lipids (diacylglycerol and triglyceride but not long chain acyl CoAs) and improved hepatic insulin sensitivity. Because Dgat catalyzes triglyceride synthesis from diacylglycerol, and because we have hypothesized that diacylglycerol accumulation triggers fat-induced hepatic insulin resistance through protein kinase C epsilon activation, we next sought to understand the paradoxical reduction in diacylglycerol in Dgat2 ASO-treated rats. Within 3 days of starting Dgat2 ASO therapy in high fat-fed rats, plasma fatty acids increased, whereas hepatic lysophosphatidic acid and diacylglycerol levels were similar to those of control rats. These changes were associated with reduced expression of lipogenic genes (SREBP1c, ACC1, SCD1, and mtGPAT) and increased expression of oxidative/thermogenic genes (CPT1 and UCP2). Taken together, these data suggest that knocking down Dgat2 protects against fat-induced hepatic insulin resistance by paradoxically lowering hepatic diacylglycerol content and protein kinase C epsilon activation through decreased SREBP1c-mediated lipogenesis and increased hepatic fatty acid oxidation.

The ATP-binding cassette transporter G1 (ABCG1) catalyzes export of cellular cholesterol from macrophages and hepatocytes. Here we identify an additional function of ABCG1 in the regulation of adiposity in screens of the Drosophila melanogaster and the New Zealand obese (NZO) mouse genomes. Insertion of modified transposable elements of the P-family upstream of CG17646, the Drosophila ortholog of Abcg1, generated lines of flies with increased triglyceride stores. In NZO mice, an Abcg1 variant was identified in a suggestive adiposity quantitative trait locus and was associated with higher expression of the gene in white adipose tissue. Targeted disruption of Abcg1 in mice resulted in reduced body weight gain (8.42+/-0.6 g in Abcg1-/- vs. 13.07+/-1.1 g in Abcg1+/+ mice) and adipose tissue mass gain (3.78+/-1.3 g in Abcg1-/- vs. 9.39+/-1.6 g in Abcg1+/+ mice) detected over a period of 12 wk. The reduction of adipose tissue mass in Abcg1-/- mice was associated with markedly decreased size of the adipocytes. In contrast to their wild-type littermates, male Abcg1-/- mice exhibited no high-fat diet-induced impairment of glucose tolerance and fatty liver. Furthermore, Abcg1-/- mice possess decreased food intake and elevated total energy expenditure (Abcg1-/- mice, 748.1+/-5.4 kJ/kg metabolic body mass; Abcg1+/+ mice, 684.3+/-5.0 kJ/kg metabolic body mass; P=0.011), body temperature (Abcg1-/- mice, 37.82+/-0.29 C; Abcg1+/+ mice, 36.83+/-0.24 C; P<0.05), and locomotor activity (Abcg1-/- mice, 3655+/-189 counts/12 h during dark phase; Abcg1+/+ mice, 2445+/-235 counts/12 h during dark phase; P<0.01). Our data indicate a previously unrecognized role of ABCG1 in the regulation of energy balance and triglyceride storage.

Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fat diet-induced insulin resistance through peroxisome proliferator-activated receptor (PPAR)-alpha activation and a subsequent decrease in intracellular lipid abundance. To directly test this hypothesis, we fed PPAR-alpha null and wild-type mice for 2 weeks with isocaloric high-fat diets containing 27% fat from either safflower oil or safflower oil with an 8% fish oil replacement (fish oil diet). In both genotypes the safflower oil diet blunted insulin-mediated suppression of hepatic glucose production (P < 0.02 vs. genotype control) and PEPCK gene expression. Feeding wild-type mice a fish oil diet restored hepatic insulin sensitivity (hepatic glucose production [HGP], P < 0.002 vs. wild-type mice fed safflower oil), whereas in contrast, in PPAR-alpha null mice failed to counteract hepatic insulin resistance (HGP, P = NS vs. PPAR-alpha null safflower oil-fed mice). In PPAR-alpha null mice fed the fish oil diet, safflower oil plus fish oil, hepatic insulin resistance was dissociated from increases in hepatic triacylglycerol and acyl-CoA but accompanied by a more than threefold increase in hepatic diacylglycerol concentration (P < 0.0001 vs. genotype control). These data support the hypothesis that n-3 fatty acids protect from high-fat diet-induced hepatic insulin resistance in a PPAR-alpha-and diacylglycerol-dependent manner.

In type 2 diabetes, glucagon levels are elevated in relation to the prevailing insulin and glucose levels. The relative hyperglucagonemia is linked to increased hepatic glucose output (HGO) and hyperglycemia. Antagonizing the effects of glucagon is therefore considered an attractive target for treatment of type 2 diabetes. In the current study, effects of eliminating glucagon signaling with a glucagon monoclonal antibody (mAb) were investigated in the diabetic ob/ob mouse. Acute effects of inhibiting glucagon action were studied by an oral glucose tolerance test (OGTT) and by measurement of HGO. In addition, the effects of subchronic (5 and 14 days) glucagon mAb treatment on plasma glucose, insulin, triglycerides, and HbA1c (A1C) levels were investigated. Glucagon mAb treatment reduced the area under the curve for glucose after an OGTT, reduced HGO, and increased the rate of hepatic glycogen synthesis. Glucagon mAb treatment for 5 days lowered plasma glucose and triglyceride levels, whereas 14 days of glucagon mAb treatment reduced A1C. In conclusion, acute and subchronic neutralization of endogenous glucagon improves glycemic control, thus supporting the contention that glucagon antagonism may represent a beneficial treatment of diabetes.

Mice lacking C3, C4 or complement receptor 1/2 (Cr) have defective germinal centers (GC). The requirement for C4 implicates complement fixation by immune complexes (IC) via the classical pathway. Yet, transgenic (Tg) mice that lack circulating antibody but still express membrane IgM (mIgM) have normal GC responses. We showed previously that cross-linking mIgM leads to the deposition of C3 on the B cell surface and that disruption of this pathway diminishes GC responses. Here, we investigate the role of Cr in this process by generating mIgM-Tg mice that lack Cr and serum Ig. These mIgM/Cr-/- mice have smaller, transient GC, with incomplete B cell receptor down-regulation and peanut agglutinin up-regulation, compared to mIgM/Crwt counterparts. BM chimera experiments showed that Cr on B cells is required for normal GC responses. These results establish that Cr ligands generated at the B cell surface are sufficient for normal GC responses and function by signaling Cr on B cells. Unexpectedly, chimera experiments also showed a critical role for Cr on follicular dendritic cells (FDC), even in the absence of IC, indicating novel functions for FDC-expressed Cr beyond the capture of C3-coated IC.

Adiponectin has insulin-sensitizing, antiatherogenic, and anti-inflammatory properties, but little is known about factors that regulate its secretion. To examine the effect of fish oil on adiponectin secretion, mice were fed either a control diet or isocaloric diets containing 27% safflower oil or 27, 13.5, and 8% menhaden fish oil. Within 15 days, fish oil feeding raised plasma adiponectin concentrations two- to threefold in a dose-dependent manner, and the concentrations remained approximately twofold higher for 7 days when the fish oil diet was replaced by the safflower oil diet. Within 24 h, fish oil markedly induced transcription of the adiponectin gene in epididymal adipose tissue but not in subcutaneous fat. The increase of plasma adiponectin by fish oil was completely blocked by administration of the peroxisome proliferator-activated receptor (PPAR)gamma inhibitor bisphenol-A-diglycidyl ether. In contrast, there was no effect of fish oil feeding on adiponectin secretion in PPARalpha-null mice. These data suggest that fish oil is a naturally occurring potent regulator of adiponectin secretion in vivo and that it does so through a PPARgamma-dependent and PPARalpha-independent manner in epididymal fat.

To further explore the nature of the mitochondrial dysfunction and insulin resistance that occur in the muscle of young, lean, normoglycemic, insulin-resistant offspring of parents with type 2 diabetes (IR offspring), we measured mitochondrial content by electron microscopy and insulin signaling in muscle biopsy samples obtained from these individuals before and during a hyperinsulinemic-euglycemic clamp. The rate of insulin-stimulated muscle glucose uptake was approximately 60% lower in the IR offspring than the control subjects and was associated with an approximately 60% increase in the intramyocellular lipid content as assessed by H magnetic resonance spectroscopy. Muscle mitochondrial density was 38% lower in the IR offspring. These changes were associated with a 50% increase in IRS-1 Ser312 and IRS-1 Ser636 phosphorylation and an approximately 60% reduction in insulin-stimulated Akt activation in the IR offspring. These data provide new insights into the earliest defects that may be responsible for the development of type 2 diabetes and support the hypothesis that reductions in mitochondrial content result in decreased mitochondrial function, which predisposes IR offspring to intramyocellular lipid accumulation, which in turn activates a serine kinase cascade that leads to defects in insulin signaling and action in muscle.

In vitro studies suggest that the mitochondrial glycerol-3-phosphate acyltransferase-1 (mtGPAT1) isoform catalyzes the initial and rate-controlling step in glycerolipid synthesis and aids in partitioning acyl-CoAs toward triacylglycerol synthesis and away from degradative pathways. To determine whether the absence of mtGPAT1 would increase oxidation of acyl-CoAs and restrict the development of hepatic steatosis, we fed wild type and mtGPAT1-/- mice a diet high in fat and sucrose (HH) for 4 months to induce the development of obesity and a fatty liver. Control mice were fed a diet low in fat and sucrose (LL). With the HH diet, absence of mtGPAT1 resulted in increased partitioning of acyl-CoAs toward oxidative pathways, demonstrated by 60% lower hepatic triacylglycerol content and 2-fold increases in plasma beta-hydroxybutyrate, acylcarnitines, and hepatic mRNA expression of mitochondrial HMG-CoA synthase. Despite the increase in fatty acid oxidation, liver acyl-CoA levels were 3-fold higher in the mtGPAT1-/- mice fed both diets. A lack of difference in CPT1 and FAS mRNA expression between genotypes suggested that the increased acyl-CoA content was not because of increased de novo synthesis, but instead, to an impaired ability to use long-chain acyl-CoAs derived from the diet, even when the dietary fat content was low. Hyperinsulinemia and reduced glucose tolerance on the HH diet was greater in the mtGPAT1-/- mice, which did not suppress the expression of the gluconeogenic genes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This study demonstrates that mtGPAT1 is essential for normal acyl-CoA metabolism, and that the absence of hepatic mtGPAT1 results in the partitioning of fatty acids away from triacylglycerol synthesis and toward oxidation and ketogenesis.

In order to investigate the role of mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase 1 (mtGPAT1) in the pathogenesis of hepatic steatosis and hepatic insulin resistance, we examined whole-body insulin action in awake mtGPAT1 knockout (mtGPAT1(-/-)) and wild-type (wt) mice after regular control diet or three weeks of high-fat feeding. In contrast to high-fat-fed wt mice, mtGPAT1(-/-) mice displayed markedly lower hepatic triacylglycerol and diacylglycerol concentrations and were protected from hepatic insulin resistance possibly due to a lower diacylglycerol-mediated PKC activation. Hepatic acyl-CoA has previously been implicated in the pathogenesis of insulin resistance. Surprisingly, compared to wt mice, mtGPAT1(-/-) mice exhibited increased hepatic insulin sensitivity despite an almost 2-fold elevation in hepatic acyl-CoA content. These data suggest that mtGPAT1 might serve as a novel target for treatment of hepatic steatosis and hepatic insulin resistance and that long chain acyl-CoA's do not mediate fat-induced hepatic insulin resistance in this model.

Signal transducer and activator of transcription (STAT)3 is widely expressed in the CNS during development and adulthood. STAT3 has been implicated in the control of neuron/glial differentiation and leptin-mediated energy homeostasis, but the physiological role and degree of involvement of STAT3 in these processes is not defined and controversial because of the lack of a direct genetic model. To address this, we created mice with a neural-specific disruption of STAT3 (STAT3(N-/-)). Surprisingly, homozygous mutants were born at the expected Mendelian ratio without apparent developmental abnormalities but susceptible to neonatal lethality. Mutants that survived the neonatal period were hyperphagic, obese, diabetic, and infertile. Administering a melanocortin-3/4 receptor agonist abrogated the hyperphagia and hypothalamic immunohistochemistry showed a marked reduction in proopiomelanocortin with an increase in neuropeptide Y and agouti-related protein. Mutants had reduced energy expenditure and became hypothermic after fasting or cold stress. STAT3(N-/-) mice are hyperleptinemic, suggesting a leptin-resistant condition. Concomitant with neuroendocrine defects such as decreased linear growth and infertility with accompanying increased corticosterone levels, this CNS knockout recapitulates the unique phenotype of db/db and ob/ob obese models and distinguishes them from other genetic models of obesity. Thus, STAT3 in the CNS plays essential roles in the regulation of energy homeostasis and reproduction.

To examine the mechanism by which fish oil protects against fat-induced insulin resistance, we studied the effects of control, fish oil, and safflower oil diets on peroxisomal content, fatty acyl-CoA, diacylglycerol, and ceramide content in rat liver and muscle. We found that, in contrast to control and safflower oil-fed rats, fish oil feeding induced a 150% increase in the abundance of peroxisomal acyl-CoA oxidase and 3-ketoacyl-CoA thiolase in liver but lacked similar effects in muscle. This was paralleled by an almost twofold increase in hepatic peroxisome content (both P < 0.002 vs. control and safflower). These changes in the fish oil-fed rats were associated with a more than twofold lower hepatic triglyceride/diacylglycerol, as well as intramuscular triglyceride/fatty acyl-CoA, content. In conclusion, these data strongly support the hypothesis that n-3 fatty acids protect against fat-induced insulin resistance by serving as peroxisome proliferator-activated receptor-alpha ligands and thereby induce hepatic, but not intramuscular, peroxisome proliferation. In turn, an increased hepatic beta-oxidative capacity results in lower hepatic triglyceride/diacylglycerol and intramyocellular triglyceride/fatty acyl-CoA content.