The effects of extracellular vesicles and their cargo on metabolism and its adaptation to physical exercise in insulin resistance and type 2 diabetes.

Lifestyle modification represents the first-line strategy for the prevention and treatment of type 2 diabetes mellitus (T2DM), which is frequently associated with obesity and characterized by defective pancreatic insulin secretion and/or insulin resistance. Exercise training is an essential component of lifestyle modification and has been shown to ameliorate insulin resistance by reducing body fat mass and by enhancing skeletal muscle mitochondrial biogenesis and insulin-independent glucose uptake. Additionally, exercising stimulates the release of exerkines such as metabolites or cytokines, but also long non-coding RNA, microRNAs, cell-free DNA (cf-DNA), and extracellular vesicles (EVs), which contribute to inter-tissue communication. There is emerging evidence that EV number and content are altered in obesity and T2DM and may be involved in several metabolic processes, specifically either worsening or improving insulin resistance. This review summarizes the current knowledge on the metabolic effects of exercise training and on the potential role of humoral factors and EV as new biomarkers for early diagnosis and tailored treatment of T2DM.

Hyperbaric oxygen rapidly improves tissue-specific insulin sensitivity and mitochondrial capacity in humans with type 2 diabetes: a randomised placebo-controlled crossover trial.

AIMS/HYPOTHESIS: Hyperbaric oxygen (HBO) therapy may improve hyperglycaemia in humans with type 2 diabetes, but underlying mechanisms are unclear. Our objective was to examine the glucometabolic effects of HBO on whole-body glucose disposal in humans with type 2 diabetes. METHODS: In a randomised placebo-controlled crossover trial located at the German Diabetes Center, 12 male individuals with type 2 diabetes (age 18-75 years, BMI <35 kg/m2, HbA1c 42-75 mmol/mol [6-9%]), randomly allocated by one person, underwent 2-h HBO, once with 100% (240 kPa; HBO) and once with 21% oxygen (240 kPa; control, CON). Insulin sensitivity was assessed by hyperinsulinaemic-euglycaemic clamps with D-[6,6-2H2]glucose, hepatic and skeletal muscle energy metabolism were assessed by 1H/31P-magnetic resonance spectroscopy, while high-resolution respirometry measured skeletal muscle and white adipose tissue (WAT) mitochondrial capacity. All participants and people assessing the outcomes were blinded. RESULTS: HBO decreased fasting blood glucose by 19% and increased whole-body, hepatic and WAT insulin sensitivity about one-third (p<0.05 vs CON). Upon HBO, hepatic γ-ATP concentrations doubled, mitochondrial respiratory control doubled in skeletal muscle and tripled in WAT (p<0.05 vs CON). HBO increased myocellular insulin-stimulated serine-473/threonine-308 phosphorylation of Akt but decreased basal inhibitory serine-1101 phosphorylation of IRS-1 and endoplasmic reticulum stress (p<0.05 vs CON). CONCLUSIONS/INTERPRETATION: HBO-mediated improvement of insulin sensitivity likely results from decreased endoplasmic reticulum stress and increased mitochondrial capacity, possibly leading to low-dose reactive oxygen species-mediated mitohormesis in humans with type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT04219215 FUNDING: German Federal Ministry of Health, German Federal Ministry of Education and Research, North-Rhine Westfalia Ministry of Culture and Science, European-Regional-Development-Fund, German-Research-Foundation (DFG), Schmutzler Stiftung.

Dietary palmitate and oleate differently modulate insulin sensitivity in human skeletal muscle.

AIMS/HYPOTHESIS: Energy-dense nutrition generally induces insulin resistance, but dietary composition may differently affect glucose metabolism. This study investigated initial effects of monounsaturated vs saturated lipid meals on basal and insulin-stimulated myocellular glucose metabolism and insulin signalling. METHODS: In a randomised crossover study, 16 lean metabolically healthy volunteers received single meals containing safflower oil (SAF), palm oil (PAL) or vehicle (VCL). Whole-body glucose metabolism was assessed from glucose disposal (Rd) before and during hyperinsulinaemic-euglycaemic clamps with D-[6,6-2H2]glucose. In serial skeletal muscle biopsies, subcellular lipid metabolites and insulin signalling were measured before and after meals. RESULTS: SAF and PAL raised plasma oleate, but only PAL significantly increased plasma palmitate concentrations. SAF and PAL increased myocellular diacylglycerol and activated protein kinase C (PKC) isoform θ (p < 0.05) but only PAL activated PKCɛ. Moreover, PAL led to increased myocellular ceramides along with stimulated PKCζ translocation (p < 0.05 vs SAF). During clamp, SAF and PAL both decreased insulin-stimulated Rd (p < 0.05 vs VCL), but non-oxidative glucose disposal was lower after PAL compared with SAF (p < 0.05). Muscle serine1101-phosphorylation of IRS-1 was increased upon SAF and PAL consumption (p < 0.05), whereas PAL decreased serine473-phosphorylation of Akt more than SAF (p < 0.05). CONCLUSIONS/INTERPRETATION: Lipid-induced myocellular insulin resistance is likely more pronounced with palmitate than with oleate and is associated with PKC isoforms activation and inhibitory insulin signalling. TRIAL REGISTRATION: ClinicalTrials.gov .NCT01736202. FUNDING: German Federal Ministry of Health, Ministry of Culture and Science of the State North Rhine-Westphalia, German Federal Ministry of Education and Research, European Regional Development Fund, German Research Foundation, German Center for Diabetes Research.

Mitochondrial respiration is decreased in visceral but not subcutaneous adipose tissue in obese individuals with fatty liver disease.

BACKGROUND & AIMS: Adipose tissue dysfunction is involved in the development of insulin resistance and is responsible for excessive lipid delivery to other organs such as the liver. We tested the hypothesis that impaired mitochondrial function is a common feature of subcutaneous (SAT) and visceral adipose tissue (VAT), but may differently contribute to adipose tissue insulin resistance (IR) in obesity, non-alcoholic fatty liver (NAFL) and steatohepatitis (NASH). METHODS: In this cross-sectional study, we analyzed tissue-specific insulin sensitivity using stable isotope dilution and hyperinsulinemic-normoglycemic clamp tests. We also assessed mitochondrial respiration, mRNA and protein expression, and tissue morphology in biopsies of SAT and VAT from obese humans without NAFL, with NAFL or with NASH (n = 22/group). RESULTS: Compared to individuals without liver disease, persons with NAFL and NASH had about 30% (p = 0.010) and 33% (p = 0.002) lower maximal mitochondrial respiration, respectively, in VAT, but not in SAT. The lower maximal mitochondrial respiration of VAT was associated with lower adipose tissue insulin sensitivity (ß = 0.985, p = 0.041) and with increased VAT protein expression of tumor necrosis factor A across all groups (ß = -0.085, p = 0.040). VAT from individuals with NASH was characterized by lower expression of oxidative phosphorylation complex IV (p = 0.042) and higher mRNA expression of the macrophage marker CD68 (p = 0.002) than VAT from participants without NAFL. CONCLUSIONS: Humans with non-alcoholic fatty liver disease have distinct abnormalities of VAT energy metabolism, which correlate with adipose tissue dysfunction and may favor progression of NAFL to NASH. LAY SUMMARY: Adipose tissue (commonly called body fat) can be found under the skin (subcutaneous) or around internal organs (visceral). Dysfunction of adipose tissue can cause insulin resistance and lead to excess delivery of fat to other organs such as the liver. Herein, we show that dysfunction specifically in visceral adipose tissue was associated with fatty liver disease. CLINICAL TRIAL NUMBER: NCT01477957.

Dietary lipid droplet structure in postnatal life improves hepatic energy and lipid metabolism in a mouse model for postnatal programming.

Early-life diets may have a long-lasting impact on metabolic health. This study tested the hypothesis that an early-life diet with large, phospholipid-coated lipid droplets (Concept) induces sustained improvements of hepatic mitochondrial function and metabolism. Young C57BL/6j mice were fed Concept or control (CTRL) diet from postnatal day 15 (PN15) to PN42, followed by western style (WSD) or standard rodent diet (AIN) until PN98. Measurements comprised body composition, insulin resistance (HOMA-IR), tricarboxylic acid (TCA) cycle- and ß-oxidation-related hepatic oxidative capacity using high-resolution respirometry, mitochondrial dynamics, mediators of insulin resistance (diacylglycerols, DAG) or ceramides) in subcellular compartments as well as systemic oxidative stress. Concept feeding increased TCA cycle-related respiration by 33% and mitochondrial fusion protein-1 by 65% at PN42 (both p 0.05). At PN98, CTRL, but not Concept, mice developed hyperinsulinemia (CTRL/AIN 0.22 ± 0.44 vs. CTRL/WSD 1.49 ± 0.53 nmol/l, p 0.05 and Concept/AIN 0.20 ± 0.38 vs. Concept/WSD 1.00 ± 0.29 nmol/l, n.s.) and insulin resistance after WSD (CTRL/AIN 107 ± 23 vs. CTRL/WSD 738 ± 284, p 0.05 and Concept/AIN 109 ± 24 vs. Concept/WSD 524 ± 157, n.s.). WSD-induced liver weight was 18% lower in adult Concept-fed mice and ß-oxidation-related respiration was 69% higher (p 0.05; Concept/WSD vs. Concept/AIN) along with lower plasma lipid peroxides (CTRL/AIN 4.85 ± 0.28 vs. CTRL/WSD 5.73 ± 0.47 µmol/l, p 0.05 and Concept/AIN 4.49 ± 0.31 vs. Concept/WSD 4.42 ± 0.33 µmol/l, n.s.) and were in part protected from WSD-induced increase in hepatic cytosolic DAG C16:0/C18:1. Early-life feeding of Concept partly protected from WSD-induced insulin resistance and systemic oxidative stress, potentially via changes in specific DAG and mitochondrial function, highlighting the role of early life diets on metabolic health later in life.

Dietary Supplementation of dl-Methionine Potently Induces Sodium-Dependent l-Methionine Absorption in Porcine Jejunum Ex Vivo.

BACKGROUND: Methionine is an essential amino acid (AA) with many fundamental roles. Humans often supplement l-Met, whereas dl-Met and dl-2-hydroxy-4-(methylthio)butanoic acid (dl-HMTBA) are more frequently used to supplement livestock. OBJECTIVES: The study aimed to investigate whether dietary Met source alters the absorptive capacity for Met isomers in the small intestine of piglets. METHODS: A total of 27 male 10-wk-old piglets in 3 feeding groups received a diet supplemented with 0.21% dl-Met, 0.21% l-Met, or 0.31% dl-HMTBA to meet the Met + cystine requirement. After ≥10 d, absorptive fluxes of d-Met or l-Met were measured at a physiological concentration of 50 µM and a high concentration of 5 mM in duodenum, middle jejunum, and ileum ex vivo. Data were compared by 2-factor ANOVA. RESULTS: Across diets, fluxes of both Met isomers at both tested concentrations increased from duodenum to ileum by a factor of ∼2-5.5 (P < 0.05). Pigs supplemented with dl-Met had greater (P < 0.085) absorptive fluxes at 50 µM l-Met (0.50, 2.07, and 3.86 nmol · cm-2 · h-1) and d-Met (0.62, 1.41, and 1.19 nmol · cm-2 · h-1) than did pigs supplemented with dl-HMTBA (l-Met: 0.28, 0.76, and 1.08 nmol · cm-2 · h-1; d-Met: 0.34, 0.58, and 0.64 nmol · cm-2 · h-1) in duodenum, jejunum, and ileum, respectively. Only in jejunum of dl-Met-fed pigs, fluxes at 50 µM l-Met were reduced by the omission of luminal Na+ (from 3.27 to 0.86 nmol · cm-2 · h-1; P < 0.05) and by a cocktail of 22 luminal AAs (to 1.05 nmol · cm-2 · h-1; P < 0.05). CONCLUSIONS: Dietary supplementation of dl-Met increases the efficiency of l-Met and d-Met absorption at physiologically relevant luminal Met concentrations along the small intestine of pigs, including a very prominent induction of an Na+-dependent transport system with preference for l-Met in the mid-jejunum. Dietary supplementation with dl-Met could be a promising tool to improve the absorption of Met and other AAs.

Human CNNM2 is not a Mg(2+) transporter per se.

CNNM2 is associated with the regulation of serum Mg concentration, and when mutated, with severe familial hypomagnesemia. The function and cellular localization of CNNM2 and its isomorphs (Iso) remain controversial. The objective of this work was to examine the following: (1) the transcription-responsiveness of CNNM2 to Mg starvation, (2) the cellular localization of Iso1 and Iso2, (3) the ability of Iso1 and Iso2 to transport Mg(2+), and (4) the complex-forming ability and spectra of potential interactors of Iso1 and Iso2. The five main findings are as follows. (1) Mg-starvation induces CNNM2 overexpression that is markedly higher in JVM-13 cells (lymphoblasts) compared with Jurkat cells (T-lymphocytes). (2) Iso1 and Iso2 localize throughout various subcellular compartments in transgenic HEK293 cells overexpressing Iso1 or Iso2. (3) Iso1 and Iso2 do not transport Mg(2+) in an electrogenic or electroneutral mode in transgenic HEK293 cells overexpressing Iso1 or Iso2. (4) Both Iso1 and Iso2 form complexes of a higher molecular order. (5) The spectrum of potential interactors of Iso1 is ten times smaller than that of Iso2. We conclude that sensitivity of CNNM2 expression to extracellular Mg(2+) depletion depends on cell type. Iso1 and Iso2 exhibit a dispersed pattern of cellular distribution; thus, they are not exclusively integral to the cytoplasmic membrane. Iso1 and Iso2 are not Mg(2+) transporters per se. Both isomorphs form protein complexes, and divergent spectra of potential interactors of Iso1 and Iso2 indicate that each isomorph has a distinctive function. CNNM2 is therefore the first ever identified Mg(2+) homeostatic factor without being a Mg(2+) transporter per se.

Gastrointestinal methionine shuttle: Priority handling of precious goods.

Methionine (Met) is a neutral, sulfur-containing, essential amino acid with biological functions in the initiation and prolongation step of protein synthesis, transmethylation reactions, the synthesis of cysteine and cystine, and as a component of antioxidant systems. Its key importance is reflected by the fact that it is usually absorbed from the diet with highest efficiency among all proteinogenic amino acids but may yet not optimally support metabolism and health. As such, crystalline Met supplements are partly used in man and heavily used in production of animal species (poultry, fish, shrimps, pigs and cattle) to provide improved health and performance. The main intention of this review is to analyze the current knowledge on transport proteins with proven or hypothetical relevance for Met absorption in the gastrointestinal tract, especially the small intestine. These transporters include Na+ -dependent B0 AT1 and ATB0,+ and the Na+ -independent exchanger b0,+ /rBAT in the apical membrane, which may be supported by the Na+ -dependent systems ASCT2 and IMINO. The basolateral exit of Met appears to be largely limited to a single uniporter protein, LAT4. Insufficient or overtaxed efflux via LAT4 may lead to significant intracellular accumulation and metabolism of Met in the absorptive state. The latter can release large amounts of homocysteine into the blood, which favors atherosclerosis and other cardiovascular, as well as neurological, diseases. When LAT4 is defective, basolateral Met exit may be compensated to a certain degree by the Met exchange proteins 4F2hc/LAT2 or 4F2hc/LAT1; while carriers 4F2hc/y+ LAT1, 4F2hc/y+ LAT2, SNAT1 and SNAT2, may serve primarily for basolateral Met import. Expression of SNAT2 is increased when amino acid supply from the lumen ceases, suggesting a key role for Met supply of enterocytes in interdigestive periods. Enterocytes themselves have a huge requirement for Met to synthesize mucins and glutathione. © 2016 IUBMB Life, 68(12):924-934, 2016.

Insulin Modulates the Na+/Mg2+ Exchanger SLC41A1 and Influences Mg2+ Efflux from Intracellular Stores in Transgenic HEK293 Cells.

BACKGROUND: Magnesium deficiency is a common complication of diabetes with an unclear molecular background. OBJECTIVE: We investigated the effect of the insulin (INS)-signaling pathway (ISP) on the regulation of Mg(2+) efflux (Mg(2+)E) conducted by solute carrier family 41, member A1 (SLC41A1; activated by protein kinase A) in transgenic human embryonic kidney (HEK) 293 cells. METHODS: HEK293 cells overexpressing SLC41A1 were loaded with the Mg(2+) fluorescent indicator mag-fura-2 and Mg(2+). Measurements of Mg(2+)E were conducted in Mg(2+)-free buffer by using fast-filter fluorescence spectrometry. We examined the effects of INS, inhibitors of ISP or p38 mitogen-activated protein kinase (p38 MAPK), an activator of adenylate cyclase (ADC), and their combinations on SLC41A1-attributed Mg(2+)E. RESULTS: The application of 400 µU/mL INS inhibited SLC41A1-mediated Mg(2+)E by up to 50.6% compared with INS-untreated cells (P < 0.001). Moreover, INS evoked the early onset of Mg(2+) release from intracellular stores. The application of 0.1 µM wortmannin or 10 µM zardaverine (both ISP inhibitors) restored SLC41A1 Mg(2+)E capacity in the presence of INS to the same levels in INS-untreated cells. The simultaneous application of 10 µM forskolin, an ADC activator, and INS resulted in a reduction of Mg(2+)E of up to 59% compared with untreated cells (P < 0.001), which was comparable to that in cells treated with INS alone. Inhibition of p38 MAPK with 10 µM SB 202190 (SB) in the absence of INS resulted in a decrease (P < 0.001) of SLC41A1-dependent Mg(2+)E (by up to 49%) compared with Mg(2+)E measured in untreated cells. Simultaneous exposure of cells to SB and INS had a stronger inhibitory effect on SLC41A1 activity than INS alone (P < 0.05). CONCLUSIONS: INS affects intracellular Mg(2+) concentration in transgenic HEK293 cells by regulating SLC41A1 activity (via ISP) and by influencing the compartmentalization and cellular distribution of Mg(2+). In addition, p38 MAPK activates SLC41A1 independently of INS action.

Loss of mitochondrial adaptation associates with deterioration of mitochondrial turnover and structure in metabolic dysfunction-associated steatotic liver disease.

BACKGROUND: Obesity and type 2 diabetes frequently have metabolic dysfunction-associated steatotic liver disease (MASLD) including steatohepatitis (MASH). In obesity, the liver may adapt its oxidative capacity, but the role of mitochondrial turnover in MASLD remains uncertain. METHODS: This cross-sectional study compared individuals with class III obesity (n = 8/group) without (control, OBE CON; NAFLD activity score: 0.4 ± 0.1) or with steatosis (OBE MASL, 2.3 ± 0.4), or MASH (OBE MASH, 5.3 ± 0.3, p < 0.05 vs. other groups). Hepatic mitochondrial ultrastructure was assessed by transmission electron microscopy, mitochondrial respiration by high-resolution respirometry, biomarkers of mitochondrial quality control and endoplasmic reticulum (ER) stress by Western Blot. RESULTS: Mitochondrial oxidative capacity was 31 % higher in OBE MASL, but 25 % lower in OBE MASH (p < 0.05 vs. OBE CON). OBE MASH showed ~1.5fold lower mitochondrial number, but ~1.2-1.5fold higher diameter and area (p < 0.001 vs. other groups). Biomarkers of autophagy (p62), mitophagy (PINK1, PARKIN), fission (DRP-1, FIS1) and fusion (MFN1/2, OPA1) were reduced in OBE MASH (p < 0.05 vs. OBE CON). OBE MASL showed lower p62, p-PARKIN/PARKIN, and p-DRP-1 (p < 0.05 vs. OBE CON). OBE MASL and MASH showed higher ER stress markers (PERK, ATF4, p-eIF2α-S51/eIF2α; p < 0.05 vs. OBE CON). Mitochondrial diameter associated inversely with fusion/fission biomarkers and with oxidative capacity, but positively with H2O2. CONCLUSION: Humans with hepatic steatosis already exhibit impaired mitochondrial turnover, despite upregulated oxidative capacity, and evidence for ER stress. In MASH, oxidative stress likely mediates progressive decline of mitochondrial turnover, ultrastructure and respiration indicating that mitochondrial quality control is key for energy metabolism and may have potential for targeting MASH. ClinGovTrial:NCT01477957.

Alterations of hepatic energy metabolism in murine models of obesity, diabetes and fatty liver diseases.

BACKGROUND: Disturbed hepatic energy metabolism contributes to non-alcoholic fatty liver (NAFLD), but the development of changes over time and obesity- or diabetes-related mechanisms remained unclear. METHODS: Two-day old male C57BL/6j mice received streptozotocin (STZ) or placebo (PLC) and then high-fat (HFD) or regular chow diet (RCD) from week 4 (W4) to either W8 or W16, yielding control [CTRL = PLC + RCD], diabetes [DIAB = STZ + RCD], obesity [OBES = PLC + HFD] and diabetes-related non-alcoholic steatohepatitis [NASH = STZ + HFD] models. Mitochondrial respiration was measured by high-resolution respirometry and insulin-sensitive glucose metabolism by hyperinsulinemic-euglycemic clamps with stable isotope dilution. FINDINGS: NASH showed higher steatosis and NAFLD activity already at W8 and liver fibrosis at W16 (all p < 0.01 vs CTRL). Ballooning was increased in DIAB and NASH at W16 (p < 0.01 vs CTRL). At W16, insulin sensitivity was 47%, 58% and 75% lower in DIAB, NASH and OBES (p < 0.001 vs CTRL). Hepatic uncoupled fatty acid oxidation (FAO)-associated respiration was reduced in OBES at W8, but doubled in DIAB and NASH at W16 (p < 0.01 vs CTRL) and correlated with biomarkers of unfolded protein response (UPR), oxidative stress and hepatic expression of certain enzymes (acetyl-CoA carboxylase 2, Acc2; carnitine palmitoyltransferase I, Cpt1a). Tricarboxylic acid cycle (TCA)-driven respiration was lower in OBES at W8 and doubled in DIAB at W16 (p < 0.0001 vs CTRL), which positively correlated with expression of genes related to lipolysis. INTERPRETATION: Hepatic mitochondria adapt to various metabolic challenges with increasing FAO-driven respiration, which is linked to dysfunctional UPR, systemic oxidative stress, insulin resistance and altered lipid metabolism. In a diabetes model, higher TCA-linked respiration reflected mitochondrial adaptation to greater hepatic lipid turnover. FUNDING: Funding bodies that contributed to this study were listed in the acknowledgements section.

Dietary methionine source alters the lipidome in the small intestinal epithelium of pigs.

Methionine (Met) as an essential amino acid has key importance in a variety of metabolic pathways. This study investigated the influence of three dietary Met supplements (0.21% L-Met, 0.21% DL-Met and 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA)) on the metabolome and inflammatory status in the small intestine of pigs. Epithelia from duodenum, proximal jejunum, middle jejunum and ileum were subjected to metabolomics analysis and qRT-PCR of caspase 1, NLR family pyrin domain containing 3 (NLRP3), interleukins IL1ß, IL8, IL18, and transforming growth factor TGFß. Principal component analysis of the intraepithelial metabolome revealed strong clustering of samples by intestinal segment but not by dietary treatment. However, pathway enrichment analysis revealed that after L-Met supplementation polyunsaturated fatty acids (PUFA) and tocopherol metabolites were lower across small intestinal segments, whereas monohydroxy fatty acids were increased in distal small intestine. Pigs supplemented with DL-HMTBA showed a pronounced shift of secondary bile acids (BA) and sphingosine metabolites from middle jejunum to ileum. In the amino acid super pathway, only histidine metabolism tended to be altered in DL-Met-supplemented pigs. Diet did not affect the expression of inflammation-related genes. These findings suggest that dietary supplementation of young pigs with different Met sources selectively alters lipid metabolism without consequences for inflammatory status.

Impaired Hepatic Mitochondrial Capacity in Nonalcoholic Steatohepatitis Associated With Type 2 Diabetes.

OBJECTIVE: Individuals with type 2 diabetes are at higher risk of progression of nonalcoholic fatty liver (steatosis) to steatohepatitis (NASH), fibrosis, and cirrhosis. The hepatic metabolism of obese individuals adapts by upregulation of mitochondrial capacity, which may be lost during the progression of steatosis. However, the role of type 2 diabetes with regard to hepatic mitochondrial function in NASH remains unclear. RESEARCH DESIGN AND METHODS: We therefore examined obese individuals with histologically proven NASH without (OBE) (n = 30; BMI 52 ± 9 kg/m2) or with type 2 diabetes (T2D) (n = 15; 51 ± 7 kg/m2) as well as healthy individuals without liver disease (CON) (n = 14; 25 ± 2 kg/m2). Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamps with d-[6,6-2H2]glucose. Liver biopsies were used for assessing mitochondrial capacity by high-resolution respirometry and protein expression. RESULTS: T2D and OBE had comparable hepatic fat content, lobular inflammation, and fibrosis. Oxidative capacity in liver tissue normalized for citrate synthase activity was 59% greater in OBE than in CON, whereas T2D presented with 33% lower complex II-linked oxidative capacity than OBE and higher H2O2 production than CON. Interestingly, those with NASH and hepatic fibrosis score ≥1 had lower oxidative capacity and antioxidant defense than those without fibrosis. CONCLUSIONS: Loss of hepatic mitochondrial adaptation characterizes NASH and type 2 diabetes or hepatic fibrosis and may thereby favor accelerated disease progression.

Insulin resistance and insulin sensitizing agents.

Insulin resistance is a common feature of obesity and type 2 diabetes, but novel approaches of diabetes subtyping (clustering) revealed variable degrees of insulin resistance in people with diabetes. Specifically, the severe insulin resistant diabetes (SIRD) subtype not only exhibits metabolic abnormalities, but also bears a higher risk for cardiovascular, renal and hepatic comorbidities. In humans, insulin resistance comprises dysfunctional adipose tissue, lipotoxic insulin signaling followed by glucotoxicity, oxidative stress and low-grade inflammation. Recent studies show that aside from metabolites (free fatty acids, amino acids) and signaling proteins (myokines, adipokines, hepatokines) also exosomes with their cargo (proteins, mRNA and microRNA) contribute to altered crosstalk between skeletal muscle, liver and adipose tissue during the development of insulin resistance. Reduction of fat mass mainly, but not exclusively, explains the success of lifestyle modification and bariatric surgery to improve insulin sensitivity. Moreover, some older antihyperglycemic drugs (metformin, thiazolidinediones), but also novel therapeutic concepts (new peroxisome proliferator-activated receptor agonists, incretin mimetics, sodium glucose cotransporter inhibitors, modulators of energy metabolism) can directly or indirectly reduce insulin resistance. This review summarizes molecular mechanisms underlying insulin resistance including the roles of exosomes and microRNAs, as well as strategies for the management of insulin resistance in humans.

Expression of proposed methionine transporters along the gastrointestinal tract of pigs and their regulation by dietary methionine sources.

BACKGROUND: Given the key role of methionine (Met) in biological processes like protein translation, methylation, and antioxidant defense, inadequate Met supply can limit performance. This study investigated the effect of different dietary Met sources on the expression profile of various Met transporters along the gastrointestinal tract (GIT) of pigs. METHODS: A total of 27 pigs received a diet supplemented with 0.21% DL-Met, 0.21% L-Met, or 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA). Changes in mRNA expression of B0AT1, ATB0,+, rBAT, ASCT2, IMINO, LAT4, y+LAT1, LAT2, and SNAT2 were evaluated in the oral mucosa, cardia, fundus, pylorus, duodenum, proximal jejunum, middle jejunum, ileum, cecum, proximal colon, and distal colon, complemented by protein expression analysis of B0AT1, ASCT2, LAT2, and LAT4. RESULTS: Expression of all investigated transcripts differed significantly along the GIT. B0AT1, rBAT, y+LAT1, LAT2, and LAT4 showed strongest mRNA expression in small intestinal segments. ASCT2, IMINO, and SNAT2 were similarly expressed along the small and large intestines but expression differed in the oral mucosa and stomach. ATB0,+ showed highest mRNA expression in large intestinal tissues, cardia, and pylorus. In pigs fed DL-Met, mRNA expression of ASCT2 was higher than in pigs fed DL-HMTBA in small intestinal tissues and mRNA expression of IMINO was lower than in pigs fed L-Met in large intestinal tissues. Dietary DL-HMTBA induced a stronger mRNA expression of basolateral uptake systems either in the small (LAT2) or large (y+LAT1) intestine. Protein expression of B0AT1 was higher in the middle jejunum and ileum in pigs fed DL-Met when compared with the other Met supplements. LAT4 expression was higher in pigs fed DL-HMTBA when compared with DL-Met (small intestine) and L-Met (small intestine, oral mucosa, and stomach). CONCLUSION: A high expression of several Met transporters in small intestinal segments underlines the primary role of these segments in amino acid absorption; however, some Met transporters show high transcript and protein levels also in large intestine, oral mucosa, and stomach. A diet containing DL-Met has potential to increase apical Met transport in the small intestine, whereas a diet containing DL-HMTBA has potential to increase basolateral Met transport in the small intestine and, partly, other gastrointestinal tissues.

Metabolic responsiveness to training depends on insulin sensitivity and protein content of exosomes in insulin-resistant males.

High-intensity interval training (HIIT) improves cardiorespiratory fitness (VO2max), but its impact on metabolism remains unclear. We hypothesized that 12-week HIIT increases insulin sensitivity in males with or without type 2 diabetes [T2D and NDM (nondiabetic humans)]. However, despite identically higher VO2max, mainly insulin-resistant (IR) persons (T2D and IR NDM) showed distinct alterations of circulating small extracellular vesicles (SEVs) along with lower inhibitory metabolic (protein kinase Cε activity) or inflammatory (nuclear factor κB) signaling in muscle of T2D or IR NDM, respectively. This is related to the specific alterations in SEV proteome reflecting down-regulation of the phospholipase C pathway (T2D) and up-regulated antioxidant capacity (IR NDM). Thus, SEV cargo may contribute to modulating the individual metabolic responsiveness to exercise training in humans.

Modulation of TRPM6 and Na(+)/Mg(2+) exchange in mammary epithelial cells in response to variations of magnesium availability.

Mammary epithelial cells (HC11) chronically adapted to grow in a low-magnesium (0.05 mM vs. 0.5 mM) or in a high-magnesium (40 mM) medium were used to investigate on the mechanisms of cell magnesium transport under conditions of non-physiological magnesium availability. Magnesium influx was higher in low-magnesium cells compared to control or high-magnesium cells, whereas magnesium efflux was higher in high-magnesium cells compared to control and low-magnesium cells. Magnesium efflux was partially inhibited by imipramine, inhibitor of the Na(+)/Mg(2+) exchange. Using a monoclonal antibody detecting a approximately 70 kDa protein associated with Na(+)/Mg(2+) exchange activity, we found that the expression levels of this protein were proportional to magnesium efflux capacity, that is, high-magnesium cells > control cells > low-magnesium cells. As for magnesium influx, this was abolished by Co(III)hexaammine, inhibitor of magnesium channels. Surprisingly, we found that cells grown in low magnesium upregulated mRNA for the magnesium channel TRPM6, but not for other channels like TRPM7 or MagT1. TRPM6 mRNA was also rapidly upregulated or downregulated in HC11 cells deprived of magnesium or in low-magnesium cells re-added with magnesium, respectively. TRPM6 protein levels, as assessed by Western blot and immunofluorescence, underwent similar changes under comparable conditions. We propose that mammary epithelial cells adapt to decreased magnesium availability by upregulating magnesium influx via TRPM6, and counteract increased magnesium availability by increasing magnesium efflux primarily via Na(+)/Mg(2+) exchange. These results show, for the first time, that TRPM6 contributes to regulating magnesium influx in mammary epithelial cells, similar to what is known for intestine and kidney.

Short-term dietary reduction of branched-chain amino acids reduces meal-induced insulin secretion and modifies microbiome composition in type 2 diabetes: a randomized controlled crossover trial.

BACKGROUND: Epidemiological studies have shown that increased circulating branched-chain amino acids (BCAAs) are associated with insulin resistance and type 2 diabetes (T2D). This may result from altered energy metabolism or dietary habits. OBJECTIVE: We hypothesized that a lower intake of BCAAs improves tissue-specific insulin sensitivity. METHODS: This randomized, placebo-controlled, double-blinded, crossover trial examined well-controlled T2D patients receiving isocaloric diets (protein: 1 g/kg body weight) for 4 wk. Protein requirements were covered by commercially available food supplemented ≤60% by an AA mixture either containing all AAs or lacking BCAAs. The dietary intervention ensured sufficient BCAA supply above the recommended minimum daily intake. The patients underwent the mixed meal tolerance test (MMT), hyperinsulinemic-euglycemic clamps (HECs), and skeletal muscle and white adipose tissue biopsies to assess insulin signaling. RESULTS: After the BCAA- diet, BCAAs were reduced by 17% during fasting (P < 0.001), by 13% during HEC (P < 0.01), and by 62% during the MMT (P < 0.001). Under clamp conditions, whole-body and hepatic insulin sensitivity did not differ between diets. After the BCAA- diet, however, the oral glucose sensitivity index was 24% (P < 0.01) and circulating fibroblast-growth factor 21 was 21% higher (P < 0.05), whereas meal-derived insulin secretion was 28% lower (P < 0.05). Adipose tissue expression of the mechanistic target of rapamycin was 13% lower, whereas the mitochondrial respiratory control ratio was 1.7-fold higher (both P < 0.05). The fecal microbiome was enriched in Bacteroidetes but depleted of Firmicutes. CONCLUSIONS: Short-term dietary reduction of BCAAs decreases postprandial insulin secretion and improves white adipose tissue metabolism and gut microbiome composition. Longer-term studies will be needed to evaluate the safety and metabolic efficacy in diabetes patients.This trial was registered at clinicaltrials.gov as NCT03261362.

Dynamic changes of muscle insulin sensitivity after metabolic surgery.

The mechanisms underlying improved insulin sensitivity after surgically-induced weight loss are still unclear. We monitored skeletal muscle metabolism in obese individuals before and over 52 weeks after metabolic surgery. Initial weight loss occurs in parallel with a decrease in muscle oxidative capacity and respiratory control ratio. Persistent elevation of intramyocellular lipid intermediates, likely resulting from unrestrained adipose tissue lipolysis, accompanies the lack of rapid changes in insulin sensitivity. Simultaneously, alterations in skeletal muscle expression of genes involved in calcium/lipid metabolism and mitochondrial function associate with subsequent distinct DNA methylation patterns at 52 weeks after surgery. Thus, initial unfavorable metabolic changes including insulin resistance of adipose tissue and skeletal muscle precede epigenetic modifications of genes involved in muscle energy metabolism and the long-term improvement of insulin sensitivity.

Overexpression of Na+/Mg2+ exchanger SLC41A1 attenuates pro-survival signaling.

The Na+/Mg2+ exchanger SLC41A1 (A1), a key component of intracellular Mg homeostasis (IMH), is the major cellular Mg2+ efflux system, and its overexpression decreases [Mg2+]intracellular. IMH plays an important role in the regulation of many cellular processes, including cellular signaling. However, whether the overexpression of A1 and the consequent drop of [Mg2+]i impact on intracellular signaling is unknown. To examine the latter, we utilized dynamic mass redistribution (DMR) assay, PathScan® RTK signaling antibody (PRSA) array, confirmatory Western blot (WB) analyses of phosphorylation of kinases selected by PRSA, and mag-fura 2-assisted fast filter spectrometry (FFS). We demonstrate here that the overexpression of A1 quantitatively and qualitatively changes the DMR signal evoked by the application of PAR-1-selective activating peptide and/or by changing [Mg2+]extracellular in HEK293 cells. PRSA profiling of the phosphorylation of important signaling nodes followed by confirmatory WB has revealed that, in HEK293 cells, A1 overexpression significantly attenuates the phosphorylation of Akt/PKB on Thr308 and/or Ser473 and of Erk1/2 on Thr202/Tyr204 in the presence of 0 or 1 mM (physiological) Mg2+ in the bath solution. The latter is also true for SH-SY5Y and HeLa cells. Overexpression of A1 in HEK293 cells significantly lowers [Mg2+]i in the presence of [Mg2+]e = 0 or 1 mM. This correlates with the observed attenuation of prosurvival Akt/PKB - Erk1/2 signaling in these cells. Thus, A1 expression status and [Mg2+]e (and consequently also [Mg2+]i) modulate the complex physiological fingerprint of the cell and influence the activity of kinases involved in anti-apoptotic and, hence, pro-survival events in cells.