Plasma insulin is required for the increase in plasma angiopoietin-like protein 8 in response to nutrient ingestion.

BACKGROUND: Plasma levels of angiopoietin-like protein 8 (ANGPTL8) are regulated by feeding and they increase following glucose ingestion. Because both plasma glucose and insulin increase following food ingestion, we aimed to determine whether the increase in plasma insulin and glucose or both are responsible for the increase in ANGPTL8 levels. METHODS: ANGPTL8 levels were measured in 30 subjects, 14 with impaired fasting glucose (IFG), and 16 with normal fasting glucose (NFG); the subjects received 75g glucose oral Glucose tolerance test (OGTT), multistep euglycaemic hyperinsulinemic clamp and hyperglycaemic clamp with pancreatic clamp. RESULTS: Subjects with IFG had significantly higher ANGPTL8 than NGT subjects during the fasting state (p < 0.05). During the OGTT, plasma ANGPTL8 concentration increased by 62% above the fasting level (p < 0.0001), and the increase above fasting in ANGPTL8 levels was similar in NFG and IFG individuals. During the multistep insulin clamp, there was a dose-dependent increase in plasma ANGPTL8 concentration. During the 2-step hyperglycaemic clamp, the rise in plasma glucose concentration failed to cause any change in the plasma ANGPTL8 concentration from baseline. CONCLUSIONS: In response to nutrient ingestion, ANGPTL8 level increased due to increased plasma insulin concentration, not to the rise in plasma glucose. The incremental increase above baseline in plasma ANGLPTL8 during OGTT was comparable between people with normal glucose tolerance and IFG.

FGF21 contributes to metabolic improvements elicited by combination therapy with exenatide and pioglitazone in patients with type 2 diabetes.

Fibroblast growth factor 21 (FGF21) is increased acutely by carbohydrate ingestion and is elevated in patients with type 2 diabetes (T2D). However, the physiological significance of increased FGF21 in humans remains largely unknown. We examined whether FGF21 contributed to the metabolic improvements observed following treatment of patients with T2D with either triple (metformin/pioglitazone/exenatide) or conventional (metformin/insulin/glipizide) therapy for 3 yr. Forty-six patients with T2D were randomized to receive either triple or conventional therapy to maintain HbA1c < 6.5%. A 2-h 75-g oral glucose tolerance test (OGTT) was performed at baseline and following 3 years of treatment to assess glucose tolerance, insulin sensitivity, and ß-cell function. Plasma total and bioactive FGF21 levels were quantitated before and during the OGTT at both visits. Patients in both treatment arms experienced significant improvements in glucose control, but insulin sensitivity and ß-cell function were markedly increased after triple therapy. At baseline, FGF21 levels were regulated acutely during the OGTT in both groups. After treatment, fasting total and bioactive FGF21 levels were significantly reduced in patients receiving triple therapy, but there was a relative increase in the proportion of bioactive FGF21 compared with that observed in conventionally treated subjects. Relative to baseline studies, triple therapy treatment also significantly modified FGF21 levels in response to a glucose load. These changes in circulating FGF21 were correlated with markers of improved glucose control and insulin sensitivity. Alterations in the plasma FGF21 profile may contribute to the beneficial metabolic effects of pioglitazone and exenatide in human patients with T2D.NEW & NOTEWORTHY In patients with T2D treated with a combination of metformin/pioglitazone/exenatide (triple therapy), we observed reduced total and bioactive plasma FGF21 levels and a relative increase in the proportion of circulating bioactive FGF21 compared with that in patients treated with metformin and sequential addition of glipizide and basal insulin glargine (conventional therapy). These data suggest that FGF21 may contribute, at least in part, to the glycemic benefits observed following combination therapy in patients with T2D.

Lactate Elicits ER-Mitochondrial Mg2+ Dynamics to Integrate Cellular Metabolism.

Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.

Mitochondrial pyruvate and fatty acid flux modulate MICU1-dependent control of MCU activity.

The tricarboxylic acid (TCA) cycle converts the end products of glycolysis and fatty acid ß-oxidation into the reducing equivalents NADH and FADH2 Although mitochondrial matrix uptake of Ca2+ enhances ATP production, it remains unclear whether deprivation of mitochondrial TCA substrates alters mitochondrial Ca2+ flux. We investigated the effect of TCA cycle substrates on MCU-mediated mitochondrial matrix uptake of Ca2+, mitochondrial bioenergetics, and autophagic flux. Inhibition of glycolysis, mitochondrial pyruvate transport, or mitochondrial fatty acid transport triggered expression of the MCU gatekeeper MICU1 but not the MCU core subunit. Knockdown of mitochondrial pyruvate carrier (MPC) isoforms or expression of the dominant negative mutant MPC1R97W resulted in increased MICU1 protein abundance and inhibition of MCU-mediated mitochondrial matrix uptake of Ca2+ We also found that genetic ablation of MPC1 in hepatocytes and mouse embryonic fibroblasts resulted in reduced resting matrix Ca2+, likely because of increased MICU1 expression, but resulted in changes in mitochondrial morphology. TCA cycle substrate-dependent MICU1 expression was mediated by the transcription factor early growth response 1 (EGR1). Blocking mitochondrial pyruvate or fatty acid flux was linked to increased autophagy marker abundance. These studies reveal a mechanism that controls the MCU-mediated Ca2+ flux machinery and that depends on TCA cycle substrate availability. This mechanism generates a metabolic homeostatic circuit that protects cells from bioenergetic crisis and mitochondrial Ca2+ overload during periods of nutrient stress.

The tumor suppressor TMEM127 regulates insulin sensitivity in a tissue-specific manner.

Understanding the molecular components of insulin signaling is relevant to effectively manage insulin resistance. We investigated the phenotype of the TMEM127 tumor suppressor gene deficiency in vivo. Whole-body Tmem127 knockout mice have decreased adiposity and maintain insulin sensitivity, low hepatic fat deposition and peripheral glucose clearance after a high-fat diet. Liver-specific and adipose-specific Tmem127 deletion partially overlap global Tmem127 loss: liver Tmem127 promotes hepatic gluconeogenesis and inhibits peripheral glucose uptake, while adipose Tmem127 downregulates adipogenesis and hepatic glucose production. mTORC2 is activated in TMEM127-deficient hepatocytes suggesting that it interacts with TMEM127 to control insulin sensitivity. Murine hepatic Tmem127 expression is increased in insulin-resistant states and is reversed by diet or the insulin sensitizer pioglitazone. Importantly, human liver TMEM127 expression correlates with steatohepatitis and insulin resistance. Our results suggest that besides tumor suppression activities, TMEM127 is a nutrient-sensing component of glucose/lipid homeostasis and may be a target in insulin resistance.

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (mCa2+) uptake, delays cytosolic Ca2+ (cCa2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid mCa2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism.

Increased lipid availability for three days reduces whole body glucose uptake, impairs muscle mitochondrial function and initiates opposing effects on PGC-1α promoter methylation in healthy subjects.

AIMS: FFA and FFA metabolites cause insulin resistance and impair beta cell function. The goal of our research was to examine whether elevation of plasma FFA impairs mitochondrial function and alters PGC-1α promoter methylation. METHODS: In this uncontrolled, change from baseline study design, insulin sensitivity and glucose-stimulated insulin secretion were measured in 9 normal glucose tolerant subjects before and after 3 day lipid infusion to elevate plasma FFA concentration. Vastus lateralis muscle biopsies were obtained and mitochondrial function, PGC-1α expression, and PGC-1α promoter methylation were quantitated. RESULTS: Increased plasma FFA (440±93 µmol/Lto 997±242 µM, p<0.001) decreased insulin-stimulated total glucose disposal (TGD) by 25% (p = 0.008), impaired suppression of endogenous glucose production (p = 0.01), and reduced mitochondrial ATP synthesis with complex 1 (34%, p<0.05) and complex 2 (30%, p<0.05) substrates. Lipid infusion had no effect on muscle PGC-1α RNA expression, total methylation or non-CpG methylation, but methylation of the alternative PGC-1α promoter decreased (1.30±0.30 to 0.84±0.15% methylated residues/patient•strand, p = 0.055). Within PGC-1α promoter there was demethylation of CpT residues (0.72±0.16 vs. 0.28±0.10 methylated residues/patient•strand) (p = 0.002), which was inversely correlated with PGC-1α mRNA expression (r = -0.94, p<0.0001) and ATP synthesis with complex 1 (r = -0.80, p<0.01) and complex 2 (r = -0.69, p<0.05) substrates. Lipid infusion increased DNMT-3B (methyltransferase associated with PGC-1α promoter non-CpG methylation) mRNA expression (0.87 ± 0.09 to 1.62 ± 0.22 arbitrary units, p = 0.005), which correlated inversely with CpT demethylation (r = 0.67, p<0.05). CONCLUSION/INTERPRETATION: Physiologic plasma FFA elevation in NGT individuals has opposing effects on PGC-1α non-CpG residue methylation (CpT demethylation and increased DNMT-3B expression), which is correlated with changes in PGC-1α expression and skeletal muscle mitochondrial function.

Sodium-glucose co-transporter (SGLT) and glucose transporter (GLUT) expression in the kidney of type 2 diabetic subjects.

The sodium-glucose co-transporters (SGLTs) are responsible for the tubular reabsorption of filtered glucose from the kidney into the bloodstream. The inhibition of SGLT2-mediated glucose reabsorption is a novel and highly effective strategy to alleviate hyperglycaemia in patients with type 2 diabetes mellitus (T2DM). However, the effectiveness of SGLT2 inhibitor therapy is diminished due, in part, to a compensatory increase in the maximum reabsorptive capacity (Tm) for glucose in patients with T2DM. We hypothesized that this increase in Tm could be explained by an increase in the tubular expression of SGLT and glucose transporters (GLUT) in these patients. To examine this, we obtained human kidney biopsy specimens from patients with or without T2DM and examined the mRNA expression of SGLTs and GLUTs. The expression of SGLT1 is markedly increased in the kidney of patients with T2DM, and SGLT1 mRNA is highly and significantly correlated with fasting and postprandial plasma glucose and HbA1c. In contrast, our data demonstrate that the levels of SGLT2 and GLUT2 mRNA are downregulated in diabetic patients, but not to a statistically significant level. These important findings are clinically significant and may have implications for the treatment of T2DM using strategies that target SGLT transporters in the kidney.

Pioglitazone inhibits mitochondrial pyruvate metabolism and glucose production in hepatocytes.

Pioglitazone is used globally for the treatment of type 2 diabetes mellitus (T2DM) and is one of the most effective therapies for improving glucose homeostasis and insulin resistance in T2DM patients. However, its mechanism of action in the tissues and pathways that regulate glucose metabolism are incompletely defined. Here we investigated the direct effects of pioglitazone on hepatocellular pyruvate metabolism and the dependency of these observations on the purported regulators of mitochondrial pyruvate transport, MPC1 and MPC2. In cultured H4IIE hepatocytes, pioglitazone inhibited [2-14 C]-pyruvate oxidation and pyruvate-driven oxygen consumption and, in mitochondria isolated from both hepatocytes and human skeletal muscle, pioglitazone selectively and dose-dependently inhibited pyruvate-driven ATP synthesis. Pioglitazone also suppressed hepatocellular glucose production (HGP), without influencing the mRNA expression of key HGP regulatory genes. Targeted siRNA silencing of MPC1 and 2 caused a modest inhibition of pyruvate oxidation and pyruvate-driven ATP synthesis, but did not alter pyruvate-driven HGP and, importantly, it did not influence the actions of pioglitazone on either pathway. In summary, these findings outline a novel mode of action of pioglitazone relevant to the pathogenesis of T2DM and suggest that targeting pyruvate metabolism may lead to the development of effective new T2DM therapies.

The mechanisms of genome-wide target gene regulation by TCF7L2 in liver cells.

In the liver Wnt-signaling contributes to the metabolic fate of hepatocytes, but the precise role of the TCF7L2 in this process is unknown. We employed a temporal RNA-Seq approach to examine gene expression 3-96 h following Tcf7l2 silencing in rat hepatoma cells, and combined this with ChIP-Seq to investigate mechanisms of target gene regulation by TCF7L2. Silencing Tcf7l2 led to a time-dependent appearance of 406 differentially expressed genes (DEGs), including key regulators of cellular growth and differentiation, and amino acid, lipid and glucose metabolism. Direct regulation of 149 DEGs was suggested by strong proximal TCF7L2 binding (peak proximity score > 10) and early mRNA expression changes (≤ 18 h). Indirect gene regulation by TCF7L2 likely occurred via alternate transcription factors, including Hnf4a, Foxo1, Cited2, Myc and Lef1, which were differentially expressed following Tcf7l2 knock-down. Tcf7l2-silencing enhanced the expression and chromatin occupancy of HNF4α, and co-siRNA experiments revealed that HNF4α was required for the regulation of a subset of metabolic genes by TCF7L2, particularly those involved in lipid and amino-acid metabolism. Our findings suggest TCF7L2 is an important regulator of the hepatic phenotype, and highlight novel mechanisms of gene regulation by TCF7L2 that involve interplay between multiple hepatic transcriptional pathways.

Independent and combined effects of acute physiological hyperglycaemia and hyperinsulinaemia on metabolic gene expression in human skeletal muscle.

Physiological hyperglycaemia and hyperinsulinaemia are strong modulators of gene expression, which underpins some of their well-known effects on insulin action and energy metabolism. The aim of the present study was to examine whether acute in vivo exposure of healthy humans to hyperinsulinaemia and hyperglycaemia have independent or additive effects on expression of key metabolic genes in skeletal muscle. On three randomized occasions, seven young subjects underwent a 4 h (i) hyperinsulinaemic (50 m-units·m⁻²·min⁻¹) hyperglycaemic (10 mmol/l) clamp (HIHG), (ii) hyperglycaemic (10 mmol/l) euinsulinaemic (5 m-units·m⁻²·min⁻¹) clamp (LIHG) and (iii) hyperinsulinaemic (50 m-units·m⁻²·min⁻¹) euglycaemic (4.5 mmol/l) clamp (HING). Muscle biopsies were obtained before and after each clamp for the determination of expression of genes involved in energy metabolism, and phosphorylation of key insulin signalling proteins. Hyperinsulinaemia and hyperglycaemia exerted independent effects with similar direction of modulation on PI3KR1 (phosphatidylinositol 3-kinase, regulatory 1), LXRα (liver X receptor α), PDK4 (pyruvate dehydrogenase kinase 4) and FOXO1 (forkhead box O1A) and produced an additive effect on PI3KR1, the gene that encodes the p85α subunit of PI3K in human skeletal muscle. Acute hyperglycaemia itself altered the expression of genes involved in fatty acid transport and oxidation [fatty acid transporter (CD36), LCAD (long-chain acyl-CoA dehydrogenase) and FOXO1], and lipogenesis [LXRα, ChREBP (carbohydrate-responseelement-binding protein), ABCA1 (ATP-binding cassette transporter A1) and G6PD (glucose-6-phosphate dehydrogenase). Surperimposing hyperinsulinaemia on hyperglycaemia modulated a number of genes involved in insulin signalling, glucose metabolism and intracellular lipid accumulation and exerted an additive effect on PI3KR1. These may be early molecular events that precede the development of glucolipotoxicity and insulin resistance normally associated with more prolonged periods of hyperglycaemia and hyperinsulinaemia.

Elevated free fatty acids attenuate the insulin-induced suppression of PDK4 gene expression in human skeletal muscle: potential role of intramuscular long-chain acyl-coenzyme A.

AIM: We investigated the effect of elevated plasma free fatty acid and insulin concentrations on PDK4 mRNA transcript and protein content and long-chain acyl-coenzyme A accumulation in human skeletal muscle. METHODS: On two occasions, 10 healthy men underwent hyperinsulinemic-euglycemic clamps for 6 h with (LIPID) and without (CON) iv Intralipid (20% at 90 ml/h) plus heparin (200 U prime + 600 U/h) infusion. RESULTS: Glucose disposal was approximately 50% lower at the end of the clamp in the LIPID compared with the CON trial (37.8 +/- 4.4 and 79.6 +/- 4.0 micromol/kg lean mass.min, respectively; P < 0.01). In the LIPID trial, muscle long-chain acyl-coenzyme A concentration increased after 6 h, but not 3 h of lipid infusion (P < 0.01). Muscle PDK4 mRNA, but not protein, was down-regulated by 2-fold within 3 h in both clamps and decreased further (6-fold; P < 0.01) at 6 h in the CON but not the LIPID clamp. The lipid-induced attenuation in the suppression of PDK4 gene expression was not dependent on the activation of the Akt/FOXO3 pathway. CONCLUSION: Accumulation of im lipids plays a more important role than impaired activation of Akt-mediated pathways in the regulation of muscle PDK4 gene expression in lipid-induced acute insulin-resistant states.