Alcoholic liver disease (ALD) is characterized by lipid accumulation and liver injury. However, how chronic alcohol consumption causes hepatic lipid accumulation remains elusive. The present study ...demonstrates that activation of the mechanistic target of rapamycin complex 1 (mTORC1) plays a causal role in alcoholic steatosis, inflammation, and liver injury. Chronic‐plus‐binge ethanol feeding led to hyperactivation of mTORC1, as evidenced by increased phosphorylation of mTOR and its downstream kinase S6 kinase 1 (S6K1) in hepatocytes. Aberrant activation of mTORC1 was likely attributed to the defects of the DEP domain‐containing mTOR‐interacting protein (DEPTOR) and the nicotinamide adenine dinucleotide–dependent deacetylase sirtuin 1 (SIRT1) in the liver of chronic‐plus‐binge ethanol‐fed mice and in the liver of patients with ALD. Conversely, adenoviral overexpression of hepatic DEPTOR suppressed mTORC1 signaling and ameliorated alcoholic hepatosteatosis, inflammation, and acute‐on‐chronic liver injury. Mechanistically, the lipid‐lowering effect of hepatic DEPTOR was attributable to decreased proteolytic processing, nuclear translocation, and transcriptional activity of the lipogenic transcription factor sterol regulatory element‐binding protein‐1 (SREBP‐1). DEPTOR‐dependent inhibition of mTORC1 also attenuated alcohol‐induced cytoplasmic accumulation of the lipogenic regulator lipin 1 and prevented alcohol‐mediated inhibition of fatty acid oxidation. Pharmacological intervention with rapamycin alleviated the ability of alcohol to up‐regulate lipogenesis, to down‐regulate fatty acid oxidation, and to induce steatogenic phenotypes. Chronic‐plus‐binge ethanol feeding led to activation of SREBP‐1 and lipin 1 through S6K1‐dependent and independent mechanisms. Furthermore, hepatocyte‐specific deletion of SIRT1 disrupted DEPTOR function, enhanced mTORC1 activity, and exacerbated alcoholic fatty liver, inflammation, and liver injury in mice. Conclusion: The dysregulation of SIRT1–DEPTOR–mTORC1 signaling is a critical determinant of ALD pathology; targeting SIRT1 and DEPTOR and selectively inhibiting mTORC1–S6K1 signaling may have therapeutic potential for treating ALD in humans. (Hepatology 2018).
When glucose–fructose dimers are supplied as the slowly digestible, completely absorbable, low glycemic index (GI) sugar isomaltulose, the detrimental effects of high GI sucrose are avoided. This ...difference requires the presence of intact glucose-induced insulinotropic peptide receptor (GIPR) and is mediated by the rapid uptake of glucose and the stimulation of GIP release from K cells in the upper small intestine. GIP promotes lipogenesis, fatty liver, insulin resistance, and postprandial inflammation, and reduces fat oxidation in skeletal muscle, partly by hypothalamic interference with energy partitioning and epigenetic programming. GIP is similarly required for the detrimental metabolic effects of other high GI carbohydrates. We therefore propose that the release of GIP in the upper small intestine is an important determinant of the metabolic quality of carbohydrates.
The GI quantifies the widely variable increases in blood glucose after ingestion of different carbohydrates, with the concept that larger increases cause the development of cardiometabolic diseases.
The metabolic effects of sugars depend primarily on the intestinal hormones GIP and GLP-1, which release insulin and regulate metabolic responses such as insulin resistance, adipose tissue subclinical inflammation, hepatic fat accumulation, and postprandial vascular responses.
The GIP-releasing K cells are located in the proximal small intestine, while GLP-1 is released by more distally located L cells. Their responses to sugars depend on the site of resorption.
GIP has mostly unfavorable, while GLP-1 has beneficial, metabolic and cardiovascular properties. The release of GIP therefore appears to be a primary mediator of the deleterious effects of sugars with a high glycemic index in metabolism.
Peritoneal macrophages (PMs) have been shown to have higher stability compared to other macrophage subtypes. However, obtaining enough PMs from a single mouse is often a limitation for metabolomics ...analysis. Here, we describe a protocol to isolate metabolites from a small number of mouse primary PMs for 13C-stable glucose tracing and metabolomics. Our protocol uses X for metabolite extraction instead of methanol. Our protocol can consistently extract metabolites from low cell number samples with fewer steps than methanol-based approaches.
For complete details on the use and execution of this protocol, please refer to De Jesus et al., (2022).
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•Streamlined isolation of murine peritoneal macrophages•Efficient metabolite extraction of limited sample amount•Detection of carbon-labeled polar metabolites from small sample size•Protocol applied to mouse primary PMs, but applicable to other cell types
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Peritoneal macrophages (PMs) have been shown to have higher stability compared to other macrophage subtypes. However, obtaining enough PMs from a single mouse is often a limitation for metabolomics analysis. Here, we describe a protocol to isolate metabolites from a small number of mouse primary PMs for 13C-stable glucose tracing and metabolomics. Our protocol uses X for metabolite extraction instead of methanol. Our protocol can consistently extract metabolites from low cell number samples with fewer steps than methanol-based approaches.
It has been shown that high fat diet (HFD) induced obesity triggers local immune cell infiltration leading to pro-inflammatory cytokine production and subsequently to a state of chronic low-grade ...inflammation in adipose and spleen tissues. However, the underlying mechanisms driving this chronic inflammation remains poorly defined. The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis to produce ATP and pyruvate or alternative pathways, such as the pentose-phosphate-pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal domain that permits mitochondrial binding, but its physiologic significance is unclear. We found that adipose and spleen tissues from HFD mice had increased cytosolic HK1, which correlated with an increase in inflammatory cytokine mRNA levels. To elucidate whether HK1 subcellular localization to the mitochondria affects the inflammatory response we generated mice lacking the HK1 mitochondrial binding domain, termed ΔE1HK1. These mice appear normal and show no change in body weight, Mendelian ratios, glucose tolerance or hexokinase activity across different tissues as compared to littermate controls. However, lipopolysaccharide challenge in these mice produced a hyper-inflammatory phenotype in vitro and in vivo. We performed steady-state and flux metabolomics in ΔE1HK1 mouse tissues and found increased glucose derived metabolites above the level of GAPDH, including PPP metabolites. Additionally, we found that GAPDH activity was attenuated in the ΔE1HK1 mice and may explain the elevated PPP flux upstream of GAPDH. Since PPP is vital for proper inflammatory cell activation, we treated cells from these mice with PPP inhibitors and found a reversal of the hyper-inflammatory phenotype observed in ΔE1HK1 mice. Our data suggests that mitochondrial dissociation of HK1 alters glucose utilization, which subsequently increases the pro-inflammatory response.
Disclosure
F. Keyhani Nejad: None. A. DeJesus: None. J.A. Geier: None. H. Ardehali: None.
The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative metabolic routes, such as the pentose phosphate pathway (PPP) to generate ...anabolic intermediates. HK1 contains an N-terminal mitochondrial binding domain (MBD), but its physiologic significance remains unclear. To elucidate the effect of HK1 mitochondrial dissociation on cellular metabolism, we generated mice lacking the HK1 MBD (ΔE1HK1). These mice produced a hyper-inflammatory response when challenged with lipopolysaccharide. Additionally, there was decreased glucose flux below the level of GAPDH and increased upstream flux through the PPP. The glycolytic block below GAPDH is mediated by the binding of cytosolic HK1 with S100A8/A9, resulting in GAPDH nitrosylation through iNOS. Additionally, human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed increased cytosolic HK1 and reduced GAPDH activity. Our data indicate that HK1 mitochondrial binding alters glucose metabolism through regulation of GAPDH.
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•HK1 mitochondrial dissociation results in GAPDH inhibition and increased PPP•Cytosolic HK1 increases macrophage-mediated inflammatory cytokine production•HK1 associates with S100A8/A9 and attenuates GAPDH activity through nitrosylation•Diabetic and aging immune cells show cytosolic HK1 and reduced GAPDH activity
De Jesus et al. describe how HK1 subcellular localization regulates the metabolic fate of its substrate, G6P, between catabolic and anabolic metabolism through inhibition of GAPDH. Immune cells from models of aging and diabetes show higher levels of cytosolic HK1 and increased inflammation.
Amino acids powerfully release glucagon but their contribution to postprandial hyperglucagonemia in type 2 diabetes remains unclear. Exogenously applied GIP stimulates, while GLP-1 inhibits, glucagon ...secretion in humans. However, their role in mixed meals is unclear, which we therefore characterized.
In three experiments, participants with type 2 diabetes and obese controls randomly received different loads of sugars and/or proteins. In the first experiment, participants ingested the rapidly cleaved saccharose (SAC) or slowly cleaved isomaltulose (ISO) which is known to elicit opposite profiles of GIP and GLP-1 secretion. In the second one participants received test meals which contained saccharose or isomaltulose in combination with milk protein. The third set of participants underwent randomized oral protein tests with whey protein or casein. Incretins, glucagon, C-peptide, and insulin were profiled by specific immunological assays.
50 g of the sugars alone suppressed glucagon in controls but slightly less in type 2 diabetes patients. Participants with type 2 diabetes showed excessive glucagon responses within 15 min and lasting over 3 h, while the obese controls showed small initial and delayed greater glucagon responses to mixed meals. The release of GIP was significantly faster and greater with SAC compared to ISO, while GLP-1 showed an inverse pattern. The glucagon responses to whey or casein were only moderately increased in type 2 diabetes patients without a left shift of the dose response curve.
The rapid hypersecretion of glucagon after mixed meals in type 2 diabetes patients compared to controls is unaffected by endogenous incretins. The defective suppression of glucagon by glucose combined with hypersecretion to protein is required for the exaggerated response.
NCT03806920, NCT02219295, NCT04564391.
Our recent studies identify that retinoic acid receptor β (RARβ), a vitamin A-related nuclear receptor, regulates mitochondrial fatty acid oxidation in hepatocytes and in vivo in a fasting-inducible ...manner, which contributes to the metabolic adaptation to fasting. Mitochondrial pyruvate carrier (MPC) has been recently identified to control gluconeogenesis in hepatocytes, which contributes to the pathogenesis of diabetes. The present study aims to determine whether RARβ is linked to the control of glucose metabolism. We observed hepatic downregulation of RARβ occurred in high fat, high sucrose (HFHS) diet-fed mice and in genetically obese ob/ob mice. Treatment with all-trans-retinoic acid (RA), a natural ligand of RARβ and an approved drug for anticancer treatment, at doses of 2.5- 5.0 mg/kg/day for 4 weeks lowered fasting glucose levels and ameliorated glucose intolerance and insulin resistance in HFHS-fed mice, which was associated with increased hepatic gluconeogenic gene expression. Moreover, adenovirus-mediated overexpression of RARβ restored hepatic impairment of RARβ and systemic glucose intolerance and decreased hepatic gluconeogenesis by obesity. Mechanistically, overexpression of RARβ decreased obesity-induced expression of MPC1 and MPC2, key regulators of gluconeogenesis from pyruvate. Increasing RARβ in ob/ob mice also inhibited the induction of alanine transaminase (ALT), a key enzyme that converts alanine into pyruvate for glucose production. Moreover, overexpression of RARβ downregulated lipogenic gene expression, upregulated fatty acid oxidation, and improved hepatic steatosis and hyperlipidemia in ob/ob mice. Hepatic and systemic insulin resistance were also reduced by overexpressing RARβ. In sum, hepatic defective RARβ may contribute to the upregulation of MPC/ALT and gluconeogenesis in diabetes and RARβ can be a novel drug target to normalize metabolic dysregulation in diabetes.
Disclosure
F. Keyhani Nejad: None. D. Lee: None. H. Chen: None. Z. Liang: None. N. Musi: None. M. Zang: None.
WISP1 (Wnt1-inducible signaling pathway protein-1, also known as CCN4) is a member of the secreted extracellular matrix-associated proteins of the CCN family and a target gene of the Wingless-type ...(WNT) signaling pathway. Growing evidence links the WNT signaling pathway to the regulation of adipogenesis and low-grade inflammation in obesity. We aimed to validate WISP1 as a novel adipokine. Human adipocyte differentiation was associated with increased WISP1 expression and secretion. Stimulation of human macrophages with WISP1 led to a proinflammatory response. Circulating WISP1 and WISP1 subcutaneous adipose tissue expression were regulated by weight changes in humans and mice. WISP1 expression in visceral and subcutaneous fat tissue was associated with markers of insulin resistance and inflammation in glucose-tolerant subjects. In patients with nonalcoholic fatty liver disease, we found no correlation among disease activity score, liver fat content, and WISP1 expression. Insulin regulated WISP1 expression in adipocytes in vitro but had no acute effect on WISP1 gene expression in subcutaneous fat tissue in overweight subjects who had undergone hyperinsulinemic clamp experiments. The data suggest that WISP1 may play a role in linking obesity to inflammation and insulin resistance and could be a novel therapeutic target for obesity.
•The induction of fatty liver and insulin resistance by sucrose requires GIP receptors and is prevented by the similarly composed but distally absorbed glucose-fructose dimer, isomaltulose, which ...releases little GIP but much GLP-1.•Hepatic insulin clearance is improved in humans by isomaltulose as compared to sucrose which correlates with a reduced GIP and increased GLP-1 response.•The most likely mechanism is a regulation of hepatic perfusion by GIP which was shown previously in fluoro-deoxy-glucose (FDG)-PET-Scans in humans as well as by GIP-receptors in the portal vein and hepatic arteries.•We therefore propose that GIP regulates hepatic insulin extraction and action.
GIP was proposed to play a key role in the development of non- alcoholic fatty liver disease (NAFLD) in response to sugar intake. Isomaltulose, is a 1,6-linked glucose-fructose dimer which improves glucose homeostasis and prevents NAFLD compared to 1,2-linked sucrose by reducing glucose-dependent insulinotropic peptide (GIP) in mice. We compared effects of sucrose vs. isomaltulose on GIP and glucagon-like peptide-1 (GLP-1) secretion, hepatic insulin clearance (HIC) and insulin sensitivity in normal (NGT), impaired glucose tolerant (IGT) and Type 2 diabetes mellitus (T2DM) participants.
A randomized crossover study was performed in 15 NGT, 10 IGT and 10 T2DM subjects.
In comparison to sucrose, peak glucose concentrations were reduced by 2.3, 2.1 and 2.5 mmol/l (all p < 0.05) and insulin levels were 88% (p < 0.01, NGT), 32% (p < 0.05, IGT) and 55% (T2DM) lower after the isomaltulose load. Postprandial GIPiAUC concentrations were decreased (56%, p < 0.01 in NGT; 42%, p < 0.05 in IGT and 40%,p < 0.001 in T2DM) whereas GLP-1iAUC was 77%, 85% and 85% higher compared to sucrose (p < 0.01), respectively. This resulted in ∼35 - 50% improved insulin sensitivity and reduced insulinogenic index after isomaltulose, which correlated closely with improved HIC, respectively (r = 0.62, r=-0.70; p < 0.001). HIC was inversely related to GIP (r=-0.44, p < 0.001) and positively related to GLP-1 levels (r = 0.40, p = 0.001).
Endogenously released GIP correlated with reduced, and GLP-1 with increased hepatic insulin extraction. Increased peripheral insulin levels may contribute to insulin resistance and obesity. We propose that the unfavorable effects of high glycemic index Western diets are related to increased GIP-release and reduced HIC.
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