Ang II (Angiotensin II)-Induced FGFR1 (Fibroblast Growth Factor Receptor 1) Activation in Tubular Epithelial Cells Promotes Hypertensive Kidney Fibrosis and Injury.

BACKGROUND: Elevated Ang II (angiotensin II) level leads to a range of conditions, including hypertensive kidney disease. Recent evidences indicate that FGFR1 (fibroblast growth factor receptor 1) signaling may be involved in kidney injuries. In this study, we determined whether Ang II alters FGFR1 signaling to mediate renal dysfunction. METHODS: Human archival kidney samples from patients with or without hypertension were examined. Multiple genetic and pharmacological approaches were used to investigate FGFR1-mediated signaling in tubular epithelial NRK-52E cells in response to Ang II stimulation. C57BL/6 mice were infused with Ang II for 28 days to develop hypertensive kidney disease. Mice were treated with either adeno-associated virus expressing FGFR1 shRNA or FGFR1 inhibitor AZD4547. RESULTS: Kidney specimens from subjects with hypertension and mice challenged with Ang II have increased FGFR1 activity in renal epithelial cells. Renal epithelial cells in culture initiate extracellular matrix programming in response to Ang II, through the activation of FGFR1, which is independent of both AT1R (angiotensin II receptor type 1) and AT2R (angiotensin II receptor type 2). The RNA sequencing analysis indicated that disrupting FGFR1 suppresses Ang II-induced fibrogenic responses in epithelial cells. Mechanistically, Ang II-activated FGFR1 leads to STAT3 (signal transducer and activator of transcription 3) activation, which is responsible for fibrogenic factor expression in kidneys. In the mouse model of hypertensive kidney disease, genetic knockdown of FGFR1 or pharmacological inhibition of its activity protected kidneys from dysfunction and fibrosis upon Ang II challenge. CONCLUSIONS: Our studies uncover a novel mechanism causing renal fibrosis in hypertension and indicate FGFR1 as a potential target to preserve renal function and integrity.

Curcumin analogue C66 attenuates obesity-induced myocardial injury by inhibiting JNK-mediated inflammation.

Obesity has been recognized as a major risk factor for the development of chronic cardiomyopathy, which is associated with increased cardiac inflammation, fibrosis, and apoptosis. We previously developed an anti-inflammatory compound C66, which prevented inflammatory diabetic complications via targeting JNK. In the present study, we have tested the hypothesis that C66 could prevent obesity-induced cardiomyopathy by suppressing JNK-mediated inflammation. High-fat diet (HFD)-induced obesity mouse model and palmitic acid (PA)-challenged H9c2 cells were used to develop inflammatory cardiomyopathy and evaluate the protective effects of C66. Our data demonstrate a protective effect of C66 against obesity-induced cardiac inflammation, cardiac hypertrophy, fibrosis, and dysfunction, overall providing cardio-protection. C66 administration attenuates HFD-induced myocardial inflammation by inhibiting NF-κB and JNK activation in mouse hearts. In vitro, C66 prevents PA-induced myocardial injury and apoptosis in H9c2 cells, accompanied with inhibition against PA-induced JNK/NF-κB activation and inflammation. The protective effect of C66 is attributed to its potential to inhibit JNK activation, which led to reduced pro-inflammatory cytokine production and reduced apoptosis in cardiomyocytes both in vitro and in vivo. In summary, C66 provides significant protection against obesity-induced cardiac dysfunction, mainly by inhibiting JNK activation and JNK-mediated inflammation. Our data indicate that inhibition of JNK is able to provide significant protection against obesity-induced cardiac dysfunction.

CDK9 inhibition improves diabetic nephropathy by reducing inflammation in the kidneys.

Diabetic nephropathy (DN) is a chronic inflammatory renal disease induced by hyperglycemia. Recent studies have implicated cyclin-dependent kinase 9 (CDK9) in inflammatory responses and renal fibrosis. In this study, we explored a potential role of CDK9 in DN by using cultured mouse mesangial cell line SV40 MES-13 and streptozotocin-induced type 1 mouse model of diabetes. We inhibited CDK9 in mice and in cultured cells by a highly selective CDK9 inhibitor, LDC000067 (LDC), and evaluated inflammatory and fibrogenic outcome by mRNA and protein analyses. Our studies show that treatment of diabetic mice with LDC significantly inhibits the levels of inflammatory cytokines and fibrogenic genes in kidney specimens. These reductions were associated with improved renal function. We also found that LDC treatment suppressed MAPK-AP1 activation. We then confirmed the involvement of CDK9 in cultured SV40 MES-13 cells and showed that deficiency in CDK9 prevents glucose-induced inflammatory and fibrogenic proteins. This protection was also afforded by suppression of MAPK-AP1. Taken together, our results how that hyperglycemia activates CDK9-MAPK-AP1 axis in kidneys to induce inflammation and fibrosis, leading to renal dysfunction. Our findings also suggest that CDK9 may serve as a potential therapeutic target for DN.

Myeloid differentiation protein 2 mediates angiotensin II-induced inflammation and mesenchymal transition in vascular endothelium.

Angiotensin II (Ang II)-induced vascular inflammation and injury entails endothelial to mesenchymal transition (EndMT). Recent studies have shown that Ang II engages toll-like receptor 4 (TLR4) in the vasculature to mediate adverse effects. Here, we aimed to investigate whether myeloid differentiation protein 2 (MD2), an extracellular molecule indispensable for TLR4 activation, mediates Ang II-induced vascular injury and EndMT. We utilized MD2 knockout mice and wildtype mice treated with a specific MD2 inhibitor to decipher its role in aortas of Ang II-challenged mice. To confirm our results and to provide mechanistic insights, we exposed cultured endothelial cells to Ang II, with or without MD2 silencing. We show that Ang II causes deleterious remodeling and EndMT in aortas of mice within two weeks. These Ang II effects were largely absent in MD2 knockout mice and in wildtype mice treated with a MD2 inhibitor. MD2 silencing in cultured endothelial cells confirmed the essential role of MD2 in Ang II-induced inflammatory factor induction, and EndMT-associated phenotypic change. We also found that Ang II-MD2-EndMT axis involves the activation of nuclear factor-κB. Our studies highlight an essential role of MD2 in Ang II-induced vascular inflammation and EndMT contributing to vascular injury. These results also imply that MD2 may be targeted to dampen inflammatory cardiovascular and EndMT-associated diseases.

TLR2 regulates angiotensin II-induced vascular remodeling and EndMT through NF-κB signaling.

Excessive vascular remodeling has been shown in hypertensive patients. In experimental models of hypertensive vascular injury, such as angiotensin II (Ang II) challenged mice, toll like receptor 2 (TLR2) initiates inflammatory responses. More recently, studies have reported atypical endothelial to mesenchymal transition (EndMT) in vascular injuries and inflammatory conditions. Here, we aimed to investigate whether TLR2 mediates Ang II-induced vascular inflammation and initiates EndMT. In a mouse model of angiotensin II-induced hypertension, we show that aortas exhibit increased medial thickening, fibrosis, and features of EndMT. These alterations were not observed in TLR2 knockout mice in response to Ang II. TLR2 silencing in cultured endothelial cells confirmed the essential role of TLR2 in Ang II-induced inflammatory factor induction, and EndMT-associated phenotypic change. Mechanistically, we found Ang II activates nuclear factor-κB signaling, inducing pro-inflammatory cytokine production, and mediates EndMT in both cultured endothelial cells and in mice. These studies illustrate a novel role of TLR2 in regulating Ang II-induced deleterious vascular remodeling through the induction of EndMT. The studies also suggest that TLR2 may be targeted to alleviate hypertension-associated vascular injury.

Inhibition of STAT3 in tubular epithelial cells prevents kidney fibrosis and nephropathy in STZ-induced diabetic mice.

Recent evidences indicate that signal transducer and activator of transcription 3 (STAT3) is one of the crucial signaling pathways in the progression of diabetic nephropathy (DN). Here, we investigated the hypothesis that pharmacological blockade of STAT3 limits the progression of DN. Treatment with selective STAT3 inhibitor, S3I-201 for 16 weeks significantly attenuated kidney injuries in streptozotocin (STZ) induced diabetic mice, associated with downregulated expression of TGF-ß1, ACE/AT1, and VEGF in diabetic mouse kidneys. Similar results were confirmed using genetic knockdown of STAT3 in mouse kidneys by injections of AAV2 expressing STAT3 shRNA in diabetic mouse. Further, STAT3 localization in kidney tissue was evaluated using immunofluorescent double-staining analysis, which indicated that STAT3 expression was mainly in the tubular epithelial cells. As expected, in renal tubular epithelial NRK-52E cells, high glucose (HG)-induced overexpression of TGF-ß1, ACE/AT1, and VEGF were abrogated by S3I-201 pretreatment, as well as by genetic knockdown of STAT3 using specific siRNA sequence. This study found that renal tubular epithelial cells contributed to STAT3-mediated progression of DN and provided the first evidence that pharmacological inhibition of STAT3 attenuates DN.

MD2 blockade prevents oxLDL-induced renal epithelial cell injury and protects against high-fat-diet-induced kidney dysfunction.

There is a strong epidemiological link between obesity, a growing worldwide concern, and kidney disease. Emerging evidence indicates that the pathogenic basis of obesity-related kidney disease may be attributed to Toll-like receptor 4 (TLR4) of the innate immune system. We hypothesized that renal epithelial cell injury in response to oxidized low-density lipoprotein (oxLDL) requires myeloid differentiation factor 2 (MD2), a co-receptor of TLR4. Moreover, we also hypothesized that renal dysfunction is MD2-dependent in the high-fat diet (HFD) mouse model. Results indicated that the MD2 selective inhibitor (L6H21) abrogated the oxLDL-induced formation of MD2-TLR4 dimerization in the renal proximal tubular epithelial cell line NRK-52E. Further, MD2 blockade in NRK-52E cells using siRNA target sequences or L6H21 prevented oxLDL-induced cell injury as indicated by expression of profibrotic molecules, autophagic activity and apoptosis. Similarly, TLR4 knockdown in NRK-52E cells using siRNA target sequences prevented oxLDL-induced cell injury. In the HFD mouse model, MD2 knockout protected against development of kidney dysfunction and renal tissue injury, corroborating the observations observed in NRK-52E cells. Thus, the oxLDL-induced renal tubular epithelial cell profibrotic responses, autophagy and apoptosis were dependent on MD2, as were the renal dysfunction and tissue impairment in HFD mice. These are new findings indicating that the MD2-TLR4 immune signaling complex is a critical pathogenic factor in the development of kidney disease related to obesity or metabolic syndrome.

Acute activation of GLP-1-expressing neurons promotes glucose homeostasis and insulin sensitivity.

OBJECTIVE: Glucagon-like peptides are co-released from enteroendocrine L cells in the gut and preproglucagon (PPG) neurons in the brainstem. PPG-derived GLP-1/2 are probably key neuroendocrine signals for the control of energy balance and glucose homeostasis. The objective of this study was to determine whether activation of PPG neurons per se modulates glucose homeostasis and insulin sensitivity in vivo. METHODS: We generated glucagon (Gcg) promoter-driven Cre transgenic mice and injected excitatory hM3Dq-mCherry AAV into their brainstem NTS. We characterized the metabolic impact of PPG neuron activation on glucose homeostasis and insulin sensitivity using stable isotopic tracers coupled with hyperinsulinemic euglycemic clamp. RESULTS: We showed that after ip injection of clozapine N-oxide, Gcg-Cre lean mice transduced with hM3Dq in the brainstem NTS downregulated basal endogenous glucose production and enhanced glucose tolerance following ip glucose tolerance test. Moreover, acute activation of PPG neuronsNTS enhanced whole-body insulin sensitivity as indicated by increased glucose infusion rate as well as augmented insulin-suppression of endogenous glucose production and gluconeogenesis. In contrast, insulin-stimulation of glucose disposal was not altered significantly. CONCLUSIONS: We conclude that acute activation of PPG neurons in the brainstem reduces basal glucose production, enhances intraperitoneal glucose tolerance, and augments hepatic insulin sensitivity, suggesting an important physiological role of PPG neurons-mediated circuitry in promoting glycemic control and insulin sensitivity.

Characterizing novel metabolic pathways of melatonin receptor agonist agomelatine using metabolomic approaches.

Agomelatine (AGM), an analog of melatonin, is a potential agonist at melatonin receptors 1/2 and a selective antagonist at 5-hydroxytryptamine 2C receptors. AGM is widely used for the treatment of major depressive episodes in adults. However, multiple adverse effects associated with AGM have been reported in clinical practice. It is little known about AGM metabolism in vitro and in vivo, although metabolism plays a pivotal role in its efficacy and safety. To elucidate metabolic pathways of AGM, we systemically investigated AGM metabolism and its bioactivation in human liver microsomes (HLM) and mice using metabolomic approaches. We identified thirty-eight AGM metabolites and adducts, among which thirty-two are novel. In HLM, we uncovered five GSH-trapped adducts and two semicarbazide-trapped aldehydes. Moreover, we characterized three N-acetyl cysteine conjugated-AGM adducts in mouse urine and feces, which were formed from the degradation of AGM_GSH adducts. Using recombinant CYP450 isoenzymes and chemical inhibitors, we demonstrated that CYP1A2 and CYP3A4 are primary enzymes contributing to the formation of AGM_GSH adducts and AGM_hydrazones. This study provided a global view of AGM metabolism and identified the novel pathways of AGM bioactivation, which could be utilized for further understanding the mechanism of adverse effects related to AGM and possible drug-drug interactions.

Metabolomics reveals the formation of aldehydes and iminium in gefitinib metabolism.

Gefitinib (GEF), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is widely used for the treatment of cancers, particularly non-small cell lung cancer. However, its clinical use is limited by multiple adverse effects associated with GEF, such as liver and lung injuries, severe nausea, and diarrhea. Although, the exact mechanism of GEF adverse effects are still unknown, xenobiotic-induced bioactivation is thought to play a significant role in GEF induced toxicity. Using a metabolomic approach, we investigated the metabolic pathways of GEF in human and mouse liver microsomes. Thirty four GEF metabolites and adducts were identified and half of them are novel. The potential reactive metabolites, two aldehydes and one iminium, were identified for the first time. The previously reported GSH adducts and primary amines were observed as well. The aldehyde and iminium pathways were further confirmed by using methoxylamine and potassium cyanide as trapping reagents. Using recombinant CYP450 isoforms, CYP3A4 inhibitor, and S9 from Cyp3a-null mice, we confirmed CYP3A is the major enzyme contributing to the formation of aldehydes, GSH adducts, and primary amines in liver. Multiple enzymes contribute to the formation of iminium. This study provided us more knowledge of GEF bioactivation and enzymes involved in metabolic pathways, which can be utilized for understanding the mechanism of adverse effects associated with GEF and predicting possible drug-drug interactions. Further studies are suggested to determine the roles of these bioactivation pathways in GEF toxicity.

The CNS glucagon-like peptide-2 receptor in the control of energy balance and glucose homeostasis.

The gut-brain axis plays a key role in the control of energy balance and glucose homeostasis. In response to luminal stimulation of macronutrients and microbiota-derived metabolites (secondary bile acids and short chain fatty acids), glucagon-like peptides (GLP-1 and -2) are cosecreted from endocrine L cells in the gut and coreleased from preproglucagonergic neurons in the brain stem. Glucagon-like peptides are proposed as key mediators for bariatric surgery-improved glycemic control and energy balance. Little is known about the GLP-2 receptor (Glp2r)-mediated physiological roles in the control of food intake and glucose homeostasis, yet Glp1r has been studied extensively. This review will highlight the physiological relevance of the central nervous system (CNS) Glp2r in the control of energy balance and glucose homeostasis and focuses on cellular mechanisms underlying the CNS Glp2r-mediated neural circuitry and intracellular PI3K signaling pathway. New evidence (obtained from Glp2r tissue-specific KO mice) indicates that the Glp2r in POMC neurons is essential for suppressing feeding behavior, gastrointestinal motility, and hepatic glucose production. Mice with Glp2r deletion selectively in POMC neurons exhibit hyperphagic behavior, accelerated gastric emptying, glucose intolerance, and hepatic insulin resistance. GLP-2 differentially modulates postsynaptic membrane excitability of hypothalamic POMC neurons in Glp2r- and PI3K-dependent manners. GLP-2 activates the PI3K-Akt-FoxO1 signaling pathway in POMC neurons by Glp2r-p85α interaction. Intracerebroventricular GLP-2 augments glucose tolerance, suppresses glucose production, and enhances insulin sensitivity, which require PI3K (p110α) activation in POMC neurons. Thus, the CNS Glp2r plays a physiological role in the control of food intake and glucose homeostasis. This review will also discuss key questions for future studies.

Central GLP-2 enhances hepatic insulin sensitivity via activating PI3K signaling in POMC neurons.

Glucagon-like peptides (GLP-1/GLP-2) are coproduced and highlighted as key modulators to improve glucose homeostasis and insulin sensitivity after bariatric surgery. However, it is unknown if CNS GLP-2 plays any physiological role in the control of glucose homeostasis and insulin sensitivity. We show that mice lacking GLP-2 receptor (GLP-2R) in POMC neurons display glucose intolerance and hepatic insulin resistance. GLP-2R activation in POMC neurons is required for GLP-2 to enhance insulin-mediated suppression of hepatic glucose production (HGP) and gluconeogenesis. GLP-2 directly modulates excitability of POMC neurons in GLP-2R- and PI3K-dependent manners. GLP-2 initiates GLP-2R-p85α interaction and facilitates PI3K-Akt-dependent FoxO1 nuclear exclusion in POMC neurons. Central GLP-2 suppresses basal HGP and enhances insulin sensitivity, which are abolished in POMC-p110α KO mice. Thus, CNS GLP-2 plays a key physiological role in the control of HGP through activating PI3K-dependent modulation of membrane excitability and nuclear transcription of POMC neurons in the brain.

GLP-2 receptor in POMC neurons suppresses feeding behavior and gastric motility.

Glucagon-like peptides (GLP-1/2) are cosecreted from endocrine L cells in the gut and preproglucagonergic neurons in the brain. Peripheral GLP-2 action is essential for maintaining intestinal homeostasis, improving absorption efficiency and blood flow, promoting immune defense, and producing efficacy in treatment of gastrointestinal diseases. However, it is unknown if CNS GLP-2 plays a physiological role in the control of energy homeostasis. Since GLP-1/2 are cotranslated from preproglucagongene and coproduced by prohormone convertase-1, it is challenging to knockout GLP-2 only. Instead, our laboratory has generated a Glp2r-floxed mouse line to dissect cell-specific GLP-2 receptor GLP-2R) action in the regulation of energy balance. Our objective was to determine if GLP-2R in the hypothalamus modulates feeding behavior and gastric emptying. We show that Glp2r mRNA and protein are highly expressed in the arcuate nucleus and dorsomedial nucleus of the mouse hypothalamus. Using the Cre-LoxP system, we generated mice that lack Glp2r expression in POMC neurons (KO; mainly in the hypothalamus). The KO mice showed hyperphagic behavior (such as increases in food intake and meal frequency), accelerated gastric emptying (assessed by [(13)C]octanoic acid breath test), and late-onset obesity, yet there was no decrease in basal metabolic rate. Infusion of GLP-2 (2.5 nmol into the 4th ventricle) suppressed food intake and gastric emptying, while GLP-2-mediated effects were abolished in the melanocortin receptor-4 (MC4R) KO mice. We conclude that Glp2r deletion in POMC neurons enhances feeding behavior and gastric motility, whereas icv GLP-2R activation suppresses food intake and gastric emptying through the MC4R signaling pathway. This study indicates that CNS GLP-2R plays a physiological role in the control of feeding behavior and gastric emptying and that this is mediated probably through the melanocortin system.

SIRT1 inhibits the mouse intestinal motility and epithelial proliferation.

Sirtuin 1 (SIRT1), a NAD(+)-dependent histone deacetylase, is involved in a wide array of cellular processes including glucose homeostasis, energy metabolism, proliferation and apoptosis, and immune response. However, it is unknown whether SIRT1 plays any physiological role in the regulation of intestinal homeostasis and motility. Thus the aim was to define SIRT1 expression and function in the gastrointestinal (GI) tract under physiological conditions. Forty 12-14-wk-old SIRT1 knockout (KO) and wild-type (WT) mice were fasted 21 h and/or refed 3 h. Fasted mice were injected intraperitoneally with bromodeoxyuridine (120 mg/kg body wt) 2 h before euthanasia. SIRT1 protein was localized to gastric and intestinal epithelial nuclei and was responsive to the nutritional status. SIRT1 was required for intestinal epithelial homeostasis. The SIRT1 KO mice showed enhanced crypt proliferation and suppressed villous apoptosis, resulting in increased intestinal villous height. In the SIRT1 KO intestine, the abundance of Forkhead box protein O1 and p53 protein decreased, whereas the subcellular localization of ß-catenin protein accumulated mainly in the crypts. The SIRT1 KO mice showed accelerated gastric emptying rate with increased abundance of ghrelin mRNA and protein in the stomach. Moreover, the SIRT1 KO mouse intestine showed enhanced ex vivo spontaneous contraction. We concluded that, SIRT1 plays a critical role in the control of intestinal homeostasis (by promoting apoptosis and inhibiting proliferation) and gastrointestinal motility (by reducing gastric emptying and intestinal contractile activity), implicating a novel role for SIRT1.

Glucagon-like peptide-2-stimulated protein synthesis through the PI 3-kinase-dependent Akt-mTOR signaling pathway.

Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive neuropeptide that exerts diverse actions in the gastrointestinal tract, including enhancing mucosal cell survival and proliferation. GLP-2 stimulates mucosal growth in vivo with an increased rate of protein synthesis. However, it was unclear whether GLP-2 can directly stimulate protein synthesis. The objective was to test critically whether GLP-2 receptor (GLP-2R) activation directly stimulates protein synthesis through a PI 3-kinase-dependent Akt-mTOR signaling pathway. HEK 293 cells (transfected with human GLP-2R cDNA) were treated with human GLP-2 with/without pretreatment of PI 3-kinase inhibitor (LY-294002) or mTOR inhibitor (rapamycin). Results show that 1) GLP-2 specifically bound to GLP-2R overexpressed in the HEK cells with K(a) = 0.22 nM and B(max) = 321 fmol/µg protein; 2) GLP-2-stimulated protein synthesis was dependent on the amount of GLP-2R cDNA and the dosage of GLP-2 and reached the plateau among 0.2-2 nM GLP-2; 3) GLP-2-stimulated protein synthesis was abolished by the PI 3-kinase inhibitor and mTOR inhibitor; and 4) GLP-2-mediated stimulation of phosphorylation on Akt and mTOR was dependent on the amount of GLP-2R cDNA transfected and the dosage of GLP-2. In addition, GLP-2-mediated action and signaling in regulation of protein synthesis were confirmed in mouse hippocampal neurons (expressing native GLP-2R). GLP-2 directly stimulated protein synthesis of primary cultured neurons in dosage-dependent, PI 3-kinase-dependent, and rapamycin-sensitive manners, which linked with activation of Akt-mTOR signaling pathway as well. We conclude that GLP-2R activation directly stimulates protein synthesis by activating the PI 3-kinase-dependent Akt-mTOR signaling pathway. GLP-2-stimulated protein synthesis may be physiologically relevant to maintaining neuronal long-term potentiation and providing secondary mediators (namely neuropeptides or growth factors).

GLP-2 potentiates L-type Ca2+ channel activity associated with stimulated glucose uptake in hippocampal neurons.

Glucagon-like peptide-2 (GLP-2) is a neuropeptide secreted from endocrine cells in the gut and neurons in the brain. GLP-2 stimulates intestinal crypt cell proliferation and mucosal blood flow while decreasing gastric emptying and gut motility. However, a GLP-2-mediated signaling network has not been fully established in primary cells. Since the GLP-2 receptor mRNA and protein were highly expressed in the mouse hippocampus, we further characterized that human (125)I-labeled GLP-2(1-33) specifically bound to cultured hippocampal neurons with K(d) = 0.48 nM, and GLP-2 acutely induced subcellular translocalization of the early gene c-Fos. Using the whole cell patch clamp, we recorded barium currents (I(Ba)) flowing through voltage-gated Ca(2+) channels (VGCC) in those neurons in the presence of GLP-2 with and without inhibitors. We showed that GLP-2 (20 nM) enhanced the whole cell I(Ba) mediated by L-type VGCC that was defined using an L-type Ca(2+) channel blocker (nifedipine, 10 microM). Moreover, GLP-2-potentiation of L-type VGCC was abolished in neurons pretreated with a PKA inhibitor (PKI(14-22), 1 microM). Finally, using a fluorescent nonmetabolized glucose analog (6-NBDG) tracing imaging, we showed that glucose was taken up directly by cultured neurons. GLP-2 increased 2-deoxy-d-[(3)H]glucose uptake that was dependent upon dosage, activation of PKA, and potentiation of L-type VGCC. We conclude that GLP-2 potentiates L-type VGCC activity through activating PKA signaling, partially stimulating glucose uptake by primary cultured hippocampal neurons. The potentiation of L-type VGCC may be physiologically relevant to GLP-2-induced neuroendocrine modulation of neurotransmitter release and hormone secretion.

alpha-Lipoic acid increases energy expenditure by enhancing adenosine monophosphate-activated protein kinase-peroxisome proliferator-activated receptor-gamma coactivator-1alpha signaling in the skeletal muscle of aged mice.

Skeletal muscle mitochondrial dysfunction is associated with aging and diabetes, which decreases respiratory capacity and increases reactive oxygen species. Lipoic acid (LA) possesses antioxidative and antidiabetic properties. Metabolic action of LA is mediated by activation of adenosine monophosphate-activated protein kinase (AMPK), a cellular energy sensor that can regulate peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a master regulator of mitochondrial biogenesis. We hypothesized that LA improves energy metabolism and mitochondrial biogenesis by enhancing AMPK-PGC-1alpha signaling in the skeletal muscle of aged mice. C57BL/6 mice (24 months old, male) were supplemented with or without alpha-LA (0.75% in drinking water) for 1 month. In addition, metabolic action and cellular signaling of LA were studied in cultured mouse myoblastoma C2C12 cells. Lipoic acid supplementation improved body composition, glucose tolerance, and energy expenditure in the aged mice. Lipoic acid increased skeletal muscle mitochondrial biogenesis with increased phosphorylation of AMPK and messenger RNA expression of PGC-1alpha and glucose transporter-4. Besides body fat mass, LA decreased lean mass and attenuated phosphorylation of mammalian target of rapamycin (mTOR) signaling in the skeletal muscle. In cultured C2C12 cells, LA increased glucose uptake and palmitate beta-oxidation, but decreased protein synthesis, which was associated with increased phosphorylation of AMPK and expression of PGC-1alpha and glucose transporter-4, and attenuated phosphorylation of mTOR and p70S6 kinase. We conclude that LA improves skeletal muscle energy metabolism in the aged mouse possibly through enhancing AMPK-PGC-1alpha-mediated mitochondrial biogenesis and function. Moreover, LA increases lean mass loss possibly by suppressing protein synthesis in the skeletal muscle by down-regulating the mTOR signaling pathway. Thus, LA may be a promising supplement for treatment of obesity and/or insulin resistance in older patients.

Mammary arteriovenous differences of glucose, insulin, prolactin and IGF-I in lactating sows under different protein intake levels.

Mammary uptake of nutrients is dependent on their availability in the circulation but the role of hormones in that process is not known. Arteriovenous differences (AVD) of glucose and key hormones across the mammary glands were therefore determined in sows fed varying levels of protein. Sixteen lactating sows (four/dietary treatment) were fed a 7.8, 13.0, 18.2 or 23.5% crude protein (CP) isocaloric diet throughout lactation and their litters were standardized to 11 pigs within 48 h of birth. The anterior main mammary vein and a carotid artery were cannulated on day 4+/-1 of lactation and blood samples were collected every 30 min over 6h on days 10, 14, 18 and 22 of lactation to measure glucose, insulin, IGF-I, and prolactin (PRL) concentrations. Amino acid data from these sows were previously published and used here to determine residual correlations. Dietary treatments had no effect on any of the insulin or PRL variables measured (P>0.1) and, on day 18 only, IGF-I AVD was greater (P=0.05) for sows on the 23.5% compared to the 18.2% diet. On days 18 and 22, sows fed the 13% CP diet had greater arterial, venous and AVD glucose concentrations than sows fed other diets (P<0.05). Total arterial amino acid concentrations were correlated to arterial insulin (P<0.001) and PRL (P<0.05) concentrations, but not to those of IGF-I (P>0.1). Mammary AVD for total (P<0.001) and essential amino acids (P<0.05) were correlated to arterial concentrations of insulin, but not to those of IGF-I (P>0.1) or PRL (P>0.1). Mammary AVD of both total (P<0.01) and essential (P<0.05) amino acids were also correlated to mammary PRL AVD. In conclusion, dietary protein level did not affect mammary AVD and circulating lactogenic hormone concentrations. Yet, amino acid utilization by the sow mammary gland seems to be regulated via both circulating insulin concentrations and PRL binding to and uptake by porcine mammary cells.

Extensive gut metabolism limits the intestinal absorption of excessive supplemental dietary glutamate loads in infant pigs.

Glutamate (Glu) is a major intestinal oxidative fuel, key neurotransmitter, and may be a useful dietary supplement to augment health of the infant gut. We quantified the metabolic fate of various supplemental dietary Glu intakes in young pigs surgically implanted with vascular, intraduodenal (ID), or intragastric (IG) catheters and a portal blood flow probe. Piglets were acutely fed a range of dietary Glu intakes using a basal milk formula (100%) supplemented with varying amounts of monosodium Glu (up to 400%) via ID or IG routes. We quantified the gastrointestinal metabolic fate of dietary Glu using [U-(13)C] Glu tracer. The Glu net absorption in the basal 100% group was low in both ID and IG groups, ranging from 13 to 17% of intake. Enteral Glu supplementation significantly increased the absolute absorption rate and arterial concentration of Glu. In both the ID and IG groups, enteral [(13)C]Glu absorption was limited (<5% tracer input) at the basal Glu intake (100%) but increased nearly 4-fold ( approximately 20% input) in the 300% intake group. A substantial fraction (33-50%) of the enteral [(13)C]Glu input was oxidized by the gut to (13)CO(2) in both the 100 and 300% intake groups. We conclude that extensive gut metabolism limits the absorption of supplemental dietary Glu even at excessive intakes.

GLP-2 rapidly activates divergent intracellular signaling pathways involved in intestinal cell survival and proliferation in neonatal piglets.

We previously demonstrated the dose-dependent glucagon-like peptide (GLP)-2 activation of intracellular signals associated with increased epithelial cell survival and proliferation in the neonatal intestine. Our current aim was to quantify the acute, temporal GLP-2 activation of these key intracellular signals and relate this to changes in epithelial cell survival and proliferation in the neonatal intestine. We studied 29 total parenteral nutrition-fed neonatal piglets infused intravenously with either saline (control) or human GLP-2 (420 micromol.kg(-1).h(-1)) for 1, 4, or 48 h. GLP-2 infusion increased small intestinal weight, DNA and protein content, and villus height at 48 h, but not at 1 or 4 h. Intestinal crypt and villus apoptosis decreased and crypt cell proliferation and protein synthesis increased linearly with duration of GLP-2 infusion, but were statistically different from controls only after 48 h. Before the morphological and cellular kinetic changes, GLP-2 rapidly activated putative GLP-2 receptor downstream signals within 1-4 h, including phosphorylation of protein kinase A, protein kinase B, extracellular signal-regulated kinase 1/2, and the transcription factors cAMP response element-binding protein and c-Fos. GLP-2 rapidly suppressed caspase-3 activation and upregulated Bcl-2 abundance within 1 h, whereas there was an increase in apoptosis inhibitors X-linked inhibitor of apoptosis at 1 h and cellular inhibitor of apoptosis-2 at 4 and 48 h. We also show that the increased c-Fos and reduced active caspase-3 immunostaining after GLP-2 infusion was localized in epithelial cells. We conclude that GLP-2-induced activation of intracellular signals involved in both cell survival and proliferation occurs rapidly and precedes the trophic cellular kinetic effects that occur later in intestinal epithelial cells.