LRP1 in GABAergic neurons is a key link between obesity and memory function.

OBJECTIVE: Low-density lipoprotein receptor-related protein-1 (LRP1) regulates energy homeostasis, blood-brain barrier integrity, and metabolic signaling in the brain. Deficiency of LRP1 in inhibitory gamma-aminobutyric acid (GABA)ergic neurons causes severe obesity in mice. However, the impact of LRP1 in inhibitory neurons on memory function and cognition in the context of obesity is poorly understood. METHODS: Mice lacking LRP1 in GABAergic neurons (Vgat-Cre; LRP1loxP/loxP) underwent behavioral tests for locomotor activity and motor coordination, short/long-term and spatial memory, and fear learning/memory. This study evaluated the relationships between behavior and metabolic risk factors and followed the mice at 16 and 32 weeks of age. RESULTS: Deletion of LRP1 in GABAergic neurons caused a significant impairment in memory function in 32-week-old mice. In the spatial Y-maze test, Vgat-Cre; LRP1loxP/loxP mice exhibited decreased travel distance and duration in the novel arm compared with controls (LRP1loxP/loxP mice). In addition, GABAergic neuron-specific LRP1-deficient mice showed a diminished capacity for performing learning and memory tasks during the water T-maze test. Moreover, reduced freezing time was observed in these mice during the contextual and cued fear conditioning tests. These effects were accompanied by increased neuronal necrosis and satellitosis in the hippocampus. Importantly, the distance and duration in the novel arm, as well as the performance of the reversal water T-maze test, negatively correlated with metabolic risk parameters, including body weight, serum leptin, insulin, and apolipoprotein J. However, in 16-week-old Vgat-Cre; LRP1loxP/loxP mice, there were no differences in the behavioral tests or correlations between metabolic parameters and cognition. CONCLUSIONS: Our findings demonstrate that LRP1 from GABAergic neurons is important in regulating normal learning and memory. Metabolically, obesity caused by GABAergic LRP1 deletion negatively regulates memory and cognitive function in an age-dependent manner. Thus, LRP1 in GABAergic neurons may play a crucial role in maintaining normal excitatory/inhibitory balance, impacting memory function, and reinforcing the potential importance of LRP1 in neural system integrity.

LRP1 mediates leptin transport by coupling with the short-form leptin receptor in the choroid plexus.

Adipocyte-derived leptin enters the brain to exert its anorexigenic action, yet its transport mechanism is poorly understood. Here we report that LRP1 (low-density lipoprotein receptor-related protein-1) mediates the transport of leptin across the blood-CSF barrier in Foxj1 expressing cells highly enriched at the choroid plexus (ChP), coupled with the short-form leptin receptor, and LRP1 deletion from ependymocytes and ChP cells leads to leptin resistance and hyperphagia, causing obesity. Thus, LRP1 in epithelial cells is a principal regulator of leptin transport in the brain.

ROCK1 regulates insulin secretion from ß-cells.

OBJECTIVE: The endocrine pancreatic ß-cells play a pivotal role in maintaining whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate ß-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homeostasis. METHODS: Mice lacking ROCK1 in pancreatic ß-cells (RIP-Cre; ROCK1loxP/loxP, ß-ROCK1-/-) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. An insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from ß-ROCK1-/- mice or ß-cell lines with knockdown of ROCK1 was also evaluated. A proximity ligation assay was performed to determine the physical interactions between PK and ROCK1. RESULTS: Mice with a deficiency of ROCK1 in pancreatic ß-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, ß-ROCK1-/- mice displayed a progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium and ATP levels and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown ß-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that glucose stimulation in ß-cells greatly enhanced ROCK1 binding to PK. CONCLUSIONS: Our findings demonstrate that ß-cell ROCK1 is essential for glucose-stimulated insulin secretion and for glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.

LRP1 regulates food intake and energy balance in GABAergic neurons independently of leptin action.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a member of LDL receptor family that plays a key role in systemic glucose and lipid homeostasis. LRP1 also regulates energy balance in the hypothalamus by mediating leptin's anorexigenic action, although the underlying neurocircuitry involved is still unclear. Because GABAergic neurons are a major mediator of hypothalamic leptin action, we studied the role of GABAergic LRP1 in energy balance and leptin action using mice lacking LRP1 in Vgat- or AgRP-expressing neurons (Vgat-Cre; LRP1loxP/loxP or AgRP-Cre; LRP1loxP/loxP). Here, we show that LRP1 deficiency in GABAergic neurons results in severe obesity in male and female mice fed a normal-chow diet. This effect is most likely due to increased food intake and decreased energy expenditure and locomotor activity. Increased adiposity in GABAergic neuron-specific LRP1-deficient mice is accompanied by hyperleptinemia and hyperinsulinemia. Insulin resistance and glucose intolerance in these mice are occurred without change in body weight. Importantly, LRP1 in GABAergic neurons is not required for leptin action, as evidenced by normal leptin's anorexigenic action and leptin-induced hypothalamic Stat3 phosphorylation. In contrast, LRP1 deficiency in AgRP neurons has no effect on adiposity and caloric intake. In conclusion, our data identify GABAergic neurons as a key neurocircuitry that underpins LRP1-dependent regulation of systemic energy balance and body-weight homeostasis. We further find that the GABAergic LRP1 signaling pathway modulates food intake and energy expenditure independently of leptin signaling and AgRP neurons.

Author Correction: Apolipoprotein J is a hepatokine regulating muscle glucose metabolism and insulin sensitivity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Apolipoprotein J is a hepatokine regulating muscle glucose metabolism and insulin sensitivity.

Crosstalk between liver and skeletal muscle is vital for glucose homeostasis. Hepatokines, liver-derived proteins that play an important role in regulating muscle metabolism, are important to this communication. Here we identify apolipoprotein J (ApoJ) as a novel hepatokine targeting muscle glucose metabolism and insulin sensitivity through a low-density lipoprotein receptor-related protein-2 (LRP2)-dependent mechanism, coupled with the insulin receptor (IR) signaling cascade. In muscle, LRP2 is necessary for insulin-dependent IR internalization, an initial trigger for insulin signaling, that is crucial in regulating downstream signaling and glucose uptake. Of physiologic significance, deletion of hepatic ApoJ or muscle LRP2 causes insulin resistance and glucose intolerance. In patients with polycystic ovary syndrome and insulin resistance, pioglitazone-induced improvement of insulin action is associated with an increase in muscle ApoJ and LRP2 expression. Thus, the ApoJ-LRP2 axis is a novel endocrine circuit that is central to the maintenance of normal glucose homeostasis and insulin sensitivity.

Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition.

Obesity is a major risk factor for developing nonalcoholic fatty liver disease (NAFLD). NAFLD is the most common form of chronic liver disease and is closely associated with insulin resistance, ultimately leading to cirrhosis and hepatocellular carcinoma. However, knowledge of the intracellular regulators of obesity-linked fatty liver disease remains incomplete. Here we showed that hepatic Rho-kinase 1 (ROCK1) drives obesity-induced steatosis in mice through stimulation of de novo lipogenesis. Mice lacking ROCK1 in the liver were resistant to diet-induced obesity owing to increased energy expenditure and thermogenic gene expression. Constitutive expression of hepatic ROCK1 was sufficient to promote adiposity, insulin resistance, and hepatic lipid accumulation in mice fed a high-fat diet. Correspondingly, liver-specific ROCK1 deletion prevented the development of severe hepatic steatosis and reduced hyperglycemia in obese diabetic (ob/ob) mice. Of pathophysiological significance, hepatic ROCK1 was markedly upregulated in humans with fatty liver disease and correlated with risk factors clustering around NAFLD and insulin resistance. Mechanistically, we found that hepatic ROCK1 suppresses AMPK activity and a ROCK1/AMPK pathway is necessary to mediate cannabinoid-induced lipogenesis in the liver. Furthermore, treatment with metformin, the most widely used antidiabetes drug, reduced hepatic lipid accumulation by inactivating ROCK1, resulting in activation of AMPK downstream signaling. Taken together, our findings establish a ROCK1/AMPK signaling axis that regulates de novo lipogenesis, providing a unique target for treating obesity-related metabolic disorders such as NAFLD.

Saturated fatty acids-induced miR-424-5p aggravates insulin resistance via targeting insulin receptor in hepatocytes.

The excessive intake of saturated fatty acids (SFA) causes obesity and liver steatosis, which are major risk factors for insulin resistance and type 2 diabetes. Although the expression of certain microRNAs (miRNAs) targeting the insulin signaling molecules are regulated aberrantly in SFA-induced obesity, their implications on hepatic insulin resistance are largely unknown. This study examined the associations of miR-424-5p, which is induced by SFA, with the development of insulin resistance. SFA palmitate (PA)-treated HepG2 cells and high fat diet (HFD)-induced obese mouse livers showed an impairment of insulin signaling due to a significant decrease in INSR and IRS-1 expression. Based on expression profiling and qRT-PCR analysis, miR-424-5p, which presumably targets the 3'UTR of INSR, was upregulated in both PA-treated HepG2 cells and the liver of HFD-fed mice. miR-424-5p was found to target the 3'UTR of INSR directly and downregulated INSR expression at the post-transcriptional step. Furthermore, the overexpression of miR-424-5p suppressed INSR expression significantly, leading to impaired insulin signaling and glycogen synthesis in hepatocytes. A novel mechanism for how SFA-induced miR-424-5p impairs insulin signaling through the targeting of INSR is reported. In addition, the crucial role and underlying mechanism of miR-424-5p in the obesity-induced hepatic insulin resistance is explained.

Data on the expression of PEPCK in HepG2 hepatocytes transfected with miR-195.

Dietary fats rich in saturated fatty acid (SFA) increase the risk of metabolic diseases, and certain microRNAs (miRNAs) dysregulated by SFA are associated with the pathogenesis of insulin resistance and type 2 diabetes. A previous study found that miR-195 is increased by SFA and impairs hepatic insulin signaling through the suppression of INSR (Yang et al., 2014) [1]. This article reports accompanying data to determine the effect of miR-195 on the expression of PEPCK, a key player in hepatic gluconeogenesis. The transfection of miR-195 in HepG2 hepatocytes was found to increase the mRNA and protein expression of PEPCK. Moreover, the insulin-stimulated reduction of PEPCK expression was attenuated drastically by miR-195. More detailed analysis and understanding of the role of miR-195 in diet-induced hepatic insulin resistance can be found in "Saturated fatty acid-induced miR-195 impairs insulin signaling and glycogen metabolism in HepG2 cells" (Yang et al., 2014) [1].

Data on the decreased expression of FOXO1 by miR-1271 in HepG2 hepatocytes.

Obesity and metabolic diseases are closely associated with insulin resistance. Obesity-induced miRNAs are also considered to be potential contributors to the development of insulin resistance and type 2 diabetes. Previously, the expression of miR-1271 was reported to be upregulated in the liver of diet-induced obese mice (Yang et al., 2016) [1]. In this data article, multiple in silico analysis predicted FOXO1 gene to be a direct target of miR-1271. Dual luciferase reporter gene analysis showed that miR-1271 suppressed FOXO1 expression by direct binding to 3'UTR. The overexpression of miR-1271 reduced the protein expression of FOXO1, thereby reducing the transcription of PEPCK, a downstream target of FOXO1. The data is related to a research article entitled "MiR-1271 upregulated by saturated fatty acid palmitate provokes impaired insulin signaling by repressing INSR and IRS-1 expression in HepG2 cells" (Yang et al., 2016) [1].

Data on the effect of miR-15b on the expression of INSR in murine C2C12 myocytes.

The ectopic expression of miR-15b is linked causally to impaired insulin signaling in human HepG2 hepatocytes through the suppression of INSR (Yang et al., 2015) [1]. In this data article, we further examined the effect of miR-15b on insulin signaling in a murine skeletal muscle cells, C2C12 myocytes. Although the 3'UTR of mouse INSR mRNA has an appropriate binding site for miR-15b based on TargetScan analysis, the ectopic expression of miR-15b did not suppress the expression and insulin-stimulated phosphorylation of insulin signaling intermediates in C2C12 myocytes. A more detailed understanding of the effects of miR-15b on hepatic insulin resistance can be found in "Obesity-induced miR-15b is linked causally to the development of insulin resistance through the repression of the insulin receptor in hepatocytes" (Yang et al., 2015) [1].

Data on the expression and insulin-stimulated phosphorylation of IRS-1 by miR-96 in L6-GLUT4myc myocytes.

Diets containing a high saturated fatty acid (SFA) increase the risk of metabolic diseases, and microRNAs (miRNAs) induced by SFA have been implicated in the pathogenesis of insulin resistance and type 2 diabetes. In a previous report, miR-96 is found to be upregulated by SFA and involved in the suppression of insulin signaling intermediates, leading to insulin resistance in hepatocytes (Yang et al., 2016) [1]. This article presents the accompanying data collected from L6-GLUT4myc myocytes to determine the effects of miR-96 on insulin signaling in skeletal muscle cells. The transfection of miR-96 decreased the expression of IRS-1 in myocytes. Accordingly, miR-96 inhibited the insulin-stimulated phosphorylation of IRS-1, which led to an impairment of insulin signaling. More detailed analysis and understanding of the roles of miR-96 in diet-induced insulin resistance can be found in "Induction of miR-96 by dietary saturated fatty acids exacerbates hepatic insulin resistance through the suppression of INSR and IRS-1" (Yang et al., 2016) [1].

Dataset on the identification of differentially expressed genes by annealing control primer-based PCR in mitochondrial DNA-depleted myocytes.

Changes in the mitochondrial DNA (mtDNA) content are believed to initiate a stress signal that leads to alterations in nuclear gene expression. This article presents data on the identification of nuclear genes that are expressed differentially in response to changes in the mtDNA content in myocytes using annealing controlled primers (ACP)-based PCR technology. The data obtained from L6 GLUT4myc myocytes showed that a total of 19 ACPs produced differentially expressed PCR amplicons in the mtDNA-depleted myocytes. Among those, 13 amplicons were cloned, sequenced, and identified successfully based on the GenBank database. To validate the efficacy of ACP-based PCR analysis, three differentially expressed genes (DEG10, 22 and 26) were confirmed by PCR using the specific primers. The further analysis and detailed results of DEG22 and its functional significance can be found in "C1q tumor necrosis factor alpha-related protein isoform 5 is increased in mitochondrial DNA-depleted myocytes and activates AMP-activated protein kinase." [1].

Spirodela polyrhiza extract modulates the activation of atopic dermatitis-related ion channels, Orai1 and TRPV3, and inhibits mast cell degranulation.

CONTEXT: Spirodela polyrhiza (L.) Schleid. (Lemnaceae), Spirodelae Herba (SH), has been known to relieve inflammation, urticaria and skin symptoms including pruritus, eczema and rash. OBJECTIVE: The effects of SH extract on two calcium ion channels, Orai1 and TRPV3, and their potential as novel therapeutics for atopic dermatitis (AD) were investigated. The regulatory role of Orai1 on mast cell degranulation was evaluated. MATERIALS AND METHODS: The dried leaves of SH were extracted by 70% methanol. Effects of SH extract (100 µg/mL) in an HEK293T cell line overexpressing human Orai1 or TRPV3 were assessed. Ion channel modulation in transfected HEK293T cells was measured using a conventional whole-cell patch-clamp technique. IgE-antigen complex-stimulated mast cell degranulation was measured by ß-hexosaminidase assay with morphological observation after treatment with 20, 50 and 100 µg/mL SH extract. RESULTS: SH extract (100 µg/mL) significantly inhibited Orai1 activity (63.8 ± 0.97%) in Orai1-STIM1 co-overexpressed HEK293T cells. SH extract significantly increased TRPV3 activity (81.29 ± 0.05% at -100 mV) compared with the positive control 2-APB (100 µM), which induced full activation. SH extract inhibited degranulation in IgE-antigen complex-stimulated RBL-2H3 mast cells by decreasing ß-hexosaminidase activity (3.14 ± 0.03, 2.56 ± 0.12 and 2.29 ± 0.08 mU/mg, respectively). CONCLUSION: Our results suggested that SH extract could treat abnormal skin barrier pathologies in AD through modulation of the activities of the calcium ion channels Orai1 and TRPV3 and inhibition of mast cell degranulation. This is the first report of an herbal effect on the modulation of ion channels associated with skin barrier disruption in AD pathogenesis.

MicroRNA expression analysis in the liver of high fat diet-induced obese mice.

A previous study indicated a causal link between certain miRNAs induced by obesity and the development of hepatic insulin resistance and type 2 diabetes. Here we provide accompanying data collected using Affymetrix GeneChip miRNAs microarrays to identify the changes in miRNAs expression in the liver of mice fed a high fat diet (HFD). Differentially expressed microRNA analyses in the liver of the HFD-fed mice revealed a range of upregulated (>1.5-fold) or downregulated (<0.5-fold) miRNAs. Among those upregulated miRNAs, in silico target analysis, such as TargetScan, PicTar, and miRWalk, identified miRNAs with the putative binding sites on the 3'UTRs of INSR and/or IRS-1. Interpretation of the data and further extensive insights into the implication of miRNAs, particularly miR-15b, in hepatic insulin resistance can be found in "Obesity-induced miR-15b is linked causally to the development of insulin resistance through the repression of the insulin receptor in hepatocytes." (W.M. Yang, H.J. Jeong, S.W. Park, W. Lee, 2015)[1].

Induction of miR-96 by Dietary Saturated Fatty Acids Exacerbates Hepatic Insulin Resistance through the Suppression of INSR and IRS-1.

Obesity is defined as the excessive accumulation of body fat that ultimately leads to chronic metabolic diseases. Diets rich in saturated fatty acids (SFA) exacerbate obesity and hepatic steatosis, which increase the risk of hepatic insulin resistance and type 2 diabetes (T2DM). Although microRNAs (miRNAs) play an important role in a range of biological processes, the implications of SFA-induced miRNAs in metabolic dysregulation, particularly in the pathogenesis of hepatic insulin resistance, are not well understood. This study investigated the implications of miR-96, which is induced strongly by SFA, in the development of hepatic insulin resistance. The liver of HFD mice and the palmitate-treated hepatocytes exhibited an impairment of insulin signaling due to the significant decrease in INSR and IRS-1 expression. According to expression profiling and qRT-PCR analysis of the miRNAs, the expression level of miR-96 was higher in hepatocytes treated with palmitate. Moreover, miR-96 was also upregulated in the liver of HFD mice. Interestingly, miR-96 targeted the 3'UTRs of INSR and IRS-1 directly, and repressed the expression of INSR and IRS-1 at the post-transcriptional level. Accordingly, the overexpression of miR-96 was found to cause a significant decrease in INSR and IRS-1 expression, thereby leading to an impairment of insulin signaling and glycogen synthesis in hepatocytes. These results reveal a novel mechanism whereby miR-96 promotes the pathogenesis of hepatic insulin resistance resulted from SFA or obesity.

Data for differentially expressed microRNAs in saturated fatty acid palmitate-treated HepG2 cells.

Certain microRNAs (miRNAs) targeting the molecules in the insulin signaling cascades are dysregulated by saturated fatty acids (SFA), which can lead to insulin resistance and type 2 diabetes. This article reports the accompanying data collected using miRNAs microarrays to identify the changes in miRNA expression in HepG2 cells treated with SFA palmitate. Differentially expressed miRNA analyses in HepG2 cells showed that a range of upregulated (>1.5-fold) or downregulated (<0.5-fold) miRNAs. Further extensive insights into the implications of miRNAs, particularly miR-1271, in HepG2 cells can be found in "MiR-1271 upregulated by saturated fatty acid palmitate provokes impaired insulin signaling by repressing INSR and IRS-1 expression in HepG2 cells" (W.M. Yang, K.H. Min, W. Lee, 2016) [1].

MiR-1271 upregulated by saturated fatty acid palmitate provokes impaired insulin signaling by repressing INSR and IRS-1 expression in HepG2 cells.

Dietary saturated fatty acids (SFA) in excess not only induce hepatic insulin resistance, but also result in type 2 diabetes (T2DM). Although microRNAs (miRNAs) participate widely in the pathogenesis of a range of diseases through the suppression of target gene expression at the post-transcriptional level, the implications of SFA-induced miRNAs in the dysregulation of metabolism, particularly in the development of insulin resistance, are largely unclear. SFA palmitate provoked an impairment of insulin signaling in HepG2 cells via a reduction in the expression of INSR and IRS-1 protein. The significant upregulation of miR-1271, which was presumed to target INSR and IRS-1 3'UTRs, was observed in the palmitate-treated HepG2 cells. Using a reporter gene assay, miR-1271 authentically targeted the 3'UTRs of INSR and IRS-1. Furthermore, the overexpression of miR-1271 caused a substantial decrease in INSR and IRS-1 expression, which led to an impairment in insulin signaling and glycogen metabolism. Therefore, these findings suggest that the induction of miR-1271 by SFA palmitate promotes the development of insulin resistance by targeting INSR and IRS-1 in hepatocytes.

C1q tumor necrosis factor α-related protein isoform 5 attenuates palmitate-induced DNA fragmentation in myocytes through an AMPK-dependent mechanism.

This article reports the data for the effects of C1q tumor necrosis factor α-related protein isoform 5 (CTRP5) on the palmitate-induced apoptosis in myocytes. The data obtained from in vitro cultured myocytes shows that the cellular treatment with the globular domain of CTRP5 (gCTRP5) significantly inhibits the palmitate-induced MTT reduction, caspase-3 activation, and DNA fragmentation in a time-dependent manner. The data presented in this article also shows that AraA, an inhibitor of AMPK, almost completely abolished the protective effect of gCTRP5 on the DNA fragmentation induced by palmitate in myocytes. Interpretation of our data and further extensive insights into the protective role of CTRP5 in palmitate-induced apoptosis in myocytes can be found in Yang and Lee (2014) [1].

Obesity-induced miR-15b is linked causally to the development of insulin resistance through the repression of the insulin receptor in hepatocytes.

SCOPE: Obesity increases intracellular lipid accumulation in key tissues or organs, which often leads to metabolic dysfunction and insulin resistance. Diets rich in saturated fatty acid (SFA) exacerbate obesity and hepatic steatosis, which accentuate the risk of insulin resistance and type 2 diabetes (T2DM). Although microRNAs (miRNAs) play a critical role in the regulation of gene expression, the implication of obesity-induced miRNAs in metabolic disorders particularly in the development of insulin resistance is largely unknown. Here, we investigated the implication of miR-15b, which is induced by SFA palmitate or obesity, in hepatic insulin resistance. METHODS AND RESULTS: Diet-induced obesity (DIO) in mice developed hyperglycemia and insulin resistance, accompanying with a reduction of insulin receptor (INSR) expression. Palmitate impaired insulin signaling as well as a decrease of INSR in hepatocytes. The expression of miR-15b was upregulated by DIO or palmitate in hepatocytes. Furthermore, the overexpression of miR-15b suppressed the protein expression of INSR through targeting INSR 3' untranslated region directly, resulting in an impairment of the insulin signaling and glycogen synthesis in hepatocytes. CONCLUSION: These results unveil a novel mechanism whereby miR-15b is linked causally to the pathogenesis of hepatic insulin resistance in SFA-induced obesity.