Differential effects of angiopoietin-like 4 in brain and muscle on regulation of lipoprotein lipase activity

The increase in Angptl4 in the hypothalamus of ob/ob type 2 diabetic mice is most likely due to insulin resistance.

“The increase in Angplt4 in type 2 diabetes in hypothalamus of ob/ob mice is most likely due to insulin resistance, since we [37,38] and others [39,40] have shown, obesity is associated with increased inflammation and mitochondrial dysfunction at the level of the hypothalamus and both of these can contribute to an altered insulin response.”

• cites [37] Kleinridders, 2014, Insulin action in brain regulates systemic metabolism and brain function, Diabetes sourceNot checked yet
• cites [38] Kleinridders, 2013, Leptin regulation of Hsp60 impacts hypothalamic insulin signaling, Journal of Clinical Investigation sourcePartly supported

  The Results (p. 2) and discussion (p. 8) link hypothalamic mitochondrial dysfunction with insulin resistance in obese diabetic mice. The paper also reports hypothalamic inflammation and insulin resistance after experimental HSP60 knockdown (p. 7), but does not show increased inflammation in the obese mice themselves. It does not address Angptl4; the proposed explanation for its increase is the citing authors’ inference, not a finding of this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [39] Thaler, 2012, Obesity is associated with hypothalamic injury in rodents and humans, Journal of Clinical Investigation sourcePartly supported

  The Discussion reports hypothalamic inflammation and injury in rodents with diet-induced obesity, supporting the inflammation portion of the cited rationale. It does not establish hypothalamic mitochondrial dysfunction or show that insulin resistance explains increased Angptl4 in ob/ob mice; the Introduction discusses insulin resistance in peripheral tissues, not that proposed mechanism. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [40] Pipatpiboon, 2011, PPARγ agonist improves neuronal insulin receptor function in hippocampus and brain mitochondria function in rats with insulin resistance induced by long term high-fat diets, Endocrinology sourceNot checked yet

STZ-induced diabetes increases LPL activity in skeletal muscle in both wildtype and Angptl4 knockout mice.

“In Angptl4-/- mice, LPL activity in skeletal muscle is increased 3-fold, and this is further increased by STZ-induced diabetes.”

• Also stated in: § Differential regulation of Angptl4 and LPL in hypothalamus and muscle · plain

  “Similarly, LPL activity was upregulated in skeletal muscle with STZ induced diabetes in both genotypes, while no differences were detected in either hypothalamus or cortex in response to diabetes or knockout of the Angptl4 gene (Figure 3B–C).”

Intracerebroventricular insulin injection into STZ diabetic mice at a dose that does not affect blood glucose levels normalizes the upregulated hypothalamic Angptl4 expression.

“Intracerebroventricular insulin injection into STZ mice at levels which have no effect on plasma glucose restores Angptl4 expression in hypothalamus.”

• Also stated in: § Regulation of hypothalamic Angptl4 in diabetes by insulin · hedged

  “Importantly, in the STZ-diabetic mice, upregulation of Angptl4 in the hypothalamus could be normalized by direct i.c.v. injection of insulin at a dose (3 mU per mouse) which does not affect blood glucose levels (Figure 1F, [19]).”

• cites [19] Suzuki, 2010, Diabetes and insulin in regulation of brain cholesterol metabolism, Cell Metabolism sourceNot checked yet

In cultured glial cells, insulin treatment does not change LPL mRNA expression.

“Consistent with the in vivo experiment, in vitro insulin treatment of glial cells causes a 50% reduction of Angptl4 and significantly increases LPL activity with no change in LPL expression.”

• Also stated in: § Differential expression of Angptl4 in neurons and glia · plain

  “Six hours of insulin treatment in vitro significantly downregulated expression of Angptl4 in glial cells, whereas LPL mRNA was unchanged (Figure 2B).”

In STZ-induced type 1 diabetic and ob/ob type 2 diabetic mice, Angptl4 expression is increased in skeletal muscle compared with controls.

“In type 1 (streptozotocin, STZ) and type 2 (ob/ob) diabetic mice, there is a ~2-fold increase of Angptl4 in the hypothalamus and skeletal muscle.”

• Also stated in: § Regulation of hypothalamic Angptl4 in diabetes by insulin · hedged

  “Indeed, a similar upregulation of Angptl4 was observed in skeletal muscle from the STZ and ob/ob models described above (Figure 1E), suggesting that Angptl4 might be regulated and act the same way centrally as in skeletal muscle.”

• Also stated in: § Regulation of LPL activity by Angptl4 and diabetes · plain

  “In the STZ-diabetic mice, there was a 3–4 fold increase in Angptl4 mRNA expression in the skeletal muscle of wildtype mice, similar to what was observed above (Figure 1E).”

LPL mRNA expression in hypothalamus and cortex is unchanged independent of Angptl4 genotype and diabetes status.

“In Angptl4 knockout mice LPL mRNA expression was upregulated in skeletal muscle and restored to control levels after induction of STZ diabetes, whereas LPL expression was unchanged in hypothalamus and cortex independent of genotype and diabetes status (Figure 3).”

STZ diabetes increases Angptl4 mRNA expression in cortex of wildtype mice.

“STZ diabetes increased mRNA expression of Angptl4 in the skeletal muscle, hypothalamus and cortex of wildtype mice (Figures 1 and 3A–C).”

Intracerebroventricular administration of Angptl4 over 5 days does not affect food intake in mice on chow or high fat diet.

“As seen in Figure 4, i.c.v. administration of Angptl4 over 5 days did not affect food intake of mice independent of whether they were on chow or high fat diet, and no change in body weight was observed compared to mice receiving vehicle (Figure 4A–B).”

In cultured glial cells, insulin treatment significantly downregulates Angptl4 mRNA expression.

“Consistent with the in vivo experiment, in vitro insulin treatment of glial cells causes a 50% reduction of Angptl4 and significantly increases LPL activity with no change in LPL expression.”

• Also stated in: § Differential expression of Angptl4 in neurons and glia · plain

  “Six hours of insulin treatment in vitro significantly downregulated expression of Angptl4 in glial cells, whereas LPL mRNA was unchanged (Figure 2B).”

Random fed blood glucose is not significantly altered after 5 days of saline or Angptl4 i.c.v. treatment in chow diet mice.

“Random fed blood glucose was only measured in the chow diet mice, and was not significantly altered after 5 days of saline or Angptl4 i.c.v. treatment (93.8 ± 6.5 vs 97.8 ± 6.8 mg/dl; saline vs Angptl4).”

Exposure to high glucose (15 mM) does not influence Angptl4 or LPL mRNA expression in glial cells after 3, 6, or 24 h.

“By contrast, exposure to high glucose (15 mM) did not influence either Angptl4 or LPL mRNA expression after 3, 6, or 24 h (Figure 2E), supporting our in vivo findings.”

STZ diabetes results in a rise in serum triglyceride levels in fasted wildtype mice.

“Although STZ diabetes did not significantly change serum triglycerides in the fed state, STZ diabetes did result in a rise in serum triglyceride levels in fasted wildtype mice as expected (1.46 ± 0.12 vs 2.42 ± 0.4 mM; p < 0.01 control vs STZ).”

Angptl4 mRNA is undetectable in muscle from Angptl4 knockout mice under normal and diabetic conditions.

“As expected, Angptl4 mRNA was totally undetectable in muscle from Angptl4 knockout mice under normal and diabetic conditions (Figure 3A).”

Angptl4 knockout mice have lower serum triglycerides than wildtype controls in fed, fasted, and STZ-diabetic states.

“Consistent with the role of Angptl4 as an LPL inhibitor, Angptl4 knockout mice had a 50% reduction in serum triglycerides levels (Angptl4+/+ 0.96 ± 0.15 vs Angptl4−/− 0.49 ± 0.07 mM, p < 0.001)”

• Also stated in: § Regulation of LPL activity by Angptl4 and diabetes · plain

  “Consistent with the increased LPL activity, Angptl4 knockout mice exhibited significantly lower serum triglycerides compared to control in both the fed (0.85 ± 0.06 mM, p < 0.001) and fasted states (0.96 ± 0.2 mM, p < 0.001), as well as after induction of diabetes (Supplementary Table 2).”

In primary cells isolated from wildtype mouse brain, Angptl4 is almost exclusively expressed in glial cells, with almost none detected in neurons.

“Isolation of cells from the brain reveals that Angptl4 is produced in glia, whereas LPL is present in both glia and neurons.”

• Also stated in: § Differential expression of Angptl4 in neurons and glia · plain

  “Interestingly, we found that Angptl4 was almost exclusively expressed in glial cells, with almost none detected in neurons (Figure 2A).”

Intracerebroventricular administration of Angptl4 over 5 days does not affect body weight compared with vehicle.

“As seen in Figure 4, i.c.v. administration of Angptl4 over 5 days did not affect food intake of mice independent of whether they were on chow or high fat diet, and no change in body weight was observed compared to mice receiving vehicle (Figure 4A–B).”

In cultured glial cells, insulin treatment significantly increases LPL activity (intrinsic and in media) after 6 h.

“Consistent with the in vivo experiment, in vitro insulin treatment of glial cells causes a 50% reduction of Angptl4 and significantly increases LPL activity with no change in LPL expression.”

• Also stated in: § Differential expression of Angptl4 in neurons and glia · plain

  “Interestingly, insulin stimulated LPL activity both intrinsic and in the media from glial cells after 6 h was significantly increased (Figure 2F, Supplementary Figure 1D), at the same time point, where mRNA expression of Angptl4 was at its lowest.”

In both STZ-induced type 1 diabetic mice and ob/ob type 2 diabetic mice, Angptl4 mRNA expression is increased in the hypothalamus compared with controls (1.5-3-fold).

“In type 1 (streptozotocin, STZ) and type 2 (ob/ob) diabetic mice, there is a ~2-fold increase of Angptl4 in the hypothalamus and skeletal muscle.”

• Also stated in: § Regulation of hypothalamic Angptl4 in diabetes by insulin · plain

  “One of the most significantly upregulated genes in the hypothalami of both STZ diabetic mice and ob/ob mice compared to controls by microarray analysis was angiopoietin-like 4 (Angptl4). Angptl4 mRNA expression was confirmed by qPCR, which revealed 1.5–3-fold increases in these two models of diabetes, respectively (Figure 1D and F).”

In Angptl4 knockout mice, skeletal muscle LPL activity is increased about 3-fold compared with wildtype in both non-diabetic and STZ-diabetic states.

“In Angptl4-/- mice, LPL activity in skeletal muscle is increased 3-fold, and this is further increased by STZ-induced diabetes.”

• Also stated in: § Regulation of LPL activity by Angptl4 and diabetes · plain

  “LPL activity in skeletal muscle was increased about 3-fold in both control and STZ diabetic Angptl4 knockout mice compared to wildtype mice (Figure 3A).”

In Angptl4 knockout mice, LPL activity in hypothalamus is not significantly different from wildtype, independent of diabetic and nutritional status.

“By contrast, Angptl4-/- mice show no significant difference in LPL activity in hypothalamus or brain independent of diabetic and nutritional status.”

• Also stated in: § Differential regulation of Angptl4 and LPL in hypothalamus and muscle · plain

  “Similarly, LPL activity was upregulated in skeletal muscle with STZ induced diabetes in both genotypes, while no differences were detected in either hypothalamus or cortex in response to diabetes or knockout of the Angptl4 gene (Figure 3B–C).”

In Angptl4 knockout mice, LPL activity in cortex/brain is not significantly different from wildtype, independent of diabetic and nutritional status.

“By contrast, Angptl4-/- mice show no significant difference in LPL activity in hypothalamus or brain independent of diabetic and nutritional status.”

• Also stated in: § Differential regulation of Angptl4 and LPL in hypothalamus and muscle · plain

  “Similarly, LPL activity was upregulated in skeletal muscle with STZ induced diabetes in both genotypes, while no differences were detected in either hypothalamus or cortex in response to diabetes or knockout of the Angptl4 gene (Figure 3B–C).”

In skeletal muscle, LPL mRNA expression is largely unchanged by STZ-induced diabetes.

“LPL mRNA expression in muscle was largely unchanged by induction of diabetes, but there was a small increase (∼30%) in the Angptl4 knockout mice, which returned toward control levels in the presence of diabetes (Figure 3A).”

STZ diabetes does not significantly change serum triglycerides in fed wildtype mice.

“Although STZ diabetes did not significantly change serum triglycerides in the fed state, STZ diabetes did result in a rise in serum triglyceride levels in fasted wildtype mice as expected (1.46 ± 0.12 vs 2.42 ± 0.4 mM; p < 0.01 control vs STZ).”

There are no changes in hypothalamic mRNA expression of NPY, AgRP, POMC, or CART between Angptl4 knockout mice and control littermates.

“In addition, we did not detect any changes on hypothalamic mRNA expression of orexigenic or anorexigenic neuropeptides; NPY, AgRP, POMC, or CART between Angptl4 knockout mice and their control littermates (Figure 4C), consistent with our findings that i.c.v. delivered Angptl4 does not affect food intake.”

Angptl4 is a known inhibitor of LPL activity in peripheral tissues.

“Angiopoietin-like (Angptl) 4 is a known inhibitor of LPL activity.”

• Also stated in: § Discussion · boosted

  “As Angptl4 is a known inhibitor of LPL activity in the peripheral tissues [4,6], we explored the role of Angptl4 as a regulator of LPL activity and a potential contributing factor in brain lipid metabolism.”

• cites [4] Kersten, 2000, Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene, Journal of Biological Chemistry sourceNot checked yet
• cites [6] Yau, 2009, A highly conserved motif within the NH2-terminal coiled-coil domain of angiopoietin-like protein 4 confers its inhibitory effects on lipoprotein lipase by disrupting the enzyme dimerization, Journal of Biological Chemistry sourceNot checked yet

The lack of change in brain LPL activity may be due to the relatively short period (overnight) of fasting, since 72 h of fasting is necessary to detect altered brain LPL activity in rats.

“The lack of change in LPL activity in the brain in our study may be due to the relatively short period (overnight) of fasting, since 72 h of fasting is necessary to detect altered brain LPL activity in rats [32].”

• cites [32] Gavin, 1987, Brain lipoprotein lipase is responsive to nutritional and hormonal modulation, Metabolism sourceNot checked yet

The experiment was conducted in random fed as well as overnight fasted mice to exclude an effect of nutritional status as an explanation for the lack of difference in central versus peripheral LPL activity.

“To exclude an effect of nutritional status as an explanation for the lack of difference in central versus peripheral LPL activity, the experiment was conducted in random fed as well as overnight fasted mice with similar results (Supplemental Figure 2).”

In contrast to the periphery, central Angptl4 does not regulate LPL activity.

“However, in contrast to the periphery, central Angptl4 does not regulate LPL activity, but appears to participate in the metabolic crosstalk between glia and neurons.”

There was no change in insulin or blood glucose levels between genotypes in the fasted state.

“There was no change in insulin or blood glucose levels between the genotypes in the fasted state, but, as expected, mice with STZ induced diabetes has significantly higher blood glucose and lower insulin levels (Supplementary Table 2).”

Central Angptl4 appears to participate in metabolic crosstalk between glia and neurons.

“However, in contrast to the periphery, central Angptl4 does not regulate LPL activity, but appears to participate in the metabolic crosstalk between glia and neurons.”

LPL affects whole body glucose homeostasis dependent on expression level in skeletal muscle.

“Since LPL is known to affect whole body glucose homeostasis dependent on expression level in skeletal muscle [16,17], this tissue was included in the analysis to compare differences between central and peripheral regulated lipid metabolism.”

• cites [16] Kim, 2001, Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance, Proceedings of the National Academy of Sciences of the United States of America sourceNot checked yet
• cites [17] Wang, 2009, Skeletal muscle-specific deletion of lipoprotein lipase enhances insulin signaling in skeletal muscle but causes insulin resistance in liver and other tissues, Diabetes sourceNot checked yet

The upregulation of hypothalamic Angptl4 in type 1 diabetes is due to lack of insulin at the level of the brain.

“The upregulation in type 1 diabetes is due to lack of insulin at the level of the brain, and i.c.v. injection of insulin can reverse this upregulation.”

Previous studies on hyperglycemia and Angptl4 expression used very high levels of glucose, much beyond those that occur in vivo in the CNS, even in diabetes.

“Although it has been suggested that hyperglycemia might regulate Angptl4 expression [33], these previous studies were done with very high levels of glucose, much beyond those that occur in vivo in the CNS, even in diabetes.”

• cites [33] Sango, 2006, High glucose-induced activation of the polyol pathway and changes of gene expression profiles in immortalized adult mouse Schwann cells IMS32, Journal of Neurochemistry sourceNot checked yet

In glial cells in culture, Angptl4 mRNA expression is regulated directly by insulin, not glucose.

“We find that in glial cells in culture, Angptl4 mRNA expression is regulated directly by insulin, not glucose, confirming our in vivo observations.”

Increased LPL activity in Angptl4 knockout mice results in a 50% reduction in serum triglyceride levels.

“Although STZ-diabetes itself had little or no effect on serum TG levels in the fed state, the increased LPL activity in Angptl4 knockout mice resulted in a 50% reduction in serum TG levels, and previous studies have shown that this is associated with increased ectopic accumulation of TG in liver [31].”

LPL in brain has been implicated in regulation of body weight and energy balance as well as cognition.

“The function of LPL in the brain still remains elusive, but it has been implicated in at least two different functions; the regulation of body weight and energy balance as well as cognition [14,15].”

• cites [14] Wang, 2012, Lipoprotein lipase in the brain and nervous system, Annual Review of Nutrition sourceNot checked yet
• cites [15] Papassotiropoulos, 2005, A cluster of cholesterol-related genes confers susceptibility for Alzheimer's disease, Journal of Clinical Psychiatry sourceNot checked yet