Cell Biol. Louis, MO. The quantification Thernogenesis protein bands was shown in the left panel. The macronutrient composition of the diet was presented elsewhere

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This pattern suggests that enhancing brain insulin signaling by intranasal administration of the hormone may act on glucose homeostasis in the body periphery by supporting hepatic insulin action. Nevertheless, given that postprandial liver glucose production accounts for approximately one-fifth to one-half of fasting values 17 , improved insulin-dependent metabolization of ingested glucose may also have contributed to the intranasal insulin-induced decrease in postprandial serum insulin levels.

Such an effect could basically be supported by the observed decrease in prandial FFA levels due to intranasal insulin inasmuch as FFAs are known to impair insulin-stimulated muscle uptake of glucose However, FFA effects on peripheral insulin-stimulated glucose uptake slowly develop over some hours 19 , which, in conjunction with the lack of a significant correlation between the decreases in prandial FFA and postprandial insulin concentrations, makes this view unlikely.

Furthermore, a contribution of enhanced noninsulin-mediated glucose disposal, i. Although the present results suggest that insulin administration to the human brain enhances the efficiency of the glucoregulatory brain-liver axis in response to nutrient intake, our observations should be corroborated in future studies that rely on more refined measurements of insulin sensitivity, e.

It is also noteworthy that most recent animal data hint at divergent effects of hypothalamic insulinergic signaling on peripheral glucose homeostasis and energy expenditure depending on the involvement of agouti-related protein or proopiomelanocortin neuronal pathways In this regard, general enhancements in brain insulin signaling as performed in our study do not permit differentiations.

Taken together, our findings indicate that intranasal insulin acutely increases postprandial thermogenesis and improves the glucoregulatory response to food intake, suggesting that boosting brain insulin signaling in humans enhances the body's ability to cope with calorie consumption 20 , Against the background of studies indicating that obesity and peripheral insulin resistance are associated with reduced central nervous insulin sensitivity 22 , — 24 , enhancing brain insulin signaling may emerge as a useful approach in the therapeutic management of disorders hallmarked by disturbed glucose homeostasis The costs of publication of this article were defrayed in part by the payment of page charges.

Section solely to indicate this fact. The funding sources had no input in the design and conduct of this study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript.

Aero Pump, Hochheim, Germany, generously provided us with precision nasal air pumps. No other potential conflicts of interest relevant to this article were reported. designed the study, analyzed the data, contributed to writing the manuscript, and collected data or performed experiments for the study.

enrolled patients and collected data or performed experiments for the study. contributed to writing the manuscript. designed the study, analyzed the data, and contributed to writing the manuscript.

All authors had full access to all of the data and take responsibility for the integrity and accuracy of the data analysis. We thank I. von Lützau, M. Grohs, and H. Ruf Department of Neuroendocrinology, University of Lübeck, Lübeck, Germany for their expert and invaluable laboratory work.

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Advanced Search. User Tools Dropdown. Sign In. Skip Nav Destination Close navigation menu Article navigation. Volume 60, Issue 1. Previous Article Next Article. RESEARCH DESIGN AND METHODS. Article Navigation. Metabolism September 28 Intranasal Insulin Enhances Postprandial Thermogenesis and Lowers Postprandial Serum Insulin Levels in Healthy Men Christian Benedict ; Christian Benedict.

Corresponding author: Christian Benedict, benedict kfg. This Site. Google Scholar. Swantje Brede ; Swantje Brede. Helgi B.

Schiöth ; Helgi B. Hendrik Lehnert ; Hendrik Lehnert. Bernd Schultes ; Bernd Schultes. Gallen, St. Gallen, Switzerland. Jan Born ; Jan Born. Manfred Hallschmid Manfred Hallschmid. Diabetes ;60 1 — Article history Received:. Get Permissions. toolbar search Search Dropdown Menu.

toolbar search search input Search input auto suggest. View large Download slide. Search ADS. Insulin signaling in the central nervous system: a critical role in metabolic homeostasis and disease from C. elegans to humans. Insulin causes hyperthermia by direct inhibition of warm-sensitive neurons.

Hypothalamic insulin signaling is required for inhibition of glucose production. Quantitative analysis of the olfactory pathway for drug delivery to the brain. Transcortical direct current potential shift reflects immediate signaling of systemic insulin to the human brain.

Differential sensitivity of men and women to anorexigenic and memory-improving effects of intranasal insulin. Comparable sensitivity of postmenopausal and young women to the effects of intranasal insulin on food intake and working memory. Divergent regulation of energy expenditure and hepatic glucose production by insulin receptor in agouti-related protein and POMC neurons.

Differential sensitivity to central leptin and insulin in male and female rats. Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats. The cerebrocortical response to hyperinsulinemia is reduced in overweight humans: a magnetoencephalographic study.

Obese men respond to cognitive but not to catabolic brain insulin signaling. Attenuation of insulin-evoked responses in brain networks controlling appetite and reward in insulin resistance: the cerebral basis for impaired control of food intake in metabolic syndrome?

Central nervous insulin resistance: a promising target in the treatment of metabolic and cognitive disorders? Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.

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Genetic Influences of Adiponectin on Insulin Resistance, Type 2 Diabetes, and Cardiovascular Disease.

Online ISSN X Print ISSN Books ShopDiabetes. C,D Protein lysates from 3T3-L1 preadipocytes Day 0 and adipocytes Day 7 after differentiation were pulled down by an anti-GRP75 antibody C and followed by detection of IP3R1 and VDAC1 using Western blot analysis D, right panel.

The MAMs formation was calculated by the ratio of interaction between GRP75 and IP3R1 and between GRP75 and VDAC1, respectively D, left panel.

To determine the importance of MAMs formation in the differentiation and function of adipocytes, we disrupted the MAMs structure by knocking down the proteins involved in MAMs formation.

Because IP3R1 and VDAC1 may have other exclusive roles in the function of ER and mitochondria, respectively, we decided to target GRP75, which is a linker between ER and mitochondria and its level was not changed during differentiation Figure 1.

We designed two different sequences of shRNA to knock down GRP75 in 3T3-L1 preadipocytes. Most importantly, the knockdown of GRP75 was sustained for 7 days after adipogenic differentiation. To determine the adipogenic differentiation capacity of 3T3-L1 with GRP75 KD, the adipocyte markers PPARγ2, aP2 known as FABP4 and adiponectin were examined.

The results revealed that the expression levels of these 3 genes were decreased after differentiation of 3T3-L1 preadipocytes with GRP75 KD Figure 3C. In addition, we stained lipid droplets of adipocytes by Oil red O. In consistency with gene markers Figure 3C , GRP75 deficiency led to the decreased formation of lipid droplets after 3T3-L1 differentiation Figure 3D.

These findings indicate that the disruption of MAMs structure by knock-down of GRP75 could impair the adipogenic differentiation of 3T3-L1 preadipocytes. FIGURE 3. Impairment of adipogenic differentiation of 3T3-L1 preadipocytes with GRP75 knockdown.

A The protein levels of GRP75 were determined in 3T3-L1 preadipocytes after delivering shRNA with scramble sequence CTL or two different sequences targeting to GRP75 KD 1 and KD 2. The quantification of protein bands was shown in the left panel. B,C The 3T3-L1 preadipocytes with CTL KD, GRP75 KD 1 and GRP75 KD 2 were induced to undergo adipogenic differentiation for 7 days.

D The 3T3-L1 preadipocytes with CTL KD, GRP75 KD 1 and GRP75 KD 2 were induced to undergo adipogenic differentiation for 7 days and were then stained by Oil red O. Next, we examined whether disruption of MAMs structure affects the mitochondrial function during adipogenic differentiation.

Surprisingly, adipocytes with GRP75 KD displayed upregulation of genes involved in mitochondrial biogenesis, such as PGC1α Figure 4A. However, the increase of mitochondrial biogenesis did not lead to the increase of mitochondrial function in adipocytes. The adipocytes with GPR75 KD expressed less MnSOD Figure 4C , which is a first-line antioxidant enzyme, leading to accumulation of intracellular reactive oxygen species ROS such as superoxide anions Figure 4D and hydrogen peroxide Figure 4E.

This may be due to the disruption of MAMs structure and mitochondrial dysfunction. FIGURE 4. Accumulation of ROS in differentiated 3T3-L1 adipocytes with GRP75 knockdown.

A—C The mRNA levels of mitochondrial biogenesis-related genes PGC1α, TFAM, NRF1 and NRF2 and antioxidant enzymes MnSOD and catalase were determined by RT-PCR in 3T3-L1 preadipocytes with CTL KD, GRP75 KD 1 and GRP75 KD 2 7 days after differentiation. To answer whether the disruption of MAMs and mitochondrial dysfunction lead to functional defects of adipocytes, we determined insulin sensitivity in adipocytes with or without GRP75 KD by monitoring the activation of insulin signaling pathway.

First, we observed that the mRNA expression levels of total AKT and IRS1 were upregulated at the basal state of adipocytes with GRP75 KD Figure 5A. Similarly, the protein level of total AKT was also increased Figures 5B,C. Since the phosphorylated AKT is the key to determine the activation of insulin signaling and glucose uptake Batista et al.

The results showed that, after insulin stimulation, AKT phosphorylation was dramatically induced in the control adipocytes CTL KD , but such induction was impaired in the adipocytes with GRP75 KD Figures 5B,D.

FIGURE 5. Insulin insensitivity of differentiated 3T3-L1 adipocytes with GRP75 knockdown. A The mRNA levels of genes involved in insulin signaling Akt2 and Irs1 were determined in 3T3-L1 preadipocytes with CTL KD, GRP75 KD 1 and GRP75 KD 2 after differentiation. B—D The 3T3-L1 preadipocytes with CTL KD and GRP75 KD 2 were induced to undergo adipogenic differentiation.

The 7-days differentiated white adipocytes were treated with nM of insulin and incubated for 30 min, and the cellular proteins were extracted to determine the levels of total AKT and phosphorylated AKT at serine p-AKT B.

The quantification of protein bands in total AKT of mature adipocytes C. Insulin sensitivity of adipocytes was determined by the ratio between p-AKT and total AKT D.

E Glucose uptake was determined by using 2-NBDG in differentiated CTL and GRP75 KD white adipocytes with or without insulin stimulation. To determine whether less activation of AKT leads to impairment of glucose uptake, we used 2-NBDG, a fluorescent glucose analog, to measure glucose uptake in adipocytes.

Although there is no difference in basal glucose uptake, GRP75 KD adipocytes displayed a decrease in insulin-stimulated glucose uptake Figure 5E , which is consistent with impaired AKT activation Figure 5D.

These findings suggest that GRP75 deficiency impairs the activation of insulin signaling pathway, which in turn causes insulin insensitivity and affects glucose utilization in mature adipocytes. For the regulation of energy metabolism, there are two types of adipose tissues in the human body, brown adipose tissue BAT and white adipose tissue WAT.

When compared with WAT, BAT contains more mitochondria and responds to the energy expenditure via thermogenesis. Thus, BAT displays a greater ability to modulate fatty acids and glucose homeostasis within the body Cannon and Nedergaard, ; Shamsi et al.

Since the mitochondrial function is more important for brown adipocytes than white adipocytes, we hypothesized that MAMs structure would play an essential role in thermogenesis of brown adipocytes.

To address this issue, we knocked down GRP75 to disrupt MAMs structure in brown preadipocytes and investigated the genes involved in fuel utilization and thermogenic function. After induction of differentiation of brown preadipocytes for 7 days, the decline of GRP75 expression Figure 6B was still maintained in the mature brown adipocytes.

Intriguingly, GRP75 deficiency led to the downregulation of glucose utilization GLUT1; Figure 6D and thermogenic program ADRB3 and UCP1; Figure 6E. In addition, decreased formation of lipid droplets revealed by Oil red O staining Figure 6F and decreased expression of fatty acid synthase FAS; Figure 6G indicate that fatty acid metabolism is impaired in brown adipocytes differentiated from GRP75 KD preadipocytes.

Similar to the findings in 3T3-L1 white adipocytes, a loss of GRP75 in brown adipocytes resulted in the upregulation of the PGC1α expression Figure 6H to compensate for the declined mitochondrial function. However, a decrease the expression of antioxidant enzymes such as MnSOD Figure 6I may cause a vicious cycle to increase intracellular ROS levels like white adipocytes.

FIGURE 6. Impaired thermogenic program of differentiated brown adipocytes with GRP75 knockdown. A The protein levels of GRP75 were determined in differentiated brown preadipocytes after delivering shRNA with scramble sequence CTL or the sequence 2 targeting to GRP75 KD.

The quantification of protein bands was shown in the right panel. B—E The brown preadipocytes with CTL KD or GRP75 KD were induced to undergo brown adipogenic differentiation. F Differentiated brown adipocytes with CTL and GRP75 KD were stained by Oil red O. G—I The mRNA expression levels of fatty acid synthase FAS; G and genes involved in the regulation of mitochondrial biogenesis PGC1α and TFAM; H and the antioxidant enzyme MnSOD; I were measured in CTL and GRP75 KD brown adipocytes 7 days after differentiation of preadipocytes.

J,K The mRNA levels of UCP1 and PGC1α J in differentiated brown adipocytes were measured after treatment with vehicle or cAMP for 4 h. The fold changes of the mRNA expression levels after cAMP stimulation in CTL and GRP75 KD brown adipocytes are presented K.

Mitochondrial biogenesis and UCP1 expression in brown adipocytes are further upregulated via β3-adrenergic receptor β3-AR signaling to promote thermogenic function during cold environment Shamsi et al. In addition to the basal status, we investigated the response of brown adipocytes to β3-AR signaling.

The differentiated brown adipocytes were treated with cAMP, a downstream second messenger of β3-AR signaling, for 4 h. UCP1 and PGC1α expression were significantly upregulated by 4. In contrast, the inductions in UCP1 and PGC1α level in response to β3-AR signaling were decreased to 2.

All the findings indicated that disruption of MAMs structure led to mitochondrial dysfunction and not only impaired insulin response of white adipocytes but also thermogenic function of brown adipocytes. This specical structure of MAMs is crucial for an accurate and efficient communication between the ER and mitochondria.

The MAMs have since been shown to be enriched in functionally diverse enzymes involved not only in lipid metabolism but also in glucose metabolism Piccini et al. In a previous study, we showed that Cisd2, located on MAMs Chen et al.

This suggests that MAMs are involved in maintaining both function and integrity of ER and mitochondria, which may play an important role in the regulation of glucose homeostasis and insulin sensitivity.

In the present study, we further demonstrated that the disruption of MAMs structure by knocking down the linker protein, GRP75, could impair the differentiation and function of 3T3-L1 adipocytes, especially in the insulin signaling pathway.

Similar to previous studies, decrease of mitochondrial function and overproduction of ROS in adipocytes with MAMs disruption may result in desensitized AKT activation and insulin insensitivity.

In addition, we found that the compensatory upregulation of mitochondrial biogenesis together with an imbalance in the expression levels of antioxidant enzymes triggered the deleterious vicious cycle of ROS accumulation. However, scavenging of ROS by treatment of adipocytes with an antioxidant N-acetyl cysteine or by overexpression of antioxidant enzymes e.

Furthermore, we did not observe the upregulation of GRP75 expression during adipogenic differentiation although the MAMs formation was increased. Further study is warranted to elucidate whether other proteins facilitate the interactions between GRP75 and VDAC or between GRP75 and IP3R.

Moreover, brown adipocytes also serve as an endocrine organ to regulate other tissues via secreted factors including proteins, lipids, metabolites, and exosomes Shamsi et al. In addition to 3T3-L1 white adipocytes, we have also demonstrated the contribution of MAMs structure in the thermogenesis of brown adipocytes.

In consistence with our results, it has been reported that the mitochondrial dysfunction and irregular formation of MAMs resulting from a defective proteasomal activity would damage the thermogenic function of BAT and led to obesity and glucose dyshomeostasis in mice Bartelt et al.

In addition, some proteins located in MAMs such as PERK Kato et al. In this study, we used GRP75, a structural protein required for the formation of MAMs, to strengthen the contribution of MAMs in the function of brown adipocytes. There are many proteins resident in the MAMs structure, some are responsible for the regulation of mitochondrial function and others are required for ER function.

In this study, we targeted the linker of the two organelles instead of the resident proteins in the ER or mitochondria. We also showed that the disruption of MAM structure impaired thermogenic function and fatty acid metabolism of brown adipocytes. However, further studies are required to unravel the mechanisms between MAM formation and brown adipocyte function.

In conclusion, our findings of this study suggest that the MAMs structure is crucial for the functions of ER and mitochondria and its disruption in adipocytes would lead to insulin insensitivity and T2D due to the overproduction and inefficient disposal of intracellular ROS.

C-HW and Y-HW have conceptualized, structured, and supervised this study. C-HW and Y-HW have written, formatted and edited the manuscript. C-HW, C-HW 2nd author , P-JH conducted the experiments and data analysis and prepared the figures and tables. This work was supported by grants from the Ministry of Science and Technology MOST of Taiwan Government MOSTB—, MOST —B—, MOST —B—, MOST —B— and MOST —B— and partly supported by intramural grants CCH-IST and CCH-MST from Changhua Christian Hospital.

was supported by a grant from MOST MOSTB—MY3 and intramural grants CMUYTY, CMUS and DMR— from China Medical University. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.

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Bukowiecki, A. Marette, J. Kearney, F. Pérusse, O. Tulp, Christian BenedictSwantje Brede insupin, Helgi Thermogeneesis. SchiöthHendrik Aids in digestive healthBernd TheromgenesisJan L-carnitine and exercise performanceManfred Hallschmid; Intranasal Insulin Enhances Postprandial Thermogenesis and Lowers Postprandial L-carnitine and exercise performance Insulin Levels Thermogenesos Healthy Men. Diabetes 1 January ; 60 1 : — Animal studies indicate a prominent role of brain insulin signaling in the regulation of peripheral energy metabolism. We determined the effect of intranasal insulin, which directly targets the brain, on glucose metabolism and energy expenditure in humans. In a double-blind, placebo-controlled, balanced within-subject comparison, 19 healthy normal-weight men 18—26 years old were intranasally administered IU human insulin after an overnight fast.