s2fieldsofstudy: Biology Chemistry

text:
SIRT-3 Modulation by Resveratrol Improves Mitochondrial Oxidative Phosphorylation in Diabetic Heart through Deacetylation of TFAM

Background and Purpose: Mitochondrial dysfunction remains the crucial cause for many heart diseases including diabetic cardiomyopathy (DCM). Sirtuin-3 (SIRT-3) is a protein deacetylase localized in the mitochondria and regulates mitochondrial function. Being a noteworthy mitochondrial protein deacetylase enzyme, the role of SIRT-3 in DCM is yet to be explored. Experimental Approach: Diabetes mellitus (Type-I, T1DM) was induced using streptozotocin (STZ, 50 mg/kg) in male Sprague Dawley (SD) rats. Rats with >200 mg/dL blood glucose levels were then divided randomly into two groups, DIA and DIA + RESV, where vehicle and resveratrol (25 mg/kg/day) were administered orally in both groups, respectively. Cardiac oxidative stress, fibrosis, and mitochondrial parameters were evaluated. H9c2 cells were transfected with SIRT-3 siRNA and shRNA, and ORF plasmid for silencing and overexpression, respectively. Key Results: After eight weeks, diabetic rat heart showed reduced cardiac cell size, increased oxidative stress and reduction of the activities of enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS). There was reduced expression and activity of SIRT-3 and mitochondrial transcription factor (TFAM) in diabetic heart. Reduced SIRT-3 expression is also correlated with increased acetylation, decreased mitochondrial DNA (mtDNA) binding activity of TFAM, and reduced transcription of mitochondrial DNA encoded genes. Administration of resveratrol prevented the decrease in SIRT-3 and TFAM activity, which was corresponding to the reduced acetylation status of TFAM. Silencing SIRT-3 using siRNA in H9C2 cells showed increased acetylation of TFAM. Conclusion and Implications: Together our data shows that resveratrol activates SIRT-3, regulates the acetylation status of TFAM and preserves the mitochondrial function along with cellular size in diabetic rat heart.

Introduction
Sirtuins are the class III histone deacetylase enzymes (HDAC) which are evolutionary conserved and possess NAD + dependent deacetylase activity. Among the seven different sirtuins, SIRT-3, SIRT-4, and SIRT-5 are mostly localized in the mitochondria. Growing evidence suggest that SIRT-3 has a crucial role in regulating metabolic homeostasis through multi-tissue coupling [1]. Reports confirmed SIRT-3 as a major protein deacetylase in mitochondria and thus explain its importance in energy metabolism. SIRT-3 is involved in regulating cellular redox status, mitochondrial energetics, biogenesis, dynamics, and apoptosis [2][3][4][5]. Cardiac mitochondrial proteins are three times more acetylated than other tissue proteins [6]. Cardiac complications remain the major cause of mortality in diabetes and explain the urgent need for mitigation. Looking at the basics of cardiac metabolic remodeling, the robust nature of cardiac cells to shift the substrate utilization based on demand is regulated by protein acetylation or deacetylation [7]. Cardiac complications in diabetes showed altered mitochondrial energetics, dysregulation of electron transport chain (ETC) complex, electron leak, and superoxide generation [8].
Mitochondrial DNA transcription encodes thirteen proteins for ETC complex assembly is regulated by TFAM, a mitochondrial transcription factor. All these thirteen proteins are essential to form a functional assembly with the nuclear encoded proteins of ETC complex. Thus, it is not surprising that altered transcriptional activity of mtDNA affects the capacity of OXPHOS. Most of the proteins/enzymes enrolled in the metabolic pathways are regulated by acetylation. Therefore, we hypothesized that altered acetylation balance in the diabetic heart may also affect mtDNA transcription.
Targeting mitochondrial energy metabolism pathways for the mitigation of disease pathology is the focus of today's drug discovery. Nowadays, sirtuins are considered as emerging druggable targets for multiple diseases including diabetes [9]. Hebert and colleagues reported that the absence of SIRT-3, a mitochondrial sirtuin, increased the acetylation modification of mitochondrial protein almost two-fold [10]. In addition, it has been shown that SIRT3 enhances mitochondrial respiration rate and ATP production by coordinated activation of mitochondrial metabolic pathways and ROS detoxification [11,12]. Recent study by Zhang and colleagues showed that resveratrol glucoside activates SIRT-3 and attenuates DCM by upregulating autophagy and improving mitochondrial function [13].
Deliberating the role of SIRT-3 in mitochondrial energetics and mitochondrial dysfunction in diabetes, we thought to find out the missing link between SITR3 and mitochondrial function in the diabetic heart. We hypothesized that SIRT-3 has a crucial role in regulating the mitochondrial function in diabetic heart through regulating TFAM and thus targeting SIRT3 could be a useful approach to overcome cardiac complications in diabetes. In the present study, we have shown that resveratrol, which directly or indirectly can activate SIRT-3 [3,14,15] reduces acetylation of TFAM and rescued the mitochondrial dysfunction in the diabetic heart.

Animals
All the experiments were performed in accordance with relevant guidelines and regulations of the Institutional Animal Ethical Committee (IAEC) and Indian Institute of Chemical Technology (IICT), Hyderabad which is consistent with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, guidelines. Male SD rats weighing 200 to 220 g and 8-10 weeks old were purchased from the National Institute of Nutrition (NIN), Hyderabad, India. Diabetes was induced by single intraperitoneal (i.p.) injection of 50 mg/kg STZ prepared in citrate buffer, pH 4.5; control rats received a similar volume of citrate buffer, pH 4.5 (i.p.). Rats were monitored for the development of hyperglycemia for the next seven days. Rats with stable increased blood glucose levels of more than 200 mg/dL were divided randomly into two groups: DIA group and DIA + RESV group (n = 12/group). Resveratrol was orally administered at a dose of 25 mg/kg/day, as per the previous study [16]. After eight weeks, rats were sacrificed and heart tissue was collected and stored in appropriate condition for downstream analysis.

Cell Culture
Rat cardiomyoblast cells (H9C2) were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM). The culture was supplemented with 10% fetal bovine serum and 100 µg/mL penicillin/streptomycin. After reaching 50 to 60% confluence, cells were treated with the respective siRNA for SIRT-1 and SIRT-3 knockdown, and ORF plasmid for SIRT3 overexpression (Dharmacon, USA) using dharmafect transfection reagent (Dharmacon, Lafayette, CO, USA) as per the manufacturer's protocol. After 48 h of transfection, cells were washed and lysed for either protein or mRNA analysis. Isolated protein or mRNA was used for downstream analysis. Similarly, the stable SIRT-3 knockdown H9C2 cell line was developed with lentiviral vector shRNA (Dharmacon, USA) and used for gene expression analysis.

Serum Biochemical Analysis
Serum samples collected from rats were analyzed with auto blood analyzer (Siemens, Qulin, MO, USA) for the measurement of triglyceride, uric acid, HDL, creatinine, cholesterol, and SGOT. Measurement of serum insulin and glycated hemoglobin (HbA1c) was carried out using Mercodias' rat insulin ELISA kit and Biosystems' glycated hemoglobin detection kit, respectively.

Measurement of Cardiac Cell Size and Histopathological Examination
Heart weight to tail length ratio was evaluated at the end of the study to demonstrate cardiac phenotypic changes. Heart tissue samples fixed in neutral buffer formalin (10%) were paraffin-embedded for histopathological analysis. Sections (5-µm) were stained with hematoxylin and eosin and masons trichrome for further analysis. Cardiac cell size was measured using Image J as described previously [17,18].

Isolation of Mitochondria
Mitochondria were isolated from equal weight of heart tissues from all groups using mitochondria isolation kit (Pierce, Thermo scientific, cat No: 89801). Isolated mitochondria were lysed and stored for downstream application.

Preparation of Heart Tissue Homogenate
Heart tissues were homogenized with ten times volume of 0.05 M phosphate buffer (pH 7.4) and centrifuged at 15,000× g for 30 min at 4 • C. Supernatant was stored at −80 • C for downstream analysis.

Electron Transport Chain Complex Assembly Activity
Enzyme activity of citrate synthase and β-hydroxyl acyl-CoA dehydrogenase was measured according to the protocol described before [23]. The specific enzymatic activity of mitochondrial ETC complex I (NADH-ubiquinone oxidoreductase), complex II (succinate-ubiquinone oxidoreductase), and complex IV (cytochrome c oxidase) was measured as described previously [24]. ATP levels were measured using luminescence based assay (Life Technology Ltd. Waltham, MA, USA).

Gene Expression Analysis
Isolation of RNA from the heart tissue (n = 5) was carried out using RNAeasy kit (Quiagen, Germantown, MD, USA). Isolated RNA was quantified using Nanodrop (ThermoFischer, Waltham, MA, USA) followed by DNA digestion (Quiagen Kit). The purified RNA was then reverse transcribed to cDNA using First Strand cDNA synthesis kit, Quiagen, USA. Synthesized cDNA was diluted for downstream analysis. Primers used for the study were designed using Primer-3 tool and standardized as per the routine protocol. For real-time PCR reaction, SYBER green chemistry was used as per manufacturer's protocol (Applied Biosystems, Waltham, MA, USA) using Step One Plus instrument (Applied Biosystems, USA). Mathematical calculation for fold change in gene expression was performed as described before [25]. RPL32 was used as reference gene [25].

Immunoblotting
Protein isolation was carried out using tissue protein extraction reagent (T-PER, Pierce, USA). Following centrifugation, resultant supernatant was collected and quantified using BCA kit, Pierce, USA. An equal amount of protein was resolved using 10 to 12% SDS-polyacrylamide gel. The resolved gel was then transferred to a polyvinyldine difluoride membrane (Thermo Scientific, Waltham, MA, USA). Blocking of the membrane was performed using 3-5% blotto, nonfat dry milk (Santa Cruz, Dallas, TX, USA) in TBS containing 0.1% Tween-20, at room temperature for 1 h. The membrane was then incubated with primary antibody at 4 • C overnight. Following three subsequent washings, the membrane was incubated with secondary anti-Rabbit (Cell signaling, Danvers, MA, USA.

Immunoprecipitation
Immunoprecipitation (IP) was carried out using Dynabeads protein G IP kit (Life Technologies, Cat No: 10007D) as per the manufacturer's instructions. An equal amount of eluted protein was resolved on the gel for assessing the acetylation status TFAM using anti-acetyl lysine antibody.

Electrophoretic Mobility Shift Assay
The DNA-binding activity of TFAM was assessed by electrophoretic mobility shift assay (EMSA) using the light-shift chemiluminescent EMSA kit (Pierce, Waltham, MA, USA). Complementary oligonucleotide probes containing the D-loop region of mtDNA were designed as binding motifs (TFAM-, 5-TTTCCTCCTAACTAAACCCTCTTTAC-3) and were end-labeled with biotin. The membrane was incubated with chemiluminescent substrate and developed using a CCD camera ChemiXRS, a chemiluminescent instrument (Bio-Rad, Hercules, CA, USA). Unlabeled oligos were added to the reaction at five-to ten-fold excess to evaluate the specificity.

Statistical Analysis
All values are expressed as the mean ± standard error (SE). One-way analysis of variance (ANOVA) test followed by Bonferoni's correction was carried out to test any differences between the mean values of all groups. Significance in group differences was assumed if p < 0.05.

Serum Biochemistry
Rats injected with STZ showed increased blood glucose levels after eight weeks compared to CON group (Table 1). In addition, there was an increased blood HbA1c levels in DIA group rats ( Table 1). Administration of resveratrol decreased the levels of blood glucose and glycated hemoglobin compared to DIA group rats. Moreover, there were reduced levels of serum insulin in DIA group rats compared to Cells 2018, 7, 235 5 of 13 CON group rats, which increased after resveratrol administration (Table 1). Increased levels of serum SGOT in DIA group rats was also reduced after resveratrol administration (Table 1). Increased levels of serum triglyceride, uric acid, and creatinine along with reduced levels of cholesterol and HDL was observed in DIA group. Administration of resveratrol normalized all these serum metabolites alteration (Table 1). HOMA analysis showed reduced beta cell efficiency which was improved by resveratrol administration (Table 1).

Cardiac Atrophy and Fibrosis
We have previously reported that the reduced ratio of heart weight to tail length in the DIA group was increased with resveratrol administration [14]. Histopathological examination revealed reduced cell size along with significant induction of cardiac fibrosis. Increased mRNA expression of β-MHC in DIA group was observed. Administration of resveratrol significantly ameliorated all of these cardiac alterations. However, there was a significant difference in mRNA expression of β-MHC between CON and resveratrol treated group (Figure 1).  (Table 1). Increased levels of serum triglyceride, uric acid, and creatinine along with reduced levels of cholesterol and HDL was observed in DIA group. Administration of resveratrol normalized all these serum metabolites alteration (Table 1). HOMA analysis showed reduced beta cell efficiency which was improved by resveratrol administration (Table 1).

Cardiac Atrophy and Fibrosis
We have previously reported that the reduced ratio of heart weight to tail length in the DIA group was increased with resveratrol administration [14]. Histopathological examination revealed reduced cell size along with significant induction of cardiac fibrosis. Increased mRNA expression of β-MHC in DIA group was observed. Administration of resveratrol significantly ameliorated all of these cardiac alterations. However, there was a significant difference in mRNA expression of β-MHC between CON and resveratrol treated group (Figure 1).

Mitochondrial Citrate Synthase, β-Hydroxy Acyl Co-A Dehydrogenase Activity, and Mitochondrial Number
There was reduced activity of citrate synthase in DIA group rat heart compared to CON group rat heart (Figure 2A). In addition, there was reduced activity of β-hydroxy acyl CoA dehydrogenase in DIA group rats. Administration of resveratrol rescued the activity of citrate synthase and β-hydroxy acyl CoA dehydrogenase (Figure 2A,B). Analysis of cardiac mitochondrial content showed reduced number of mitochondria in DIA group compared to CON group. Administration of resveratrol prevented the decline in mitochondrial content compared to DIA group ( Figure 2H). were observed for histopathology examination and twenty cells per image were analyzed for cell size measurement.

Mitochondrial Citrate Synthase, β-Hydroxy Acyl Co-A Dehydrogenase Activity, and Mitochondrial Number
There was reduced activity of citrate synthase in DIA group rat heart compared to CON group rat heart (Figure 2A). In addition, there was reduced activity of β-hydroxy acyl CoA dehydrogenase in DIA group rats. Administration of resveratrol rescued the activity of citrate synthase and βhydroxy acyl CoA dehydrogenase (Figure 2A,B). Analysis of cardiac mitochondrial content showed reduced number of mitochondria in DIA group compared to CON group. Administration of resveratrol prevented the decline in mitochondrial content compared to DIA group ( Figure 2H).

Activity of ETC Complex Assembly and ATP Generation
Diabetic heart showed reduced activity of complexes I, II, and IV along with reduced levels of ATP generation compared to the CON group ( Figure 2). Administration of resveratrol prevented decline in the activity of complexes I, II, IV, V, and ATP content compared to the DIA group. In addition, ATP level was significantly higher in the resveratrol-treated group as compared to the CON group ( Figure 2).

Reactive Oxygen Species Levels and Antioxidant Defense
Increased levels of ROS and TBARS in DIA group compared to CON group were observed ( Table 2). In addition, there was reduced activity of SOD, catalase, and DPPH along with reduced levels of glutathione (GSH) in the DIA group compared to the CON group ( Table 2). Administration of resveratrol prevented the increase in ROS levels as well as the decline in SOD, catalase, DPPH activity, and levels of GSH (Table 2).

Activity of ETC Complex Assembly and ATP Generation
Diabetic heart showed reduced activity of complexes I, II, and IV along with reduced levels of ATP generation compared to the CON group ( Figure 2). Administration of resveratrol prevented decline in the activity of complexes I, II, IV, V, and ATP content compared to the DIA group. In addition, ATP level was significantly higher in the resveratrol-treated group as compared to the CON group ( Figure 2).

Reactive Oxygen Species Levels and Antioxidant Defense
Increased levels of ROS and TBARS in DIA group compared to CON group were observed ( Table 2). In addition, there was reduced activity of SOD, catalase, and DPPH along with reduced levels of glutathione (GSH) in the DIA group compared to the CON group ( Table 2). Administration of resveratrol prevented the increase in ROS levels as well as the decline in SOD, catalase, DPPH activity, and levels of GSH (Table 2).

mRNA Expression of Mitochondrial Encoded Genes
Reduced expression of all thirteen mitochondrial encoded genes in DIA group hearts compared to CON group hearts was observed. Administration of resveratrol restored the expression of all these genes except MT-CYB. In addition, mitochondrial CYB and CO-1 gene expression was significantly low in resveratrol treated group as compared to CON (Figure 3).

Expression and Activity of TFAM with Increased Acetylation Status
Reduced expression and activity of TFAM in diabetic heart compared to CON group heart was observed ( Figure 4A,B). Resveratrol administration improved the expression and activity of TFAM compared to DIA group heart. Transcript level of TFAM mRNA was increased with SIRT-3 overexpression in H9C2 cells (Supplementary Figure S1C). In addition, there was increased acetylation status of TFAM in DIA group heart compared to CON group heart. However, this increased acetylated TFAM levels was reduced with resveratrol administration ( Figure 4C). Table 2.
Cardiac antioxidant and oxidative stress markers in diabetic rats and effect of resveratrol administration.

mRNA Expression of Mitochondrial Encoded Genes
Reduced expression of all thirteen mitochondrial encoded genes in DIA group hearts compared to CON group hearts was observed. Administration of resveratrol restored the expression of all these genes except MT-CYB. In addition, mitochondrial CYB and CO-1 gene expression was significantly low in resveratrol treated group as compared to CON (Figure 3).

Expression and Activity of TFAM with Increased Acetylation Status
Reduced expression and activity of TFAM in diabetic heart compared to CON group heart was observed ( Figure 4A, B). Resveratrol administration improved the expression and activity of TFAM compared to DIA group heart. Transcript level of TFAM mRNA was increased with SIRT-3 overexpression in H9C2 cells (Supplementary Figure S1C). In addition, there was increased acetylation status of TFAM in DIA group heart compared to CON group heart. However, this increased acetylated TFAM levels was reduced with resveratrol administration ( Figure 4C).

Expression and Activity of SIRT-1 and SIRT-3
The reduced expression and activity of SIRT-1 and SIRT-3 in type-I diabetic heart compared to the CON group heart has already been reported by us. Administration of resveratrol prevented the decline in expression and activity of both SIRT-1 and SIRT-3 in diabetic heart. However, SIRT-1 and SIRT-3 protein expression was significantly higher in the resveratrol treated group compared to the

Expression and Activity of SIRT-1 and SIRT-3
The reduced expression and activity of SIRT-1 and SIRT-3 in type-I diabetic heart compared to the CON group heart has already been reported by us. Administration of resveratrol prevented the decline in expression and activity of both SIRT-1 and SIRT-3 in diabetic heart. However, SIRT-1 and SIRT-3 protein expression was significantly higher in the resveratrol treated group compared to the CON group ( Figure 5) [14].

TFAM Acetylation Is Regulated by SIRT-3
There was no change in the expression of TFAM and NRF-1 in SIRT-1 and SIRT-3 silenced H9C2 cells ( Figure 6A). In addition, we did not observe any change in the mRNA transcript level of NRF-1 and TFAM in SIRT-3 (shRNA) silenced H9C2 cells (Supplementary Figure S1D, E). However, there was reduced activity of TFAM in SIRT-3 silenced cell, but not in SIRT-1 silenced cells ( Figure 6B). Immunoprecipitation followed by immunoblotting with anti-acetyl lysine of TFAM showed increased acetylation status of TFAM in SIRT-3 silenced cells ( Figure 6C). Thus, our data indicates that lack of SIRT-3 reduces TFAM activity via hyperacetylation.

TFAM Acetylation Is Regulated by SIRT-3
There was no change in the expression of TFAM and NRF-1 in SIRT-1 and SIRT-3 silenced H9C2 cells ( Figure 6A). In addition, we did not observe any change in the mRNA transcript level of NRF-1 and TFAM in SIRT-3 (shRNA) silenced H9C2 cells (Supplementary Figure S1D,E). However, there was reduced activity of TFAM in SIRT-3 silenced cell, but not in SIRT-1 silenced cells ( Figure 6B). Immunoprecipitation followed by immunoblotting with anti-acetyl lysine of TFAM showed increased acetylation status of TFAM in SIRT-3 silenced cells ( Figure 6C). Thus, our data indicates that lack of SIRT-3 reduces TFAM activity via hyperacetylation.

Discussion
Diabetes is a complex metabolic disease that leads to cardiac complications as the disease progresses. Hyperglycemia induces multiple molecular and phenotypic perturbations, leading to the development of cardiac dysfunction [26]. Although the incidence of T1DM is only 5 to 10% in the diabetic populace, its severity is comparatively higher than type II diabetes mellitus. Hyperglycemia remains the common culprit in both types of diabetes and contributes most to the pathology of cardiac complications. In the present study, we have selected the T1DM rat model to study the effect of hyperglycemia and lack of insulin on cardiac mitochondrial energetics and transcription.
Our study showed increased blood glucose and reduced serum insulin levels in STZ injected rats. The hyperglycemic condition sustained throughout the study duration, as indicated by increased HbA1c levels. In addition, there was increased level of serum triglyceride, serum creatinine, uric acid, and SGOT in diabetic rats along with reduced levels of serum HDL and total cholesterol. All these altered metabolite profiles were normalized with resveratrol administration. Thus all the above serum metabolite changes indicate the development of T1DM, which is a well-known phenomenon in this model [27].
Cardiac complications in this T1DM model revealed that there was reduced heart weight to tail length ratio, called cardiac atrophy. This was supported by histopathological examination and a reduction in cardiac cell size. We observed the incidence of cardiac atrophy in T1DM, contrast with T2DM, where cardiac hypertrophy is the predominant phenotype [28]. Previous reports also showed similar outcomes and indicate the effect of reduced insulin and starved condition [29][30][31]. Administration of resveratrol prevented reduction in the cell size as well as heart weight to tail length ratio. Moreover, the reduced cardiac cell size may be one of the reasons of increased apoptosis in cardiomyocytes as described before [30]. These dead cells are then replaced by fibrosis as a natural repair mechanism, which we have observed in diabetic rat heart in our present study.

Discussion
Diabetes is a complex metabolic disease that leads to cardiac complications as the disease progresses. Hyperglycemia induces multiple molecular and phenotypic perturbations, leading to the development of cardiac dysfunction [26]. Although the incidence of T1DM is only 5 to 10% in the diabetic populace, its severity is comparatively higher than type II diabetes mellitus. Hyperglycemia remains the common culprit in both types of diabetes and contributes most to the pathology of cardiac complications. In the present study, we have selected the T1DM rat model to study the effect of hyperglycemia and lack of insulin on cardiac mitochondrial energetics and transcription.
Our study showed increased blood glucose and reduced serum insulin levels in STZ injected rats. The hyperglycemic condition sustained throughout the study duration, as indicated by increased HbA1c levels. In addition, there was increased level of serum triglyceride, serum creatinine, uric acid, and SGOT in diabetic rats along with reduced levels of serum HDL and total cholesterol. All these altered metabolite profiles were normalized with resveratrol administration. Thus all the above serum metabolite changes indicate the development of T1DM, which is a well-known phenomenon in this model [27].
Cardiac complications in this T1DM model revealed that there was reduced heart weight to tail length ratio, called cardiac atrophy. This was supported by histopathological examination and a reduction in cardiac cell size. We observed the incidence of cardiac atrophy in T1DM, contrast with T2DM, where cardiac hypertrophy is the predominant phenotype [28]. Previous reports also showed similar outcomes and indicate the effect of reduced insulin and starved condition [29][30][31]. Administration of resveratrol prevented reduction in the cell size as well as heart weight to tail length ratio. Moreover, the reduced cardiac cell size may be one of the reasons of increased apoptosis in cardiomyocytes as described before [30]. These dead cells are then replaced by fibrosis as a natural repair mechanism, which we have observed in diabetic rat heart in our present study.
We have observed the reduced mitochondrial activity of citrate synthase and β-hydroxyacyl coA dehydrogenase in diabetic rat heart. In addition, there was reduced activity of ETC complex assembly and ATP levels. All these outcomes may be associated with reduced cardiac size because of deficient mitochondrial energetics. Along with these changes, we have also observed reduced cardiac mitochondrial content in diabetic rats. Administration of resveratrol ameliorated these reduced enzyme activities, along with increasing the levels of ATP and mitochondrial content. Data showed that resveratrol improved cardiac phenotype with increased mitochondrial efficiency and content. Increased SIRT3 expression and activity in the diabetic heart after resveratrol administration might be responsible for improved mitochondrial ETC activity. Although, we have not explored the exact mechanism of SIRT-3 activation by resveratrol in this study, previous work showed the involvement of estrogen-related receptor alpha (ERRα) for SIRT-3 expression [32,33]. Apart from regulating mitochondrial energy metabolism, SIRT-3 plays a critical role in attenuating cardiac fibrosis. Chen et al. recently showed that activation of SIRT-3 by resveratrol improved cardiac function and attenuated cardiac fibrosis by inhibiting TGF β/Smad pathway [15]. We also observed increased fibrosis in diabetic rat heart in our present study. Administration of resveratrol reduced cardiac fibrosis in diabetic rat heart. SIRT-3 also plays a critical role in regulating autophagy during diabetes cardiomyopathy. Previously, it has been shown that resveratrol promotes autophagy by upregulating SIRT-3 expression and phosphorylating AMP-activated protein kinase (AMPK) [34]. However, in the present study, we are more focused on understanding the regulation of cellular metabolism by SIRT-3 in mitochondria.
We evaluated whether the improvement of mitochondrial efficiency through resveratrol is due to enhancement of mitochondrial biogenesis. TFAM, a mitochondrial transcription factor, is involved in the transcriptional control of mtDNA, whereas NRF-1 is involved in the control of nuclear DNA transcription for the mitochondrial proteins. We have observed reduced expression and activity of TFAM with no change in NRF-1 (data not shown) in diabetic heart. This suggests that the nuclear trigger for mitochondrial biogenesis is not affected in this diabetic heart and the problem persists mostly at the mitochondrial levels. As TFAM is the only mitochondrial transcription factor involved in transcription of mtDNA that encodes proteins for ETC complex, reduced activity of the ETC complex and its activity is expected in diabetic heart. We have also tried to elucidate if the altered TFAM activity is due to reduced expression and activity of SIRT-1 and SIRT-3 in diabetic heart as observed in the present study and previously [14]. SIRT-1 is involved in deacetylating PGC-1α, which regulates the action of NRF-1 as reported earlier [35,36]; whereas, SIRT-3, a major mitochondrial deacetylase, controls mitochondrial health [37]. To find out the underlying mechanism of the regulation of TFAM activity, we have silenced SIRT-1 and SIRT-3 in H9C2 cells. We found no change in the expression of TFAM and NRF-1. However, there was reduced activity of TFAM in SIRT-3 silenced cells but not in SIRT-1 silenced cells. Next, we demonstrated whether SIRT-3 controls the acetylation status of TFAM and its activity. We found that silencing SIRT-3 increases the acetylation of TFAM and reduces its DNA binding activity, which was not observed in SIRT-1 silenced cells. This reinforces that SIRT-3 is crucial in regulating the acetylation status of TFAM and also its activity. As observed in in vitro cell line, we have also observed the acetylation status of TFAM in diabetic rat heart in vivo. Reduced activity of SIRT-3 in diabetic rat heart was associated with increased acetylation of TFAM, which leads to reduced activity. Administration of resveratrol prevented the decline in SIRT-3 activity and TFAM acetylation as well as its activity in diabetic rat heart.

Conclusions
In conclusion, we have observed that reduced SIRT-3 activity in diabetic heart is associated with increased acetylation and reduced activity of TFAM, which corresponds to reduced activity of ETC complex assembly and ATP levels and decreased cardiac size. Administration of resveratrol prevented all these cardiac changes and improves cardiac health. Our data indicates the potential role of SIRT-3 in Type-I diabetic heart providing an opportunity for future therapeutic intervention.


extfieldsofstudy: Medicine Chemistry

text:
Macropinosomes as units of signal transduction

Macropinosome formation occurs as a localized sequence of biochemical activities and associated morphological changes, which may be considered a form of signal transduction leading to the construction of an organelle. Macropinocytosis may also convey information about the availability of extracellular nutrients to intracellular regulators of metabolism. Consistent with this idea, activation of the metabolic regulator mechanistic target of rapamycin complex-1 (mTORC1) in response to acute stimulation by growth factors and extracellular amino acids requires internalization of amino acids by macropinocytosis. This suggests that macropinocytosis is necessary for mTORC1-dependent growth of metazoan cells, both as a route for delivery of amino acids to sensors associated with lysosomes and as a platform for growth factor-dependent signalling to mTORC1 via phosphatidylinositol 3-kinase (PI3K) and the Akt pathway. Because the biochemical signals required for the construction of macropinosomes are also required for cell growth, and inhibition of macropinocytosis inhibits growth factor signalling to mTORC1, we propose that signalling by growth factor receptors is organized into stochastic, structure-dependent cascades of chemical reactions that both build a macropinosome and stimulate mTORC1. More generally, as discrete units of signal transduction, macropinosomes may be subject to feedback regulation by metabolism and cell dimensions. This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’.

Introduction
Macropinosomes are transient endocytic organelles which form spontaneously or in response to chemical or physical stimulation. Although the details of macropinosome formation vary among different cell types [1], the process always involves cell surface protrusions that enclose extracellular fluids into plasma membrane-derived intracellular vesicles, which may range in diameter from 0.2 mm to larger than 8 mm (macropinosomes are distinguished from micropinosomes, which are smaller than 0.2 mm, the limit of resolution for light microscopy). In macrophages and fibroblasts, macropinosomes originate from actin-rich, cell surface ruffles that reorganize into cups or circular dorsal ruffles, then close at their distal margins and separate into the cytoplasm from the plasma membrane as vacuoles. The profiles of these nascent macropinosomes are often irregularly shaped, but mature within seconds into rounded, swollen-looking vacuoles. Rounded macropinosomes frequently fuse with other macropinosomes, migrate centripetally from the cell periphery, and shrink in size (figure 1a; electronic supplementary material, Video S1). In macrophages, macropinosomes mature by acquiring markers of early endosomes then late endosomes before fusing with lysosomes, all within 15 min of their formation [3]. They may form intravacuolar vesicle inclusions reminiscent of multivesicular bodies [4]. Fusion between macropinosomes and lysosomes occurs by direct and complete fusion, or by transient association and dissociation of macropinosomes and lysosomes, a process called 'pyranhalysis' [5] or 'kissand-run' [6]. Such transient interactions can lead to molecular size-dependent, differential delivery of solute contents between macropinosomes and lysosomes (figure 2; electronic supplementary material, Video S2) [6,7]. In some cells, macropinosomes recycle directly to the cell surface without fusing with lysosomes [8].
Compared to smaller clathrin-coated vesicles (0.15 mm diameter), macropinosomes internalize relatively large quantities of plasma membrane, water and solutes [9]. Cells can maintain high rates of macropinocytosis for hours by returning membrane to the cell surface via small recycling vesicles with accompanying water flux across membranes and out of the cell [10]. Internalized solutes concentrate into lysosomes, where they may accumulate or be degraded by acid hydrolases. Thus, because of their large size and mechanism of maturation, macropinosomes can be considered as mechanical pumps that move water through cytoplasm and efficiently deliver extracellular solutes into lysosomes [7]. This efficient delivery of extracellular solutes into lysosomes may explain  how, in actively macropinocytic cells, cell growth can be supported by the ingestion of extracellular proteins [11][12][13][14].
In conventional models of signal transduction, an extracellular stimulus initiates changes in intracellular chemistry, which lead to alterations of cell behaviour or metabolism. Typically, ligand binding to cell surface receptors initiates chemical reactions that modify phospholipid chemistry and protein function, and those modifications reach intracellular targets by diffusion or membrane traffic. The cellular response may be localized to a region of the surface, as, for example, during phagocytosis; it may occur throughout the cell, as in metabolic regulation; or it may be graded along the length of the cell, as in chemotactic migration [15]. Intracellular signals can also originate independently of cell surface receptors, leading to self-organized patterns of cellular motility responses [15,16].
Macropinosomes can form in response to receptor-mediated signals or as self-organized structures formed independently of external stimuli [17,18]. Whether macropinocytosis is spontaneous or induced, the construction of the organelle requires a sequence of intracellular signals that organize the sequence of different movements: actin polymerization for ruffle protrusion followed by contractile activities that close the organelle into the cell. The signals associated with macropinocytosis may be strictly limited to organizing the construction and disposal of this transient organelle. However, they may also influence cell function by conveying additional information. For example, as a mechanism for internalizing large portions of plasma membrane, macropinocytosis could regulate levels of cell surface receptors or transport proteins. Accordingly, clathrinindependent endocytosis, of which macropinocytosis is a subset, can internalize plasma membrane proteins that are not cleared by concentration into clathrin-coated vesicles [19].
Here we consider the roles for signalling molecules in the formation and maturation of macropinosomes and the evidence that macropinosomes convey signals that modulate cell metabolism and growth. We consider two kinds of signal: (i) those chemical activities which organize membranes and the cytoskeleton for macropinosome formation and trafficking and (ii) activities associated with macropinocytosis which have separate functions related to metabolism. We propose that macropinosomes serve as discrete, independent units of signalling for cell growth, whose magnitude may be modulated by feedback stimulation or inhibition.

Localized signals essential to macropinosome formation and maturation
Macropinosome formation exploits biochemical pathways associated with many kinds of receptor, including growth factor receptors, chemoattractant receptors and Fc receptors [18,20]. Upon binding their cognate ligands, these receptors recruit from cytoplasm various adapter proteins and enzymes, including tyrosine kinases, phospholipase C-g1 (PLCg1) and isoforms of class I phosphatidylinositol 3-kinase (PI3K), which lead to various cellular responses. PI3K synthesizes phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) from phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P 2 ). In non-transformed cells, plasma membrane concentrations of PIP 3 are maintained low by the activity of the 3 0 -phosphatase PTEN [21] (Many kinds of cancer cell have elevated levels of PIP 3 due to activating mutations of PI3K or to inactivating mutations of PTEN [22].) Elevated concentrations of PIP 3 in membranes activate proteins with PIP 3 -binding domains, including Akt, PLCg1, 3 0 -phosphoinositide-dependent protein kinase-1 (PDK1) [23], and guanine-nucleotide exchange factors (GEFs) that activate the small GTPases Rac1 and Ras [24,25]. These signalling proteins figure prominently in signalling associated with plasma membrane receptors. PI3K is required for most forms of macropinocytosis. In some cells, inhibition of PI3K inhibits ruffling, likely through inhibition of PIP 3 -activated Rac1 GEFs [26]. In most cells, however, general PI3K inhibitors do not reduce ruffling in response to the growth factors, but rather inhibit a contractile activity that closes macropinocytic cups into macropinosomes [27,28]. Fluorescent reporters of PIP 3 expressed in living cells reveal PIP 3 -rich membrane within macropinocytic cups [2,29] (figure 1b). The increased concentration of PIP 3 in cups is transient and precedes cup closure, consistent with a role for a PI3K-dependent activity in the closure process [2].
Other activities associated with receptor signalling are necessary for macropinosome formation. Rac1, a GTPase that regulates the actin cytoskeleton, and its effectors p21-activated kinase-1 (Pak1) and Ctb1/BARS are required for macropinocytosis [30,31]. Rac1 is transiently activated in macropinocytic cups, coincident with the PIP 3 spike [2]. Rac1 supports ruffling and cup formation, but must be converted to its inactive GDPbound form for cup closure to occur [32]. PLCg1, which hydrolyses PI(4,5)P 2 to inositol trisphosphate and diacylglycerol (DAG), is required for macropinosome formation [33,34]. DAG activates protein kinase C (PKC), which directs cup closure by an unknown mechanism. PLCg contains a PIP 3 -binding domain required for membrane binding and activity [35]; and PLCg1 may be activated by the elevated concentrations of PIP 3 in cups. Macropinocytosis occurs independently of class I PI3K in macrophages stimulated by phorbol myristate acetate (PMA) [34], which is a DAG mimetic. The PI3K-independence of PMA-stimulated macropinocytosis suggests that the DAG-dependent activities of macropinosome closure function downstream of PI3K in the signalling pathway. This sequence is supported by fluorescence microscopic studies localizing both PIP 3 (Btk-PH) and DAG (C1d-PH) in single cells [34]. PMA-stimulated macropinocytosis is sensitive to PI3K inhibitors with broader specificity, which suggests a role for other classes of PI3K acting downstream of DAG and PKC. Other molecules necessary for macropinosome formation and other kinds of signal transduction are DAG kinase [36], Rab5a [37], Cdc42 [38], Arf6 [39], RhoG [40], Trio [41] and phospholipase D [42]. Because signalling for macropinocytosis has been analysed thoroughly in only a few cell types, it remains possible that the contributions of various signalling molecules to macropinosome formation may be different in other cells or in different kinds of macropinocytic response (e.g. constitutive versus induced).

(a) Signal cascades are restricted to macropinocytic cups
Macrophages stimulated acutely with macrophage colonystimulating factor (M-CSF) show a striking asynchrony in the timing of the signals that appear at their surfaces. Rac1 activity and PIP 3 concentrations increase initially at cell margins, but then settle into a sustained pattern of transient spikes of activity, localized to macropinocytic cups and appearing at different times after stimulation [2] (figure 1b; electronic supplementary material, Video S1). Moreover, a full sequence of biochemical responses to M-CSF unfolds during the formation of each macropinosome. These responses include transient increases in PI(4,5)P 2 , DAG, PI(3,4)P 2 , PI3P and recruitment of Rab5a, Rab20, Rab21, Ras and PKCa [2,43,44] (figure 3). Maekawa et al. [45] described a similar sequence of activities required for macropinocytosis in Caenorhabditis elegans cells and identified essential roles for phosphoinositide phosphatases MTMR6 and INPP4P, and a Ca þþ -activated K þ channel (KCa3.1) in macropinosome formation. In macrophages, the irregular timing of these signal cascades relative to when M-CSF was added, and the correlation of the signal cascades with closure of ruffles into cups, indicate that signal propagation after receptor activation is contingent on the formation of cup domains in plasma membrane. Thus, the signals used to build macropinosomes are confined to cups and the closing macropinosome. inhibited the lateral diffusion of GFP into or out of those cups [46]. This suggests that the circular barrier of the cup facilitates signal propagation, perhaps through feedback amplification of PI3K or other signal molecules. However, physical barriers may not be required for the construction of large subdomains of plasma membrane. Axenic strains of the soil ameba Dictyostelium discoideum exhibit increased macropinocytosis due to mutations in the Ras GAP neurofibromin, which allows them to grow in liquid medium [11]. Macropinosome formation in these cells is organized by PI3K and Ras, whose activities are confined to large patches of plasma membrane surrounded by a narrow rim of the actin-regulatory protein SCAR/WAVE at the advancing edges of macropinocytic cups. Veltman et al., [16] showed that PIP 3 /Ras patch formation in D. discoideum occurred in cells whose actin cytoskeleton was disrupted by latrunculin A/B, which suggests that this large domain of plasma membrane does not require an actin-based diffusion barrier to maintain lateral heterogeneity. Other possible mechanisms of restricting lateral diffusion of plasma membrane molecules have been described [47]. At present, however, the physical properties and molecular components of cups essential for maintaining the sharp boundary of cup domains (PIP 3 spikes and Ras patches) remain largely undefined.
(c) Signalling for macropinocytosis is similar to signalling for phagocytosis Ingestion of particles by phagocytosis entails activities of the actin cytoskeleton which are much like the movements of macropinocytosis. In Fc receptor-mediated phagocytosis by macrophages, PI3K is required for ingestion of particles larger than 3 mm diameter [27,48]. Inhibition of PI3K does not prevent phagocytic cup formation, but rather inhibits a contractile activity that closes the cup into a phagosome [27]. Fluorescence microscopic methods showed PIP 3 and active Rac1 localizing to phagocytic cups throughout the ingestion process [49]. Förster resonance energy transfer-based microscopy of other small GTPases showed two distinct kinds of cytoskeletal regulation associated with phagosome formation: active Cdc42 and Arf6 localized to early stages of cup formation and to distal regions of cups, whereas active Rac2 and Arf1 appeared later and localized to the proximal, basal regions of cups [49]. This spatially organized conversion of one category of signals to another (Rac1/Cdc42/Arf6 to Rac1/Rac2/Arf1) within phagocytic cups was named the 'signal transition' [50]. The signal transition during phagocytosis is regulated by PI3K. Inhibition of PI3K does not limit early signals or cup initiation, but blocks the appearance of the late signals, indicating that PI3K is required for the transition from early to late signals. The signal transition during phagocytosis may be regulated by a PIP 3 concentration threshold. Macrophages fed particles with different surface densities of opsonizing IgG showed all-or-none ingestion responses. Over a wide range of IgG densities on particle surfaces, all particles stimulated actin-dependent movements over the particle surface and generation of PIP 3 in the plasma membrane near the particle, but the ingestion of particles with low IgG densities stalled [51]. Particles with higher densities of IgG were ingested completely, generated more PIP 3 in phagocytic cups, and recruited the late stage protein PKCe as the cups closed into phagosomes. These studies indicated that a threshold concentration of PIP 3 in phagosomal membranes must be exceeded to allow completion of phagocytosis. The PIP 3 concentration threshold could regulate the signal transition from early activities (actin polymerization and cup extension) to late activities (cup contraction and closure).
In macropinocytosis as in phagocytosis, distinct activities must be organized spatially on a relatively large scale, first to assemble the cup, then to close the cup into a macropinosome. Like phagocytosis, macropinosome formation may be modulated by the magnitude of PI3K signalling, in determining either the size of the macropinosome or the ability of cups to close into macropinosomes. Ruffling is a kind of exploratory behaviour, in which actin-rich protrusions extend and withdraw into the cell, and only occasionally close into macropinosomes. This behaviour is regulated by the readily reversible chemistry of PIP 3 synthesis (PI3K versus PTEN), similar to the excitable signal transduction networks associated with chemotaxis [15]. By contrast, the later stages of macropinosome formation require the hydrolysis of PI(4,5)P 2 to DAG, which may, by its irreversible nature, signal an all-ornone commitment to completing macropinosome closure. Thus, a cell's ability to make a macropinosome may be modulated by feedback that affects its ability to create a patch of the membrane with sufficiently high concentrations of PIP 3 to trigger the late signals necessary for cup closure.

(d) Downstream effectors of PI3K may be regulated differentially by PIP 3 concentration thresholds
The various PIP 3 -binding domains of signalling proteins bind 3 0 -phosphoinositides with different characteristic specificities and affinities. Some, such as the pleckstrin-homology (PH) domain of Bruton's tyrosine kinase (Btk) bind to PIP 3 specifically and with high affinity [52]. The PH domains of the different isoforms of Akt bind to both PIP 3 and PI(3,4)P 2 , but exhibit widely different affinities for those phospholipids [53]. Thus, the quality and quantities of PIP 3 -binding proteins recruited into a membrane-associated signal cascade will likely vary according to the concentrations of PIP 3 generated in membranes by the stimulus. Phosphorylation of Akt requires the recruitment to membranes of Akt and two additional PIP 3 -binding proteins, PDK1 [54,55] and mSIN1 [56], which is a component of mechanistic target of rapamycin complex 2 (mTORC2). Because Akt phosphorylation requires three different proteins to bind to PIP 3 near each other [57], Akt activation may require higher local concentrations of PIP 3 than does recruitment of Btk or activation of PLCg1. PI3K activity is stimulated by receptor-associated, PIP 3 -binding adapter proteins Gab1 and Gab2 [58,59]; their spatially organized activities could facilitate PIP 3 concentration increases confined to cup domains of macropinosomes or phagosomes. Moreover, different PIP 3 concentration thresholds within cups could define a sequence of PIP 3 -dependent effector activities associated with macropinosome formation.

(e) Traffic of macropinosomes into lysosomes or back to the cell surface is regulated
Macropinosome closure creates an intracellular vacuole with enclosed water and solutes from outside the cell. The membrane bounding the nascent macropinosome differs from plasma membrane in several respects: Rab5a, PI3P and DAG are enriched in the membrane, whereas PI(4,5)P 2 is depleted. The Rab5-positive macropinosome eventually matures to a Rab20/Rab21-positive organelle before acquiring Rab 7 [3,44,60,61]. Active Rab5a promotes the formation of tubulovesicular extensions [37], which are rich in PI3P-binding sorting nexins (Snx), especially Snx5 [62]. These morphological rearrangements alter the surface-to-volume ratio of the macropinosome vacuole and likely increase hydrostatic pressure inside the organelle [62]. Inhibition of Rab5 activity promotes macropinosome recycling to the plasma membrane [37]. Studies of PI3P dynamics during phagocytosis showed that concentrations of PI3P are integrated over phagosome membranes [63]. Thus, the concentrations of PI3P in macropinosome membranes and the attendant Rab5a activity could regulate whether macropinosomes recycle to the plasma membrane (low PI3P or Rab5 activity) or fuse with lysosomes (high PI3P or Rab5 activity). Thus, macropinosome formation and maturation occurs by a regular sequence of cytoskeletal activities and changes in membrane chemistry. The networks of molecular interactions that organize these activities and the transitions from one activity to the next are likely to be affected by positive and negative feedback regulation.

Macropinocytosis-associated signals that modulate cell metabolism
To what extent are the chemical reactions organized to build the organelle also conveying other information that cells use? Ingestion of extracellular albumin by macropinocytosis and its subsequent digestion in lysosomes is sufficient to support the growth of K-Ras-transformed cells [13]. In this way, the macropinosome delivers a nutrient required for cell growth. Moreover, recent studies demonstrated that amino acids internalized by macropinocytosis can signal their presence in lysosomes [6] and that macropinocytic cups and macropinosomes localize essential elements of growth factor signalling which are unrelated to the process of macropinosome formation [64,65]. These studies suggest that macropinocytosis is more than an aberrant process of nutrient acquisition used by transformed cells, but is rather an essential component of signalling for normal cell growth.

(a) Activation of mTORC1 requires ingestion of amino acids by macropinocytosis
The mechanistic target-of-rapamycin complex-1 (mTORC1) is a cytosolic protein complex that regulates metabolism in response to various inputs, including nutrient status and growth factor receptor signalling [66]. mTORC1 phosphorylates S6 kinase, 4EBP1 and other proteins which increase anabolic metabolism. Regulation of mTORC1 activity occurs on the cytosolic face of the lysosomal membrane. In nutrientrich conditions, Rag GTPases associated with lysosomes recruit mTORC1 from cytosol onto those membranes, where mTORC1 can be activated by the GTPase Rheb [67,68]. The ability of Rag GTPases to recruit mTORC1 to lysosomes is regulated by a complex of lysosome-associated proteins called Ragulator, which integrates detection of amino acid concentrations in cytosol and lysosomes [69,70]. Elevated lumenal concentrations of the amino acid leucine in the lysosome signal to Ragulator through the lysosomal membrane proteins V-ATPase and SLC38A9 [71,72]. Ragulator then recruits mTORC1 from cytosol [73]. Macropinocytosis provides a route for delivery of extracellular amino acids into lysosomes for the rapid activation of mTORC1. One biochemical model for measuring mTORC1 activation by growth factors or amino acids measures increased mTOR kinase activity following transient deprivation of growth factors or amino acids [74]. Re-addition of essential amino acids leads to activation of mTORC1 within 20 min in fibroblasts and HEK293 cells, which suggests that amino acids reach the lysosome lumen from outside the cell within that time period. Macropinocytosis can deliver solutes into lysosomes that quickly [6]. Growth factor stimulation enhances the activation of mTORC1 by amino acids. Moreover, activation of mTORC1 by amino acids also requires proteins that regulate endocytic membrane traffic, including Rab5a, Arf1 and enzymes which increase levels of the endolysosomal phospholipid PI(3,5)P 2 [75][76][77]. Inhibition of macropinocytosis in macrophages and fibroblasts, by the sodium-proton antiporter inhibitor ethylisopropylamiloride, by the cytoskeletal inhibitors jasplakinolide and blebbistatin, or by depletion of Rac1, leads to decreased phosphorylation of S6K in response to leucine and growth factors [6]. Upregulation of macropinocytosis by acute growth factor stimulation increases the rate and efficiency of extracellular leucine delivery into lysosomes, permitting the recruitment and activation of mTORC1 on lysosomes ( figure 4). This macropinocytosis-dependent pathway of mTORC1 activation serves as a vesicular mechanism of growth factor receptor signal transduction. Discovery of this dependency of mTORC1 activity on macropinocytosis led to the proposal that anabolic metabolism leading to cell growth is supported by amino acids internalized by macropinocytosis, or by similarly internalized proteins which are degraded to amino acids within lysosomes [6].
However, this concept of macropinocytosis-dependent cell growth was not supported by experimental models measuring cell proliferation instead of acute stimulation of mTORC1. Cells transformed by oncogenic Ras proteins exhibit increased macropinocytosis [78], which is required for protein synthesis and cell proliferation when albumin is provided as a source of amino acids [13,14]. By contrast, macropinocytosis is not required for proliferation of fibroblasts or Ras-transformed cells when amino acids are provided instead of albumin [79]. These studies suggest that amino acids reach mTORC1 and amino acid-detection mechanisms in cytosol by direct import of amino acids through transport proteins in the plasma membrane. The different results are not easily reconciled, especially for fibroblasts, which showed macropinocytosis-dependent activation of mTORC1 by amino acids [6] but macropinocytosis-independent proliferation in amino acids [79]. We speculate that the difference is due to mechanisms of cell growth or proliferation that are independent of mTORC1 activity [14,80]. Nonetheless, activation of mTORC1 by macropinocytosisdependent delivery of leucine into lysosomes is clearly detectable in acute responses to stimulation. Whether other amino acids are capable of this macropinocytosis-mediated signalling to mTORC1 remains unknown.

(b) The actin cytoskeleton differentially stimulates receptor-dependent phosphorylation of Akt
In addition to Rag GTPases, growth factor receptor signalling to mTORC1 requires that lysosome-associated Rheb be in its active, GTP-bound form. An essential component of Rheb activation is the kinase Akt, which is required for the activation of mTORC1 but not for macropinosome formation [34]. Rheb activity on lysosome membranes is maintained at low levels by the GTPase-activating protein (GAP) complex TSC1/2 [81], which also locates at the lysosomes. Growth factor receptor signalling activates Rheb via PI3K, which synthesizes PIP 3 in membranes. The PIP 3 -rich membrane recruits Akt and the kinases PDK1 and mTORC2, which phosphorylate Akt [57]. Phospho-Akt phosphorylates TSC2, leading to the dissociation of TSC1/2 complex from lysosome membranes and separation from its substrate Rheb [82,83]. Thus, Rheb activity toward mTORC1 is increased by signalling that activates Akt to displace the Rheb GAP TSC1/2. Whether macropinocytosis affects growth factor receptor signalling to Rheb via Akt remains largely unresolved. In our initial characterization of signalling in macrophages and fibroblasts stimulated by M-CSF and platelet-derived growth factor (PDGF), respectively, we observed that inhibition of macropinocytosis blocked S6K phosphorylation (a measure of mTORC1 activity) but did not reduce Akt phosphorylation [6]. This indicated the existence of a cytoskeleton-independent pathway from growth factor receptors to Rheb via PI3K, Akt and TSC1/2. However, a requirement of the actin cytoskeleton for growth factor-induced Akt phosphorylation has been demonstrated previously [84], and subsequent studies of macrophages stimulated with CXCL12 showed that cytoskeletal inhibition reduced phosphorylation of not only S6K, but also Akt and TSC2 [65]. Thus, CXCL12 initiates a cytoskeleton-dependent pathway leading to phosphorylation of Akt and TSC2. This suggests that ruffles or macropinocytic cups formed in response to CXCL12 amplify PI3K activity or otherwise locally stimulate phosphorylation of Akt.
Why is the actin cytoskeleton required for Akt phosphorylation in response to CXCL12 but not to M-CSF or PDGF? The signals generated by CXCR4, the G-protein-coupled receptor that binds CXCL12, may be qualitatively different from the signals generated by the M-CSF receptor, such that Akt phosphorylation occurs by distinct biochemical pathways which are differentially dependent on the actin cytoskeleton. However, we observed in macrophages that the maximal Akt phosphorylation response to CXCL12 was significantly lower than the maximal response to M-CSF [65]. We hypothesized that receptor signalling which generates low concentrations of PIP 3 in membranes may be enhanced by cytoskeletondependent amplification of PI3K activity, such as occurs in macropinocytic cups. Consistent with this model, stimulation of macrophages with lower concentrations of M-CSF (140 pM), which generated lower levels of Akt phosphorylation than did receptor-saturating concentrations of M-CSF (6.9 nM), could be reduced by cytoskeleton inhibitors [65]. A similar relationship between the maximal levels of Akt phosphorylation responses and sensitivity to cytoskeletal inhibition was also observed in fibroblasts stimulated with PDGF, which generates a high maximal Akt response, and epidermal growth factor, which generates a low maximal Akt response [85]. These studies indicate that the actin cytoskeleton can amplify PI3K activity locally, and suggest that ruffles, macropinocytic cups or macropinosomes may serve as platforms for localized amplification of Akt phosphorylation by both PDK1 (generating pAkt(308)) and mTORC2 ( pAkt(473)) [85]. Receptors with lower potential for generating PIP 3 in membranes may be PIP 3 in macropinocytic cups facilitates phosphorylation of Akt ( pAkt) and the activation of PLCg. pAkt is not required for macropinosome formation, but may be internalized on macropinosome membranes. PLCg generates DAG in the cup (green line), leading to PKC-dependent pathways that close the macropinosome. Extracellular proteins or amino acids internalized by the macropinosomes are delivered rapidly into lysosomes through fusion reactions. Nutrient transfer from macropinosomes to lysosomes leads to activation of Rag GTPases (black to red), followed by mTORC1 recruitment from cytoplasm onto lysosomal membranes. pAkt generated in plasma membrane or macropinocytic cups may reach its substrate TSC1/2 via macropinosome traffic to lysosomes. Phosphorylation of TSC2 by pAkt leads to dissociation of pTSC1/2 from lysosome membrane, displacing a GAP activity that allows GTP loading of Rheb on lysosome membranes (black to red). GTP-bound Rheb activates mTORC1 on the lysosomal membranes (black to red).
better suited to modulation of PIP 3 concentrations by the actin cytoskeleton and therefore better suited for organizing cytoplasmic signalling spatially. We proposed that macropinocytic cups or dorsal ruffles create large subdomains of the plasma membrane which localize signal amplification and propagation [86]. Such domains may restrict diffusion of enzymes, substrates and/or products, which could lead to locally elevated concentrations of PIP 3 . Alternatively or additionally, Akt phosphorylation may be directly increased by Rac1-dependent cytoskeletal activity. The Rac1 effector PAK, which is also required for macropinosome formation, can enhance Akt phosphorylation by serving as a scaffold for both Akt and PDK1 [87].

Possibilities
Whether macropinosomes form spontaneously or in response to receptor ligation, the spatially confined biochemical reactions leading to their formation are a kind of signal transduction. Macropinosomes are also capable of signalling to mTORC1 by delivering leucine or other amino acids into lysosomes, and macropinocytic cups provide platforms for localized activation of Akt. Additional roles for macropinocytosis in cell signalling, and for cell metabolism to feedback regulate signalling for macropinocytosis, are largely speculation. The distinct molecular constituents of macropinosome membranes may render them as platforms for signalling from internalized receptors [64,88]. The increased volume of ingested water and its subsequent flux across membranes and out of the cell could regulate cell volume or convey other information about extracellular conditions. Several additional possibilities are offered here.
(a) Akt phosphorylated at the plasma membrane could reach its substrate TSC2 on lysosomal membranes via macropinocytosis As described above, activation of PI3K by growth factor receptors leads to Akt phosphorylation in plasma membranes and eventually to Rheb activation on lysosomal membranes. The Rheb GAP TSC1/2 dissociates from lysosomal membranes when TSC2 is phosphorylated by phospho-Akt, thereby permitting activation of mTORC1 by Rheb [89]. It is not yet known how phospho-Akt at the plasma membrane accesses TSC2 on lysosomal membranes, but the enzyme and its substrate may be brought together via macropinocytosis ( figure 4). Akt is present in macropinocytic cups [90] and has been localized to macropinosomes [64,65], where it may be en route to lysosomes.

(b) Signalling in constant concentrations of growth factors may be stochastic and contingent on the formation of ruffles and cups
How important is macropinocytosis to receptor-regulated growth in metazoan cells? We propose that, in cells which are proliferating in constant concentrations of growth factor, receptor signalling is confined to cups and amplified by the cytoskeleton. As a mechanism to coordinate cell proliferation with organogenesis, growth factor-mediated regulation of cell growth may have co-opted macropinocytosis as an ancient mechanism of nutrient acquisition and feedback regulation. However, not all cells are as robustly macropinocytic as macrophages and tumor cells, and it remains to be determined whether macropinosomes form frequently enough or convey sufficiently strong signals to explain growth factor-dependent growth in other metazoan cells. Maybe when PI3K is active constitutively then other forms of endocytosis, less efficient than macropinocytosis, can deliver sufficient amino acids into lysosomes to activate mTORC1 and to support slow cell growth. Alternatively, amino acid transport proteins in plasma membrane of some cells may obviate the need for endocytic mechanisms of activating mTORC1. In that case, macropinocytosis may be unnecessary or function simply as a mechanism for clearing solutes and membranes.
(c) Nutrient status or cellular dimensions may feedback regulate macropinocytosis Cellular metabolic status can modulate macropinocytosis. Inhibition of mTORC1 or Akt increases rates of protein degradation in lysosomes without affecting rates of macropinosome formation [79], which suggests that nutritional status, signalling through mTORC1 or Akt, affects macropinosomelysosome fusion. More generally, these observations suggest that starvation or nutrient sufficiency regulates the signalling pathways that initiate or complete macropinosome assembly or that mediate macropinosome fusion with lysosomes. As a mechanism for scavenging extracellular nutrients, macropinocytosis is likely to be regulated by cellular metabolic status, just as metabolic status regulates degradation of cytoplasm by autophagy [91]. The large-scale ingestion of membrane and extracellular fluid that occurs during macropinosome formation changes cellular dimensions by decreasing cell surface area and increasing cell volume. In earlier experiments, we examined the effects of cell dimensions on pinocytosis by asking whether macrophages recognize satiety. We reasoned that if cells can monitor their dimensions, then they should be capable of responding to the enlargement of their lysosomal compartment that follows phagocytosis. We determined that expansion of the lysosomal compartment by phagocytosis of a latex bead meal or by ingestion of sucrose did not affect rates of basal pinocytosis but significantly inhibited pinocytosis in macrophages stimulated by phorbol myristate acetate (PMA), which we later determined to be macropinocytosis [9]. Moreover, the more cells ate, the less they drank [92,93]. The mechanism by which expansion of the lysosomal compartment or overall cell size inhibits macropinocytosis remains unknown, but it indicates the existence of a feedback regulation of macropinocytosis by cellular dimensions. We speculate that PI3K, PTEN and PIP 3 are components of a mechanism that translates cell dimensions into feedback regulation of cell behaviour, including macropinocytosis, phagocytosis and chemotaxis.

Conclusion
At the very least, macropinosomes organize and propagate signals necessary for their own construction. Their formation and maturation are subject to regulation by other inputs, and the distinct features of macropinosomes may influence other cell activities. The same signals that stimulate cell growth and oriented cell motility also direct macropinosome formation, which suggests that macropinocytosis itself is essential to cell growth. Although macropinosomes are capable of activating mTORC1 through delivery of amino acids into lysosomes, it is not yet clear whether these activities are required for cell growth. Macropinosomes may serve transient regulatory functions associated with their distinct compartmental identity within cytoplasm. That is, cells may be responsive to the abrupt appearance in cytoplasm of large acidic organelles with bounding membranes similar to plasma membrane but depleted of PI(4,5)P 2 and rich in PI3P, which possibly also maintain a membrane electrical potential or high internal hydrostatic pressure. Moreover, cells may call on such organelles to deal with unusual situations, such as the appearance in cytoplasm of pathogens or other disruptive particles [94].
Data accessibility. This article has no additional data. Authors' contributions. Both authors wrote, discussed and revised the manuscript. Y.S. performed the microscopy shown in figure 1. J.A.S. performed the microscopy shown in figure 2.