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. 2018 Jul;67(7):1272-1284.
doi: 10.2337/db17-1226. Epub 2018 May 10.

Regulation of Insulin Receptor Pathway and Glucose Metabolism by CD36 Signaling

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Free PMC article

Regulation of Insulin Receptor Pathway and Glucose Metabolism by CD36 Signaling

Dmitri Samovski et al. Diabetes. .
Free PMC article

Abstract

During reduced energy intake, skeletal muscle maintains homeostasis by rapidly suppressing insulin-stimulated glucose utilization. Loss of this adaptation is observed with deficiency of the fatty acid transporter CD36. A similar loss is also characteristic of the insulin-resistant state where CD36 is dysfunctional. To elucidate what links CD36 to muscle glucose utilization, we examined whether CD36 signaling might influence insulin action. First, we show that CD36 deletion specific to skeletal muscle reduces expression of insulin signaling and glucose metabolism genes. It decreases muscle ceramides but impairs glucose disposal during a meal. Second, depletion of CD36 suppresses insulin signaling in primary-derived human myotubes, and the mechanism is shown to involve functional CD36 interaction with the insulin receptor (IR). CD36 promotes tyrosine phosphorylation of IR by the Fyn kinase and enhances IR recruitment of P85 and downstream signaling. Third, pretreatment for 15 min with saturated fatty acids suppresses CD36-Fyn enhancement of IR phosphorylation, whereas unsaturated fatty acids are neutral or stimulatory. These findings define mechanisms important for muscle glucose metabolism and optimal insulin responsiveness. Potential human relevance is suggested by genome-wide analysis and RNA sequencing data that associate genetically determined low muscle CD36 expression to incidence of type 2 diabetes.

Figures

Figure 1
Figure 1
Characterization of the mouse with conditional muscle CD36 deletion. A: Induction of smCd36−/− using the HSA-rtTA Cre reduced Cd36 gene expression in the predominantly slow-twitch oxidative diaphragm (Dia) and in the mixed (slow-fast twitch, oxidative-glycolytic) gastrocnemius (Gastro) but not in the mixed quadriceps (Quad) or abdominal muscle (rectus abdominus, Abdom). CD36 expression in the heart, as expected, was not reduced. Tissues were obtained from smCd36−/− and floxed littermate controls (Cd36fl/fl). All mice were given doxycycline, followed by a 7-day washout period (see research design and methods for details). Cd36 gene expression by quantitative PCR was normalized to 36B4 and to Cd36 in gastrocnemius of Cd36fl/fl mice set as 1 (n = 3–4 males/genotype). **P < 0.01. B: Representative Western blots show reduced CD36 protein in diaphragm (Dia) but not in quadriceps (Quad) muscle. Graph: means ± SE of CD36/β-actin expression (n = 3/genotype, males). C: Immunostaining of CD36 in gastrocnemius sections of Cd36fl/fl and smCd36−/− mice show CD36 depletion in muscle fibers of smCd36−/−. Representative images of five to six fields (n = 3/genotype, males). Scale bar: 100 μm. D: Body composition of Cd36fl/fl and smCd36−/− mice. Shown are means ± SE of fat and lean mass (n = 4/genotype, males). E: Muscle CSA for Cd36fl/fl and smCd36−/− mice. Hematoxylin and eosin–stained gastrocnemius sections were scanned and analyzed. Representative areas are shown (scale bar: 100 μm). Graph shows means ± SE of 228 individual muscle fibers for Cd36fl/fl and 359 for smCd36−/− (n = 3/genotype, males). Fasting plasma levels of free FA (FFA) (F), glucose (G), and TG (H) in Cd36fl/fl and smCd36−/− mice. Shown are means ± SE (n = 4–8/genotype, n = 3–4 females, 4–5 males). I: Diaphragm (Dia) and quadriceps (Quad) TG content (means ± SE (n = 3/genotype, males). J: Reduced muscle content of ceramide species in diaphragm of smCd36−/− mice. The decrease correlates with CD36 knockdown and is not observed in quadriceps (Supplementary Fig. 1A) (n = 4/genotype, males). *P < 0.05 **P < 0.01. K: Diacylglycerol species in diaphragm (see also Supplementary Fig. 1B) (n = 4/genotype, males).
Figure 2
Figure 2
Muscle CD36 deletion alters glucose metabolism in vivo. A: Cd36 deficiency in gastrocnemius reduced expression of genes for insulin signaling (insulin receptor substrate-1 [IRS1] and phosphatidylinositol 3-kinase regulatory-α [PI3KR1]) and glucose metabolism (hexokinase [HK], phosphofructokinase-2 [PFK2], pyruvate dehydrogenase B1 [PDHB1], aldolase A [ALDOA], and ACLy) while pyruvate dehydrogenase kinase 4 (PDK4) and FOXO1 (FoxO1) increased. FA metabolism genes, FA transport protein 1 (FATP1), carnitine palmitoyl transferase 2 (CPT2), and FA binding protein 3 (FABP3) were unchanged. Mitochondrial genes (Cox2, Porin1, and CPT1B) (Supplementary Fig. 1C) and protein levels of electron transfer chain complexes (C1–C5) (Supplementary Fig. 1D) were also unaltered. Gene expression by quantitative PCR normalized to 36B4. Shown are means ± SE (n = 7–14 mice/genotype, males). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with Cd36fl/fl mice. B: Glucose disposal is not affected by muscle CD36 depletion. Intraperitoneal glucose tolerance test (ipGTT) for Cd36fl/fl and smCd36−/− mice (n = 4 males and 3 females/genotype). Shown are means ± SE of glucose as percentage of basal level (see also Supplementary Fig. 2AC). C: FDG uptake is suppressed in hind-limb muscle but not in heart or abdominal (Abdom) muscle of smCd36−/− mice compared with Cd36fl/fl. Data are percentages of injected dose (%ID) per organ shown as means ± SE of cumulative tracer uptake at 60 min (n = 4/genotype, females). D: Insulin-stimulated glucose oxidation and glycolysis is suppressed in diaphragm explants from smCd36−/− mice. Glucose oxidation and glycolysis were assayed by Seahorse XF24. Shown are means ± SE AUC (n = 3/genotype, males, five technical replicates). OCR, oxygen consumption rate. *P < 0.05 and **P < 0.01 compared with Cd36fl/fl.
Figure 3
Figure 3
Skeletal muscle CD36 is important for postprandial glucose disposal. A: Glucose disposal after a fatty meal is impaired in smCd36−/− mice. Cd36fl/fl and smCd36−/− mice were given intragastric palm oil (1:1 with skim milk) or skim milk (vehicle). Glucose was administered i.p. 2 h later, and glucose disposal was measured in tail blood. Shown are means ± SE of % change in glucose related to level before i.p. glucose injection (n = 6–8/genotype). *P < 0.05 and **P < 0.01 compared with Cd36fl/fl controls (n = 6–8/genotype, 1–2 females, 6 males). B: AUC (means ± SE) for glucose disposal in mice given palm oil. Plasma TG (C) and free FA (FFA) (D) in mice after palm oil feeding. Cd36fl/fl (n = 3 males and 3 females) and smCd36−/− (n = 8, 5 males and 3 females) mice were given intragastric palm oil or skim milk (vehicle), followed 2 h later by glucose i.p. and blood collection. Shown are means ± SE of plasma TG and free FA. E: Muscle insulin signaling after a fatty meal is impaired in smCd36−/− mice. Representative immunoblots of diaphragm (Dia) and gastrocnemius (Gastroc) muscle from Cd36fl/fl and smCd36−/− mice. Mice were given palm oil as in A, followed 2 h later by i.p. insulin, and tissues were harvested after 15 min. Quantification of pAKT in diaphragm (Dia), gastrocnemius (Gastro), and quadriceps (Quad) (F) and of pGSK3 and pACLy in diaphragm (G) (n = 4–5/genotype, 2–3 males, 2 females). *P < 0.05 compared with Cd36fl/fl controls. HK: Muscle CD36 knockdown does not protect against HFD-induced insulin resistance. H: Weight gain at the end of 5 weeks of chow or HFD feeding. Groups were initially weight matched (n = 4/diet/genotype, males). ***P < 0.001. Weight (means ± SE) HFD compared with chow controls. I: TG content (means ± SE) of diaphragm (Dia) and quadriceps (Quad) (n = 4/genotype, males). *P < 0.05 compared with Cd36fl/fl controls. J: Intraperitoneal insulin tolerance test (ipITT) for Cd36fl/fl and smCd36−/− mice. Mice fasted for 4 h were given 0.75 IU/kg insulin, and blood glucose was monitored. Shown are means ± SE of glucose as percentage of basal before insulin (n = 4/genotype, males, representative of two experiments). (See also Supplementary Fig. 2D and E.) K: FDG uptake is similar in hind limb and heart of Cd36fl/fl and smCd36−/− mice fed the HFD. Shown are means ± SE of organ tracer uptake at 60 min, expressed as percentage of injected dose (%ID) per organ (n = 4/genotype, males, representative of two experiments).
Figure 4
Figure 4
CD36 modulates insulin signaling in HSMMs. A: Insulin (Ins) induces membrane CD36 translocation in differentiated HSMMs. Myotubes serum-starved for 16 h were incubated with or without insulin (100 nmol/L, 15 min) and processed for immunostaining: red, mouse monoclonal anti-CD36; blue, DAPI (nuclei). Images are representative of multiple fields from three experiments. C, control. Scale bar: 10 μm. B: Representative bright-field images of differentiated myotubes after treatment with CD36-targeted siRNA (siCD36-1) or with a nonspecific control siRNA (siC). Western blot shows expression of myosin heavy-chain and β-actin in siC and siCD36 myotubes, representative of two experiments. C: CD36 depletion decreases insulin (Ins) signaling in myotubes. Immunoblot shows insulin signaling in siC and siCD36 HSMM. Insulin (100 nmol/L) was added for 5 min. Blots were probed for CD36, pAKTT308, S473, total AKT, pGSK3 α/βS21/9 (pGSK3), pACLyS455, and β-actin. Blots are representative of four experiments. D: Quantification of CD36 knockdown normalized to β-actin. Shown are means ± SE of five experiments, as percentage of siC. ***P < 0.001. E: Quantification of insulin-induced pAKTT308, S473, pGSK3βS21/9 (bottom band), and pACLyS455. Shown are means ± SE of fold change compared with insulin-treated siC myotubes (n = 4 experiments). *P < 0.05, **P < 0.01. F: Acute CD36 inhibition by SSO suppresses insulin signaling: HSMMs were serum-starved and incubated with or without SSO (20 μmol/L, 15 min), followed by insulin as in C. Immunoblots of cell lysates (triplicates from different wells), representative of two experiments. G: Quantification of insulin-induced pAKTT308 and pGSK3βS21/9 with or without SSO. Shown are means ± SE from two experiments with triplicate samples. *P < 0.05 vs. controls (no SSO). H: Glycolysis was measured (Seahorse XF96 analyzer) from the ECAR in siCD36 or siC HSMMs. The following were added when indicated: (A) glucose ± insulin, (B) oligomycin, and (C) 2DG. Shown are means ± SE of 32 wells from one experiment representative of two more. I: Glycolytic measurements (means ± SE) from three experiments. *P < 0.05, ***P < 0.001 compared with respective unstimulated siC. J: CD36 depletion suppresses insulin-induced glucose uptake. siCD36 or siC HSMMs were incubated (60 min) with 0.1 mmol/L 2DG (1 μCi/mL) in PBS in the presence or absence of insulin. Disintegrations per minute (DPM) were normalized by WGA staining. Shown are means ± SE of four experiments. **P < 0.01 vs. untreated siC. K: CD36 depletion suppresses insulin-induced glycogen synthesis. siCD36 and siC HSMMs were incubated (2.5 or 5 h) with [3H]glucose (1 μCi/mL) in presence or absence of insulin. Insulin-induced fold change in radiolabel incorporation, normalized by WGA staining, is shown. Means ± SE of three experiments, three to five replicates per experiment. **P < 0.01 compared with siC. L: SSO (as in F) inhibits glucose uptake by HSMMs. Shown are means ± SE of three experiments. **P < 0.01 compared with untreated controls, #P < 0.05 compared with insulin-treated controls.
Figure 5
Figure 5
CD36 mediates FA effects on insulin (Ins) signaling in myotubes. A: Saturated MA and PA but not monounsaturated OA suppress insulin signaling. Top: HSMMs were incubated 15 min with OA, PA, or MA (200 μmol/L at 2:1 BSA), followed by addition of insulin (100 nmol/L, 5 min). Cells were lysed, and pAKT(S) and β-actin were detected by immunoblotting. Data are representative of two experiments. Bottom: HSMMs were incubated with different MA concentrations, followed by insulin, lysed, and immunoblotted. Data are representative of two experiments. C, control. B: CD36 mediates suppression of insulin signaling by PA. ICW assay showing effect of CD36 depletion on insulin signaling with and without FA. HSMMs were incubated with OA or PA (2:1 BSA, 200 μmol/L, 15 min), followed by incubation with insulin (100 nmol/L, 5 min), fixation, and processing for ICW. pAKTS473 quantification normalized by WGA staining. Shown are means ± SE (n = 24 samples per condition, representative of three experiments). ***P < 0.001 vs. no insulin, ###P < 0.001 vs. insulin-treated siC. C: CD36/IR/+CD36 (+CD36) or vehicle (V) control CHO/IR cells were incubated with PA, followed by insulin, fixed, and processed for ICW. Graph shows pAKTS473 quantification normalized by WGA staining. Shown are means ± SE for n = 24 samples per condition, representative of two experiments. **P < 0.01 vs. insulin-stimulated +CD36 cells. D: Unsaturated FA do not suppress insulin signaling. Vector (V) or +CD36 CHO cells were treated with EPA, PO, M16, OA, PA, or LA, followed by insulin (100 nmol/L, 5 min). Cells were lysed and immunoblotted as indicated. Data are representative of two experiments. E: Schematic summary of key steps in regulation of glucose metabolism by insulin. Activation of IR leads to Akt phosphorylation and enhanced glucose uptake. Insulin induces phosphorylation of GSK3, which promotes glycogen synthesis. Glucose metabolized via glycolysis can be further oxidized in the citric acid cycle. Insulin signaling also promotes FA synthesis by enhancing phosphorylation of the key lipogenic enzyme ACLy, which converts citrate to cytosolic acetyl-CoA.
Figure 6
Figure 6
CD36 interacts with IRβ and enhances its tyrosine phosphorylation. A and B: CD36 and IRβ co-IP. A: CHO/IR/CD36 cells (+CD36) or empty vector controls (V) were lysed, and equal concentrations of lysate protein were used to IP IRβ. B: Reciprocal IP using anti-CD36 antibody. Data are representative of three experiments. C: CHO/IR/+CD36 cells were stained and processed for PLA. CD36-only staining (no anti-IRβ antibody) was used as the negative control. Nuclei were visualized by DAPI (blue). Data are representative of three experiments. Scale bar: 10 μm. D: CD36 expression facilitates IRβ tyrosine phosphorylation and P85 recruitment. Vector or +CD36 cells were incubated with or without insulin (100 nmol/L, 5 min) and lysed. Equal lysate protein was used to IP IRβ, and IPs were probed for pY100 (pIRβ), P85, and IRβ. Quantification of insulin-induced pIRβ/IRβ data are means ± SE from three experiments. *P < 0.05. E: Equal amounts of lysate protein (3.5 mg/sample) from gastrocnemius (Gastroc) and quadriceps (Quad) muscles were subjected to IRβ IP and resolved by SDS-PAGE. The IPs were immunoblotted for CD36 and IRβ. Data are representative of two experiments. F: Proximity ligation assay of gastrocnemius (Gastroc) paraffin-embedded sections stained with anti-CD36 and anti-IRβ antibodies. Combined CD36 and IRβ staining resulted in appearance of typical punctate patterns of amplified DNA. Nonspecific IgGs, isotype-matched to CD36 and IRβ antibodies, were used as negative controls. Images are representative of three mice. Scale bar: 35 μm.
Figure 7
Figure 7
CD36 facilitates Fyn recruitment to plasma membrane and interaction with IRβ. A and B: Combined Fyn and CD36 expression enhances insulin-stimulated IRβ phosphorylation. CHO/IR+CD36 or vector (V) control (C) cells were transiently transfected with or without Fyn. Cells were incubated with or without insulin (Ins; 100 nmol/L, 5 min), lysed, and equal cell lysate protein was used to IP IRβ. Data are representative of three experiments. C: Top panels: V or +CD36 CHO/IR cells transiently transfected with Fyn were incubated with or without insulin, fixed, and stained with anti-Fyn antibody. Bottom panel: +CD36 cells transiently transfected with Fyn were incubated with insulin, fixed, and imaged for Fyn and IRβ. Image shows overlay between IRβ (red) and Fyn (green). Image magnification shows distinct punctate yellow structures, suggesting IRβ-Fyn colocalization. Data are representative of two experiments. Scale bar: 10 μm. D and E: PA suppresses Fyn association with CD36 and IRβ. +CD36 or V CHO/IR cells were preincubated with PA (200 μmol/L, 2:1 BSA, 15 min), then with insulin (100 nmol/L, 5 min), and lysates were used to IP CD36. Resolved IPs and inputs were immunoblotted for CD36, Fyn, and pFynY416. F and G: Differentiated C2C12 myotubes, treated as in D, were processed for IP of IRβ, and IPs were probed for IRβ and Fyn. Data are representative of two experiments.
Figure 8
Figure 8
COOH-terminal CD36 mutation suppresses IRβ-Fyn interaction and blunts insulin signaling. A: CHO/IR/+CD36 and CHO/IR/+CD36K/A cells were incubated with insulin and cell lysates and immunoblotted for the indicated proteins. Data are representative of two experiments. B: CHO/IR/+CD36 and CHO/IR/+CD36K/A cells were incubated with or without insulin, fixed, and processed for ICW assay. Quantification of pAKTS473 normalized by WGA. Shown are means ± SE of 24 samples per condition, representative of three experiments. ***P < 0.001 vs. +CD36. C and D: +CD36 and +CD36K/A cells were incubated with or without insulin (100 nmol/L, 5 min), lysed, and lysates were used for IP of IRβ. IP (C) and cell lysates (D) were immunoblotted as indicated. Data are representative of two experiments. E: Insulin-induced glycolysis is reduced in the CD36K/A mutant. ECAR was measured in +CD36 and +CD36K/A cells. The following compounds were added when indicated: (A) glucose/glucose + insulin, (B) oligomycin, and (C) 2DG. Shown are means ± SE of 32 wells from one experiment representative of two more. F: Proposed mechanism for regulation of glucose metabolism by CD36 and FA. Left panel: Insulin stimulates IR signaling and Akt phosphorylation to drive glucose uptake and utilization. CD36 association with IR promotes Fyn recruitment to IR to increase its tyrosine phosphorylation (pY). This enhances IR signaling and muscle glucose utilization. Right panel: Saturated FA (SFA) trigger Fyn dissociation from CD36 and IR, reducing IR phosphorylation and signaling and diminishing glucose uptake. Thus, CD36 enhances glucose metabolism in skeletal muscle and its saturated FA sensing spares glucose, when FA and glucose are both available.

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