Insulin/IGF-1 signaling, including class II/III PI3Ks, β-arrestin and SGK-1, is required in C. elegans to maintain pharyngeal muscle performance during starvation

Abstract

In C. elegans, pharyngeal pumping is regulated by the presence of bacteria. In response to food deprivation, the pumping rate rapidly declines by about 50-60%, but then recovers gradually to baseline levels on food after 24 hr. We used this system to study the role of insulin/IGF-1 signaling (IIS) in the recovery of pharyngeal pumping during starvation. Mutant strains with reduced function in the insulin/IGF-1 receptor, DAF-2, various insulins (INS-1 and INS-18), and molecules that regulate insulin release (UNC-64 and NCA-1; NCA-2) failed to recover normal pumping rates after food deprivation. Similarly, reduction or loss of function in downstream signaling molecules (e.g., ARR-1, AKT-1, and SGK-1) and effectors (e.g., CCA-1 and UNC-68) impaired pumping recovery. Pharmacological studies with kinase and metabolic inhibitors implicated class II/III phosphatidylinositol 3-kinases (PI3Ks) and glucose metabolism in the recovery response. Interestingly, both over- and under-activity in IIS was associated with poorer recovery kinetics. Taken together, the data suggest that optimum levels of IIS are required to maintain high levels of pharyngeal pumping during starvation. This work may ultimately provide insights into the connections between IIS, nutritional status and sarcopenia, a hallmark feature of aging in muscle.

Figure 1. Reduction of function in DAF-2 prevents recovery of pharyngeal pumping.

Young adult animals (15 per group) were grown overnight at the temperatures indicated (♦ 15°C; ▪ 20°C, or ▴ 25°C) and pharyngeal pumping was then evaluated on food (0 hr time point) and 2, 3.5, 6 and 24 hr after removal from bacteria. The data are expressed as the average pumps per minute (PPM). Standard deviations are represented by the error bars. Significant differences from the N2 controls are indicated by asterisks, *p<0.05; **p<0.01 as determined by ANOVA and a Newman-Keuls test. Results are shown for temperature-sensitive strains, A) daf-2(e1370) and B) daf-2(e1371).

Figure 2. Severity of daf-2 allele affects recovery of pharyngeal pumping during starvation.

Young adults (20 per group) from the various strains indicated in A) & B) were grown overnight at 25°C and pharyngeal pumping was measured as described above. To simplify the figure, we have only shown the 99% confidence intervals around the means of the N2 control groups. Experimental group means lying outside these intervals are significantly different from the controls with a p value <0.01.

Figure 3. Identification of insulins involved in pumping recovery.

A) Young adults were grown overnight at the temperatures indicated and recovery of pharyngeal pumping was assayed as before. Pumping in the daf-28 strain (25°C) at the 3.5 and 24 hr time points was significantly (p<0.01) faster than the control. B) For these experiments, young adults from the various strains were grown at 15°C (the standard condition) and recovery of pumping during starvation was measured as before. The 99% confidence intervals around the control means have been depicted here.

Figure 4. Effect of mutations in genes that potentially regulate insulin/neuropeptide release.

A) & B) Young adults (20 per group) from the various strains indicated in the figure were evaluated for recovery of pumping during food deprivation under standard conditions. The 99% confidence intervals for the control data are indicated as before.

Figure 5. Effect of mutations in genes encoding signaling molecules downstream of DAF-2 on the recovery response.

A) & B) The strains listed here were evaluated under standard conditions described above; gf and lf refer to gain-of-function and loss-of-function mutations, respectively. C) For these experiments, young adults (20 per group) from the N2 and daf-16; age-1 strains were incubated on plates with food and either DMSO (0.8%; vehicle control) or LY294002 (LY; 150 µM) for 1.5 hr at 15°C prior to food deprivation and evaluation of pharyngeal pumping. The 0-hr data point represents pumping on food in the presence of either DMSO or LY294002. The 99% confidence intervals for the control data are depicted for comparison.

Figure 6. Role of potential downstream targets of IIS in recovery of pharyngeal pumping.

A) The strains shown here were evaluated under standard conditions described above. For R09B5.11(ok1759) experiments, the XA7401 strain was used. B) Young adults (20 per group) from the N2 or XA7401(R09B5.11) strains were grown overnight on plates with food and 2-deoxyglucose (2-DOG; 5 mM final concentration) or dilute acetic acid (control; CON). The next day, they were evaluated for recovery of pharyngeal pumping off food. The 99% confidence interval for control means (N2 or R09B5.11) are shown as before.

Figure 7. Model of how IIS affects recovery of pharyngeal pumping during starvation.

This scheme is discussed in the text. Although the neuron is depicted as close by to the pharyngeal muscle, in reality, the neuronal signal (e.g., insulin and/or neuropeptides) may act more like a neurohormone over a greater distance. The depiction of the DAF-2 receptor is intended to show different binding modes for insulin molecules (e.g., INS-1 vs. DAF-28). Signaling downstream of DAF-2 bifurcates into Path 1, mediated by PIKI-1 and ARR-1, and Path 2, the classical pathway mediated by AGE-1 and AKT-1. INS-?? refers to additional C. elegans insulins that participate in this response.