It could be that the user identity is outdated, especially if you’ve tried starting a stopped app pool and the next request again fails.
In IIS, go to the Application Pools under the Server, then find the correct Application Pool for your web site, and click on it. On the Advanced Settings menu to the right, select Identity and change it and enter new user and password. Click on your Application Pool again, and select Recycle to restart it.
You can also try looking at the error message in Event Viewer, under Windows Logs, Application, Details tab.