As already was said, RegisterApiControllers is an extension method, only virtual members can be intercepted by NSubstitute.
But it's not that bad, it sticks you to have more clear and loosely coupled design through tests. With tools like Fakes or JustMock you can mock everything, and they don't force you to make the system loosely coupled.
I think your test is useless, the thing that you registered the controllers does not mean you can resolve them. I would suggest to test that you can resolve them. Something like this:
[Test]
public void ResolvesServiceController()
{
// Arrange
var configuration = new HttpConfiguration();
var containerBuilder = new ContainerBuilder();
DependencyResolverConfig.Register(configuration, containerBuilder);
// Act
var controller = configuration.DependencyResolver.GetService(typeof (ServiceController));
// Assert
Assert.IsNotNull(controller);
}
Then you can make further refactorings, ie create an Autofac module where you register your controllers and their dependencies, then test exact this module.